Compositions for modifying T cells

Abstract

Provided is a composition for modifying T cells, the composition comprising: a protein complex comprising a polynucleotide-modifying enzyme domain, a T cell membrane-binding domain, and an endosomal escape domain; a guide oligonucleotide specific to the T cell receptor constant (TRAC) gene of the T cell; and donor DNA comprising two homology arms at each end of the donor DNA that are homologous to exon 1 of the TRAC gene, and encoding a chimeric antigen T cell receptor between the two homology arms that comprises a translocation signal for translocation to the cell membrane of the T cell; a transmembrane domain; an intracellular signaling domain; and an extracellular antigen-binding domain.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to U.S. Provisional Application No. 63 / 397150, filed August 11, 2022, and incorporated herein by reference in its entirety.

[0002] Technical Field The present disclosure relates to the field of T cell gene modification, particularly the production of chimeric antigen receptor T cells (CAR-T) and vectors for CAR gene editing. [Background technology]

[0003] Chimeric antigen receptors (CARs) typically contain an extracellular target-binding domain, a hinge region, a transmembrane domain that anchors the CAR to the cell membrane, and one or more intracellular domains that transmit activation signals through a signal cascade. The transmembrane domain is generally a hydrophobic helix that spans the thickness of the cell membrane. CARs are generally classified based on the number of their costimulatory domains: first generation (CD3z only), second generation (one costimulatory domain and CD3z), and third generation (more than one costimulatory domain and CD3z). The purpose of introducing CAR molecules into T cells is to redirect T cells to the desired specificity and provide the necessary signals to drive overall T cell activation. For example, T cells can be genetically engineered into CAR-T cells with specificity for antigens presented by specific cancer cells. One particular area of ​​clinical interest for CAR-T cells is the treatment of blood cancer types. Antigen recognition by CAR-T cells is driven by the binding of target-binding single-chain variable fragments (scFvs) to surface antigens.

[0004] The hinge, also referred to as the spacer, is the extracellular structural region of the CAR that separates the binding unit from the transmembrane domain. Most CAR T cells are designed with an immunoglobulin (Ig)-like domain hinge. These spacers generally provide stability for efficient CAR expression and activity. The hinge also provides flexibility for accessing the targeted antigen. The length of the hinge affects the binding efficiency of the CAR. For example, a long spacer provides flexibility and therefore improved access to membrane-proximal epitopes or complex glycosylated antigens, while a short space is more effective in binding to distal epitopes. The length of the spacer provides sufficient intercellular distance for immunological synapse formation. Thus, the hinge can affect the overall performance of the CAR-T cell.

[0005] Cell therapy using CAR-T cells is an immunotherapeutic tool for combating conditions, such as blood disorders. Natural killer (NK) cells are a type of T cell that can also be engineered into specific target-bearing CAR-T cells. CD8 T cells, like NK cells, have the ability to target and kill specific cells, while CD4 T cells generally mediate immune responses by directing other killer cells. NK and CD8 T cells have the ability to target and kill cells based on the specificity of the CAR inserted into them. The use of CAR-T cells can greatly improve cancer treatment and patient outcomes. Allogeneic situations require universal CAR-T cells that can kill targeted tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. On the other hand, in autologous therapy, the patient's own T cells are harvested, then genetically engineered into CAR-T cells and administered to the patient.

[0006] Modifying T cells into CAR-T cells requires a delivery vehicle to provide the gene editing tools as well as the genetic material encoding the CAR. Viral vectors have been used to manufacture CAR-T cells. Examples of viral vectors include lentiviral vectors, gamma-retroviral vectors, and recombinant adeno-associated viral vectors. In the case of retroviral transduction, insertional mutagenesis has a detrimental effect on the survival of primary T cells, which is a major limitation for the clinical application of retroviruses. Lentiviral-based transduction is a safer option due to its lower genotoxicity and insertional mutagenesis. Unfortunately, the efficiency of lentiviral transduction in primary T cells is low, often requiring multiple rounds of transduction, thus limiting its potential in clinical settings. Further disadvantages of retroviruses include the fact that they can only integrate into dividing cells in the mitotic stage, and gene integration is not targeted. An additional disadvantage of lentiviral vectors is that they are non-integrating. In general, viral vectors present a risk of mutagenesis, which is a major concern in clinical settings.

[0007] Electroporation has also been used to deliver gene editing materials and CAR constructs into T cells.However, electroporation leads to high cell death rate and difficult genome integration.Overall, electroporation technology has low efficacy and is undesirable in clinical situations, especially when primary cells are involved.In addition, gene transduction approach usually leads to random integration of DNA into target cell genome, resulting in the potential risk of off-target effects, such as the silencing of essential genes or tumor suppressor genes, which can trigger cell apoptosis or malignant transformation.

[0008] Thus, improvements in transduction efficiency and targeting are desirable, particularly for primary cells, which would greatly improve the use of CAR-T cell therapy involving primary T cells, for example, in the treatment of cancer. Summary of the Invention

[0009] In one embodiment, a composition for modifying T cells is provided, the composition comprising: a protein complex comprising a polynucleotide-modifying enzyme domain, a T cell membrane-binding domain, and an endosomal escape domain; a guide oligonucleotide specific to the T cell receptor alpha constant (TRAC) gene of a T cell; and donor DNA, the donor DNA comprising two homologous arms at each end homologous to exon 1 of the TRAC gene, and encoding a chimeric antigen T cell receptor comprising, between the two homologous arms, a translocation signal for translocation to the T cell membrane; a transmembrane domain; an intracellular signaling domain; and an extracellular antigen-binding domain. In some embodiments, the protein complex further comprises a hapten-binding domain, preferably, the donor DNA is conjugated to a hapten, and the hapten binds to the hapten-binding domain. In some embodiments, the protein complex further comprises a nuclear localization sequence. In some embodiments, the chimeric antigen T cell receptor further comprises a CD8 hinge region. In some embodiments, the chimeric antigen T cell receptor further comprises a B cell lymphoma recognition domain. In some embodiments, the guide oligonucleotide is complementary to a sequence located between 250 nucleotides before the start codon of exon 1 of the TRAC gene and 250 nucleotides after the start codon of exon 1 of the TRAC gene. In some embodiments, the polynucleotide-modifying enzyme domain is covalently linked to an endosomal escape domain. In some embodiments, the T cell membrane-binding domain is a cationic peptide. In some embodiments, the T cell membrane-binding domain is a cell recognition domain. In some embodiments, the cell recognition domain targets CD4, CD8, CD16, or CD56. In some embodiments, the cell recognition domain is covalently linked to the endosomal escape domain. In some embodiments, the cell recognition domain is a display domain, which is a peptide recognition sequence 3 to 20 amino acids long located in a loop or alpha helix on the outer surface of the polynucleotide-modifying enzyme domain.In some embodiments, the peptide recognition sequence is a complementarity determining region (CDR). In some embodiments, the cell recognition domain is located in a loop on the outer surface of the polynucleotide modifying enzyme, such as a Fab, a single domain antibody (sdAb), a V. HH or a camelid antibody domain. In some embodiments, the polynucleotide modifying domain is Type II Cas, a functional analog thereof, a variant thereof, or a derivative thereof. In some embodiments, the Type II Cas is Cas9, a functional analog thereof, a variant thereof, or a derivative thereof. In some embodiments, the polynucleotide modifying domain is Type V Cas, a functional analog thereof, a variant thereof, or a derivative thereof. In some embodiments, the extracellular antigen-binding domain is specific for a cancer-specific antigen.

[0010] In one embodiment, the compositions of the present disclosure are provided for use in cell therapy, for example, in the treatment of cancer.

[0011] In one embodiment, there is provided the use of the compositions of the present disclosure in cell therapy, for example, in the treatment of cancer.

[0012] In one embodiment, provided is a method of performing cell therapy for a subject in need thereof, the method comprising providing ex vivo allogeneic T cells, modifying the genome of the T cells with a composition of the present disclosure to obtain chimeric antigen receptor (CAR) T cells, and administering the CAR T cells to the subject.

[0013] In one embodiment, there is provided a method of performing cell therapy for a subject in need thereof, the method comprising providing ex vivo allogeneic T cells, modifying the genome of the T cells to obtain CAR-T cells with a composition of the present disclosure by binding the composition to the cell membrane of the T cells and allowing cellular internalization to occur, and administering the CAR T cells to the subject.

[0014] In one embodiment, provided is a method of treating cancer for a subject in need thereof, the method comprising providing allogeneic T cells, modifying the genome of the T cells with a composition of the present disclosure to obtain CAR T cells, and administering the CAR T cells to the subject.

[0015] In one embodiment, provided is a method of performing cell therapy for a subject in need thereof, the method comprising delivering a composition of the present disclosure to a T cell in vivo in the subject to modify the genome of the T cell and obtain a chimeric antigen receptor (CAR) T cell in vivo.

[0016] In one embodiment, provided is a method of treating cancer for a subject in need thereof, the method comprising delivering a composition of the present disclosure to a T cell in vivo in the subject to modify the genome of the T cell and obtain a chimeric antigen receptor (CAR) T cell in vivo.

[0017] In one embodiment, a method of manufacturing a CAR-T cell is provided, the method comprising internalizing a composition of the present disclosure by binding to the cell membrane of the T cell, and incubating the T cell to allow the composition to edit the genome of the T cell.

[0018] In one embodiment, the polynucleotide modifying enzyme comprises a functional nuclease domain comprising a nuclease catalytic pocket; a Fab, a single domain antibody (sdAb), a V in a loop located on the outer surface of the polynucleotide modifying enzyme; HH

[0003] Provided herein is a polynucleotide modifying enzyme comprising: an antigen-binding domain selected from a VHH, a VHH, or a camelid antibody domain, wherein the antigen-binding domain recognizes a target cell receptor of a target cell and enables cellular internalization of the polynucleotide modifying enzyme into the target cell; and a linker of 0 to 30 amino acids upstream of the antigen-binding domain. In some embodiments, the nanobody is a VHH. The linker sequence is preferably 16 to 23 amino acids. The nuclease catalytic pocket is preferably a Cas nuclease catalytic pocket, a recombinase catalytic pocket, or a meganuclease catalytic pocket. The Cas can be a type II Cas, such as Cas9, a functional analog, a variant, or a derivative thereof. In some embodiments, the nuclease catalytic pocket comprises an HNH nuclease domain. In some embodiments, the Cas is a type V Cas, such as Cas12, a functional analog, a variant, or a derivative thereof. In some embodiments, the Cas is a type VI Cas, such as Cas13, a functional analog, a variant, or a derivative thereof. In some embodiments, the Cas is Cas14, a functional analog thereof, a variant thereof, or a derivative thereof. In some embodiments, the nuclease catalytic pocket comprises a RuvC nuclease domain. In one aspect, a vector encoding a polynucleotide modifying enzyme is provided, the vector comprising: an encoded functional nuclease domain at the 5'-end and 3'-end of the nuclease enzyme, and between the 5'-end and 3'-end of the nuclease enzyme; an encoded antigen-binding domain; and a linker sequence upstream of the 5'-end of the encoded antigen-binding domain, encoding a linker.

[0019] Many further features and combinations of the present improvement will be apparent to those skilled in the art upon reading this disclosure. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram of delivery of nuclease protein complexes to edit T cells. [Figure 2] FIG. 2 is a diagram showing the mechanism of action of receptor-mediated delivery of nuclease complexes targeting the CD4 receptor of T cells for intracellular delivery. [Figure 3] FIG. 3 is a map of the CAR construct. [Figure 4] Figure 4 shows the vector map of M7 Mav anti CD8. [Figure 5] Figure 5 shows the vector map of M7-Mav-anti CD4. [Figure 6] Figure 6 shows the vector map of C9mAur. [Figure 7] Figure 7 shows the vector map of C9mC4. [Figure 8] FIG. 8 shows the vector map of C9C4 anti CD4n1. [Figure 9A] FIG. 9A is an image of a gel electrophoresis showing the expression of the engineered nuclease "Zero." [Figure 9B] FIG. 9B is a graph showing the expression of the engineered nuclease "Zero," with the labeled numbers on the graph corresponding to the lane numbers on the gel in FIG. 9A. [Figure 9C] FIG. 9C is an image of a gel electrophoresis showing the expression of the modified nuclease "L1." [Figure 9D] FIG. 9D is a graph showing the expression of the modified nuclease "L1," with the labeled numbers on the graph corresponding to the lane numbers on the gel in FIG. 9C. [Figure 9E] FIG. 9E is an image of a gel electrophoresis showing the expression of the modified nuclease "L2." [Figure 9F] FIG. 9F is a graph showing the expression of the modified nuclease "L2," with the labeled numbers on the graph corresponding to the lane numbers on the gel in FIG. 9E. [Figure 10A] FIG. 10A is a gel electrophoresis showing the cleavage activity of Zero, L1, and L2 on a 100 bp DNA template. [Figure 10B] FIG. 10B is a graph showing the cleavage activity of Zero, L1, and L2 against a 100 bp DNA template over time. [Figure 11] Figure 11 is an image of a gel electrophoresis of biotinylated CAR donor construct and biotin-CAR donor bound to Md7-MAV-CD47. [Figure 12A] Figure 12A is a bright-field microscopy image of Jurkat cells gene-edited with M7-Mav-CD4 (20x magnification). [Figure 12B] Figure 12B is a fluorescent microscope image for green fluorescent protein showing Jurkat cells gene-edited with M7-Mav-CD4(GFP) (20x magnification). [Figure 13] FIG. 13 is an image of a gel electrophoresis showing the CAR-DNA donor without C9mAur and the biotinylated DNA donor complexed to C9mAur. [Figure 14A] FIG. 14A is a fluorescent microscopy image of cells given only C9 without donor DNA (i.e., control condition). [Figure 14B] Figure 14B is a fluorescent microscope image of cells receiving C9 along with sgRNA1 guide and donor DNA. [Figure 14C] Figure 14C is a fluorescent microscopy image of cells receiving C9 along with sgRNA3 guide and donor DNA. [Figure 14D] Figure 14D is a fluorescent microscopy image of cells receiving C9 along with sgRNA11 guide and donor DNA. [Figure 15] Figure 15 is a gel electrophoresis image of polymerase chain reaction (PCR) using insert confirmation primers (Jurkat cell triplicate samples J1, J2, and J3 using sgRNA11 as the guide RNA). [Figure 16] FIG. 16 is a graph of raw read counts sequenced by next-generation sequencing (NGS) using three biological replicates (samples 1, 2, and 3). [Figure 17]FIG. 17 is a graph of the percent reads sequenced by NGS using three biological replicates (samples 1, 2, and 3). [Figure 18] FIG. 18 shows the mass spectrometry spectrum of the synthesized RL peptide. [Figure 19] FIG. 19 is a graph of high performance liquid chromatography (HPLC) performed on the synthesized RL peptide. [Figure 20] FIG. 20 is a gel electrophoresis of the retardation assay showing that Zero, L1, L2, and L3 all bound to the donor DNA (L3 is labeled as C9C4 on the gel; these labels are equivalent). [Figure 21A] FIG. 21A is a fluorescence microscopy image showing that Zero bound to the cell membrane of CD4+ primary T cells. [Figure 21B] FIG. 21B is a fluorescence microscopy image showing that L1 bound to the cell membrane of CD4+ primary T cells. [Figure 21C] FIG. 21C is a fluorescence microscopy image showing that L2 bound to the cell membrane of CD4+ primary T cells. [Figure 21D] FIG. 21D is a fluorescence microscopy image showing that L2 bound to the cell membrane of CD4+ primary T cells. [Figure 22A] FIG. 22A is a flow cytometry graph showing events (×1000) in function of TAMRA detection for control conditions (no nuclease, 1 hour incubation) in Jurkat CD4+ T cells. [Figure 22B] FIG. 22B is a flow cytometry graph showing events (x1000) in function of TAMRA detection in Jurkat CD4+ T cells 1 hour after receiving Zero. [Figure 22C] FIG. 22C is a flow cytometry graph showing events (×1000) in function of TAMRA detection in Jurkat CD4+ T cells 1 hour after receiving L1. [Figure 22D]FIG. 22D is a flow cytometry graph showing events (×1000) in function of TAMRA detection in Jurkat CD4+ T cells 1 hour after receiving L2. [Figure 22E] FIG. 22E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in Jurkat CD4+ T cells 1 hour after receiving L3. [Figure 23] FIG. 23 is a graph showing green fluorescent protein (GFP) fluorescence intensity counts in cells treated with L1 over time. [Figure 24A] FIG. 24A is a flow cytometry graph showing events (x1000) in function of TAMRA detection for control conditions (no nuclease, 1 hour incubation) in human primary CD4+ T cells. [Figure 24B] FIG. 24B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in human primary CD4+ T cells 1 hour after receiving L1. [Figure 24C] FIG. 24C is a flow cytometry graph showing events (×1000) in function of TAMRA detection in human primary CD4+ T cells 1 hour after receiving L2. [Figure 24D] FIG. 24D is a flow cytometry graph showing events (x1000) in function of TAMRA detection in human primary CD4+ T cells 1 hour after receiving L3. [Figure 25A] FIG. 25A is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (no nuclease provided during 48 hour Jurkat T cell incubation). [Figure 25B] FIG. 25B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (no nuclease provided during 48 hour Jurkat T cell incubation). [Figure 25C]FIG. 25C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (no nuclease provided during 48 hour Jurkat T cell incubation). [Figure 26A] FIG. 26A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26B] FIG. 26B is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26C] FIG. 26C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (Zero at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26D] FIG. 26D is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26E] FIG. 26E is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26F] FIG. 26F is a flow cytometry graph showing events (x1000) in function of GFP detection in control conditions (Zero at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26G] FIG. 26G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26H]FIG. 26H is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26I] FIG. 26I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (Zero at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26J] FIG. 26J is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26K] FIG. 26K is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 26L] FIG. 26L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (Zero at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27A] FIG. 27A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 0.33 ng / μL during 48 hour Jurkat T cell incubation). [Figure 27B] FIG. 27B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27C] FIG. 27C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27D]FIG. 27D is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27E] FIG. 27E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27F] FIG. 27F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27G] FIG. 27G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 33.3 ng / μL during 48 hour Jurkat T cell incubation). [Figure 27H] FIG. 27H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27I] FIG. 27I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27J] FIG. 27J is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27K] FIG. 27K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 27L]FIG. 27L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 28A] FIG. 28A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 0.33 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28B] FIG. 28B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 0.33 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28C] FIG. 28C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 28D] FIG. 28D is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 16 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28E] FIG. 28E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 16 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28F] FIG. 28F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 28G] FIG. 28G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 33.3 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28H]FIG. 28H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 33.3 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28I] FIG. 28I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 28J] FIG. 28J is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 66 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28K] FIG. 28K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 66 ng / μL during 48 hour Jurkat T cell incubation). [Figure 28L] FIG. 28L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 29A] FIG. 29A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 0.33 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29B] FIG. 29B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 0.33 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29C] FIG. 29C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 0.33 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 29D]FIG. 29D is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 16 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29E] FIG. 29E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 16 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29F] FIG. 29F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 16 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 29G] FIG. 29G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 33.3 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29H] FIG. 29H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 33.3 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29I] FIG. 29I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 33.3 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 29J] FIG. 29J is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 66 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29K] FIG. 29K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 66 ng / μL during 48 hour Jurkat T cell incubation). [Figure 29L]FIG. 29L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 66 ng / μL during 48 hours of Jurkat T cell incubation). [Figure 30A] FIG. 30A is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (no nuclease provided during 72 hour Jurkat T cell incubation). [Figure 30B] FIG. 30B is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (no nuclease provided during 72 hour Jurkat T cell incubation). [Figure 30C] FIG. 30C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (no nuclease provided during 72 hour Jurkat T cell incubation). [Figure 31A] FIG. 31A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31B] FIG. 31B is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31C] FIG. 31C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (Zero at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31D] FIG. 31D is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31E]FIG. 31E is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31F] FIG. 31F is a flow cytometry graph showing events (x1000) in function of GFP detection in control conditions (Zero at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31G] FIG. 31G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31H] FIG. 31H is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31I] FIG. 31I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (Zero at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31J] FIG. 31J is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (Zero at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31K] FIG. 31K is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (Zero at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 31L] FIG. 31L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (Zero at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32A]FIG. 32A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 0.33 ng / μL during 72 hour Jurkat T cell incubation). [Figure 32B] FIG. 32B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32C] FIG. 32C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32D] FIG. 32D is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32E] FIG. 32E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32F] FIG. 32F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32G] FIG. 32G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 33.3 ng / μL during 72 hour Jurkat T cell incubation). [Figure 32H] FIG. 32H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32I]FIG. 32I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32J] FIG. 32J is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32K] FIG. 32K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 32L] FIG. 32L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 33A] FIG. 33A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 0.33 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33B] FIG. 33B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 0.33 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33C] FIG. 33C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 33D] FIG. 33D is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 16 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33E]FIG. 33E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 16 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33F] FIG. 33F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 33G] FIG. 33G is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 33.3 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33H] FIG. 33H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 33.3 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33I] FIG. 33I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 33J] FIG. 33J is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 66 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33K] FIG. 33K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 66 ng / μL during 72 hour Jurkat T cell incubation). [Figure 33L] FIG. 33L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 34A]FIG. 34A is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 0.33 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34B] FIG. 34B is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 0.33 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34C] FIG. 34C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 0.33 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 34D] FIG. 34D is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 16 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34E] FIG. 34E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 16 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34F] FIG. 34F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 16 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 34G] FIG. 34G is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 33.3 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34H] FIG. 34H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 33.3 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34I]FIG. 34I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 33.3 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 34J] FIG. 34J is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 66 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34K] FIG. 34K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 66 ng / μL during 72 hour Jurkat T cell incubation). [Figure 34L] FIG. 34L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 66 ng / μL during 72 hours of Jurkat T cell incubation). [Figure 35] Figure 35 is an image of a gel electrophoresis of the insert-specific primers. [Figure 36A] FIG. 36A is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (no nuclease provided during 48 hour primary T cell incubation). [Figure 36B] FIG. 36B is a flow cytometry graph showing events (x1000) in function of TAMRA detection in control conditions (no nuclease provided during 48 hour primary T cell incubation). [Figure 36C] FIG. 36C is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (no nuclease provided during 48 hour primary T cell incubation). [Figure 36D] FIG. 36D is a flow cytometry graph showing the detection of TAMRA in function of GFP in control conditions (L1 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36E] FIG. 36E is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L1 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36F] FIG. 36F is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L1 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36G] FIG. 36G is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L2 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36H] FIG. 36H is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L2 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36I] FIG. 36I is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L2 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36J] FIG. 36J is a flow cytometry graph showing detection of TAMRA in function of GFP in control conditions (L3 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36K] FIG. 36K is a flow cytometry graph showing events (×1000) in function of TAMRA detection in control conditions (L3 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 36L] FIG. 36L is a flow cytometry graph showing events (×1000) in function of GFP detection in control conditions (L3 at a concentration of 66 ng / μL during 48 hours of primary T cell incubation). [Figure 37A]Figure 37A is a flow cytometry graph of analyzed peripheral T cells obtained from control (no L1 or L2) mice and stained with APC-antiCD4 to assess T cell populations at the 3 hour post-injection stage. [Figure 37B] FIG. 37B is a flow cytometry graph of analyzed peripheral T cells obtained from mice treated with TAMRA-labeled L1 and stained with APC-antiCD4 to assess T cell populations at the 3-hour post-injection stage. [Figure 37C] Figure 37C is a flow cytometry graph of analyzed peripheral T cells obtained from TAMRA-labeled L2-treated mice and stained with APC-antiCD4 to assess T cell populations at the 3-hour post-injection stage. [Figure 37D] Figure 37D is a flow cytometry graph of analyzed peripheral T cells obtained from control (no L1 or L2) mice and stained with APC-antiCD4 to assess T cell populations at the 24 hour post-injection stage. [Figure 37E] Figure 37E is a flow cytometry graph of analyzed peripheral T cells obtained from mice treated with TAMRA-labeled L1 and stained with APC-antiCD4 to assess T cell populations at 24 hours post-injection stage. [Figure 37F] Figure 37F is a flow cytometry graph of analyzed peripheral T cells obtained from TAMRA-labeled L2-treated mice and stained with APC-antiCD4 to assess T cell populations at 24 hours post-injection stage. [Figure 37G] Figure 37G is a flow cytometry graph of analyzed peripheral T cells obtained from control (no L1 or L2) mice and stained with APC-antiCD4 to assess T cell populations at the 48 hour post-injection stage. [Figure 37H]FIG. 37H is a flow cytometry graph of analyzed peripheral T cells obtained from mice treated with TAMRA-labeled L1 and stained with APC-antiCD4 to assess T cell populations at 48 hours post-injection stage. [Figure 37I] Figure 37I is a flow cytometry graph of analyzed peripheral T cells obtained from mice treated with TAMRA-labeled L2 and stained with APC-antiCD4 to assess T cell populations at 48 hours post-injection stage. [Figure 38A] FIG. 38A is a flow cytometry graph of T cells obtained from the mouse in FIG. 37A showing CD19 events (detection). [Figure 38B] FIG. 38B is a flow cytometry graph of T cells obtained from the mouse in FIG. 37B showing CD19 events (detection). [Figure 38C] FIG. 38C is a flow cytometry graph of T cells obtained from the mouse in FIG. 37C showing CD19 events (detection). [Figure 39A] Figure 39A is a bioluminescence image of a control mouse given control vehicle (labeled as C), a mouse given L1 (labeled as L1), and a mouse given L2 (labeled as L2) 192 hours after Raji injection. [Figure 39B] FIG. 39B is a graph showing bioluminescence for the control, L1-fed, and L2-fed mice of FIG. 39A. [Figure 40A] FIG. 40A is a gel showing the results of streptavidin-aptamer modification of a donor template. [Figure 40B] FIG. 40B is a gel showing off-target analysis for cells treated with L2. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description definition As used herein, the term "cell recognition domain" (or "CRD") refers to a natural or synthetic peptide or nucleic acid domain capable of specific, non-covalent association with a cell surface antigen or receptor.

[0022] As used herein, the term "polynucleotide modifying enzyme" (or "PNME") refers to a peptide enzyme that has the ability to cleave the phosphodiester backbone of a nucleic acid (e.g., DNA or RNA) or to alter the identity of one or more nitrogenous bases within a nucleic acid.

[0023] As used herein, the term "endosomal escape domain" (or "EE domain") refers to a peptide sequence that, when associated with a molecular cargo, facilitates diffusion of the cargo from the endosomal compartment to the cytosol and / or alters the steady-state distribution of the cargo between the endosomal compartment and the cytosol in favor of the cytosol.

[0024] As used herein, the term "display domain" refers to a peptide sequence capable of specific non-covalent association with a cell surface antigen or receptor. The display domain is incorporated into the PNME and does not interfere with the activity of the functional nuclease domain. The display domain can be sized and / or positioned in the sequence of the PNME so that the nuclease catalytic pocket is not obstructed and retains at least 50%, at least 60%, at least 70%, preferably at least 80%, more preferably at least 90% of its cleavage activity. For example, the three-dimensional conformation of the nuclease catalytic pocket can substantially correspond to the three-dimensional conformation that would be obtained without the insertion of the display domain into the PNME (e.g., the same alpha helix and the same beta sheet).

[0025] As used herein, the term "hapten" refers to a small molecule that, when combined with a larger carrier, such as a protein, has the ability to bind with high affinity to an antibody or antibody mimic (the "hapten-binding domain"). In some embodiments, the molecular weight of the organic compound is less than 500 daltons. In some embodiments, the affinity (K) of the hapten for the hapten-binding domain is D ) is 10 -6 In some embodiments, the affinity (K) of the hapten for the peptide or nucleic acid aptamer D ) is 10 -7 In some embodiments, the affinity (K) of the hapten for the peptide or nucleic acid aptamer D ) is 10 -8 In some embodiments, the affinity (K) of the hapten for the peptide or nucleic acid aptamer D ) is 10 -9 The concentration is less than molar. As used herein, the term "linker," "linker group," or "linker domain" refers to a group capable of linking one chemical moiety to another. In some embodiments, the linker is a chemical bond. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is a cleavable linker, e.g., the linker comprises a linkage that can be cleaved upon exposure to a cleavage activity, such as UV light or a hydrolase, e.g., a lysosomal protease. In some embodiments, the linker may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more amino acids. In some embodiments, the peptide linker comprises a repeat of the tri-peptide Gly-Gly-Ser, e.g., the sequence (GGS) n(n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more repeats). In some embodiments, the linker can comprise at least two polyethylene glycol (PEG) residues. In some embodiments, the PEG linker comprises 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more PEG residues. In some embodiments, the PNME described herein comprises a linker joining two or more domains described herein, e.g., any combination of two or more of an endosomal escape domain, a nuclear localization sequence, or a PNME domain.

[0026] The term "tracrRNA" or "tracr sequence," as used herein, refers to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., a tracrRNA from S. pyogenes, S. aureus, etc.). A tracrRNA can refer to a nucleic acid having up to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence. A tracrRNA can also refer to modified forms of a tracrRNA, which can contain nucleotide changes, such as deletions, insertions, or substitutions, variants, mutations, or chimeras. A tracrRNA may refer to a nucleic acid that can be at least about 60% identical to a wild-type exemplary tracrRNA sequence over a stretch of at least six contiguous nucleotides. For example, a tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical to a wild-type exemplary tracrRNA sequence over a stretch of at least six contiguous nucleotides.

[0027] As used herein, "guide nucleic acid" refers to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid may preferably be RNA or DNA. The guide nucleic acid may be programmed to specifically bind to a nucleic acid having a unique sequence. The targeted nucleic acid, or target nucleic acid, may comprise nucleotides. The guide nucleic acid may comprise nucleotides. A portion of the target nucleic acid may be complementary to a portion of the guide nucleic acid. A strand of a double-stranded target polynucleotide that is complementary to and hybridizes with a guide nucleic acid may be referred to as a complementary strand. A strand of a double-stranded target polynucleotide that is complementary to a complementary strand and therefore may not be complementary to the guide nucleic acid may be referred to as a non-complementary strand. A guide nucleic acid may comprise a polynucleotide strand and may be referred to as a "single guide nucleic acid." A guide nucleic acid may comprise two polynucleotide strands and may be referred to as a "double guide nucleic acid." Unless otherwise specified, the term "guide nucleic acid" may be inclusive and refer to both single guide nucleic acids and double guide nucleic acids. The guide nucleic acid may comprise a nucleic acid targeting segment (e.g., crRNA) and a protein binding sequence. The guide nucleic acid may include a protein-binding sequence, a transactivating RNA (e.g., tracrRNA), and a transactivating RNA (e.g., tracrRNA). In some cases, the guide RNA described herein includes a sequence of n nucleotides, counting from the first nucleotide at the 5' end to the nth nucleotide at the 3' end, wherein one or more of the nucleotides at positions 1, 2, n-1, and n are phosphorothioate-modified nucleotides. The guide nucleic acid may include one or more bridged nucleotides in the seed region of the guide oligonucleotide.

[0028] The guide nucleic acid may include a segment that may be referred to as a "nucleic acid targeting segment," "nucleic acid targeting sequence," or "seed sequence." In some embodiments, the sequence is 19-21 nucleotides in length. In some embodiments, the "nucleic acid targeting segment" or "nucleic acid targeting sequence" comprises a crRNA. The nucleic acid targeting segment may include a subsegment that may be referred to as a "protein binding segment," "protein binding sequence," or "Cas protein binding segment."

[0029] A "host cell" generally includes an individual cell or cell culture that can be or has been a recipient for a subject vector, such as a vector described herein, into which exogenous nucleic acid has been introduced. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic representation of the entire DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of the invention.

[0030] The term "derivative," when referring to a protein, means that the protein has been modified by adding or removing sequences while retaining its function. The term "functional analog" means a different sequence that performs the same function. The term "variant" means that the protein has been mutated while retaining or enhancing its function.

[0031] CAR-T cells and gene editing tools T cells are immune cells that express a T cell receptor encoded by the T cell receptor alpha constant (TRAC) gene. T cells include CD4 T cells, CD8 T cells, and NK cells, each of which characteristically expresses CD4, CD8, and CD16-CD56 on their cell surface, respectively. Natural killer (NK) cells and CD8 cytotoxic T cells are two types of immune cells that can kill target cells through similar cytotoxic mechanisms. CD4 and CD8 CAR T cell therapy, unlike NK cells, has become a major focus of research and development because NK cell immunotherapy approaches require efficient gene transfer methods in primary NK cells, which current gene editing methods cannot achieve. CD16 and / or CD56 can be used as targets for specific NK cell modification. CD56 and CD16 are key differentiation clusters that define natural killer cells within leukocyte populations, and they can be targeted, for example, by antibodies, as a means to enrich, identify, and characterize NK cells. Bispecific targeting can be achieved by targeting both CD16 and CD56, particularly CD56 Bright, which is the most active NK cell population with respect to anti-cancer activity.

[0032] The present disclosure achieves significant improvements in the efficiency of CAR-T gene editing by providing protein-based interactions to bind to the T cell membrane and internalize genetic material and PNME. The present disclosure achieves transduction efficiencies of greater than 75%, preferably greater than 80%, more preferably greater than 90%, and even more preferably greater than 95%. In contrast, traditional methods only achieve efficiencies on the order of 25-30%. The efficiencies commonly reported in the literature are post-selection efficiencies following steps such as cell sorting, antibiotic selection, or magnetic separation. The present disclosure, on the other hand, reports direct efficiencies without any steps that artificially increase efficiency. Efficiency is calculated based on the entire starting cell population, not just the vector-fed cell population.

[0033] The improved efficiency of the present invention means that the CAR-T cell methods described herein can be applied to NK T cells. The advantages of NK cells are that they generally offer improved safety to subjects receiving them (i.e., lack or minimal cytokine release syndrome and neurotoxicity) and multiple mechanisms for activating cytotoxicity.

[0034] The present disclosure contemplates all types of CAR T cell receptors, including multi-targeting CAR configurations such as: Dual CARs, which co-express two different CARs in one cell; Tandem CARs containing two different scFvs in a single CAR molecule that can be stacked in series or as a loop-like structure. Combinatorial CARs combine two constructs: one carrying a CD3z signaling motif and the other carrying a costimulatory signaling domain. Synthetic Notch (syn-Notch) receptors induce CAR transcription after antigen recognition of their cognate antigens, and Inhibitory CARs (iCARs) inhibit T cell activation after antigen recognition in normal cells.

[0035] The present disclosure provides a protein complex comprising a polynucleotide-modifying enzyme domain (having a functional nuclease catalytic pocket) and an endosomal escape domain, a guide oligonucleotide targeting TRAC, and donor DNA.In some cases, the PNME enzyme is a programmable nuclease.Such nuclease is preferably engineered to target specific DNA or RNA sequence for cleavage.The nuclease is, for example, a CRISPR endonuclease, such as Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, and Cas14.For the purpose of CAR T cell therapy, the CRISPR endonuclease is preferably selected from Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Cas12d, and Cas12e. In some embodiments, CRISPR endonuclease is class II CRISPR endonuclease.In some cases, CRISPR endonuclease is class II, type II, V or VI endonuclease.In preferred embodiments for the purpose of CAR T cell manufacturing, CRISPR endonuclease is type II or type V Cas.In some cases, such nuclease comprises at least one nuclease-deficient nuclease domain.In some embodiments, the CRIPSR endonuclease is encoded by a sequence having at least at least 75% identity, at least 78% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 1, 3, 5, 7, or 9. In some embodiments, the CRIPSR endonuclease has at least 75% identity, at least 78% identity, at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 2, 4, 6, 8 or 10. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0036] In some embodiments, the PNME of the present disclosure is linked to a cationic peptide adapted for binding to negatively charged cell membranes. The cationic peptide imparts a non-covalent positive charge to the protein complex. Physical adsorption of the cationic peptide to the surface of oppositely charged proteins on the cell membrane allows for cationic transfection of the cell membrane. Thus, in such embodiments, cell targeting and internalization are not specific to T cells. However, cationic peptides can be used in vitro when T cells are in culture and specific cell targeting is not required. The cationic peptide can be 10 to 20 amino acids long and can contain repeating or non-repeating positively charged amino acids, such as R and L. In one example, the cationic peptide is SEQ ID NO: 11, which is RRRRRRRLLLLLLLL. On the other hand, in vivo applications of CAR T gene editing generally require specific targeting of T cells. Thus, in other embodiments, the PNME is engineered to have a domain that targets and binds to T cells or specific subtypes of T cells.

[0037] Thus, in some aspects, the present disclosure provides a method for the production of a polynucleotide-modifying polypeptide comprising: Provided is a PNME, wherein an endosomal escape domain is covalently linked to a cell recognition domain, which targets a T cell marker, such as CD4, CD8, CD16, or CD56.

[0038] The cell recognition domain can be a natural or synthetic peptide or nucleic acid domain capable of specific, non-covalent association with a cell surface antigen or receptor. The cell recognition domain can bind to an epitope of a cell surface antigen or receptor. In some embodiments, the cell recognition domain is an antibody or an antigen-binding fragment thereof, or an antibody mimetic. The antibody includes a camelid antibody. The antigen-binding fragment includes a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a fragment produced by a Fab expression library, an Fd fragment, an Fv fragment, a disulfide-linked Fv (dsFv) domain, a single-chain antibody (e.g., scFv) domain, a VHH domain, or a single-domain antibody. Antibody mimetics are non-antibody-derived peptides or nucleic acids that bind with affinity similar to antibodies, and include affibodies, affilins, affimers, affitins, alphabodies, anticalins, atrimers, avimers, aptamers, DARPins, finomers, knottins, Kunitz domain peptides, monobodies, nanoCLAMPs, and linear peptides of 6 to 20 amino acids. Suitable antibody mimetics can be derived from mammalian cells, bacterial cells, or bacteriophage display by systematic evolution of ligands by exponential enrichment (SELEX™) or by a DNA-encoded library approach, for example, involving immobilization of a given antigen on a surface followed by binding selection. In some cases, the cell recognition domain is an aptamer oligonucleotide, e.g., a polyribonucleotide or polydeoxyribonucleotide; Such oligonucleotide aptamers can include non-canonical nucleotides, such as 2'-OMe, 2'-F, or 4'-S nucleotides, 2'-FANA, HNA, or locked nucleic acid residues. In some embodiments, the cell recognition domain comprises a chemical ligand having a molecular weight of less than about 800 Da.Such ligands include small molecule ligands of cell surface receptors, such as folate (which binds to folate receptors), piperidine carboxamide (which binds to FSHR), phenylpyrazole or thienopyrimidine compounds (which bind to LHR), cinacalcet or analogs (which bind to CRF1), or nitrobenzoxadiazole compounds (which bind to EGFR). Such ligands also include protein ligands of cell surface receptors, such as IL2 (which binds to IL2 alpha receptors), EGF (which binds to EGFR), or HFG (which binds to HFGR). In some cases, the cell recognition domain does not directly associate with a cell surface antigen, but rather has the ability to bind to a cell surface receptor or a carbohydrate-selective protein ligand. In some cases, the cell recognition domain comprises a cell surface receptor or a carbohydrate-selective protein ligand. In some cases, the cell surface receptor or carbohydrate-selective protein ligand comprises 5 to 15 amino acids in length. In some cases, the protein ligand is a peptide growth hormone. In some cases, the protein ligand has a globular or cyclic structure.

[0039] In some embodiments, the PNME of the present disclosure is modified to incorporate a display domain to achieve display on the external surface of the PNME targeting a T cell marker, such as CD4, CD8, CD16, or CD56. The PNME can therefore be considered a single protein delivery platform in such embodiments. In some embodiments, "single protein" means that the entire sequence of a single protein is contained between the N-terminus and C-terminus, and no linkage or fusion occurs at the N- or C-terminus. In some embodiments, the display domain of the present disclosure is positioned at least 25 amino acids after the N-terminus or at least 25 amino acids before the C-terminus of the polynucleotide modifying enzyme. In some embodiments, the display domain is positioned at least 30, at least 40, at least 50, at least 75, or at least 100 amino acids after the N-terminus, or at least 30, at least 40, at least 50, at least 75, or at least 100 amino acids before the C-terminus. Cell-penetrating peptides have been used as platforms for biomolecule delivery. However, cell-penetrating peptides generally do not have the same specificity and success as delivery platforms involving immunoglobulin approaches. An exemplary immunoglobulin approach can be one in which antibodies or antibody mimics are first screened against a defined biological target, such as a receptor, and then verified for target recognition. CRISPR proteins are fused with peptides, such as RGB, SV40NLS, at the C- and N-termini of the protein, or are charged-associated with CRISPR RNPs that affect non-specific cell entry. To affect organ tropism or preferential tissue accumulation, receptor-specific binding is preferably a feature of PNMEs that act as cell-penetrating peptides. In some embodiments, the cell recognition domain is the peptide sequence of SEQ ID NO: 12:QQYYSYRT, which targets CD4.

[0040] In some embodiments, the PNMEs of the present disclosure have been modified to include an antigen-binding domain in a loop located on the outer surface of the PNME. The PNME of this embodiment is also a single protein because the antigen-binding domain is inserted into an external loop between the N- and C-termini of the PNME. It has surprisingly been found that large domains (e.g., more than 20 amino acids, more than 50 amino acids, more than 100 amino acids, 100-200 amino acids, or 136-156 amino acids) can be incorporated into the loop of the PNME without interfering with the folding of the catalytically active nuclease pocket of the PNME. Indeed, antigen-binding domains have been found in Fabs, single-domain antibodies (sdAbs), Vs, and other polynucleotide-modifying enzymes located in loops on the outer surface of the PNME. HH , or a camelid antibody domain. A linker domain is preferably included upstream of the antigen-binding domain, which helps the three-dimensional conformation of the PNME maintain its catalytic activity while providing specific targeting to the desired cell type. In some embodiments, the linker domain has a size of 0-30, 8-30, 10-30, 12-28, 16-25, or 18-23 amino acids. In some embodiments, the antigen-binding domain targets a T cell marker, although the antigen-binding domain may target any other cell type or cell receptor. For example, the antigen-binding domain can target any of the targets provided in Table 2 or any of the epitopes in Table 3 for cancer. In one example, the PNME is spCas9, and the linker and antigen-binding domain are inserted at ser1154. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 3-1] [Table 3-2] [Table 3-3]

[0041] In some embodiments, CRISPR-modified nucleases, such as C9mAur or M7ma, are modified with peptides for general delivery (cationic and nonspecific cell entry), or grafted with CDR peptide sequences into the loops of a C9-modified (C9m) scaffold (receptor-specific binding and delivery via receptor-mediated endocytosis), or C9m is fused to an anti-CD4 nanobody, either chemically or as part of a single protein expressed in a protein expression system. A generalized transfection option is provided by complexing any of the CRISPR enzyme derivatives described herein with cationic peptides. In the formulation, the protein complex is provided by complexing a chimeric antigen receptor (sequence) and a biotinylated donor encoding a polynucleotide-modifying enzyme through monoavidin:biotin interaction.

[0042] Figure 1 shows that homology-directed repair (HDR) enhanced formulation and delivery. Generalized delivery is defined herein as driven by cationic peptide complexation of PNME, where the nuclease is Cas9, Cas12, or a type II or type V CRISPR system enzyme capable of generating double-strand breaks. Delivery is cationic in nature, interacting nonspecifically with oppositely charged cell membranes. Receptor-mediated delivery requires that the PNME possess a domain capable of recognizing a cellular receptor or marker so that the PNME can be selectively internalized.

[0043] In some embodiments, the PNMEs of the present disclosure can be combined with an endosomal escape domain to form a fusion polypeptide. The endosomal escape domain allows the fusion peptide to exit the endosome and enter the cytoplasm after endocytosis. The endosomal escape domain can be incorporated into the sequence of the PNME or can be linked at the N- or C-terminus of the PNME. Table 4 details non-limiting examples of endosomal escape (EE) domains. [Table 4]

[0044] Double-strand break caused by CRISPR can be repaired by non-homologous end joining (NHEJ), or through homology-directed repair (HDR) or single-strand annealing.For CRISPR editing, NHEJ is preferred, because for most of the cell cycle, NHEJ is the dominant method of resolving double-strand breaks through the action of Ku70 / 80, artemis, DNA-Pk and lig4, which results in small insertions and deletions that can inactivate genes.When donor template exists and cell cycle is permissive (S1 G2), homologous recombination can guide repair, where donor provides the template for resolving double-strand breaks.Unfortunately, HDR with CRISPR is very inefficient due to many factors, such as the availability of donor DNA at the position of double-strand break formation and the limited period of cell cycle when HDR is preferred. To eliminate donor availability, donor DNA can be linked to a fusion CRISPR protein via biotin interaction, and the nuclease is expressed with an attached monoavidin domain. This is advantageous compared to other gene delivery systems, such as viral delivery (e.g., AAV), due to packaging volume limitations. In fact, the CRISPR nuclease and the donor must be delivered separately, and the benefits of colocalization are lost in both time and spatial colocalization. Other iterations of colocalization use SNAP tags, aptamers, and nanoparticle systems. The advantage of this system is the use of proteins with additional endosomal escape function and optional delivery via generalized cationic or preferential receptor-mediated delivery.

[0045] In some embodiments, the PNME further comprises a hapten-binding domain for linking additional protein or nucleic acid ligands to the PNME. A "hapten-binding domain" is a peptide or oligonucleotide domain that binds to a hapten. A "hapten" refers to a small molecule that, when combined with a larger carrier, such as a protein, has the ability to bind with high affinity to an antibody or antibody mimetic (the "hapten-binding domain"). In some embodiments, the hapten / hapten-binding domain pair is derived from a natural protein or an engineered variant thereof, such as a biotin / avidin pair or an amylose / MBP pair. Engineered alternatives for biotin include D-desthiobiotin. Alternatives for avidin include streptavidin, neutravidin, and CaptAvidin.In some embodiments, the hapten / hapten-binding domain pair is a synthetically engineered pair, such as 3-methylindole / anti-3-methylindole monoclonal antibody (e.g., 14G8, 3F12, 4A1G, 8F2, or 8H1 monoclonal antibody), fumonisin B1 / anti-fumonisin antibody, 1,2-naphthoquinone / anti-1,2-naphthoquinone antibody, 15-acetyldeoxynivalenol / anti-15-acetyldeoxynivalenol antibody, (2-(2,4-dichlorophenyl)-3(1H-1,2,4-triazol-1-yl)propanol) / anti-(2-(2,4-dichlorophenyl)-3(1H-1,2,4-triazol-1-yl)propanol) antibody, 22-oxacalcitriol / anti-22-oxacalcitriol antibody, (24,25(OH)2D3) / anti-(24, 25(OH)2D3) antibody, 2,4,5-trichlorophenoxyacetic acid / anti-2,4,5-trichlorophenoxyacetic acid antibody, 2,4,6-trichlorophenol / anti-2,4,6-trichlorophenol antibody, 2,4,6-trinitrotoluene / anti-2,4,6-trinitrotoluene antibody, 2,4-dichlorophenoxyacetic acid / anti-2,4-dichlorophenoxyacetic acid antibody, 2-hydroxybiphenyl / anti-2-hydroxybiphenyl antibody, 3,5,6-trichloro-2-pyridinol / anti-3,5,6-trichloro-2-pyridinol antibody, 3-acetyldeoxynivalenol / anti-3-acetyldeoxynivalenol antibody, 3-phenoxybenzoic acid / anti-3-phenoxybenzoic acid antibody, digoxin / anti-digoxin antibody, fluorescein / anti-fluorescein antibody, or hexahistidine / Ni-NTA. The hapten-binding domain can be located N- or C-terminal to the PNME, or both. The hapten-binding domain can be separated from another domain described herein by a linker or can be fused directly to the domain sequence without intervening amino acids. In some cases, the hapten-binding domain is within a linker domain that separates two other domains of the PNME. In some cases, the PNME comprises at least one, at least two, at least three, at least four, or at least five or more hapten-binding domains.

[0046] In some embodiments, a composition is provided comprising a PNME and a hapten-binding domain. The composition may further comprise a peptide, protein, oligonucleotide, or polynucleotide linked to a corresponding hapten. The oligonucleotide may comprise deoxyribonucleotides or ribonucleotides. The oligonucleotide may comprise a single-stranded or double-stranded oligonucleotide.

[0047] In some embodiments where the PNME comprises a hapten-binding domain and a programmable or site-specific nuclease, the PNME further comprises a nucleic acid (e.g., a repair template or donor DNA) having a homology arm complementary to a region adjacent to the target site for the programmable or site-specific nuclease. In this manner, the nuclease can be delivered to the cell in the vicinity of the site to be cleaved. In some cases, the repair template or donor DNA is a single-stranded or double-stranded DNA repair template or donor DNA, comprising, from 5' to 3', a first homology arm comprising a sequence of at least about 20 nucleotides 5' to the target sequence, an insert DNA sequence or a region of at least about 10 nucleotides, and a second homology arm comprising a sequence of at least about 20 nucleotides 3' to the target sequence. In some embodiments, the first or second homology arm comprises a sequence of at least about 20, 40, 50, 80, 120, 150, 200, 300, 500, or 1000 nucleotides. In some embodiments, the 5' and 3' homology regions have different lengths. In some embodiments, the 5' and 3' homology regions have the same length. In some embodiments, the repair template or donor DNA is a single-stranded polynucleotide, and the 5' homology region comprises 50-100 nucleotides, and the 3' homology region comprises 20-60 nucleotides. In some embodiments, the 3' end of the 5' homology region is homologous to a sequence within 5 nucleotides of the double-stranded break. In some embodiments, the 5' end of the 3' homology region is homologous to a sequence within 5 nucleotides of the double-stranded break. The insert region can include the coding sequence of a gene that contains an exon, an intron, a transgene, a stop codon (e.g., a stop codon in-frame with the gene ORF into which it is inserted), at least one nonsense or missense mutation, or a mutation that eliminates activity of a PAM site near the sequence targeted by the PNME CRISPR enzyme.Exemplary transgenes include selectable markers, such as BlaS, HSV-tk, puromycin N-acetyl-transferase, or Tn5 NEO genes, which can be used to select for cells that have undergone recombination with the donor DNA or repair template. Exemplary transgenes also include detectable labels, such as fluorescent enzymes, protein sequences that allow high-affinity detection with antibodies, epitope tags, or fluorescent proteins.

[0048] In one example, PNME is constructed on the C9m scaffold, Cas9 is fused with a monoavidin domain, and a peptide sequence or antigen-binding domain is grafted onto the loop domain identified above. The antigen-binding domain (e.g., VHH) can be selected as a binder that targets a specific receptor, such as CD4, CD8, CD16, or CD56. The grafting of the antigen-binding domain can be achieved by inserting the corresponding DNA sequence into an expression vector encoding C9m.

[0049] In some embodiments, the PNME can comprise a nuclear localization sequence (NLS). The NLS can be located at the N- or C-terminus of the PNME, or both. The NLS can be separated from the PNME peptide sequence by a linker or fused directly to the PNME sequence without intervening amino acids. In embodiments, the PNME comprises at least one, at least two, at least three, at least four, or at least five or more NLSs. In some embodiments, the NLS comprises 7 to 25 amino acid residues. In some embodiments, the NLS is derived from a mammalian nuclear entry protein, such as a splicing factor or transcription factor. In some embodiments, the NLS interacts with an importin. In some embodiments, the NLS is a bipartite NLS, in which the amino acids in the N-terminal portion of the NLS involved in importin recognition and the amino acids in the C-terminal portion of the NLS involved in importin recognition are separated by an amino acid sequence not involved in importin recognition. In some embodiments, the NLS comprises at least one sequence depicted in Table 5 below, or a combination of sequences from Table 5 (i.e., SEQ ID NOS: 22-37), a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a sequence set forth in Table 5, or a sequence identical to any of the sequences in Table 5. When more than one NLS is included in a PNME or PNME composition, the NLSs may comprise the same sequence or different sequences. In some embodiments, more than one NLS sequence is included (e.g., an SV40 NLS), and the NLS sequence may be disposed in a linker between the PNME and the monoavidin domain. [Table 5]

[0050] In some embodiments, the PNME is bispecific, i.e., it has two domains or peptide sequences that can recognize the same or different cell receptors (e.g., T cell receptors). The cell recognition domain, display domain, and / or antigen-binding domain can be combined to form bi- or multi-specific protein complexes. Thus, bispecific PNMEs can be generated, effectively having two receptor-binding domains (e.g., a display domain such as a CDR and a cell recognition domain, or a display domain such as a CDR and an antigen-binding domain such as an inserted VHH).

[0051] In some embodiments, a CRISPR system is provided for introducing a CAR by first formulating a CRISPR protein complex via either a cationic or receptor-mediated delivery mechanism using a TRAC-specific sgRNA molecule. The intention is to introduce a chimeric antigen receptor by forming a double-stranded break in the early exon of the TRAC receptor, eliminating its native expression, and placing the CAR under the control of the endogenous promoter of the TRAC gene. Figure 2 illustrates the mechanism of action of the system with respect to receptor-mediated delivery of a protein complex that targets the CD4 receptor for delivery. The anti-CD4 domain binds to the cellular receptor and is internalized by receptor-mediated endocytosis. Next, endosomal escape is mediated by an endosomal escape domain (e.g., an endosomal peptide escape sequence), and nuclear translocation is achieved by an NLS. In the nucleus, homologous recombination occurs using genomic DNA and repair template homologous sequences (left and right homology arms) flanking the CAR insertion on the donor DNA.

[0052] To achieve co-delivery, once the protein complex is formed (e.g., CRISPR nuclease + guide RNA), the relationship between biotin and monoavidin is exploited by biotinylating the CAR-encoding donor DNA molecule using a 5', 3', or internal biotin label. Mixing CRISPR nucleases in equimolar parts allows binding of the donor to the protein complex. At this point, the enhanced HDR CRISPR complex is ready for delivery into cells. If a CRISPR protein complex is used without an anti-CD4 binding domain and a cationic peptide is used, this allows for non-specific delivery via cationic interactions of the peptide with the oppositely charged cell membrane. If an anti-CD4 domain is present, delivery to the cell is achieved by interaction with the CD4 receptor on T cells or T cell models.

[0053] Compared to viral vectors, the described gene delivery system has low immunogenicity, increased biosafety, reduced manufacturing costs, and the ability to transduce large gene fragments greater than 100 kb in length, making it an advantageous means for CAR insertion into the genome of T cells, including NK cells, with long-term persistent expression.

[0054] In some embodiments, a vector is provided that comprises a nucleotide sequence encoding PNME. In some cases, the vector further comprises an endosomal escape domain and a hapten-binding domain in the same open reading frame (ORF) as PNME. A "vector" is a nucleic acid sequence that has the ability to transfer other operably linked heterologous or recombinant nucleic acid sequences into target cells. In some examples, the vector is a minicircle, a plasmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a cosmid, a phagemid, a bacteriophage genome, or a baculovirus genome. Suitable vectors also include vectors derived from bacteriophages or plant, invertebrate, or animal (including human) viruses, such as CELiD vectors, adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or pseudotyped combinations thereof, such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV or its self-inactivating or SIN version, or a pseudotyped version thereof), herpesvirus (e.g., HSV- or EBV-based), lentiviral vectors (e.g., HIV-, FIV-, or EIAV-based, or a pseudotyped version thereof), adenoviral vectors (e.g., Ad5-based; including replication-deficient, replication-competent, or helper-dependent versions thereof), or baculoviral vectors (suitable for transfecting insect cells as described herein). In some embodiments, the vector is a replication-competent virus-derived vector.

[0055] Thus, in some aspects, the present disclosure also provides a host cell comprising any of the vectors described herein. In some embodiments, the host cell is an animal cell. The term "animal cell" encompasses any animal cell, including, but not limited to, invertebrate, non-mammalian vertebrate (e.g., bird, reptile, and amphibian), and mammalian cells. Many mammalian cell lines are suitable host cells for recombinant expression of a polypeptide of interest. Mammalian host cell lines include, for example, COS, PER.C6, TM4, VERO076, MDCK, BRL-3A, W138, Hep G2, MMT, MRC 5, FS4, CHO, 293T, A431, 3T3, CV-1, C3H10T1 / 2, Colo205, 293, HeLa, L cells, BHK, HL-60, FRhL-2, U937, HaK, Jurkat cells, Rat2, BaF3, 32D, FDCP-1, PC12, M1x, mouse myelomas (e.g., SP2 / 0 and NS0), and C2C12 cells, as well as transformed primate cell lines, hybridomas, normal diploid cells, and cell lines derived from in vitro culture of primary tissues and primary explants. Any eukaryotic cell capable of expressing recombinant and / or transgenic proteins may be used in the disclosed cell culture methods. Numerous cell lines are available from commercial sources, such as the American Type Culture Collection (ATCC). The host cell can be a CHO cell. In some embodiments, the host cell is a bacterial cell suitable for protein expression, such as a derivative of the E. coli K12 strain. In some embodiments, the host cell comprises a plant cell into which a gene has been introduced via a vector, the single-stranded RNA virus, tobacco mosaic virus. The "host cell" can be an insect cell, which is utilized to produce large quantities of polypeptides according to the present disclosure. In some embodiments, a baculovirus system, which offers all the advantages of higher eukaryotes, is utilized.Host cells for the baculovirus system include, but are not limited to, the Spodoptera frugiperda ovarian cell lines SF9 and SF21 and the Trichoplusia ni egg-derived cell line High Five.

[0056] In some embodiments, the PNME described herein is delivered to cells (e.g., in vitro or in vivo) via a specifically designed pharmaceutical composition or dosage form. The pharmaceutical composition may include sterile water, along with pharmaceutically acceptable excipients and optional electrolytes to ensure the composition is isotonic. Because the PNME described herein and pharmaceutical compositions comprising it do not require chemical transfection agents to enter cells, in some embodiments, the liquid formulation for delivery does not include polyetherimide (PEI), polyethylene glycol (PEG), polyamidoamine (PAMAM), or sugar (dextran) derivative polymers containing more than three subunits.

[0057] The CAR-T cells of the present disclosure can be engineered to target diverse antigens, to enhance proliferation and persistence in vivo, to increase infiltration into solid tumors, to overcome resistant tumor microenvironments, and ultimately to achieve effective anti-tumor responses.

[0058] In some embodiments, the T cell target is a CD8 T cell. Cytotoxic CD8 T cells can eliminate tumor cells through the recognition of peptide epitopes presented on major histocompatibility complex class I (MHC-I) molecules by the alpha-beta T cell receptor (αβTCR). T cells can recognize peptides derived from tumor-associated antigens, cancer-testis antigens, viral antigens (in the case of virally derived tumors), and neoantigens. Neoantigens are peptides derived from mutated "self" proteins that the immune system detects as "non-self." Many neoantigens are "private" to individual tumors, and immunity to these antigens can be exploited with immunotherapy, such as checkpoint blockade or personalized vaccines. Some neoantigens are derived from common or "hotspot" mutations, such as those occurring in RAS proteins and p53. The RAS family of small GTPases (H, N, and KRAS) are among the most commonly mutated oncogenes in cancer. Among them, the G12D mutation in KRAS occurs most frequently.

[0059] In some embodiments, a safety check is introduced into the CAR T cells. This can be done by including a suicide gene to destroy the CAR cells in the event of a cytokine storm. This can be achieved by packaging the gene instead of GFP on the CAR and then minimizing the donor size in base pairs. In such embodiments, interchangeable CAR heads can be obtained by substituting a monoavidin domain for the scFV, allowing generic CAR T cells to be created and easy targeting exchange. Anti-CD4 targeting DNA complexes can be used with standard CAR designs but with the exchange of scFV with monoavidin (MAV) to carry and introduce donor DNA. After generation of CAR-CD4+ T cells, activation of the cells toward specific cell markers can be achieved by intravenously injecting biotinylated scFVs, nanobodies, cyclic peptides, or antibody mimics that then bind to the CAR-CD4+ T cells. As long as the biotinylated ligand is in excess, expansion occurs due to MAV:biotinylated ligand interaction with the target cells; as expansion occurs, the new cells lack the biotinylated target. These cells arise in a situation where the biotinylated ligand is in excess and are distributed systemically, readily associating with the ligand and targeting the appropriate cells. A conceptual advantage of such an embodiment is the ability to selectively bind the ligand. Because selection pressure results in the selection of cancer cells lacking the original target receptor (e.g., CD19), the subsequently retained receptor can be readily selected for. For example, CD19 targeting can be switched to CD22 targeting by intravenous injection of a CD22-biotinylated VHH, which allows targeting switching without further genetic manipulation.

[0060] Autologous CAR-T cell therapy can include several steps. First, T cells are isolated from the blood of a patient or donor. Subsequently, the cells are transduced with a gene encoding a CAR using the protein complex described herein. The CAR-modified immune cells are expanded until a sufficient number of cells is achieved, and then adoptively transferred into the patient to fight malignant cells. Prior to the infusion of CAR-modified immune cells, lymphocyte depletion is performed in most therapeutic situations to enable efficient cell engraftment.

[0061] Conventional cancer treatments include radiation therapy, chemotherapy, and surgery. These are associated with poor efficacy and serious side effects. Therefore, new strategies with higher efficacy and fewer complications, such as CAR-T-based immunotherapy, have been developed. Immunotherapy is the modification and enhancement of the host immune system to fight different pathologies, such as cancer. Adoptive cell therapy (ACT) is a type of immunotherapy that involves the application of immune cells to treat cancer, of which CAR T-cell therapy is an example.

[0062] In some embodiments, CAR-T cell therapy can be combined with other therapies, such as chemotherapy, radiation therapy, and immune checkpoint blockade. CAR-T cell therapy can allow for a reduction in the dose of chemotherapy or radiation therapy when used in combination, which would reduce the side effects suffered by patients receiving these traditional treatments.

[0063] The evolution of resistance in cancer populations is a major factor limiting patient remission and cure. One way to mitigate the occurrence of resistance is to create bispecific CAR T cells; in this way, if one receptor is counterselected, the other receptor can replace it and be relied upon for target cell binding. The situation in which the two receptors of bispecific CAR T cells are counterselected and cancer evolves to prevent or reduce their expression has a much lower probability than the case in which mutational changes in a single receptor are selected in the case of monospecific CAR T cells. One alternative approach described above is MAV-CAR, in which interchangeability is achieved in a "headless or exchange platform" by the addition of a biotinylated receptor binding agent. Yet another approach to address cancer resistance is to evaluate patients for changes in cancer cell expression (e.g., by flow cytometry) and then inject the protein complex of the present disclosure with donors targeting an alternative receptor verified to be expressed on the cancer cells. This approach may also benefit cancer patients, who have been used to pre-select suitable binders from libraries of VHHs, antibody mimetics, or peptides to provide robust validation prior to treatment of cell recognition.

[0064] Examples of diseases that can be treated with the present CAR T cell therapy and some exemplary cellular targets for each disease are provided as follows: multiple myeloma (MM) (CD138, CS1), glioblastoma (EGFR, EGFRVIII, CD73, HER2), lymphoma (CD22, CD19, CD4), acute lymphocytic leukemia (ALL) (CD7, CD19, CD5, FLT3), acute myeloid leukemia (AML) (CD33, CD19, CD4, CD123), chronic lymphocytic leukemia (CLL) (CD19), breast cancer (HER2, EpCAM, TF, EGFR), colorectal cancer (HER2, EpCAM, NKG2D, MUC1), and leukemia (HER2, EpCAM, TF, EGFR). 1), ovarian cancer (HER2, mesothelin), renal cell carcinoma (RCC) (HER2, EGFR), prostate (PSMA), neuroblastoma (GD2, CD244, CD276), melanoma (GPA7), Ewing's sarcoma (GD2), hepatocellular carcinoma (HCC) (GPC3), pancreatic cancer (MUC 1), gastric cancer (MUC 1), non-small cell lung cancer (MUC 1), hepatocellular carcinoma (MUC 1), glioma (MUC 1), triple-negative breast cancer (TNBC) (MUC 1), and B-cell malignancies (CD19, CD20).

[0065] In some embodiments, allogeneic CAR NK cells are produced using the delivery and gene editing described herein. Allogeneic CAR NK cells generally have a reduced risk of graft-versus-host disease (GVHD). Furthermore, cytokine release syndrome (CRS) and neurotoxicity are less likely to occur in CAR-NK immunotherapy, partly due to the different spectrum of cytokines secreted: activated NK cells typically produce IFN-γ and GM-CSF, while CAR-T cells primarily induce cytokines highly associated with CRS and severe neurotoxicity, such as IL-1a, IL-1Ra, IL-2, IL-2Ra, IL-6, TNF-α, MCP-1, IL-8, IL-10, and IL-15.

[0066] In some embodiments, subsequent genetic modification can be performed after the introduction of the CAR construct with the protein complex of the present disclosure.For example, the genetic ablation of PD1 can improve T cell function, and in the case of cancer treatment, can also improve tumor targeting and treatment efficacy.In the embodiment where NK cells are CAR T cells, B2M can be genetically ablated or other genetic modification can be made to interfere with HLA presentation.

[0067] Allograft rejection is mainly driven by CD8 T cells, CD4 T cells, NK cells, and to a lesser extent, macrophages.However, in the context of CAR T cell therapy, the relative contribution of these cell types to allograft rejection may vary depending on their absolute number and reconstitution kinetics after preconditioning regimen.In some embodiments, dual targeting approach and adaptor CAR are used to avoid treatment resistance caused by antigen loss. [Example]

[0068] material The following reagents were purchased from Wisent™: Dulbecco's Modified Eagle's Medium (DMEM), heat-inactivated fetal bovine serum (FBS) Premium, penicillin / streptomycin (Pen / Strep), F12, Luria Bertani (LB), peptone, yeast extract, and super broth. The following reagents were purchased from Biobasic: ethanol, isopropanol, phosphate-buffered saline (PBS), DNA ladder 1 kb, DNA ladder 100 bp, protein ladder 250 kda, 33:1 Acrylamide premix, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (TRIS), glucose, arabinose, NaCl, KCl, HCl, ammonium hydroxide, calcium chloride, SOC broth, ethylenediaminetetraacetic acid (EDTA), agar, agarose, Tris-acetate-EDTA (TAE) 50X buffer, micropipette tips, serological pipettes (10 ml, 25 ml, 5 ml), 15 ml sterile tubes, 1.5 ml sterile tubes, 50 ml sterile tubes, PCR tubes, culture plates (6-well, 12-well, 24-well, 96-well flat-bottom, 96-well round-bottom, 10 cm plates), and plastic Petri dishes. The following Monarch RNA Cleanup Columns were purchased from BioLabs™: Monarch DNA, RNA, Plasmid Prep Kit and restriction enzymes, T7 endonuclease I (and buffer NEB 2.0), protease K, hifi assembly mix, and PCR enzymes. PCR enzymes were obtained from Transgen™, and primers from Biocorp™. Mutagenesis services were provided by ABM™. Primers and gblocks were obtained from IDT™. Large DNA synthesis was performed using TwistBio™. The following reagents were purchased from Thermofischer™: pierce dye removal columns, 4 ml bacterial culture tubes, PCR enzymes (DIrect Phire / Phusion), various fluorescent dyes (DAPI, NHS fluorescent), photoprobes: Cy5.5 NHS ester and TAMRA nhs ester.NiNTA beads and endotox kit were purchased from Genscript™.

[0069] Chimeric antigen receptor DNA donor construct The donor DNA for the CAR antigen receptor was coded in the following manner to contain the domains required for CAR function: Left homology arm (LHA): a sequence homologous to the exon 1 TRAC locus located around the guide position that directs the position of the double-strand break; CD28 signal peptide: Translocation to the cell membrane, Three Flag tags: for identification of the CAR construct; Anti-CD19 scFV: for recognition of CD19 on B-cell lymphoma cells; CD8 hinge region: connects anti-CD19 to the transmembrane domain and allows signal transduction through the CAR protein construct; presentation of the anti-CD19 domain and receptor expression are also influenced by the CD8 hinge region sequence and amino acid length. CD28 transmembrane domain: allows receptor presentation in lipid bilayers, Cd3z: Initiation of intracellular signaling and T cell anticancer function, P2A: a cleavage domain that removes the downstream peptide sequence from the remainder of the CAR construct, in this case releasing the eGFP fluorescent protein tag; eGFP: a green fluorescent protein tag to confirm both in-frame CAR insertion into the genome and full-length receptor expression; the presence of the P2A cleavage sequence prevents eGFP from becoming part of the final CAR receptor. A post-transcriptional regulatory element (WPRE) sequence that is in-frame with GFP to improve RNA stability and protein yield, and Right homology arm (RHA): a sequence homologous to the exon 1 TRAC locus located around the guide position that directs the location of the double-strand break.

[0070] A map of the CAR construct is shown in Figure 3.

[0071] Vector Generation Proteins were expressed in E. coli using bacterial expression vectors employing the T7 promoter. Inserts were synthesized to encode the pNME and complementary sequences. The vectors incorporate a pB322 origin, a repressor-of-primer (ROP) element for low copy number, a kanamycin or ampicillin resistance gene, a Lac repressor to inhibit transcription until isopropyl β-D-1-thiogalactopyranoside (IPTG) is introduced, a T7 promoter, and a ribosome binding site, completing the basic structure of the expression vector. Vectors and inserts were either ordered through commercial suppliers or prepared from libraries of DNA segments for each component and assembled using either Golden Gate Assembly or Gibson Assembly. Base C9m and M7 bacterial expression vectors were synthesized by assembly cloning.

[0072] Site-directed mutagenesis services from commercial suppliers were used to graft inserts of less than 15 amino acids or less than 30 DNA base pairs into the vector sequence, where the base C9m and M7 nuclease expression vectors were template vectors, and Inserts were determined by the desired amino acid sequence required at the insertion site, optionally labeled "SP3." The SP3 site is located on an external loop of spCas9 and was identified as a suitable location for insertion of an antigen-binding domain. More specifically, site SP3 is ser1154, located as part of the external and non-hidden loop domain. The determined designs included "Zero" - no linker, "L1" - an N-terminal linker to improve VHH presentation, and "L2" - an alternative linker sequence of 23 amino acids to improve VHH presentation and provide greater flexibility in VHH presentation. [Table 6]

[0073] Creation of a common backbone for insertion of all fragments The common C9mAur fragment was amplified using the following primers: C9m_fwd and C9m_rev. The template for amplification was the C9mAur vector. The product size was confirmed by gel electrophoresis. The fragment linearized the plasmid and cleaved C9mAur at the position of the loop domain we intended to clone into. A Dpn1 treatment was performed to digest the template plasmid. Dpn1 was first inactivated (PCR cleanup column and quantification of the fragment) and Dpn1 digestion was confirmed by DH5a transformation resulting in zero colonies.

[0074] Creating a "Zero" Linker Insert: C4n C4n1 amplified fragments were performed using primers minC4n_fwd and commonC4n_rev, and the template for amplification was the C4n vector. Product size was confirmed by gel electrophoresis. Dpn1 treatment was performed to digest the template plasmid. Dpn1 was first inactivated (PCR cleanup column and quantification of the fragment), and Dpn1 digestion was confirmed by DH5a transformation resulting in zero colonies. The resulting fragments were purified and quantified, and then used for cloning.

[0075] Addition of a linker (L) to C4n Two linkers (L1 and L2) were added to C4n, "Zero" since it contains zero linkers. Primers L1 For and L2 For from Table 7 were used to add 5' linkers to C4n to obtain L1-C4n and L2-C4n (i.e., the template for amplification was the C4n vector). A common reverse primer, common C4n_rev, was used in the amplification. The amplification created a reverse overhang using the common primer and introduced a linker at the 5' end of the C4n VHH sequence. PCR clean-up purification of both L1-C4n and L2-C4n was then performed. [Table 7]

[0076] Introduction of overhangs for Gibson cloning using the C9mAur fragment Fragments with overhang primers were amplified to introduce overhangs into the C9m fragment. Amplification of L1-C4n was performed using the L1_c4n2c9m_fwd forward primer, and amplification of L2-C4n was performed using the L2_C4n2c9m_fwd forward primer. The common reverse primer C4n_Comm_rev was used in the amplification. Product size was confirmed by gel electrophoresis. Dpn1 treatment was performed to digest the template plasmid. Dpn1 was first inactivated (PCR cleanup column and quantification of the fragment), and Dpn1 digestion was confirmed by DH5a transformation resulting in zero colonies. The resulting fragments were purified and quantified, and then used for cloning.

[0077] The resulting vector The synthesized vectors are shown in Figures 4 to 8. More specifically, Figure 4 shows a vector map for M7 Mav anti CD8. Figure 5 shows a vector map for M7-Mav-anti CD4. Figure 6 shows a vector map for C9mAur. Figure 7 shows a vector map for C9mC4. Figure 8 shows a vector map for C9C4 anti CD4n1. [Table 8-01] [Table 8-02] [Table 8-03] [Table 8-04] [Table 8-05]

Table 8-06

Table 8-07

Table 8-08

Table 8-09

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

[0078] Assembly The assembly consisted of the following steps: 1) PCR amplification, 2) Gibson assembly, and 3) colony screening. PCR amplification was performed with the following steps repeated for 32 cycles: a) 98°C for 30 seconds, b) 98°C for 8 seconds, c) 72°C for 30 seconds, followed by 72°C for 5:50 minutes and 82°C for 2 minutes. The PCR mix was prepared by mixing 10 μL of 5x Q5 buffer, 1 μL of 10 mM dNTPs, 2 μL of 5x Q5 enhancer, 2 μL of template (C9mAur-30 ng / μL), 0.5 μL of Q5 High-Fidelity Polymerase, 1 μL of forward and reverse primers (25 μM), and 32.5 ml of deionized water. The PCR product (11 kb) was treated with Dpn1 (Thermo Scientific™) twice for 1 hour at 37°C, and then purified using a Qiagen™ purification kit. Gibson assembly was performed using a solution with concentrations of 80 ng / μL vector, 200 ng / μL C4n, 290 ng / μL for L1, and 280 ng / μL for L2. The Gibson assembly master mix was obtained by mixing 1 μL vector, 1.5 μL of 200 ng / μL C4n, and 1.5 μL of 290 ng / μL L1 and 280 ng / μL L2. The Gibson master mix was completed by adding water to a total volume of 20 μL. The mix was incubated at 50°C for 30 minutes. Transformation was performed using dh5alpha and plated on agar plates.

[0079] To perform colony screening, PCR amplification was performed using 18.2 μL of water, 3 μL of Taq buffer, 2.4 μL of 25 mM MgCl2, 3 μL of 2 mM dNTPs, 2.5 μL of 10x enhancer, 0.3 μL of T7 promoter (600 μg / mL), 0.3 μL of T7 terminator (600 μg / mL), and 0.3 μL of Taq polymerase (5 μL / μL). The PCR program was 94°C for 2 minutes, followed by 29 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 68°C for 1 minute / kb, followed by a temperature hold at 72°C for 10 minutes, with a final temperature of 4°C. The resulting fragments were sequenced by Sanger sequencing using the c9mfwdscreen forward primer and c9mrevscreen reverse primer.

[0080] Sequence characterization for engineered nucleases "Zero," "L1," "L2," and "L3." The VHH domain was inserted without a linker, and the resulting protein was labeled "Zero." The VHH domain was inserted with a 16 amino acid linker, L1, and the resulting protein was labeled "L1." The VHH domain was inserted with a 23 amino acid linker, L2, and the resulting protein was labeled "L2." The complementarity determining region (CDR) QQYYSYRT (SEQ ID NO: 63) was inserted, and the resulting protein was labeled "L3." The sequences for Zero, L1, L2, and L3 are provided in Table 9 below.

[0081] The sequence was generated by Gibson assembly / NEB hifi assembly, in which the C9m backbone was opened at position ser1174, and homology arms were generated by PCR in the anti-CD4 VHH encoded in the DNA. Linkers were simultaneously added by overhang PCR. Fragments were purified by silica spin column and assembled by Gibson assembly using fragments representing the Zero, L1, and L2 designs. Sequence verification of the reassembly plasmid and inserts indicated that all inserts were in-frame. Test gel filtration confirmed that the protein was purifiable and that tobacco etch virus (TEV) protease cleavage was functional. [Table 9-01] [Table 9-02] [Table 9-03] [Table 9-04] [Table 9-05] [Table 9-06] [Table 9-07] [Table 9-08] [Table 9-09]

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

[0082] Figures 9A and 9B show the expression of Zero, Figures 9C and 9D show the expression of L1, and Figures 9E and 9F show the expression of L2, as well as their purification by fast protein liquid chromatography (FLPC). Gel images are used to indicate the protein content and molecular weight of fractions collected from FLPC gel filtration. Fractions labeled "1" in Figures 9A, 9C, and 9E are eluted fractions. Fractions labeled "2" are fractions obtained after TEV protease treatment. Fractions labeled "3" and above are fractions obtained from FLPC on Superdex200™ using 0.5 M KCl, 20 mM HEPES, pH 7.5. Briefly, proteins were expressed in E. coli (De3 BL21 strain) using standard IPTG or autoinduction methods for T7 promoter-controlled expression. Overnight expression at 18°C ​​was followed by cell lysis using sonication and mild detergent lysis, followed by the first step of his-tag purification using a Ni-nitriloacetic acid (NTA) column. After TEV cleavage of the MBP domain and buffer exchange, the concentrated protein fraction was loaded onto a Superdex200™ gel filtration column for size-based purification and cleanup.

[0083] The enzymatic DNA cleavage activity of Zero, L1, and L2 was measured over a 3-hour time course by incubating the enzymes with a 100-bp DNA template at 37°C. 0.5 μL of proteinase K was added to the incubation to quench the reaction at different time points (30 minutes, 1 hour, 1 hour 30 minutes, 2 hours, and 3 hours). A control was used in which the 100-bp template was incubated and proteinase K was added at the appropriate time point. Figures 10A and 10B show the cleavage results demonstrating that L1 and L2 are functional nucleases, but the cleavage activity of Zero was reduced (Figure 10B).

[0084] Protein Expression Test The sequence-characterized expression vector was transformed into chemically competent BL21(DE3). For transformation, either commercial chemically competent BL21, based on the calcium chloride method, or homemade competent cells were used. The transformation buffer was prepared by first preparing a 1 M calcium chloride solution by dissolving 1.1 g in 10 mL of water, then transferring 1 mL of this solution to a fresh tube and adding 9 mL of distilled water. It was then filter-sterilized into a fresh tube, labeled "Transformation Buffer." For improved results, pre-cool the buffer in a refrigerator for at least 1 hour before use.

[0085] The day before the transformation protocol, 10 mL of LB broth was inoculated with BL21 cells or any other E. coli species in a 15 mL tube. It was placed in a rotating / shaking incubator at 37°C and grown overnight. 100 μl of the overnight solution was inoculated into 10 mL of fresh LB and grown for 2 hours. The pelleted cells were harvested by centrifugation at 4,500 rpm for 2-3 minutes. The supernatant was discarded, and the pellet was resuspended in 1 mL of transformation buffer. The resuspension was transferred to a 1.5 mL tube and centrifuged again at 12,000 rpm for 30 seconds. The supernatant was discarded. The pellet was resuspended using 1 mL of transformation buffer by gentle pipetting. The centrifugation / resuspension process was repeated twice. 100 μl of transformation buffer was added to the resuspension for high-efficiency transformation. 50-400 ng of DNA was added, and the mixture was then incubated on ice for 30 minutes. A heat block was preheated to 42°C and a 45-second heat shock was performed for BL21 and derivatives, or a 30-second heat shock for T7. The heat-shocked solution was immediately cooled on ice for 2 minutes. 650 μL of fresh SOC was added and incubated at 37°C for 4 hours with shaking / rotation at 250 rpm (especially for Kan resistance vectors). When using DH5, 100 μl was plated onto the appropriate antibiotic selection plate. When using BL21 (shuffle and derivatives), pelleted cells were obtained by centrifugation at 12,000 rpm for 10 seconds. The entire pellet was plated with an additional 100 μL of medium. The pellet was spread using a sterile spreader or inoculation loop. The plate was incubated at 37°C for 2-3 days until colonies emerged.

[0086] All vectors and constructs were expressed in BL21(DE3) in 2x yeast extract tryptone (2xYT) or Luria Bertani (LB) medium under 0.2–1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Initial protein expression studies were performed in 4 mL culture volumes prior to scale-up purification, as detailed below.

[0087] Up to 100 ng of vector was used to transform chemically competent BL21(DE3) E. coli with the protein expression vector. After plating the cells on appropriate antibiotic restriction plates, single colonies were picked and grown in 2xYT medium in 4 mL cultures, induced with 1 mM IPTG for 24 h at 18 °C, and rotated at 150 rpm. Expression was confirmed by performing a centrifugation with . Once confirmed, a starting culture was initiated based on the desired total volume of the scale-up culture. The scale-up culture was grown at 37°C until the optical density (OD) at 600 nm reached (0.6-0.8). The cells were then immediately cold-shocked by placing the culture vessel in ice water for 15 minutes to induce chaperone expression. Once complete, induction could be performed with IPTG at a concentration of 0.2-1 mM, and incubation was completed for 18-24 hours at 18°C. Cells were harvested by centrifugation at 5000 rpm at 4°C. Lysis was performed in 500 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (TRIS), 10 mM imidazole supplemented with 1 mg / mL lysozyme and 0.5% Triton X100. Enzymatic digestion with lysozyme was performed with shaking for 1 hour at 4°C with the addition of non-ethylenediaminetetraacetic acid (EDTA)-containing protease inhibitors. After 1 hour, Dnase 1 and RNase (both at 0.25 mg / ml) and MgCl2 up to 5 mM were added to disrupt bacterial nucleic acids. Lysis was completed by either freeze-thawing, sonication, or homogenization to increase the culture volume / pellet mass.

[0088] The lysate was clarified by centrifugation at 9000 rpm for 30 minutes at 4°C. All subsequent chromatography steps were performed at 4°C. Approximately 1.5 mL of the lysate was spun onto 2 x 5 mL HisTrap™ High Performance columns overnight at 4°C to ensure maximum binding. -1The clarified lysate was loaded in parallel using a peristaltic pump. Parallel columns with bound protein were attached to an AKTA™ FPLC liquid chromatography system. The columns were washed with 10 column volumes of wash buffer (20 mM Tris-Cl, pH 8.0, 250 mM NaCl, 5 mM imidazole, pH 8.0, 1.5 mL min) until the absorbance again nearly reached baseline. -1 After washing, the column was eluted with a 0-500 mM imidazole gradient (elution buffer: 20 mM Tris-Cl, pH 8.0, 250 mM NaCl, pH 8.0, 0-500 mM imidazole) and collected in 2 mL fractions. Fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0089] Certain proteins require removal of maltose-binding protein (MBP), which is achieved using 0.5 mg of tobacco etch virus (TEV) protease per 50 mg of protein. The nuclease sample is then dialysis buffer (20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)-KOH, pH 7.5, 150 mM KCl, 10% (v / v) glycerol, 1 mM dithiothreitol (DTT), 1 mM EDTA) to approximately 1 mg mL. -1 The sample was diluted to 1000 kDa and dialyzed in dialysis tubing with a molecular weight cutoff (MWCO) of 12-14 kDa against 2 L of dialysis buffer overnight at 4 °C. Dialysis buffer (without DTT and glycerol) can be prepared as a 10x stock, but DTT should be added immediately before use. The collected dialyzed sample was centrifuged at 3900 rpm (approximately 3200 x g) for 5 min at 4 °C to remove any precipitate. TEV protease cleavage was confirmed using SDS-PAGE.

[0090] All resulting proteins (with or without TEV cleavage) were then placed in size-exclusion chromatography (SEC) buffer (20 mM HEPES-KOH, pH 7.5, 500 mM KCl, 1 mM DTT) using a 30,000 MWCO ultracentrifugal filter to concentrate the protein to a volume of less than 1.5 mL, and filtered through a 0.22 μm filter prior to loading into an injection column for gel filtration on a HiLoad™ 26 / 600 Superdex 200 prep-grade gel filtration column (GE Healthcare) equilibrated with SEC buffer. The concentrated SEC buffer solution was injected into the column using a 10 mL sample loop. The column was run for 1 mL min. -1 The protein was eluted with 320 mL of SEC buffer at a flow rate of 100 s, and 2 mL fractions were collected. Peak fractions were analyzed using SDS-PAGE. SDS-PAGE was also performed on concentrated fractions. The final sample was exchanged into a storage buffer based on the following composition: 25 mM Na phosphate, pH 7.25, 300 mM NaCl, 200 mM trehalose (with or without DTT or glycerol depending on short- or long-term storage requirements). The protein was aliquoted and stored at a concentration of 10 mg / mL.

[0091] Protein characterization Proteins Zero, L1, L2, and L3 were purified by gel filtration followed by fast flow liquid chromatography (FPLC) fraction collection. Tables listing the identities of the fractions are provided below (Tables 10 and 11). Proteins Zero, L1, L2, and L3 were also confirmed by mass spectrometry (spectrum not shown). The measured molecular weights for L1 and L2 were 189.3 kDa and 190.3 kDa, respectively. 50 μg of each protein (Zero, L1, L2, and L3) was purified and digested using trypsin. The resulting peptide fragments were analyzed using nanoflow HPLC and Orbitrap™ mass spectrometry (quadrupole ion trap). The sequences of the peptide fragments were predicted based on the mass spectrometry. [Table 10-01]

Table 10-02

Table 10-03

Table 10-04

Table 10-05

Table 10-06

Table 10-07

Table 10-08

Table 10-09

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 11-01

Table 11-02

Table 11-03

Table 11-04

Table 11-05

Table 11-06

Table 11-07

Table 11-08

Table 11-09

Table 11-10

Table 11-11

Table 11-12

Table 11-13

Table 11-14

Table 11-15

Table 11-16

Table 11-17

Table 11-18

Table 11-19

Table 11-20

Table 11-21

Table 11-22

Table 11-23

Table 11-24

Table 11-25

Table 11-26

[0092] Synthesis of sgRNA / gRNA sgRNAs (Cas9 derivatives, Table 12) were either purchased as single-piece guides from commercial suppliers or synthesized in-house by in vitro synthesis (IVT). The IVT synthesis method for synthesizing sgRNAs involves the synthesis of ssDNA in the following format (following the NEB sgRNA guide synthesis method):

[0093] The T7 polymerase promoter sequence was followed by a cr DNA sequence with an overlap for the reverse complement encoding the tr:RNA backbone as DNA.

[0094] The NEB™ EnGen™ Synthesis Kit was used for IVT synthesis. DNA strands were added to the premixed reaction mixture according to the manufacturer's instructions (recommended 2 micrograms of template DNA in the form: T7 promoter-GG-XXXXXXXX seed sequence and backbone) and incubated at 37°C for 12 hours for maximum yield of short templates. Bleach agarose gel or urea polyacrylamide gel electrophoresis was used to verify the RNA. The Zymo™ Clean and Concentrate Kit was used to remove impurities from the RNA according to the manufacturer's instructions. Quantification was performed by UV / VIS and, after adding RNas inhibitors (various manufacturers), the samples were stored at -80°C.

[0095] Guide RNA for Cas12 was purchased as a single guide from Horizon™ or IDT™ or synthesized by overlap PCR to generate a double-stranded DNA template. The double-stranded template contained a T7 promoter sequence followed by the tr gRNA backbone for Cas12a and terminated with cr RNA (as the DNA sequence for the guide). The sequence was converted to RNA using the NEB™ T7 Transcription Kit, and all subsequent steps of purification, quantification, and storage were identical to those for the synthesis of the Cas9 derivative sgRNA guide described above.

[0096] T7 endonuclease assay for evaluating gene editing The principle of the T7 endonuclease I assay is to demonstrate indel formation in gene-edited loci. The first step was PCR amplification from extracted genomic DNA, followed by purification of the PCR amplicon. According to the NEB protocol, the amplicon was heated to 95°C for 2–5 min and then gradually cooled to allow heteroduplex formation between the wild-type and edited strands. This mismatch causes a bulge in the DNA that is recognized by T7 endonuclease I, which cleaves the strand. Incubation is typically at 37°C for 20–30 min. After removal of the endonuclease by 1 M EDTA or, preferably, proteinase K treatment at 56°C for 5 min, the sample was ready for gel analysis on a 1.5% agarose gel in TAE buffer, run at 100 V for 20 min.

[0097] The goal was to demonstrate indel formation in a rapid and cost-effective manner: the formation of truncated products or, in the case of large indel formation, degradation of the original amplicon are clear determinants of achieved gene editing.

[0098] All T7 assays were preceded by DNA extraction from sample cells using a silica column DNA extraction and purification method as per the manufacturer's instructions (Biobasic Genomic DNA extraction kit) or alternatively using Thermofisher direct PCR with Protease K & detergent cell lysis.

[0099] TRAC-specific guide RNA molecules used by M7 Cas12 derivative nucleases (e.g., C9m or Cas9) are presented in Table 12. [Table 12]

[0100] PCR amplification was performed using Kras G12s primers, which also amplify the WT Ras sequence, and was used in DNA samples from A549 and H2228. Thermofisher™ Direct PCR or KDplus™ (transgen) PCR amplifications could be used with either silica column-purified DNA or direct PCR samples; reactions were set up according to the respective manufacturer's specifications, and the temperature setting for the primers was 58°C.

[0101] The amplicons can be used directly in T7 endonuclease assays, but a PCR cleanup is preferred. Quantification of the purified products was achieved by UV / VIS spectroscopy.

[0102] To prepare the T7 reaction, add 200 ng of PCR product, 2 μL of 10x NEB™ Buffer 2.0, and HO to a volume of 19 μL. The reaction was performed in a 0.2 mL PCR tube.

[0103] The PCR products were annealed to form heteroduplexes using a PCR thermocycler by the following steps: Initial denaturation was performed at 95°C for 5 minutes. Annealing was performed from 95 to 85°C with a temperature ramp of 2°C / s to 85°C and then at a rate of 0.1°C / s to 25°C.

[0104] T7 was added to the annealed DNA sample (1 μL of T7 endonuclease) and incubation was carried out at 37°C for 1 hour. The reaction was stopped by the addition of proteinase K and incubated at 37°C for 20 minutes to remove the T7 endonuclease from the cleaved DNA product. The product was diluted to 1.5 with the addition of 4 μL of a fluorescent DNA dye (sybr). Run on a 2% gel and imaged with Chemi Doc™.

[0105] Fluorescent labeling with pHab dye Fluorescent labeling was performed to visualize nuclease localization, binding, and cellular internalization. pHab is a pH-sensitive dye manufactured by Promega™ in both N-hydroxysuccinimide ester (NHS) or maleimide formats for bioconjugation. Bioconjugation to PNME proteins was achieved by following the manufacturer's instructions for amide coupling of the N-succinimide pHaB dye to primary amines on the protein. Briefly, protein (5–10 mg) was aliquoted into a 1.5 ml tube, and the dye was dissolved in DMSO (200 microliters per mg) to obtain 24 microliters to provide an excess of dye:protein of at least 5:1, depending on the protein molecular weight. The reaction mixture was incubated on ice for 4 hours, protected from light. Purification of the protein from unconjugated dye involved a two-step quenching of remaining NHS groups (either 1 M Tris or ethanolamine) and gel extraction of remaining small molecules (Pierce G25 spin column). The purified protein was then tagged with a pH-sensitive dye. When internalized into cells, a decrease in pH leads to an increase in fluorescence. Protocols for the attachment of other NHS ester dyes, such as Cy5.5 NHS or Tamra NHS, were accomplished in the same way.

[0106] Generalized fusion protein preparation For functional CRISPR nuclease, a ratio of 1:1 to 1:9 (nuclease:sgRNA) can be used. Generally, an equimolar formulation is appropriate if the protein is of good quality and has been successfully stored. For example, 1 μM nuclease protein was pipetted into a 0.2 mL polymerase chain reaction (PCR) tube, 1 μM guide RNA was added, and gentle pipetting was performed. Complexation was complete within 15–20 minutes at room temperature. Unless otherwise specified, all conditions followed this sgRNA complexation method.

[0107] When nucleases were used in combination with either biotinylated donors or biotinylated aptamers or biotinylated scFvs or biotinylated peptides, the biotin-modified components were added to the protein complex in equimolar ratios.

[0108] In vitro cleavage protocol Cleavage (i.e., nuclease function) was first assessed in vitro to confirm that the insertion of the display domain did not affect nuclease cleavage function. PNME and sgRNA / gRNA were first thawed on ice. PCR product cleavage template (Kras g12s amplicon synthesized by PCR from A549 cells) was thawed. The PCR composition detailed in Table 13 was prepared, mixed by pipetting, and then incubated at 37°C for 45 minutes. To prepare the PCR composition, gRNA and nuclease were first mixed in buffer and allowed to complex at room temperature for 20 minutes, after which the template was added. [Table 13]

[0109] Blank reactions were prepared as described above, but without the primer to prevent template cleavage. The template was added (and mixed by pipetting) to both the blank and test reactions (with the primer). The resulting mixture was incubated at 37°C for 45 minutes in a thermocycler. After incubation, 1 microliter of proteinase K (10-20 mg / mL) was added, mixed, and incubated at 37°C for 15 minutes. A 4 μL loading of fluorescent DNA dye (i.e., Sybr) was added to all reactions in the wells. Results were analyzed by running a 1.5-2% agarose gel to confirm cleavage. All reactions were run with a negative control to compare the template. The negative control did not contain any nuclease; the nuclease was replaced with an additional volume of HO.

[0110] The gel was used to calculate the guiding efficiency (in vitro). Quantification was based on relative band intensity. The indel percentage was determined by the following formula:

number

[0111] Synthesis of biotinylated donor constructs Biotinylated donor CAR DNA constructs were prepared in the following manner. A double-stranded DNA construct containing the key elements of the CAR construct, along with the LHA and RHA arms detailed above, was synthesized. PCR primers were designed to provide homologous arms (LHA / RHA) of equal length for gel retardation assays. Primers for the full-length 400 bp arm were used, resulting in a product of approximately 3167 bp, and 100 bp arm primers were used for cellular assays where a smaller construct is preferred. In some conditions, biotin was introduced into the amplicon as a 5' modification on the forward or reverse primer.

[0112] PCR was performed using high-fidelity polymerase (Kdplus) with appropriate primers (tm58°C). A total of less than 1 ng of double-stranded template was kept in the reaction and amplified through 35 PCR cycles. PCR reaction volumes and primer concentrations were used as per the manufacturer's instructions (Transgen™ biotech). PCR products were verified on a 1.5% agarose gel and purified with a PCR clean-up column (Favorgen™) prior to use in either gel retardation assays or cell evaluations. Quantification was performed using UV / Vis spectroscopy and stored at -80°C until required.

[0113] Cellular methods The cells used were Jurkat-lucia immortalized T cells (Invivogen™), referred to as Jurkat, or primary CD4 T cells from a single donor. + T cells (HemaCare™). Cells were obtained from Roswell Park Memorial The cells were cultured in RPMI Medium (Pen / strep with 10% FBS, sodium pyruvate, and glutamate) at 37°C and 5% CO2. Medium was purchased from Wisent™. Cultures were passaged when they reached 90% confluence or every 3 days.

[0114] For cell experiments evaluating CAR insertion, 50,000 Jurkat T cells were plated as suspension cultures in 4 mL of 10% FBS Roswell Park Memorial Institute (RPMI) medium supplemented with penicillin / streptomycin (pen / strep), glutamate, and sodium pyruvate in each well of a 6-well plate. Twelve hours after seeding in the 6-well plate, nuclease was prepared. For each well, nuclease was prepared in the following manner: 5 μg of nuclease was complexed with equimolar sgRNA and donor DNA (1:1:1 ratio) for 15 minutes. When generalized cationic delivery was performed, 2.5 μg or RL peptide was added to a 0.2 mL tube and mixed well by pipetting. When using the receptor-mediated method, RL peptide was not added. After 20 minutes, an additional 100 μL of RPMI medium was introduced into the tube and into each well and mixed with the medium by gentle rotation. Cells were incubated at 37°C / 5% CO2 for 72 hours. Suspension cultures were sampled periodically over the next 72 hours using fluorescence microscopy to observe green fluorescent protein (GFP) signal as an indicator of successful integration. Cells sampled from the medium (200 μL volume) were spun down and fixed with 4% paraformaldehyde or ice-cold 100% methanol. After 10 minutes, cells were spun down, washed with ice-cold phosphate-buffered saline (PBS), and added dropwise to a microscope slide with microscope slide fixant. A coverslip was added, and microscopic images were taken using bright-field and fluorescence microscopy. [Table 14]

[0115] After 72 hours, cells were harvested by collecting the cell culture medium and centrifuging at 500 rpm to pellet the cells. The cell pellet was washed twice with ice-cold PBS and prepared for DNA extraction. [Table 15]

[0116] Gel retardation assay A gel retardation assay was used to confirm the binding of biotinylated DNA donors to C9m derivative nucleases (C9mAur, C9C4, and other derivatives). To perform the assay, biotinylated donors prepared by PCR amplification were thawed and a volume equivalent to 1 μM was added to a 0.2 mL PCR tube. To achieve a 1:1 ratio between protein and biotinylated donor, 1 μM of C9mAur derivative or M7Mav derivative (containing the MA domain for biotin binding) was added to the tube, for a final volume of 20 μL. To verify unbound DNA, a control sample containing only the donor was prepared. The control and protein:DNA samples were incubated at room temperature for a minimum of 15 minutes to allow biotinylation to occur. 4 μL of fluorescent DNA dye was added to visualize the DNA components, and the samples were run on a 1.5% agarose gel at 100 V for 20 minutes. DNA was visualized using Biorad™ ChemiDoc, and retardation of DNA was observed via fluorescence in samples using proteins containing the MA domain; in their absence, DNA migrated appropriately to its length in BP and was visualized with a standard 1 kb size marker (Transgen™ 1 kb-plus marker). Complete complexation at a 1:1 ratio occurred with all C9m and M7ma proteins. The complex was stable, and MA folded properly.

[0117] Gel retardation assays were performed by mixing the MA protein construct at equimolar concentrations with the biotinylated donor construct and incubating for 15 minutes (1 μM of each biomolecule in a volume of 20 μL). Complete complexation occurred within 15 minutes at room temperature, at which point a non-toxic DNA dye (sybr) was added to the mixture to bind to DNA. Ten microliters were sampled and run on a 1.5% agarose gel in Tris-acetate-EDTA (TAE) buffer at 100 V for 30 minutes. Imaging was achieved using a BioRad™ chemi doc.

[0118] First, M7MAV-CD8 and M7MAV-CD4 were formed as complexed proteins and then introduced into Jurkat cell cultures in 6-well plates. For M7-MAV-CD4 and CD8, it was observed that the CD4-modified construct was the only one that produced a GFP signal, while the anti-CD8-modified construct did not. Since Jurkat is a CD4-positive cell line, editing resulted in a GFP signal for the anti-CD4 M7MAv protein construct, demonstrating selectivity. It was later observed that the extended GGGS linker domain in the amino acid sequence of each construct led to poor stability of the protein construct and attenuated translation during protein synthesis. M The consequences of the poor stability and maintenance of the fusion between AV and the nuclease domain can be seen in the limited gel retardation of the complexes (Figure 11) and in the microscopy images in Figures 12A and 12B. Development of these constructs was discontinued in favor of C9m derivatives that conferred better stability. The improved gel retardation of DNA bound to MAV in C9m-derived constructs prompted further investigation.

[0119] We hypothesized that improvements in protein stability, endosomal escape, and donor complexation would improve overall CAR production rates, and these were demonstrated in experiments performed with the C9m derivative as well as without the use of the GGGS linker, an additional endosomal escape sequence, and increased stability. To confirm the improved stability of the MAV domain in complex with the C9m derivative, we performed gel retardation assays again with the C9mAur derivative to confirm the improved retardation of the biotinylated donor. This time, the donor was well retained in the gel, as the protein's MW (approximately 185 kDa) does not readily migrate in agarose gels (Figure 13), demonstrating gel retardation upon complexation with the donor (C9m).

[0120] GFP knock-in evaluation was performed using the donor construct genomic insertion. Three guide RNAs (sgRNA1, sgRNA3, and sgRNA11, Table 16) that closely group on TRAC exon 1 with a non-optimal donor fragment were used for gene editing. GFP knock-in was performed using a C9m derivative. Cells were harvested at the 24-hour stage, fixed with paraformaldehyde, and then immobilized on glass slides for fluorescence microscopy imaging. Increased stability clearly increased GFP signal and therefore HDR gene editing, as observed in a greater number of cells for M7MAVCD4 (Figures 14A-14D). Significant GFP signal could only be generated if the inserted CAR was inserted in-frame with the endogenous promoter at the TRAC locus. If the CAR was present in cells without an insert, there was no possibility of a signal because the donor construct lacked a promoter. Imaging (Figures 14A-14D) showed that approximately 80-90% of cells produced GFP signals (Figures 14B-14D), compared to no fluorescence detected in the control (Figure 14A). Sample cells were collected for further analysis by PCR and NGS. Results indicated that sg11 was the preferred guide for CAR transduction. [Table 16]

[0121] The insertion site was evaluated to further confirm the edit formation and insertion into the genome, and the CAR sequence was confirmed. Splice site analysis was performed, in which one primer targeted the genomic TRAC target upstream of the donor homology arm, and the reverse primer targeted the anti-CD19 scFV sequence of the CAR donor. Successful insertion was confirmed by PCR. Figure 15 shows the gel run after insert confirmation PCR. PCR was performed using the insert confirmation primers (Table 17). [Table 17]

[0122] 0.5 mL of medium containing GFP-expressing Jurkat cells was collected (three biological replicates in which the sg11 guide was used). The cells were spun down at 500 rpm for 1 minute, and the medium was removed. The cells were lysed, and DNA was extracted using a silica column (Biobasic™ One 4 all Genomic DNA extraction kit). Genomic DNA was amplified using TRAC / CAR-specific primers to confirm the insertion. The insertion was confirmed, and the amplicon was purified using a PCR cleanup column. Quantification was performed, and samples were normalized for the amplicon sequence. Paired-end amplicon sequencing, including quantification, library preparation, and insert analysis, was performed using Genewiz™. It was observed that the CAR was successfully spliced ​​into the TRAC genome, and the splice site contained the partial TRAC locus sequence and the anti-CD19 scFV sequence of the insert. The consensus sequence for the insert position in TRAC exon 1 and the 5' end of the CAR construct is shown in the annotated SEQ ID NO:353 below. The genomic TRAC sequence is underlined and the insertion breakpoints are indicated by lines. [ka]

[0123] NGS sequencing allowed for the estimation of the percentage of CAR insertion into the genome of the cell population subjected to gene editing. The correlation between the NGS-estimated HDR CAR insertion rate was strong, with GFP reporter signal expression of approximately 90% (Figures 16 and 17). More specifically, HDR insertion of CAR was estimated to be 88-92% across three biological replicates.

[0124] Generalized cationic cell delivery using RL peptides To achieve cationic peptide-based delivery, a peptide containing arginine and lysine repeat residues, i.e., "RL," SEQ ID NO: 11:RRRRRRRLLLLLLLL was used. Peptide synthesis was completed by a commercial supplier (Biomatik™) and characterized by mass spectrometry to determine the exact sequence (Figure 18). Purification was performed by high-performance liquid chromatography (HPLC) (Figure 19) and lyophilized. The peptide was resuspended at 1 mg / mL in MilliQ™ and stored at -80°C until required. Nuclease conjugation was achieved by mixing the nuclease with a biotinylated donor at a ratio of 5 μg of peptide for every 10 μg of protein used in nuclease formation. The peptide was added to the nuclease at room temperature and mixed by gentle pipetting. Conjugation was complete after 15 minutes, and the conjugate was ready to be added to cells at the appropriate concentration.

[0125] primary cells The following experiments were performed using human primary cells. Primary cells are important because they are a direct clinical analog of the method's performance. In fact, when performing cell therapy, primary cells collected from a subject are the cells that are gene-edited into CAR-T cells for treatment.

[0126] To enable rapid validation of the delivery of the engineered nucleases (Zero, L1, L2, and L3), the engineered nucleases were labeled with tetramethylrhodamine via an NHS amine coupling mechanism and purified by gel filtration. When complexed with a TRAC-specific sgRNA of known potency, a double-stranded break was generated, mediated by intracellular delivery, endosomal escape, and nuclear localization of the engineered nuclease constructs via anti-CD4 binding. In the HDR system required for CAR generation, the engineered nuclease also possesses the ability to bind to a hapten-modified donor DNA construct encoding an anti-CD19 CAR with a P2A cleavage site, followed by GFP expression as a signal of successful integration into the genome.

[0127] The modified nucleases (Zero, L1, L2, and L3) were subjected to a gel retardation assay of biotin DNA donors. More specifically, all modified nucleases were subjected to a QA biotin donor binding experiment. When donor DNAs were bound by the modified nucleases, they migrated slower in the gel and spent more time in the well (in other words, they were retarded). Figure 20 shows the gel electrophoresis for the gel retardation assay. As can be seen in Figure 20, all modified nucleases were retarded and barely migrated in the gel, indicating that they bound to the DNA donor via biotin interactions.

[0128] Membrane association in primary T cells Fluorescence microscopy was performed to determine and visualize the association of the engineered nucleases (Zero, L1, L2, and L3) with the plasma membrane of primary T cells. All engineered nucleases were labeled with NHS-TAMRA fluorophores and purified using a dye-exclusion column. Five micrograms of each engineered nuclease was incubated with CD4+ primary T cells for 24 hours, followed by fixation with 5% formalin in solution and subsequent transfer to microscopy coverslips. Fluorescence microscopy was achieved at 40x oil immersion using a BX51 and TAMRA filter set (ex 550 nm, em 578-600 nm). All engineered nucleases were found to bind strongly to the plasma membrane and initiate internalization (Figures 21A-21D).

[0129] Validation of engineered nuclease delivery into Jurkat and primary T cells Jurkat CD4+ T-cell leukemia cells were maintained in RMP1™ 1640 medium supplemented with 10% fetal bovine serum (FBS). 25,000 cells were seeded into wells of a 96-well plate. Each well was treated with 10 micrograms of each protein (Zero, L1, L2, or L3) labeled with the TMRA fluorophore and incubated for 1 hour. Cells were then harvested, washed, fixed in 5% formalin for 1 hour, centrifuged, and resuspended in PBS + 1% FBS. Cells were stored at 4°C until analysis using a Sony™ Cell spectroanalyzer ID7000 flow cytometer. Fluorescence was detected in the TAMRA fluorescence range (TAMRA excitation 555 nm, emission 580 nm) with autofluorescence subtraction, and full fluorescence spectra were acquired. Control cells were cultured under the same conditions but without nuclease. All proteins achieved cell binding with L3 producing the most significant fluorescent signal, reflecting a higher degree of cell binding and accumulation (Figures 22A-22E). Greater than 99% delivery was achieved for each of Zero, L1, L2, and L3, indicating efficient internalization of the modified nucleases described herein.

[0130] GFP monitoring was performed using an in vivo imaging system (IVIS) as a time course assay using Jurkat T cells treated with the L1 protein construct (for GFP genome integration), assessed over a 48-hour period. The GFP signal consistently increased over the duration of the experiment, indicating that the CAR donor had integrated via HDR (Figure 23). To assess the degree of integration across the cell population, all constructs were evaluated by flow cytometry. Human primary CD4+ T cells (seeded at 20,000 cells per well, 96-well plate) were maintained in RMP1™ 1640 + 10% FBS and treated with 10 micrograms of each protein. GFP signal was monitored at 18, 24, 45, and 48 hours to track the performance of the L1 construct. The successful observation of increasing GFP signal (Figure 23) suggested successful HDR due to the CAR template construct. Because the CAR construct lacks a promoter and is driven from the endogenous promoter at the TRAC locus, GFP can only be expressed when the entire CAR construct is inserted into the genome. As can be seen in Figure 23, the GFP signal nearly doubled from 18 to 48 hours, indicating gene editing that resulted in GFP expression.

[0131] The validation delivery experiment was repeated in a clinically relevant model: primary T cells. 20,000 human primary CD4+ T cells were seeded into wells of a 96-well plate. Cells were maintained in RMP1™ 1640 + 10% FBS and treated with 10 micrograms of each protein (Zero, L1, L2, or L3) labeled with the TMRA fluorophore and incubated for 1 hour. Cells were then harvested, washed, and fixed in 5% formalin for 1 hour, then spun down and resuspended in PBS + 1% FBS. Cells were stored at 4°C until analysis using a Sony™ Cell spectroanalyzer ID7000 flow cytometer. Fluorescence was detected in the TAMRA fluorescence range (TAMRA excitation 555 nm, emission 580 nm) with autofluorescence subtraction, and full fluorescence spectra were acquired. Controls included cells cultured under the same conditions but without nuclease. All proteins (Zero, L1, L2, or L3) achieved cell binding with L3 and produced the most significant fluorescent signal, reflecting a higher degree of cell binding and accumulation (FIGS. 24A-24D).

[0132] Using spectral flow cytometry, delivery and CAR integration were assessed in the TAMRA and GFP channels, respectively. Efficient delivery (>90%) into Jurkat cells was assessed for modified nucleases (Zero, L1, L2, and L3) at various nuclease protein concentrations (0.33, 16, 33.3, and 66 ng / μL) and for incubation times of 48 and 72 hours. As the nuclease protein concentration increased, the efficiency of CAR-GFP donor integration was observed to increase with a corresponding increase in GFP-expressing cells for all protein constructs after 48 and 72 hours of incubation. (See Figures 25A-25C, 26A-26L, 27A-27L, 28A-28L, 29A-29L, 30A-30C, 31A-31L, 32A-32L, 33A-33L, 34A-34L, and Table 18 below.) 4With TAMRA detection exceeding log intensity, the likelihood of highly efficient editing increases. Over 72 hours, GFP increased in the wells due to two mechanisms: cell expansion and HDR editing to insert the CAR template. When considering flow results, the GFP representative fraction continued to increase, indicating that editing persisted throughout 72 hours, although the overall increase observed at 48 hours was lower. Cell viability at the completion of the experiment remained good, exceeding 90% for all conditions, and cell numbers expanded from an initial seeding of 25,000 to approximately 75,000-80,000 cells. [Table 18]

[0133] The greatest success was seen with L1 and L2, which achieved 87 and 90% GFP signal at the 48-hour stage. Further evaluation at the 72-hour stage revealed further improvement. Editing efficiency was measured using a GFP reporter encoded downstream of the CAR receptor insert with increasing concentrations of the modified nuclease. Zero was found to be a reduced nuclease in that its enzymatic activity was reduced by the placement of the delivery domain, which affected its ability to form double-strand breaks. Zero activity improved with increasing concentration. The presence of a linker domain in L1 and L2 provided favorable protein orientation, position, and folding. In L3, a small CDR was inserted into the SP3 site in the loop, so the overall protein folding and shape were not significantly altered. L3 performance was better than Zero. Overall, a high frequency of gene delivery resulting in highly efficient gene editing was demonstrated. The GFP percentage reported in Table 18 above is based on linear gating of the histogram rather than binary division. The binary division with TAMRA and GFP as the Y and X axes allows for a high degree of impact on the delivery of the modified nuclease and its GFP signal. 26A, FIG. 26D, FIG. 26G, FIG. 26J, FIG. 27A, FIG. 27D, FIG. 27G, FIG. 27J, FIG. 28A, FIG. 28D, FIG. 28G, FIG. 28J, FIG. 29A, FIG. 29D, FIG. 29G, FIG. 29J, FIG. 30A, FIG. 31A, FIG. 31D, FIG. 31G, FIG. 31J, FIG. 32A, FIG. 32D, FIG. 32G, FIG. 32J, FIG. 33A, FIG. 33D, FIG. 33G, FIG. 33J, FIG. 34A, FIG. 34D, FIG. 34G, and FIG. 34J.

[0134] Verification of the insertion was achieved by PCR using primers (Table 17) that bridge the C-terminus of the CAR receptor CD3z to the GFP sequence after P2A site amplification. Genomic DNA was extracted from Jurkat cells treated at 66 ng / μL at the 72-hour stage. Using insert-specific primers, a 600-bp fragment of the insert was amplified from genomic DNA extracted from control (untreated Jurkat), Z-10, L1-10, L2-10, and L3-10 (where 10 represents a concentration of 66 ng / μL). Lack of insert amplification excludes CAR insertion, while the presence of a PCR product confirms insertion. For Zero, L1, L2, and L3, products (approximately 700 bp) were confirmed by agarose gel (1.5%, TAE buffer), while control Jurkat cells did not exhibit an insert (Figure 35).

[0135] Spectral flow cytometry analysis performed on Jurkat T cells was repeated in primary T cells with donor CAR and TRAC sgRNAs without nuclease as a control and with 66 ng / µL L1, L2, or L3. At 24 hours, GFP signal was detected, indicating cell proliferation. At 48 hours, 99% of primary T cells were successfully delivered with L1 or L2, and gene editing was achieved in over 63-71.6% of cases (Figures 36A-L).

[0136] In vivo CD4 T cell delivery Injections into NOD scid gamma (NSG) mice were performed at 1 / 12 of the maximum tolerated dose (MTD) (i.e., 150 μg) of L1 or L2 protein complexes in a 1:1:1 molar ratio of protein:sgRNA:biotinylated donor CAR in 150 μL of PBS as a carrier. L1 and L2 proteins had previously been labeled with TAMRA NHS reagent and purified by gel filtration. Injected mice were obtained from JAX laboratories. Briefly, engraftment was achieved in the following manner: female mice were transfected with human hematopoietic stem cells (hu-CD34 + ) and mature CD45 + Cells were identified prior to delivery. A single human donor was used. Injections were performed via the tail vein, and the first blood sample was collected 3 hours after injection to first assess CD4+ T cells and second assess whether delivery had occurred. L1 was selected to assess early delivery. NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl) CD34+ human stem cell graft mice were pretreated with interleukin 7 (IL-7) on days -4 and -1 prior to L1 or L2 injection, as well as 1 day after injection. The purpose of this was to boost T cell numbers after arrival of the NSG mice and to equalize engraftment variability. At 3, 24, and 48 hours after injection, 20 microliters of whole blood was collected from the tail into heparin capillary tubes to prevent clotting. The capillary tubes were spun at 14k revolutions per minute (14k RPM) for 4 minutes to separate serum and whole blood. Red blood cells (RBCs) were lysed with 400 μL of RBC lysis buffer (1x) for 20 minutes at room temperature, followed by centrifugation at 3500 RPM for 4.5 minutes to separate white blood cells, and the supernatant was discarded. White blood cells were resuspended in PBS + 1% FBS and stained with 5 μg of APC anti-CD4 antibody (Biolegend™) for 15 minutes, followed by centrifugation and washing with PBS + 1% FBS before resuspension and analysis by flow cytometry. Control samples were derived from untreated NSG mice and L1 samples from L1-treated mice. Flow cytometry analysis was performed with autofluorescence subtraction, with a minimum of 5000 cells analyzed. The analysis was performed using double gating for APC and TAMRA on a Sony™ Spectral Analyzer flow cytometer. The presence of CD4+ and Tamra-positive cell populations at 3 hours for L1 verified that targeting of CD4+ T cells was achieved in vivo (Figures 37A and 37B). Furthermore, L2 also achieved CD4+ T cell delivery at 3 hours, reaching 24.5% delivery (Figures 37A and 37C). At 3 hours, NSG mice treated with L1 and independently with L2 achieved 13.22% and 24.5% of the analyzed populations, respectively, showing positive TAMRA signals, associated with delivery of L1 or L2 Tamra-labeled proteins into the analyzed cell populations. It is noted that at 1 / 12 of the MTD, there is significant margin for improvement in delivery to the entire T cell population. At 24 and 48 hours (Figures 37D-37I), a decrease in the overall intensity of TAMRA was observed as L1 or L2 cleared from serum. L1 maintained a 15% positive signal, but the overall intensity decreased to 10%. 2.5 To assess the clearance of L1 and L2 from cells, the TAMRA signal was considered to be cleared when it decreased below that of the control (i.e., 2%).

[0137] Along with the clearance of L1 or L2 TAMRA from CD4+ T cells at 48 h, the spectral analysis channel was opened for PE-CD19 (phycoerythrin or PE, fluorophore-tagged CD19) staining to assess CAR expression. Given the demonstration that L1 and L2 were cleared at 48 h, it was therefore appropriate to assess L1 and L2 internalization and gene editing through PE-CD19 expression at 48 h. The sample protocol for blood collection, cell preparation, and washing was followed, except that PE-CD19 was added simultaneously with APC-anti-CD4 at the same concentration. Incubation was performed at room temperature for 15 min and washed prior to analysis. PE-CD19 filters were used for analysis on a Sony™ spectral analyzer flow cytometer, and the results presented represent 48 samples from representative L1- and L2-treated animals, as well as untreated controls (Figures 38A-38C). With an increase in CD19 staining for both L1 and L2 treated animals, it was demonstrated that CD19-expressing CAR-T cells could be produced in vivo. At this stage, a challenge was introduced into the animals to stimulate the action and expansion of in vivo generated anti-CD19 CAR CD4+ T cells. This was achieved by administering 1x10 6 Raji-Luc CD19+ (B-cell lymphocytes of Burkitt's lymphoma origin adapted with a luciferase expression cassette to allow bioluminescent assessment of in vivo survival) were delivered via tail vein injection. Bioluminescence was assessed by intraperitoneal injection of 10 μg / mL luciferin into each animal, and imaging was performed 7 minutes after injection using an IVIS whole animal imager (0.5-second exposure, fstop 1, binning factor 8). Following injection, mice were evaluated every two weeks, and their torsos were shaved weekly to limit scattering and absorption caused by hair, allowing accurate quantification of bioluminescence. Xenograft engraftment and expansion were verified 4 days after injection; by day 7, bioluminescence was found to be reduced in L1- and L2-treated animals compared with control vehicle (untreated PBS-injected) animals.

[0138] 192 hours after injection of Raji cells, proliferation and cell survival of Raji cells were observed to be hindered in NSG mice compared to untreated control Raji (Figures 39A and 39B). Biological replicates were n=4 for control (PBS vehicle) (labeled as C), n=3 for L1 (labeled as L1), and n=4 for L2 (labeled as L2). Injections were 1x10 in 150 μL. 6 The expression of anti-CD19 chimeric antigen receptor was monitored by flow cytometry. Bioluminescence intensity was measured by intraperitoneal injection of luciferin, which luciferase-expressing Raji cells converted into a bioluminescent signal to measure cell survival and proliferation. Animals treated with L1 or L2 and expressing CD19-positive CD4+ T cells were then injected intraperitoneally. It was observed to interfere with the proliferation of CD19-expressing Raji cells in vivo. Signal was detected by IVIS luminescence imaging using the following conditions: 0.5 seconds, Fstop: 1, binning 8, and 7 minutes after injection of 100 μL (10 μg / mL) IP.

[0139] Dfd for streptavidin aptamer modification of the donor template for CAR insertion. Importantly, the donor no longer requires biotinylation modification, and the aptamer can be used to bind to the MAV domain of 6.0 nuclease nabs. The sequences of the primers used to amplify the donor with the addition of the streptavidin aptamer are also provided in the figure.

[0140] Initial CAR donor synthesis involved PCR amplification of a linear template using a forward primer that adds biotin to the 5' end of the donor. While this can be scaled to production, two simpler strategies can be applied: Amplification of the donor on a plasmid, followed by plasmid purification, restriction digestion, purification and enzymatic addition of biotin, and final purification, or Amplification of a donor carrying an avidin-binding sequence at the 5' end on a plasmid, followed by plasmid preparation, restriction digestion, and final gel filtration. The addition of DNA-encoded avidin binding eliminates the need for additional purification and enzyme treatment. Effectively, a fully functional donor can be directly encoded into DNA. [Table 19]

[0141] A gel retardation assay (Figure 40A) shows four double-stranded linear DNA donors, each 2.4 kb in length. One donor has biotin, and the other has the Strep-Apt DNA aptamer, which binds to avidin (Table 19). The donors migrate relative to their size in the gel. When complexed with a protein avidin-binding domain through either biotin or the strep-Apt aptamer, the donors were retarded in the wells and prevented from migrating through the gel. A 1:1 molar ratio of DNA to PNME is maintained for the standard donor and the three strep-apt donor variants (Table 19).

[0142] Off-target analysis was also performed on L2-treated cells. The top 10 most likely sites were selected and amplified using PCR. If insertions occurred frequently, a larger increase in amplicon length would be observed, since the donor is 2.4 kb long. The amplicon is centered on the predicted off-target cleavage site and is 750-800 bp long in the absence of an insertion. If an insertion is present, the length should increase substantially. The selected samples were from a 48-hour stage Jurkat control sample (untreated) and an ex vivo cell experiment using Jurkat treated with L2 at the highest concentration (66 ng / µL). DNA was extracted from each sample and PCR was performed using locus-specific primers. Figure 40B shows the chromosomes on which the off-targets are located. The amplicons for each sample are presented side-by-side. Consistent amplicon sizes and the absence of multiple bands between control and L2-treated cells were observed, strongly suggesting that frequent insertions are unlikely (approximately 750-800 bp). Sanger confirmation of the sequence confirmed the absence of an insertion at the cleavage site. Indel analysis showed no indels below the 1.5% certainty threshold (Tracking of Indels by Decomposition (TIDE)). Equivalence of control and L2 samples with respect to indels and insertions is indicated by a "Y" below each sample on the gel.

Claims

1. A composition for modifying T cells, wherein the composition is A protein complex containing a polynucleotide-modifying enzyme domain, a T cell membrane-binding domain, and an endosomal escape domain; Guide oligonucleotides specific to the T cell receptor α constant (TRAC) gene of the T cell; and Donor DNA comprising two homology arms at each end of the donor DNA homologous to exon 1 of the TRAC gene, and between the two homology arms, A transition signal for the migration of the T cell to the cell membrane; Transmembrane domain; Intracellular signaling domains; and Extracellular antigen-binding domain The donor DNA encoding a chimeric antigen T cell receptor containing the donor DNA The composition comprising the above.

2. The composition according to claim 1, wherein the protein complex further comprises a hapten-binding domain.

3. The composition according to claim 2, wherein the donor DNA is conjugated to a hapten, and the hapten binds to the hapten-binding domain.

4. The composition according to claim 1, further comprising a nuclear localization sequence within the protein complex.

5. The composition according to claim 1, wherein the chimeric antigen T cell receptor further comprises a CD8 hinge region.

6. The composition according to claim 1, further comprising a chimeric antigen T cell receptor and a B cell lymphoma recognition domain.

7. The guide oligonucleotide consists of 250 nucleotides prior to the start codon of exon 1 of the TRAC gene and the nucleotides following the start codon of exon 1 of the TRAC gene. The composition according to claim 1, wherein the sequence is complementary to the sequence located between 250 nucleotides.

8. The composition according to claim 1, wherein the polynucleotide-modifying enzyme domain is covalently linked to the endosomal escape domain.

9. The T cell membrane-binding domain is a cationic peptide, or a cell recognition domain that targets CD4, CD8, CD16, or CD56. i) The cell recognition domain is covalently linked to the endosomal escape domain, ii) The cell recognition domain is a display domain which is a peptide recognition sequence of 3 to 20 amino acids in length located in a loop or alpha-helix on the outer surface of the polynucleotide-modifying enzyme domain, and the peptide recognition sequence is a complementarity-determining region (CDR), or iii) The cell recognition domain is an antigen-binding domain selected from Fab, a single-domain antibody (sdAb), VHH, or a camelid antibody domain, located in a loop on the outer surface of the polynucleotide-modified enzyme. The composition according to claim 1.

10. The composition according to claim 1, wherein the polynucleotide modification domain is type II Cas, a functional analog thereof, a variant thereof, or a derivative thereof.

11. The composition according to claim 10, wherein the type II Cas is Cas9, its functional analog, its variant, or a derivative thereof.

12. The composition according to claim 1, wherein the polynucleotide modification domain is type V Cas, a functional analog thereof, a variant thereof, or a derivative thereof.

13. The composition according to claim 1, wherein the extracellular antigen-binding domain is specific to a cancer-specific antigen.

14. The composition according to claim 1 for use in cell therapy.

15. The composition according to claim 14 for use in the treatment of cancer.

16. A polynucleotide modifying enzyme, A functional nuclease domain containing a nuclease catalyst pocket; In the loop arranged on the outer surface of the polynucleotide-modified enzyme, Fab, a single-domain antibody (sdAb), and V ＨＨ , or an antigen-binding domain selected from camelid antibody domains, wherein the antigen-binding domain recognizes a target cell receptor on a target cell, thereby enabling intracellular translocation of the polynucleotide-modified enzyme into the target cell; and A linker of 0 to 30 amino acids upstream of the antigen-binding domain. The polynucleotide-modifying enzyme comprising the above.

17. The polynucleotide-modified enzyme according to claim 16, wherein the nanobody is VHH.

18. The polynucleotide modifying enzyme according to claim 16, wherein the linker sequence comprises 16 to 23 amino acids.

19. The nuclease catalyst pocket is a Cas nuclease catalyst pocket, a recombinase A catalyst pocket or meganuclease catalyst pocket, wherein the Cas is a) Cas9, its functional analog, its variant or derivative thereof, wherein the nuclease catalyst pocket contains an HNH nuclease domain. b) Cas12, its functional analogs, its variants or derivatives, c) Cas13, its functional analog, its variant or derivative, or d) Cas14, its functional analogs, its variants or derivatives, The polynucleotide modifying enzyme according to claim 16.

20. The polynucleotide modified enzyme according to claim 16, wherein the nuclease catalyst pocket contains a RuvC nuclease domain.

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