Methods for producing genetically modified cells

Abstract

The present disclosure relates to a new modular approach for the generation of genetically modified cells, in particular immune cells and iPSCs, that uses a common Cas9 element to simultaneously enable precise editing of defined nucleic acid targets (knock-out) and introduction of selected exogenous sequences at desired loci (knock-in).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC § 119(e) to U.S. Provisional Patent Application No. 63 / 203,996, filed August 6, 2021, which is incorporated by reference herein in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates to new methods, cells, systems, kits, and other embodiments for generating genetically engineered cells using a Clustered Interspaced Regularly Short Palindromic Repeat (CRISPR)-based gene editing system for introducing multiple genetic modifications into cells. [Background technology]

[0003] Precise genetic modulation of primary human cells has multiple applications for the treatment of human diseases, including in the fields of immunotherapy, autoimmunity, and enzymopathies. For example, genetic modulation of a patient's immune cells is an attractive route of therapy due to the durability of the changes made to the immune cells and the low risk of rejection of such cells by the patient. One approach for gene editing of immune cells uses the clustered regularly interspaced short palindromic repeats (CRISPR) system to induce double-strand breaks (DSBs) within the gene of interest, which are subsequently repaired by the efficient but error-prone non-homologous end joining (NHEJ) pathway or the less efficient but high fidelity homology-directed repair (HDR) pathway. The NHEJ repair pathway is the most active repair mechanism, often resulting in a small number of nucleotide insertions or deletions (indels) at the DSB site, resulting in amino acid deletions, insertions, or frameshift mutations that lead to premature stop codons or nonsense mutations within the open reading frame (ORF) of the target gene. Moreover, inducing multiple DSBs during multiplexed gene editing procedures may cause the formation of large chromosomal translocations with undesirable genotoxic and potentially carcinogenic properties. More precise gene editing can be achieved by the use of engineered nucleases (e.g., Cas9 nickase) that retain only one active nuclease domain and generate DNA nicks rather than blunt-end DSBs. A mutant of SpCas9, variant Cas9 D10A, retains only HNH nuclease activity and generates staggered DSBs in the presence of two guide RNAs (gRNAs) targeting opposite DNA strands, thus enhancing target specificity.

[0004] Chimeric antigen receptor-T (CAR-T) cell immunotherapy is a novel method that involves genetic modification of a patient's own T cells to express a CAR specific for tumor antigens. This method is a personalized treatment that involves ex vivo expansion of genetically modified cells and then reinjecting them into the patient. This therapy has shown impressive results in blood cancers, with anti-CD19 CAR-T therapy approved for the treatment of CD19-positive leukemia or lymphoma (Yescarta™, Kymriah™, Tecartus™, and Breyanzi™), and anti-BCMA CAR-T therapy approved for multiple myelomas (Abecma™). Despite promising results in some patients, the application of CAR-T causes several acute side effects, such as cytokine release syndrome and neurotoxicity, which in some cases leads to the death of the patient.

[0005] Long-term safety outcomes, such as immunogenicity and adverse effects on the proliferation and development of genetically modified T cells, remain concerns regarding this therapy. Therefore, there is a demand to develop improved CAR-T cell therapies with reduced side effects and health risks to patients. Furthermore, given the level of complexity associated with the personalized approach currently required, there is a need to develop "universal" or allogeneic CAR-T cell therapies to address issues related to time to treatment, manufacturing, quality and cost.

[0006] CARs are typically transduced into patient T cells using random integration vectors, which can result in oncogenesis, expression of diverse transgenes, and transcriptional silencing. In recent years, advances in genome editing have enabled efficient and targeted gene delivery. Directing a CD19-specific CAR to the T cell receptor alpha constant (TRAC) locus allows expression of the CAR under the control of endogenous TRAC regulatory elements, which enhances T cell potency and delays attrition. Summary of the Invention

[0007] In a first aspect, the present disclosure provides a method for making multiple genetic modifications to a cell, comprising: a) a CRISPR system for integrating an exogenous sequence into a first target nucleic acid sequence, i) a first gRNA and a second gRNA that are complementary to opposite strands of a first target nucleic acid sequence; and ii) a donor nucleic acid sequence comprising an exogenous sequence CRISPR systems, including b) a base editing system for introducing a genetic modification into a second target nucleic acid sequence, i) an RNA scaffold comprising a guide RNA sequence complementary to a second target nucleic acid sequence and a recruitment RNA motif; and ii) an effector fusion protein comprising an RNA-binding domain capable of binding to a recruitment RNA motif and an effector domain comprising a base-modifying enzyme; A base editing system comprising the c) an RNA-guided nickase capable of interacting with the first and second gRNAs of the CRISPR system and the RNA scaffold of the base editing system; into a cell and / or express in the cell; Culturing the cells to generate cells containing multiple genetic modifications. The present invention provides a method comprising:

[0008] In either embodiment, the method can be performed using only one RNA-guided nickase (also referred to herein as a single RNA-guided nickase or a common RNA-guided nickase), since the RNA-guided nickase can interact with both the CRISPR system and the RNA scaffold of the base editing system. This can be advantageous as it reduces the number of components that need to be provided and delivered to the cell.

[0009] In some embodiments, the base modifying enzyme has cytosine deamination activity, adenosine deamination activity, DNA methyltransferase activity, or demethylase activity.

[0010] In some embodiments, the RNA-guided nickase can be a CRISPR type II or V enzyme. In some embodiments, when the RNA-guided nickase is a CRISPR type II enzyme, the enzyme is a Cas9 nickase. In one embodiment, the RNA-guided nickase is nCas9 with one or two uracil glycosylase inhibitors (UGI).

[0011] In some embodiments, the first and second gRNAs may be provided as sgRNAs.

[0012] In some embodiments, the RNA scaffold used in the methods, cells, systems and kits herein may comprise tracrRNA. In CRISPR type II system, the maturation of precursor crRNA (pre-crRNA) requires the participation of trans-acting CRISPR (tracr)RNA. However, in CRISPR type V system, tracrRNA has not been identified at all, and pre-crRNA processing is mediated by type V effector protein itself.

[0013] In some embodiments, the RNA scaffolds used in the methods, cells, systems and kits herein can be introduced into a cell as chemically synthesized RNA and can include one or more chemical modifications.

[0014] In some embodiments, the methods, cells, systems, and kits provided herein can utilize one or more recruitment RNA motifs, which in some embodiments are located at the 3' end of the RNA scaffold. The recruitment RNA motif can be an MS2 aptamer, which in some embodiments can be an MS2 aptamer with an extended stem, for example, an extended stem comprising 2-24 nucleotides.

[0015] In some embodiments, the methods, cells, systems, and kits provided herein can use effector domains with cytosine deamination activity or cytidine deamination activity (these terms are used interchangeably), such as wild-type or engineered versions of AID, CDA, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, or other APOBEC family enzymes.

[0016] In some embodiments, the methods, cells, systems, and kits provided herein can use effector domains with adenine deamination activity or adenosine deamination activity (these terms are used interchangeably), e.g., wild-type or engineered versions of ADA, ADAR family enzymes, or tRNA adenosine deaminase.

[0017] In some embodiments, the methods, cells, systems, and kits provided herein can use effector domains with DNA methyltransferase activity, such as wild-type or engineered versions of Dnmt1, Dnmt3a, or Dnmt3b.

[0018] In some embodiments, the methods, cells, systems, and kits provided herein can use effector domains with demethylase activity, such as wild-type or engineered versions of Tet1, Tet2, or TDG.

[0019] In some embodiments, the methods, cells, systems, and kits provided herein may use a first gRNA and a second gRNA that are complementary to opposite strands of the TRAC or B2M locus.

[0020] In some embodiments, the methods, cells, systems, and kits can use a modular system that includes multiple base editing systems that can bind to different target nucleic acid sequences to genetically modify multiple different genetic loci.

[0021] In some embodiments, the CRISPR system used in the method herein can introduce a donor nucleic acid sequence that includes a CAR or TCR coding sequence flanked by specific homology arms to a first target nucleic acid sequence.In some embodiments, the CAR or TCR coding sequence is integrated into the TRAC or B2M locus.The expression of the CAR or TCR coding sequence can be driven by endogenous TRAC or B2M promoter.

[0022] In some embodiments, the nucleic acid encoding each of CRISPR system, base editing system and RNA-guided nickase can be introduced into cell in a single transfection step.In some embodiments, donor nucleic acid sequence can be introduced into cell using viral vector, for example, AAV.Alternatively, donor nucleic acid sequence can be introduced into cell in a single transfection step.

[0023] In some embodiments, the methods, cells, systems, and kits provided herein include a base editing system that introduces one or more genetic modifications that correct a genetic mutation, inactivate expression of a gene, change the expression level of a gene, or change intron-exon splicing. In other embodiments, the genetic modification introduced by the base editing system can be a point mutation, and optionally, the point mutation introduces a premature stop codon, destroys a start codon, destroys a splice site, or corrects a genetic mutation. In some embodiments, the guide RNA sequence used in the methods provided herein can include a splice acceptor-splice donor site (SA-SD) sequence.

[0024] In some embodiments, the methods, cells, systems and kits provided herein can target different genes in cells.For example, the base editing system can introduce gene modifications that result in the downregulation of any one or more of TRAC, TRBC1, TRBC2, PDCD1, CD52 and B2M.

[0025] In some embodiments, the methods, cells, systems, and kits provided herein can be used to provide multiple genetic modifications that occur simultaneously.

[0026] In some embodiments, the methods provided herein can be used to modify any cell, particularly immune cells or human pluripotent stem cells (hPSCs). Immune cells can include T cells, natural killer (NK) cells, B cells, myeloblasts, lymphoblasts, and CD34+ hematopoietic stem progenitor cells (HSPCs).

[0027] In one particular embodiment, the immune cells are primary T cells.

[0028] In one particular embodiment, the cells are induced pluripotent stem cells (iPSCs).

[0029] In a second aspect, the present disclosure provides a genetically modified cell obtained by the method described herein.In some embodiments, the genetically modified cell comprises an exogenous CAR or TCR coding sequence at the endogenous TRAC or B2M locus and at least one point mutation in three or more genes.In other embodiments, the genetically modified cell comprises an exogenous CAR or TCR coding sequence at the endogenous TRAC or B2M locus and at least one point mutation in three or more genes selected from the group consisting of TRAC, TRBC1, TRBC2, PDCD1, CD52, and B2M, resulting in functional knockout of said genes.

[0030] In a third aspect, the present disclosure provides an allogeneic T cell obtained by the methods described herein.

[0031] In a fourth aspect, the present disclosure provides a system for genetically modifying a cell comprising i) a CRISPR system, ii) a base editing system, and iii) an RNA-guided nickase, or one or more nucleic acids encoding i), ii), and iii) as described herein, or one or more expression vectors encoding i), ii), and iii) as described herein.

[0032] In a fifth aspect, the present disclosure provides a kit for genetically modifying a cell, comprising i) a CRISPR system, ii) a base editing system, and iii) an RNA-guided nickase, or one or more nucleic acids encoding i), ii), and iii) as described herein, or one or more expression vectors encoding i), ii), and iii) as described herein. The kit may further comprise one or more components for introducing nucleic acid or polypeptide into a host cell. In some embodiments, the one or more components are selected from the group consisting of a viral vector, a non-integrating viral particle, an extracellular vesicle, a nanoparticle, a cell-penetrating peptide, and a donor nucleic acid sequence.

[0033] The disclosed methods, systems, kits, and other aspects are described with reference to the following drawings. [Brief description of the drawings]

[0034] [Figure 1]1A and 1B show an example of a schematic diagram illustrating a strategy of knocking out a desired locus by base editing technology and simultaneously knocking in an exogenous gene at the desired locus (e.g., TRAC locus) while knocking out one or more genes. FIG. 1A shows a schematic diagram illustrating a strategy of knocking out a desired locus (e.g., TRAC) by a double nick introduced by nCas9-UGI-UGI, together with knocking in an exogenous gene (e.g., CAR gene) at the locus. The enzyme (nCAS9-UGI-UGI) in this CRISPR system is directed to the knock-in locus by the first and second gRNAs. The donor template DNA for the integration of the exogenous gene is delivered by a viral vector, e.g., adeno-associated virus (e.g., AAV6), or delivered by other methods. FIG. 1B shows a schematic diagram illustrating a strategy for base editing knockout of one or more genes (e.g., B2M or CD52). The enzyme in this CRISPR system (a common RNA-guided nickase), in this example nCAS9-UGI-UGI, is also directed to a specific gene or genes by an RNA scaffold (sgRNA-aptamer) that contains an RNA aptamer linked to a gRNA. The enzyme complexed with the sgRNA-aptamer recruits the deaminase component of the base editing system (MCP-deaminase) to the site where base conversion is required. [Diagram 2] Figure 2 shows an example of a linear schematic of a CAR construct used in certain embodiments. In this example, the CAR construct comprises an anti-CD19 scFv (FMC63 scFV) derived from FMC63 mouse hybridoma, a portion of the human CD28 molecule (the hinge extracellular portion, the transmembrane domain, and the entire intracellular domain) (black box in the figure), and the entire domain of the CD3-zeta chain. [Diagram 3]Figure 3 shows a schematic diagram of an example of a suitable CD19 CAR delivery strategy. The enzymes in the CRISPR system of the present disclosure induced the integration of CD19 CAR into the TRAC locus. The donor construct (AAV6) contained the CAR gene flanked by homology sequences (LHA and RHA). The CD19-CAR gene was integrated into the TRAC exon 1 locus. Once integrated, CAR expression was driven by the endogenous TCR alpha promoter, while the TRAC locus was disrupted. P2A: self-cleaving porcine teschovirus 2A sequence. pA: bovine growth hormone polyA sequence. [Figure 4] Figures 4A, 4B, 4C, and 4D show the analysis of targeted integration of a sequence encoding GFP into the TRAC locus. A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, and Cas9-UGI-UGI mRNA were co-delivered into CD3-positive T cells via electroporation. This was followed by transduction with the viral vector AAV6-TRAC-GFP, in which a sequence encoding GFP is flanked by HA for the TRAC locus. The levels of GFP integration and TCRα / β functional knockout were determined by flow cytometry 4-7 days after delivery and compared to cells not transduced with the virus. Control cells (i.e., cells not electroporated with Cas9 and sgRNA) were also analyzed. Figure 4A shows the levels of GFP-positive cells on the live population. Figure 4B shows the levels of TCRα / β-positive cells on the live population. Figure 4C shows the distribution of TCR- / GFP+, TCR+ / GFP-, TCR+ / GFP+ and TCR- / GFP+ cell populations on the live population, and Figure 4D shows the viability of cells under the above conditions. [Diagram 5]Figures 5A, 5B, 5C, and 5D show the analysis of targeted integration of sequences encoding CD19-CAR into the TRAC locus. A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, and Cas9-UGI-UGI mRNA were co-delivered into CD3-positive T cells via electroporation. This was followed by transduction with a viral vector, AAV6-TRAC-CAR, in which sequences encoding CD19-CAR are flanked by HA for the TRAC locus. The levels of CAR integration and TCRα / β functional knockout were determined by flow cytometry 4-7 days after delivery and compared to cells not transduced with the virus. Control cells (i.e., cells not electroporated with Cas9 and sgRNA) were also analyzed. Figure 5A shows the levels of CAR-positive cells on live cells. Figure 5B shows the levels of TCRα / β-positive cells on live cells. Figure 5C shows the distribution of TCR- / CAR+, TCR+ / CAR-, TCR+ / CAR+ and TCR- / CAR+ cell populations on live cells. Figure 5D shows the viability of cells under the above conditions. [Figure 6]Figures 6A, 6B, 6C, and 6D compare the base editing efficiency and functional KO generation of B2M and CD52 genes in non-transduced and AAV6-transduced cells. A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing targeting of B2M and CD52, and nCas9-UGI-UGI and Apobec1-MCP mRNA were co-delivered into CD3-positive T cells via electroporation. This was followed by transduction with the viral vector AAV6-TRAC-CAR. Four days after delivery, the base editing efficiency and functional knockout generation for B2M and CD52 were evaluated by Sanger sequencing and flow cytometry, respectively. Control cells (i.e., cells not electroporated with Cas9 and sgRNA) were also analyzed. Figures 6A and 6B show the editing efficiency for B2M and CD52, respectively, as determined by Sanger sequencing 4 days after delivery. Figures 6C and 6D show the percentage of B2M and CD52 positive cells, respectively, on live cells as measured by flow cytometry 4 days after delivery. [Figure 7]Figures 7A, 7B, and 7C show the knock-in of the CAR gene at the TRAC locus and the knock-out of TRAC, B2M, and CD52, which are simultaneously achieved by the base editing technology of the present disclosure. A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing targeting of B2M and CD52, and nCas9-UGI-UGI and Apobec1-MCP mRNA were simultaneously delivered to CD3 positive T cells via electroporation. This was followed by transduction with the viral vector AAV6-TRAC-CAR. Four days after delivery, functional knock-out generation for B2M, CD52, and TCRa / b, as well as CAR integration, were evaluated by flow cytometry. Figure 7A shows the level of TCRa / b positive cells on live cells measured by flow cytometry. Figure 7B shows the level of CAR positive cells on live cells measured by flow cytometry. Figure 7C shows flow cytometry data showing the percentage of cells KO or non-edited at one, two, and three genes within the CAR positive population (single KO (TRAC KO+B2M KO+CD52 KO), double KO (TRAC-B2M KO+TRAC-CD52 KO+B2M-CD52 KO), and triple KO (TRAC-B2M-CD52 KO)). [Figure 8]Figures 8A, 8B, 8C, 8D, 8E, and 8F show the base editing efficiency by cytidine base editing technology at B2M, CD52, and PDCD1 loci in non-transduced and AAV6-transduced samples (Figures 8A, 8B, 8C) and the efficiency of indel formation by wt Cas9 (Figures 8D, 8E, 8F). A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing targeting of B2M, CD52, and PDCD1, and nCas9-UGI-UGI and Apobec1-MCP mRNA were co-delivered into CD3-positive T cells via electroporation. The Cas9 samples were electroporated with wild-type Cas9 mRNA and normal sgRNA. This was followed by transduction with the viral vector AAV6-TRAC-CAR. Four days after delivery, base editing efficiency and indel formation efficiency were assessed by Sanger in non-transduced and transduced samples. [Figure 9] Figures 9A, 9B, and 9C show functional KO generation of B2M, CD52, and PDCD1 genes by cytidine base editing (nCas9-UGI-UGI / Apobec) and wt Cas9 in non-transduced and AAV6-transduced samples. A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing targeting of B2M, CD52, and PDCD1, and nCas9-UGI-UGI and Apobec1-MCP mRNA were co-delivered into CD3-positive T cells via electroporation. Cas9 samples were electroporated with wild-type Cas9 mRNA and normal sgRNA. This was followed by transduction with the viral vector AAV6-TRAC-CAR. Four days after delivery, functional knockout generation of B2M, CD52, and PDCD1 genes was evaluated by flow cytometry (Figures 9A, 9B, and 9C, respectively). Control samples represent samples that were mock electroporated and left untransduced, or transduced with AAV6-TRAC-CAR. [Figure 10]Figures 10A and 10B show the knock-in of CAR at the TRAC locus and the knock-out of TRAC when three additional genes were knocked out simultaneously. A pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing targeting of B2M, CD52 and PDCD1, as well as nCas9-UGI-UGI and Apobec1-MCP mRNA were co-delivered via electroporation into CD3 positive T cells. Cas9 samples were electroporated with wild-type Cas9 mRNA and regular sgRNA. This was followed by transduction with the viral vector AAV6-TRAC-CAR. The levels of CAR integration and TCRa / b functional knock-out were determined by flow cytometry 4-7 days after delivery. Figure 10A shows the levels of CAR positive cells on live cells. Figure 10B shows the levels of TCRa / b positive cells on live cells. [Figure 11]Figure 11 shows the tumor-killing ability of CAR-T cells generated using base editing technology. For the generation of CAR-T cells, a pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing of B2M, CD52 and PDCD1 genes, and nCas9-UGI-UGI and Apobec1-MCP mRNA were co-delivered into CD3-positive T cells via electroporation. Cas9 samples were electroporated with wild-type Cas9 mRNA and normal sgRNA. This was followed by transduction with the viral vector AAV6-TRAC-CAR. Approximately 7 days after electroporation, CD3+ cells were depleted from the culture, and the resulting allogeneic CAR-T cells were incubated with CD19-positive Raji cells preloaded with calcein AM at 1:1 and 5:1 CAR-T:Raji cell ratios for 4 hours. After incubation, the medium was collected and analyzed for fluorescence emission as a measure of Raji cell lysis. The percentage of target cell killing was calculated as [(mean of test condition-mean of negative control condition) / (mean of positive control condition-mean of negative control condition)] x 100, where the negative control condition was Raji cells without CAR-T cells and the positive control condition was Raji cells exposed to 2% Triton to achieve complete lysis. [Figure 12] Figure 12 shows an example of a linear schematic of a scHLA-E trimer used in certain embodiments. In this example, the scHLA-E trimer construct includes a leader peptide of human B2M (hB2M lp), an HLA-E binding peptide antigen, a 15 amino acid linker ((G4S)3), mature human B2M (hB2M), a 20 amino acid linker ((G4S)4), and a mature HLA-E heavy chain. [Figure 13]Figure 13 shows an example of a schematic diagram of a circular double-stranded DNA used in certain embodiments. The exogenous DNA template was flanked by homology arms (right homology arm, RHA and left homology arm, LHA) from the B2M locus. In the circular form, the exogenous DNA template with homology arms was flanked on both sides (A) or not (B) by sequences of gRNA pairs targeting the B2M genomic locus (CTS or CRISPR / Cas9 targeting sequence or sgRNA B2M targeting sequence), so that once the circular double-stranded DNA was co-delivered together with the CRISPR components in the cell, the donor nucleic acid sequence was released from the circular dsDNA as linear DNA following cleavage by CRISPR / Cas. pA: bovine growth hormone polyA sequence [Figure 14] FIG. 14 shows a schematic diagram of an example of a suitable scHLA-E trimer delivery method. The enzymes in the CRISPR system of the present disclosure induced the integration of scHLA-E trimers into the B2M locus. The exogenous DNA template contained a sequence encoding the scHLA-E trimer flanked by homologous sequences (LHA and RHA). The scHLA-E trimer transgene was integrated into the B2M exon 1 locus. Once integrated, expression of the scHLA-E trimer was driven by the endogenous B2M promoter, while the B2M locus was disrupted. pA: bovine growth hormone polyA sequence [Figure 15]Figure 15A, 15B, 15C and 15D show the analysis of the targeted integration of the sequence encoding tGFP into the B2M locus and base editing at the CIITA locus when the exogenous DNA template is delivered as a circular double-stranded DNA. A pair of synthetic sgRNAs targeting exon 1 of the B2M locus, sgRNA-aptamer for base editing targeting of CIITA, nCas9-UGI-UGI and Apobec1-MCP mRNA, and circular double-stranded DNA containing the GFP coding sequence with homology arms to the B2M gene were simultaneously delivered into iPSCs via electroporation. In the circular form, the exogenous DNA template with homology arms was flanked on both sides by sgRNA B2M targeting sequences (respectively CTS_B2M_tGFP and B2M_tGFP in the graph) (CTS represents CRISPR / Cas9 target sequence) or not. The level of GFP incorporation was determined by flow cytometry after 48 hours of treatment with interferon-γ 5-7 days after delivery and compared to cells that did not receive exogenous DNA template. Base editing efficiency at the CIITA locus was assessed by Sanger sequencing 5-7 days after delivery. Control cells (i.e., cells that did not receive electroporation of Cas9 and sgRNAs) were also analyzed. Figure 15A shows the base editing efficiency of the CIITA gene as determined by Sanger sequencing. Figure 15B shows the level of B2M-positive cells on live cells. Figure 15C shows the level of GFP-positive cells on live cells. Figure 15D shows the distribution of GFP- / B2M+, GFP- / B2M+, GFP+ / B2M+, and GFP+ / B2M- cell populations on live cells. [Figure 16]Figure 16A, 16B, 16C and 16D show the analysis of the targeted integration of the sequence encoding tGFP into B2M locus and base editing in CIITA gene when exogenous DNA template is delivered as linear double-stranded DNA.A pair of synthetic sgRNAs targeting exon 1 of B2M locus, sgRNA-aptamer for base editing targeting CIITA gene, nCas9-UGI-UGI and Apobec1-MCP mRNA, and linear double-stranded DNA comprising the tGFP coding sequence with homology arms to B2M gene are delivered simultaneously into iPSCs via electroporation. In the linear form, the exogenous DNA template with homology arms was either flanked or not flanked on both sides by sgRNA B2M targeting sequences (LinearCTS_B2M_tGFP and LinearB2M_tGFP, respectively, in the graphs) (CTS represents the CRISPR / Cas9 target sequence). The level of GFP incorporation was determined by flow cytometry after 48 h of treatment with interferon-γ 5-7 days after delivery and compared to cells that did not receive an exogenous DNA template. Base editing efficiency at the CIITA gene was assessed by Sanger sequencing 5-7 days after delivery. Control cells (i.e., cells that did not receive electroporation of Cas9 and sgRNA) were also analyzed. Figure 16A shows the base editing efficiency of CIITA determined by Sanger sequencing. Figure 16B shows the level of B2M-positive cells on live cells. Figure 16C shows the level of GFP-positive cells on live cells. FIG. 16D shows the distribution of GFP- / B2M+, GFP- / B2M+, GFP+ / B2M+, and GFP+ / B2M- cell populations on live cells. [Figure 17]Figure 17A, 17B and 17C show the analysis of the targeted integration of the sequence encoding scHLA-E trimer into B2M locus and base editing in CIITA gene when exogenous DNA template is delivered as circular double-stranded DNA.A pair of synthetic sgRNAs targeting exon 1 of B2M locus, sgRNA-aptamer for base editing targeting CIITA, nCas9-UGI-UGI and Apobec1-MCP mRNA, and circular double-stranded DNA comprising the sequence encoding scHLA-E trimer with homology arms to B2M gene are delivered simultaneously into iPSCs via electroporation. In the circular form, the exogenous DNA template with homology arms was either flanked or not flanked on both sides by sgRNA B2M targeting sequences (linearCTS_B2M_scHLA-E_trimer and linearB2M_scHLA-E_trimer, respectively, in the graphs) (CTS represents the CRISPR / Cas9 target sequence). The level of scHLA-E_trimer integration was determined by flow cytometry after 48 h of treatment with interferon-γ 5-7 days after delivery and compared to cells that did not receive an exogenous DNA template. Base editing efficiency at the CIITA gene was assessed by Sanger sequencing 5-7 days after delivery. Control cells (i.e., cells that did not receive electroporation of Cas9 and sgRNA) were also analyzed. Figure 17A shows the base editing efficiency of the CIITA gene as determined by Sanger sequencing. Figure 17B shows the level of B2M-positive cells on live cells. FIG. 17C shows the levels of scHLA-E_trimer positive cells on live cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present disclosure relates to a new modular approach for the generation of genetically modified cells, in particular immune cells and iPSCs, that uses a common Cas9 element to simultaneously enable precise editing of defined nucleic acid targets (knock-out) and introduction of selected exogenous sequences at desired loci (knock-in).

[0036] The present inventors have developed a new modular method for the generation of genetically modified cells, particularly immune cells and iPSCs, which allows for the precise editing of defined nucleic acid targets (knockout) and the introduction of selected exogenous sequences (knockin) at desired loci simultaneously using common CRISPR / Cas9 targeting elements. Advantageously, it is shown herein that the method and system according to the present disclosure can be used to simultaneously knock in exogenous genes, such as CAR or TCR, and base edit multiple genes to generate functional knockouts.

[0037] The methods provided herein can target different genes in cells, particularly immune cells. For example, the base editing components used in the methods can be used to introduce genetic modifications that result in desired base changes that result in the subsequent phenotypic loss of any of the above proteins encoded by the genes TRAC, TRBC1, TRBC2, PDCD1, CD52, CIITA, NKG2A, and B2M. The methods can be used to edit one or both alleles of a target gene in a cell, for example, an immune cell or an iPS cell. The methods provided herein can be used to edit multiple different genes (multiple base editing) to successfully edit one or both alleles of a target gene. For example, multiple RNA scaffolds containing different guide RNA sequences can be used in the methods to perform genetic modifications (base editing) of multiple different loci (e.g., 2-10). Advantageously, it is shown herein that the methods and systems can be used to simultaneously knock in an exogenous gene, for example, a sequence encoding a CAR or TCR, and base edit multiple genes to produce a functional knockout.

[0038] The method according to the present disclosure can be configured to generate genetically engineered cells, particularly immune cells, as well as stem and progenitor cells that can differentiate into immune cells. Immune cells include T cells, natural killer (NK) cells, B cells, myeloblasts, lymphoid dendritic cells, myeloid dendritic cells, macrophages, eosinophils, neutrophils, basophils, and CD34+ hematopoietic stem progenitor cells (HSPCs). HSPCs can give rise to common myeloid and common lymphoid progenitor cells that can differentiate into T cells, dendritic cells, natural killer (NK) cells, B cells, myeloblasts, and other immune cells, erythroblasts, megakaryoblasts, and mast cells. In addition, hPSCs (human pluripotent stem cells), which are human-derived pluripotent stem cells and include hESCs (human embryonic stem cells) and induced pluripotent stem cells (iPSCs), can be used to derive immune cells. hPSCs and, for example, iPSCs can be genetically engineered prior to differentiation into a population of a desired cell type, or the iPSCs can be subsequently genetically engineered after differentiation into a population of a desired cell type.

[0039] In some embodiments, the immune cell is a T cell, for example, a CAR-T / TCR-T cell. The engineered T cell can be derived from primary T cells or differentiated from stem cells that are suitable as "universally acceptable" cells for therapeutic applications. Suitable stem cells include, but are not limited to, mammalian stem cells, such as human stem cells, including, but not limited to, hematopoietic stem cells (HSC), embryonic and induced pluripotent stem cells (iPSC) derived from neural, mesenchymal, mesodermal, hepatic, pancreatic, muscle, and retinal stem cells. Other stem cells include, but are not limited to, mammalian stem cells, such as mouse stem cells, for example, mouse embryonic stem cells.

[0040] The CRISPR-based platform of the present disclosure can be used to integrate exogenous DNA sequence into one or more target nucleic acid sequences of cells, particularly T cells or iPSCs.Exogenous DNA can include CAR or TCR sequences, or code therapeutic proteins, or correct point mutations / indels in genomes.

[0041] In one embodiment, the present disclosure is based on the application of a CRISPR-based platform for the generation of CAR-T cells with one or more site-specific mutations that result in functional loss of a target gene (Figure 1). The present system can be used in multiple ways to generate CAR-T cells with advantageous properties, such as prevention of immunosuppressive side effects, graft-versus-host disease and host-versus-graft disease. The present disclosure may be particularly suitable for the development of allogeneic universal therapeutics.

[0042] definition As used herein, the term "about" refers to + / - 10%.

[0043] As used herein, the term "antisense" refers to a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules can be produced by any method, including synthesis by ligating a gene of interest in a reverse orientation to a viral promoter that allows the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with the natural sequence produced by the cell to form a duplex. These duplexes then block either further transcription or translation.

[0044] "Cell," as defined herein, includes any type of cell, prokaryotic or eukaryotic, whether isolated or not, cultured or not, differentiated or not, as well as higher levels of cellular organization, such as tissues, organs, organisms or parts thereof. Exemplary cells include, but are not limited to, vertebrate cells, mammalian cells, human cells, plant cells, animal cells, invertebrate cells, nematode cells, insect cells, stem cells, and the like.

[0045] "Complement" or "complementary" as used herein means Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. Full complement or fully complementary can mean 100% complementary base pairing between the nucleotides or nucleotide analogs of a nucleic acid molecule. Partially complementary can mean less than 100% complementarity, e.g., less than 80% complementarity. "Complementary" as used herein means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions.

[0046] "Delivery vector" or "delivery vectors" refers to any delivery vector that can be used in the present invention to place the required agents / chemicals and molecules (proteins or nucleic acids) in contact with cells or deliver them into cells or intracellular compartments. This includes, but is not limited to, transduction vectors, liposomal delivery vectors, plasmid delivery vectors, viral delivery vectors, bacterial delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other suitable transfer vectors. These delivery vectors allow the delivery of molecules, chemicals, macromolecules (genes, nucleic acids, proteins), or other vectors such as plasmids and T-DNA. These delivery vectors are molecular carriers.

[0047] A "donor nucleic acid" is defined herein as any nucleic acid that is supplied to an organism or receptacle to be inserted or recombined, in whole or in part, into a target sequence by either DNA repair mechanisms, homologous recombination (HR), or non-homologous end joining (NHEJ).

[0048] A "gene" as used herein may be a natural (e.g., genomic) or synthetic gene that includes transcriptional and / or translational regulatory sequences and / or coding regions and / or non-translated sequences (e.g., introns, 5'- and 3'-non-translated sequences). The coding region of a gene may be a nucleotide sequence that codes for an amino acid sequence, or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA, or antisense RNA. A gene may also be an mRNA or cDNA that corresponds to the coding region (e.g., exons and miRNA), optionally linked with 5'- or 3'-non-translated sequences. A gene may also be an amplified nucleic acid molecule produced in vitro that includes all or a portion of the coding region and / or linked with 5'- or 3'-non-translated sequences.

[0049] "Gene targeting" is used herein as any genetic technique that induces permanent changes to a target nucleic acid sequence, including deletions, insertions, mutations, and replacements of nucleotides in the target sequence.

[0050] A "target nucleic acid" or "target sequence," as used herein, is any desired predetermined nucleic acid sequence on which an action is to be performed, including, but not limited to, coding or non-coding sequences, genes, exons or introns, regulatory sequences, intergenic sequences, synthetic sequences, and intracellular parasite sequences. In some embodiments, the target nucleic acid is present within a target cell, tissue, organ, or organism. The target nucleic acid includes a target site that includes one or more nucleotides within the target sequence, which nucleotides are modified to any extent by the methods and compositions disclosed herein. For example, the target site can include one nucleotide. For example, the target site can include 1-300 nucleotides. For example, the target site can include about 1-100 nucleotides. For example, the target site can include about 1-50 nucleotides. For example, the target site can include about 1-35 nucleotides. In some embodiments, the target nucleic acid can include multiple target sites, which may be the same or different.

[0051] "Genomic or genetic modification" is used herein as any modification made to the genome or chromosomal or extrachromosomal or organellar DNA of an organism as a result of gene targeting or gene function modification.

[0052] "Mutant" as used herein refers to a sequence in which at least a part of the function of the sequence has been lost, for example, a change in the sequence in a promoter or enhancer region would at least partially affect the expression of a coding sequence in an organism. As used herein, the term "mutation" refers to any change in sequence in a nucleic acid sequence, which may result from deletion, addition, substitution, or rearrangement, etc. Mutations may also affect one or more steps in which the sequence is involved. For example, changes in a DNA sequence may result in the synthesis of altered mRNAs and / or proteins that are active, partially active, or inactive.

[0053] An "exogenous" sequence, as used herein, refers to a sequence that is not normally present in the genome of a particular cell, but which can be introduced into the cell by the methods of the present disclosure.

[0054] The term "% indels" as used herein refers to the percentage of insertions or deletions of several nucleotides in a target sequence of a genome.

[0055] As used herein, the term "variant" refers to a polynucleotide or polypeptide that has a sequence substantially similar to that of a reference polynucleotide or polypeptide. In the case of a polynucleotide, a variant may have one or more nucleotide deletions, substitutions, additions at the 5' end, 3' end, and / or at one or more internal sites compared to the reference polynucleotide. The sequence similarity and / or difference between a variant and a reference polynucleotide can be detected using conventional techniques known in the art, such as polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated using site-directed mutagenesis. In general, variants of polynucleotides, including but not limited to DNA, may have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a reference polynucleotide, as determined by sequence alignment programs known to those skilled in the art. In the case of polypeptides, variants may have one or more amino acid deletions, substitutions, additions compared to the reference polypeptide. Similarities and / or differences in sequence between variants and reference polypeptides can be detected using conventional techniques known in the art, such as Western blots. Generally, a variant of a polypeptide can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88% about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to a reference polypeptide as determined by sequence alignment programs known to those of skill in the art.

[0056] The exogenous sequence to be incorporated (e.g., CAR or scHLA-E) In some embodiments, the donor nucleic acid sequence comprising the exogenous sequence is a sequence encoding a protein of interest. In some embodiments, the donor nucleic acid sequence is selected from the group consisting of a CAR nucleic acid construct, a TCR nucleic acid, and scHLA-E.

[0057] A "chimeric antigen receptor" (CAR) may also be referred to as a "chimeric receptor," "T-body," or "chimeric immune receptor" (CIR). As used herein, the term "chimeric antigen receptor" (CAR) refers to an artificially constructed hybrid protein or polypeptide that includes an extracellular antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) operably linked to a transmembrane domain and at least one intracellular domain. In general, the antigen-binding domain of a CAR has specificity for a particular antigen expressed on the surface of a target cell of interest. For example, T cells can be engineered to express a CAR specific for CD19 on a B-cell lymphoma.

[0058] First generation CAR constructs include a binding domain (scFv antibody), a hinge region, a transmembrane domain and an intracellular signaling domain (Liu et al., 2019, Frontiers in Immunology, the entire contents of which are incorporated herein by reference).

[0059] Yescarta™ (axicabtagene ciloleucel) was approved for use in 2017 for the treatment of large B-cell lymphomas that have failed conventional treatments, and was one of the first therapies of its kind. It employs a binding domain that targets CD19, a protein expressed by normal B cells, B-cell leukemias, and lymphomas. The second generation CAR used in this therapy (Kochenderfer et al., 2009, J Immunotherapy, the entire contents of which are incorporated herein by reference) consists of an anti-CD19 scFv derived from the FMC63 mouse hybridoma (Nicholson et al., 1997, Mol Immunology, the entire contents of which are incorporated herein by reference), a portion of the human CD28 molecule (the hinge extracellular portion, the transmembrane domain, and the entire intracellular domain), and the entire domain of the CD3-zeta chain.

[0060] In some embodiments, the exogenous sequence incorporated into the target nucleic acid sequence comprises a sequence encoding a CAR. In some embodiments, the CAR construct comprises a binding domain, a hinge region, a transmembrane domain and an intracellular signaling domain. The CAR construct used in some of the embodiments of the present disclosure is shown in Figure 2.

[0061] In some embodiments, the binding domain is an scFv antibody. In some embodiments, the scFv antibody comprises an anti-CD19 scFv from FMC63 mouse hybridoma (FMC63 scFv). In one particular embodiment, the binding domain is an anti-CD19 scFv. In another embodiment, the binding domain is an anti-B cell maturation antigen (BCMA) scFv.

[0062] In some embodiments, the CAR construct comprises a portion of the human CD28 molecule (e.g., the hinge extracellular portion, the transmembrane domain, and the entire intracellular domain).

[0063] In some embodiments, the CAR construct comprises the entire domain of the CD3-zeta chain.

[0064] In some embodiments, the exogenous sequence incorporated into the target nucleic acid sequence comprises a CAR coding sequence including FMC63 scFV, the CD28 hinge extracellular portion, transmembrane domain, and the entire intracellular domain, and the CD3 Z chain.

[0065] The intracellular signaling domain causes signal transduction within the cell through phosphorylation of CD3-zeta after antigen binding. The cytoplasmic domain of CD3-zeta is routinely used as the main CAR endodomain component. In addition to CD3 signaling, other costimulatory molecules are also required for T cell activation, so CAR receptors typically contain costimulatory molecules, including CD28, CD27, CD134 (Ox40) and CD137 (4-1BB).

[0066] Examples of first, second, third and fourth generation CARs are described in Subklewe M et al., Transfusion Medicine and Hemotherapy. 2019, Feb;46(1):15-24, the contents of which are incorporated by reference in their entirety. In some embodiments, the exogenous sequence is a CAR sequence of a first, second, third or fourth generation CAR.

[0067] In one specific embodiment, the intracellular signaling domain is the entire intracellular domain of the CD3-zeta chain. In one embodiment, the intracellular signaling domain further comprises the 41BB-CD3-zeta chain or the CD28-CD3-zeta chain.

[0068] The hinge region is typically a small structural spacer located between the binding domain and the cell outer membrane. Ideally, this increases the flexibility of the scFv to reduce the spatial constraint between the CAR and its target antigen. The design of the hinge region has been described in the art and is typically based on sequences that are membrane proximal regions from other immune molecules, such as IgG, CD8 and CD28 (Chandran, SS et al., 2019, Immunological Reviews, 290(1):127-147 and Qin L et al., 2017, Journal of Hematologic Oncology, 10(1)68, the contents of which are incorporated herein by reference in their entirety).

[0069] The transmembrane domain is a structural element consisting of a hydrophobic alpha helix that spans the cell membrane. It functions by anchoring the CAR to the plasma membrane, thereby bridging the hinge region and binding domain with the intracellular signaling domain. The CD28 transmembrane domain is typically used in CARs and is known to result in a stably expressed receptor.

[0070] In some embodiments, the CAR nucleic acid construct comprises a binding domain that targets CD19 and an intracellular signaling domain that comprises the entire intracellular domain of the CD3-zeta chain and a portion of the CD28 costimulatory molecule.

[0071] Genetic modification of CAR T cells can be performed through virus-based gene transfer methods or non-viral methods, such as DNA-based transposons, CRISPR / Cas9 technology, or direct introduction of in vitro transcribed mRNA by electroporation. Gene transfer techniques allow integration into specific loci of interest or random or pseudorandom integration into the genome. Random or pseudorandom genome integration gene transfer methods include, but are not limited to, transposon, lentivirus, retrovirus, and adenovirus methods. Site-specific integration approaches have the advantage of being more predictable, since they may replace regions of the genome and insert exogenous genetic material precisely. In some embodiments, the CAR nucleic acid is integrated into the genome or chromosome or extrachromosomal DNA or organelle DNA of an organism as a result of gene targeting.

[0072] In other embodiments, the donor nucleic acid sequence is a TCR gene.

[0073] In other embodiments, the donor nucleic acid sequence is a scHLA-E trimer, which is a chimeric protein that includes the following elements: (a) a leader peptide of B2M, (b) VMAPRTLIL (HLA-E binding peptide, SEQ ID NO:1), (c) a 15 amino acid linker (G4S)3, (d) mature human B2M, (e) a 20 amino acid linker (G4S)4, and (f) a mature HLA-E heavy chain.

[0074] In some embodiments, the scHLA-E trimer nucleic acid sequence comprises the sequence set forth in Accession No. AY289236.1 (SEQ ID NO:2): In some embodiments, the AY289236.1 sequence is modified at certain nucleotides to avoid recognition by the sgRNA pair used to target the endogenous locus.The transgene is also flanked by homology arms (e.g., B2M homology arms) that originate from the locus into which it is integrated and surround the CRISPR / Cas9 cleavage site.The resulting sequence that is intended to be integrated into the B2M locus is called scHLA-E_trimerB2M-900HA (SEQ ID NO:3): In some embodiments, the scHLA-E trimer transgene flanked by homology arms is flanked by sgRNA targeting sequences for the desired locus, such that the donor nucleic acid sequence, once co-delivered into the cell with the CRISPR components, is released from the plasmid as linear DNA after cleavage by CRISPR / Cas. In some embodiments, the scHLA-E trimer transgene flanked by homology arms is flanked by sequences of a gRNA pair targeting the B2M locus. The resulting sequence is referred to as scHLA-E_trimer_B2M-900HAs_CTS (SEQ ID NO: 4): Graft rejection due to alloreactivity is a complication associated with the use of donor-derived allogeneic cells / tissues. HLA proteins are antigen-presenting receptors present on cell membranes that interact with T cell receptors (TCRs) to mediate immune surveillance by the adaptive immune system.

[0075] Class 1 HLA proteins (HLA-A / -B / -C / -E / -F / -G), encoded at the major histocompatibility complex 1 (MHC-1) locus, form heterodimeric receptors with beta2-microglobulin (B2M) to present intracellular antigens on the surface of most cells. In the case of allogeneic transplants, antigens presented by HLA class 1 proteins are recognized as foreign by host CD8+ cytotoxic T cells via the TCR complex, leading to direct cytolytic attack and loss of the infused cells. In addition to recognizing antigens presented by HLA receptors, the TCR also directly engages and recognizes the HLA receptor itself, identifying it as either "self" or "non-self".

[0076] Class 2 HLA (HLA-DR / -DQ / -DP), encoded by the MHC-2 locus, forms heterodimers composed of alpha and beta chains that present extracellular antigens and are constitutively expressed by specialized antigen-presenting cells, e.g., macrophages and dendritic cells, as well as other cell types, including microglia, endothelial, and epithelial cells, in response to inflammatory cytokines. Foreign extracellular antigens presented by class 2 HLA activate CD4+ / TCR-mediated responses of CD4+ helper T cells that recruit cytotoxic T cells and NK cells through secreted chemokines. Recognition of an allogeneic graft as non-self, either through HLA protein mismatch or through foreign antigen presentation, leads to alloreactivity of host T cells, resulting in graft rejection through inflammatory and cytolytic attack.

[0077] Therefore, HLA matching is essential for successful cell and tissue transplantation. However, the genes encoding HLA-A, HLA-B, HLA-C and HLA-DQ are some of the most highly polymorphic coding loci in the human population, and therefore HLA matching of allogeneic grafts is a major challenge to overcome in both conventional cell and tissue donation and the application of iPSC-derived cell therapy drugs.

[0078] Because HLA class 1 proteins depend on dimerization with B2M for cell surface presentation, genetic modification of the B2M locus may mask allogeneic cells from cytotoxic CD8+ T cells and evade elimination by the patient's immune system. Similarly, disrupting the function of class 2 transactivator protein (CIITA) silences HLA class 2 protein expression and masks cells from CD4+ helper T cells. However, although such an approach avoids T cell-mediated immune surveillance, complete loss of HLA expression induces an NK cell-mediated "loss of self" response. This "loss of self" response can be prevented by forced expression of minimally polymorphic HLA-E molecules. To obtain inducible and regulated surface expression of HLA-E without surface expression of HLA-A, B, or C, a single-chain HLA-E trimer (scHLA-E trimer) containing HLA-E, B2M, and an antigenic peptide can be knocked in to the B2M locus. Without wishing to be bound by this theory, it is believed that this approach (depletion of B2M and forced expression of HLA-E) confers resistance to NK-mediated killing, while the cells are not recognized as foreign by host CD8+ T cells.

[0079] To demonstrate a simultaneous knock-in-knock-out strategy in iPSCs, this disclosure generates a base editing knockout of the CIITA gene and a simultaneous knock-in of the scHLA-E trimer sequence at the B2M locus with knockout of the B2M gene.

[0080] The general method for site-specific integration is to use CRISPR-Cas technology to target and cut the desired site in the presence of exogenous DNA sequence with complementary region to genome and the region that is desired to be modified.The DNA sequence for insertion by CRISPR-Cas technology can be provided by several methods, including the method of transduction by non-integrated adeno-associated virus (AAV) or by providing DNA template.The exogenous template for insertion into genome by CRISPR-Cas requires the insertion sequence to be flanked by the specific region that is cut by CRISPR-Cas technology, and these are generally known as "homology arms".

[0081] In some embodiments, the exogenous DNA template or exogenous sequence is provided as single-stranded and double-stranded DNA. Here, the DNA can be an open linear structure with exposed 5' and 3' ends of the DNA, or a closed structure with no exposed ends of the DNA. Closed DNA molecules include, but are not limited to, circular dsDNA, linear dsDNA, plasmid, minicircle, circularized ssDNA, and doggybone DNA (dbDNA). In some embodiments, the exogenous sequence is delivered as linear dsDNA. In some embodiments, the exogenous sequence is delivered in circular dsDNA. In some embodiments, when circular dsDNA is used to deliver the exogenous sequence, the exogenous DNA is flanked by homology arms from the locus to be inserted. In some embodiments, when circular dsDNA is used to deliver the exogenous sequence, the exogenous DNA flanked by homology arms from the locus to be inserted is flanked on both sides by sgRNA target sequences that target the locus to be inserted.

[0082] In some embodiments, the exogenous sequence is introduced into the cell using a viral vector. In some embodiments, the viral vector is selected from the group consisting of lentivirus, retrovirus, adeno-associated virus (AAV) and adenovirus. There are various serotypes of AAV, which have different preferences for tissue types. Examples of AAV that can be used in the present disclosure include, but are not limited to, AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAV-DJ, AAV-DJ9. In some embodiments, the viral vector is AAV serotype 6 (AAV6). AAV6 vectors have been shown to provide improved transduction efficiency in the field of CAR-T cell generation (Wang et al., Nucleic Acid Research 2016, the contents of which are incorporated herein by reference). AAV is a small, icosahedral, non-enveloped virus with a diameter of 25 nm that contains a single-stranded DNA genome, and has become an essential therapeutic gene delivery tool in recent years. It is frequently used to deliver genetic material to target cells in vivo for the treatment of disease and is widely used in clinical applications in academia and industry (Hamieh, M et al., Nature 2019, 568(7750)112-116, the contents of which are incorporated herein by reference).

[0083] In one embodiment, the method of exogenous gene delivery of exogenous sequence (e.g., CAR, TCR or scHLA-E) sequence is by AAV6. In another embodiment, the method of extracellular gene delivery of CAR, TCR or scHLA-E sequence is by using plasmid or linear DNA. The integration site at a specific locus will disrupt the endogenous gene (e.g., TRAC, B2M or CISH) while inserting the exogenous DNA fragment containing the transgene (e.g., CAR, TCR or scHLA-E gene). In some embodiments, the exogenous sequence is inserted under the control of an endogenous promoter. In other embodiments, the exogenous sequence is controlled by its own promoter.

[0084] In some embodiments, the disclosed methods or systems include the incorporation of two or more exogenous sequences. In some embodiments, multiple CAR exogenous sequences are incorporated, where these multiple CARs recognize different antigens, for example to generate dual-targeting CAR-T cells, limiting antigen escape during treatment.

[0085] RNA-guided nickase As used herein, Cas protein, CRISPR-associated protein, or CRISPR protein are used interchangeably and refer to proteins of or derived from CRISPR-Cas type I, II, or III systems that have an RNA-guided DNA binding domain. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasS), Csel ... E), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. Non-limiting examples of RNA-guided nickases that can interact with the first and second sgRNAs of the CRISPR system and the RNA scaffold of the base editing system include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Css1, Css2, Css3, Css4, Css5, Css6, Css7, Css8, Css9, Css1, Css1, Css2, Css1, Css2, Css1, Css2, Css3, Css4, Css5, Css6, Css7, Css8, Css1, Css1, Css1, Css2, Css1, Css1, Css2, Css1, Css1, Css2, Css1, Css2, Css3 ...3, Css1, Css2, Css1, Css1, Css2, Css1, Css1, Css2, Css1, Css2, Css3, Css1, C These include, but are not limited to, se2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.See, e.g., Koonin and Makarova, 2019, Origins and Evolution of CRISPR-Cas systems, Review Philos Trans R Soc Lond B Biol Sci. 2019 May, 13;374(p. 1772); for type V, Yan et al., Science, 363 88-92, 2019; and for mini-Cas14, Harrington et al., 2018, Science, vol 362 839-842, the contents of which are incorporated by reference in their entireties.

[0086] The sequence targeting component of the methods and systems provided herein typically utilizes the Cas protein of a bacterial species-derived CRISPR / Cas system as an RNA-guided nickase. In some embodiments, the Cas protein is from a type II CRISPR system. See, for example, Makarova, KS, Wolf, YI, Iranzo, J. et al., Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol 18, 67-83 (2020), the contents of which are incorporated herein by reference in their entirety.

[0087] In one embodiment, the Cas protein is derived from type II CRISPR-Cas system. In an exemplary embodiment, the Cas protein is or is derived from Cas9 protein. In some embodiments, the RNA-guided nickase is or is derived from Cas9 protein or comprises a mutation compared to WT Cas9 protein. Cas9 protein is a virulent pathogen that causes Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, and Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.), Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium ebestigatum evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp.), Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Legionella pneumophila, Francisella novicida, Gammaproteobacteria proteobacterium HTCC5015, Parasutterella excrementihominis, Sutterella wadsworthensis, Sulfurospirillum sp. SC ADC, Ruminobacter sp. RM87, Burkholderiales bacterium 1 1 47, Bacteroidetes oral taxon 274 str.F0058, Wolinella succinogenes, Burkholderiales YL45, Ruminobacter amylophilus, Campylobacter sp.) P0111, Campylobacter sp.RM9261, Campylobacter lanienae strain RM8001, Campylobacter lanienae strain P0121, Turicimonas muris, Legionella londiniensis, Salinivibrio sharmensis, Leptospira sp. isolate FW.030, Moritella sp. isolate NORP46, Endozoicomonas sp. S-B4-1U, Tamilnaduibacter salinus, Vibrio natriegens natriegens, Arcobacter skirrowii, Francisella philomiragia, Francisella hispaniensis, or Parendozoicomonas haliclonae.

[0088] Cas protein or RNA-guided nickase can be obtained as a recombinant fusion polypeptide, for example, as a fusion protein with glutathione-s-transferase (GST), 6x-His epitope tag, or M13 Gene 3 protein, by methods known in the art, and expressed in a suitable host cell. Alternatively, Cas protein or RNA-guided nickase can be chemically synthesized (see, for example, Creighton, "Proteins: Structures and Molecular Principles", WH Freeman & Co., NY, 1983), or produced by recombinant DNA technology as described herein. For further guidance, the skilled artisan can refer to Frederick M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003; and Sambrook et al., "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001), the entire contents of each of which are incorporated herein by reference in their entirety.

[0089] In another embodiment, the RNA-guided nickase is a nuclease-deficient nickase nCas9 from Streptococcus pyogenes D10A mutant (Cas9(D10A)). In another embodiment, the RNA-guided nickase is a nuclease-deficient nickase nCas9 H840A mutant. In another embodiment, the RNA-guided nickase is a nuclease protein that cleaves only one DNA strand. In some embodiments, the RNA-guided nickase comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the RNA-guided nickase consists of the sequence set forth in SEQ ID NO: 19.

[0090] Table 1 lists examples of Cas9 and a non-exhaustive list of their corresponding PAM requirements. Synthetic Cas surrogates can also be used, such as those described in Rauch et al., Programmable RNA-Guided RNA Effector Proteins Built from Human Parts. Cell vol. 178, no. 1, 27 June 2019, pp. 122-134, e12, the entire contents of which are incorporated herein by reference. In some embodiments, the RNA-guided nickase is a functional variant or fragment of a Cas protein or synthetic Cas surrogate described herein. The functional variant or fragment has at least about 70% (e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%) homology to the Cas protein or synthetic Cas surrogate.

[0091] [Table 1]

[0092] In some aspects of the present disclosure, the sequence targeting component comprises a fusion of (a) an RNA-guided nickase and (b) a uracil-DNA glycosylase (UNG) inhibitor peptide (UGI). For example, in some embodiments, the RNA-guided nickase comprises a Cas protein, e.g., a Cas9 protein, fused with one or more UGIs. Such a fusion protein may exhibit increased nucleic acid editing efficiency compared to a fusion protein that does not include a UGI domain. In some embodiments, the UGI comprises a wild-type UGI sequence or one having the following amino acid sequence: Protein Accession Number: sp|P14739|UNGI_BPPB2: Uracil-DNA glycosylase inhibitor (UGI). In some embodiments, the UGI peptide comprises the following amino acid sequence: MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 5). In another embodiment, the UGI peptide consists of the sequence set forth in SEQ ID NO: 5.

[0093] In some embodiments, the UGI protein provided herein includes fragments of UGI and proteins homologous to UGI or UGI fragments. For example, in some embodiments, UGI includes a fragment of the amino acid sequence described above. In some embodiments, UGI includes an amino acid sequence homologous to the amino acid sequence described above, or an amino acid sequence homologous to a fragment of the amino acid sequence described in the UGI sequence described above. In some embodiments, a protein including UGI or a fragment of UGI, or a homolog of UGI or UGI fragment, is referred to as a "UGI variant." A UGI variant has homology to UGI or a fragment thereof. For example, a UGI variant includes at least about 70% sequence identity (e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%) compared to wild-type UGI, which may be the UGI sequence described above (SEQ ID NO:5).

[0094] Suitable UGI protein and nucleotide sequences are provided herein, and additional suitable UGI sequences will be known to those of skill in the art, including, for example, those published in Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J Biol. Chem. 264:1163-1171 (1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J Biol. Chem. 272:21408-21419 (1997); Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor.

[0095] In one embodiment, the RNA-guided nickase is a nuclease-deficient nickase nCas9 from Streptococcus pyogenes (D10A mutant) fused with UGI. In another embodiment, the RNA-guided nickase is a nuclease-deficient nickase nCas9 from Streptococcus pyogenes (D10A mutant) fused with two UGI peptides (referred to herein as nCas9-UGI-UGI).

[0096] nCas9-UGI-UGI-SEQ ID NO:195: In some embodiments, the RNA-guided nickase that can interact with the first and second gRNA of CRISPR system and the RNA scaffold of base editing system is a single molecule, that is, the same nickase molecule interacts with the first and second gRNA of CRISPR system and interacts with the RNA scaffold of base editing system.Therefore, the RNA-guided nickase simultaneously cuts one strand of the first target nucleic acid sequence and base edits the second / third / fourth target nucleic acid sequence.

[0097] gRNA The CRISPR-Cas system has been used to perform genome editing in cells of various organisms. The specificity of this system is defined by the base pairing between the target DNA and a custom-designed guide RNA (gRNA). By manipulating and adjusting the base pairing properties of the guide RNA, any nucleic acid sequence of interest can be targeted, provided that a PAM sequence is present in the target sequence. Thus, the first target nucleic acid sequence or locus into which the exogenous sequence is integrated is any locus flanked by the PAM sequences recognized by the first and second gRNAs.

[0098] The CRISPR system of the present disclosure can be used to integrate donor nucleic acid sequences, including exogenous DNA sequences, into target nucleic acid sequences of cells, particularly immune cells, T cells or iPSCs.In some embodiments, exogenous DNA can include sequences that code CAR, TCR or scHLA-E trimers, or code therapeutic proteins, or correct point mutations / indels in genomes.

[0099] According to one embodiment of the present disclosure, the first target nucleic acid sequence represents the CAR integration site in host cell, particularly T cell or iPSC.As mentioned above, the CAR gene to be integrated into the target nucleic acid sequence of the cell can be delivered into the host cell using a viral vector.In some embodiments, the CAR gene to be integrated into the target nucleic acid sequence of the cell can be delivered into the host cell using AAV.In some embodiments, the AAV is AAV6.

[0100] According to one embodiment of the present disclosure, the first target nucleic acid sequence represents the TCR integration site in host cell, particularly T cell or iPSC.As mentioned above, the CAR gene to be integrated into the target nucleic acid sequence of the cell can be delivered into the host cell using a viral vector.In some embodiments, the TCR gene to be integrated into the target nucleic acid sequence of the cell can be delivered into the host cell using AAV.In some embodiments, the AAV is AAV6.

[0101] According to one embodiment of the present disclosure, the first target nucleic acid sequence represents the scHLA-E trimer integration site in host cell, particularly T cell or iPSC.As mentioned above, the scHLA-E trimer gene to be integrated into the target nucleic acid sequence of the cell can be delivered into the host cell using a viral vector.In some embodiments, the scHLA-E trimer gene to be integrated into the target nucleic acid sequence of the cell can be delivered into the host cell using AAV.In some embodiments, the AAV is AAV6.

[0102] For type II CRISPR systems, the two components of gRNA are crRNA and tracrRNA, which together form a CRISPR / Cas-based module for sequence targeting and recognition. The crRNA provides target specificity and contains a region that is complementary to and can hybridize with a preselected target site of interest (guide RNA sequence). The tracrRNA is the region of gRNA that interacts with Cas protein.

[0103] In various embodiments, the crRNA comprises from about 10 nucleotides to more than about 25 nucleotides. In some embodiments, the region of base pairing between the guide sequence and the corresponding target site sequence (crRNA) is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides long. In an exemplary embodiment, the crRNA is about 17-20 nucleotides long, e.g., 20 nucleotides.

[0104] The tracrRNA component of the gRNA specifically binds to the Cas protein and guides the Cas protein to the target DNA or RNA sequence. In some embodiments, the tracrRNA is derived from Streptococcus pyogenes. In some embodiments, the tracrRNA comprises about 10 nucleotides to about 50 nucleotides. In some embodiments, the tracrRNA is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 30, 35, 40, 45 nucleotides in length, or more than 50 nucleotides in length.

[0105] One requirement for selecting a suitable target nucleic acid is that it has a 3' protospacer adjacent motif (PAM) site / sequence. Each target sequence and its corresponding PAM site / sequence are referred to herein as Cas target sites. The type II CRISPR system, one of the most thoroughly characterized systems, requires only the Cas9 protein and a crRNA complementary to the target sequence to effect target cleavage. As an example, the Streptococcus pyogenes type II CRISPR system uses a target site with N12-20NGG, where NGG represents the PAM site from Streptococcus pyogenes and N12-20 represents the 12-20 nucleotides located immediately 5' to the PAM site. Examples of other PAM site sequences from other bacterial species include, but are not limited to, NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC. See, for example, US20140273233, WO2013176772, Cong et al. (2012), Science 339(6121):819-823, Jinek et al. (2012), Science 337(6096):816-821, Mali et al. (2013), Science 339(6121):823-826, Gasiunas et al. (2012), Proc Natl Acad Sci US A.109(39):E2579-E2586, Cho et al. (2013) Nature Biotechnology 31, 230-232, Hou et al., Proc Natl Acad Sci US A.109(39):E2579-E2586 A. 2013 Sep 24;110(39):15644-9, Mojica et al., Microbiology. 2009 Mar;155(Pt 3):733-40, and www.addgene.org / CRISPR / , the contents of which are incorporated herein by reference in their entireties.

[0106] In one embodiment, if two or more nickase cleavage sites are required, the PAM sites are designed such that they are 20-200 bp apart. In other embodiments, if two or more nickase cleavage sites are required, the PAM sites are designed such that they are 40 and 70 bp apart. In some embodiments, if two or more nickase cleavage sites are required, the PAM sites are on the outside (known as a "PAM-out" configuration).

[0107] In some embodiments, the target nucleic acid is ssDNA, dsDNA, ssRNA or dsRNA. In some embodiments, the target nucleic acid is one of the two strands on a double-stranded nucleic acid in a host cell. In some embodiments, the target nucleic acid is a single-stranded nucleic acid. Examples of target nucleic acids include, but are not limited to, genomic DNA, host cell chromosomes, mitochondrial DNA, or stably maintained plasmids. However, it should be understood that the method can be carried out on other target nucleic acids present in a host cell, such as unstable plasmid DNA, viral DNA, and phagemid DNA, regardless of the nature of the host cell dsDNA, as long as a Cas target site exists. The method can also be carried out on RNA.

[0108] In some embodiments, the gRNA is a hybrid RNA molecule in which the above crRNA is fused with tracrRNA, mimicking the natural crRNA:tracrRNA duplex. As used herein, the active portion of tracrRNA retains the ability to form a complex with a Cas protein, e.g., Cas9 or dCas9 or nCas9. See, e.g., WO2014144592. Methods for generating crRNA-tracrRNA hybrid RNAs (also known as single guide RNAs or sgRNAs) are known in the art. In embodiments in which the crRNA and tracrRNA are provided as a single gRNA (sgRNA), the two components are typically linked to each other via a tetraloop (also called a repeat:anti-repeat). See, e.g., WO2014099750, US20140179006, and US20140273226. The contents of these documents are incorporated herein by reference in their entirety.

[0109] In some embodiments, the gRNA used in the methods herein can be introduced into cells as a chemically synthesized RNA molecule. The gRNA can include one or more modifications as described herein below. For example, the guide RNA sequence can be chemically modified to include a 2'-(9-methylphosphorthioate) modification on at least one 5' nucleotide and / or at least one 3' nucleotide of the guide RNA sequence. The gRNA can be synthesized as a single molecule (sgRNA) or can be synthesized or expressed as two separate components, where the first component includes (a) the crRNA and the second component includes (b) the tracrRNA, as appropriate. The two components can then be hybridized before being introduced into the cell.

[0110] According to the first aspect of the present disclosure, the method uses a first gRNA and a second gRNA. They function simultaneously to guide RNA-guided nickase to the opposite strand of the target DNA site to generate (alternate) DSB. Therefore, the design of the first and second gRNA defines the exact position of DSB. The position of DSB and the design of the homology arm of donor nucleic acid affect the exact position of the integration of donor sequence.

[0111] The disclosure also provides a method for making multiple genetic modifications in a cell, the method comprising: a) a CRISPR system for incorporating an exogenous sequence into a first target nucleic acid sequence, the CRISPR system comprising a first sgRNA and a second sgRNA complementary to opposite strands of the first target nucleic acid sequence, and a donor nucleic acid sequence comprising the exogenous sequence; and a base editing system for introducing genetic modifications into a second target nucleic acid sequence, the base editing system comprising a guide RNA sequence complementary to the second target nucleic acid sequence, an RNA scaffold comprising a recruitment RNA motif, and an effector fusion protein comprising an RNA binding domain capable of binding to the recruitment RNA motif, and an effector domain comprising a base-modifying enzyme; and introducing into and / or expressing in the cell a single RNA-guided nickase capable of interacting with the first and second sgRNAs of the CRISPR system, and the RNA scaffold of the base editing system, and culturing the cell to generate a cell comprising the multiple genetic modifications.

[0112] In one embodiment, the first gRNA and the second gRNA bind to opposite strands of a first target nucleic acid sequence.

[0113] In one embodiment, the exogenous sequence is integrated into the TRAC locus. In another embodiment, the CAR coding sequence is integrated into the TRAC locus. In one embodiment, the exogenous sequence is integrated into the B2M locus. In another embodiment, the CAR coding sequence is integrated into the B2M locus. In one embodiment, the exogenous sequence is integrated into the PDCD1 locus. In another embodiment, the CAR coding sequence is integrated into the PDCD1 locus. In one embodiment, the exogenous sequence is integrated into the TRBC2 locus. In another embodiment, the CAR coding sequence is integrated into the TRBC2 locus. In one embodiment, the exogenous sequence is integrated into the TRBC1 locus. In another embodiment, the CAR coding sequence is integrated into the TRBC1 locus. In one embodiment, the exogenous sequence is integrated into the TRBC1 / 2 locus. In another embodiment, the CAR coding sequence is integrated into the TRBC1 / 2 locus. In one embodiment, the exogenous sequence is integrated into the CD52 locus. In another embodiment, the CAR coding sequence is integrated into the CD52 locus. In one embodiment, the exogenous sequence is integrated into the CISH locus. In another embodiment, the CAR coding sequence is integrated into the CISH locus.

[0114] In another embodiment, the expression of the exogenous sequence is driven by the endogenous promoter of the locus in which it is integrated.In another embodiment, the expression of the exogenous sequence is driven by its own promoter.In another embodiment, the CAR expression is driven by the TRAC endogenous promoter.In another embodiment, the CAR expression is driven by its own promoter.

[0115] Figure 3 shows a schematic diagram of an example of a suitable CD19 CAR delivery strategy. In this example, the CAR-encoding sequence was inserted into exon 1 of the TRAC locus, in frame with the upstream TRAC locus, before the transmembrane domain of the T cell receptor alpha chain variable region. The CAR-encoding sequence in the AAV6 vector was flanked by left homology arm (LHA) and right homology arm (RHA) adjacent to the left and right sequences of the double nick in TRAC exon 1. The 2A peptide (P2A) from porcine teschovirus-1 was introduced to avoid interference from the T cell receptor alpha chain variable region. Typically, the 2A peptide has approximately 20 amino acids, and the "self-cleavage" occurs between the last two amino acids, glycine (G) and proline (P).

[0116] The present disclosure can further include additional modular components including multiple base editing systems. When multiple modules are used, each guide RNA sequence is complementary to a unique sequence that allows editing of multiple nucleic acid sites. The modular system provides tools for targeting multiple loci (e.g., 2-10), so that multiple genes can be knocked out simultaneously or sequentially.

[0117] Thus, the methods of the present disclosure can use a modular system, where each module comprises a base editing system for introducing a genetic modification into a second or additional target nucleic acid sequence, i.e., a plurality of base editing systems that can bind to different target nucleic acid sequences to genetically modify a plurality of different loci. Each module present in the modular system can: a) i) a guide RNA sequence complementary to a second or further target nucleic acid sequence; ii) containing a recruitment RNA motif; RNA scaffolds, and b) an effector fusion protein comprising an RNA-binding domain capable of binding to the recruitment RNA motif; Includes.

[0118] The second or additional target nucleic acid sequence is distinct from the first target nucleic acid sequence.

[0119] The RNA scaffold may further comprise a tracrRNA capable of binding to an RNA-guided nickase.

[0120] The data provided herein compares the disclosed system with another base editing system (referred to herein as alternative fusion cytidine base editing (CBE) system) that uses direct fusion of Cas protein with effector protein (e.g., deaminase). The modular design of the disclosed system allows flexible system engineering. The modules are interchangeable, and many combinations of different modules can be achieved by simply exchanging the nucleotide sequence of the RNA scaffold. On the other hand, recruitment of effectors by direct fusion or direct interaction with protein components of sequence targeting units always requires re-engineering of new fusion proteins, which is technically more challenging and results are less predictable. The system described herein is based on the base editing (BE) method developed to take advantage of the DNA targeting ability of Cas9, which lacks double-strand cleavage activity, and is combined with the DNA editing ability of deaminases such as APOBEC-1, an enzyme member of the APOBEC family of DNA / RNA cytidine deaminases. By directly fusing deaminase effectors with Cas proteins lacking double-stranded break activity, such as dCas9 or nCas9 proteins, these tools, called base editors, can introduce targeted point mutations into genomic DNA or RNA without generating DSBs and without the need for HDR activity.In essence, the present BE system utilizes the CRISPR / Cas9 complex lacking double-stranded break activity as a DNA targeting mechanism, where mutant Cas9 serves as an anchor for recruiting cytidine or adenine deaminase through direct protein-protein fusion.As previously described in GB2015204.7 and GB2010692.8 (the entire contents of which are incorporated herein by reference) and used herein, the RNA component (scaffold) of the CRISPR / Cas9 complex serves as an anchor for effector recruitment by including RNA motifs (aptamers) in the RNA molecule. RNA aptamers recruit effectors, such as base-editing enzymes, that are fused to the RNA aptamer ligand.

[0121] The methods provided herein can target different genes in immune cells to introduce genetic modifications that result in, for example, a desired base change and / or subsequent phenotypic loss of a protein. Examples of genes that can be targeted include, but are not limited to, any of the following genes: TRAC, TRBC1, TRBC2, PDCD1, CD52, CISH, CIITA, and B2M. The methods can be used to edit one or both alleles of a target gene in a cell. The methods provided herein can be used to edit multiple different genes (multiple base editing) to successfully edit one or both alleles of a target gene. For example, multiple RNA scaffolds containing different guide RNA sequences can be used in the methods to genetically modify (base edit) multiple different loci (e.g., 2-10). Advantageously, it is shown herein that the system can be used to simultaneously base edit multiple genes to create a functional knockout, for example by introducing point mutations in one or both alleles of a target gene, and to introduce exogenous sequences into a cell.

[0122] RNA scaffolds The RNA scaffold can be either a single RNA molecule or part of a complex of multiple RNA molecules. For example, the crRNA, optionally the tracrRNA, and the recruiting RNA motif can be three segments of one long single RNA molecule. Alternatively, one, two, or three of them can be in separate molecules. In the latter case, the three components can be linked together to form the scaffold through covalent or non-covalent linkage or bond, including, for example, Watson-Crick base pairing.

[0123] In one embodiment, the RNA scaffold comprises two separate RNA molecules. The first RNA molecule may comprise a programmable crRNA and a region capable of forming a stem duplex structure with a complementary region. The second RNA molecule may comprise a complementary region in addition to the tracrRNA and the RNA motif. Through this stem duplex structure, the first and second RNA molecules form the RNA scaffold of the present disclosure. In one embodiment, the first and second RNA molecules each comprise a sequence (of about 6 to about 20 nucleotides) that base pairs with the other sequence. In some embodiments, the tracrRNA and the RNA motif are present on different RNA molecules and may be combined into another stem duplex structure. In some embodiments, the crRNA and the tracrRNA are part of a single RNA molecule.

[0124] The RNA and related scaffolds of the present disclosure can be produced by various methods known in the art, including cell-based expression, in vitro transcription, and chemical synthesis.The ability to chemically synthesize relatively long RNA (200mer or longer) using TC-RNA chemistry (see, for example, U.S. Patent No. 8,202,983) allows RNA to be produced with special characteristics that are superior to those possible with the four basic ribonucleotides (A, C, G, and U).

[0125] Cas protein-guide RNA scaffold complex can be produced by recombinant technology using host cell system or in vitro translation-transcription system known in the art.Details of such systems and technologies can be found, for example, in WO2014144761, WO2014144592, WO2013176772, US20140273226 and US20140273233, the contents of which are incorporated herein by reference in their entirety.The complex can be isolated or purified, at least to some extent, from the cellular material of cells or the in vitro translation-transcription system in which they are produced.

[0126] RNA motifs The base editing system and method provided herein is based on RNA scaffold-mediated effector protein recruitment. More specifically, the platform utilizes various recruitment RNA motif / RNA binding protein binding pairs. To this end, the RNA scaffold is designed such that an RNA motif (e.g., MS2 operator motif) that specifically binds to an RNA binding protein (e.g., MS2 coat protein, MCP) is linked to the gRNA-CRISPR scaffold. As a result, this RNA scaffold component of the platform disclosed herein is a designed RNA molecule that includes a crRNA for specific DNA / RNA sequence recognition and a guide RNA sequence that optionally includes a CRISPR RNA motif (tracrRNA) for Cas protein binding. The RNA scaffold component also includes an RNA motif for effector recruitment (also referred to as a recruitment RNA motif). In this way, the effector protein fusion can be recruited to the site through its ability to bind to the RNA motif. A non-exhaustive list of examples of recruitment RNA motif / RNA binding protein pairs that could be used in the methods and systems provided herein is summarized in Table 2. In some embodiments, the recruitment RNA motif is an RNA aptamer and the RNA binding protein is an aptamer-binding protein. In some embodiments, the recruitment RNA motif is an MS2 phage operator stem-loop and the RNA binding protein is an MS2 coat protein (MCP).

[0127] As will be clear to those skilled in the art, chemically modified versions and / or sequence variants of the RNA motif and its binding partner can also be utilized.Further examples of recruiting RNA motif / RNA binding protein pairs can be found, for example, in Pumpens P et al., Intervirology; 2016; 59: 74-110, and Tars K. (2020); Biocommunication of Phages, 261-292, the contents of which are incorporated herein by reference in their entirety.

[0128] [Table 2]

[0129] The sequences of the above binding pairs are listed below.

[0130] 1. Telomerase Ku-binding motif / Ku heterodimer a. Ku-binding hairpin 5'-UUCUUGUCGUACUUAUAGAUCGCUACGUUAUUUCAAUUUUGAAAAUCUGAGUCCUGGGAGUGCGGA-3' (SEQ ID NO: 6) b Ku heterodimer (SEQ ID NO:7) MVRSGNKAAVVLCMDVGFTMSNSIPGIESPFEQAKKVITMFVQRQVFAENKDEIALVLFGTDGTDNPLSGGDQYQNITVHRHLMLPDFDLLEDIESKIQPGSQQADFLDALIVSMDVIQHETI GKKFEKRHIEIFTDLSRFSKSQLDIIIHSLKKCDISERHSIHWPCRLTIGSNLSIRIAAYKSILQERVKKTWTVVDAKTLKKEDIQKETVYCLNDDDETEVLKEDIIQGFRYGSDIVPFSKV DEEQMKYKSEGKCFSVLGFCKSSQVQRRFFMGNQVLKVFAARDDEAAAVALSSLIHALDDLDMVAIVRYAYDKRANPQVGVAFPHIKHNYECLVYVQLPFMEDLRQYMFSSLKNSKKYAPTEAQLNAVDALIDSMSLAKKDEKTDTLEDLFPTTKIPNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTTKSQIPLSKIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPTAK (SEQ ID NO: 8) 2. Telomerase Sm7 binding motif / Sm7 homoheptamer a.Sm consensus site (single stranded) 5'-AAUUUUUGGA-3' (SEQ ID NO: 9) B Monomeric Sm-like protein (Archaea) GSVIDVSSQRVNVQRPLDALGNSLNSPVIIKLKGDREFRGVLKSFDLHMNLVLNDAELEDGEVTRRLGTVLIRGDNIVYISP (SEQ ID NO: 10) 3. MS2 phage operator stem loop / MS2 coat protein a. MS2 phage operator stem loop 5'-GCGCACAUGAGGAUCACCCAUGUGC-3' (SEQ ID NO: 11) B MS2 coat protein MASNFTQFVLVDNGGTGDVTVAPSNFANGIAEWISSNSRSQAYKVTCSVRQSSAQNRKYTIKVEVPKGAWRSYLNMELTIPIFATNSDCELIVKAMQGLLKDGNPIPSAIAANSGIY (SEQ ID NO: 12) 4. PP7 phage operator stem loop / PP7 coat protein a. PP7 phage operator stem loop 5'-aUAAGGAGUUAUAUGGAACCUUA-3' (SEQ ID NO: 13) b. PP7 coat protein (PCP) MSKTIVLSVGEATRTLTEIQSTADRQIFEEKVGPLVGRLRLTASLRQNGAKTAYRVNLKLDQADVVDCSTSVCGELPKVRYTQVWSHDVTIVANSTEASRKSLYDLTKSLVATSQVEDLVVNLVPLGR. (SEQ ID NO: 14) 5. SfMu Com stem-loop / SfMu Com binding protein a.SfMu Com stem loop 5'-CUGAAUGCCUGCGAGCAUC-3' (SEQ ID NO: 15) b. SfMu Com binding protein MKSIRCKNCNKLLFKADSFDHIEIRCPRCKRHIIMLNACEHPTEKHCGKREKITHSDETVRY (SEQ ID NO: 16) The RNA motif may be located at various positions of the RNA scaffold. In some embodiments, the RNA motif is located at the 3' end of the guide RNA, in particular at the 3' end of the tracrRNA (if present), the tetraloop of the gRNA, the stem loop 2 of the tracrRNA (if present), or the stem loop 3 of the tracrRNA (if present). In some embodiments, the MS2 aptamer is located at the 3' end of the gRNA. In particular, the MS2 aptamer may be located at the 3' end of the tracrRNA (if present), the tetraloop of the gRNA, the stem loop 2 of the tracrRNA (if present), and the stem loop 3 of the tracrRNA (if present). The positioning of the RNA motif (e.g., the MS2 aptamer) is crucial in view of steric hindrance that may result from bulky loops. In some embodiments, the MS2 aptamer is at the 3' end of the gRNA. Advantageously, the positioning of the MS2 aptamer at the 3' end of the gRNA thus reduces steric hindrance with other bulky loops of the RNA scaffold.

[0131] In some embodiments, the recruiting RNA motif can be linked to the guide RNA (particularly the tracrRNA, if present) via a linker sequence. The linker sequence can be 2, 3, 4, 5, 6, 7 nucleotides, or more than 7 nucleotides. Advantageously, the linker sequence provides flexibility to the RNA scaffold. The linker sequence can be a GC-rich sequence.

[0132] Modification can be made to the recruiting RNA motif. In one particular embodiment, the modification to the MS2 aptamer is the substitution of 2-aminopurine (2-AP) for the adenine at position 10. Advantageously, the substitution induces conformational changes that result in greater affinity.

[0133] The nucleic acid targeting motif or guide RNA sequence, including the crRNA and optionally the CRISPR RNA motif (tracrRNA), can be provided as a single guide RNA (sgRNA). In some embodiments, the two components (crRNA and optionally the tracrRNA) are linked via a "repeat:anti-repeat" or "tetraloop". The upper stem of the repeat:anti-repeat can be extended to increase the flexibility, correct folding and stability of the loop. The tetraloop can be extended by 2, 3, 4, 5, 6, 7 bp, or more than 7 bp.

[0134] In some embodiments, the RNA scaffold can have one or more of the above modifications.The one or more modifications can be on different components of the RNA scaffold, such as the extension of the tetraloop of the sgRNA and the extension of the RNA motif, or on the same component of the RNA scaffold, such as the extension of the RNA motif and the substitution of the nucleotide of the RNA motif.In some embodiments, the modification can be two or more, three or more, four or more, or five or more nucleotides.In one embodiment, the modification can be the extension of the RNA motif and / or the substitution of one or more nucleotides.

[0135] An example of a recruitment RNA motif, as used herein, is the MS2 aptamer. The MS2 aptamer specifically binds to the MS2 bacteriophage coat protein (MCP). In one embodiment, the MS2 aptamer is a wild-type MS2 aptamer (SEQ ID NO:11), a mutant MS2 aptamer, or a variant thereof. In another embodiment, the MS2 aptamer comprises a C-5 and / or an F-5 mutation. In some embodiments, the MS2 aptamer can be single copy (i.e., one MS2 aptamer) or double copy (i.e., two MS2 aptamers). In some embodiments, the RNA motif is a single copy RNA motif. In other embodiments, the RNA motif comprises one or more copies.

[0136] Effector fusion proteins The effector fusion protein comprises two components: an RNA binding domain that can bind to the recruiting RNA motif, and an effector domain that comprises a base-modifying enzyme.In some embodiments, the base-modifying enzyme has an activity selected from the group consisting of cytosine deamination activity, adenosine deamination activity, DNA methyltransferase activity and demethylase activity.The terms cytosine and cytidine are used interchangeably for deaminase and deamination activity, and the same is true for the terms adenine and adenosine.

[0137] RNA-binding domain In some embodiments, the RNA binding domain is not the RNA binding domain of a Cas protein (e.g., Cas9) or a variant thereof (e.g., dCas9 or nCas9). Examples of suitable RNA binding domains, including RNA motif-RNA binding pairs, are listed in Table 2. Due to the flexibility of RNA scaffold-mediated recruitment, functional monomers of RNA binding domains, as well as dimers, tetramers, or oligomers, can be formed relatively easily near target DNA or RNA sequences.

[0138] Effector Domain The effector domain or effector protein comprises a base-modifying enzyme with cytidine deaminase activity (e.g., AID, APOBEC1, APOBEC3G) or adenosine deaminase activity (e.g., ADA and tadA) or DNA methyltransferase activity (e.g., Dnmt1 and Dnmt3a) or demethylase activity (e.g., Tet1 and Tet2). In some embodiments, the effector is modified to induce or improve DNA editing activity, for example, in the case of ADA and tadA, which require modification to edit DNA. In some embodiments, the base-modifying enzyme is a wild-type or engineered version of AID, CDA, APOBEC1, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, or other APOBEC family enzymes.

[0139] In some embodiments, the base modifying enzyme is a cytosine deaminase, such as APOBEC1. In some embodiments, the base modifying enzyme is APOBEC1 and comprises the sequence set forth in SEQ ID NO: 17 below: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFI YIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK The effector domain is linked with the RNA binding domain to generate an effector fusion protein. This may be by chemical modification, peptide linker, chemical linker, covalent or non-covalent bond, or protein fusion, or by any means known to those skilled in the art. The linkage may be permanent or reversible. For example, see U.S. Patent Nos. 4,625,014, 5,057,301 and 5,514,363, U.S. Patent Application Nos. 20150182596 and 20100063258, and WO2012142515. The contents of which are incorporated herein by reference in their entirety. In some embodiments, the effector domain is linked with the RNA binding domain by a peptide linker.

[0140] In some embodiments, the effector fusion protein can contain other domains apart from the RNA binding domain and the effector domain. In certain embodiments, the effector fusion protein can contain at least one nuclear localization signal (NLS). Generally, an NLS consists of a chain of basic amino acids. Nuclear localization signals are known in the art (see, for example, Lange et al., J.Biol.Chem., 2007, 282:5101-5105, the entire contents of which are incorporated herein by reference). The NLS can be located at the N-terminus, C-terminus, or internal position of the fusion protein.

[0141] In some embodiments, the fusion protein can comprise at least one cell-penetrating domain to facilitate the delivery of the protein to target cells.In one embodiment, the cell-penetrating domain can be a cell-penetrating peptide sequence.Various cell-penetrating peptide sequences are known in the art, examples of which include HIV-1 TAT protein, TLM of human HBV, Pep-1, VP22, and polyarginine peptide sequence.

[0142] In still other embodiments, the fusion protein can include at least one marker domain.Non-limiting examples of marker domain include fluorescent protein, purification tag, and epitope tag.In some embodiments, the marker domain can be a fluorescent protein.In other embodiments, the marker domain can be a purification tag and / or an epitope tag.See, for example, US20140273233, the entire contents of which are incorporated herein by reference.

[0143] In one embodiment, AID is used as an effector domain and as an example to show how the system works. AID is a cytidine deaminase that can catalyze the deamination of cytidine in the context of DNA or RNA. When delivered to the target site, AID changes the C base to a U base. In dividing cells, this can lead to a C to T point mutation. Alternatively, the C to U change can trigger the cell's DNA repair pathways, primarily the excision repair pathway, which removes the mismatched UG base pair and replaces it with a TA, AT, CG or GC pair. As a result, a point mutation is thought to be generated at the target CG site. Since the excision repair pathway is present in most, if not all, somatic cells, recruitment of AID to the target site can correct the CG base pair to something else. In that case, if the CG base pair is the underlying disease-causing genetic mutation in somatic tissues / cells, the above approach can be used to correct the mutation and thereby treat the disease.

[0144] In another embodiment, APOBEC is used as an effector domain and as an example to show how the system works. APOBEC is also a cytidine deaminase that can catalyze the deamination of cytidine in the context of DNA or RNA.

[0145] In another embodiment, adenosine deaminase is used as an effector domain and as an example to show how the system works. Adenosine deaminase can catalyze the deamination of adenosine in the context of DNA or RNA. For the same reason, if the underlying disease causing gene mutation is AT base pair at a specific site, the same approach can be used to recruit adenosine deaminase to a specific site, where adenosine deaminase can correct the AT base pair to another. See, for example, David Liu-US10113163, the entire contents of which are incorporated herein by reference. Other effector enzymes are expected to produce other types of changes in base pairing. Non-exhaustive examples of examples of base-modifying enzymes are detailed in Table 3.

[0146] In some embodiments, the effector proteins provided herein can include functional variants, e.g., fragments of effector proteins as described in Table 3, and proteins homologous to the fragments or proteins. A functional variant will have, for example, at least about 70% (e.g., at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%) homology to the effector protein or fragment thereof, as compared to the wild-type effector protein.

[0147] [Table 3]

[0148] The three specific components above constitute the technology platform. Each component can be selected from the lists in Tables 1-3 to achieve a specific therapeutic / utility goal.

[0149] In one embodiment, an RNA scaffold-mediated recruitment system was constructed using (i) Cas9, dCas9 or nCas9 from Streptococcus pyogenes as the RNA-guided nickase, (ii) an RNA scaffold comprising a guide RNA sequence, a tracrRNA and a recruitment RNA motif comprising an MS2 phage operator stem-loop, and (iii) an effector fusion protein comprising human AID fused to an MS2 phage operator stem-loop binding protein (MCP). The sequences of the components are listed below.

[0150] Streptococcus pyogenes dCas9 protein sequence (SEQ ID NO:18)

[0151] [ka]

[0152] Cas9 D10A protein (underlined residues: D10A, SEQ ID NO: 19) (nCas9)

[0153] [ka]

[0154] DNA encoding Cas9 D10A protein (SEQ ID NO:20) RNA scaffold expression cassette (Streptococcus pyogenes) (SEQ ID NO:21) containing a 20 nucleotide programmable sequence, a tracrRNA motif, and an MS2 phage operator stem-loop motif:

[0155] [ka]

[0156] The above RNA scaffold contains one MS2 loop (1xMS2). Shown below is an RNA scaffold containing two MS2 loops (2xMS2), where the MS2 scaffold is underlined:

[0157] [ka]

[0158] Effector fusion protein comprising an effector AID-MCP fusion (SEQ ID NO:23):

[0159] [ka]

[0160] Effector fusion protein comprising the effector Apobec1-MCP fusion (SEQ ID NO: 24):

[0161] [ka]

[0162] As with the Cas protein described herein, this effector fusion protein can also be obtained as a recombinant polypeptide. Techniques for producing recombinant polypeptides are known in the art. For example, see Creighton, "Proteins: Structures and Molecular Principles", WH Freeman & Co., NY, 1983; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003; and Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001, the entire contents of which are incorporated herein by reference.

[0163] As described herein, the recruitment of AID to off-target sites can be reduced by mutating Ser38 in AID to Ala. Listed below are the DNA and protein sequences of both wild-type AID and AID_S38A (phosphorylation null, pnAID).

[0164] wtAID cDNA (Ser38 codon in bold and underlined, SEQ ID NO:25):

[0165] [ka]

[0166] wtAID protein (bold and underlined Ser38, SEQ ID NO:26):

[0167] [ka]

[0168] AID_S38A cDNA (bold and underlined is S38A mutation, SEQ ID NO:27)

[0169] [ka]

[0170] AID_S38A protein (bold and underlined is S38A mutation, SEQ ID NO:28)

[0171] [ka]

[0172] The above three components of the platform / system disclosed herein can be expressed using one, two or three expression vectors.The system can be programmed to target virtually any DNA or RNA sequence.In addition to the above second generation base editors, similar second generation base editors can be generated by varying the modular components of the system, including any suitable Cas orthologs, deaminase orthologs and other DNA modifying enzymes.

[0173] In some embodiments, the second target nucleic acid sequence is B2M and / or CD52. In this embodiment, the method comprises two modules, one targeting the B2M gene and the other targeting CD52.

[0174] The inventors have shown that the system described herein is significantly more effective at generating gene knock-ins compared to the alternative fusion CBE system (Figure 7). Similarly, the efficiency of gene knock-out by double nicking according to the present disclosure is 6-fold higher than using the alternative fusion CBE system (Figure 7).

[0175] The present disclosure can also be used to knock out or modify (i) genes involved in fratricide of immune cells, e.g., T cells and NK cells, or (ii) genes that alert the immune system of a subject or animal that a foreign cell, particle, or molecule has entered the subject or animal, e.g., the B2M gene, or (iii) genes that code for proteins that are current therapeutic targets used to reduce or enhance immune responses, e.g., the CD52 and PDCD1 genes. For example, for chimeric antigen receptor (CAR) T therapy for CD7+ leukemia (e.g., AML), CAR T cells need to be genetically modified to not contain CD7 in order to avoid fratricide.

[0176] In various embodiments, the present disclosure can be used to generate gene knockouts or modify or increase the expression of single or multiple genes in various types of cells or cell lines, including but not limited to cells from mammals. The present systems and methods can be applied to multiple gene modifications, including genetically modifying multiple genes or multiple targets within the same gene, as known in the art. This technology can be used for many applications, including but not limited to knocking out genes to prevent graft-versus-host disease by making non-host cells non-immunogenic to the host, or knocking out genes to prevent host-versus-graft disease by making non-host cells resistant to attack by the host. These approaches are also suitable for generating allogeneic (universal) or autologous (patient-specific) cell-based therapeutics.Such knockout genes include genes involved in T cell receptors (TRAC, TRBC1, TRBC2, TRDC, TRGC1, TRGC2), major histocompatibility complex (MHC class I and class II) genes including B2M, coreceptors (HLA-F, HLA-G), genes involved in the innate immune response (MICA, MICB, HCP5, STING, DDX41 and Toll-like receptors (TLRs)), inflammation (NKBBiL, LTA, TNF, LTB, LST1, NCR3, AIF1), heat shock proteins (HSPA1L, HSPA1A, HSPA1B), complement cascade, regulatory receptors (NOTCH family members), antigen processing (TAP, HLA-DM, HLA-DO), increased potency or persistence (e.g., PD-1, CTLA-4 and checkpoint inhibitors). other members of the B7 family of cytokines), genes involved in immunosuppressive immune cells (e.g., FOXP3 and interleukin (IL)-10), genes involved in the interaction of T cells with the tumor microenvironment (including, but not limited to, receptors for cytokines such as TGFB, IL-4, IL-7, IL-2, IL-15, IL-12, IL-18, IFN gamma, etc.), genes that contribute to cytokine release syndrome (including, but not limited to, IL-6, IFN gamma, IL-8 (CXCL8), IL-10, GM-CSF, MIP-1α / β, MCP-1 (CCL2), CXCL9, and CXCL10 (IP-10)), genes that encode the antigen targeted by the CAR / TCR (e.g., endogenous CS1 against which the CAR is designed), or genes that encode the antigen targeted by the CAR / TCR (e.g., endogenous CS1 against which the CAR is designed). These include, but are not limited to, other genes found to be beneficial for T / TCR-T (e.g., TET2, ARG2, NR4A1, NR4A2, NR4A3, TOX and TOX2) or other cell-based therapeutics, including, but not limited to, CAR-NK, CAR-B, etc. See, for example, DeRenzo et al., Genetic Modification Strategies to Enhance CAR T Cell Persistence for Patients With Solid Tumors. Front. Immunol., February 15, 2019, the entire contents of which are incorporated herein by reference.

[0177] One application of the methods and systems provided herein is to manipulate the HLA alleles of bone marrow cells or bone marrow cells differentiated from iPS cells to enhance haplotype compatibility. The engineered cells can be used in bone marrow transplantation to treat leukemia. Another application is to manipulate the negative regulatory element of the fetal hemoglobin gene in hematopoietic stem cells to treat sickle cell anemia and beta-thalassemia. Mutating the negative regulatory element reactivates the expression of the fetal hemoglobin gene in hematopoietic stem cells to compensate for the loss of function caused by mutations in the adult alpha or beta hemoglobin gene. A further application is to manipulate iPS cells to generate allogeneic therapeutic cells for various degenerative diseases, including Parkinson's disease (neuronal cell loss), type 1 diabetes (pancreatic beta cell loss). Other exemplary uses include engineering T cells resistant to HIV infection by inactivating the CCR5 gene and other genes that encode receptors necessary for HIV to enter cells, removing the premature stop codon in the DMD gene to restore expression of dystrophin, and correcting cancer driver mutations, e.g., p53 Y163C.

[0178] Delivery of components into cells In the embodiments provided herein, the guide RNA molecule can be delivered to the target cell through various methods, including but not limited to the following: First, the synthetic RNA molecule (whether sgRNA, crRNA, or tracrRNA and their modifications) is directly introduced into the cell of interest by electroporation, nucleofection, transfection, via nanoparticles, via virus-mediated RNA delivery, via non-virus-mediated delivery, via extracellular vesicles (e.g., exosomes and microvesicles), via eukaryotic cell transfer (e.g., by recombinant yeast), and other methods that can package the RNA molecule and deliver it to the target living cell without changing the genome landscape. Other methods for the introduction of guide RNA molecules include non-integrative transient transfer of DNA polynucleotides containing relevant sequences for protein recruitment so that the molecule can be transcribed into the target guide RNA molecule, including, but not limited to, DNA-only vehicles (e.g., plasmids, minicircles, minivectors, ministrings, protelomerase-generated DNA molecules (e.g., doggybone), artificial chromosomes (e.g., HACs), cosmids), DNA vehicles via nanoparticles, extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., via recombinant yeast), transient viral transfer via AAV, non-integrative viral particles (e.g., lentivirus and retrovirus-based systems), cell-penetrating peptides, and other techniques that may mediate the introduction of DNA into cells without directly integrating into the genomic landscape.Another method for the introduction of guide RNA includes the use of integrative gene transfer techniques for stable introduction of machinery for guide RNA transcription into the genome of target cells, which can be controlled via constitutive or promoter-inducible systems for attenuating guide RNA expression, which can also be designed to remove the system after the utility is met (e.g., introduction of the Cre-Lox recombination system); such techniques for stable gene transfer include, but are not limited to, integration of viral particles (e.g., lentivirus, adenovirus, and retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty and Piggybac), utilization of non-homologous repair pathways introduced by DNA breaks (e.g., utilization of CRISPR and TALEN) techniques and surrogate DNA molecules, as well as other techniques that facilitate integration of target DNA into cells of interest.

[0179] The method for delivering effector fusion protein and CRISPR targeting component is often mediated by the same technology. In some situations, it is advantageous to mediate the delivery of effector fusion protein by one method and the delivery of CRISPR targeting component by another method. Applicable methods are listed below, but are not limited to them. First, direct introduction of mRNA and protein molecules into the cell of interest by electroporation, nucleofection, transfection, via nanoparticles, via virus-mediated package delivery, via extracellular vesicles (e.g., exosomes and microvesicles), via eukaryotic cell transfer (e.g., by recombinant yeast), and other methods that can package macromolecules and deliver them to target living cells without being integrated into the genome landscape. Other methods for the introduction of the coding sequence of effector fusion protein include non-integrative transient transfer of DNA polynucleotides that contain relevant sequences for protein recruitment, so that one or more molecules can be transcribed and translated into target protein molecules. This includes, but is not limited to, DNA-only vehicles (e.g., plasmids, minicircles, minivectors, ministrings, protelomerase-generated DNA molecules (e.g., doggybone), artificial chromosomes (e.g., HACs), cosmids), DNA vehicles via nanoparticles, extracellular vesicles (e.g., exosomes and microvesicles), eukaryotic cell transfer (e.g., via recombinant yeast), transient viral transfer via AAV, non-integrating viral particles (e.g., lentivirus- and retrovirus-based systems), and other techniques that may mediate the introduction of DNA into cells without directly integrating into the genomic landscape.Another method for the introduction of effector fusion proteins (e.g., deaminases) and / or CRISPR targeting components includes the use of integrative gene transfer techniques for stable introduction of transcription and translation machinery into the genome of target cells, which can be controlled via constitutive or inducible promoter systems to attenuate expression of one or more molecules, which can also be designed to allow removal of the system after the utility is met (e.g., introduction of the Cre-Lox recombination system); such techniques for stable gene transfer include, but are not limited to, integration of viral particles (e.g., lentivirus, adenovirus and retrovirus-based systems), transposase-mediated transfer (e.g., Sleeping Beauty and Piggybac), utilization of non-homologous repair pathways introduced by DNA breaks (e.g., utilizing CRISPR and TALEN) techniques and surrogate DNA molecules, as well as other techniques that facilitate integration of target DNA into cells of interest.

[0180] Expression system The nucleic acid encoding the RNA scaffold, effector fusion protein or nickase can be cloned into one or more intermediate expression vectors for introduction into prokaryotic or eukaryotic cells for replication and / or transcription. The intermediate vector is typically a prokaryotic vector, e.g., a plasmid, or a shuttle vector, or an insect vector, for storage or manipulation of the nucleic acid encoding the RNA scaffold or protein component for production of the RNA scaffold or protein component. The nucleic acid can also be cloned into one or more expression vectors for administration to plant cells, animal cells. In some embodiments, the nucleic acid can be cloned into one or more expression vectors for administration to mammalian or human cells, fungal cells, bacterial cells, or protozoan cells. Thus, the present disclosure provides a nucleic acid encoding any of the above RNA scaffolds or proteins. In some embodiments, the nucleic acid is isolated and / or purified.

[0181] The present disclosure also provides recombinant constructs or vectors having sequences encoding one or more of the above RNA scaffolds or proteins. Examples of constructs include vectors, such as plasmids or viral vectors, into which the nucleic acid sequences of the present disclosure are inserted in forward or reverse orientation. In one embodiment, the construct further comprises a regulatory sequence, including a promoter, operably linked to the sequence. Many suitable vectors and promoters are known to those skilled in the art and are commercially available. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press), the entire contents of which are incorporated herein by reference.

[0182] Cell culture The method of the present disclosure further includes maintaining the cell under suitable conditions such that the guide RNA directs the effector protein to a target site in the target sequence and the effector domain modifies the target sequence. Generally, the cell can be maintained under suitable conditions for the growth and / or maintenance of the cell. Suitable cell culture conditions are well known in the art and are described, for example, in "Current Protocols in Molecular Biology", Ausubel et al., John Wiley & Sons, New York, 2003, or "Molecular Cloning: A Laboratory Manual", Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd ed., 2001), Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al. (2007) Nat. Biotechnology 25:1298-1306, the entire contents of each of which are incorporated herein by reference. Those skilled in the art will recognize that methods of culturing cells are known in the art and can and will vary depending on the cell type, in any case routine optimization can be used to determine the best approach for a particular cell type.

[0183] Cells useful for the methods provided herein can be freshly isolated primary cells or can be obtained from frozen aliquots of primary cell cultures. In some embodiments, cells are electroporated for incorporation of gRNA and base editing fusion proteins. As described in the examples below, electroporation conditions for some assays (e.g., for T cells) can include 1600 volts, 10 millisecond pulse width, 3 pulses. After electroporation, electroporated T cells are allowed to recover in cell culture medium and subsequently cultured in T cell expansion medium. In some cases, electroporated cells are allowed to recover in cell culture medium for about 5 to about 30 minutes (e.g., about 5, 10, 15, 20, 25, 30 minutes). In one embodiment, the recovery cell culture medium does not contain antibiotics or other selection agents. In some cases, the T cell expansion medium is complete CTS OpTmizer T-cell Expansion or Immunocult-XT Expansion medium.

[0184] Various illustrative embodiments of the compositions and methods according to the present disclosure are described in the following examples.

[0185] [Example] [Example 1] Promoterless transgene integration into the TRAC locus in T cells with concomitant expression of the transgene driven by the TRAC endogenous promoter and disruption of TCRa / b expression In this example, primary human pan-T lymphocytes were used to demonstrate the utility of the CRISPR system targeting module (nCas9-UGI-UGI) for specific integration of promoterless transgenes into the TRAC locus. Pan-T cells were activated using anti-CD3 and anti-CD28 antibodies, followed by electroporation using nCas9-UGI-UGI component mRNA and two sgRNAs targeting opposite strands in the first exon of the TRAC gene. Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs resulted in alternating double-strand breaks (DSBs). Following electroporation, cells were transduced with AAV6 virus, which was used to drive integration of the GFP coding sequence in frame with the TRAC gene. Integration of the transgene by homology-directed repair (HDR) or non-homologous end joining (NHEJ) induced by a DSB at this locus resulted in efficient knockout of the TRAC gene and disruption of the TCRa / b complex. Following transduction, cells were incubated for 4–7 days and cells were examined by flow cytometry for GFP expression and surface knockout of TCRa / b.

[0186] The data show that the disclosed technique can efficiently induce the loss of TCRα / β from the surface (FIG. 4B) and generate good levels of GFP incorporation (FIG. 4A). GFP expression was not observed in control cells. Control cells were cells that did not receive the targeting components, i.e., cells that did not receive electroporation with the nCas9-UGI-UGI components and the two sgRNAs targeting the TRAC gene. GFP expression was observed only in cells in which the TRAC locus was cleaved and thus GFP was integrated and transcriptionally controlled by the TRAC promoter. As expected, T cells expressing GPF lost expression of TCRa / b (FIG. 4C). Furthermore, the combination of electroporation of the targeting components and AAV transduction did not affect viability when compared to transduction alone, indicating that this technique is not harmful to the cells (FIG. 4D).

[0187] material and method Knock-in guide RNA To disrupt the TRAC locus and place GFP under its transcriptional control, we designed an sgRNA pair targeting the 5' end of the first exon of TRAC and an AAV vector with homology arms to the target locus and encoding a self-cleaving P2A peptide followed by GFP cDNA. The sgRNA targeting the TRAC locus was designed following the rules of PAM-out configuration (PAM site facing outside the target region) and with cleavage sites spaced 40-70 bp apart. The knock-in guide was designed to not contain the 1xMS2 aptamer. The sgRNA was synthesized by Horizon Discovery (formerly Dharmacon).

[0188] Synthetic sgRNA sequence (SEQ ID NO:29) mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues N (spacer) = G, U, A or C 5' sgRNA sequence (SEQ ID NO:30) mG*mA*GAAUCAAAAUCGGUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U 3' sgRNA sequence (SEQ ID NO:31) mA*mA*CAAAUGUGUCACAAAGUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U Messenger RNA The messenger RNA molecule was custom synthesized by TriLink Biotechnologies using the modified nucleotides pseudouridine and 5-methyl-cytosine. This mRNA component was translated into the following protein: nCas9=NLS-nCas9-UGI-UGI-NLS.

[0189] Construction of AAV plasmids AAV plasmids were custom synthesized by GenScript, Inc. Based on the pAAV backbone, we designed pAAV-TRAC-GFP, which contains, in order: a 0.9 kb left homology arm of genomic TRAC flanked by a 5' gRNA targeting sequence, a GSG (gly-ser-gly) peptide followed by a self-cleaving P2A peptide in frame with the first exon of TRAC, a GFP coding sequence, a bovine growth hormone polyA signal (bGHpA), and a 0.9 kb right homology arm of genomic TRAC flanked by a 3' gRNA targeting sequence.

[0190] cell CD3+ T cells were isolated from whole blood or outsourced to Hemacare. Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (SepMate PBMC isolation tubes, STEMCELL Technologies), followed by purification of T lymphocytes using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies). Cells were cultured at 37°C and 5% CO2 for 48 h with Dynabeads (1:1 beads:cells) Human T-Activator CD3 / CD28 (ThermoFisher) in Immunocult XT T Cell Expansion medium (STEMCELL Technologies) supplemented with 100U / ml IL-2 (STEMCELL Technologies) and 1× penicillin / streptomycin (ThermoFisher) at 37°C and 5% CO2 for 10 min. 6After activation, the beads were removed by placing on a magnet and the cells were placed back in culture.

[0191] Electroporation of T cells 48-72 hours after activation, T cells were electroporated using a Neon Electroporator (Thermofisher). The conditions for the Neon Electroporator were 1600v / 10ms / 3 pulses using a 10μl tip containing 250K cells, combined with a total amount of 1-5μg of mRNA and 2μM of each targeting gRNA. After electroporation, cells were transferred to Immunocult XT medium containing 100U / ml IL-2, 100U / ml IL-7 and 100U / ml IL-15 (STEMCELL Technologies) and cultured at 37℃ and 5% CO2 for 48-72 hours.

[0192] Transduction of T cells Recombinant AAV6 particles were produced by Vigene Biosciences. When applicable, recombinant AAV6 particles carrying the GFP coding sequence were injected at 1 × 10 per cell 2–4 h after electroporation. 6 The edited cells were then cultured at 37°C and 5% CO2 for 96 h, at a concentration of approximately 1 x 10 cells per ml. 6 The density of individuals was maintained.

[0193] Flow cytometry T cell identity and QC were confirmed by CD3 antibody staining (Biolegend). T cell activation was confirmed by CD25 staining. GFP positive cells were measured by flow cytometry 7 days after electroporation / transduction. Levels of TCR- / GFP+ cells were assessed by flow cytometry using TCRα / β antibody (Biolegend) 7 days after electroporation / transduction. All phenotypic data were reported as percentage of live cells confirmed by viability staining.

[0194] [Example 2] Integration of a CAR gene into the TRAC locus in T cells results in transgene expression driven by the TRAC endogenous promoter and disruption of TCR expression In this example, primary human pan-T lymphocytes were used to demonstrate the utility of the enzyme, targeting module, (nCas9-UGI-UGI) in the CRISPR system for specific integration of the CAR gene into the TRAC locus. Pan-T cells were activated using anti-CD3 and anti-CD28 antibodies, followed by electroporation with the nCas9-UGI-UGI component mRNA and two sgRNAs targeting opposite strands in the first exon of the TRAC gene. Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs resulted in alternating double-strand breaks (DSBs). Following electroporation, cells were transduced with AAV6 virus, which was used to drive the integration of the CAR coding sequence in frame with the TRAC gene. Integration of the transgene by homology-directed repair (HDR) or non-homologous end joining (NHEJ) induced by a DSB at this locus resulted in efficient knockout of the TRAC gene and disruption of the TCR complex. Following transduction, cells were incubated for 4–7 days and cells were examined by flow cytometry for CAR expression and surface knockout of the TCR.

[0195] The data show that the disclosed technique can efficiently induce the loss of TCRα / β from the surface and generate good levels of CAR integration (Figure 5A-B). CAR expression was not observed in control cells (cells that did not receive the two sgRNAs targeting nCas9-UGI-UGI and TRAC gene components) and was only observed in cells in which the TRAC locus was cleaved and thus the CAR was integrated and transcriptionally controlled by the TRAC promoter. As expected, CAR-expressing T cells lost expression of the TCR (Figure 5C). Furthermore, the combination of electroporation of the targeting components with AAV transduction did not affect viability when compared to transduction alone, indicating that this technique is not harmful to the cells (Figure 5D).

[0196] material and method Knock-in guide RNA To disrupt the TRAC locus and place the CD19-specific 1928z CAR gene under its transcriptional control, we designed an sgRNA pair targeting the 5' end of the first exon of TRAC and an AAV vector with homology arms to the target locus and encoding the CAR cDNA followed by a self-cleaving P2A peptide. The sgRNA targeting the TRAC locus was designed according to the rules of PAM-out configuration (the PAM site faces outside the target region) and the cleavage sites were spaced 40-70 bp apart. The knock-in guide was designed to not contain the 1xMS2 aptamer. The sgRNA was synthesized by Horizon Discovery (formerly Dharmacon).

[0197] Synthetic sgRNA sequence (SEQ ID NO:29) mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues 5' sgRNA sequence (SEQ ID NO:30) mG*mA*GAAUCAAAAUCGGUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U 3' sgRNA sequence (SEQ ID NO:31) mA*mA*CAAAUGUGUCACAAAGUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U Messenger RNA The messenger RNA molecules were custom synthesized by TriLink Biotechnologies using the modified nucleotides pseudouridine and 5-methyl-cytosine. The mRNA components were translated into the following protein: nCas9=NLS-nCas9-UGI-UGI-NLS.

[0198] Construction of AAV plasmids AAV plasmids were custom synthesized by GenScript, Inc. Based on the pAAV backbone, we designed pAAV-TRAC-1928Z_CAR and pAAV-TRAC-GFP, which contain, in order: a 0.9 kb left homology arm of genomic TRAC flanked by a 5' gRNA targeting sequence, a GSG (gly-ser-gly) peptide followed by a self-cleaving P2A peptide in frame with the first exon of TRAC, the 1928z CAR or GFP coding sequence used in Yescarta™ therapy, the bovine growth hormone polyA signal (bGHpA), and a 0.9 kb right homology arm of genomic TRAC flanked by a 3' gRNA targeting sequence. Briefly, CD19-CAR (Kochenderfer et al., 2009, J Immunotherapy) was composed of a single-chain variable fragment (scFV) specific for human CD19 derived from the FMC63 murine hybridoma (Nicholson et al., 1997, Mol Immunology), a portion of the human CD28 molecule (the entire hinge extracellular portion, transmembrane domain, and intracellular domain) and the entire domain of the CD3-zeta chain (Figure 2).

[0199] cell CD3+ T cells were isolated from whole blood or outsourced to Hemacare. Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (SepMate PBMC isolation tubes, STEMCELL Technologies), followed by purification of T lymphocytes using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies). Cells were cultured at 37°C and 5% CO2 for 48 h with Dynabeads (1:1 beads:cells) Human T-Activator CD3 / CD28 (ThermoFisher) in Immunocult XT T Cell Expansion medium (STEMCELL Technologies) supplemented with 100U / ml IL-2 (STEMCELL Technologies) and 1× penicillin / streptomycin (ThermoFisher) at 37°C and 5% CO2 for 10 min.6 After activation, the beads were removed by placing on a magnet and the cells were placed back in culture.

[0200] Electroporation of T cells 48–72 hours after activation, T cells were electroporated using a Neon Electroporator (Thermofisher). The Neon Electroporator conditions were 1600v / 10ms / 3 pulses using a 10μl tip containing 250K cells combined with a total amount of 1–5μg of mRNA and 2μM of each targeting gRNA. After electroporation, cells were transferred to Immunocult XT medium containing 100U / ml IL-2, 100U / ml IL-7 and 100U / ml IL-15 (STEMCELL Technologies) and cultured at 37°C and 5% CO2 for 48–72 hours.

[0201] Transduction of T cells Recombinant AAV6 particles were produced by Vigene Biosciences. Where applicable, recombinant AAV6 particles carrying the CD19-CAR coding sequence were delivered to cells at 1 × 10 cells / cell 2–4 h after electroporation. 6 The edited cells were then cultured at 37°C and 5% CO2 for 96 h, with a concentration of approximately 1 x 10 cells per ml. 6 The density of individuals was maintained.

[0202] Flow cytometry T cell identity and QC were confirmed by CD3 antibody staining (Biolegend). T cell activation was confirmed by CD25 staining. CD19-CAR positive cells were detected 96 hours after electroporation / transduction by flow cytometry using anti-FMC 63 scFv antibody (AcroBiosystem). Levels of TCR- / CAR+ cells were assessed by combined staining with TCRa / b antibody (Biolegend). All phenotypic data were reported as percentage of live cells confirmed by viable cell staining.

[0203] [Example 3] Generation of universal CAR-T cells by an aptamer-based base editing system In this example, primary human pan-T lymphocytes were used to demonstrate the usefulness of the CRISPR-aptamer-based gene editing system for specific integration of CAR gene into TRAC locus and simultaneous knockout of TRAC, B2M and CD52 genes by cytosine base editing system. The disclosed method achieves base editing guided knockout through the recruitment of deaminase to target site by sgRNA-aptamer. CAR integration and the resulting TRAC knockout are achieved by the same enzyme used in the CRISPR system in combination with sgRNA. The advantage over previous systems is that both modifications are achieved using one CRISPR enzyme, or a single RNA-guided nickase, i.e., the same enzyme is used for knocking in CAR gene and knocking out TRAC, B2M and CD52 genes.

[0204] Pan-T cells were activated using anti-CD3 and anti-CD28 antibodies, followed by electroporation using the following components: (i) mRNA encoding deaminase-MCP, (ii) mRNA encoding nCas9-UGI-UGI protein, (iii) two sgRNAs targeting opposite strands in the first exon of the TRAC gene, and (iv) sgRNA-aptamers against two different genes. Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs resulted in alternating double-strand breaks (DSBs). Following electroporation, cells were transduced with AAV6 virus, which was used to drive integration of the CAR coding sequence in frame with the TRAC gene by homology-directed repair (HDR). Integration of the transgene by HDR or non-homologous end joining (NHEJ) induced by DSBs at this locus resulted in efficient knockout of the TRAC gene and disruption of the TCR complex. Following transduction, cells were incubated for 4-7 days and subsequently examined by flow cytometry for CAR expression and surface knockout of TCR, B2M and CD52. Base conversion was measured by targeted PCR amplification and Sanger sequencing. Multiplex KO levels within the CAR+ population were confirmed by flow cytometry using a multi-antibody panel.

[0205] When the base editing components were delivered to cells, high levels of base conversion were observed at the two targeted loci, B2M and CD52, and the efficiency of editing was not compromised by viral vector delivery (Figure 6A-B). The data show that sgRNA-aptamer-based base editing was comparable in efficiency to a conventional CRISPR-assisted base editing system that fuses a deaminase with a Cas protein. Functional KO information generated by flow cytometry correlated with base conversion (Figure 6C-D). However, loss of TCRα / β from the surface and integration of the CAR were efficiently achieved only by the enzymes of the disclosed CRISPR system, not by the alternative fusion CBE system (Figure 7A-B). High levels of multi-gene KO (triple KO in this example) were achieved in the CAR-expressing population by the present technology (Figure 7C). The data show that the present technology generated CAR-T cells with functional knockouts in multiple genes and functioning as universal CAR-T cells. This data also demonstrates the superiority of the disclosed CRISPR-based gene editing system that achieves HDR-guided integration compared to the alternative fusion CBE system.

[0206] material and method Guide RNA for base editing Internally generated data was used to identify base editing windows calculated at set distances from the PAM motif (NGG). This data was used to develop algorithms to predict guide sequences applicable for phenotype or gene KO for the following genes: TRAC, TRBC1, TRBC2, PDCD-1, B2M, and CD52 (Table 4). sgRNAs were designed to contain 1x MS2 aptamers. Guide RNA sequences were synthesized by Horizon Discovery (formerly Dharmacon) and Agilent.

[0207] Synthetic 1x MS2 sgRNA sequence (SEQ ID NO:32): mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGCGCACAUGAGGAUCACCCAUGUGCUUUUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues

[0208] [Table 4-1]

[0209] [Table 4-2]

[0210] [Table 4-3]

[0211] [Table 4-4]

[0212] [Table 4-5]

[0213] [Table 4-6]

[0214] Knock-in guide RNA To disrupt the TRAC locus and place the CD19-specific 1928z CAR gene under its transcriptional control, we designed an sgRNA pair targeting the 5' end of the first exon of TRAC and an AAV vector with homology arms to the target locus and encoding the CAR cDNA followed by a self-cleaving P2A peptide. The sgRNA targeting the TRAC locus was designed according to the rules of PAM-out configuration (the PAM site faces outside the target region) and the cleavage sites were spaced 40-70 bp apart. The knock-in guide was designed to not contain the 1xMS2 aptamer. The sgRNA was synthesized by Horizon Discovery (formerly Dharmacon).

[0215] Synthetic sgRNA sequence (SEQ ID NO:29) mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues 5' sgRNA sequence (SEQ ID NO:30) mG*mA*GAAUCAAAAUCGGUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U 3' sgRNA sequence (SEQ ID NO:31) mA*mA*CAAAUGUGUCACAAAGUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U Messenger RNA Messenger RNA molecules were custom synthesized by TriLink Biotechnologies using the modified nucleotides pseudouridine and 5-methyl-cytosine. The mRNA components were translated into the following proteins: deaminase Apobec1 = NLS-rApobec1-linker-MCP and nCas9 = NLS-nCas9-UGI-UGI-NLS.

[0216] Construction of AAV plasmids AAV plasmids were custom synthesized by GenScript, Inc. Based on the pAAV backbone, we designed pAAV-TRAC-1928Z_CAR, which contains, in order: a 0.9 kb left homology arm of genomic TRAC flanked by a 5' gRNA targeting sequence, a GSG (gly-ser-gly) peptide followed by a self-cleaving P2A peptide in frame with the first exon of TRAC, the 1928z CAR used in Yescarta™ therapy, the bovine growth hormone polyA signal (bGHpA), and a 0.9 kb right homology arm of genomic TRAC flanked by a 3' gRNA targeting sequence. Briefly, CD19-CAR (Kochenderfer et al., 2009, J Immunotherapy) was composed of a single chain variable fragment scFV specific for human CD19 derived from the FMC63 murine hybridoma (Nicholson et al., 1997, Mol Immunology), a portion of the human CD28 molecule (the entire hinge extracellular portion, transmembrane domain, and intracellular domain) and the entire domain of the CD3-zeta chain. A second AAV vector was designed and cloned in which the CD19-CAR CDS was replaced with the turboGFP coding sequence.

[0217] cell CD3+ T cells were isolated from whole blood or outsourced to Hemacare. Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (SepMate PBMC isolation tubes, STEMCELL Technologies), followed by purification of T lymphocytes using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies). Cells were cultured at 37°C and 5% CO2 for 48 h with Dynabeads (1:1 beads:cells) Human T-Activator CD3 / CD28 (ThermoFisher) in Immunocult XT T Cell Expansion medium (STEMCELL Technologies) supplemented with 100U / ml IL-2 (STEMCELL Technologies) and 1× penicillin / streptomycin (ThermoFisher) at 37°C and 5% CO2 for 10 min. 6 After activation, the beads were removed by placing on a magnet and the cells were placed back in culture.

[0218] Electroporation of T cells 48-72 hours after activation, T cells were electroporated using a Neon Electroporator (Thermofisher). The Neon Electroporator conditions were 1600v / 10ms / 3 pulses using a 10μl tip containing 250K cells for both deaminase-MCP and nCas9-UGI-UGI, combined with a total amount of 1-5μg mRNA and 2μM of each targeting gRNA, where applicable. After electroporation, cells were transferred to Immunocult XT medium containing 100U / ml IL-2, 100U / ml IL-7 and 100U / ml IL-15 (STEMCELL Technologies) and cultured at 37°C and 5% CO2 for 48-72 hours.

[0219] Transduction of T cells Recombinant AAV6 particles were produced by Vigene Biosciences. When applicable, recombinant AAV6 particles were delivered to cells at 1 × 10 cells per well 2–4 h after electroporation. 6 The edited cells were then cultured at 37°C and 5% CO2 for 96 h, with approximately 1 × 10 cells per ml. 6 The density of individuals was maintained.

[0220] Flow cytometry T cell identity and QC were confirmed by CD3 antibody staining (Biolegend). T cell activation was confirmed by CD25 staining. CD19-CAR+ cells were detected 96 hours after electroporation / transduction by flow cytometry using anti-FMC63 scFv antibody (AcroBiosystem). Multiple KO of phenotypic genes was assessed 96 hours after electroporation / transduction: TRAC by TCRab antibody staining (Biolegend), B2M by B2M antibody (Biolegend), and CD52 by CD52 antibody (Biolegend), and all phenotypic data were reported as percentage of live cells confirmed by viable cell staining.

[0221] Genomic DNA analysis Genomic DNA was released from lysed cells 96 hours after electroporation. Loci of interest were amplified by PCR and the products were subsequently subjected to Sanger sequencing (Genewiz). Data were analyzed by proprietary in-house software.

[0222] [Table 5]

[0223] [Example 4] Generation of iPSC cell lines for allogeneic CAR therapy by a CRISPR-aptamer-based gene editing system In this example, we use induced pluripotent stem cells (iPSCs) to demonstrate the utility of CRISPR-aptamer-based gene editing system for specific integration of CAR gene into TRAC locus or B2M locus, and simultaneous knockout of TRAC, B2M and CIITA genes. The method disclosed herein achieves base editing guided knockout through the recruitment of deaminase to target site by sgRNA-aptamer. The integration of CAR and the resulting knockout of B2M are achieved using the same enzyme (combined with sgRNA) and base editing system used for CRISPR system. The advantage over previous systems is that all modifications are achieved using one enzyme, or a single RNA-guided nickase, i.e., the same enzyme is used for knocking in CAR gene and knocking out TRAC and CIITA genes.

[0224] iPSCs are cultured in cell line-specific medium, dissociated, and subsequently electroporated using deaminase-MCP, nCas9-UGI-UGI components, two sgRNAs targeting opposite strands in the first exon of the B2M gene, sgRNA-aptamers for two different genes (TRAC and CIITA), and mRNA components for both exogenous dsDNA templates. Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs result in alternating double-strand breaks (DSBs). The exogenous dsDNA template contained homology arms associated with the DNA cleavage target site on the B2M locus and also contained a CAR transgene cassette. Following electroporation, cells are incubated for 4-7 days and subsequently examined by flow cytometry for CAR expression and knockout of B2M and CIITA. Base conversion is also measured by targeted PCR amplification and Sanger sequencing for TRAC and CIITA.Multiple KO levels within the CAR+ population are confirmed by flow cytometry using a multi-antibody panel.

[0225] This technology has the potential to generate iPSC lines that contain both transgenes at highly specific loci and multiple simultaneous edits, which may be superior to currently available techniques. These edited iPSCs can then be used to differentiate or program into clinically relevant iPSC-derived allogeneic CAR T cells.

[0226] [Example 5] Generation of improved NK cells for allogeneic CAR therapy by a CRISPR-aptamer-based gene editing system In this example, NK cells are used to demonstrate the usefulness of CRISPR-based gene editing methods for specific integration of CAR into CISH locus and simultaneous knockout of PD1 and NKG2A.This system achieves base editing guided knockout through the recruitment of deaminase to target site by sgRNA-aptamer.The integration of CAR and the resulting CISH knockout are achieved using the same enzymes used for CRISPR system and base editing system.The advantage over previous systems is that both modifications are achieved using one enzyme or one RNA-guided nickase.

[0227] NK cells are electroporated with deaminase-MCP, nCas9-UGI-UGI components, two sgRNAs targeting opposite strands in the first exon of the CISH gene, and both mRNA components against sgRNA-aptamers against two different genes (PD1 and NKG2A). Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs result in alternating double-strand breaks (DSBs). Following electroporation, cells are transduced with AAV6 virus, which is used to drive integration of the CAR coding sequence in frame with the CISH gene by homology-directed repair (HDR). Integration of the transgene by HDR or non-homologous end joining (NHEJ) induced by DSBs at this locus results in efficient knockout of the CISH gene. Following transduction, cells are incubated for 4-7 days and then examined for CAR expression and CISH, PD1 and NKG2A knockout by flow cytometry. Base conversion is also measured by targeted PCR amplification and Sanger sequencing for PD1 and NKG2A. A multi-antibody panel is used to confirm multiple KO levels within the CAR+ population by flow cytometry.

[0228] Thus, this technology has the potential to generate NK cells that contain both transgenes at highly specific loci and multiple simultaneous edits, which may be superior to currently available technologies. These edited NK cells can be used as improved CAR-NK cells.

[0229] [Example 6] Generation of universal CAR-T cells with four gene knockouts by a CRISPR-aptamer-based gene editing system In this example, primary human pan-T lymphocytes were used to demonstrate the utility of the CRISPR-based gene editing system for specific integration of CAR gene into TRAC locus and simultaneous knockout of TRAC, B2M, CD52 and PDCD1 genes by cytosine base editing system. The disclosed method achieves base editing guided knockout through the recruitment of deaminase to target site by sgRNA-aptamer. The integration of CAR gene and the resulting TRAC knockout are achieved using the same enzyme used in CRISPR system in combination with sgRNA. The advantage over previous systems is that both modifications are achieved using one CRISPR enzyme, or a single RNA-guided nickase.

[0230] Pan-T cells were activated using anti-CD3 and anti-CD28 antibodies, followed by electroporation using the following components: deaminase-MCP, nCas9-UGI-UGI protein, two sgRNAs targeting opposite strands in the first exon of the TRAC gene, and an sgRNA-aptamer mRNA component for three different genes. Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs result in alternating double-strand breaks (DSBs). Following electroporation, cells were transduced with AAV6 virus, which was used to drive integration of the CAR coding sequence in frame with the TRAC gene by homology-directed repair (HDR). Integration of the transgene by HDR or non-homologous end joining (NHEJ) induced by DSBs at this locus resulted in efficient knockout of the TRAC gene and disruption of the TCR complex. After transduction, cells were incubated for 4-7 days and then examined by flow cytometry for CAR expression and surface knockout of TCRa / b, B2M, CD52 and PD1. Base conversion was measured by targeted PCR amplification and Sanger sequencing.

[0231] When base editing constructs were delivered to cells, high levels of C to T conversions were observed at the three targeted loci, B2M, CD52 and PDCD1, and base editing efficiency was not compromised by viral vector delivery (Figure 8A-C). In contrast, similar levels of editing with indel formation were observed at the three targeted loci, B2M, CD52 and PDCD1, by wild-type Cas9 (Figure 8D-F). Functional KOs for B2M, CD52 and PD1 genes generated by flow cytometry correlated with base conversion or indel formation and were comparable to knockouts generated by wt Cas9 (Figure 9A-C). CAR integration, measured as CAR-positive or TCRa / b-positive cells, was efficiently achieved using the present base editing system and was comparable to the levels observed using wild-type Cas9 (Figure 10A-B).

[0232] Allogeneic CAR-T cells were generated using the base editing system of the present disclosure. To generate CAR-T cells, a pair of synthetic sgRNAs targeting exon 1 of the TRAC locus, sgRNA-aptamers for base editing targeting of B2M, CD52 and PDCD1, and nCas9-UGI-UGI and Apobec1-MCP mRNA were co-delivered into CD3-positive T cells. Cas9 samples were electroporated with wild-type Cas9 mRNA and normal sgRNA. This was followed by transduction with the viral vector AAV6-TRAC-CAR. Approximately 7 days after electroporation, CD3+ cells were depleted from the culture, and the resulting CAR-T cells were incubated with CD19-positive Raji cells preloaded with Calcein AM at CAR-T:Raji cell ratios of 1:1 and 5:1 for 4 hours. As shown in Figure 11, the allogeneic CAR-T cells of the present disclosure efficiently killed antigen-positive cancer cells (CD19-positive Raji cells), which was comparable to the results of wtCas9. This data indicates that this technology can generate CAR-T cells with functional knockouts of multiple genes and function efficiently as universal CAR-T cells.

[0233] material and method Guide RNA for base editing: Internally generated data was used to identify base editing windows calculated at set distances from the PAM motif (NGG). This data was used to develop algorithms predicting guide sequences applicable for phenotype or gene KO for the following genes: TRAC, TRBC1, TRBC2, PDCD-1, B2M and CD52 (Table 4). sgRNAs were designed to contain 1x MS2 aptamers. Guide RNA sequences were synthesized by Horizon Discovery (formerly Dharmacon) and Agilent.

[0234] Synthetic 1x MS2 sgRNA sequence (SEQ ID NO:32): mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGCGCACAUGAGGAUCACCCAUGUGCUUUUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues Knock-in guide RNA: To disrupt the TRAC locus and place the CD19-specific 1928z CAR gene under its transcriptional control, we designed an sgRNA pair targeting the 5' end of the first exon of TRAC and an AAV vector with homology arms to the target locus and encoding the CAR cDNA followed by a self-cleaving P2A peptide. The sgRNA targeting the TRAC locus was designed according to the rules of PAM-out configuration (the PAM site faces outside the target region) and the cleavage sites were spaced 40-70 bp apart. The knock-in guide was designed to not contain the 1xMS2 aptamer. The sgRNA was synthesized by Horizon Discovery (formerly Dharmacon).

[0235] Synthetic sgRNA sequence (SEQ ID NO:29): mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues 5' sgRNA sequence (SEQ ID NO:30) mG*mA*GAAUCAAAAUCGGUGAAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U 3' sgRNA sequence (SEQ ID NO:31) mA*mA*CAAAUGUGUCACAAAGUAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U Messenger RNA Messenger RNA molecules were custom synthesized by TriLink Biotechnologies using the modified nucleotides pseudouridine and 5-methylcytosine. The mRNA components were translated into the following proteins: deaminase Apobec1 = NLS-rApobec1-linker-MCP and nCas9 = NLS-nCas9-UGI-UGI-NLS.

[0236] Construction of AAV plasmids AAV plasmids were custom synthesized by GenScript, Inc. Based on the pAAV backbone, we designed pAAV-TRAC-1928Z_CAR: a 0.9 kb left homology arm of genomic TRAC flanked by a 5' gRNA targeting sequence, a self-cleaving P2A peptide in frame with a GSG (gly-ser-gly) peptide followed by the first exon of TRAC, the 1928z CAR used in Yescarta™ therapy, the bovine growth hormone polyA signal (bGHpA), and a 0.9 kb right homology arm of genomic TRAC flanked by a 3' gRNA targeting sequence. Briefly, CD19-CAR (Kochenderfer et al., 2009, J Immunotherapy) was composed of a single chain variable fragment scFV specific for human CD19 derived from the FMC63 murine hybridoma (Nicholson et al., 1997, Mol Immunology), a portion of the human CD28 molecule (the entire hinge extracellular portion, transmembrane domain, and intracellular domain) and the entire domain of the CD3-zeta chain. A second AAV vector in which the CD19-CAR CDS was replaced with the turboGFP coding sequence was designed and cloned.

[0237] cell CD3+ T cells were isolated from whole blood or outsourced to Hemacare. Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (SepMate PBMC isolation tubes, STEMCELL Technologies), followed by purification of T lymphocytes using the EasySep™ Human T Cell Isolation Kit (STEMCELL Technologies). Cells were cultured at 37°C and 5% CO2 for 48 h with Dynabeads (1:1 beads:cells) Human T-Activator CD3 / CD28 (ThermoFisher) in Immunocult XT T Cell Expansion medium (STEMCELL Technologies) supplemented with 100U / ml IL-2 (STEMCELL Technologies) and 1× penicillin / streptomycin (ThermoFisher) at 37°C and 5% CO2 for 10 min.6 After activation, the beads were removed by placing on a magnet and the cells were placed back in culture.

[0238] Electroporation of T cells 48-72 hours after activation, T cells were electroporated using a Neon Electroporator (Thermofisher). The Neon Electroporator conditions were 1600v / 10ms / 3 pulses using a 10μl tip containing 250K cells for both deaminase-MCP and nCas9-UGI-UGI, combined with a total amount of 1-5μg mRNA and 2μM of each targeting gRNA, where applicable. After electroporation, cells were transferred to Immunocult XT medium containing 100U / ml IL-2, 100U / ml IL-7 and 100U / ml IL-15 (STEMCELL Technologies) and cultured at 37°C and 5% CO2 for 48-72 hours.

[0239] Transduction of T cells Recombinant AAV6 particles were produced by Vigene Biosciences. When applicable, recombinant AAV6 particles were delivered to cells at 1 × 10 cells per well 2–4 h after electroporation. 6 The edited cells were then cultured at 37°C and 5% CO2 for 96 h, and approximately 1 × 10 cells were added to the culture. 6 The density was maintained at 100 / ml.

[0240] Flow cytometry For detection of PD1 by flow cytometry, cells were stimulated with PMA (50 ng / ml) and ionomycin (250 ng / ml) for 48 h prior to analysis.

[0241] T cell identity and QC were confirmed by CD3 antibody staining (Biolegend). T cell activation was confirmed by CD25 staining. CD19-CAR+ cells were detected 96 hours after electroporation / transduction using anti-FMC63 scFv antibody (AcroBiosystem). Multiple KO of phenotypic genes was assessed 96 hours after electroporation / transduction: TRAC by TCRab antibody staining (Biolegend), B2M by B2M antibody (Biolegend), CD52 by CD52 antibody (Biolegend), and PD1 by PD1 antibody (Biolegend), and all phenotypic data were reported as percentage of live cells confirmed by viable cell staining.

[0242] Genomic DNA analysis Genomic DNA was released from lysed cells 96 hours after electroporation. Loci of interest were amplified by PCR and the products were subsequently subjected to Sanger sequencing (Genewiz). Data were analyzed by proprietary in-house software.

[0243] Killing assay To test the functionality of CAR-T cells generated using base editing technology, the modified CAR-T cells were first depleted of CD3 positive cells using EasySep™ Human CD3 Positive Selection Kit II (Stemcell) and then incubated with CD19 positive Raji cells at a CAR-T:Raji cell ratio of 1:1 or 5:1. Prior to incubation, the Raji cells were loaded with Calcein AM. After 4 hours of incubation, the supernatants from the cultures were collected and analyzed for fluorescence emission using a plate reader with an excitation / emission of 494 / 517. The level of fluorescence is proportional to the level of target Raji cell killing. The percentage of target cell killing was calculated as [(mean value of test condition-mean value of negative control condition) / (mean value of positive control condition-mean value of negative control condition)]×100, where the negative control condition is Raji cells without CAR-T cells and the positive control condition is Raji cells exposed to 2% Triton to achieve complete lysis.

[0244] [Example 7] Promoter-less transgene integration into the B2M locus in iPSCs with transgene expression driven by the B2M endogenous promoter, as well as disruption of B2M expression and base editing of the CIITA gene In this example, iPSCs were used to demonstrate the utility of the CRISPR-aptamer-based gene editing system for specific integration of promoterless transgene (GFP) into the B2M locus and simultaneous knockout of B2M and CIITA genes by cytosine base editing system. The disclosed method achieved base editing guided knockout through the recruitment of deaminase to the target site by sgRNA-aptamer. The integration of GFP and the resulting knockout of B2M was achieved by the same enzyme used in the CRISPR system in combination with sgRNA. The advantage over previous systems is that both modifications were achieved using one CRISPR enzyme, or a single RNA-guided nickase, i.e., the same enzyme was used for knocking in the transgene and knocking out the B2M and CIITA genes.

[0245] In this example, exogenous DNA template was delivered in the form of circular or linear double-stranded DNA. Exogenous DNA template was flanked by homology arms from B2M locus. Exogenous DNA template with homology arms was flanked or not flanked by sgRNA B2M targeting sequence (CRISPR / Cas9 target sequence (CTS)) (Figure 13A and B, respectively). In some cases, GFP transgene flanked by homology arms was flanked by the sequence of gRNA pair targeting B2M locus, so that when circular double-stranded DNA was co-delivered with CRISPR components into cells, donor nucleic acid sequence was released from circular dsDNA as linear DNA after cleavage by CRISPR / Cas.

[0246] iPSCs were electroporated with the following components: (i) mRNA encoding deaminase-MCP (SEQ ID NO:XX), (ii) mRNA encoding nCas9-UGI-UGI protein, (iii) two sgRNAs targeting opposite strands in the first exon of the B2M gene, (iv) an sgRNA-aptamer to the CIITA gene, and (v) a circular or linear double-stranded DNA containing a GFP coding sequence with homology arms to the B2M gene. In the circular and linear forms, the homology arms can be flanked (CTS_B2M_tGFP-SEQ ID NO:136) or not (B2M_tGFP-SEQ ID NO:135) by the sgRNA B2M targeting sequence (CRISPR / Cas9 target sequence (CTS)).

[0247] B2M-tGFP - SEQ ID NO: 135: CTS__B2M_tGFP - SEQ ID NO: 136: Two single nicks generated by nCas9-UGU-UGI at two target loci recognized by sgRNAs in B2M exon 1 resulted in alternating double-strand breaks (DSBs). Integration of the GFP transgene was prompted by homology arms to the B2M locus by homology-directed repair (HDR). Transgene integration by HDR or non-homologous end joining (NHEJ) induced by DSBs at this locus resulted in efficient knockout of the B2M gene. B2M expression levels were low in pluripotent stem cells, including iPSCs, and could be induced by treatment with interferon-γ. To detect functional knockout of B2M and successful GFP knock-in at the B2M locus, edited cells were treated with interferon-γ for 48 h 2 or 4 days after electroporation and subsequently analyzed by flow cytometry. Base conversion at the CIITA locus was measured by targeted PCR amplification and Sanger sequencing 4–6 days after electroporation.

[0248] When circular double-stranded DNA containing the base editing components and a tGFP coding sequence with homology arms to B2M was delivered to cells, a C to T conversion was observed at the targeted CIITA locus, and base editing efficiency was not compromised by delivery of the donor DNA (Figure 15A). Efficient B2M knockout was observed (Figure 15B). Integration of tGFP at the B2M locus was achieved, measured as tGFP-positive cells in cells treated with interferon-γ (Figure 15C). The highest degree of integration was achieved using the donor template flanked on both sides by the sgRNA B2M targeting sequence (CTS_B2M_tGFP). As expected, the majority of GFP-positive cells are B2M-negative (Figure 15D).

[0249] When linear double-stranded DNA containing the base editing components and a tGFP coding sequence with homology arms to B2M was delivered to cells, a C to T conversion was observed at the targeted CIITA locus, and base editing efficiency was not compromised by the delivery of donor DNA (Figure 16A). Efficient B2M knockout was observed (Figure 16B). Integration of tGFP at the B2M locus was achieved, measured as tGFP-positive cells in cells treated with interferon-γ (Figure 16C). The highest degree of integration was achieved using the donor template flanked on both sides by the sgRNA B2M targeting sequence (CTS_B2M_tGFP). As expected, the majority of GFP-positive cells are B2M-negative (Figure 16D).

[0250] [Example 8] Generation of universal iPSCs by a CRISPR-aptamer-based gene editing system In this example, we used iPSCs to demonstrate the utility of the CRISPR-aptamer-based gene editing system for specific integration of scHLA-E trimer transgene into the B2M locus (see FIG. 12 for a schematic of the construct) and simultaneous knockout of B2M and CIITA genes by a cytosine base editing system. The disclosed method achieved base editing guided knockout via the recruitment of deaminase to the target site by sgRNA-aptamer. The integration of scHLA-E trimer and the resulting knockout of B2M was achieved by the same enzyme used in the CRISPR system in combination with sgRNA. The advantage over previous systems is that both modifications were achieved using one CRISPR enzyme, or a single RNA-guided nickase, i.e., the same enzyme was used for the knock-in of scHLA-E trimer transgene and the knockout of B2M and CIITA genes.

[0251] In this embodiment, exogenous DNA template is delivered in the form of circular double-stranded DNA.Exogenous DNA template is flanked by homology arms from B2M locus, and flanked on both sides by sgRNA B2M targeting sequence or not (Figure 13).In some cases, the scHLA-E trimer transgene flanked by homology arms is flanked by the sequence of gRNA pair that targets B2M locus, so that when circular double-stranded DNA is co-delivered with CRISPR components into cells, donor nucleic acid sequence is released from plasmid as linear DNA after cleavage by CRISPR / Cas.

[0252] Figure 14 shows a schematic diagram of an example of a suitable scHLA-E trimer delivery method. In this example, the scHLA-E trimer gene was integrated into exon 1 of the B2M locus, in frame with the upstream B2M locus. The exogenous DNA template contained the scHLA-E trimer coding sequence flanked by homology sequences (LHA and RHA). Once integrated, expression of the scHLA-E trimer was driven by the endogenous B2M promoter, while the B2M locus was disrupted.

[0253] iPSCs were electroporated with a circular double-stranded exogenous DNA template containing the following components: (i) mRNA encoding deaminase-MCP, (ii) mRNA encoding nCas9-UGI-UGI protein, (iii) two sgRNAs targeting opposite strands in the first exon of the B2M gene, (iv) an sgRNA-aptamer against the CIITA gene, and (v) an scHLA-E trimer coding sequence. Two single nicks generated by nCas9-UGU-UGI at the two target loci recognized by the sgRNAs resulted in alternating double-strand breaks (DSBs). Integration of the scHLA-E trimer transgene was prompted by the homology arms to the B2M locus by homology-directed repair (HDR). Integration of the transgene by HDR or non-homologous end joining (NHEJ) induced by DSBs at this locus resulted in efficient knockout of the B2M gene. B2M expression levels are low in pluripotent stem cells, including iPSCs, and can be induced by treatment with interferon-γ. To detect functional knockout of B2M and successful scHLA-E trimer knock-in at the B2M locus, edited cells were treated with interferon-γ for 48 h 2 or 4 days after electroporation and subsequently analyzed by flow cytometry. Base conversion at the CIITA locus was measured by targeted PCR amplification and Sanger sequencing 4–6 days after electroporation.

[0254] When circular double-stranded DNA containing the base editing components and the scHLA-E trimer coding sequence with homology arms to B2M was delivered to cells, C to T conversion was observed at the target CIITA locus, and base editing efficiency was not compromised by the delivery of donor DNA (Figure 17A). Efficient B2M knockout was observed (Figure 17B). Integration of scHLA-E trimer at the B2M locus was achieved, measured as scHLA-E trimer positive cells in cells treated with interferon-γ (Figure 17C). The highest degree of integration was achieved using the donor template flanked on both sides by the sgRNA B2M targeting sequence (CTS_B2M_scHLA-trimer).

[0255] These data indicate that the base editing system can efficiently induce site-specific integration of transgenes and functional knockout of B2M while achieving high levels of base editing at other loci (CIITA). These genetic modifications enable the generation of universal iPSCs with low immunogenicity.

[0256] Materials and Methods for Examples 7 and 8 Guide RNA for base editing: Using internally generated data, we identified base editing windows calculated at set distances from the PAM motif (NGG). We used this data to develop an algorithm to predict guide sequences applicable for phenotypes or gene KO for CIITA (Table X). sgRNAs were designed containing the 1x MS2 aptamer. Guide RNA sequences were synthesized by Horizon Discovery (formerly Dharmacon) and Agilent.

[0257] Synthetic 1x MS2 sgRNA sequence (SEQ ID NO:32): mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGCGCACAUGAGGAUCACCCAUGUGCUUUUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues

[0258] [Table 6-1]

[0259] [Table 6-2]

[0260] Knock-in guide RNA: To disrupt the B2M locus and place the scHLA-E trimer gene under its transcriptional control, we designed an sgRNA pair targeting the 5' end of the first exon of B2M and a donor DNA template with homology arms to the target locus and encoding the scHLA-E trimer. The sgRNA targeting the B2M locus was designed according to the rules of PAM-out configuration (PAM site facing outside the target region) and with the cleavage sites spaced 40-70 bp apart (Table 7). The knock-in guide was designed to not contain the 1xMS2 aptamer. The sgRNA was synthesized by Horizon Discovery (formerly Dharmacon).

[0261] Synthetic sgRNA sequence (SEQ ID NO:29): mN*mN*NNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U (m) 2-O methyl and (*) phosphorothioate modified residues 5' sgRNA sequence 2 (SEQ ID NO: 185) mC*mG*CGAGCACAGCUAAGGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U 3' sgRNA sequence 2 (SEQ ID NO: 186) mA*mC*UCUCUCUUUCUGGCCUGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmU*mU*U

[0262] [Table 7]

[0263] Messenger RNA Messenger RNA molecules were custom synthesized by TriLink Biotechnologies using the modified nucleotides pseudouridine and 5-methylcytosine. The mRNA components were translated into the following proteins: deaminase Apobec1 = NLS-rApobec1-linker-MCP and nCas9 = NLS-nCas9-UGI-UGI-NLS.

[0264] Construction of donor DNA template The donor DNA template was custom synthesized by GenScript and cloned into a pUC19 cloning plasmid. The donor DNA template encoding the scHLA-E trimer contained, in order: a 0.9 kb left homology arm of genomic B2M flanked by a 5' gRNA targeting sequence, a scHLA-E trimer coding sequence, a bovine growth hormone polyA signal (bGHpA), and a 0.9 kb right homology arm of genomic B2M flanked by a 3' gRNA targeting sequence. Briefly, the scHLA-E trimer is a chimeric protein that contains the following elements: (a) the leader peptide of B2M, (b) VMAPRTLIL (HLA-E binding peptide), (c) a 15 amino acid linker (G4S)3, (d) mature human B2M, (e) a 20 amino acid linker (G4S)4, and (f) a mature HLA-E heavy chain. The donor DNA template with homology arms was either flanked on both sides by sgRNA targeting sequences targeting the B2M locus or not. A second donor DNA template was designed and cloned in which the scHLA-E trimer coding sequence was replaced with the turboGFP coding sequence. To obtain a linear double-stranded form of the donor DNA template, the above plasmid was digested by specific restriction enzymes to excise the donor DNA template from the plasmid. The correctly sized fragment was then purified after gel electrophoresis using a gel purification kit.

[0265] Culture of human iPSCs Frozen human iPSCs were obtained from ThermoFisher Scientific (Gibco line). Cells were thawed and cultured on non-adherent cell culture plasticware (Greiner Bio-One) coated with Vitronectin-XF (STEMCELL Technologies) in mTesr-PLUS medium (STEMCELL Technologies) at 37C and 5% CO2. Upon confluence, cells were passaged as clumps using Versene dissociation reagent (ThermoFisher Scientific). Medium was changed at 1-3 day intervals and cells were passaged as needed at 3-5 day intervals.

[0266] Electroporation and post-electroporation culture of human iPSCs 2–4 h prior to electroporation, iPSCs were fed with fresh mTesr-PLUS culture medium (STEMCELL Technologies) containing 10 μM Y-27632 (STEMCELL Technologies) and subsequently dissociated into single cells using Accutase (ThermoFisher Scientific). 150k–200k cells were resuspended in 20 μl of buffer P3 (Lonza) and combined with 1–4 μg of modified mRNA encoding the deaminase-MCP and nCas9-UGI proteins (Trilink), 1–4 μM of each sgRNA (Agilent) and 1–2 ug of donor DNA. Electroporation was performed using a 4D Nucleofector (Lonza) in 16 × 20 μl multiwell cuvettes using programs CM138 or DN100. After electroporation, cells were seeded in mTesr-PLUS (STEMCELL Technologies) containing 10 μM of the Rho kinase inhibitor Y-27632 (STEMCELL Technologies) on Geltrex (ThermoFisher Scientific)-coated cell culture plasticware (Corning) and placed in medium for 24 hours after electroporation to promote cell survival. Two or four days after electroporation, cells were treated with interferon-γ at a concentration of 100 ng / ml in mTesr-PLUS culture medium (STEMCELL Technologies) for 48 hours before flow cytometry analysis.

[0267] Flow cytometry scHLA-E trimer positive cells were detected using anti-HLA-E antibody (Biolegend) after 48 h of treatment with interferon-γ. Phenotypic knockout of B2M was assessed using B2M-antibody (Biolegend) after 48 h of treatment with interferon-γ, and all phenotypic data were reported as the percentage of live cells confirmed by viability staining.

[0268] Genomic DNA analysis Genomic DNA was released from lysed cells 96 hours after electroporation. Loci of interest were amplified by PCR and the products were subsequently subjected to Sanger sequencing (Genewiz). Data were analyzed by proprietary in-house software.

Claims

1. 1. A method for making multiple genetic modifications to a cell, comprising: (a) introducing the following (i), (ii), and (iii) into a cell and / or expressing them in a cell: (i) a CRISPR system for integrating an exogenous sequence into a first target nucleic acid sequence, comprising: (a) a first gRNA and a second gRNA that are complementary to opposite strands of the first target nucleic acid sequence; and (b) a donor nucleic acid sequence comprising the exogenous sequence; a CRISPR system comprising: (ii) a base editing system for introducing a genetic modification into a second target nucleic acid sequence, (a) an RNA scaffold comprising (i) a gRNA sequence that is complementary to the second target nucleic acid sequence, and (ii) a recruitment RNA motif; and (b) an effector fusion protein comprising (i) an RNA-binding domain capable of binding to the recruitment RNA motif; and (ii) an effector domain comprising a base-modifying enzyme. base editing system including (iii) an RNA-guided nickase capable of interacting with the first and second gRNAs of the CRISPR system and the RNA scaffold of the base editing system; and (b) culturing the cells to produce cells comprising a plurality of genetic modifications. A method comprising:

2. 2. The method of claim 1, wherein the base-modifying enzyme has cytosine deamination activity, adenosine deamination activity, DNA methyltransferase activity, or demethylase activity.

3. 2. The method of claim 1, wherein the RNA-guided nickase is a CRISPR type II or type V enzyme.

4. The method of any one of claims 1 to 3, wherein the RNA-guided nickase is Cas9 nickase.

5. The method of any one of claims 1 to 3, wherein the RNA scaffold comprises tracrRNA.

6. The method of any one of claims 1 to 3, wherein a modular system is used comprising multiple base editing systems that can bind to different target nucleic acid sequences and genetically modify multiple different loci.

7. The method according to any one of claims 1 to 3, wherein the cells are immune cells or human PSCs.

8. 8. The method of claim 7, wherein the hPSCs are iPSCs.

9. 8. The method of claim 7, wherein the cell is an immune cell selected from the group consisting of a T cell, a natural killer cell (NK cell), a B cell, a myeloblast, a lymphoblast, and a CD34+ hematopoietic stem progenitor cell (HSPC).

10. 4. The method of any one of claims 1 to 3, wherein the first gRNA and second gRNA are complementary to opposite strands of a locus selected from the group consisting of TRAC, B2M, and CISH.

11. The method of any one of claims 1 to 3, wherein the CRISPR system is used to introduce a donor nucleic acid sequence comprising a sequence encoding a CAR, a TCR, or a scHLA-E trimer, and the donor nucleic acid sequence is flanked by homology arms specific for a target locus.

12. 12. The method of claim 11, wherein a sequence encoding a CAR, a TCR, or a scHLA-E trimer is integrated into the TRAC, B2M, or CISH locus.

13. 13. The method of claim 12, wherein expression of the sequence encoding the CAR, TCR, or scHLA-E trimer is driven by an endogenous TRAC, B2M, or CISH promoter.

14. The method of any one of claims 1 to 3, wherein the multiple genetic modifications occur simultaneously.

15. The method of any one of claims 1 to 3, wherein nucleic acids encoding each of the CRISPR system, the base editing system, and the RNA-guided nickase are introduced into the cell in a single delivery step.

16. The method of any one of claims 1 to 3, wherein the donor nucleic acid sequence is introduced into the cell using a viral vector.

17. 17. The method of claim 16, wherein the viral vector is AAV.

18. 4. The method of any one of claims 1 to 3, wherein the base editing system introduces one or more genetic modifications that correct a gene mutation, inactivate expression of a gene, alter the expression level of a gene, or alter intron-exon splicing.

19. The method of any one of claims 1 to 3, wherein the genetic modification introduced by the base editing system is a point mutation.

20. 20. The method of claim 19, wherein the point mutation introduces a premature stop codon, disrupts a start codon, disrupts a splice site, or corrects a genetic mutation.

21. 4. The method of any one of claims 1 to 3, wherein the genetic modifications introduced by the base editing system result in reduced expression of any one or more genes selected from the group consisting of TRAC, TRBC1, TRBC2, PDCD1, CD52, CIITA, NKG2A, and B2M.

22. The method of any one of claims 1 to 3, wherein the RNA scaffold is introduced into the cell as chemically synthesized RNA.

23. The method of any one of claims 1 to 3, wherein the RNA scaffold comprises one or more chemical modifications.

24. The method of any one of claims 1 to 3, wherein the recruitment RNA motif is located at the 3' end of the RNA scaffold.

25. The method of any one of claims 1 to 3, wherein the RNA scaffold comprises two or more recruitment RNA motifs.

26. The method of any one of claims 1 to 3, wherein the recruitment RNA motif is an RNA aptamer.

27. The method of any one of claims 1 to 3, wherein the recruitment RNA motif is an MS2 aptamer.

28. 4. The method of any one of claims 1 to 3, wherein the RNA-guided nickase is nCas9 with one or two UGIs, and the recruitment RNA motif is a single MS2 aptamer located at the 3' end of the RNA scaffold.

29. The method of any one of claims 1 to 3, wherein the introduced genetic modification results in the generation of allogeneic T cells.

30. A genetically modified cell obtained by the method according to any one of claims 1 to 3.

31. A genetically modified cell obtained by the method of any one of claims 1 to 3, wherein the cell comprises a sequence encoding an exogenous CAR or TCR integrated into an endogenous TRAC or B2M locus and contains at least one point mutation in two or more genes.

32. 32. The genetically modified cell of claim 31 , wherein said two or more genes are selected from the group consisting of TRAC, TRBC1, TRBC2, PDCD1, CD52, CIITA, NKG2A and B2M, resulting in a functional knockout of said genes.

33. 1. A system for genetically modifying a cell, comprising: (i) a CRISPR system for integrating an exogenous sequence into a first target nucleic acid sequence, comprising: (a) a first gRNA and a second gRNA that are complementary to opposite strands of the first target nucleic acid sequence; and (b) a donor nucleic acid sequence comprising the exogenous sequence; a CRISPR system comprising: (ii) a base editing system for introducing a genetic modification into a second target nucleic acid sequence, (a) an RNA scaffold comprising (i) a gRNA sequence that is complementary to the second target nucleic acid sequence, and (ii) a recruitment RNA motif; and (b) an effector fusion protein comprising (i) an RNA-binding domain capable of binding to the recruitment RNA motif; and (ii) an effector domain comprising a base-modifying enzyme. A base editing system comprising: (iii) an RNA-guided nickase capable of interacting with the first and second gRNAs of the CRISPR system and the RNA scaffold of the base editing system; Including, the system.

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