Systems and methods for cell modification

Abstract

A system for modifying cells is provided. Methods of using the system for modifying cells are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of PCT / CN2021 / 141667, filed December 27, 2021, the entirety of which is incorporated herein by reference.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Background technology]

[0003] Cell-based therapies, such as cell transplantation or therapeutic drug delivery, are becoming more successful. However, cells used in cell-based therapies may express cell surface markers that induce innate immunity. For example, CAR-T cell therapy has shown promising therapeutic effects but is associated with severe inflammatory side effects in patients. Another example is cell transplantation, which results in graft-versus-host disease (GVHD). As a result, the majority of current clinical trials rely on autologous cells as the primary cell source for cell-based therapies. Summary of the Invention [Problem to be solved by the invention]

[0004] However, this reliance on autologous cells is costly and cumbersome.There is still a need for systems and methods that allow cells to be modified for cell-based therapy, so that the modified cells can have knockdown or knockout expression of cell surface molecules such as major histocompatibility complex (MHC), so that the modified cells do not induce innate immune responses after being administered to a subject in need of the cells.There is also still a need for systems and methods for modifying cells to express therapeutic agents by modified cells. [Means for solving the problem]

[0005] In some aspects, systems are described herein that include a guide nucleic acid, a genetically modified portion, and a polynucleotide that includes at least one expression sequence and at least one genetically modified portion targeting fragment, and that is circular. In some aspects, systems are described herein that include a guide nucleic acid, a genetically modified portion, and a polynucleotide that includes at least one expression sequence and at least one genetically modified portion targeting fragment, where (i) the polynucleotide further includes at least one covalently closed circular end, and / or (ii) the polynucleotide is linear DNA. In some embodiments of any one of the systems disclosed herein, the polynucleotide includes double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). In some embodiments of any one of the systems disclosed herein, the polynucleotide is a vector. In some embodiments of any one of the systems disclosed herein, the polynucleotide is a minicircle. In some embodiments of any one of the systems disclosed herein, at least one covalently closed circular end is at the 5' end of the polynucleotide. In some embodiments of any one of the systems disclosed herein, at least one covalently closed circular end is at the 3' end of the polynucleotide. In some embodiments of any one of the systems disclosed herein, the polynucleotide comprises at least one covalently closed circular end at the 5' end and at least one covalently closed circular end at the 3' end of the polynucleotide. In some embodiments of any one of the systems disclosed herein, the polynucleotide is a circular single-stranded DNA. In some embodiments of any one of the systems disclosed herein, the polynucleotide is a linear single-stranded DNA. In some embodiments of any one of the systems disclosed herein, the polynucleotide is a linear single-stranded DNA with modified ends. In some embodiments of any one of the systems disclosed herein, at least one covalently closed circular end comprises a telomeric end.In some embodiments of any one of the systems disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 5' end of the polynucleotide. In some embodiments of any one of the systems disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 3' end of the polynucleotide. In some embodiments of any one of the systems disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 5' end of the polynucleotide and at least one genetically modified portion targeting fragment near the 3' end of the polynucleotide. In some embodiments of any one of the systems disclosed herein, the guide nucleic acid comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to a genomic sequence in the cell. In some embodiments of any one of the systems disclosed herein, the genomic sequence comprises a genomic locus of the cell. In some embodiments of any one of the systems disclosed herein, the genomic locus comprises T cell receptor alpha chain constant (TRAC), beta-2-microglobulin (B2M), cluster of differentiation 38 (CD38), cytokine-induced SH2-containing protein (CISH), programmed cell death protein 1 (PD-1), cluster of differentiation 70 (CD70). In some embodiments of any one of the systems disclosed herein, the genomic locus comprises TRAC or B2M. In some embodiments of any one of the systems disclosed herein, the guide nucleic acid complexes with the genetic modification moiety and guides it to the genomic sequence in the cell. In some embodiments of any one of the systems disclosed herein, the at least one genetic modification moiety targeting fragment comprises about 10 nucleotide base pairs (bps) to about 100 nucleotide bps.In some embodiments of any one of the systems disclosed herein, the at least one genetically modified portion targeting fragment comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to the guide nucleic acid. In some embodiments of any one of the systems disclosed herein, the at least one genetically modified portion targeting fragment comprises at least one mismatch, at least two mismatches, at least three mismatches, at least four mismatches, at least five mismatches, at least six mismatches, at least seven mismatches, at least eight mismatches, at least nine mismatches, or at least ten mismatches compared to the guide nucleic acid. In some embodiments of any one of the systems disclosed herein, the at least one genetically modified portion targeting fragment is complexed with the genetically modified portion, thereby bringing the genetically modified portion into close proximity with the polynucleotide. In some embodiments of any one of the systems disclosed herein, the at least one genetically modified portion targeting fragment does not induce the enzymatic activity of the genetically modified portion when complexed with the genetically modified portion. In some embodiments of any one of the systems disclosed herein, the genetically modified portion comprises a Cas protein or an mRNA encoding a Cas protein. In some embodiments of any one of the systems disclosed herein, the genetically modified portion comprises a Cas / RNP. In some embodiments of any one of the systems disclosed herein, the genetically modified portion comprises a Cas9 / RNP. In some embodiments of any one of the systems disclosed herein, at least one expression sequence encodes a chimeric receptor.In some embodiments of any one of the systems disclosed herein, the chimeric receptor is selected from the group consisting of CD1a, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44v6, CD47, CD52, CD56, CD57, CD58, CD70, CD79a, CD79b, CD80, CD81, CD86, CD99, CD117, CD123, CD133, CD135, CD137, CD151, CD171, CD276, BAFF-R, BC In some embodiments of any one of the systems disclosed herein, the chimeric receptor comprises an antigen binding domain that binds to MA, B7H4, CEA, CEACM6, Claudin18.2, CLL-1, c-Met, CS-1, CTLA-4, EGFRvIII, GPC2, GPC3, GPRC5, HER2, HER3, HER4 / ErbB4, HVEM, MAGE-A, MAGE3, MSLN, MUC-1, MUC-16, NY-ESO-1, OX40, PD-1, PD-L1, PD-L2, PMSA, ROR1, TCRa, TCRb, TLR7, TLR9, VEGFR-2, WT-1, or a fragment thereof. In some embodiments of any one of the systems disclosed herein, the chimeric receptor comprises a transmembrane domain. In some embodiments of any one of the systems disclosed herein, the chimeric receptor comprises a signaling domain. In some embodiments of any one of the systems disclosed herein, the cell comprises an immune cell or a stem cell. In some embodiments of any one of the systems disclosed herein, the immune cells are lymphocytes. In some embodiments of any one of the systems disclosed herein, the lymphocytes are B cells. In some embodiments of any one of the systems disclosed herein, the lymphocytes are T cells. In some embodiments of any one of the systems disclosed herein, the T cells are selected from the group consisting of cytotoxic T cells, alpha-beta T cells, gamma-delta T cells, natural killer T cells, regulatory T cells, and T helper cells. In some embodiments of any one of the systems disclosed herein, the immune cells comprise ILCs. In some embodiments of any one of the systems disclosed herein, the immune cells are derived from iPSCs.In some embodiments of any one of the systems disclosed herein, the immune cells are iPSC-derived T cells. In some embodiments of any one of the systems disclosed herein, the immune cells are iPSC-derived natural killer T cells. In some embodiments of any one of the systems disclosed herein, the immune cells are iPSC-derived macrophages. In some embodiments of any one of the systems disclosed herein, the stem cells are hematopoietic stem cells. In some embodiments of any one of the systems disclosed herein, the stem cells are iPSCs. In some embodiments of any one of the systems disclosed herein, the systems described herein further comprise a polymer, wherein the polymer comprises an overall anionic charge. In some embodiments of any one of the systems disclosed herein, the polymer comprises an anionic polynucleotide comprising polyglutamic acid (PGA) or polyaspartic acid (PASA).

[0006] In some aspects, compositions comprising the systems described herein are described herein.

[0007] In some aspects, cells or cell lines comprising the systems described herein are described herein.

[0008] In some aspects, described herein are pharmaceutical compositions comprising the system described herein or the cells described herein. In some embodiments of any one of the pharmaceutical compositions disclosed herein, the pharmaceutical composition comprises a unit dose form. In some embodiments of any one of the pharmaceutical compositions disclosed herein, the pharmaceutical composition is formulated to be administered to a subject in need thereof intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, intratumorally, intrapulmonary, intratracheal, intraperitoneally, intravesically, intravaginally, intrarectally, orally, sublingually, transdermally, by inhalation, by inhalation spray form, by intraluminal gastrointestinal route, or in combinations thereof. In some embodiments of any one of the pharmaceutical compositions disclosed herein, the pharmaceutical composition comprises at least one additional active agent. In some embodiments of any one of the pharmaceutical compositions disclosed herein, the at least one additional active agent comprises a cytokine, a growth factor, a hormone, an enzyme, a small molecule, a compound, or a combination thereof.

[0009] In some aspects, described herein are kits comprising a system described herein, a cell described herein, or a pharmaceutical composition described herein and a container.

[0010] In some aspects, methods are described herein that include contacting a cell with any one of the systems disclosed herein, where the system knocks in a polynucleotide in a genomic sequence in the cell, thereby expressing a chimeric receptor encoded by the polynucleotide in the cell. In some aspects, methods are described herein that include contacting a cell population with a system described herein, where the system knocks in a polynucleotide in a genomic sequence in the cell population, thereby expressing a chimeric receptor encoded by the polynucleotide in the cell population. In some embodiments of any one of the methods disclosed herein, the system described herein increases the knock-in efficiency of the polynucleotide in the cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the Cas protein-mediated knock-in efficiency of the polynucleotide in the absence of at least one genetically modified partial targeting fragment in a comparable cell population. In some embodiments of any one of the methods disclosed herein, the system described herein increases expression of the polynucleotide in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to expression of the polynucleotide in the absence of at least one genetically modified partial targeting fragment knocked into an equivalent cell population by a Cas protein. In some embodiments of any one of the methods disclosed herein, the system described herein increases viability of the cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to viability of an equivalent cell population modified by Cas protein-mediated knock-in of the polynucleotide in the absence of at least one genetically modified partial targeting fragment.

[0011] In some aspects, methods are described herein that comprise contacting a cell with a first system comprising any one of the systems disclosed herein, the first system comprising a first guide nucleic acid complexed with a first genetic modification moiety and a first chimeric polynucleotide comprising at least one genetic modification moiety targeting fragment, and a second system comprising any one of the systems disclosed herein, the second system comprising a second guide nucleic acid complexed with a second genetic modification moiety and a second chimeric polynucleotide comprising at least one genetic modification moiety targeting fragment, wherein the first system introduces the first chimeric polynucleotide into a first genomic sequence in the cell, and the second system introduces the second chimeric polynucleotide into a second genomic sequence in the cell.

[0012] In some aspects, methods are described herein that comprise contacting a cell population with a first system comprising any one of the systems disclosed herein, the first system comprising a first guide nucleic acid complexed with a first genetic modification moiety and a first chimeric polynucleotide comprising at least one genetic modification moiety targeting fragment, and a second system comprising any one of the systems disclosed herein, the second system comprising a second guide nucleic acid complexed with a second genetic modification moiety and a second chimeric polynucleotide comprising at least one genetic modification moiety targeting fragment, wherein the first system introduces the first chimeric polynucleotide into a first genomic sequence in the cell population, and the second system introduces the second chimeric polynucleotide into a second genomic sequence in the cell population. In some embodiments of any one of the methods disclosed herein, the first system and the second system increase the knock-in efficiency of the first and second polynucleotides in the cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the Cas protein-mediated knock-in efficiency of the first and second polynucleotides in the absence of at least one genetically modified partial targeting fragment in a comparable cell population. In some embodiments of any one of the methods disclosed herein, the first system and the second system increase the expression of the first and second polynucleotides in the cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the expression of the first and second polynucleotides in the absence of at least one genetically modified partial targeting fragment knocked in by the Cas protein into a comparable cell population. In some embodiments of any one of the methods disclosed herein, the first system and the second system increase the viability of the cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the viability of an equivalent cell population modified by Cas protein-mediated knock-in of the first and second polynucleotides in the absence of the at least one genetically modified partial targeting fragment.

[0013] In some aspects, methods are described herein that include contacting a cell with a polynucleotide that includes a guide nucleic acid, a genetic modification portion, and at least one expression sequence and at least one genetic modification portion targeting fragment, and is circular. In some aspects, methods are described herein that include contacting a cell with a polynucleotide that includes a guide nucleic acid, a genetic modification portion, and at least one expression sequence, at least one genetic modification portion targeting fragment, and at least one covalently closed circular end. In some embodiments of any one of the methods disclosed herein, the polynucleotide includes double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). In some embodiments of any one of the methods disclosed herein, the polynucleotide is a vector. In some embodiments of any one of the methods disclosed herein, the polynucleotide is a minicircle. In some embodiments of any one of the methods disclosed herein, at least one covalently closed circular end is at the 5' end of the polynucleotide. In some embodiments of any one of the methods disclosed herein, at least one covalently closed circular end is at the 3' end of the polynucleotide. In some embodiments of any one of the methods disclosed herein, the polynucleotide comprises at least one covalently closed circular end at the 5' end of the polynucleotide and at least one covalently closed circular end at the 3' end. In some embodiments of any one of the methods disclosed herein, at least one covalently closed circular end comprises a telomeric end. In some embodiments of any one of the methods disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 5' end of the polynucleotide. In some embodiments of any one of the methods disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 3' end of the polynucleotide.In some embodiments of any one of the methods disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 5' end of the polynucleotide and at least one genetically modified portion targeting fragment near the 3' end of the polynucleotide. In some embodiments of any one of the methods disclosed herein, the at least one genetically modified portion targeting fragment comprises about 10 nucleotide base pairs (bps) to about 100 nucleotide bps. In some embodiments of any one of the methods disclosed herein, the at least one genetically modified portion targeting fragment comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to the guide nucleic acid. In some embodiments of any one of the methods disclosed herein, the at least one genetically modified portion targeting fragment comprises at least one mismatch, at least two mismatches, at least three mismatches, at least four mismatches, at least five mismatches, at least six mismatches, at least seven mismatches, at least eight mismatches, at least nine mismatches, or at least ten mismatches compared to the guide nucleic acid. In some embodiments of any one of the methods disclosed herein, the at least one genetically modified portion targeting fragment is complexed with the genetically modified portion, thereby bringing the genetically modified portion into proximity with the polynucleotide. In some embodiments of any one of the methods disclosed herein, the at least one genetically modified portion targeting fragment does not induce the enzymatic activity of the genetically modified portion when complexed with the genetically modified portion.

[0014] In some aspects, described herein are polynucleotides that include at least one expression sequence and at least one genetically modified partial targeting fragment and are circular. In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide is a vector. In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide is a minicircle.

[0015] In some aspects, described herein are polynucleotides comprising at least one expression sequence, at least one genetically modified partial targeting fragment, and at least one covalently closed circular end. In some embodiments of any one of the polynucleotides disclosed herein, the at least one covalently closed circular end is at the 5' end of the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the at least one covalently closed circular end is at the 3' end of the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide comprises at least one covalently closed circular end at the 5' end and at least one covalently closed circular end at the 3' end of the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the at least one covalently closed circular end comprises a telomeric end. In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide comprises double stranded DNA (dsDNA) or single stranded DNA (ssDNA). In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 5' end of the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 3' end of the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the polynucleotide comprises at least one genetically modified portion targeting fragment near the 5' end of the polynucleotide and at least one genetically modified portion targeting fragment near the 3' end of the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the at least one genetically modified portion targeting fragment comprises from about 10 nucleotide base pairs (bps) to about 100 nucleotide bps.In some embodiments of any one of the polynucleotides disclosed herein, the at least one genetically modified portion targeting fragment is complexed with the genetically modified portion, thereby bringing the genetically modified portion into close proximity with the polynucleotide. In some embodiments of any one of the polynucleotides disclosed herein, the at least one genetically modified portion targeting fragment, when complexed with the genetically modified portion, does not induce the enzymatic activity of the genetically modified portion.

[0016] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0017] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0018] [Figure 1]1 illustrates the knock-in of a New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1) targeting TCR into cells by the system described herein. The knock-in was mediated by contacting the cells with a polynucleotide containing a modified partial targeting fragment as described herein at both the 5' and 3' ends. The polynucleotide was not covalently closed at both the 5' and 3' ends of the polynucleotide. The knock-in resulted in a 34.4% population of cells expressing a TCR knock-in (TCRVβ13.1) at the TRAC locus. "Column-derived template" refers to the polynucleotide obtained from column purification. "Bead-derived template" refers to the polynucleotide obtained from magnetic bead purification. For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.25 μg, 0.50 μg, 0.70 μg, or 1.00 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37° C., followed by addition of various amounts of polynucleotides and further incubation at 37° C. for 5 min, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). Flow cytometry analysis showed that CD3 was knocked out in 94.9% of cells, and 0.18% of cells were positive for both TCR Vβ13.1 and CD3. [Diagram 2]1 shows the knock-in of CD19 or mesothelin-targeted CAR into cells by the system described herein. The knock-in was mediated by contacting cells with a polynucleotide containing modified partial targeting fragments described herein at both the 5' and 3' ends. The polynucleotide was not covalently closed at both the 5' and 3' ends of the polynucleotide. The knock-in resulted in 31.4% of the cell population expressing CD19-targeted CAR (CD-19-CAR) at the TRAC locus, 33.4% of the cell population expressing humanized CD19-targeted CAR (HCD-19-CAR) at the TRAC locus, and 33.8% of the cell population expressing mesothelin-targeted CAR (Meso-CAR-T) at the TRAC locus. For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg or 0.40 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37°C, after which various amounts of chimeric polynucleotides were added and incubated for an additional 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Diagram 3]1 shows knock-in of dual CARs (two different CARs targeting CD19 and CD20 in the same cell, shown as GC020; or two different CARs targeting CD19 and BCMA in the same cell, shown as GC012L) into the TRAC genomic locus of cells by the system described herein. Knock-in was mediated by contacting cells with a polynucleotide comprising modified partial targeting fragments as described herein at both the 5' and 3' ends. The polynucleotide was not covalently closed at both the 5' and 3' ends of the polynucleotide. The knock-in resulted in 41.1% of the cell population expressing dual CD19 and CD20 CARs via knock-in at the TRAC genomic locus (82.7% of cells showing TRAC knockout) and 22.5% of the cell population expressing dual CD19 CAR via knock-in at the TRAC genomic locus (90.1% of cells showing TRAC knockout). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg or 0.40 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of the polynucleotide were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 4A]1 shows knock-in of HLA-E into cells by the system described herein. Knock-in was mediated by contacting cells with a polynucleotide comprising a modified partial targeting fragment as described herein at both the 5' and 3' ends. The polynucleotide was further modified to comprise both a covalently closed 5' end and a covalently closed 3' end. Knock-in mediated by a polynucleotide comprising a modified partial targeting fragment as described herein at both ends, but not a covalently closed 5' end and 3' end, resulted in 37.7% of the cell population expressing HLA-E (HLA-E KI). Knock-in mediated by a polynucleotide comprising a modified partial targeting fragment as described herein at both ends and having a covalently closed 5' end and 3' end, resulted in 54.9% of the cell population expressing HLA-E (ds-HLA-E KI). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg, 0.30 μg or 0.40 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of the polynucleotide were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 4B] 13A-C depict cell viability measurements showing that cells modified with a polynucleotide comprising a modified partial targeting fragment at both termini and having covalently closed 5' and 3' termini had equal or greater viability over an 8 day period than cells modified with a polynucleotide comprising a modified partial targeting fragment as described herein at both termini, but not having covalently closed 5' and 3' termini. [Figure 5A]1 shows the knock-in of a dual CAR (GC012HL, Dual-CAR-T) targeting CD19 and BCMA into cells by the system described herein. The knock-in was mediated by contacting the cells with a polynucleotide (in an amount of 0.5 μg, 1.0 μg, 1.5 μg, or 2.0 μg) containing a modified partial targeting fragment at both the 5' and 3' ends and a covalently closed 5' end and a covalently closed 3' end. 34.6% of the cells expressed the dual CAR. For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.50 μg, 1.0 μg, 1.5 μg, or 2.0 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37°C, after which various amounts of chimeric polynucleotides were added and incubated for an additional 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 5B] Cell viability after 3 days of knock-in cells is shown. [Figure 6] Shown is the knock-in of both dual-CAR (Dual-CAR-T, GC012HL) and HLA-E at the TRAC and B2M loci, respectively, resulting in 21.7% of cells expressing HLA-E and 50.1% of cells expressing dual-CAR. For this knock-in experiment, 10 million cells (in a final volume of 100 μl) were contacted with 1.0 μg, 1.25 μg, or 1.50 μg of polynucleotides. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of polynucleotides were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 7]Shown is knock-in of both Dual-CAR-T and HLA-E at the B2M and TRAC loci, respectively, resulting in 31.8% of cells expressing HLA-E R. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of chimeric polynucleotides were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 8] 1 shows that knock-in of both Dual-CAR-T and HLA-E at the TRAC and B2M loci resulted in 96.57% knockout of TRAC and 92.48% knockout of B2M. For this knock-in experiment, 10 million cells (in a final volume of 100 μl) were contacted with 1.0 μg, 1.25 μg, or 1.50 μg of polynucleotides. Cells were incubated with Cas9 / RNP for 15 minutes at 37° C., after which various amounts of polynucleotides were added and incubated for an additional 5 minutes at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 9] Figure 1 shows knock-in of polynucleotides in which the polynucleotides were double stranded DNA (dsDNA) containing modified partial targeting fragments as described herein and were covalently closed at both 5' and 3' ends (telomeric end, top), not covalently closed at both 5' and 3' ends (middle) and circular (minicircle, bottom). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg, 0.40 μg or 0.80 μg of polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of polynucleotide were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 10]Knock-in of polynucleotides that were single-stranded (either sense or antisense) DNA (ssDNA) is shown. On day 8, 47.2% of cells expressed dual CAR (GC012HL). For this knock-in experiment, 10 million cells (in a final volume of 100 μl) were contacted with 1.0 μg, 1.25 μg, or 1.50 μg of polynucleotides. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of polynucleotides were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). [Figure 11A] 1 shows knock-in of both dual CAR (targeting both CD19 and BCMA) and HLA-E into cells by the system described herein. [Figure 11B] Figure 1 shows the cell killing activity of cells with dual CARs knocked in by the system described herein. At day 8, cells with CARs targeting CD19 or BCMA showed cell killing activity to kill B cell precursor leukemia cells (Nalm6, which expressed CD19) and cancer cells (MM.1S, RPMI-8226 and JeKo-1 cells, all of which expressed BCMA). [Figure 12-1]12A shows non-viral knock-in of polynucleotides as described herein to generate CAR-T cells (Dual-CAR-T targeting CD19 and BCMA). FIG. 12A shows modified cells that were positive for CAR presentation on day 2. FIG. 12B shows the percentage change of modified cells that were positive for knock-in presentation. FIG. 12C shows cell viability of knock-in modified cells (left) and cells after thawing from cell freezing (right; 1.0: cells contacted with 1.0 μg polynucleotides; KO: control TRAC knockout only T cells; T: unmodified T cells) over a 3-day period. FIG. 12D shows the viability and percentage change of modified cells that were positive for knock-in presentation on day 1 after thawing from cell freezing. NT-Ctrl: no template control. Top: modified cells displaying CD19-targeted CAR. Bottom: modified cells displaying CD3. Figure 12E shows the cell-killing activity of modified cells (Dual-CAR-T, ZAP-CAR-T) against cells expressing CD19 (Nalm6 cells) or BCMA (JeKo-1 and MM.1S cells). [Figure 12-2] 12A shows non-viral knock-in of polynucleotides as described herein to generate CAR-T cells (Dual-CAR-T targeting CD19 and BCMA). FIG. 12A shows modified cells that were positive for CAR presentation on day 2. FIG. 12B shows the percentage change of modified cells that were positive for knock-in presentation. FIG. 12C shows cell viability of knock-in modified cells (left) and cells after thawing from cell freezing (right; 1.0: cells contacted with 1.0 μg polynucleotides; KO: control TRAC knockout only T cells; T: unmodified T cells) over a 3-day period. FIG. 12D shows the viability and percentage change of modified cells that were positive for knock-in presentation on day 1 after thawing from cell freezing. NT-Ctrl: no template control. Top: modified cells displaying CD19-targeted CAR. Bottom: modified cells displaying CD3. Figure 12E shows the cell-killing activity of modified cells (Dual-CAR-T, ZAP-CAR-T) against cells expressing CD19 (Nalm6 cells) or BCMA (JeKo-1 and MM.1S cells). [Figure 12-3]12A shows non-viral knock-in of polynucleotides as described herein to generate CAR-T cells (Dual-CAR-T targeting CD19 and BCMA). FIG. 12A shows modified cells that were positive for CAR presentation on day 2. FIG. 12B shows the percentage change of modified cells that were positive for knock-in presentation. FIG. 12C shows cell viability of knock-in modified cells (left) and cells after thawing from cell freezing (right; 1.0: cells contacted with 1.0 μg polynucleotides; KO: control TRAC knockout only T cells; T: unmodified T cells) over a 3-day period. FIG. 12D shows the viability and percentage change of modified cells that were positive for knock-in presentation on day 1 after thawing from cell freezing. NT-Ctrl: no template control. Top: modified cells displaying CD19-targeted CAR. Bottom: modified cells displaying CD3. Figure 12E shows the cell-killing activity of modified cells (Dual-CAR-T, ZAP-CAR-T) against cells expressing CD19 (Nalm6 cells) or BCMA (JeKo-1 and MM.1S cells). [Figure 13A] 1 shows knock-in of dual CARs for targeting CD19 or CD20 at a single locus of TRAC by the system described herein. 41.1% of cells showed positive expression of dual CARs for targeting CD19 or CD20. [Figure 13B] 1 shows knock-in of dual CARs for targeting CD19 or BCMA at a single locus of TRAC by the system described herein. 41.1% of cells showed positive expression of dual CARs for targeting CD19 or BCMA. [Figure 14] Showing knock-in of polynucleotides by the system described herein, the polynucleotides knocked in at loci such as TRAC or B2M were greater than 5,000 base pairs (bps). Approximately 20% of modified cells were positive for knock-in presentation. Flow cytometry studies show that 25.2% of cells expressed CD19 CAR. 5.77% of cells expressed both CD19 CAR and HLA-E. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

[0020] overview In some aspects, a system for modifying a cell is described herein. In some embodiments, the system modifies a cell by knocking in a polynucleotide at a genomic locus of the cell, where the polynucleotide encodes a chimeric receptor. In some embodiments, instead of encoding a chimeric receptor, the polynucleotide encodes a major histocompatibility complex (MHC) protein. Expression of the MHC protein by the modified cell may reduce the induction of an innate immune response by the modified cell in the subject. In some embodiments, the system comprises a guide nucleic acid. In some embodiments, the system comprises a genetically modified portion, where the genetically modified portion is complexed with the guide nucleic acid. Upon complexing with the guide nucleic acid, the genetically modified portion is directed to the genomic locus to mediate knock-in of the polynucleotide. In some embodiments, the polynucleotide comprises at least one genetically modified portion targeting fragment, where the at least one genetically modified portion targeting fragment can be complexed with the genetically modified portion. Complexing the at least one genetically modified portion targeting fragment with the genetically modified portion allows the genetically modified portion and the at least one genetically modified portion targeting fragment to be in close proximity to each other. Such close proximity between the genetic modification portion and at least one genetic modification portion targeting fragment may result in increased knock-in efficiency of the polynucleotide. In some embodiments, the polynucleotide is circular. In some embodiments, the circular polynucleotide is a vector. In some embodiments, the circular polynucleotide is a minicircle. In some embodiments, the polynucleotide is linear. In some embodiments, the linear polynucleotide comprises at least one covalently closed circular end at either the 5' or 3' end of the polynucleotide. In some embodiments, the linear polynucleotide comprises covalently closed circular ends at both the 5' and 3' ends of the polynucleotide.Polynucleotides that are circular or have at least one covalently closed end may reduce degradation of the polynucleotide (e.g., by nucleases in the cell being modified). Reduced degradation of the polynucleotide may result in increased abundance of the polynucleotide for knock-in at a genomic locus. In some embodiments, polynucleotides that are circular or have at least one covalently closed end increase the knock-in efficiency of the polynucleotide.

[0021] In some aspects, cells modified with the systems described herein are described herein. In some embodiments, cells modified with the systems described herein can be cells in a cell line. In some embodiments, cells modified with the systems described herein can be formulated in a composition, pharmaceutical composition, kit, or combination thereof. In some embodiments, cells modified with the systems described herein, compositions, pharmaceutical compositions, kits, or combinations thereof can be used to treat a disease or condition in a subject in need of such treatment. In some embodiments, the disease or condition is a neoplasm or cancer.

[0022] In some aspects, methods of modifying cells are described herein. In some embodiments, the methods include contacting the cells with a system described herein. In some embodiments, the methods include contacting the cells with one or any combination of the components of the system described herein. In some embodiments, the methods include contacting the cells with a first system and a second system described herein, each of which knocks in a chimeric polynucleotide. In such a configuration, two chimeric polyuronides (each encoding the same or different chimeric receptors) can be knocked into the same cell. In some embodiments, the methods increase the knock-in efficiency of a transgene (e.g., a chimeric receptor encoded by a chimeric polynucleotide) of a cell compared to the knock-in efficiency of the same transgene mediated by other methods that do not use the chimeric polynucleotides described herein. In some embodiments, the methods increase the knock-in efficiency of a transgene (e.g., a chimeric receptor encoded by a chimeric polynucleotide) of a cell compared to the knock-in efficiency of the same transgene mediated by other methods that do not use the chimeric polynucleotides described herein. In some embodiments the methods increase the viability (as determined by viability) of cells modified by the described system compared to the viability of cells modified by other means (e.g., other systems that do not use the polynucleotides described herein).

[0023] system In some embodiments, a system for modifying a cell as described herein is described. In some embodiments, the system modifies a cell by knock-in in a polynucleotide, the polynucleotide comprising at least one expression sequence. In some embodiments, the at least one expression sequence encodes a transgene or a fragment thereof. In some embodiments, the transgene comprises a chimeric receptor. In some embodiments, the transgene comprises a major histone compatibility complex (MHC) protein. In some embodiments, the transgene comprises an MCH-I protein. In some embodiments, the transgene comprises an MCH-II protein. In some embodiments, the cell can be modified by at least a first system and at least a second system, the polynucleotides of the first system and the second system each encoding a transgene. The transgenes can be the same or different. For example, the first system can knock-in a first chimeric receptor that is the same or different from a second chimeric receptor knocked in by the second system. In some embodiments, the cells may be modified with the first system, the second system, or any further number of systems, and the modified cells may subsequently express the first transgene, the second transgene, or any further number of transgenes. In some embodiments, the system comprises a polymer that comprises an overall anionic charge. In some embodiments, the polymer may be polyglutamic acid (PGA) or polyaspartic acid (PASA). The inclusion of an anionic polymer can block excess cationic charge of other components of the system, thereby increasing the knock-in efficiency of the polynucleotides.

[0024] Guide Nucleic Acid In some embodiments, the system includes a guide nucleic acid. In some embodiments, the guide nucleus includes two separate nucleic acid molecules, which may be referred to as a double guide nucleic acid, or a single nucleic acid molecule (e.g., sgRNA), which may be referred to as a single guide nucleic acid. In some embodiments, the guide nucleic acid is a single guide nucleic acid that includes a fused CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid that includes a crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid that includes a crRNA but not a tracrRNA. In some embodiments, the guide nucleic acid is a double guide nucleic acid that includes a non-fused crRNA and a tracrRNA. Exemplary double guide nucleic acids may include crRNA-like molecules and tracrRNA-like molecules. Exemplary single guide nucleic acids may include crRNA-like molecules. Exemplary single guide nucleic acids may include fused crRNA-like molecules and tracrRNA-like molecules. The crRNA may include the nucleic acid targeting segment (e.g., spacer region) of the guide nucleic acid and a stretch of nucleotides that can form one half of a double-stranded duplex of the Cas protein binding segment of the guide nucleic acid. The tracrRNA may include a stretch of nucleotides that forms the other half of the double-stranded duplex of the Cas protein binding segment of the gRNA. The stretch of nucleotides of the crRNA is complementary to and can hybridize with a stretch of nucleotides of the tracrRNA to form a double-stranded duplex of the Cas protein binding domain of the guide nucleic acid. The crRNA and tracrRNA can hybridize to form the guide nucleic acid. The crRNA may also provide a single-stranded nucleic acid targeting segment (e.g., a spacer region) that hybridizes with the target nucleic acid recognition sequence (e.g., a protospacer). The sequence of the crRNA or tracrRNA molecule, including the spacer region, may be designed to be specific for the species in which the guide nucleic acid is used. In some embodiments, the nucleic acid targeting region of the guide nucleic acid may be 18-72 nucleotides in length.The nucleic acid targeting region of the guide nucleic acid (e.g., the spacer region) can have a length of about 12 nucleotides to about 100 nucleotides. For example, the nucleic acid targeting region of the guide nucleic acid (e.g., the spacer region) can have a length of about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 40 nt, about 12 nt to about 30 nt, about 12 nt to about 25 nt, about 12 nt to about 20 nt, about 12 nt to about 19 nt, about 12 nt to about 18 nt, about 12 nt to about 17 nt, about 12 nt to about 16 nt, or about 12 nt to about 15 nt. Alternatively, the DNA targeting segment can have a length of about 18 nt to about 20 nt, about 18 nt to about 25 nt, about 18 nt to about 30 nt, about 18 nt to about 35 nt, about 18 nt to about 40 nt, about 18 nt to about 45 nt, about 18 nt to about 50 nt, about 18 nt to about 60 nt, about 18 nt to about 70 nt, about 18 nt to about 80 nt, about 18 nt to about 90 nt, about 18 nt to about 100 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, about 20 nt to about 60 nt, about 20 nt to about 70 nt, about 20 nt to about 80 nt, about 20 nt to about 90 nt, or about 20 nt to about 100 nt. The length of the nucleic acid targeting region can be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides. The length of the nucleic acid targeting region (e.g., spacer region) can be up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.

[0025] In some embodiments, the nucleic acid targeting region of the guide nucleic acid (e.g., a spacer) is 20 nucleotides long. In some embodiments, the nucleic acid targeting region of the guide nucleic acid is 19 nucleotides long. In some embodiments, the nucleic acid targeting region of the guide nucleic acid is 18 nucleotides long. In some embodiments, the nucleic acid targeting region of the guide nucleic acid is 17 nucleotides long. In some embodiments, the nucleic acid targeting region of the guide nucleic acid is 16 nucleotides long. In some embodiments, the nucleic acid targeting region of the guide nucleic acid is 21 nucleotides long. In some embodiments, the nucleic acid targeting region of the guide nucleic acid is 22 nucleotides long.

[0026] The nucleotide sequence of the guide nucleic acid that is complementary to the nucleotide sequence of the target nucleic acid (target sequence) can have a length of, for example, at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt. The nucleotide sequence of the guide nucleic acid that is complementary to the nucleotide sequence of the target nucleic acid (target sequence) can have a length of, for example, about 12 nucleotides (nt) to about 80 nt, about 12 nt to about 50 nt, about 12 nt to about 45 nt, about 12 nt to about 40 nt, about 12 nt to about 35 nt, about 12 nt to about 30 nt, about 12 nt to about 25 nt, about 12 nt to about 20 nt, about 12 nt to about 19 nt, about 19 nt to about 20 ... It may have a length of about nt to about 25 nt, about 19 nt to about 30 nt, about 19 nt to about 35 nt, about 19 nt to about 40 nt, about 19 nt to about 45 nt, about 19 nt to about 50 nt, about 19 nt to about 60 nt, about 20 nt to about 25 nt, about 20 nt to about 30 nt, about 20 nt to about 35 nt, about 20 nt to about 40 nt, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt.

[0027] The protospacer sequence of a target polynucleotide can be identified by identifying a PAM within the region of interest and selecting a region of desired size upstream or downstream of the PAM as the protospacer. The corresponding spacer sequence can be designed by determining the complementary sequence of the protospacer region.

[0028] Spacer sequences can be identified using a computer program (e.g., machine-readable code) that can use variables such as predicted melting temperature, secondary structure formation and predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC%, frequency of genomic occurrence, methylation status, presence of SNPs, etc.

[0029] The Cas protein binding segment of the guide nucleic acid may comprise two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to each other. The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to each other may be covalently linked by an intervening nucleotide (e.g., a linker in the case of a single guide nucleic acid). The two stretches of nucleotides (e.g., crRNA and tracrRNA) that are complementary to each other may hybridize to form a double-stranded RNA duplex or hairpin of the Cas protein binding segment, resulting in a stem-loop structure. The crRNA and tracrRNA may be covalently linked via the 3' end of the crRNA and the 5' end of the tracrRNA. Alternatively, the tracrRNA and crRNA may be covalently linked via the 5' end of the tracrRNA and the 3' end of the crRNA.

[0030] The Cas protein binding segment of the guide nucleic acid can have a length of about 10 nucleotides to about 100 nucleotides, such as about 10 nucleotides (nt) to about 20 nt, about 20 nt to about 30 nt, about 30 nt to about 40 nt, about 40 nt to about 50 nt, about 50 nt to about 60 nt, about 60 nt to about 70 nt, about 70 nt to about 80 nt, about 80 nt to about 90 nt, or about 90 nt to about 100 nt. For example, the Cas protein binding segment of the guide nucleic acid can have a length of about 15 nucleotides (nt) to about 80 nt, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or about 15 nt to about 25 nt.

[0031] The dsRNA duplex of the Cas protein binding segment of the guide nucleic acid can have a length of about 6 base pairs (bp) to about 50 bp. For example, the dsRNA duplex of the protein binding segment can have a length of about 6 bp to about 40 bp, about 6 bp to about 30 bp, about 6 bp to about 25 bp, about 6 bp to about 20 bp, about 6 bp to about 15 bp, about 8 bp to about 40 bp, about 8 bp to about 30 bp, about 8 bp to about 25 bp, about 8 bp to about 20 bp, or about 8 bp to about 15 bp. For example, the dsRNA duplex of the Cas protein binding segment can have a length of about 8 bp to about 10 bp, about 10 bp to about 15 bp, about 15 bp to about 18 bp, about 18 bp to about 20 bp, about 20 bp to about 25 bp, about 25 bp to about 30 bp, about 30 bp to about 35 bp, about 35 bp to about 40 bp, or about 40 bp to about 50 bp.

[0032] Guide nucleic acids of the present disclosure may contain modifications or sequences that provide additional desirable characteristics (e.g., altered or modulated stability, intracellular targeting, tracking by fluorescent labels, binding sites for proteins or protein complexes, etc.). Examples of such modifications include, for example, 5' caps (e.g., 7-methylguanylate caps (m7G)), 3' polyadenylation tails (3' poly(A) tails), riboswitch sequences (e.g., to allow for regulated stability and / or regulated accessibility by proteins and / or protein complexes), stability control sequences, sequences that form dsRNA duplexes (hairpins), modifications or sequences that target the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.), modifications or sequences that allow tracking (e.g., direct conjugation to fluorescent molecules, conjugation to moieties that facilitate fluorescent detection, sequences that allow fluorescent detection, etc.), modifications or sequences that provide binding sites for proteins (e.g., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and combinations thereof). Guide nucleic acids can include one or more modifications (e.g., base modifications, backbone modifications) to provide new or enhanced characteristics (e.g., improved stability) to the nucleic acid. The guide nucleic acid may include a nucleic acid affinity tag. A nucleoside may be a base-sugar combination. The base portion of a nucleotide may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide may be a nucleoside that further comprises a phosphate group, which is covalently linked to the sugar portion of the nucleoside. In the case of nucleosides that comprise a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3' or 5' hydroxyl portion of the sugar. In forming a guide nucleic acid, the phosphate group may covalently link adjacent nucleosides to each other to form a linear polymeric compound. The respective ends of this linear polymeric compound may then be further joined to form a circular compound. In general, however, linear compounds are preferred.In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold to produce fully or partially double-stranded compounds. Furthermore, within a guide nucleic acid, the phosphate groups may generally be referred to as forming the internucleoside backbone of the guide nucleic acid. The linkage or backbone of the guide nucleic acid may be a 3'→5' phosphodiester bond.

[0033] The guide nucleic acid may include modified backbones and / or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified guide nucleic acid backbones that contain a phosphorus atom therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs, and those with reverse polarity, where one or more internucleotide linkages are 3'→3', 5'→5' or 2'→2' linkages. A suitable guide nucleic acid with reverse polarity may contain a single 3'→3' linkage at the 3'-most internucleotide linkage (e.g., a single reverse nucleoside residue lacking a nucleobase or having a hydroxyl group instead). Various salts (e.g., potassium chloride or sodium chloride), mixed salts and free acid forms may also be included.

[0034] The guide nucleic acid may contain one or more substituted sugar moieties. Suitable polynucleotides may contain sugar substituents selected from OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2 and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Sugar substituents can be selected from C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of the guide nucleic acid or groups for improving the pharmacodynamic properties of the guide nucleic acid and other substituents with similar properties. Suitable modifications can include 2'-methoxyethoxy (2'-O-CH2CHOCH3, 2'-O-(2-methoxyethyl) or 2'-MOE, also known as an alkoxyalkoxy group).

[0035] Guide nucleic acids may also include nucleobase (or "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C) and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine ... Mention may be made of tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0036] In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to a genomic sequence of a genomic locus of the cell. In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is 100% identical to a genomic sequence of a genomic locus of the cell. In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more complementary to a genomic sequence of a genomic locus of the cell. In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is 100% complementary to a genomic sequence of a genomic locus of the cell. In some embodiments, the genomic locus of the cell comprises a T cell receptor alpha chain constant (TRAC) locus. In some embodiments, the genomic locus of the cell comprises a beta-2-microglobulin (B2M) locus. In some embodiments, the genomic locus may also comprise a genomic locus of CD38, CISH, PD-1 or CD70.

[0037] Genetically modified part In some embodiments, the system comprises at least one genetic modification moiety. In some embodiments, the genetic modification moiety may be complexed with at least one guide nucleic acid described herein. In some embodiments, the genetic modification moiety is directed to a genomic locus when complexed with a guide nucleic acid, and the genetic modification moiety cleaves the genomic locus. In some embodiments, the cleavage mediated by the genetic modification moiety results in a double-stranded break in the genomic locus of the cell. In some embodiments, the cleavage mediated by the genetic modification moiety results in a single-stranded break or nicking in the genomic locus of the cell. In some embodiments, the cleavage mediated by the genetic modification moiety may then be repaired by endogenous repair mechanisms, during which a polynucleotide described herein is introduced or knocked-in to the cleaved genomic locus of the cell. For example, the cleavage mediated by the genetic modification moiety may induce homology-directed repair (HDR) in the cell to be modified, and HDR inserts the polynucleotide into the cleaved genomic locus of the cell.

[0038] In some embodiments, the genetically modified portion comprises a nucleic acid-guided nuclease. In some embodiments, the genetically modified portion comprises a CRISPR-Cas polypeptide. In some embodiments, the genetically modified portion can be, for example, a class 1 CRISPR-associated (Cas) polypeptide, a class 2 Cas polypeptide, a Cas type I polypeptide, a Cas type II polypeptide, a Cas type III polypeptide, a Cas type IV polypeptide, a Cas type V polypeptide and a type VI, a CRISPR-associated RNA-binding protein or a functional fragment thereof. Suitable Cas polypeptides for use with the present disclosure include, but are not limited to, Cas9, Cas12, Cas13, Cpf1 (or Cas12a), C2C1, C2C2 (or Cas13a), Cas13b, Cas13c, Cas13d, C2C3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Csnl, Csxl2, Cas10, Cas10d, CaslO, CaslOd, CasF, CasG, CasH, Cs yl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 or Cul966, any derivative thereof, any variant thereof or any fragment thereof. In some embodiments, Cas13 may include, but is not limited to, Cas13a, Cas13b, Cas13c, and Cas13d (e.g., CasRx). In some embodiments, the genetically modified portion comprises a Cas / RNP. In some embodiments, the genetically modified portion comprises a Cas9 / RNP. In some embodiments, the genetically modified portion comprises a Cas protein or an mRNA encoding a Cas protein.

[0039] Any suitable nuclease (eg, endonuclease) can be used as the genetic modification moiety. Suitable nucleases include CRISPR-associated (Cas) proteins or Cas nucleases, such as CRISPR type I associated (Cas) polypeptides, CRISPR type II associated (Cas) polypeptides, CRISPR type III associated (Cas) polypeptides, CRISPR type IV associated (Cas) polypeptides, CRISPR type V associated (Cas) polypeptides, and CRISPR type VI associated (Cas) polypeptides; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregorii (Natronobacterium gregoryi) Argonaute (NgAgo); Adenosine deaminase acting on RNA (ADAR); CIRT, PUF, homing endonuclease or any functional fragment thereof, any derivative thereof, any variant thereof, and any fragment thereof.

[0040] In some embodiments, the genetic modification portion can be complexed with the polynucleotide described herein through at least one genetic modification portion targeting fragment.In some embodiments, the at least one genetic modification portion targeting fragment allows the polynucleotide and the genetic modification portion to be in close proximity when complexed with the genetic modification portion.This arrangement makes it easy to insert the polynucleotide into the genome locus when the genome locus is cleaved by the genetic modification portion.

[0041] Chimeric polynucleotides In some embodiments, the system comprises a polynucleotide. In some embodiments, the polynucleotide comprises double stranded DNA (dsDNA). In some embodiments, the polynucleotide comprises single stranded DNA (ssDNA). In some embodiments, the polynucleotide comprises a combination of double stranded DNA (dsDNA) and single stranded DNA (ssDNA). In some embodiments, the polynucleotide comprises at least one genetic modification moiety targeting fragment that complexes with the genetic modification moiety. In some embodiments, the polynucleotide comprises at least one expression sequence. In some embodiments, the at least one expression sequence encodes at least one transgene or a fragment thereof. In some embodiments, the transgene may encode any one of the chimeric receptors described herein. In some embodiments, the at least one expression sequence may be flanked by at least one homology arm, the homology arm comprising a nucleic acid sequence that is homologous to a nucleic acid sequence of a genomic locus, such as the TRAC locus or the B2M locus. In some embodiments, the genomic loci may also include the genomic loci of CD38, CISH, PD-1, or CD70.

[0042] In some embodiments, the chimeric polynucleotide comprises from about 1,000 base pairs to about 6,500 base pairs. In some embodiments, the chimeric polynucleotide comprises from about 1,000 base pairs to about 1,500 base pairs, from about 1,000 base pairs to about 2,000 base pairs, from about 1,000 base pairs to about 2,500 base pairs, from about 1,000 base pairs to about 3,000 base pairs, from about 1,000 base pairs to about 3,500 base pairs, from about 1,000 base pairs to about 4,000 base pairs, from about 1,000 base pairs to about 4,500 base pairs, from about 1,000 base pairs to about 5,000 base pairs, from about 1,000 base pairs to about 5,500 base pairs, from about 1,000 base pairs to about 6,000 base pairs, from about 1,000 base pairs to about 6 ,500 base pairs, about 1,500 base pairs to about 2,000 base pairs, about 1,500 base pairs to about 2,500 base pairs, about 1,500 base pairs to about 3,000 base pairs, about 1,500 base pairs to about 3,500 base pairs, about 1,500 base pairs to about 4,000 base pairs, about 1,500 base pairs to about 4,500 base pairs, about 1,500 base pairs to about 5,000 base pairs, about 1,500 base pairs to about 5,500 base pairs, about 1,500 base pairs to about 6,000 base pairs, about 1,500 base pairs to about 6,500 base pairs, about 2,000 base pairs to about 2,500 base pairs, about 2,000 base pairs ~ about 3,000 base pairs, about 2,000 base pairs to about 3,500 base pairs, about 2,000 base pairs to about 4,000 base pairs, about 2,000 base pairs to about 4,500 base pairs, about 2,000 base pairs to about 5,000 base pairs, about 2,000 base pairs to about 5,500 base pairs, about 2,000 base pairs to about 6,000 base pairs, about 2,000 base pairs to about 6,500 base pairs, about 2,500 base pairs to about 3,000 base pairs, about 2,500 base pairs to about 3,500 base pairs, about 2,500 base pairs to about 4,000 base pairs, about 2,500 base pairs to about 4,500 base pairs, about 2,500 base pairs to about 5,000 base pairs, about 2,500 base pairs to about 5,500 base pairs, about 2,500 base pairs to about 6,000 base pairs, about 2,500 base pairs to about 6,500 base pairs, about 3,000 base pairs to about 3,500 base pairs, about 3,000 base pairs to about 4,000 base pairs, about 3,000 base pairs to about 4,500 base pairs, about 3,000 base pairs to about 5,000 base pairs, about 3,000 base pairs to about 5,500 base pairs, about 3,000 base pairs to about 6,000 base pairs, about 3,000 base pairs to about 6,500 base pairs, about 3,500 base pairs to about 4,000 base pairs, about 3,500 base pairs to about 4,500 base pairs, about 3,500 base pairs to about 5,000 base pairs, about 3,500 base pairs to about 5,500 base pairs, about 3,500 base pairs to about 6,000 base pairs, about 3,500 base pairs to about 6,500 base pairs, about 4,000 base pairs to about 4,500 base pairs, about 4,000 base pairs to about 5,000 base pairs, about 4,000 base pairs to about 5,500 base pairs, about 4,000 base pairs to about 6,000 base pairs, about 4,000 base pairs to about 6,500 base pairs, about 4, The range includes 500 base pairs to about 5,000 base pairs, about 4,500 base pairs to about 5,500 base pairs, about 4,500 base pairs to about 6,000 base pairs, about 4,500 base pairs to about 6,500 base pairs, about 5,000 base pairs to about 5,500 base pairs, about 5,000 base pairs to about 6,000 base pairs, about 5,000 base pairs to about 6,500 base pairs, about 5,500 base pairs to about 6,000 base pairs, about 5,500 base pairs to about 6,500 base pairs, or about 6,000 base pairs to about 6,500 base pairs. In some embodiments, the polynucleotide comprises about 1,000 base pairs, about 1,500 base pairs, about 2,000 base pairs, about 2,500 base pairs, about 3,000 base pairs, about 3,500 base pairs, about 4,000 base pairs, about 4,500 base pairs, about 5,000 base pairs, about 5,500 base pairs, about 6,000 base pairs, or about 6,500 base pairs. In some embodiments, the polynucleotide comprises at least about 1,000 base pairs, about 1,500 base pairs, about 2,000 base pairs, about 2,500 base pairs, about 3,000 base pairs, about 3,500 base pairs, about 4,000 base pairs, about 4,500 base pairs, about 5,000 base pairs, about 5,500 base pairs, or about 6,000 base pairs. In some embodiments, the polynucleotide comprises at most about 1,500 base pairs, about 2,000 base pairs, about 2,500 base pairs, about 3,000 base pairs, about 3,500 base pairs, about 4,000 base pairs, about 4,500 base pairs, about 5,000 base pairs, about 5,500 base pairs, about 6,000 base pairs, or about 6,500 base pairs.

[0043] In some embodiments the polynucleotide is circular. In some embodiments the circular polynucleotide is a vector. In some embodiments the circular polynucleotide is a minicircle. In some embodiments the polynucleotide is linear. In some embodiments the linear polynucleotide comprises at least one covalently closed circular end at either the 5' or 3' end of the polynucleotide. In some embodiments the linear polynucleotide comprises covalently closed circular ends at both the 5' and 3' ends of the polynucleotide. In some embodiments the covalently closed circular end comprises a telomeric end. In some embodiments the linear polynucleotide comprises a telomeric end at the 5' end of the polynucleotide. In some embodiments the linear polynucleotide comprises a telomeric end at the 3' end of the polynucleotide. In some embodiments the linear polynucleotide comprises a telomeric end at both the 5' end of the polynucleotide and the 3' end of the polynucleotide. Polynucleotides that are circular or have at least one covalently closed end may reduce degradation of the polynucleotide (e.g., by nucleases in the cell being modified). Reduced degradation of the polynucleotide may result in increased abundance of the polynucleotide for knock-in at a genomic locus. In some embodiments, polynucleotides that are circular or have at least one covalently closed end increase the knock-in efficiency of the polynucleotide.

[0044] In some embodiments, at least one genetically modified partial targeting fragment is located near the 5' end of the linear polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment is located at the 5' end of the linear polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment is located at least about 10 base pairs (bps), at least about 50 base pairs (bps), at least about 100 base pairs (bps), at least about 200 base pairs (bps), at least about 500 base pairs (bps), at least about 1000 base pairs (bps), or at least about 1500 base pairs (bps) from the 5' end of the linear polynucleotide.

[0045] In some embodiments, at least one genetically modified partial targeting fragment is located near the 3' end of the linear chimeric polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment is located at the 3' end of the linear chimeric polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment is located at least about 10 base pairs (bps), at least about 50 base pairs (bps), at least about 100 base pairs (bps), at least about 200 base pairs (bps), at least about 500 base pairs (bps), at least about 1000 base pairs (bps), or at least about 1500 base pairs (bps) from the 3' end of the linear chimeric polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment can be located near both the 5' end of the linear chimeric polynucleotide and the 3' end of the linear chimeric polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment is located near the expressed sequence of the chimeric receptor-encoding polynucleotide. In some embodiments, at least one genetically modified partial targeting fragment is located near the expressed sequence of the circular polynucleotide encoding the chimeric receptor. In some embodiments, at least one genetically modified partial targeting fragment is located at least about 10 base pairs (bps), at least about 50 base pairs (bps), at least about 100 base pairs (bps), at least about 200 base pairs (bps), at least about 500 base pairs (bps), at least about 1000 base pairs (bps), or at least about 1500 base pairs (bps) from the expressed sequence of the polynucleotide.

[0046] In some embodiments, at least one genetically modified portion targeting fragment comprises from about 5 base pairs to about 200 base pairs.In some embodiments, at least one genetically modified portion targeting fragment is from about 5 base pairs to about 10 base pairs, from about 5 base pairs to about 20 base pairs, from about 5 base pairs to about 30 base pairs, from about 5 base pairs to about 40 base pairs, from about 5 base pairs to about 50 base pairs, from about 5 base pairs to about 60 base pairs, from about 5 base pairs to about 70 base pairs, from about 5 base pairs to about 80 base pairs, from about 5 base pairs to about 90 base pairs, from about 5 base pairs to about 100 base pairs, from about 5 base pairs to about 200 base pairs, from about 10 base pairs to about 20 base pairs, from about 10 base pairs to about 30 base pairs, from about 10 base pairs to about 40 base pairs, from about 10 base pairs to about 5 ... base pairs to about 60 base pairs, about 10 base pairs to about 70 base pairs, about 10 base pairs to about 80 base pairs, about 10 base pairs to about 90 base pairs, about 10 base pairs to about 100 base pairs, about 10 base pairs to about 200 base pairs, about 20 base pairs to about 30 base pairs, about 20 base pairs to about 40 base pairs, about 20 base pairs to about 50 base pairs, about 20 base pairs to about 60 base pairs, about 20 base pairs to about 70 base pairs, about 20 base pairs to about 80 base pairs, about 20 base pairs to about 90 base pairs, about 20 base pairs to about 100 base pairs, about 20 base pairs to about 200 base pairs, about 30 base pairs to about 40 base pairs, about 30 base pairs to about 50 base pairs, about 3 ... about 60 base pairs, about 30 base pairs to about 70 base pairs, about 30 base pairs to about 80 base pairs, about 30 base pairs to about 90 base pairs, about 30 base pairs to about 100 base pairs, about 30 base pairs to about 200 base pairs, about 40 base pairs to about 50 base pairs, about 40 base pairs to about 60 base pairs, about 40 base pairs to about 70 base pairs, about 40 base pairs to about 80 base pairs, about 40 base pairs to about 90 base pairs, about 40 base pairs to about 100 base pairs, about 40 base pairs to about 200 base pairs, about 50 base pairs to about 60 base pairs, about 50 base pairs to about 70 base pairs, about 50 base pairs to about 80 base pairs, about 50 base pairs to about 90 base pairs, about 50 base pairs to about 1 00 base pairs, about 50 base pairs to about 200 base pairs, about 60 base pairs to about 70 base pairs, about 60 base pairs to about 80 base pairs, about 60 base pairs to about 90 base pairs, about 60 base pairs to about 100 base pairs, about 60 base pairs to about 200 base pairs, about 70 base pairs to about 80 base pairs, about 70 base pairs to about 90 base pairs, about 70 base pairs to about 100 base pairs, about 70 base pairs to about 200 base pairs, about 80 base pairs to about 90 base pairs, about 80 base pairs to about 100 base pairs, about 80 base pairs to about 200 base pairs, about 90 base pairs to about 100 base pairs, about 90 base pairs to about 200 base pairs, or 100 base pairs to about 200 base pairs.In some embodiments, at least one genetically modified portion targeting fragment comprises about 5 base pairs, about 10 base pairs, about 20 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, about 60 base pairs, about 70 base pairs, about 80 base pairs, about 90 base pairs, about 100 base pairs, or about 200 base pairs. In some embodiments, at least one genetically modified portion targeting fragment comprises at least about 5 base pairs, about 10 base pairs, about 20 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, about 60 base pairs, about 70 base pairs, about 80 base pairs, about 90 base pairs, or about 100 base pairs. In some embodiments, at least one genetically modified portion targeting fragment comprises at most about 10 base pairs, about 20 base pairs, about 30 base pairs, about 40 base pairs, about 50 base pairs, about 60 base pairs, about 70 base pairs, about 80 base pairs, about 90 base pairs, about 100 base pairs, or about 200 base pairs.

[0047] In some embodiments, at least one genetically modified partial targeting fragment and the guide nucleic acid share about 60% to about 100% nucleic acid sequence identity. In some embodiments, at least one genetically modified partial targeting fragment and the guide nucleic acid share about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 96%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 60% to about 100%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 70% to about 100%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 96%, about 75% to about 97%, about 75% to about 98%, about 75% to about 99%, about 75% to about 100%, about 80% to about 85%, about 80% to about 9 0%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 85% to about 100%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 9 9%, about 90% to about 100%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 95% to about 100%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 96% to about 100%, about 97% to about 98%, about 97% to about 99%, about 97% to about 100%, about 98% to about 99%, about 98% to about 100%, or about 99% to about 100% nucleic acid sequence identity. In some embodiments, at least one genetically modified partial targeting fragment and the guide nucleic acid share between about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% nucleic acid sequence identity.In some embodiments, at least one genetically modified partial targeting fragment and the guide nucleic acid share at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% nucleic acid sequence identity. In some embodiments, at least one genetically modified partial targeting fragment and the guide nucleic acid share up to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% nucleic acid sequence identity. In some embodiments, at least one genetically modified partial targeting fragment comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to the guide nucleic acid. In some embodiments, at least one genetically modified partial targeting fragment comprises at least one mismatch, at least two mismatches, at least three mismatches, at least four mismatches, at least five mismatches, at least six mismatches, at least seven mismatches, at least eight mismatches, at least nine mismatches, or at least ten mismatches compared to the guide nucleic acid.

[0048] In some embodiments, at least one genetic modification portion targeting fragment is complexed with the genetic modification portion, thereby bringing the genetic modification portion into close proximity with the polynucleotide.In some embodiments, at least one genetic modification portion targeting fragment does not induce the enzymatic activity of the genetic modification portion when complexed with the genetic modification portion.For example, at least one genetic modification portion targeting fragment can be complexed with the genetic modification portion (e.g., in a manner similar to that of a guide nucleic acid complexed with the genetic modification portion) without inducing the cleavage event mediated by the genetic modification portion.

[0049] In some embodiments, the polynucleotides comprise at least one expression sequence encoding a transgene. In some embodiments, the polynucleotides encode a transgene comprising a chimeric receptor as described herein. In some embodiments, the polynucleotides encode a transgene comprising an MHC protein as described herein. In some embodiments, the MHC protein includes HLA-A, HLA-E, HLA-DM, HLA-DO, HLA-DR, HLA-DQ, HLA-DP, or a combination thereof.

[0050] Chimeric Receptors In some aspects, cells modified by the system described herein for knocking in a transgene encoded by a polynucleotide are described herein. In some embodiments, cells may be modified by a first system and a second system for knocking in two separate transgenes, which may be the same or different. In some embodiments, the transgene encoded by the polynucleotide is a chimeric receptor. In some aspects, the chimeric receptor may be a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, cells modified by the system described herein may express a transgene. In some embodiments, cells modified by the system described herein may express a chimeric receptor described herein. In some embodiments, cells modified by the system described herein may express a first transgene and a second transgene. In some embodiments, cells modified by the system described herein may express a first chimeric receptor and a second chimeric receptor described herein. In some embodiments, cells modified by the systems described herein may express a first transgene, a second transgene, or any further number of transgenes. In some embodiments, cells modified by the systems described herein may express a first chimeric receptor, a second chimeric receptor, or any further number of chimeric receptors described herein.

[0051] In some aspects, the cells provided herein comprise one or more CAR-containing chimeric receptors. The CAR may comprise an extracellular domain, a transmembrane domain, or an intracellular signaling domain. The extracellular domain may comprise a target-specific binding element (also known as an antigen-binding domain). The intracellular domain may comprise a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that may be required for efficient response of lymphocytes to antigens. A spacer domain may be incorporated between the extracellular domain and the transmembrane domain of the CAR or between the cytoplasmic domain and the transmembrane domain of the CAR.

[0052] As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that serves to link a transmembrane domain to either the extracellular or cytoplasmic domain of a polypeptide chain. A spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. In relation to a transmembrane domain, a CAR may be designed to contain a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains of the CAR is used. Optionally, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins and minimize interactions with other members of the receptor complex.

[0053] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in the present disclosure may be derived from the alpha, beta or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4 (e.g., including at least the transmembrane region thereof). Alternatively, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A particularly suitable linker is provided by a glycine-serine doublet. The cytoplasmic domain or other intracellular signaling domain of the CAR of the present disclosure may be responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell.

[0054] The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits a signal for effector function and instructs the cell to perform a specialized function. The entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits a signal for effector function. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit a signal for effector function.

[0055] Examples of intracellular signaling domains for use in the CAR of the present disclosure include cytoplasmic sequences of TCR and co-receptor that coordinately initiate signaling after antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequences with the same functional capabilities. Signals generated through the TCR alone may be insufficient for full activation of T cells, and secondary and / or costimulatory signals may be included. Thus, activation of T cells may be mediated by two different classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0056] The primary cytoplasmic signaling sequence can regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that include primary cytoplasmic signaling sequences of particular use in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present disclosure includes a cytoplasmic signaling sequence derived from CD3 zeta. In some embodiments, the cytoplasmic domain of the CAR can be designed to include a CD3 zeta signaling domain by itself or can be combined with any other desired cytoplasmic domain useful in connection with the CAR of the present disclosure. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. A costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule.

[0057] Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that may be required for an efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. Thus, although the present disclosure is occasionally exemplified using 4-1BB as a costimulatory signaling element, other costimulatory elements are within the scope of the present disclosure.

[0058] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present disclosure may be linked to each other randomly or in a specific order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the linkage. A glycine-serine doublet provides a particularly suitable linker. In some embodiments, the cytoplasmic domain is designed to include the signaling domain of CD3 zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3 zeta and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3 zeta and the signaling domains of CD28 and 4-1BB.

[0059] The CARs provided herein may comprise one or more antigen binding domains. In some cases, the CARs provided herein comprise an antigen binding domain that can target both immune cell antigens (e.g., for inhibiting the killing activity of T cells or NK cells) and disease-associated antigens (e.g., tumor-associated antigens). For example, antigen binding domains that target both immune cell antigens and cancer antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD69, CD100, CD122, CD132, CD137, CD161, CD159a, CD159c, CD279, CD314, CD319 (CS1), and TCR. In some cases, the CARs provided herein comprise two antigen binding domains, such that each CAR is a bispecific CAR that targets two different antigens. In the case of a bispecific CAR, one antigen-binding domain can target an immune cell antigen, and the other antigen-binding domain can target a disease-associated antigen. The two antigen-binding domains of a bispecific CAR can have a tandem, parallel, or loop structure. For example, a CAR can target a tumor cell marker and CD3. A CAR can have a structure of the formula I: L-scFyl-I-scFv2-H-TM-C-CD3(I), where each "-" is independently a linker peptide or peptide bond, L is optionally a signaling peptide sequence, I is a flexible linker, H is optionally a hinge region, TM is a transmembrane domain, C is a costimulatory domain, CD3 is a cytoplasmic signaling sequence derived from CD3t, and one of scFv1 and scFv2 is an antigen-binding domain that targets a tumor cell marker, and the other is an antigen-binding domain that targets CD3.The CAR can have the structure of formula II or II': L-VL-scFv-VH-H-TM-C-CD3(II), L-VH-scFv-VL-H-TM-C-CD3(II'), where each "-" is independently a linker peptide or a peptide bond, and the elements L, H, TM, C and CD3 are as described above, and the scFv is an antigen binding domain targeting a tumor cell marker, the VH is an anti-CD3 antibody heavy chain variable region and the VL is an anti-CD3 antibody light chain variable region, or the scFv is an antigen binding domain targeting CD3, the VH is an anti-tumor cell marker antibody heavy chain variable region and the VL is an anti-tumor cell marker antibody light chain variable region. Optionally, the CAR may comprise the structure EGFRt-CD3 scFv-CD19 scFv-hinge-TM-CD28 / 41BB-CD3, where EGFRt is a truncated EGFR as a safety switch (e.g., an inducible cell death portion), CD3 scFv is a svFCv fragment of the heavy and light chain variable regions of monoclonal antibodies OKT3 or UCHT1 linked by a GS linker, and CD19 scFv fragment is the heavy and light chain variable regions of monoclonal antibodies linked by a GS linker. This CAR structure may further comprise a hinge, a transmembrane region, a costimulatory signaling region of CD28 or 41BB, and / or a CD3 intracellular domain. In the present disclosure, the nucleic acid construct of EGFRt-CD3 scFv-CD19 scFv-hinge-TM-CD28 / 41BB-CD3 may be inserted into a vector (e.g., a lentiviral vector). This vector may be packaged into 293T cells. T cells can be selected from PBMCs, and after activation, TCR and PD-1 genes can be knocked out by CRISPR / CAS technology. T cells can then be infected with vectors to express CAR. The prepared CAR-T cells can be used to detect the infection efficiency and gene editing efficiency of CAR by flow cytometry.

[0060] The immune cell markers in the above examples, such as CD3, can be replaced with other immune cell markers, such as CD7 and CD137. Optionally, a CAR comprising two antigen binding domains is arranged in tandem. In some embodiments, the first antigen binding domain and the second antigen binding domain are arranged from amino terminus to carboxyl terminus as follows: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; (iv) VL1-VH1-VH2-VL2; (v) VH2-VL2-VL1-VH1; (vi) VH2-VL2-VH1-VL1 (vii) VH1-VL1-VL2-VH2; or (viii) VH1-VL1-VH2-VL2, where VH1 is the heavy chain variable domain of the first antigen-binding domain, VL1 is the light chain variable domain of the first antigen-binding domain, VH2 is the heavy chain variable domain of the second antigen-binding domain, and VL2 is the light chain variable domain of the second antigen-binding domain. For example, a CAR may have a structure represented by the following formula IV or IV': L3-scFv1-R-scFv2-H3-TM3-C3-CD3(IV); L3-scFv2-R-scFv1-H3-TM3-C3-CD3(IV'), where each "-" is independently a linker peptide or a peptide bond, L3 is an optional signal peptide sequence, scFv1 is an antigen binding domain targeting a tumor cell marker, R is a rigid or flexible binding site, scFv2 is an antigen binding domain (e.g., an antibody single chain variable region sequence) targeting a T cell and NK cell consensus marker, H3 is an optional hinge region, TM3 is a transmembrane domain, C3 is a costimulatory domain, and CD3 is a cytoplasmic signaling sequence derived from CD3. Optionally, a CAR comprising two antigen binding domains is arranged in a loop configuration.Optionally, the first antigen-binding domain and the second antigen-binding domain are arranged, from amino-terminus to carboxyl-terminus, as follows: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; (iv) VH1-VL2-VH2-VL1; (v) VL2-VL1-VH1-VH2; (vi) VH2-VH1-VL1-VL2; (vii) VL1-VL2-VH2-VH1; or (viii) VH1-VH2-VL2-VL1, where VH1 is the heavy chain variable domain of the first antigen-binding domain, VL1 is the light chain variable light domain of the first antigen-binding domain, VH2 is the heavy chain variable domain of the second antigen-binding domain, and VL2 is the light chain variable domain of the second antigen-binding domain. For example, a CAR can have the structure of the following formula VI, VI', VI'', or VI'''L8-VL1-VH2-I-VL2-VH1-H8-TM8-C8-CD3(VI); L8-VH1-VL2-I-VH2-VL1-H8-TM8-C8-CD3(VI'); L8-VL2-VH1-I-VL1-VH2-H8-TM8-C8-CD3(VI''); L8-VH2-VL1-I-VH1-VL2-H8-TM8-C8-CD3(VI''), where each "-" is independently a linker peptide or a peptide bond, and L8 is an optional signal is a peptide sequence, VH1 is an anti-tumor cell marker antibody heavy chain variable region, VL1 is an anti-tumor cell marker antibody light chain variable region, VH2 is an anti-T cell and NK cell consensus marker (such as CD7 or CD2) antibody heavy chain variable region, VL2 is an anti-T cell and NK cell consensus marker (such as CD7 or CD2) antibody light chain variable region, I is a flexible binding site, H8 is an optional hinge region, TM8 is a transmembrane domain, C8 is a costimulatory domain, and CD3 is a cytoplasmic signaling sequence derived from CD3.

[0061] In some cases, a CAR comprising two antigen binding domains is arranged in a side-by-side configuration. The side-by-side configuration may include a complete construct of a first CAR with a first antigen binding domain bound to a complete construct of a second CAR with a second antigen binding domain. An example of a side-by-side configuration may be tEGFR-CD19 scFv-CD28-CD3-CD3 scFv-41BB-CD3. The tEGFR shown here can function as a safety switch, which can be replaced with other safety switches described in this disclosure. As described herein, CD19 scFv and CD3 scFv are two examples of antigen binding domains, which can be replaced with various antigen binding domains described in this disclosure. CD28 is an example of a transmembrane domain, which can be replaced with other transmembrane domains described herein. 41BB is an example of a costimulatory domain, which can be replaced with other costimulatory domains described herein. In some cases, a linker is used to connect the first CAR to the second CAR. The linker can be a cleavable linker. The cleavable linker can be a self-cleaving peptide, for example a 2A self-cleaving peptide.

[0062] Additionally, nucleic acid molecules encoding CARs or bispecific CARs are also contemplated in the present disclosure. The nucleic acid may comprise a first sequence encoding a chimeric antigen receptor (CAR), wherein the CAR may comprise a binding moiety comprising (i) a first antigen binding domain that, when administered to a subject, suppresses or reduces the subject's immune response to the cells described herein, and (ii) a second antigen binding domain capable of binding to a disease-associated antigen, wherein each CAR of the one or more CARs may further comprise a transmembrane domain and an intracellular signaling domain. The first antigen-binding domain is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD The second antigen-binding domain can target an immune cell antigen selected from the group consisting of CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, and SLAMF7. The second antigen-binding domain can target a disease-associated antigen, such as CD19.Other non-limiting examples of disease associated antigens include BCMA, VEGFR2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3. HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Fltl, KDR, Flt4, CD44V6, CEA, CA 125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A, Mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robl, Frizzled, OX40, CD79b, claudin 18.2, folate receptor alpha, folate receptor (3, GPC2, CD70, BAFF-R, and Notch-1-4.

[0063] The nucleic acid molecule may further comprise a second sequence encoding an enhancer portion, which when expressed in a cell may enhance one or more activities of the CAR. The enhancer portion may be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors thereof, functional fragments thereof, functional variants thereof, and combinations thereof. The nucleic acid molecule may further comprise a second sequence encoding an inducible cell death portion, which when expressed in a cell may result in the death of the cell upon contact with a cell death activator. The inducible cell death moiety may be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ACD20, mTMPK, ACD19, RQR8 and EGFRt.

[0064] The nucleic acid molecule may further comprise a third sequence flanked by the first and second sequences, and the third sequence may encode a cleavable linker. The cleavable linker may be a self-cleaving peptide. The nucleic acid molecule may further comprise a regulatory sequence that regulates the expression of the first sequence and / or the second sequence. The present disclosure also contemplates a kit comprising the nucleic acid molecules described herein. Optionally, the nucleic acid encoding the CAR described herein may be delivered into an immune cell to express the CAR and generate an engineered cell.

[0065] Methods for modifying cells In some aspects, methods of modifying cells with the systems described herein are described. In some aspects, the cells are modified to express at least one chimeric receptor described herein. In some aspects, the cells are modified to express at least one MHC protein described herein. In some aspects, the cells are modified by knocking in a polynucleotide described herein, the polynucleotide encoding a chimeric receptor or MHC protein. In some embodiments, the cells may be modified with at least a first system and at least a second system, the polynucleotides of the first system and the second system each encoding a transgene. The transgenes of the first system and the second system may be the same or different. For example, the first system may knock in a first transgene comprising a chimeric receptor or MHC protein, and the second system may knock in a second transgene comprising a chimeric receptor or MHC protein that is different from the first chimeric receptor or the first MHC protein. In some embodiments, the cells may be modified with the first line, the second line, or any further number of lines, and the modified cells may subsequently express the first chimeric receptor, the second chimeric receptor, or any further number of chimeric receptors.

[0066] In some aspects, the method comprises contacting a cell with any component of the system or system described herein or any combination of system components. In some aspects, the method comprises contacting a cell with a polynucleotide, which after knock-in encodes or expresses a chimeric receptor or MHC protein. In some aspects, any component of the system or system described herein or any combination of system components can be readily introduced into a cell, such as a mammalian cell, a bacterial cell, a yeast cell, or an insect cell, by any method in the art. For example, any component of the system or system described herein or any combination of system components can be transfected into a cell by physical, chemical, or biological means. In some embodiments, any component of the system or system described herein or any combination of system components can be delivered into a cell by physical methods, such as calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, electroporation, etc.

[0067] Physical methods for introducing the systems described herein or any component of the systems or any combination of the system components into cells may include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene guns, electroporation, etc. One method for introducing the systems described herein or any component of the systems or any combination of the system components into cells is calcium phosphate transfection.

[0068] Chemical means for introducing the system described herein or any component of the system or any combination of components of the system into cells may include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes, or lipid nanoparticles. An exemplary colloidal system for use in vitro and in vivo as a delivery vehicle is a liposome (e.g., artificial membrane vesicle). Other prior art targeted delivery methods of nucleic acids are available, such as the delivery of non-natural polynucleotides or vectors encoding gene modification moieties using targeted nanoparticles.

[0069] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations to introduce the system described herein or any component of the system or any combination of the components of the system into cells (in vitro, ex vivo, or in vivo) is contemplated. In another aspect, the system described herein or any component of the system or any combination of the components of the system can be associated with lipid. The system described herein or any component of the system or any combination of the components of the system associated with lipid can be encapsulated in the aqueous interior of a liposome, can be interspersed within the lipid bilayer of a liposome, can be attached to a liposome via a linking molecule associated with both the liposome and the genetically modified portion or the heterologous polynucleotide encoding the genetically modified portion, can be entrapped in a liposome, can be complexed with a liposome, can be dispersed in a solution containing lipid, can be mixed with a lipid, can be combined with a lipid, can be contained as a suspension in a lipid, can be contained in or complexed with a micelle, or can be otherwise associated with a lipid. The compositions involving lipids, lipids / DNA or lipids / expression vectors are not limited to any particular structure in solution. For example, in some embodiments, they exist in bilayer structures, as micelles or in "collapsed" structures. Instead, they may simply be dispersed in the solution, forming aggregates that are sometimes not uniform in size or shape. Lipids are fatty substances, which in some embodiments are natural or synthetic lipids. For example, lipids include the naturally occurring lipid droplets in the cytoplasm, as well as classes of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, fatty alcohols, fatty amines, fatty amino alcohols and fatty aldehydes.

[0070] Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are often stored at about -20°C. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by creating sealed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement to form a closed structure, entrapping water and dissolved solutes between the lipid bilayers. However, compositions with structures in solution that differ from the normal vesicular structure are also encompassed. For example, lipids may in some embodiments adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0071] In some cases, non-viral delivery methods include lipofection, nucleofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid:cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced uptake of polypeptides or DNA.

[0072] In some embodiments, the system described herein or any component of the system or any combination of components of the system can be delivered to cells via biological methods, such as the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors, in some embodiments, can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc. Exemplary viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV vectors), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors or herpes simplex virus vectors (HSV). In some cases, retroviral vectors include gamma retroviral vectors, such as vectors derived from Moloney murine leukemia virus (MoMLV, MMLV, MuLV or MLV) or mouse steam cell virus (MSCV) genomes. In some cases, retroviruses also include lentiviral vectors, such as those derived from the human immunodeficiency virus (HIV) genome. In some cases, the AAV comprises a serotype comprising AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. Based on these initial serotypes, the AAV capsid of each serotype can be engineered to be more suitable for biological function, tissue, or cell selection.

[0073] In some aspects, the systems described herein or any component of the systems or any combination of components of the systems include various gene editing methods used in the present disclosure to modify cells described herein, such as CRISPR, RNA interference technology, TALENs (transcription activator-like (TAL) effector nucleases) and zinc finger nucleases (ZFNs).

[0074] In some cases, the CRISPR / Cas9 system is used to edit genes in immune cells. For example, the CRISPR / Cas9 system can be used to knock out endogenous TCR or cell surface markers (e.g., CS1, CD7, CD137) in immune cells to generate the cells described herein for T cell therapy. The CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system is a natural immune system inherent to prokaryotes that are resistant to viruses or exogenous plasmids. The CRISPR / Cas system type II has been applied to many eukaryotic and prokaryotic organisms as a direct genome-directed genome editing tool. The development of the CRISPR / Cas9 system has revolutionized the ability to edit DNA sequences and regulate the expression levels of target genes, providing a powerful tool for precise genome editing in organisms. A simplified CRISPR / Cas9 system can include a Cas9 protein and a gRNA. Its principle of action is that gRNA forms a Cas9-gRNA complex with Cas9 protein through its own Cas9 handle, and the base-complementary pairing sequence of gRNA in the Cas9-gRNA complex is paired with the target sequence of the target gene by the principle of base-complementary pairing. Cas9 cleaves the target DNA sequence using its own endonuclease activity. Compared with traditional genome editing technology, the CRISPR / Cas9 system has some distinct advantages, including ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes simultaneously. In some embodiments, the genetic modification portion comprises a gRNA that complexes with Cas to form a Cas / RNP. In some aspects, the genetic modification portion is a Cas9 / RNP.

[0075] Proteins that bind to guide RNA and are guided by the guide RNA to induce sequence-specific cleavage or otherwise bind to nucleic acid sequences in a sequence-specific manner to induce non-specific cleavage are consistent with the present disclosure. Such proteins include programmable endonucleases, such as programmable Cas endonucleases. Examples of programmable Cas endonucleases that are consistent with the present disclosure include Cas12a (or Cpf1), Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas14, Cas9, etc. Optionally, the site-specific endonuclease can include Cas12a (Cpf1), including any derivative thereof, any variant thereof, or any fragment thereof. Cas12a is classified as a class II, type V CRISPR / Cas effector protein with approximately 1,300 amino acids. Cas12a is smaller than Cas9. Cas12a contains two main domains, including REC and RuvC domains. Cas12a does not have an HNH endonuclease domain like Cas9. Cas12a cleaves double-stranded DNA (dsDNA) immediately downstream of the T-rich (5'-TTTN-3') PAM. Cas12a generates a 4-5 nt long 5'-overhang 20 nucleotides away from the T-rich PAM. In some cases, the sticky end generated by Cas12a improves the efficiency of DNA replacement during HR. In some cases, the site-specific endonuclease can include Cas13a (C2c2). Alternatively, in some cases, the site-specific endonuclease can include Cas13b. Cas13 is an RNA-targeting endonuclease that exhibits promiscuous RNA activity as a side effect upon target recognition.

[0076] In some embodiments, the method includes contacting a cell with a system or any component of the system or any combination of components of the system described herein, and the cell includes an immune cell or stem cell. In some aspects, the cell is an immune cell described herein (e.g., a lymphocyte, a B cell, or a T cell). Optionally, the T cell can be a cytotoxic T cell, an alpha-beta T cell, a gamma-delta T cell, a natural killer T cell, a regulatory T cell, or a T helper cell. In some aspects, the immune cell includes an ILC. In some aspects, the stem cell is a hematopoietic stem cell or an iPSC. In some aspects, the iPSC can be derived from an immune cell or a T cell.

[0077] The cells described herein may be isolated from a sample from a donor who is not a subject in need of treatment for a disease or condition described herein. The sample may be a bodily fluid or tissue, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and tumor. Optionally, the sample comprises NK cells, NKT cells, T cells, or T cell precursors. For example, optionally, the sample is a cord blood sample, a peripheral blood sample (e.g., mononuclear cell fraction), or a sample from a subject that comprises pluripotent cells. In some embodiments, the sample from the subject can be cultured to generate induced pluripotent stem (iPS) cells, which are used to produce NK cells, NKT cells, or T cells. The cell sample may be cultured directly from the subject and may be cryopreserved prior to use. In some embodiments, obtaining the cell sample comprises harvesting the cell sample. In other aspects, the sample is obtained from a third party. In still further aspects, a sample from a subject may be processed to purify or enrich T cells or T cell precursors in the sample. For example, the sample may be subjected to gradient purification, cell culture selection and / or cell sorting (e.g., via fluorescence activated cell sorting (FACS)). The cells may be NK cells. The NK cells may be obtained from peripheral blood, umbilical cord blood or other sources described herein. The NK cells may be derived from induced pluripotent stem cells. In some embodiments, the cells that may be used in the methods provided herein may be positive or negative for a given factor. In some aspects, the cells may be prepared from a cell line.

[0078] In some embodiments, the cells provided herein are CD3+ cells, CD3- cells, CD5+ cells, CD5- cells, CD7+ cells, CD7- cells, CD14+ cells, CD14- cells, CD8+ cells, CD8- cells, CD103+ cells, CD103- cells, CD11b+ cells, CD11b- cells, BDCA1+ cells, BDCA1- cells, L-selectin+ cells, L-selectin- cells, CD25+, CD25- cells, CD27+, CD27- cells, CD28+ cells, CD28- cells, CD44 The cell may be a CD4+ cell, a CD44- cell, a CD56+ cell, a CD56- cell, a CD57+ cell, a CD57- cell, a CD62L+ cell, a CD62L- cell, a CD69+ cell, a CD69- cell, a CD45RO+ cell, a CD45RO- cell, a CD127+ cell, a CD127- cell, a CD132+ cell, a CD132- cell, an IL-7+ cell, an IL-7- cell, an IL-15+ cell, an IL-15- cell, a lectin-like receptor G1 positive cell, a lectin-like receptor G1 negative cell, or a differentiated or dedifferentiated cell thereof. The examples of factors expressed by the cells are not intended to be limiting, and one of skill in the art will understand that the cells may be positive or negative for any factor known in the art. In some embodiments, the cells may be positive for more than one factor. For example, the cells may be CD4+ and CD8+.

[0079] In some embodiments, the cells can be negative for two or more factors. For example, the cells can be CD25-, CD44-, and CD69-. In some embodiments, the cells can be positive for one or more factors and negative for one or more factors. For example, the cells can be CD4+ and CD8-. In some embodiments, the cell markers provided herein can be used to select, enrich, or deplete a cell population. In some embodiments, enriching includes selecting a monocyte fraction. In some embodiments, enriching includes sorting a population of immune cells from the monocyte fraction. In some embodiments, the cells can be selected for having or not having one or more given factors (e.g., the cells can be separated based on the presence or absence of one or more factors). In some embodiments, the selected cells can also be transduced and / or expanded in vitro. The selected cells can be expanded in vitro prior to injection. In some embodiments, the selected cells can be transduced with a vector provided herein. It is understood that the cells used in any of the methods disclosed herein can be a mixture of any of the cells disclosed herein (e.g., two or more different cells). For example, the disclosed method may include cells, where the cells are a mixture of CD4+ cells and CD8+ cells. In another example, the disclosed method may include cells, where the cells are a mixture of CD4+ cells and naive cells. Optionally, the cells may be stem memory TCM cells consisting of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and IL-7Ra+, where the stem memory cells may also express CD95, IL-2R13, CXCR3, and LFA-1, and exhibit many functional properties characteristic of memory cells. The cells provided herein may also be central memory TCM cells, including L-selectin and CCR7, where the central memory cells can, for example, secrete IL-2, but cannot secrete IFNγ or IL-4. The cells may also be effector memory TEM cells that contain L-selectin or CCR7 and are capable of producing effector cytokines such as IFNγ and IL-4.

[0080] In some aspects, the cells described herein can be immune cells or stem cells. In some embodiments, the immune cells are lymphocytes, such as T cells, B cells, natural killer (NK) cells, or macrophages. In some aspects, the T cells are cytotoxic T cells, natural killer T cells, regulatory T cells, or T helper cells. In some embodiments, the cells to be modified are immune cells, including innate lymphoid cells (ILCs). In some aspects, the cells modified by the methods described herein are induced pluripotent stem cell (iPSC)-derived immune cells. In some embodiments, the immune cells are iPSC-derived T cells. In some embodiments, the immune cells are iPSC-derived natural killer T cells. In some embodiments, the cells modified by the methods described herein are stem cells. In some aspects, the stem cells can be hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs).

[0081] In some embodiments, the cells described herein comprise a cell surface marker. The cell surface marker can be an immune cell antigen. The genes encoding the immune cell antigen of the immune cells used to prepare the cells described herein can be inactivated. Examples of immune cell antigens include, but are not limited to, CD2, CD3, CD4, CDS, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRa, TCRf3, and SLAMF7. For example, optionally the gene encoding CD7 on the immune cell is inactivated.

[0082] In some embodiments, methods using the system described herein or any component of the system or any combination of the system components increase the knock-in efficiency of the polynucleotides described herein in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the nuclease (e.g., Cas) protein-mediated knock-in efficiency of an equivalent polynucleotide in an equivalent cell population in the absence of a chimera that is circular, has at least one covalently closed circular end, or has at least one genetically modified partial targeting fragment.

[0083] In some embodiments, methods using the system described herein or any component of the system or any combination of the components of the system increase the knock-in efficiency of the polynucleotides described herein in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the Cas protein-mediated knock-in efficiency of the same polynucleotide in the absence of at least one genetically modified partial targeting fragment in a comparable cell population.

[0084] In some embodiments, methods using the system described herein or any component of the system or any combination of the system components increase expression of the polynucleotides described herein in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to nuclease (e.g., Cas) protein-mediated expression of equivalent polynucleotides in an equivalent cell population in the absence of a chimera that is circular, has at least one covalently closed circular end, or has at least one genetically modified partial targeting fragment.

[0085] In some embodiments, methods using the system described herein or any component of the system or any combination of the system components increase expression of the polynucleotides described herein in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to Cas protein-mediated expression of the polynucleotides of the same polynucleotides in the absence of at least one genetically modified partial targeting fragment in a comparable cell population.

[0086] In some embodiments, methods using the system described herein or any component of the system or any combination of the system components increase the viability of the polynucleotides described herein in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the nuclease (e.g., Cas) protein-mediated viability of an equivalent polynucleotide in an equivalent cell population in the absence of a chimera that is circular, has at least one covalently closed circular end, or has at least one genetically modified partial targeting fragment.

[0087] In some embodiments, methods using the system described herein or any component of the system or any combination of the system components increase the viability of the polynucleotides described herein in a cell population by at least 10%, at least 20%, at least 30%, at least 40% or more compared to the Cas protein-mediated viability of the same polynucleotide in the absence of at least one genetically modified partial targeting fragment in a comparable cell population.

[0088] In some cases, the cells described herein may exhibit (i) improved survival by maintaining in vitro viability in the presence of cells xenogeneic to the cells described herein, including, but not limited to, xenogeneic T cells, xenogeneic NK cells, and mixtures of xenogeneic T cells and xenogeneic NK cells, (ii) improved expansion, or (iii) improved cytotoxicity against target cells comprising an antigen, as compared to additional engineered immune cells comprising one or more CARs in which the TCR, MHC molecule, and / or immune cell antigen has not been inactivated. In some cases, the cells described herein may be characterized by exhibiting two or more of (i) improved survival by maintaining in vitro viability in the presence of cells xenogeneic to the cells described herein, including, but not limited to, xenogeneic T cells, xenogeneic NK cells, and mixtures of xenogeneic T cells and xenogeneic NK cells, (ii) improved expansion, or (iii) improved cytotoxicity.

[0089] In some embodiments, the cells described herein may also include an enhancer portion capable of enhancing one or more activities of the cells described herein. The enhancer portion may be configured to constitutively upregulate one or more intracellular signaling pathways of the cells described herein. The one or more intracellular signaling pathways may be one or more cytokine signaling pathways. The enhancer portion may be autoactivating by self-oligomerization. The enhancer portion may be autoactivating by self-dimerization. The enhancer portion may be a cytokine or a cytokine receptor. The enhancer portion may be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, a receptor thereof, a functional fragment thereof, a functional variant thereof, and a combination thereof.

[0090] In some aspects, the cells described herein further comprise an inducible cell death moiety, which upon contact with a cell death activator can cause the cells described herein to commit suicide. The inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ACD20, mTMPK, ACD19, RQR8, and EGFRt. Optionally, the inducible cell death moiety is EGFRt and the cell death activator is an antibody or antigen-binding fragment thereof that binds to EGFRt. Optionally, the inducible cell death moiety is HSV-TK and the cell death activator is GCV. Optionally, the inducible cell death moiety is iCasp9 and the cell death activator is AP1903. The cell death activator can include a nucleic acid, a polynucleotide, an amino acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof.

[0091] In some aspects, the cells described herein provided herein may comprise a chimeric polypeptide comprising (i) an enhancer portion capable of enhancing one or more activities of the cells described herein, and (ii) an inducible cell death portion capable of causing the death of the cells described herein when the chimeric polypeptide and a cell death activator contact, the enhancer portion being linked to the inducible cell death portion. Optionally, the enhancer portion and the inducible portion may be linked by a linker. The linker may be a cleavable linker, for example, a self-cleaving peptide.

[0092] In some embodiments, the cells described herein may further comprise at least one heterologous polypeptide comprising at least one heterologous receptor. In some aspects, the heterologous receptor is a chimeric polypeptide receptor (CPR) comprising a binding moiety, the binding moiety comprising (i) a first antigen binding domain that, when administered to a subject, suppresses or reduces the subject's immune response to the cells described herein, and (ii) a second antigen binding domain capable of binding to a disease-associated antigen. An individual CPR of the one or more CPRs may comprise (i) a first antigen binding domain, (ii) a second antigen binding domain, or (iii) both the first antigen binding domain and the second antigen binding domain. A CPR of the one or more CPRs may further comprise a transmembrane domain and an intracellular signaling region. Optionally, one or more CPRs in the cells described herein are one or more chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs). Optionally, the cells described herein comprise both a CAR and an engineered TCR. In some embodiments, the at least one heterologous receptor comprises at least one chimeric antigen receptor (CAR), and each CAR of the at least one CAR comprises a hinge, a transmembrane domain, a costimulatory signaling region, and an intracellular signaling region.

[0093] The engineered TCR may be a TCR fusion protein. For example, the TCR fusion protein may comprise a heterologous antigen-binding domain fused to one or more subunits of a TCR complex. Optionally, the TCR fusion protein may comprise a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain, and an antibody domain comprising an antigen-binding domain, wherein the TCR subunit and the antibody domain are associated. The TCR fusion protein may be incorporated into a TCR complex upon expression in a T cell. Optionally, the TCR fusion protein may further comprise a TCR transmembrane domain. The TCR extracellular domain, the TCR intracellular domain or the TCR transmembrane domain may be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, CD3 epsilon, CD3 gamma, CD3 delta or CD3 zeta. Optionally, the endogenous TCR of the cell described herein comprising the engineered TCR is inactivated.

[0094] In some cases, the cell described herein that comprises inactivated endogenous TCR may not cause GVHD.For example, the gene that codes for endogenous TCR subunits can be inactivated.In another example, the gene that codes for endogenous TCR subunits can be mutated so that endogenous TCR cannot be formed.

[0095] CARs may include an extracellular antigen recognition region, such as an scFv (single chain variable fragment), a transmembrane region, and an intracellular costimulatory signal region. The extracellular domain of CARs can recognize a specific antigen and then transmit a signal via the intracellular domain to cause T cell activation and proliferation, cytolytic toxicity, and secretion of cytokines, thereby eliminating the target cell. The patient's autologous T cells (or a xenogeneic donor) can be first isolated, activated, and genetically engineered to produce CAR-T cells, which can then be infused back into the same patient. In this way, the possibility of graft-versus-host disease can be reduced, and antigens can be recognized by T cells in a non-MHC-restricted manner. Furthermore, CAR-T can treat any cancer that expresses the antigen.

[0096] In some aspects, the cells described herein can target both disease-associated antigens (e.g., tumor-associated antigens or tumor cell markers) and immune cell antigens (e.g., CD3, CD7, or CD137) via bispecific or multivalent CARs. For example, the present disclosure provides engineered immune cells capable of targeting tumor cell markers and immune cell antigens such as CD3. Endogenous TCRs can be inactivated (e.g., destroyed, inhibited, knocked out, or silenced). The CAR-T of the present disclosure targeting tumor cell markers and immune cell antigens can avoid host rejection (HVG) by eliminating positive tumor cells and depleting host immune cell antigen positive T cells and NK cells. In the present disclosure, the endogenous TCR of the cells described herein can be knocked out, which can prevent graft-versus-host disease (GVHD), thereby preparing universal or universal CAR-T (UCAR-T) cells. The cells described herein can be derived from autologous or allogeneic T cells. Additionally, the cells described herein may include a cell suicide element (e.g., an inducible cell death moiety), and the CAR-T may be inactivated / removed at any time to reduce side effects. Optionally, the cells described herein may further include an enhancer moiety. The enhancer moiety may modulate one or more activities of the cells described herein when the cells described herein are administered to a subject. For example, the enhancer moiety may be a cytokine (e.g., IL-5 or IL-7) or a cytokine receptor (e.g., IL-5R or IL-7R). The enhancer moiety may enhance a signaling pathway in the cells described herein, such as the STAT5 signaling pathway. In some embodiments, the cells described herein include a bispecific CAR that targets both CD19 and CD3. The cells described herein shown in this example may further include an inducible cell death moiety, such as a truncated epidermal growth factor receptor (EGFRt or tEGFR, which may be used interchangeably herein). The inducible cell death moiety or the enhancer moiety may be introduced into the immune cell via a separate expression vector.Optionally, the inducible cell death portion and the enhancer portion can be introduced into the immune cell via an expression vector that contains sequences encoding both portions. Optionally, the inducible cell death portion and the enhancer portion are combined and expressed as a chimeric polypeptide. The application of the cells described herein can be used in cell therapy to treat a disease or condition of interest (e.g., cancer), and can be prepared in large scale in advance to avoid GVHD and HvG, reduce treatment costs, inactivate CAR-T at any time if necessary, reduce side effects of immunotherapy, and ensure product safety.

[0097] Treatment method In some embodiments, disclosed herein are methods of using the systems described herein, the polynucleotides described herein, or the modified cells described herein. In some embodiments, the methods comprise administering to the subject a system described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein. In some embodiments, administration can be by any suitable method of administration, for example, systemic administration (e.g., intravenous, inhalation, etc.). In some embodiments, the subject is a human.

[0098] In some embodiments, the system described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein are administered at least once within a given period of time (e.g., once every other day, twice a week, once a week, every week, three times a month, twice a month, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, once a year). In some embodiments, the composition is administered more than once (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times) within a given period of time.

[0099] In some embodiments, the systems described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein are administered in therapeutically effective amounts via a variety of forms and routes, including, for example, oral or topical administration. In some embodiments, the compositions may be administered, e.g., injected or infused, parenterally, intravenously, subcutaneously, intramuscularly, intradermally, intraperitoneally, intracerebrally, intrathecally, intraocularly, intrasternally, ophthalmic, endothelial, topically, intranasally, intrapulmonary, intrarectally, intraarterially, intrathecally, inhaled, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, subarachnoid, or intraspinal. In some embodiments, the compositions may be administered by absorption through epithelial or mucosal linings (e.g., oral mucosal, rectal, intestinal mucosal administration). In some embodiments, the compositions are delivered via multiple routes of administration.

[0100] In some embodiments, the system described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein are administered by intravenous infusion. In some embodiments, the composition is administered by slow continuous infusion for an extended period of time, for example, greater than 24 hours. In some embodiments, the composition is administered as an intravenous injection or short-term infusion.

[0101] The systems described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein may be administered locally, for example, by injecting the agent directly into an organ, optionally in a depot or sustained release formulation or implant. The compositions may be provided in the form of a fast release formulation, a sustained release formulation, or an intermediate release formulation. The fast release form may provide an immediate release. The sustained release formulation may provide a controlled release or a sustained delayed release. In some embodiments, a pump may be used to deliver the composition. In some embodiments, a pen delivery device may be used, for example, for subcutaneous delivery of the compositions of the present disclosure.

[0102] The systems described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein may be administered in combination with other therapies, such as antiviral therapy, chemotherapy, antibiotics, cell therapy, cytokine therapy, or anti-inflammatory agents. In some embodiments, the cyclic polyribonucleotides or antibodies or antigen-binding fragments thereof described herein may be used alone or in combination with one or more therapeutic agents as components of a mixture. In some embodiments, the linear polyribonucleotides or antibodies or antigen-binding fragments thereof described herein may be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0103] The systems described herein, polynucleotides described herein, modified cells described herein, or pharmaceutical compositions described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of the composition comprising a therapeutic agent can vary. In some cases, the cells or pharmaceutical compositions can be used as prophylactics and can be administered continuously to subjects (e.g., vaccination subjects or treatment subjects) susceptible to infection by a pathogen or prone to a condition or disease associated with a pathogen. Prophylactic administration can reduce the likelihood of an infection, disease, or condition occurring, or can reduce the severity of an infection, disease, or condition.

[0104] The system described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein may be administered to a subject before symptoms appear. The composition may be administered to a subject (e.g., a vaccinated subject or a treated subject) after (e.g., as soon as possible thereafter) a test result, such as a test result providing a diagnosis, a test indicating the presence of coronavirus in the subject (e.g., a vaccinated subject or a treated subject) or a test indicating the progression of a condition, such as a decrease in blood oxygen levels. The therapeutic agent may be administered after (e.g., as soon as possible thereafter) the onset of a disease or condition is detected or suspected. The therapeutic agent may be administered after potential exposure to coronavirus, such as after (e.g., as soon as possible thereafter) the subject (e.g., a vaccinated subject or a treated subject) comes into contact with an infected subject or learns that they have come into contact with an infected subject that may be contagious by contact.

[0105] The actual dosage levels of the agents of the present disclosure (e.g., a system described herein, a polynucleotide described herein, a modified cell described herein, or a pharmaceutical composition described herein) may be varied to obtain an amount of agent sufficient to achieve the desired therapeutic response for a particular subject, composition, or method of administration without toxicity to the subject (e.g., a subject to be vaccinated or treated). The dosage level selected may vary depending on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention being used, the route of administration, the timing of administration, the rate of excretion, the duration of treatment, other agents, compounds and / or materials used in combination with the particular composition being used, the age, sex, weight, condition, overall health and past medical history of the patient being treated, and similar factors well known in the medical arts.

[0106] Dosage regimens may be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased, as indicated based on the exigencies of the treatment situation. It is particularly advantageous to formulate parenteral compositions in unit dosage forms for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to a physically discrete unit suitable as a unitary administration to a subject (e.g., a subject for vaccination or a subject for treatment), each unit containing a predetermined amount of active agent calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of the unit dosage forms of the present disclosure are determined by, and may depend directly on, (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active agents to treat individual susceptibility. The dose may be determined by reference to the plasma or local concentration of the cyclic polyribonucleotide or the antibody or antigen-binding fragment thereof. Dosage can be determined by reference to plasma or local concentrations of the linear polyribonucleotide or the antibody or antigen-binding fragment thereof.

[0107] The system described herein, the polynucleotides described herein, the modified cells described herein, or the pharmaceutical compositions described herein may be in the form of a unit dosage form suitable for single administration of a precise dosage amount. In the unit dosage form, the formulation may be divided into unit doses containing an appropriate amount of the composition. In the unit dosage form, the formulation may be divided into unit doses containing an appropriate amount of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosage form may be in the form of a package containing a discrete amount of the formulation. Non-limiting examples are packaged injection solutions, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-resealable containers. Resealable containers for multiple doses may be used, for example, with or without a preservative. The injectable formulations disclosed herein may be in unit dosage form, for example, ampoules or multi-dose containers containing a preservative.

[0108] In some embodiments, the dose may be based on the number of cells per kilogram of subject body weight. In some embodiments, the dose may be administered at a dosage of about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight. About 1,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose is from about 1,000 cells / kg body weight to about 1000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 10,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 100,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 1,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 10,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 100,000,000 cells / kg body weight, from about 1,000 cells / kg body weight to about 100,000,000 cells / kg body weight, 00 cells / kg body weight ~ approx. 1,000,000,000 cells / kg body weight, approx. 1,000 cells / kg body weight ~ approx. 10,000,000,000 cells / kg body weight, approx. 1,000 cells / kg body weight ~ approx. 100,000,000,000 cells / kg body weight, Approx. 1,000 cells / kg body weight ~ approx. 1,000,000,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 100,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 1,000,000 cells / kg body weight, approx. 10 ,000 cells / kg body weight ~ approx. 10,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 100,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 1,000,000,000 cells / kg body weight, approx. 10, 000 cells / kg body weight ~ approx. 10,000,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 100,000,000,000 cells / kg body weight, approx. 10,000 cells / kg body weight ~ approx. 1,000,000,000,000 cells cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 10,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 100,000,000 cells / kg body weight kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 100,000,000,000 cells / kg body weight, approximately 100,000 cells / kg body weight ~ approximately 1,000,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 10,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 100,00 0,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 1,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 10,000,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 100,000 ,000,000 cells / kg body weight, approximately 1,000,000 cells / kg body weight ~ approximately 1,000,000,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight ~ approximately 100,000,000 cells / kg body weight, approximately 10,000,000 cells / kg body weight ~ approximately 1 ,000,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight~about 10,000,000,000 cells / kg body weight,about 10,000,000 cells / kg body weight~about 100,000,000,000 cells / kg body weight,about 10,000,000 cells / k g body weight ~ approx. 1,000,000,000,000 cells / kg body weight, approx. 100,000,000 cells / kg body weight ~ approx. 1,000,000,000 cells / kg body weight, approx. 100,000,000 cells / kg body weight ~ approx. 10,000,000,000 cells / kg body weight, approx. 000,000 cells / kg body weight ~ approx. 100,000,000,000 cells / kg body weight, approx. 100,000,000 cells / kg body weight ~ approx. 1,000,000,000,000 cells / kg body weight, approx. 00 cells / kg body weight, about 1,000,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight to about 100,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight to about 1,000,000,000,000 cells / kg body weight or about 100,000,000,000 cells / kg body weight to about 1,000,000,000 cells / kg body weightIn some embodiments, the dose may be administered at a dosage of about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight, about 1,000 cells / kg body weight, about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, about 100,000,000,000 cells / kg body weight, or about 1,000,000,000,000 cells / kg body weight. In some embodiments, the dose may be administered in a dosage of about 1000 cells / kg body weight to about 1000000000000 cells / kg body weight, at least about 1,000 cells / kg body weight, about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight or about 100,000,000,000 cells / kg body weight. In some embodiments, doses may be administered in dosages of about 1,000 cells / kg body weight to about 1000,000,000 cells / kg body weight, up to about 10,000 cells / kg body weight, about 100,000 cells / kg body weight, about 1,000,000 cells / kg body weight, about 10,000,000 cells / kg body weight, about 100,000,000 cells / kg body weight, about 1,000,000,000 cells / kg body weight, about 10,000,000,000 cells / kg body weight, about 100,000,000,000 cells / kg body weight or about 1,000,000,000,000 cells / kg body weight. In some embodiments, the anucleated cells are administered to the subject twice within at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, or 4 years.

[0109] In some embodiments, the methods described herein treat a disease or condition in a subject, the methods comprising administering a cell or pharmaceutical composition described herein without inducing an innate immune response or GVHD in the subject being treated. In some individuals, the cells or pharmaceutical composition treat a disease or condition in a subject in need of treatment, the disease or condition being cancer. Non-limiting examples of cancer include acute lymphoblastic leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenal carcinoma, adrenocortical carcinoma, adult leukemia, AIDS-related lymphoma, amyloidosis, anal cancer, astrocytoma, ataxia telangiectasia, atypical mole syndrome, atypical teratoma / rhabdomyosarcoma, basal cell carcinoma, cholangiocarcinoma, Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor ( gastrointestinal tract), carcinoma of unknown primary site, cardiac (heart) tumor, cervical cancer, bile duct cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, skin cell lymphoma, ductal carcinoma, germinoma, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, fibrous histiocytoma of bone, malignant tumors and osteosarcoma, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal Stromal tumors (GIST), germ cell carcinoma, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, HER2 positive breast cancer, histiocytosis, Langerhans cell, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, juvenile polyposis syndrome, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia lip and oral cancer, liver cancer, lobular carcinoma, lung cancer (non-small cell and small cell), lymphoma, malignant fibrous histiocytoma and osteosarcoma of bone, malignant glioma, melanoma, intraocular Melanoma, meningioma, Merkel cell carcinoma, mesothelioma, malignant tumor, metastatic cancer, metastatic squamous cell cervical cancer of unknown primary, midline duct carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma, plasmacytoma, mycosis fungoides, myelodysplastic syndrome (MDS), myeloproliferative neoplasms, chronic, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma neuroendocrine tumor, non-Hodgkin's lymphoma, oral cavity cancer, lip and oral cavity cancer and oropharyngeal cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic neuroendocrine tumor, papillomatosis,Paraganglioma, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Peritoneal cancer, Peutz-Jeghers syndrome, Pheochromocytoma, Pituitary tumor, Plasmacytoma / multiple myeloma, Pleuropulmonary blastoma, Polycythemia vera, Pregnancy and breast cancer, Primary central nervous system (CNS) lymphoma, Primary peritoneal cancer, Prostate cancer, Rectal cancer, Recurrent cancer, Renal cell carcinoma, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sezary syndrome, Skin Skin cancer, small intestine cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma of the skin, squamous cell cervical carcinoma of unknown primary, metastasis, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, rare childhood cancer, ureter and renal pelvis, transitional cell carcinoma, urethral cancer, uterine (endometrial) cancer, uterine sarcoma, vaginal cancer, vascular tumors, vulvar cancer, Wilms' tumor, or a combination thereof.

[0110] In some embodiments, the cells described herein (e.g., modified cells expressing at least one transgene described herein) can target cancer or tumor cells to treat a disease or condition in a subject. Non-limiting examples of cancer or tumor cells for use in the present disclosure include acanthoma, acinic cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrohidrosis, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, amelomatosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK cell leukemia, AIDS-related cancer, AIDS-related lymphoma, cystic leukemia ... soft part sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendix cancer, astrocytoma, atypical teratoid rhabdomyoid tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brain stem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer , carcinoma of unknown primary site, carcinosarcoma, Castleman's disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, bile duct carcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Dego's disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, dysembryoplastic neuroepithelial tumor, embryonal Carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, nasal neuroblastoma, Ewing's tumor family, Ewing's sarcoma family, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, extramammary Paget's disease, fallopian tube cancer, inclusion malformation fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglioglioma, gangliocytoma, gastric cancer, gastric lymphoma, gastrointestinal cancer,Gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell cancer, germ cell tumor, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, cerebral glioma, glomus tumor, glucagon-producing tumor, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, cardiac cancer, hemangioblastoma, hemangiopericytoma, hemangioendothelioma, hematologic malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell cystic cyst, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma kidney cancer, Krukkin's tumor, Krukenberg's tumor, laryngeal cancer, laryngeal cancer, lentigo maligna melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteinoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdomyoid tumor, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell cervical carcinoma of unknown primary, metastatic urothelial carcinoma, mixed mullerian tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder, myxoma, nasal cancer, nasopharyngeal carcinoma, nasopharyngeal carcinoma, neoplasia, neurofibromatosis, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, oligoastrocytoma, oligodendroglioma, eosinophilic granular cell tumor, optic nerve sheath meningioma, oral cavity cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasmacytoma,Pleuropulmonary blastoma, polyembryoma, precursor T-lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver carcinoma, primary peritoneal carcinoma, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, airway cancer involving the NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation , sacrococcygeal teratoma, salivary gland carcinoma, sarcoma, schwannoma, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord stromal tumor, Sezary syndrome, solid tumor, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatin-producing tumor, sooty wart, spinal cord tumor, spinal cord The cancer cells may include medullary tumors, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, superficial epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratocarcinoma end stage lymphoma, testicular cancer, theca cell tumor, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal cancer, urethral cancer, genitourinary tumors, uterine sarcoma, uveal melanoma, vaginal cancer, Verner-Morrison syndrome, verrucous carcinoma, optic tract glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof. In some embodiments, the target cancer cells represent a subpopulation within the cancer cell population, such as cancer stem cells. In some embodiments, the cancer is a hematopoietic cancer, e.g., lymphoma.

[0111] Pharmaceutical Compositions Pharmaceutical compositions comprising a therapeutic agent are described herein (e.g., a system described herein, a polynucleotide described herein, or a modified cell described herein). In some aspects, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier, excipient, or diluent. In some aspects, the pharmaceutical compositions described herein comprise at least one additional active agent other than the cells described herein. In some aspects, the at least one additional active agent is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an anti-hormonal agent, an anti-angiogenic agent, or a checkpoint inhibitor.

[0112] In practicing the methods of treatment or use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease, disorder or condition to be treated, such as cancer. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors. The therapeutic agents and optionally compositions described herein can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0113] The pharmaceutical compositions described herein may be administered to a subject by any suitable route of administration, including, but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal. The compositions described herein may include, but are not limited to, aqueous dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed formulations of immediate release and controlled release formulations.

[0114] Pharmaceutical compositions containing a therapeutic agent may be manufactured in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes.

[0115] The pharmaceutical composition may include at least one exogenous therapeutic agent as an active ingredient in free acid or free base form, or in pharma- ceutically acceptable salt form. Additionally, the methods and compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, and active metabolites of these compounds with the same type of activity. In some embodiments, the therapeutic agent is present in a nonsolvated form or a solvated form with a pharma- ceutically acceptable solvent, such as water, ethanol, etc. The solvated forms of the therapeutic agent are also considered to be disclosed in this disclosure.

[0116] In certain embodiments, the pharmaceutical compositions provided herein include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merphen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0117] In some embodiments, the pharmaceutical compositions described herein benefit from antioxidants, metal chelators, thiol-containing compounds, and other common stabilizers. Examples of such stabilizers include: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, about 0.01% to about 2% w / v ascorbic acid, (f) about 0.003% to about 0.02% w / v polysaccharides, (g) about 0.01% to about 0.02% w / v polysaccharides, (h) about 0.01% to about 0.02% w / v polysaccharides, (i) about 0.01% to about 0.02% w / v polysaccharides, (j) about 0.01% to about 0.02% w / v polysaccharides, (k) about 0.01% to about 0.02% w / v polysaccharides, (l ... (g) about 0.001% w / v to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0118] The pharmaceutical compositions described herein may be formulated into any suitable dosage form, including, but not limited to, oral dispersions, solutions, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and combinations of immediate release and controlled release formulations. In one aspect, the therapeutic agents described herein, for example, therapeutic agents, may be formulated into pharmaceutical compositions suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (olive oil, etc.), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispensing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0119] For intravenous injection or infusion or infusion, the pharmaceutical compositions described herein are formulated in aqueous solutions, preferably physiologically compatible buffers, such as Hanks' solution, Ringer's solution or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably containing physiologically compatible buffers or excipients.

[0120] Parenteral injection may involve bolus injection or continuous infusion. Injectable pharmaceutical compositions may be provided in unit dosage form, for example in ampoules or multi-dose containers with added preservatives. The pharmaceutical compositions described herein may be in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0121] When administered by inhalation, the therapeutic agent is formulated for use as an aerosol, mist or powder. The pharmaceutical composition described herein is conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. By way of example only, capsules and cartridges of gelatin or the like for use in an inhaler or insufflator can be formulated to contain a powder mix of the therapeutic agent described herein and a suitable powder base, such as lactose or starch. The formulation containing the composition is prepared as a solution in saline with benzyl alcohol or other suitable preservatives, fluorocarbons and / or other solubilizing or dispersing agents known in the art. Preferably, these compositions and formulations are prepared using suitable non-toxic pharmaceutically acceptable ingredients. The selection of suitable carriers depends on the exact nature of the nasal dosage form (e.g., solution, suspension, ointment or gel) desired. Nasal dosage forms generally contain a large amount of water in addition to the active ingredient. Small amounts of other ingredients, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents or buffers, and other stabilizers and solubilizers, are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0122] Oral medicaments are obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally grinding the resulting mixture, adding suitable auxiliaries as necessary, and then processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients include, for example, sugars including lactose, sucrose, mannitol or sorbitol, fillers such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose sodium, and other cellulose preparations, such as polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If necessary, disintegrants such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, are added. In some embodiments, dyes or pigments are added to the tablet or dragee coating to identify or characterize various combinations of doses of active therapeutic agents.

[0123] In some aspects, the pharmaceutical composition of the exogenous therapeutic agent is in capsule form, including push-fit capsules made of gelatin and soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules contain a filler such as lactose mixed with the active ingredient, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In the soft capsule, the active therapeutic agent is dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some aspects, stabilizers are added. The capsule can be prepared, for example, by placing a bulk blend of the therapeutic agent formulation inside the capsule. In some aspects, the formulation (non-aqueous suspensions and solutions) is placed in a soft gelatin capsule. In other embodiments, the formulation is placed in a standard gelatin capsule or a non-gelatin capsule such as a capsule containing HPMC. In other embodiments, the formulation is placed in a sprinkle capsule and the capsule is swallowed whole, or the capsule is opened and its contents are sprinkled on food before eating.

[0124] Pharmaceutical compositions for oral administration may be in dosages suitable for such administration. In one aspect, solid oral dosage forms are prepared by mixing the composition with one or more of the following: antioxidants, flavoring agents, and carrier substances, such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some aspects, the solid dosage forms disclosed herein are in the form of tablets (including suspension tablets, fast dissolving tablets, bite-disintegrating tablets, fast disintegrating tablets, effervescent tablets, or caplets), pills, powders, capsules, solid dispersions, solid solutions, biodegradable dosage forms, controlled release formulations, pulsatile release formulations, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the composition is in the form of a powder. Compressed tablets are solid dosage forms prepared by compressing bulk blends of the above formulations. In various embodiments, the tablets include one or more flavoring agents. In other embodiments, the tablets include a film that surrounds the final compressed tablet. In some aspects, the film coating can provide a delayed release of the therapeutic agent from the formulation. In other embodiments, the film coating aids in patient compliance. The film coating typically ranges from about 1% to about 3% of the tablet weight. In some aspects, solid dosage forms, such as tablets, effervescent tablets, and capsules, are prepared by mixing particles of the therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. This bulk blend is easily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some aspects, the individual unit dosage forms include a film coating. These formulations are manufactured by conventional formulation techniques.

[0125] In another embodiment, the dosage form comprises a microencapsulated formulation.In some embodiments, one or more other compatible materials are present in the microencapsulated material.Non-limiting examples of materials include pH adjusters, erosion promoters, antifoaming agents, antioxidants, flavoring agents, and carrier materials, such as binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0126] The liquid formulation dosage form for oral administration is optionally an aqueous suspension selected from the group including, but not limited to, pharma- ceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form optionally includes additives such as (a) a disintegrant, (b) a dispersant, (c) a wetting agent, (d) at least one preservative, (e) a thickening agent, (f) at least one sweetener, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersion further includes a crystal formation inhibitor.

[0127] In some aspects, the pharmaceutical compositions described herein may be self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are generated by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to excess water without the need for any external mechanical dispersion or agitation. The advantage of SEDDS is that droplets can be distributed throughout the solution with only gentle mixing. Furthermore, water or aqueous phase is optionally added just prior to administration, thereby ensuring stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some aspects, SEDDS improves the bioavailability of hydrophobic active ingredients.

[0128] Buccal formulations are administered using various formulations known in the art. In addition, the buccal dosage forms described herein may further comprise a biodegradable (hydrolyzable) polymeric carrier that also serves to attach the dosage form to the buccal mucosa. For buccal or sublingual administration, the composition may take the form of a tablet, lozenge or gel that is formulated in a conventional manner.

[0129] For intravenous injection, the pharmaceutical composition is optionally formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution or physiological saline buffer. For transmucosal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably containing physiologically compatible buffers or excipients.

[0130] Parenteral injections optionally involve bolus injection or continuous infusion. Injectable preparations are optionally provided in unit dosage form, for example in ampoules or multi-dose containers with added preservatives. In some aspects, the compositions described herein are in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle, and include formulation agents such as suspending agents, stabilizing agents and / or dispersing agents. Compositions for parenteral administration include aqueous solutions of agents that modulate the activity of the carotid body in water-soluble form. Additionally, suspensions of agents that modulate the activity of the carotid body are optionally prepared as needed (e.g., suspensions in oily injections).

[0131] In some embodiments, a pharmaceutical composition may be provided that includes particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a patient. The formulation may be a powder and / or granules for suspension, which upon mixing with water results in a substantially uniform suspension.

[0132] In addition, the pharmaceutical compositions optionally contain one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride, etc. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0133] In addition, the pharmaceutical composition optionally contains one or more salts in an amount necessary to bring the osmolality of the pharmaceutical composition into an acceptable range, including those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0134] In one embodiment, the aqueous suspensions and dispersions described herein remain homogenous for at least 4 hours. In one embodiment, the aqueous suspension is resuspended into a homogenous suspension by physical agitation lasting less than 1 minute. In yet another embodiment, no agitation is required to maintain a homogenous aqueous dispersion.

[0135] kit In some aspects, described herein are kits that use the systems described herein, polynucleotides described herein, or modified cells described herein to practice the methods described herein. In some aspects, the kits disclosed herein may be used to treat a disease or condition of a subject. In some aspects, the kits include a combination of materials or components apart from the cells.

[0136] In some aspects, the kits include components for assaying the number of units of a biomolecule (e.g., a therapeutic agent) synthesized on a surface and / or released or expressed by the cells described herein. In some aspects, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR and qPCR. The exact nature of the components configured in the kit will vary depending on its intended purpose. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein (e.g., cancer) in a subject. In some aspects, the kits are specifically configured for treating a mammalian subject. In some aspects, the kits are specifically configured for treating a human subject.

[0137] The kit may include instructions for use. In some embodiments, the kit includes instructions for administering the cells to a subject in need thereof. In some embodiments, the kit includes instructions for further manipulating the composition to express a biomolecule (e.g., a therapeutic agent). In some embodiments, the kit includes instructions for thawing or otherwise restoring biological activity of cells that may have been preserved during storage or transport. In some embodiments, the kit includes instructions for measuring the viability of preserved cells to ensure efficacy for their intended purpose (e.g., therapeutic efficacy when used to treat a subject).

[0138] Optionally, the kit also includes other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, dispensing or metering devices, dressings, or other useful paraphernalia. The materials or components assembled in the kit may be stored and provided to the practitioner in any convenient and suitable manner that preserves their operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form, and they may be provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging materials.

[0139] The use of absolute or sequential terms such as "shall", "will not", "shall", "shall not", "must", "must not", "first", "firstly", "next", "then", "before", "after", "lastly" and "finally" are not intended to limit the scope of the embodiments disclosed herein but are exemplary.

[0140] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "including," "having," "having," "comprising," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in a similar manner as the term "comprising."

[0141] As used herein, the terms "at least one," "one or more," and "and / or" are open-ended expressions that operate both conjunctively and disjunctively. For example, each of the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and C together.

[0142] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a particular meaning.

[0143] Any systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of action.

[0144] The term "about" when referring to a number or range of values ​​means that the number or range of values ​​referred to is approximate within experimental variability (or within statistical experimental error), and that the number or range of values ​​may vary, for example, by 1% to 15% of the stated number or range of values. For example, the term "about" refers to ±10% of the stated number or value.

[0145] The terms "increased," "increasing," or "increase" are used herein to generally mean an increase by a statistically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to 100%, or any increase between 10-100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0146] The terms "reduced," "reducing," or "reducing" are used herein to generally mean a statistically significant amount of reduction. In some embodiments, "reduced" or "reducing" means a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a decrease of 100% or less (e.g., no or undetectable levels compared to a reference level), or any decrease between 10% and 100% compared to a reference level. In the context of a marker or condition, these terms mean a statistically significant reduction in such levels. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and preferably down to a level that is accepted as within the normal range for an individual without a given disease.

[0147] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, modifications and alternatives will occur to those skilled in the art without departing from the present invention. It is further understood that all aspects of the present invention are not limited to the specific depictions, configurations or relative proportions set forth herein, which depend upon a variety of conditions and variables. It is understood that various variations of the embodiments of the present invention described herein can be used in practicing the present invention. It is therefore contemplated that the present invention encompasses such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby. EXAMPLES

[0148] The following illustrative examples are representative of embodiments of the stimulation systems and methods described herein, but are not intended to be limiting in any way.

[0149] Example 1. Cellular modification To knock in the chimeric receptor or MHC protein, the cells described herein were contacted with any of the components of the system or any combination of the components of the system described herein. Modified cells expressing the chimeric receptor or MHC protein were examined and illustrated in Figures 1-14. For knock-in or knock-out experiments, the cells to be modified were first activated on day 0. The knock-in or knock-out experiment was performed on day 2. Flow cytometry was performed on day 10 to determine the efficiency of the knock-in or knock-out experiment. Table 1 shows the ratio between the RNPs of the system described herein and various other components for modifying cells. Table 2 shows an exemplary amount of polynucleotides contacted with cells to generate the data in Figures 1-14. Table 3 shows exemplary materials for practicing the methods described herein. Table 4 shows an exemplary amount of polynucleotides contacted with 10 million cells to be modified. Table 5 shows an exemplary electroporation protocol for the Lonza 4D-Nucleofector protocol. Table 6 shows an exemplary maxcyte program.

[0150] By using CRISPR / Cas9 to knock-in the polynucleotides described herein (via homology directed repair (HDR)), the percentage of knock-in (KI) single CAR gene was about 30%-50% based on the data of knock-in at the TRAC and B2M loci. Simultaneous knock-in of two polynucleotides (e.g., TRAC and B2M loci) was more than 20% effective. The percentage of knock-in of NY-ESO-1 specific TCR at the TRAC locus via the use of polynucleotides comprising double-stranded DNA with at least one genetically modified partial targeting fragment was 34.4%. Using a polynucleotide comprising double-stranded DNA with at least one genetically modified partial targeting fragment, the knock-in efficiency of the CD19-specific CAR gene at the TRAC locus was 31.4%, the knock-in efficiency of the humanized CD19-specific CAR gene at the TRAC locus was 33.4%, and the knock-in efficiency of the mesothelin-specific CAR gene at the TRAC locus was 33.8%. The knock-in efficiency of GC020 at the TRAC locus was 41.1%. The knock-in efficiency of GC012 at the TRAC locus was 22.5%. The knock-in efficiency of HLA-E at the B2M locus using a polynucleotide comprising double-stranded DNA with at least one genetically modified partial targeting fragment was 37.7%, and the efficiency was up to 54.9% when using a polynucleotide comprising double-stranded DNA with at least one genetically modified partial targeting fragment and a telomeric end. The knock-in efficiency of GC012HL at the TRAC locus using a chimeric polynucleotide comprising a double-stranded DNA template with at least one gene-modified partial targeting fragment and with a telomeric end was 34.6%. The knock-in efficiency of GC012HL at the TRAC locus and HLA-E at the B2M locus was 21.7% and 50.1% for GC012HL. The knock-in efficiency of GC012HL at the TRAC locus and HLA-E at the B2M locus was 31.8%.The polynucleotides knocked in by the systems and methods described herein can be large DNA templates (e.g., greater than 5,000 nucleotide base pairs).

[0151] [Table 1]

[0152] [Table 2]

[0153] [Table 3]

[0154] [Table 4]

[0155] [Table 5]

[0156] [Table 6]

[0157] Example 2. Knock-in of NY-ESO-1-targeting TCR at the TRAC locus To detect the knock-in efficiency of NY-ESO-1 specific TCR at the TRAC locus, T and TRAC knockout T cells were used as negative controls. "Column-derived template" refers to the polynucleotide obtained from column purification. "Bead-derived template" refers to the polynucleotide obtained from magnetic bead purification. For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.25 μg, 0.50 μg, 0.70 μg or 1.00 μg of polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of polynucleotide were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). Flow cytometry analysis showed that CD3 was knocked out in 94.9% of cells, and 0.18% of cells were positive for both TCR Vβ13.1 and CD3.

[0158] As shown in Figure 1, knock-in efficiency was detected by flow cytometry at specific days after electroporation, and the CD3 knock-out efficiency was 94.9%, while the percentage of TCR Vβ13.1 and CD3 double positive was 0.18% of the TRAC knock-out control. The highest knock-in efficiency of NY-ESO-1 specific TCR using 1 μg of chimeric polynucleotide was 34.4%.

[0159] Example 3. Knock-in of CD19-targeted CAR at the TRAC locus The knock-in was mediated by a polynucleotide containing a modified partial targeting fragment at both the 5' and 3' ends as described herein. The polynucleotide was not covalently closed at both the 5' and 3' ends of the polynucleotide. The knock-in resulted in 31.4% of the cell population expressing a CD19-targeted CAR at the TRAC locus (CD-19-CAR), 33.4% of the cell population expressing a humanized CD19-targeted CAR at the TRAC locus (HCD-19-CAR), and 33.8% of the cell population expressing a mesothelin-targeted CAR at the TRAC locus (Meso-CAR-T). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg or 0.40 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37°C, after which various amounts of chimeric polynucleotides were added and incubated for an additional 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115).

[0160] The knock-in efficiency was detected by flow cytometry on a specific day after electroporation. As shown in Figure 2, the highest knock-in efficiency of CD19-targeted CAR and humanized CD19 CAR and mesothelin CAR was 31.4%, 33.4%, and 33.8%, respectively.

[0161] Example 4. Knock-in of GC020 or GC012 in TRAC The knock-in was mediated by a polynucleotide containing modified partial targeting fragments at both the 5' and 3' ends as described herein. The polynucleotide was not covalently closed at both the 5' and 3' ends of the polynucleotide. The knock-in resulted in 41.1% of the cell population expressing dual CARs of CD19 and CD20 via knock-in at the TRAC genomic locus (82.7% of cells showing TRAC knockout) and 22.5% of the cell population expressing dual CARs of CD19 via knock-in at the TRAC genomic locus (90.1% of cells showing TRAC knockout). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg or 0.40 μg of the polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37°C, and then various amounts of polynucleotides were added and incubated for another 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). Knock-in efficiency was detected at specific days after electroporation by flow cytometry. As shown in Figure 3, the knock-in efficiency of GC020 was 41.1% and that of GC012 was 22.5%, with knock-out efficiencies of 82.7% and 90.1%, respectively.

[0162] Example 5. Knock-in of HLA-E FIG. 4A shows knock-in of HLA-E into cells by the system described herein. Knock-in was mediated by a polynucleotide containing a modified partial targeting fragment as described herein at both the 5' and 3' ends. The polynucleotide was further modified to contain both a covalently closed 5' end and a covalently closed 3' end. Knock-in mediated by a polynucleotide containing a modified partial targeting fragment as described herein at both ends, but without a covalently closed 5' end and 3' end, resulted in a 37.7% cell population expressing HLA-E (HLA-E KI). Knock-in mediated by a polynucleotide containing a modified partial targeting fragment as described herein at both ends and with a covalently closed 5' end and 3' end, resulted in a 54.9% cell population expressing HLA-E (ds-HLA-E KI). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg, 0.30 μg, or 0.40 μg of polynucleotide. Cells were incubated with Cas9 / RNP for 15 minutes at 37° C., after which various amounts of polynucleotide were added and incubated for an additional 5 minutes at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). FIG. 4B illustrates cell viability measurements showing that cells modified with polynucleotides containing modified partial targeting fragments at both ends and having covalently closed 5' and 3' ends had equal or greater viability over a period of 8 days than cells modified with polynucleotides containing modified partial targeting fragments as described herein at both ends, but without covalently closed 5' and 3' ends. Knock-in efficiency was detected by flow cytometry at specific days following electroporation. As shown in Figure 4A, the highest knock-in efficiency of HLA-E was 37.7% at a polynucleotide concentration of 0.4 μg, and the highest knock-in efficiency of ds-HLA-E was up to 54.9% at a polynucleotide concentration of 0.3 μg. The viability of knock-in ds-HLA-E was higher than HLA-E (Figure 4B).

[0163] Example 6. Knock-in of GC012HL at the TRAC locus Knock-in was mediated by polynucleotides (at amounts of 0.5 μg, 1.0 μg, 1.5 μg, or 2.0 μg, FIG. 5A) containing modified partial targeting fragments at both the 5' and 3' ends and covalently closed 5' and 3' ends. 34.6% of the cells expressed dual CARs. For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.50 μg, 1.0 μg, 1.5 μg, or 2.0 μg of polynucleotides. Cells were incubated with Cas9 / RNP for 15 min at 37° C., after which various amounts of polynucleotides were added and incubated for an additional 5 min at 37° C., followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). FIG. 5B shows the cell viability after 3 days of knock-in cells.

[0164] Example 7. Simultaneous knock-in of GC012HL at the TRAC locus and HLA-E at the B2M locus Figure 6 shows knock-in of both Dual-CAR (Dual-CAR-T) and HLA-E at the TRAC and B2M loci, respectively, resulting in 21.7% of cells expressing HLA-E and 50.1% of cells expressing dual-CAR. For this knock-in experiment, 10 million cells (in a final volume of 100 μl) were contacted with 1.0 μg, 1.25 μg, or 1.50 μg of polynucleotides. Cells were incubated with Cas9 / RNP for 15 minutes at 37°C, after which various amounts of polynucleotides were added and incubated for an additional 5 minutes at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115).

[0165] Example 8. Simultaneous knock-in of GC012HL at the TRAC locus and HLA-E at the B2M locus Figure 7 shows knock-in of both Dual-CAR (Dual-CAR-T) and HLA-E at the B2M and TRAC loci, respectively, resulting in 31.8% of cells expressing HLA-E R. Cells were incubated with Cas9 / RNP for 15 min at 37°C, after which various amounts of polynucleotides were added and incubated for another 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). A series of concentrations of polynucleotides were used, ranging from 1 μg to 1.5 μg per 100 μl reaction per 5 or 10 million activated T cells. The RNPX1.2 group was used with 1.2-fold more RNP compared to the RNP group. The general operation of the experiment was to mix sgRNA and Cas9 protein with PGA.

[0166] Knock-in efficiency was detected by flow cytometry on the 8th day after electroporation. As shown in FIG. 7, the HLA-E+B2M(high) population was endogenous HLA-E and the HLA-E+B2M(low) population was exogenous, i.e., knock-in of HLA-E. The cell group contacted with 1.5 μg of chimeric polynucleotide per 10 million cells had the highest knock-in efficiency of 31.8%.

[0167] Example 9. Double knock-in had little effect on knock-out efficiency Figure 8 shows that knock-in of both Dual-CAR (Dual-CAR-T) and HLA-E at the TRAC and B2M loci resulted in a knock-out of TRAC at 96.57% and a knock-out of B2M at 92.48%. For this knock-in experiment, 10 million cells (in a final volume of 100 μl) were contacted with 1.0 μg, 1.25 μg or 1.50 μg of polynucleotides. Cells were incubated with Cas9 / RNP for 15 min at 37°C, after which various amounts of polynucleotides were added and incubated for an additional 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). A range of concentrations of polynucleotides were used, from 1 μg to 1.5 μg per 100 μl reaction per 5 or 10 million activated T cells. The RNPX1.2 group was used with 1.2 times the amount of RNP compared to the RNP group.

[0168] Example 10. Chimeric polynucleotides containing telomeric ends Figure 9 shows knock-in of polynucleotides where the polynucleotides were double stranded DNA containing modified partial targeting fragments as described herein and were covalently closed at both 5' and 3' ends (telomeric end, top), not covalently closed at both 5' and 3' ends (middle) and circular (minicircle, bottom). For this knock-in experiment, 1 million cells (in a final volume of 20 μl) were contacted with 0.20 μg, 0.40 μg or 0.80 μg of polynucleotide. Cells were incubated with Cas9 / RNP for 15 min at 37°C, after which various amounts of polynucleotide were added and incubated for another 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). In this experiment, T cells served as a negative control and GC012HL was knocked into the TRAC locus. Knock-in efficiency was detected by flow cytometry at specific days after electroporation. As shown in FIG. 9, the knock-in efficiency of polynucleotides comprising minicircles was similar to that of linear polynucleotides comprising at least one genetically modified partial targeting fragment without a telomeric end, but was lower than that of polynucleotides comprising at least one genetically modified partial targeting fragment with a telomeric end.

[0169] Example 11. Knock-in using single-stranded DNA (ssDNA) as a chimeric polynucleotide Figure 10 shows knock-in of polynucleotides that were single stranded (either sense or antisense). At day 8, 47.2% of cells expressed dual CAR (GC012HL). For this knock-in experiment, 10 million cells (in a final volume of 100 μl) were contacted with 1.0 μg, 1.25 μg or 1.50 μg of polynucleotide. After incubating cells with Cas9 / RNP for 15 min at 37°C, various amounts of polynucleotide were added and incubated for another 5 min at 37°C, followed by electroporation (Lonza 4D-Nucleofector, protocol EH-115). Knock-in efficiency was detected by flow cytometry at specific days after electroporation. The CAR positive percentage of the ssDNA sense strand knock-in group at day 8 was 47.2%.

[0170] Example 12. Knock-in of GC012HL at the TRAC locus and HLA-E at the B2M locus Figure 11A shows knock-in of both dual CARs (targeting both CD19 and BCMA) and HLA-E into cells by the system described herein. Figure 11B shows the cell killing activity of cells with dual CARs knocked in by the system described herein. At day 8, cells with CARs targeting CD19 or BCMA showed cell killing activity to kill B cell precursor leukemia cells (Nalm6, which expressed CD19) and cancer cells (MM.1S, RPMI-8226 and JeKo-1 cells, all of which expressed BCMA).

[0171] Example 13. Autologous knock-in of CAR Figures 12A-E show non-viral knock-in of the polynucleotides described herein to generate CAR-T cells (ZAR-CAR-T). T cells were activated for 48 hours before electroporation and CAR positive percentage and viability were detected 1, 2 and 3 days after electroporation. CAR expression and viability after thawing were also detected. After thawing, an in vitro tumor cell killing assay was performed. The data shows that ZAR-CAR-T had stable CAR expression even after thawing, with up to 80% viability 3 days after electroporation. CAR-T showed sufficient cell killing after 6 hours. Figure 12A shows modified cells that were positive for CAR presentation on day 2. Figure 12B shows the percentage change in modified cells that were positive for knock-in presentation. Figure 12C shows cell viability of knock-in modified cells (left) and cells after thawing from cell freezing (right) over a 3-day period. Figure 12D shows the viability and percentage change of modified cells positive for knock-in presentation 1 day after thawing from cell freezing. NT-Ctrl: no template control. Top: modified cells displaying CD19-targeted CAR. Bottom: modified cells displaying CD3. Figure 12E shows the cell killing activity of modified cells (ZAP-CAR-T) against CD19 (Nalm6 cells) or BCMA (JeKo-1 and MM.1S cells).

[0172] Example 14. Knock-in efficiency of GC012HL and HLA-E at the TRAC and B2M loci FIG. 13A shows knock-in of dual CARs for targeting CD19 or CD20 at a single locus of TRAC by the system described herein. 41.1% of cells showed positive expression of dual CARs for targeting CD19 or CD20. FIG. 13B shows knock-in of dual CARs for targeting CD19 or BCMA at a single locus of TRAC by the system described herein. 41.1% of cells showed positive expression of dual CARs for targeting CD19 or BCMA. FIG. 14 shows knock-in of polynucleotides by the system described herein, where polynucleotides knocked in at loci such as TRAC or B2M were greater than 5,000 bps. Approximately 20% of modified cells were positive for knock-in presentation. Flow cytometry studies show that 25.2% of cells expressed CD19 CAR. 5.77% of cells expressed both CD19 CAR and HLA-E.

[0173] Although the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made therein without departing from the scope of the disclosure. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application and / or other document was individually and individually indicated to be incorporated by reference for all purposes.

Claims

1. a) a guide nucleic acid; b) a genetically modified portion; and c) a chimeric polynucleotide comprising at least one expression sequence and at least one genetically modified partial targeting fragment, and further comprising at least one covalently closed circular end or being circular; A system containing

2. 10. The system of claim 1, wherein said chimeric polynucleotide is linear DNA.

3. 10. The system of claim 1, wherein said polynucleotide comprises double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA).

4. 10. The system of claim 1, wherein said polynucleotide is a vector or a minicircle.

5. 2. The system of Claim 1, wherein said at least one covalently closed circular end is at the 5' end of said polynucleotide or the 3' end of said polynucleotide.

6. 2. The system of Claim 1, wherein said polynucleotide comprises said at least one covalently closed circular end at the 5' end of said polynucleotide and said at least one covalently closed circular end at the 3' end of said polynucleotide.

7. The system of claim 1 , wherein the at least one covalently closed end comprises a telomeric end.

8. 2. The system of Claim 1, wherein said polynucleotide comprises said at least one genetically modified partial targeting fragment near the 5' end of said polynucleotide or near the 3' end of said polynucleotide.

9. 2. The system of Claim 1, wherein said polynucleotide comprises said at least one genetically modified partial targeting fragment near the 5' end of said polynucleotide and said at least one genetically modified partial targeting fragment near the 3' end of said polynucleotide.

10. 2. The system of claim 1, wherein the guide nucleic acid comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to a genomic sequence in a cell, wherein the genomic sequence comprises a genomic locus of the cell, wherein the genomic locus comprises T-cell receptor alpha chain constant (TRAC), beta-2-microglobulin (B2M), cluster of differentiation 38 (CD38), cytokine-induced SH2-containing protein (CISH), programmed cell death protein 1 (PD-1), or cluster of differentiation 70 (CD70).

11. (1) the at least one genetically modified partial targeting fragment comprises about 10 nucleotide base pairs (bps) to about 100 nucleotides bps, and / or (2) the at least one genetically modified partial targeting fragment comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to the guide nucleic acid, and / or (3) the at least one genetically modified partial targeting fragment has at least one mismatch, at least two mismatches, at least three mismatches, or at least three mismatches compared to the guide nucleic acid.

2. The system of claim 1, wherein the at least one genetically modified portion targeting fragment comprises at least four matches, at least four mismatches, at least five mismatches, at least six mismatches, at least seven mismatches, at least eight mismatches, at least nine mismatches, or at least ten mismatches; and / or (4) the at least one genetically modified portion targeting fragment complexes with the genetically modified portion, thereby bringing the genetically modified portion into proximity with the polynucleotide; and / or (5) the at least one genetically modified portion targeting fragment, when complexed with the genetically modified portion, does not induce enzymatic activity of the genetically modified portion.

12. The system of claim 1 , wherein the genetically modified portion comprises a Cas protein or an mRNA encoding the Cas protein.

13. The system of claim 12, wherein the genetic modification portion comprises Cas / RNP.

14. The system of claim 1 , wherein the at least one expression sequence encodes a chimeric receptor, wherein the chimeric receptor comprises an antigen-binding domain, a transmembrane domain, and / or a signaling domain.

15. The system of claim 1, further comprising a polymer, wherein the polymer comprises an overall anionic charge.

16. A cell comprising a system described in any one of claims 1 to 15.

17. The cell of claim 16 , wherein the cell comprises an immune cell or a stem cell.

18. A composition comprising a system according to any one of claims 1 to 15.

19. A kit comprising the system of any one of claims 1 to 15 and a container.

20. 16. A method comprising contacting a cell with the system of any one of claims 1 to 15, wherein said system knocks in said polynucleotide in a genomic sequence in said cell, thereby expressing in said cell a chimeric receptor encoded by at least one expression sequence of said polynucleotide.

21. Cells, a) a first system comprising the system of any one of claims 1 to 15, the first system comprising a first guide nucleic acid complexed with a first genetic modification moiety and a first chimeric polynucleotide comprising said at least one genetic modification moiety targeting fragment; and b) A second system comprising the system of any one of claims 1 to 15, the second system comprising a second guide nucleic acid complexed with a second genetic modification moiety and a second chimeric polynucleotide comprising the at least one genetic modification moiety targeting fragment. wherein said first system introduces said first polynucleotide into a first genomic sequence in said cell, and said second system introduces said second polynucleotide into a second genomic sequence in said cell.

Images