Vectors and methods for in vivo antibody production

Abstract

The present invention relates to compositions and methods capable of targeting B cells and / or hematopoietic stem cells (HSCs) to engineer those cells to express specific antibodies ex vivo or in vivo and to become part of a host's long-lived immune repertoire.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to U.S. Provisional Application No. 63 / 339,665, filed on May 9, 2022, the disclosure of which is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present invention relates to compositions and methods that can target B cells and / or hematopoietic stem cells (HSCs) in order to engineer those cells to express specific antibodies ex vivo or in vivo and become part of the host's long-lived immune repertoire. Background of the Invention

[0005] In general, the success of immunization depends on the host's ability to react to a given immunogen and to produce an appropriate response. In some populations (such as young children, the elderly, immunocompromised persons, etc.), vaccines sometimes fail to correctly induce the desired reaction. For several infectious agents, immunogens have not been successful in producing sufficiently extensive and effective immune responses even in normal healthy populations. In addition, for some pathogens (such as dengue fever), vaccination may actually lead to the enhancement (ADE) of infection rather than protection, depending on individual vaccine reactions, such as the isotype of the antibody induced. Monoclonal antibodies can be selected or designed to overcome many of these problems, but compared with vaccines, the antibody life of passive delivery is short, and lifelong immunity will need to be frequently re-administered. In the past few years, methods for expressing monoclonal antibodies in vivo have been sought. Summary of the invention

[0006] As described in the background section, there is a great need in the art to express monoclonal antibodies in vivo. The present invention addresses this need and other needs by providing compositions and methods that can target B cells and / or hematopoietic stem cells (HSCs) to engineer those cells to express specific antibodies ex vivo or in vivo and become part of the host's long-lived immune system repertoire.

[0007] In one aspect, the present invention provides a system for producing an antibody or an antigen-binding fragment thereof in a subject, the system comprising:

[0008] a) a first component comprising a polynucleotide molecule, wherein the polynucleotide molecule comprises a sequence encoding an antibody or an antigen-binding fragment thereof; and

[0009] b) A second component, which comprises a gene editing molecule or a polynucleotide molecule containing a sequence encoding the gene editing molecule.

[0010] In some embodiments, administration of the first component and the second component to the subject results in integration of sequences encoding the antibody or antigen-binding fragment thereof into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), resulting in production of the antibody or antigen-binding fragment in the subject.

[0011] In some embodiments, ex vivo administration of the first component and the second component to B cells and / or hematopoietic stem cells (HSCs) results in integration of sequences encoding the antibody or antigen-binding fragment thereof into the DNA of the cells to produce modified B cells or modified HSCs, thereby resulting in production of the antibody or antigen-binding fragment thereof in the subject upon administration of the modified B cells or HSCs to the subject.

[0012] In some embodiments, the B cell is a B1 B cell. In some embodiments, the B cell is a B2 B cell.

[0013] In some embodiments, the first component and / or the second component are independently selected from viral vectors, virus-like particles (VLPs), liposomes, lipid nanoparticles (LNPs), and ribonucleoprotein (RNP) complexes.

[0014] In some embodiments, the first component and the second component are both viral vectors. In some embodiments, the viral vectors are derived from the same viral species. In other embodiments, the viral vectors are derived from different viral species.

[0015] In some embodiments, the first component or the second component further comprises a guide RNA (gRNA) molecule or a sequence encoding the gRNA molecule.

[0016] In some embodiments, the first component comprises a polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof and a sequence encoding the gRNA.

[0017] In some embodiments, the first component comprises (i) a first polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof; and (ii) a second polynucleotide molecule comprising a sequence encoding the gRNA.

[0018] In some embodiments, the first component comprises (i) a first polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof; and (ii) the gRNA molecule.

[0019] In some embodiments, the second component comprises the gRNA molecule or a sequence encoding the gRNA molecule.

[0020] In one aspect, the present invention provides a vector system for producing a cell population capable of producing antibodies or antigen-binding fragments thereof in vivo, the system comprising: a first viral vector comprising a sequence encoding a target antibody or a fragment thereof and a sequence encoding a guide RNA (gRNA), a second viral vector comprising a sequence encoding a gene editing molecule, wherein the vector system integrates the sequence encoding the target antibody or the antigen-binding fragment thereof into the DNA of the cells, causing the cells to produce the antibody or the antigen-binding fragment thereof.

[0021] In some embodiments, the cell population is a human cell population. In some embodiments, the cell population is a B cell population (e.g., comprising B1 B cells and / or comprising B2 B cells). In some embodiments, the cell population is a hematopoietic stem cell (HSC) population.

[0022] In some embodiments, one or both of the viral vectors used in the system of the present invention are adeno-associated virus (AAV) vectors. In some embodiments, the AAV vector is derived from AAV1, AAV2, AAV6, AAV9 or AAV9.PHP. In some embodiments, the AAV vector capsid comprises one or more mutations, wherein the one or more mutations eliminate the natural tropism of the AAV vector. In some embodiments, the AAV vector capsid is derived from AAV1 or AAV6 and comprises mutations Y445F and / or V473D. In some embodiments, the AAV vector capsid is derived from AAV9 and comprises mutation W503A.

[0023] In some embodiments, one or both of the viral vectors used in the systems of the invention are retroviral vectors, such as lentiviral vectors.

[0024] In some embodiments, the viral vector for the system of the present invention further comprises a targeting moiety. In some embodiments, the targeting moiety is expressed on the outer surface of the viral capsid. In some embodiments, the targeting moiety is attached to the outer surface of the viral capsid by a joint.

[0025] In some embodiments, the viral vector is an AAV vector, and the targeting moiety is inserted into the protein forming the viral capsid, or covalently or non-covalently attached to the protein forming the viral capsid. In some embodiments, the targeting moiety is attached to the viral capsid by the first member and the second member of the binding pair. The first member and the second member can form an isopeptide bond.

[0026] In some embodiments, the viral vector is a lentiviral vector, and the targeting moiety is covalently or non-covalently attached to the fusogen.

[0027] In some embodiments, the targeting moiety is attached to the outer surface of the viral capsid by a SpyTag: SpyCatcher system. In some embodiments, the targeting moiety is a targeting antibody or an antigen binding fragment thereof. Non-limiting examples of useful antibodies include, for example, anti-CD19, anti-CD20, anti-CD34, anti-CD38, anti-CD40, anti-CD117, anti-CD22, anti-CD79, anti-CD180, anti-CD5, anti-B cell receptor (BCR) (e.g., IgM, IgD, IgG), anti-B cell activation factor (BAFF) and anti-Sca-1 antibodies, or antigen binding fragments thereof.

[0028] In some embodiments, the gene editing molecule is a Cas nuclease, such as a Cas9 nuclease.

[0029] In various embodiments, the gRNA is complementary to a sequence at an IgH locus, a J chain locus, or an Igκ locus. In some embodiments, the gRNA is complementary to a sequence at the J chain locus. In one embodiment, the gRNA is complementary to a sequence in the 4th exon of the J chain locus. In one embodiment, the gRNA is complementary to a sequence in the first intron of the J chain locus.

[0030] In some embodiments, the gRNA encoded by the gRNA coding sequence is complementary to the sequence in the V13 region of the encoding antibody. In some embodiments, the gRNA is selected from gRNA1, gRNA2, gRNA3, gRNA4, gRNA5, gRNA6, gRNA7, gRNA8, gRNA9, gRNA10 and gRNA12.

[0031] In some embodiments, the sequence encoding the antibody or its antigen-binding fragment comprises a sequence encoding a light chain variable region and an optional light chain constant region of the antibody. In some embodiments, the sequence encoding the antibody or its fragment comprises a sequence encoding a heavy chain variable sequence of the antibody.

[0032] In some embodiments, the sequence encoding the antibody or antigen-binding fragment thereof is integrated at the IgH locus in the genomic region downstream of the last J gene but upstream of the Eμ enhancer.

[0033] In some embodiments, integration of the sequence encoding the antibody or antigen-binding fragment thereof into the DNA of the B cell or HSC results in disruption of the kappa light chain constant region.

[0034] In some embodiments, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a 5'IgH homology region, a splice acceptor, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a splice donor sequence, and a 3'IgH homology region.

[0035] In some embodiments, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a 5' J chain exon 4 homology region, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a sequence encoding the heavy chain constant region of the antibody, and a 3' J chain exon 4 homology region, wherein the heavy chain sequence and the light chain sequence can be placed in any order.

[0036] In some embodiments, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a sequence encoding a guide RNA (gRNA) sequence, a splice acceptor sequence, a 2A sequence, a sequence encoding the light chain of the target antibody, a 2A sequence, a sequence encoding the heavy chain variable sequence of the target antibody, and a splice donor sequence.

[0037] In some embodiments, the sequence encoding the antibody or its antigen binding fragment does not contain a promoter sequence. When integrated into the DNA of the B cell or HSC, the sequence encoding the antibody or its antigen binding fragment may be under the transcriptional control of an endogenous promoter. In one embodiment, when the sequence encoding the antibody or its antigen binding fragment is integrated into the DNA of the B cell or HSC, the sequence is under the transcriptional control of an endogenous heavy chain promoter in the B cell or HSC. In one embodiment, when the sequence encoding the antibody or its antigen binding fragment is integrated into the DNA of the B cell or HSC, the sequence is under the transcriptional control of an endogenous J chain promoter in the B cell or HSC.

[0038] In some embodiments, the sequence encoding the antibody or its antigen-binding fragment comprises a promoter sequence. In some embodiments, the promoter is a B cell-specific promoter or a HSC-specific promoter. Non-limiting examples of B cell-specific promoters or HSC-specific promoters include Hg38-mCP promoters and spleen focus forming virus (SFFV) promoters or fragments thereof.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof binds to an antigen associated with a disease or condition. The disease or condition may include, but is not limited to, infection, cancer, autoimmune disease, cardiovascular disease, musculoskeletal disorder, or neurodegenerative disease. In some embodiments, the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. In some embodiments, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor-associated antigen (TAA).

[0040] In various embodiments of the systems described herein, the subject is a human.

[0041] In various embodiments of the systems described herein, the subject is a laboratory animal, such as a mouse or rat.

[0042] In a related aspect, provided herein is a modified B cell or a modified hematopoietic stem cell (HSC) comprising the system of any embodiment described herein.

[0043] In another aspect, provided herein is a pharmaceutical composition comprising the system of any embodiment described herein and a pharmaceutically acceptable carrier or excipient.

[0044] In another aspect, provided herein is a kit comprising (i) a system according to any embodiment described herein and optionally (ii) a container and / or instructions for use.

[0045] In another aspect, provided herein is a method for producing a modified B cell or a modified hematopoietic stem cell (HSC) that produces an antibody or an antigen-binding fragment thereof. The method may include ex vivo transduction of a B cell or HSC with an effective amount of a system of any embodiment described herein, wherein a first component and a second component of the system are administered to the cell simultaneously or in any order, and wherein the administration of the first component and the second component results in the integration of a sequence encoding the antibody or an antigen-binding fragment thereof into the DNA of the cell, wherein the cell becomes a modified cell.

[0046] In some embodiments of the ex vivo methods described above, the first component and the second component of the system are administered to the cell simultaneously as two separate compositions.

[0047] In some embodiments of the above ex vivo methods, the first component and the second component of the system are administered to the cell simultaneously as a composition.

[0048] In various embodiments of the above ex vivo methods, wherein the B cells or HSCs are present in a heterogeneous cell population during transduction.

[0049] In various embodiments of the above ex vivo methods, the B cells have been isolated from the spleen, peritoneum or peripheral blood.

[0050] In some embodiments, the B cells are primary B cells.

[0051] In various embodiments of the above ex vivo methods, the B cell is a B2 B cell.

[0052] In various embodiments of the above ex vivo methods, the B cell is a B1 B cell. In some embodiments, the B cell is a B1a B cell (CD19+ / CD5+ / CD23-) or a B1b B cell (CD19+ / CD5- / CD23-).

[0053] In various embodiments of the above ex vivo methods, said B cells are cultured under stimulating conditions prior to and / or after said transduction.

[0054] In some embodiments, the stimulatory conditions promote B cell activation without differentiation.

[0055] In various embodiments of the above-described ex vivo methods, the B cells are cultured in the presence of a CD40 agonist and / or a CD180 agonist before and / or after the transduction. In some embodiments, the B cells are cultured in the presence of a CD40 agonist and a CD180 agonist before and / or after the transduction. In some embodiments, the CD40 agonist is a CD40 ligand (CD40L) or an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the CD180 agonist is an anti-CD180 antibody or an antigen-binding fragment thereof.

[0056] In some embodiments, before and / or after the transduction, the B cells are cultured in the presence of about 20 ng / ml or less of a CD40 agonist (e.g., CD40L) and / or about 100 ng / ml or less of a CD180 agonist (e.g., anti-CD180 antibody). In some embodiments, the CD40 agonist (e.g., CD40L) used in the B cell culture is about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20 ng / ml, or about 0-5 ng / ml, about 4-8 ng / ml, about 5-10 ng / ml, about 6-12 ng / ml, about 8-15 ng / ml, about 10-15 ng / ml, about 12-18 ng / ml, or about 15-20 ng / ml. In some embodiments, the CD180 agonist (e.g., anti-CD180 antibody) used in the B cell culture is about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 85, about 90, about 95, about 100 ng / ml, or about 0-5 ng / ml. In some embodiments, the B cells are cultured in the presence of about 20 ng / ml of a CD40 agonist (e.g., CD40L) and about 20 ng / ml of a CD180 agonist (e.g., anti-CD180 antibody). In some embodiments, the B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 4 days or less prior to the transduction. In some embodiments, the B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 1 day, about 36 hours, about 2 days, about 60 hours, about 3 days, about 84 hours, or about 4 days prior to the transduction.In some embodiments, prior to the transduction, the B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 2 days. In some embodiments, after the transduction, the B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 2 days.

[0057] In some embodiments of the above ex vivo method, the method further comprises culturing the modified B cells or modified HSCs under differentiation conditions to promote differentiation of the modified B cells or modified HSCs into modified plasma cells.

[0058] In various embodiments of the above-mentioned ex vivo method, the method further comprises introducing the modified B cell or the modified HSC or the modified plasma cell into the subject. In some embodiments, the modified B cell or HSC or the modified plasma cell is introduced into the subject intraperitoneally. In some embodiments, before the introduction of the modified B cell or HSC, the CD20+ cells of the subject have been exhausted. In some embodiments, after the modified B cell or HSC or plasma cell is introduced into the subject, the modified B cell or HSC or plasma cell is amplified in vivo by administering an antigen to the subject, and the antigen is recognized by the antibody or its antigen-binding fragment produced by the modified B cell or HSC or plasma cell. In some embodiments, the subject is autologous for the modified B cell or HSC or plasma cell. In some embodiments, the subject is allogeneic for the modified B cell or HSC or plasma cell. In some embodiments, the subject is a human. In some embodiments, the subject is an experimental animal.

[0059] In a related aspect, provided herein are modified B cells or modified HSCs or populations thereof produced by the method of any of the embodiments described above. In a related aspect, provided herein are modified plasma cells produced by the methods described above.

[0060] On the other hand, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof in a subject in need thereof, the method comprising transducing an in vitro B cell and / or hematopoietic stem cell (HSC) isolated from the subject or donor with an effective amount of any of the above-mentioned systems, wherein the first component (e.g., a viral vector) and the second component (e.g., a viral vector) are sequentially administered simultaneously or in any order, and then the transduced cells are reintroduced into the subject. In some embodiments, the B cells include B1 B cells. In some embodiments, the B cells include B2 B cells. In some embodiments, the B cells include primary B cells.

[0061] In a related aspect, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the above-described systems, wherein the first component (e.g., a viral vector) and the second component (e.g., a viral vector) are administered sequentially simultaneously or in any order. In some embodiments, administration of the first component and the second component to the subject results in the sequence encoding the antibody or its antigen-binding fragment being integrated into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), resulting in the production of the antibody or its antigen-binding fragment in the subject.

[0062] In some embodiments of the above-described in vivo methods, the first component and the second component of the system are administered to the subject simultaneously as two separate compositions.

[0063] In some embodiments of the in vivo methods described above, the first component and the second component of the system are administered to the subject simultaneously as a composition.

[0064] In some embodiments of the in vivo methods described above, the first component and / or the second component of the system is administered intraperitoneally to the subject.

[0065] In some embodiments of the in vivo method described above, the method further comprises administering to the subject an effective amount of a CD180 agonist and / or a CD40 agonist prior to administering the system to the subject. In some embodiments, the CD40 agonist is a CD40 ligand (CD40L) or an anti-CD40 antibody or an antigen-binding fragment thereof. In some embodiments, the CD180 agonist is an anti-CD180 antibody or an antigen-binding fragment thereof.

[0066] In some embodiments, the method comprises administering to the subject an effective amount of a CD180 agonist (e.g., an anti-CD180 antibody) and a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 250 μg or less of a CD180 agonist (e.g., an anti-CD180 antibody) and / or about 50 μg or less of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200 , about 210, about 220, about 230, about 240, about 250, or about 0-5, about 4-8, about 5-10, about 6-12, about 8-15, about 10-15, about 12-18, about 15-20, about 20-25, about 20-30, about 25-40, about 30-50, about 40-60, about 50-75, about 60-80, about 70-90, about 80-100, about 100-120, about 120-150, about 140-160, about 150-180, about 175-200, about 200-225, about 225-250 μg of a CD180 agonist (e.g., an anti-CD180 antibody). In some embodiments, the method comprises administering to the subject about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, or about 0-5, about 4-8, about 5-10, about 6-12, about 8-15, about 10-15, about 12-18, about 15-20, about 20-25, about 20-30, about 25-35, about 30-40, about 40-50 μg of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In one embodiment, the method comprises administering to the subject about 12.5 μg of a CD180 agonist (eg, an anti-CD180 antibody) and not administering a CD40 agonist (eg, an anti-CD40 antibody).In one embodiment, the method comprises administering to the subject about 12.5 μg of a CD40 agonist (eg, an anti-CD40 antibody) without administering a CD180 agonist (eg, an anti-CD180 antibody).

[0067] In some embodiments, the method comprises administering to the subject an effective amount of a CD180 agonist (e.g., an anti-CD180 antibody) and a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 8.5 mg / kg body weight or less of a CD180 agonist (e.g., an anti-CD180 antibody) and / or about 1.8 mg / kg body weight or less of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.33, about 8.5, or about 0-0.5, about 0.4-0.8, about 0.5-1, about 1-2, about 1.5-2.5, about 2-4, about 3-5, about 4-6, about 5-7, about 6-8, or about 7.5-8.5 mg / kg body weight of a CD180 agonist (e.g., an anti-CD180 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, or about 0-0.5, about 0.2-0.6, about 0.4-0.8, about 0.5-1, about 0.6-1.2, about 0.8-1.5, about 1-1.5, about 1.2-1.6, about 1.5-1.8 mg / kg body weight of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In one embodiment, the method comprises administering to the subject about 0.4 mg / kg body weight of a CD180 agonist (e.g., an anti-CD180 antibody). In one embodiment, the method comprises administering to the subject about 0.4 mg / kg body weight of a CD40 agonist (e.g., an anti-CD40 antibody). In some embodiments, the method comprises administering to the subject a CD180 agonist without administering a CD40 agonist. In some embodiments, the method comprises administering to the subject a CD40 agonist without administering a CD180 agonist.

[0068] In some embodiments, the method comprises administering a CD180 agonist (e.g., an anti-CD180 antibody) and / or a CD40 agonist (e.g., an anti-CD40 antibody) to the subject about 7 days or less before administering the system to the subject. In some embodiments, the method comprises administering a CD180 agonist (e.g., an anti-CD180 antibody) and / or a CD40 agonist (e.g., an anti-CD40 antibody) to the subject about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 1 day, about 36 hours, about 2 days, about 60 hours, about 3 days, about 84 hours, about 4 days, about 5 days, about 6 days, or about 7 days before administering the system to the subject. In some embodiments, the method comprises administering a CD180 agonist (e.g., an anti-CD180 antibody) and / or a CD40 agonist (e.g., an anti-CD40 antibody) to the subject about 2-3 days before administering the system to the subject.

[0069] In some embodiments of the above-mentioned in vivo methods, the method further comprises administering to the subject an effective amount of an antigen that is recognized by the antibody or its antigen-binding fragment, wherein the antigen is administered before and / or after the first and / or second components of the system are administered. In some embodiments, the antigen has a low affinity for the antibody or its antigen-binding fragment. In some embodiments, the antigen has a high affinity (e.g., picomolar range) for the antibody or its antigen-binding fragment. In some embodiments, the method comprises administering an antigen with a low affinity for the antibody or its antigen-binding fragment before administering the first and second components of the system, and administering an antigen with a high affinity (e.g., picomolar range) for the antibody or its antigen-binding fragment after administering the first and second components of the system.

[0070] In some embodiments of the above-mentioned in vivo methods, the method comprises administering to the subject an effective amount of a first antigen, and administering to the subject an effective amount of a second antigen; wherein the first antigen has a low affinity for the antibody or antigen-binding fragment thereof, and wherein the first antigen is administered before administration of the first component and the second component of the system; wherein the second antigen has a high affinity for the antibody or antigen-binding fragment thereof, and wherein the second antigen is administered after administration of the first component and the second component of the system.

[0071] In some embodiments of the above-described in vivo methods, the subject is a human.

[0072] In some embodiments of the above-described in vivo methods, the subject is an experimental animal.

[0073] In another aspect, the present invention provides a method for treating or reducing the likelihood of a disease or condition in a subject in need thereof, the method comprising implementing the ex vivo method of any of the above embodiments or the in vivo method of any of the above embodiments, wherein the method results in the production of an effective amount of the antibody or antigen-binding fragment thereof in the subject.

[0074] In yet another aspect, the present invention provides a method for treating a disease or condition in a subject in need thereof or reducing the likelihood thereof, comprising administering to the subject an effective amount of any of the above-described systems, wherein the first component (e.g., viral vector) and the second component (e.g., viral vector) are administered simultaneously or sequentially in any order, wherein administration of the system results in the production of an effective amount of the antibody or antigen-binding fragment thereof in the subject. The antibody or antigen-binding fragment thereof produced binds to an antigen associated with the disease or condition.

[0075] In yet another aspect, the present invention provides a method for treating a disease in a subject in need thereof, the method comprising transducing an in vitro B cell and / or hematopoietic stem cell (HSC) isolated from the subject or donor with an effective amount of any of the above systems, wherein the first component (e.g., viral vector) and the second component (e.g., viral vector) are sequentially administered simultaneously or in any order, and then the transduced cells are reintroduced into the subject, wherein administration of the system results in the production of an effective amount of the antibody or antigen-binding fragment thereof in the subject. The antibody or antigen-binding fragment thereof produced binds to an antigen associated with a disease or condition. In some embodiments, the B cell comprises a primary B cell. In some embodiments, the B cell comprises a B1 B cell. In some embodiments, the B cell comprises a B2B cell.

[0076] In some embodiments of any of the above methods of treatment, the disease is an infection, cancer, an autoimmune disease, a cardiovascular disease, a musculoskeletal disorder, or a neurodegenerative disease. In some embodiments, the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

[0077] In some embodiments of any of the methods described above, the first component (eg, viral vector) and the second component (eg, viral vector) are administered in the same composition.

[0078] In some embodiments of any of the methods described above, the subject is a human.

[0079] In some embodiments of any of the methods described above, the subject is an experimental animal, such as a mouse or a rat.

[0080] These and other aspects described herein will be apparent to those of ordinary skill in the art from the following description, claims and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 An overview of AAV-mediated delivery of transgenes to mice for episomal expression is described.

[0082] Figure 2 An overview of AAV plus cas 9 / guide RNA (gRNA)-mediated insertion of transgenes into a mouse liver genomic locus is described.

[0083] Figure 3 Results showing in vitro neutralization with episomal and liver-inserted anti-PcrV monoclonal antibodies (mAbs) from mouse serum to within 2- to 5-fold of CHO-purified mAbs are described.

[0084] Figure 4 Described are results showing that episomal and liver-inserted anti-PcrV mAbs provide protection against lethal infection in an in vivo challenge model with P. aeruginosa.

[0085] Figure 5A An overview of the ex vivo strategy for adaptive antibody vaccination in mice is described. Figure 5B Exemplary AAV vectors carrying antibody heavy and light chain genes are described.

[0086] Figure 6 Depicted are results showing the percentage of B cells expressing the introduced target B cell receptor (BCR) for mock control, RNP control, and RNP+AAV1.

[0087] Figure 7A-7C An overview of engineering B cell specificity by inserting antibody genes into the heavy chain locus of peripheral B cells is described. Fig. 7A Depletion of mouse B cells modified by Cas9 / gRNA via AAV delivery is described. Figure 7B The VI3 heavy chain, gRNA cleavage site, and BCR insert are depicted. Figure 7C The BCR insert spliced ​​into the VI3 heavy chain is depicted.

[0088] Figure 8A-B ULC pairing and full-length BCR insertion are described. Fig. 8A The two BCR variants used are depicted: ULC ligated against BCMA (top) and against PcrV (bottom). Figure 8BDepicted are the results for the two variants in experimental conditions with AAV only and RNP only controls, plus AAV+RNP. For each condition, antigen binding is given as a percentage.

[0089] Figure 9A-9B Insertion of the mCherry construct designed for the testing template is described. Fig.9A Two mCherry variants are depicted: T2A-mCherry (top) and pV h3-23-mCherry (bottom). Fig. 9B Results for both variants, no virus and no RNP controls are depicted. Results were analyzed 3 days post infection. Positive mCherry expression in both variants using 150 pmol Cas9 and 400 pmol gRNA3.

[0090] Figure 10A-10B The use of multiple gRNA targeting sites for VI3 insertion is described. Fig. 10A The insertion positions of the eight different gRNAs used are depicted. Fig. 10B Depicted are the results of T2A-mCherry expression using each of eight different gRNAs.

[0091] Figure 11A-11B Multiple gRNA targeting sites available in the Igκ locus that disrupt expression of endogenous light chains and support insertion of full-length antibodies are described. Fig.11A Seven different gRNAs used for comparison are described: gRNA4, gRNA6, gRNA7, gRNA8, gRNA9, gRNA10, and gRNA4+6. Fig. 11B Results showing gRNA7 (which cuts at the splice acceptor site and does not require recoding of constant kappa in the AAV template, with low mlgλ and mlgκ expression of 91.7%) as well as gRNA10 (92.2) and gRNA4+6 (92.0) are described. Other results: mock control (5.42), gRNA4 (82.4), gRNA6 (85.7), gRNA8 (37.3) and gRNA9 (81.7).

[0092] Figure 12A-12B Results are described for mouse splenic B cells cultured with the following growth factors: 1) CD40L-HA, anti-HA, and IL-4 (as before), 2) anti-CD180, 3) CD40L-HA, anti-HA, and BAFF, 4) anti-CD180 and BAFF, and 5) CD40L-HA, anti-HA, anti-CD180, and BAFF. Three million cells were nucleofected at 24 hours with 50 pmol Cas9, 400 pmol gRNA, and AAV6-VI3-gRNA1-T2A-mCherry ( Fig. 12A). 500,000 cells were infected with AAV6 at 2.5e5 vg / cell and analyzed 3 days after infection. Fig. 12B As shown, mCherry expression was strongest under condition 1 (19.5) and condition 5 (10.8).

[0093] Figure 13A-13B Results are described for mouse splenic B cells cultured with 1) CD40L-HA, anti-HA, and IL-4, or 2) CD40L-HA, anti-HA, and anti-CD180. Three million cells were nucleofected with 150 pmol Cas9, 400 pmol total gRNA (BCR-gRNA1 and mlgK-gRNA7), and full-length H1H29338 antibody ( Fig.13A ). 500,000 cells were infected with AAV1 at 2e5 vg / cell and analyzed 2 days after infection. Both conditions were effective, with 8.24% and 3.64% for condition 1 and condition 2, respectively. Fig. 13B shown.

[0094] Figure 14A-14B Results of transfer and immunization experiments with anti-PcrV edited B cells are described. B cells from CHC WT mice were grown in CD40L-HA, anti-HA, and anti-CD180, followed 24 hours later by RNP nucleofection and AAV1 infection with h1h29339 anti-PcrV full-length antibody ( Fig.14A Antibodies from CHCWT littermates performed well compared to standard non-insertion VI3 / ULC (13.7 to 9.42, respectively). Fig. 14B ). 1) All results for PcrV binding of CHC WT littermates: mock control (0.28), RNP only (0.13), RNP+AAV1 (13.7); and 2) All results for PcrV binding of VI3 / ULC: mock control (0.25), RNP only (0.54), RNP+AAV1 (9.42).

[0095] Figure 15A-15B The results of B cells edited to express anti-PcrV BCRs that matured both in vitro and in vivo to produce anti-PcrV antibodies following adoptive transfer and immunization into mice are described. In vitro, analysis of supernatants from B cells edited for PcrVBCR and cultured in LPS for 7 days for PcrV antibodies showed antibody production ( Fig.15A In vivo, B cells were edited against the PcrV BCR and transferred into Flu-CHC mice as described previously. Serum analysis was performed approximately one week after immunization, and mouse sera again produced antibodies ( Fig. 15B ).

[0096] Fig.16 The adoptive transfer of donor B cells from HA antigen-immunized mice into CD20-depleted naive mouse recipients is described, with donor ex vivo activated B cells showing a dramatic expansion one week after transfer but not persisting after one month.

[0097] Fig.17A The SFFV promoter, "B cell core" and the putative core promoter (predicted based on the location of the TATA box) are depicted with subsequences 1, 2, 3, 4 indicated. Fig. 17B The expression and cell type specificity of the enriched transcription factors are described. The expression data were used to select B cell-specific transcription factors. Fig. 17C Described are consensus sites that can be engineered into enhancer backbones to enhance expression.

[0098] Figure 18A-18B The overview and results of reporter construct generation are described. Fig.18A An overview of representative reporter constructs in the AAV context is described. The SFFV subsequence is paired with the MLP and cloned upstream of the eGFP coding sequence. The overview describes only the sequence between the AAV ITRs, which will be paired with bacterial sequences (such as ampicillin resistance) for propagation in Stbl2 cells. Fig.18B Depicted is GFP expression (x-axis) following infection of primary murine B cells with AAV encoding five SFFV subsequences as promoter constructs. All subsequences were paired with the adenovirus major late promoter.

[0099] Fig.19 Results are described for full-length SFFV-eGFP and variants (including SFFV-core-mCP-GFP, SFFV1-mCP-eGFP, SFFV2-mCP-eGFP, SFFV3-mCP-eGFP, and SFFV4-mCP-eGFP) transfected into Ramos and HEK293-HZ cells. SFFV4 showed activity in Ramos and HEK cells at a length of 121 bp.

[0100] Fig. 20 Depicted are results demonstrating that HS-B is a 180 bp B cell specific Pax5 enhancer as shown by luciferase expression in a mouse B cell line. Note the right shift in GFP in the top row of pro-B cells, pre-B cells, immature B cells, and mature B cells compared to controls.

[0101] Fig.21AResults of AAV-GFP testing of 120-170 base pair promoters in primary B cells and HEK293-HZ cells are described, with three promoters used with mCP-eGFP: 1) HS-B, 2) hg38HS-B, and 3) SFFV4. Cells were cultured and transfected with 5e5 vg of AAV6 crude virus preparation per cell, as well as CD40L-HA, anti-HA, and IL-4. Fig.21B Depicted is the ranking of HS-B based on B cell enrichment in the Corces et al. ATAC-seq dataset: #286 / 589,844 (#5344 if ranked by B cell signaling).

[0102] Fig. 22 A schematic representation of SpyTag:SpyCatcher used to attach mAbs to the surface of AAV capsids for retargeting purposes is depicted.

[0103] Fig.23A It was shown that AAV2 and AAV6 could be conjugated to the hCD20 mAb at similar levels as the ASGR1 control antibody. Fig. 23B Depicted are results showing that AAV2 (top) and AAV6 (bottom) can target HEK293 cells expressing CD20. AAV2 or AA6 with anti-hCD20 mAb attached via SpyTag:SpyCatcher accurately targeted HEK293-hCD20 cells.

[0104] Fig.24 Depicted are results showing that AAV2 (top row) and AAV6 (bottom row) can target Ramos cells expressing CD20. AAV2 or AA6 with anti-hCD20 mAb attached via SpyTag:SpyCatcher accurately targeted Ramos3-hCD20 cells. The result graph (bottom) shows slight off-targeting of AAV6, but no off-targeting of AAV2.

[0105] Fig.25A Human B cells cultured under various stimulation conditions are described. CD19+ B cells were isolated from human peripheral blood and cultured under various stimulation conditions: 1) IL-4 alone, 2) IL-4, CD40L-HA and anti-HA mAb, and 3) IL-4 and anti-CD40 mAb. Cells were infected with AAV2 / CD20 or AAV6 / CD20, and virally delivered eGFP was measured by flow cytometry at day 4 post-infection. Fig.25B Depicted are results showing that AAV2 (top row) and AAV6 (bottom row) can target human B cells expressing CD20. The results demonstrate that while both AAV2 and AAV6 can target primary human B cells via CD20, AAV6 / CD20 exhibits a significant enhancement in transduction.

[0106] Fig.26 Retargeting of HEK 293CD20(-), HEK 293CD20(+), Jurkat T cells, and Duadi B cell lines with AAV1 WT, AAV1 detargeting mutants, and AAV1-hCD20 (all packaged with SSF-eGFP) is described. Results show that AAV1 detargeting mutants still transduce, and antibody conjugation slightly reduces off-target transduction, while the retargeted virus is comparable to WT in the Daudi cell line.

[0107] Fig. 27 Describes retargeting of HEK 293CD20(-), HEK 293CD20(+), Jurkat T cells and Duadi B cell lines with AAV2 WT, AAV2 detargeting mutants and AAV2-hCD20 (all packaged with SSF-eGFP). Results show that AAV2-CD20 exhibits gain of function on the Daudi cell line.

[0108] Fig.28 Retargeting of HEK 293CD20(-), HEK 293CD20(+), Jurkat T cells, and Duadi B cell lines with AAV6 WT, AAV6 detargeting mutants, and AAV6-hCD20 (all packaged with SSF-eGFP) is described. Results show that AAV6 retargeting mutants do not completely detarget, non-binding mAbs reduce off-target transduction, and AAV6-CD20 exhibits gain of function in the 293hCD20(+) cell line.

[0109] Fig.29 The authors describe the retargeting of HEK 293CD20(-), HEK 293CD20(+), Jurkat T cells, and Duadi B cell lines with AAV9 WT, AAV9 detargeting mutants, and AAV9-hCD20 (all packaged with SSF-eGFP). The results show that AAV9-CD20 has gain-of-function and reduced off-target transduction in hCD20(+) cell lines. Fig.29 ).

[0110] Fig.30 A schematic overview of the methods used to translate ex vivo B cell targeting and editing technologies into in vivo applications by in vivo delivery of viral vectors to mediate BCR insertion is described.

[0111] Fig.31 Depicted are diagrams showing that human stem cells are upstream of immune cells and represent targets for a range of viral transductions, including AAV viruses and lentiviruses.

[0112] Fig.32Described are results showing that in AAV6, attached to anti-hCD34 (My10) antibody via the SpyTag:SpyCatcher system, different promoters were attached to GFP for infection of human cord blood cells and primary mouse B cells. The results indicate that SFFV is a preferred promoter over CAG and EF1.

[0113] Fig.33 Retargeting of HSCs with AAV2-hCD34 packaged with SFFV-eGFP is described. Results show that natural tropism exceeds the retargeting antibody on human umbilical cord blood cells, while non-binding mAbs reduce off-target transduction, and anti-CD34 mAbs can retarget AAV2 HBM mutants in 293 / hCD34 and human umbilical cord blood cells.

[0114] Fig.34 Retargeting of HSCs with AAV9-hCD34 packaged with SFFV-eGFP is described. Results demonstrate gain of function on the 293hCD34+ cell line in the presence of CD34 antibodies; low off-target transduction; and poor transduction of human umbilical cord blood cells by AAV9+ / - anti-hCD34 antibodies.

[0115] Fig.35 Retargeting of HSCs with AAV6-hCD34 packaged with SFFV-eGFP is described. Results show that native tropism again outweighs the retargeting antibody on human cord blood cells, but anti-CD34 mAbs can robustly retarget AAV6 HBM mutants in 293 / hCD34 cells and moderately in human cord blood cells.

[0116] Fig.36 Described are results showing that mAb-conjugated lentiviral vectors were specifically retargeted to cells expressing CD34 compared to anti-CD34, with mAb-dependent transduction efficacy. 10,000 cells were seeded per plate (96-well plate), and LV-SINmuZZ EF1a-FLuc of 2E+08VG was mixed with 2-fold serial dilutions of CHOt supe (starting from 100ul) in DMEM. After incubation at 37°C for 30min, the LV-CHOt mixture was added to the cells and incubated at 37°C. Fluc readout was performed 4 days after transduction. Results are shown for the following 9 cases: 1) 9C5(CD34)-SpyC, 2) My1C(CD34)-SpyC, 3) 563(CD34)-SpyC, 4) CD20-SpyC, 5) 9C5, 6) CD20, 7) BSTpro MOCK, 8) VLP alone, and 9) NT. The experiment was repeated for HEK293 cells, 293-hCD20 cells, and 293-hCD34 cells.

[0117] Fig.37 Described are results showing that mAb-conjugated SpyTagged AAV2 can also specifically retarget CD34-expressing cells with mAb-dependent transduction efficacy compared to anti-CD34-SpyCatcher. Results are shown for the following 9 conditions: 1) 9C5(CD34)-SpyC, 2) My1C(CD34)-SpyC, 3) 563(CD34)-SpyC, 4) CD20-SpyC, 5) 9C5, 6) CD20, 7) BSTpro MOCK, 8) VLPs alone, and 9) NT. The experiment was repeated for HEK293 cells, 293-hCD20 cells, and 293-hCD34 cells.

[0118] Fig.38 Described are results showing that the optimization of chimerism revealed that AAV2HBM-mix 1 / 4 resulted in higher transduction in the HEK293T / hCD34 cell line. Screening for platform gene delivery for CD34 was started by seeding 10,000 cells per well in a 96-well black wall clear bottom plate with three cell types (293, 293-hCD20, 293-hCD34). Next, AAV2 1 / 8SpyTag / HBM SFFV-Fluc of 5E+09VG was mixed with 2-fold serial dilutions of CHOt supe (starting from 100 μl) in DMEM and incubated at 37°C for 1.5 hr. Then, the AAV2-CHOt mixture was added to the cells and incubated at 37°C. Three days later, the cells were collected for flow analysis. Fig.34 The results shown in are for different types of HBM mixes. AAV2 HBM-mix 1 / 4 had higher transduction in 293-CD34 cells, followed by AAV2 HBM-mix 1 / 2.

[0119] Figure 39A-Figure 39C Described are results showing the following: Results of lentiviral vector retargeting of anti-CD117 and anti-Sca-1 transduced cell lines expressing those corresponding target antigen receptors in vitro. 1E+04 cells were seeded in 100ul of DCM containing 4ug / ml polybrene in a 96-well black well clear bottom plate. Cells were transduced with 2E+04 VG / cell in a 100μl aliquot of DCM containing 4ug / ml polybrene. After 2 days, fluorescence imaging was performed ( Fig.39A ) and analyzed by flow cytometry for GFP( Fig.39B and Fig.39C ). The results showed that the vectors successfully retargeted cell lines expressing their respective target antigens by imaging and flow cytometry.

[0120] Fig.40Results showing that surface expression of CD117 and Sca-1 was detected on mouse HSPCs are described. Mouse HSPCs were isolated from collected bone marrow on day 0. Cells were cultured in progenitor cell culture medium with SFEM+SCF (100 ng / mL), TPO (100 ng / mL), Flt3L (100 ng / mL), IL-6 (50 ng / mL), and IL-3 (30 ng / mL). Two days after isolation, cells were observed for CD117 and Sca-1 and then transduced with pseudoparticles having an SFFV-GFP reporter gene. Two days after transduction (day 4 after isolation), GFP expression was read out by FACS. The results show that CD117 and Sca-1 are expressed on the surface of mouse HSPCs.

[0121] Fig.41 Results showing mouse HSPCs transduced with lentiviral vectors pseudotyped with anti-mouse CD117 mAb and SINmu are depicted. Conditions were no additive, vectofusin-1, or lentiboost (left to right). Cells were either not transduced or transduced with LV-VSVg, LV-ahASGR1+SINmu, or LV-amCD117+SINmu (top to bottom). LVs pseudotyped with α-CD117+SINmu can transduce expanded mouse primary HSPCs with very low efficiency. This is an entry problem because LVs pseudotyped with VSVg can efficiently transduce expanded mouse HSPCs.

[0122] Figure 42A-42B Described are results showing that SpyTagged AAV2 was efficiently retargeted to cell lines expressing CD117 or Sca-1 in vitro. Retargeted AAV2-HBM 1 / 8 chimeras with CD117, Sca-1, hCD34, or hCD20 successfully retargeted HEK293 cell lines expressing these markers at 5E+05 VG / cell, as demonstrated by fluorescence imaging ( Fig.42A ) and GFP analysis by flow cytometry ( Fig.42B ) as shown. Figure 42C-Figure 42D The design principles of SpyTagged AAV2 with anti-ScaI antibody are described.

[0123] Fig.43 A proposed strategy for B1 B cell antibody engineering is shown.

[0124] Fig.44 A proposed strategy for ectopic engineered antibody expression in B1 B cells is shown.

[0125] Fig.45 It was shown that B1a B cells activated with CD40L / aCD180 and transferred intraperitoneally had enhanced recovery at 14 and 32 days.

[0126] Fig.46 CD180 stimulation of B1a cells was shown to induce proliferation without differentiation into plasmablasts / plasma cells (PCs).

[0127] Fig.47 Different transduction efficiencies between B1 and B2 peritoneal (PerC) B cell subsets were demonstrated.

[0128] Fig.48 It was shown that pan B cells from the peritoneum could be edited, but not as efficiently as B2 splenocytes.

[0129] Figure 49A-49B Results are described showing in vitro culture conditions that facilitate re-engraftment of ex vivo cultured mouse B cells. In vitro B cell culture with low levels of aCD40 and / or aCD180 facilitates re-engraftment of cells in mice, whereas high levels of B cell activation are detrimental to long-term re-engraftment ( Fig.49A CD45.1 B cells cultured in vitro for 3 days were adoptively transferred into CD45.2 mice ( Fig.49B ).

[0130] Figures 50A-50D Results showing ex vivo AAV transduction / editing and transfer of cultured non-differentiated Cas9 mouse B cells into SRG mice are described. Fig.50A An overview of ex vivo AAV transduction / editing and transfer of cultured undifferentiated Cas9 mouse B cells into SRG mice is described. Fig.50B A J chain locus exon 4 insertion is described. Fig.50C ROSA insertion is described. Fig.50D Described is the measurement of luciferase signaling in vivo over time using IVIS technology.

[0131] Figure 51A-Figure 51B Exemplary editing strategies for editing different murine loci for different modes are described. Fig.51A Exemplary "immune system repertoire enhancement" paradigms are described. Fig.51B An exemplary "protein factory" schema is described.

[0132] Figure 52A-Figure 52B An exemplary mouse J chain locus insertion strategy for high expression of a protein of interest in plasma cells is described. Fig.51A An exemplary strategy for using the endogenous J chain promoter and preserving the J chain is described. Fig.52B An exemplary strategy for using the endogenous J chain promoter and eliminating the J chain is described.

[0133] Figure 53A-Figure 53BWe show that the generation of memory B cells is critical for the successful in vivo B cell editing of adaptive antibodies and protein factory models. Fig.53A An exemplary protocol for expansion of BCR-edited B cells via a new BCR is described. Fig.53B An exemplary protocol for the expansion of non-BCR-edited B cells via "ligation specificity" to priming Ag is described.

[0134] Figure 54A-Figure 54D Describes outcomes showing that modulation of “pan-B cell” stimulation of Cas9 mice enables AAV editing of B cells and Ab production. Fig.54A The VI3 heavy chain, gRNA cleavage site, and BCR insert are depicted. Fig.54B Described are results showing synergistic effects of aCD40 and aCD180 stimulation in vivo on B cell editing and antibody expression. Fig.54C Described are results demonstrating that Ab1 Ab expression is transient in "pan-B" primed / edited mice, but show evidence of Ab recall following subsequent Ag challenge. Fig.54D Described are results of in vivo B cell editing in mice showing "pan-B" stimulation to generate edited B cells that can be recruited into subsequent responses to Ag challenge.

[0135] Figure 55A-Figure 55D Described are results demonstrating that priming and boosting with suboptimal BCR:Ag interactions promotes the generation of Ab1 memory B cells compared to Ab-producing PCs.

[0136] Fig.55A It was shown that suboptimal BCR:Ag interactions ("low affinity") are expected to bias edited B cells toward the memory compartment. "High affinity" BCRs lead to high B cell activation and enhance Tfh interactions, which drive cells to become Ab-producing plasma cells. B cells with "low affinity" BCRs have lower activity and cannot fully engage with Tfh cells. This neglect leads to a bias toward memory B cells. Fig.55B Results showing that neutralization of the F490L spike by Ab1 was reduced 90-fold are described. Fig.55C Described are demonstrations that priming and boosting with F490L spike Ag fail to induce the production of Abl Ab from Abl edited B cells. Fig.55D Described are results demonstrating that Abl BCR editing into mice primed with a "low affinity" Ag resulted in reproducible induction of Ab expression following boosting with WT "high affinity" Ag at d28.

[0137] Figure 56A-Figure 56CDescribed are results demonstrating that the addition of aCD180 to Ag priming increases the number of edited B cells that can be recalled 1 and 3 months after editing. Figure 56A-Figure 56B An exemplary experimental workflow is described. Fig.56C It was shown that the Ag-primed group alone resulted in lower Ab levels after the “high-affinity” Ag boost compared to the aCD180-treated group, and the increase in Ab titer in the aCD180 group after the Ag boost was evidence of the initial generation of more edited B cells.

[0138] Figure 57A-Figure 57C Results demonstrating long-term persistence of in vivo edited B cells (non-IgH loci) in Ag-primed mice are shown. Fig.57A An exemplary workflow is described for programming B cell-specific promoter-driven luciferase into the Rosa locus of Ag-primed Cas9 mice. Fig.57B Depicted are the results of IVIS imaging of B cell-specific luciferase expression in edited mice. Fig.57C Longitudinal analysis of luciferase signaling demonstrating persistence of in vivo edited B cells in mice is described.

[0139] Figure 58A-Figure 58B Described are results demonstrating that editing of AAV "Nluc-Abl" into the IgH locus enables tracking of BCR-edited cells in vivo over time. Fig.58A An exemplary workflow is described. Fig.58B Described are results showing tracking of BCR-edited cells over time.

[0140] Figure 59A-Figure 59C Described are results showing peritoneal B cell editing achieved by AAV IP delivery into unprimed Cas9Ready mice. Fig.59A An exemplary workflow is described. Fig.59B It is described that luciferase signals are readily observed in all draining lymph nodes of the peritoneal cavity of Cas9Ready mice edited with B cell-specific luciferase. Fig.59C It is described that nLuc-positive signals were observed mainly in B1b and B1a B cells in the peritoneal cavity.

[0141] Detailed Description

[0142] definition

[0143] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0144] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or which will become apparent to those skilled in the art upon reading this disclosure.

[0145] The terms "protein", "polypeptide" and "peptide" used interchangeably herein include polymeric forms of amino acids of any length, including coded and non-coded amino acids and amino acids modified or derivatized chemically or biochemically. The term also includes polymers that have been modified, such as polypeptides with modified peptide backbones.

[0146] The terms "nucleic acid" and "polynucleotide" used interchangeably herein include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified forms thereof. They include single-stranded, double-stranded, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derived nucleotide bases.

[0147] Nucleic acids are said to have a "5' end" and a "3' end" because mononucleotides are reacted in such a way that the 5' phosphate of one mononucleotide pentose ring is attached to its adjacent 3' oxygen in one direction via a phosphodiester linkage to produce an oligonucleotide. If the 5' phosphate of an oligonucleotide is not linked to the 3' oxygen of a mononucleotide pentose ring, then the end of the oligonucleotide is called the "5' end". If the 3' oxygen of an oligonucleotide is not linked to the 5' phosphate of another mononucleotide pentose ring, then the end of the oligonucleotide is called the "3' end". A nucleic acid sequence, even within a larger oligonucleotide, can be considered to have a 5' and 3' end. In a linear or circular DNA molecule, discrete elements are referred to as "upstream" or 5' of the "downstream" or 3' element.

[0148] The term "wild type" includes an entity having a structure and / or activity found in a normal (eg, as opposed to mutated, diseased, altered, etc.) state or environment. Wild-type genes and polypeptides typically exist in multiple different forms (eg, alleles).

[0149] The term "B cell" as used herein refers to a cell of the B cell lineage. In some embodiments, the B cells used in the compositions and methods of the present invention include, but are not limited to, B1 B cells, B2 B cells, memory B cells, plasmablasts, or plasma cells, or a combination thereof. In some embodiments, the B cells used in the compositions and methods of the present invention may be primary B cells.

[0150] The term "antibody" includes immunoglobulin molecules containing four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds). Each heavy chain contains a heavy chain variable domain (V H ) and the heavy chain constant region (C H The heavy chain constant region consists of at least three domains: C H 1. C H 2. C H 3 and optionally CH4. Each light chain comprises a light chain variable domain (C H ) and the light chain constant region (C L ). The heavy and light chain variable domains can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). The typical tetrameric antibody structure contains two identical antigen binding domains, each of which is bound by a V H With V L The domains are formed by association, and each domain is associated with the corresponding C H and C L The domains together form the antibody Fv region. Single-domain antibodies contain a single antigen-binding domain, such as V H or V LAs used herein, the term "antibody" encompasses B cell receptors (BCR) and secretory antibodies, etc. The term "antibody" also encompasses monoclonal antibodies, multispecific (e.g., bispecific) antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, single-domain antibodies, Fab fragments, F (ab') fragments, disulfide-linked Fv (sdFv), intracellular antibodies, mini antibodies, diabodies, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies for antigen-specific TCRs) and epitope-binding fragments of any of the above antibodies. The terms "antibody" and "antibodies" also refer to covalent diabodies (such as those disclosed in U.S. Patent Application Publication 2007 / 0004909) and Ig-DARTS (such as those disclosed in U.S. Patent Application Publication 2009 / 0060910). Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0151] The antigen binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to an antigenic epitope, is also referred to as a "paratope." It is a small region (5-10 amino acids) of the Fv region of an antibody, a portion of the antigen binding fragment (Fab region), and may include a portion of the heavy and / or light chains of an antibody. When a paratope binds to an epitope with high affinity, then the paratope specifically binds to the epitope. The term "high affinity" antibody refers to an antibody that has about 10 amino acids relative to its target epitope. -9 M or less (e.g., about 1×10 -9 M, 1×10 -10 M, 1×10 -11 M or about 1×10 -12 M) K D In one embodiment, K D By surface plasmon resonance (BIACORE TM ) measurement; in another embodiment, K D Measured by ELISA.

[0152] The phrase "complementarity determining region" or the term "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin gene, which is usually (i.e., in a wild-type animal) present between two framework regions in a light chain or heavy chain variable region of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). CDRs may be encoded by, for example, germline sequences or rearranged or unrearranged sequences, and are, for example, encoded by initial or mature B cells or T cells. CDRs may be somatically mutated (e.g., different from sequences encoded in animal germlines), humanized, and / or modified by amino acid substitutions, additions, or deletions. In some cases (e.g., for CDR3), CDRs may be encoded by two or more sequences (e.g., germline sequences) that are not adjacent (e.g., in unrearranged nucleic acid sequences) but adjacent in a B cell nucleic acid sequence (e.g., as a result of splicing or connecting sequences (e.g., VDJ recombination to form heavy chain CDR3)).

[0153] "Epitope" is a part of a macromolecule recognized by the immune system, particularly by antibodies, B cells or cytotoxic T cells. Although epitopes are generally considered to be derived from non-self proteins, sequences that can be identified from hosts are also classified as epitopes. An epitope has at least 4 amino acids, preferably 4 to 30 amino acids, more preferably 5 to 20 amino acids, especially 5 to 15 amino acids in length. An epitope can be linear or three-dimensional (usually formed by amino acids that are away from each other in the primary protein structure but become adjacent to related amino acids in the secondary and / or tertiary structure). An epitope specifically recognized by a B cell is referred to as a B-cell epitope.

[0154] The phrase "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa and lambda light chains and VpreB, and alternative light chains, unless otherwise specified. Unless otherwise specified, a light chain variable domain typically includes three light chain CDRs and four framework (FR) regions. Typically, a full-length light chain includes a variable domain and a light chain constant region from the amino terminus to the carboxyl terminus, and the variable domain includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The light chain variable domain is encoded by a light chain variable region gene sequence, which typically includes V L and J LSegments, which are derived from the V and J segment libraries present in the germline. The sequences, positions and nomenclature of the V and J light chain segments of various organisms can be found in the IMGT database at imgt.org. Light chains include, for example, light chains that do not selectively bind to the first or second epitopes that are selectively bound by the epitope-binding protein in which they are located. Light chains also include light chains that bind to and recognize one or more epitopes that are selectively bound by the epitope-binding protein in which they are located or that help the heavy chain or another light chain bind to and recognize the one or more epitopes. Common or universal light chains include light chains derived from human Vκ1-39Jκ genes or human Vκ3-20Jκ genes, and include somatic mutations (e.g., affinity maturation) thereof. Exemplary human V L Segments include human Vκ1-39 gene segments, human Vκ3-20 gene segments, human Vλ1-40 gene segments, human Vλ1-44 gene segments, human Vλ2-8 gene segments, human Vλ2-14 gene segments, and human Vλ3-21 gene segments, and include somatic mutations (e.g., affinity matured) thereof. A light chain comprising a variable domain from one organism (e.g., a human or a rodent, such as a rat or a mouse; or a bird, such as a chicken) and a constant region from the same or different organism (e.g., a human or a rodent, such as a rat or a mouse; or a bird, such as a chicken) can be prepared.

[0155] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences from any organism, including immunoglobulin heavy chain constant region sequences. Unless otherwise indicated, a heavy chain variable domain includes three heavy chain CDR regions and four FR regions. A heavy chain fragment includes CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has CDRs after the variable domain (from N-terminus to C-terminus). H 1 domain, hinge region, C H 2 domains and C H The functional fragment of the heavy chain includes a heavy chain that is capable of specifically recognizing an epitope (e.g., with a K in the micromolar, nanomolar or picomolar range). D The heavy chain variable domain is encoded by a variable region nucleotide sequence, which generally comprises a V region derived from a V region present in the germline. H , D H and J H Segment Library V H , D H and J H Segments. The sequences, locations and nomenclature of the V, D and J heavy chain segments of various organisms can be found in the IMGT database, which is available via the Internet at the URL "imgt.org" on the World Wide Web (www).

[0156] The terms "heavy chain-only antibody", "heavy chain-only antigen binding protein", "single domain antigen binding protein", "single domain binding protein" and the like refer to monomeric or homodimeric immunoglobulin molecules comprising immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region, which is incapable of associating with a light chain because the heavy chain constant region generally lacks a functional C H 1 domain. Thus, the terms "heavy chain-only antibody", "heavy chain-only antigen binding protein", "single domain antigen binding protein", "single domain binding protein" and the like encompass (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chains comprising a heavy chain-only antibody and a heavy chain-only antigen binding protein lacking a functional C H 1 domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each chain comprising a variable domain that lacks a functional C H In various aspects, the homodimeric single-domain antigen binding protein comprises two identical immunoglobulin-like chains, each chain comprising a variable domain that lacks a functional C H In addition, each immunoglobulin-like chain of the single-domain antigen binding protein comprises a variable domain, which can be derived from a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segments (e.g. V L , J L ) or a combination thereof, and a heavy chain constant region (C H ) gene sequence, wherein the heavy chain constant region gene sequence is in the C of a heavy chain constant region gene (eg, IgG, IgA, IgE, IgD or a combination thereof). H 1 coding sequence (and optionally the hinge region) contains a deletion or inactivating mutation. A single domain antigen binding protein comprising a variable domain derived from a heavy chain gene segment may be referred to as a "V H -single domain antibody" or "V H -single domain antigen binding proteins", see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876, 20150197553, 20150197554, 20150197555, 20150196015, 20150197556 and 20150197557, each of which is incorporated by reference in its entirety. A single domain antigen binding protein comprising a variable domain derived from a light chain gene segment may be referred to as a "V L -single domain antibody" or "V L-Single domain antigen binding protein", see, e.g., U.S. Publication No. 20150289489 (incorporated by reference in its entirety).

[0157] The term "about" or "approximately" includes within a statistically significant numerical range. Such a range can be within the order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5%. The permissible variation encompassed by the term "about" or "approximately" depends on the specific system under study and can be easily understood by those of ordinary skill in the art.

[0158] The term "affinity tag" includes a polypeptide sequence that is a member of a specific binding pair, for example, it specifically binds to another polypeptide sequence, such as an antibody epitope, with high affinity. Exemplary and non-limiting affinity tags include hexahistidine tags, FLAG tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S-tags, HA tags, and c-Myc tags. (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; incorporated herein by reference).

[0159] The term "capsid protein" includes proteins that are part of the viral capsid. For adeno-associated virus (AAV), capsid proteins are commonly referred to as VP1, VP2 and / or VP3, and are each encoded by a single cap gene. For AAV, three AAV capsid proteins are produced from the cap open reading frame (ORF) in an overlapping manner by selective mRNA splicing and / or selective translation initiation codons, although all three proteins use a common stop codon. Warrington et al. (2004) J.Virol 78: 6595 (incorporated herein by reference in its entirety). The VP1 of AAV2 is usually translated from the ATG start codon (amino acid M1) on 2.4kb mRNA, while the VP2 and VP3 of AAV2 are produced by a smaller 2.3kb mRNA, using a weaker ACG start codon to produce VP2 (amino acid T138), and read-through translation to the next available ATG codon (amino acid M203) to produce the most abundant capsid protein VP3. Warrington, supra; Rutledge et al. (1998) J. Virol 72:309-19 (incorporated herein by reference in its entirety). The amino acid sequences of adeno-associated virus capsid proteins are well known in the art and are generally conserved, particularly for dependoparvoviruses. See Rutledge et al., supra. For example, Rutledge et al. (1998) (supra) Figure 4 A comparison of the amino acid sequences of the VP1, VP2 and VP3 capsid proteins of AAV2, AAV3, AAV4 and AAV6 is provided in B, wherein the start sites of each of the VP1, VP2 and VP3 capsid proteins are indicated by arrows and the variable domains are marked with boxes. Thus, while the amino acid positions provided herein may be provided relative to the VP1 capsid protein of AAV, a skilled person will be able to separately and easily determine the positions of the same amino acids in the VP2 and / or VP3 capsid proteins of AAV, as well as the corresponding positions of the amino acids in different serotypes. In addition, a skilled person will be able to swap domains between capsid proteins of different AAV serotypes to form "chimeric capsid proteins".

[0160] Domain swapping between two capsid protein constructs for producing "chimeric AAV capsid proteins" has been described, see, e.g., Shen et al. (2007) Mol. Therapy 15(11): 1955-1962 (incorporated by reference in its entirety). "Chimeric AAV capsid proteins" include AAV capsid proteins that contain amino acid sequences (e.g., domains) from two or more different AAV serotypes and are capable of forming and / or forming AAV-like viral capsids / viral particles. Chimeric AAV capsid proteins are encoded by chimeric AAV capsid genes, e.g., nucleotides containing multiple (e.g., at least two) nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a different AAV serotype, and the multiple nucleic acid sequences together encode a functional chimeric AAV capsid protein. Reference to a chimeric capsid protein associated with a specific AAV serotype means that the capsid protein contains one or more domains from the capsid protein of that serotype and one or more domains from the capsid protein of a different serotype. For example, an AAV2 chimeric capsid protein includes a capsid protein comprising one or more domains of an AAV2 VP1, VP2 and / or VP3 capsid protein and one or more domains of a VP1, VP2 and / or VP3 capsid protein of a different AAV.

[0161] A "chimeric capsid" comprises at least two sets of VP1, VP2 and / or VP3 proteins, each set being encoded by a different cap gene.

[0162] In some embodiments, the chimeric capsid described herein comprises a recombinant VP1, VP2 and / or VP3 protein encoded by a cap gene genetically modified by insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprises a VP1, VP2 and / or VP3 protein encoded by a reference cap gene, wherein the reference cap gene is, for example, a wild-type reference cap gene encoding a wild-type VP1, VP2 and / or VP3 protein having the same AAV serotype as the recombinant VP1, VP2 and / or VP3 protein, a control reference cap gene encoding a VP1, VP2 and / or VP3 protein that is identical to the recombinant VP1, VP2 and VP3 protein but lacks the heterologous epitope, a mutated wild-type reference cap gene encoding a substantially wild-type VP1, VP2 and / or VP3 protein having the same AAV serotype as the recombinant VP1, VP2 and / or VP3 protein but having a mutation (e.g., insertion, substitution, deletion), wherein the mutation preferably reduces the tropism of the wild-type VP1, VP2 and VP3 protein. In some embodiments, the reference capsid protein is a chimeric reference protein comprising at least one domain of a VP1, VP2 and / or VP3 protein of the same AAV serotype as the recombinant VP1, VP2 and / or VP3 protein. In some embodiments, the reference cap gene encodes a chimeric VP1, VP2 and / or VP3 protein.

[0163] The term "recombinant capsid protein" includes a capsid protein having at least one mutation compared to the corresponding capsid protein of a wild-type virus, which can be a reference and / or control virus for comparative studies. Recombinant capsid protein includes a capsid protein containing a heterologous epitope, which can be inserted into and / or displayed by the capsid protein. "Heterologous" in this specification refers to being heterologous compared to the virus from which the capsid protein is derived. The inserted amino acid can simply be inserted between two given amino acids of the capsid protein. The insertion of amino acids can also be accompanied by the deletion of a given amino acid of the capsid protein at the insertion site, for example, one or more capsid protein amino acids are replaced by 5 or more heterologous amino acids.

[0164] "Retargeting" or "redirecting" may include situations where a wild-type vector targets several cells in a tissue and / or several organs in an organism, where the general targeting of said tissue or organ is reduced or eliminated by inserting a heterologous epitope, and where the retargeting of more specific cells in a tissue or a specific organ in an organism is achieved by a targeting ligand that binds to a marker expressed by a specific cell. Such retargeting or redirecting may also include situations where a wild-type vector targets a tissue, where the targeting of said tissue is reduced or eliminated by inserting a heterologous epitope, and where the retargeting of a completely different tissue is achieved by a targeting ligand.

[0165] The phrase "inverted terminal repeats" or "ITRs" includes symmetrical nucleic acid sequences in the adeno-associated virus (AAV) genome that are required for efficient replication. ITR sequences are located at both ends of the AAV DNA genome. ITRs serve as replication origins for viral DNA synthesis and are essential cis elements for the production of AAV integration vectors.

[0166] "Codon optimization" utilizes the degeneracy of codons, as shown by the diversity of three base pair codon combinations of specified amino acids, and generally includes replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of host cells and / or target cells, while maintaining the native amino acid sequence, to modify the nucleic acid sequence to enhance the process of expression in a specific host cell (e.g., packaging cell) and / or target cell. For example, the nucleic acid encoding the Cas9 protein can be modified to replace a codon with a higher frequency of use in a given prokaryotic or eukaryotic cell (including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host and / or target cell) compared to a naturally occurring nucleic acid sequence. Codon usage tables can be easily obtained, for example, in a "codon usage database". These tables can be modified in a variety of ways. See Nakamura et al. (2000) Nucleic Acids Research 28: 292 (incorporated herein in its entirety by reference for various purposes). Computer algorithms for codon optimization of a specific sequence for expression in a specific host and / or target are also available (see, e.g., Gene Forge).

[0167] "Promoter" is a regulatory region of DNA, which generally includes a TATA box capable of guiding RNA polymerase II to initiate RNA synthesis at an appropriate transcription start site of a specific polynucleotide sequence. The promoter may additionally include other regions that affect the transcription start rate. As used herein, the term "promoter" encompasses enhancers. The promoter sequence disclosed herein regulates the transcription of operably linked polynucleotides. The promoter may be active in one or more cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single-cell embryos, differentiated cells, or a combination thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-restricted promoter (e.g., a developmentally regulated promoter) or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). RNA Pol III promoters are often used to express small RNAs, such as the guide RNA sequences used in small interfering RNA (siRNA) / short hairpin RNA (shRNA) and CRISPR-Cas9 systems. Examples of RNA Pol III promoters that can be used in the present invention include, but are not limited to, human U6 promoter, rat U6 polymerase III promoter or mouse U6 polymerase III promoter and HI promoter, which are described, for example, in Goomer and Kunkel, Nucl. Acids Res., 20(18):4903-4912 (1992) and Myslinski et al., Nucleic Acids Res., 29(12):2502-9 (2001). Examples of promoters can be found, for example, in WO 2013 / 176772 (incorporated herein by reference in its entirety for all purposes).

[0168] Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol regulated promoters (e.g., alcohol dehydrogenase (alcA) gene promoters), tetracycline regulated promoters (e.g., tetracycline responsive promoters, tetracycline operator sequences (tetO), tet-On promoters or tet-Off promoters), steroid regulated promoters (e.g., rat glucocorticoid receptor, promoters of estrogen receptors or promoters of ecdysone receptors) or metal regulated promoters (e.g., metalloprotein promoters). Physically regulated promoters include, for example, temperature-controlled promoters (e.g., heat shock promoters) and light-regulated promoters (e.g., light-inducible promoters or light-repressible promoters).

[0169] The tissue-specific promoter can be, for example, a neuron-specific promoter, a glial cell-specific promoter, a muscle cell-specific promoter, a cardiac cell-specific promoter, a kidney cell-specific promoter, a bone cell-specific promoter, an endothelial cell-specific promoter, or an immune cell-specific promoter (e.g., a B cell promoter or a T cell promoter).

[0170] Developmentally regulated promoters include, for example, promoters that are active only during embryonic stages of development or only in adult cells.

[0171] A "self-cleaving peptide" or "self-cleaving sequence" encoding a self-cleaving domain is a peptide or coding sequence that induces ribosome skipping during protein translation, thereby causing cleavage. Suitable protease cleavage sites and self-cleaving peptides are known to the skilled person (see, for example, Ryan et al. (1997) J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites are potyvirus NIa proteases (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, byovirus RNA-2 encoded protease, foot-and-mouth disease virus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C proteases, cowpea mosaic virus group 24K protease, nematode-transmitted polyhedron virus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (European parsnip yellow spot virus) 3C-like protease, thrombin, factor Xa and enterokinase cleavage sites. The TEV (tobacco etch virus) protease cleavage site is particularly preferred due to its high cleavage stringency. In some embodiments, the isolated nucleic acid includes a self-cleaving peptidyl sequence encoding a self-cleaving peptidyl domain between the heavy chain sequence and the light chain sequence. Preferred self-cleaving peptides (also known as "cis-acting hydrolysis elements", CHYSEL; see deFelipe (2002) Curr. Gene Ther. 2, 355-378) are derived from potyvirus and cardiovirus 2A peptides. Particularly preferred self-cleaving peptides are selected from 2A peptides derived from FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus and porcine teshchina virus.

[0172] In some embodiments, the self-cleaving peptidyl linker sequence used herein is a 2A sequence. In some embodiments, the self-cleaving peptidyl linker sequence is a T2A sequence, a P2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the self-cleaving peptidyl linker sequence is a foot-and-mouth disease virus sequence. In some embodiments, the self-cleaving peptidyl linker sequence is PVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 6). In some embodiments, the self-cleaving peptidyl linker sequence is a horse rhinitis A virus sequence. In some embodiments, the self-cleaving peptidyl linker sequence is QCTNYALLKLAGDVESNPGP (SEQID NO: 7). In embodiments, the self-cleaving peptidyl linker sequence is a porcine Teschovirus 1 sequence. In embodiments, the self-cleaving peptidyl linker sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 8). In some embodiments, the self-cleaving peptidyl linker sequence is a Thosea asigna virus sequence. In some embodiments, the self-cleaving peptidyl linker sequence is EGRGSLLTCGDVESNPGP (SEQ ID NO: 9). In some embodiments, the light chain sequence is located 3' to the heavy chain sequence. In some embodiments, the light chain sequence is located 5' to the heavy chain sequence.

[0173] As used herein, the phrase "operably linked" includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or otherwise coordinate with each other to participate in biological events, which juxtaposition achieves or allows such interaction and / or coordination. To cite just one example, when a control sequence (e.g., an expression control sequence) in a nucleic acid is positioned relative to a coding sequence so that its presence or absence affects the expression and / or activity of the coding sequence, the control sequence is considered to be "operably linked" to the coding sequence. In many embodiments, "operable linkage" involves covalent linkage of related components or elements to each other. However, it will be readily appreciated by those skilled in the art that in some embodiments, covalent linkage is not required to achieve effective operable linkage. For example, in some embodiments, a nucleic acid control sequence operably linked to a coding sequence controlled therewith is continuous with a target nucleotide. Alternatively or additionally, in some embodiments, one or more such control sequences act in trans or remotely to control a target coding sequence. In some embodiments, the term "expression control sequence" as used herein refers to a polynucleotide sequence that is necessary and / or sufficient to affect the expression and processing of a coding sequence connected thereto. In some embodiments, expression control sequences may be or include appropriate transcription initiation, termination, promoter and / or enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and / or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are preferentially or exclusively active in a particular host cell or organism or type thereof. To cite just one example, in prokaryotes, control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, in many embodiments, control sequences typically include promoters, enhancers, and / or transcription termination sequences. One of ordinary skill in the art will understand from the context that in many embodiments, the term "control sequence" refers to components whose presence is necessary for expression and processing, and in some embodiments, includes components whose presence is conducive to expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0174] A "specific binding pair", "protein:protein binding pair", etc. includes two proteins (e.g., a first member (e.g., a first polypeptide) and a second homologous member (e.g., a second polypeptide)) that interact to form a covalent isopeptide bond under conditions that enable or promote isopeptide bond formation, wherein the term "homologous" refers to components that work together (i.e., react together) to form an isopeptide bond. Thus, two proteins that efficiently react together to form an isopeptide bond under conditions that enable or promote isopeptide bond formation may also be referred to as a "complementary" peptide linker pair. Specific binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506, and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, isopeptag:Piliin C, SnoopTag:SnoopCatcher, SnoopTagJr:DogTag, etc. In general, a peptide tag refers to a member of a protein:protein binding pair that is typically less than 30 amino acids in length and forms a covalent isopeptide bond with a second homologous protein, where the second homologous protein is typically larger, but may also be less than 30 amino acids in length, such as in the SpyTag:KTag system.

[0175] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, at least one of which is not derived from the protein backbone or is not considered to be part of the protein backbone. An isopeptide bond can be formed within a single protein, or between two peptides or between a peptide and a protein. Therefore, an isopeptide bond can be formed within a single protein molecule, or between molecules, i.e., between two peptide / protein molecules, such as between two peptide linkers. Typically, an isopeptide bond can be present between a lysine residue and an asparagine, aspartic acid, glutamine or glutamic acid residue or a terminal carboxyl group of a protein or peptide chain, or between the α-amino terminal of a protein or peptide chain and asparagine, aspartic acid, glutamine or glutamic acid. Each residue in the residue pair participating in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond can be formed between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond can be present between the side chain amine of lysine and the carboxamide group of asparagine or the carboxyl group of aspartic acid.

[0176] The SpyTag:SpyCatcher system is described in U.S. Pat. No. 9,547,003, Zakeri et al. (2012) PNAS109:E690-E697 and WO2019006046 (each of which is incorporated herein by reference in its entirety), and is derived from the CnaB2 domain of the fibronectin FbaB of Streptococcus pyogenes. By cleaving the domain, Zakeri et al. obtained a peptide "SpyTag" with the sequence AHIVMVDAYKPTK (SEQ ID NO: 13), which forms an amide bond with its homologous protein "SpyCatcher". (Zakeri (2012), supra). Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of Spy ligase. (Fierer (2014) PNAS 111: E1176-1181) Spy ligases were engineered by cleaving the beta strand containing a reactive lysine from SpyCatcher, generating KTag, a 10-residue peptide tag with the amino acid sequence ATHIKFSKRD (SEQ ID NO: 14). The SpyTag002: SpyCatcher002 system is described in Keeble et al. (2017) Angew Chem Int Ed Engl 56: 16521-25, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK (SEQ ID NO: 15) and binds SpyCatcher002.

[0177] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. The D4 Ig-like domain of the adhesin RrgA from Streptococcus pneumoniae is cleaved to form SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher results in specific spontaneous isopeptide bonds between complementary proteins. Veggiani (2016)), supra.

[0178] Isopeptide tag: Pilin-C specifically binds to Spy0128, the major pilin protein from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132: 4526-27). The isopeptide tag has the amino acid sequence TDKDMTITFTNKKDAE (SEQ ID NO: 16) and binds to Pilin-C (residues 18-299 of spy0128). Incubation of SnoopTag and SnoopCatcher results in a specific spontaneous isopeptide bond between complementary proteins. Zakeir and Howarth (2010), supra.

[0179] The term "peptide tag" includes (1) a polypeptide heterologous to the protein tagged with the peptide tag, (2) a member of a specific protein:protein binding pair capable of forming an isopeptide bond, and (3) a polypeptide not exceeding 50 amino acids in length.

[0180] The term "detectable marker" includes a polypeptide sequence that is a member of a specific binding pair, for example, it specifically binds to another polypeptide sequence (e.g., an antibody paratope) with high affinity through a non-covalent bond. Exemplary and non-limiting detectable markers include hexahistidine tags, FLAG tags, Strep II tags, streptavidin binding peptide (SBP) tags, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S-tags, HA tags, and c-myc. (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; incorporated herein by reference). A common detectable marker for primate AAV is the B1 epitope. The non-primate AAV capsid protein of the present invention (which does not naturally contain the B1 epitope) may be modified herein to contain the B1 epitope. Typically, the non-primate AAV capsid protein may contain a sequence having substantial homology to the B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, the non-primate AAV capsid protein of the invention can be modified with one but less than five point mutations within the last 10 amino acids of the capsid protein such that the AAV capsid protein comprises a B1 epitope.

[0181] In various embodiments, the Fc domain can be modified to have a changed Fc receptor binding, which in turn affects effector function. In some embodiments, the engineered heavy chain constant region (CH) comprising the Fc domain is chimeric. Therefore, the chimeric CH region combines CH domains from more than one immunoglobulin isotype. For example, the chimeric CH region comprises part or all of the CH2 domains derived from human IgG1, human IgG2 or human IgG4 molecules combined with part or all of the CH3 domains derived from human IgG1, human IgG2 or human IgG4 molecules. In some embodiments, the chimeric CH region comprises a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 216 to 227 according to EU numbering; amino acid residues at positions 226 to 240 according to Kabat numbering) and a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 228 to 236 according to EU numbering; amino acid positions 241 to 249 according to Kabat numbering). In some embodiments, the chimeric hinge region comprises amino acid residues derived from an upper hinge of human IgG1 or human IgG4 and amino acid residues derived from a lower hinge of human IgG2.

[0182] In some embodiments, the Fc domain can be engineered to activate all, some, or no normal Fc effector functions without affecting the desired pharmacokinetic properties of Fc-containing proteins (e.g., antibodies). For examples of proteins comprising chimeric CH regions and having altered effector functions, see WO2014022540, which is incorporated herein in its entirety.

[0183] The terms "transduction", "transfection" or "infection" are used interchangeably herein and refer to the introduction of nucleic acids into target cells, for example, by viral vectors. The term efficiency related to transduction, for example, "transduction efficiency" refers to the fraction (e.g., percentage) of cells expressing the target nucleotide after incubation with a certain number of viral vectors containing the target nucleotide. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting of cells transduced with fluorescent reporter genes, PCR for expression of the target nucleotide, and the like.

[0184] As used herein, the term "wild type" includes entities with structures and / or activities found in nature in a "normal" (as opposed to mutated, diseased, altered, etc.) state or environment. It will be understood by those of ordinary skill in the art that wild-type viral vectors, such as AAV vectors comprising wild-type capsid proteins, can be used as reference viral vectors in comparative studies. In general, the reference viral capsid protein / capsid / vector is identical to the test viral capsid protein / capsid / vector except that the effect of the test viral capsid protein / capsid / vector is to be tested. For example, in order to determine the effect of inserting a heterologous epitope into a test viral vector, such as the effect on transduction efficiency, the transduction efficiency of the test viral vector (in the absence or presence of a suitable binding molecule) can be compared with the transduction efficiency of the reference viral vector (in the absence or presence of a suitable binding molecule, if necessary), except that the test viral vector has a heterologous epitope, the reference viral vector is identical to the test viral vector in each case (e.g., additional mutations, target nucleotides, the number of viral vectors and target cells, etc.).

[0185] "Complementarity" or "complementarity" of nucleic acids means that the nucleotide sequence in one strand of a nucleic acid forms hydrogen bonds with another sequence on the opposite nucleic acid strand due to the orientation of its nucleobase groups. Complementary bases in DNA are typically A to T and C to G. In RNA, they are typically C to G and U to A. Complementarity can be complete or substantially / sufficient. Complete complementarity between two nucleic acids means that the two nucleic acids can form a duplex, wherein each base in the duplex is bonded to a complementary base by Watson-Crick pairing. "Substantially" or "sufficiently" complementary means that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposite strand, but sufficient bonding occurs between the bases on the two strands, thereby forming a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequence and standard mathematical calculations to predict the Tm (melting temperature) of the hybridized strand, or by empirically determining Tm using conventional methods. Tm includes the temperature at which the population of hybrid complexes formed between two nucleic acid chains is 50% denatured (i.e., half of the population of double-stranded nucleic acid molecules is dissociated into single strands). At temperatures below Tm, the formation of hybrid complexes is favored, while at temperatures above Tm, the melting or separation of the chains in the hybrid complex is favored. The Tm of a nucleic acid with a known G+C content in a 1M NaCl aqueous solution can be estimated by using, for example, Tm=81.5+0.41 (%G+C), although other known Tm calculations take into account the structural features of the nucleic acid.

[0186] "Hybridization conditions" include an environment in which one nucleic acid strand interacts and hydrogen bonds with a second nucleic acid strand to produce a hybrid complex. Such conditions include the chemical components of the aqueous or organic solution containing the nucleic acid and their concentrations (e.g., salts, chelating agents, formamide), and the temperature of the mixture. Other factors, such as the length of the incubation time or the size of the reaction chamber may also have an impact on the environment. See, for example, Sambrook et al., Olecular Cloning, A Laboratory Manual, 2.sup.nd ed., pp. 1.90-1.91, pp. 9.47-9.51, pp. 11.47-11.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), which is incorporated herein by reference in its entirety for all purposes.

[0187] Hybridization requires that two nucleic acids contain complementary sequences, although there may be mispairing between bases. The conditions suitable for hybridization between two nucleic acids depend on the length and complementary degree of nucleic acid, and these variables are well known. The greater the complementary degree between two nucleotide sequences, the greater the melting temperature (Tm) value of the nucleic acid hybrid with these sequences. For hybridization between nucleic acids with short complementary segments (for example, more than 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotide complementarity), the position of mispairing becomes important (see Sambrook et al., the same, 11.7-11.8). Usually, the length of hybridizable nucleic acids is at least about 10 nucleotides. The illustrative minimum length of hybridizable nucleic acids includes at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides and at least about 30 nucleotides. In addition, temperature and washing solution salt concentration can be adjusted as necessary according to factors such as the length and complementary degree of complementary regions.

[0188] The sequence of the polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid / target locus in order to specifically hybridize. In addition, the polynucleotide can hybridize on one or more segments so that the intervening or adjacent segments do not participate in hybridization events (such as loop structures or hairpin structures). Polynucleotides (such as gRNA) can have at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence complementarity with the target region in the target nucleic acid / target locus sequence they target. For example, 18 of the 20 nucleotides are complementary to the target region, so the gRNA that specifically hybridizes will represent 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed in complementary nucleotides, and do not need to be adjacent to each other or adjacent to complementary nucleotides.

[0189] The percent complementarity between specific nucleic acid sequence segments in a nucleic acid can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al. (1990) J. Mol. Biol. 215:403-410; Zhang and Madden (1997) Genome Res. 7:649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 (for Unix), Genetics Computer Group, University Research Park, Madison Wis.) (using default settings, which uses the algorithm of Smith and Waterman (Adv Appl. Math., 1981, 2, 482-489)).

[0190] In the context of two polynucleotides or polypeptide sequences, "sequence identity" or "identity" refers to the same residues in two sequences when the maximum correspondence is compared within a specified comparison window. When the percentage of sequence identity is used to refer to a protein, the residue positions that are not identical are usually different due to conservative amino acid substitutions, in which the amino acid residues are replaced by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), thus not changing the functional properties of the molecule. When the sequence difference lies in conservative substitutions, the percentage of sequence identity can be adjusted upward to correct the conservative nature of the substitution. Sequences that differ in such conservative substitutions are referred to as having "sequence similarity" or "similarity". Methods for such adjustments are well known. Generally, this includes scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Therefore, for example, when the same amino acid score is 1 and the non-conservative substitution score is zero, the conservative substitution score is between 0 and 1. The score of the conservative substitution is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0191] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences (maximum number of fully matched residues) within a comparison window, wherein for optimal alignment of the two sequences, a portion of the polynucleotide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions). The percentage is calculated by the following process: determining the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a connected heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared.

[0192] Unless otherwise indicated, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 using the following parameters: for nucleotide sequences, % identity and % similarity, using a gap weight of 50 and a length weight of 3 and the nwsgapdna.cmp scoring matrix; for amino acid sequences, % identity and % similarity, using a gap weight of 8 and a length weight of 2 and the BLOSUM62 scoring matrix; or any equivalent programs thereof. "Equivalent programs" include any sequence comparison program that, for any two of the sequences, produces an alignment with identical nucleotide or amino acid residue matches and identical sequence identity percentages when compared to the corresponding alignments produced by GAP version 10.

[0193] The term "conservative amino acid substitution" refers to the substitution of amino acids normally present in a sequence with different amino acids of similar size, charge or polarity. Examples of conservative substitutions include the substitution of non-polar (hydrophobic) residues such as isoleucine, valine or leucine to another non-polar residue. Similarly, examples of conservative substitutions include the substitution of a polar (hydrophilic) residue to another residue (such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine). In addition, the substitution of a basic residue such as lysine, arginine or histidine to another residue, or the substitution of an acidic residue such as aspartic acid or glutamic acid to another acidic residue are other examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine or methionine to polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid or lysine and / or the substitution of polar residues to non-polar residues. Typical amino acid classifications are summarized as follows.

[0194]

[0195]

[0196] The term "in vitro" includes artificial environments and processes or reactions that occur in artificial environments (e.g., test tubes). The term "in vivo" includes natural environments (e.g., cells or organisms or the body) and processes or reactions that occur in natural environments. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur in such cells.

[0197] A composition or method that "comprising," "having," or "including" one or more recited elements may include additional elements not specifically recited. For example, a composition that "comprising" or "including" a protein may contain the protein alone or in combination with other ingredients. The transition phrase "consisting essentially of" means that the scope of the claim should be interpreted to encompass the specified elements recited in the claim and those elements that do not materially affect one or more of the basic and novel characteristics of the claimed invention. Therefore, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as being equivalent to "comprising."

[0198] "Individual" or "subject" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In some embodiments, the subject is a human.

[0199] The term "treat" or "treatment" of a condition, disorder, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the development of at least one clinical or subclinical symptom of a condition, disorder, or condition in a subject who may have or be susceptible to the condition, disorder, or condition but does not yet experience or exhibit clinical or subclinical symptoms of the condition, disorder, or condition; or (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom thereof; or (3) ameliorating the disease, i.e., causing regression of the condition, disorder, or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant or at least perceptible to the patient or physician.

[0200] The term "effective," as applied to dosage or amount, refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is effective when administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs used, the mode of administration, and the like.

[0201] The phrase "pharmaceutically acceptable" used in conjunction with the compositions described herein refers to the molecular entities and other ingredients of such compositions that are physiologically tolerable and generally do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in mammals, especially humans.

[0202] In light of the disclosure herein, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are fully explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (hereinafter "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Vols. I and II (DN Glover, ed. 1985); Oligonucleotide Synthesis (MJ Gait, ed. 1984); Nucleic Acid Hybridization [BD Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [BD Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [RI Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); Ausubel, FM et al. (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis as described in Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985), U.S. Pat. No. 5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263:357-360 (1999); Kim and Maas, BioTech. 28:196-198 (2000); Parikh and Guengerich, BioTech. 24:4 28-431 (1998); Ray and Nickoloff, BioTech. 13:342-346 (1992); Wang et al., BioTech.19:556-559 (1995); Wang and Malcolm, BioTech.26:680-682 (1999); Xu and Gong, BioTech.26:639-641 (1999), U.S. Pat. Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3:74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270 and 6,242,222, Angag and Schutz, Biotech.30:486-488 (2001), Wang and Wilkinson, Biotech.29:976-978 (2000), Kang et al., Biotech.20:44-46 (1996), Ogel and McPherson, Protein Engineer. 5:467-468 (1992), Kirsch and Joly, Nucl. Acids. Res. 26:1848-1850 (1998), Rhem and Hancock, J. Bacteriol. 178:3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22:541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57:229-237, and Pons et al., Meth. Molec. Biol. 67:209-218.

[0203] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0204] The present invention provides (among other things) a system for producing an antibody or an antigen-binding fragment thereof in a subject, comprising: a) a first component comprising a polynucleotide molecule, wherein the polynucleotide molecule comprises a sequence encoding the antibody or an antigen-binding fragment thereof; and b) a second component comprising a gene-editing molecule or a polynucleotide molecule containing a sequence encoding the gene-editing molecule.

[0205] In some embodiments, administration of the first component and the second component to a subject results in integration of sequences encoding the antibody or antigen-binding fragment thereof into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), resulting in production of the antibody or antigen-binding fragment in the subject.

[0206] In some embodiments, administration of the first component and the second component to B cells and / or hematopoietic stem cells (HSCs) isolated ex vivo from a subject results in integration of sequences encoding the antibody or antigen-binding fragment thereof into the DNA of the cells, thereby generating modified B cells or modified HSCs, resulting in production of the antibody or antigen-binding fragment thereof in the subject when the modified B cells or HSCs are administered to the subject.

[0207] In some embodiments, the first component and / or the second component are independently selected from a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a liposome, and a ribonucleoprotein (RNP) complex.

[0208] Recombinant viral capsid protein, viral vector and nucleic acid

[0209] In some embodiments, the first component and the second component are both viral vectors. In some embodiments, the viral vectors are derived from the same viral species. In other embodiments, the viral vectors are derived from different viral species.

[0210] Viral vectors that can be used in the compositions and methods of the present application include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, retroviruses (e.g., lentiviruses), baculovirus vectors, herpes virus vectors, cytomegalovirus (CMV), Epstein-Barr virus (EBV), mouse mammary tumor virus (MMTV), human polyomavirus 2 (JC virus or John Cunningham virus), hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus 1 (HIV-1), influenza virus, norovirus, measles virus, polyomavirus, rhabdovirus (e.g., vesicular stomatitis virus), or variants thereof.

[0211] In some embodiments, one or both of the viral vectors used in the system of the present invention is an adeno-associated virus (AAV) vector. "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. AAV is a small, non-enveloped single-stranded DNA virus. Typically, the wild-type AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (rhabdovirus): rep and cap. The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD ("Rep78"), 68 kD ("Rep68"), 52 kD ("Rep52"), and 40 kD ("Rep 40"). Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. These proteins play a major role in regulating the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VP) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). More than 80% of the total protein in the AAV virion (capsid) comprises VP3; in mature virions, VP1, VP2, and VP3 are found in a relative abundance of approximately 1:1:10, although a ratio of 1:1:8 has been reported. Padron et al. (2005) J. Virology 79:5047-58.

[0212] The genomic sequences of various serotypes of AAV, as well as the sequences of native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank Accession Nos. C_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated herein by reference for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; U.S. Patent Publication No. 20170130245; International Patent Publication No. WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; U.S. Patent No. 6,156,303, each of which is incorporated herein by reference in its entirety. Table 2 herein provides the sequences of various non-primate AAVs.

[0213] "AAV" encompasses all subtypes known in the art, as well as naturally occurring and modified forms. AAV includes primate AAV (e.g., AAV 1 (AAV1), primate AAV 2 (AAV2), primate AAV 3 (AAV3B), primate AAV 4 (AAV4), primate AAV 5 (AAV5), primate AAV 6 (AAV6), primate AAV 7 (AAV7), primate AAV 8 (AAV8), primate AAV 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV rh10 (AAV rh10), AAV h10 (AAV h10), AAV hu11 (AAV hu11), AAV rh32.33 (AAV rh32.33), AAV retro (AAV AAV PHP.S, AAV2 / 8, etc., non-primate AAV (e.g., avian AAV (AAAV)) and other non-primate AAVs such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, horse AAV, goat AAV, and sheep AAV, etc.), squamates AAV (e.g., snake AAV, bearded dragon AAV), etc. "Primate AAV" refers to AAVs that are generally isolated from primates. Similarly, "non-primate AAV" refers to AAVs isolated from non-primates.

[0214] In some embodiments, the AAV vector is derived from AAV1, AAV2, AAV6, AAV9, or AAV9.PHP.

[0215] Also included herein are recombinant viral particles that are genetically modified to display a heterologous amino acid sequence comprising a first member of a specific binding pair, wherein the amino acid sequence is less than 50 amino acids in length, and wherein the recombinant viral capsid / particle protein exhibits reduced to eliminated native tropism. In some embodiments, the viral particle further comprises a second homologous member of the specific binding pair, wherein the first member and the second member are covalently bonded, and wherein the second member is fused to a targeting ligand.

[0216] In some embodiments, the heterologous amino acid sequence comprises a first member of a specific binding pair and one or more linkers. In some embodiments, the heterologous amino acid sequence comprises a first member of a specific binding pair flanked by linkers, for example, the heterologous amino acid sequence comprises a first linker, a first member of a specific binding pair, and a second linker from the N-terminus to the C-terminus. In some embodiments, the length of the first linker and the second linker is each independently at least one amino acid. In some embodiments, the first linker and the second linker are identical.

[0217] Typically, a heterologous amino acid sequence as described herein, e.g., comprising a first member of a specific binding pair alone or in combination with one or more linkers, is between about 5 amino acids and about 50 amino acids in length. In some embodiments, the length of the heterologous amino acid sequence is at least 5 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 6 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 7 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 8 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 9 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 10 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 11 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 12 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 13 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 14 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 15 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 16 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 17 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 18 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 19 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 20 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 21 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 22 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 23 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 24 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 25 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 26 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 27 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 28 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 29 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 30 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 31 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 32 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 33 amino acids. In some embodiments, the heterologous amino acid sequence is 34 amino acids in length. In some embodiments, the heterologous amino acid sequence is 35 amino acids in length. In some embodiments, the heterologous amino acid sequence is 36 amino acids in length. In some embodiments, the heterologous amino acid sequence is 37 amino acids in length. In some embodiments, the heterologous amino acid sequence is 38 amino acids in length.In some embodiments, the length of the heterologous amino acid sequence is 39 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 40 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 41 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 42 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 43 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 44 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 45 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 46 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 47 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 48 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 49 amino acids. In some embodiments, the length of the heterologous amino acid sequence is 50 amino acids.

[0218] In some embodiments, the specific binding pair is a SpyTag:SpyCatcher binding pair, wherein the first member is a SpyTag, and wherein the second homologous member is a SpyCatcher. In some embodiments, the specific binding pair is a SpyTag:KTag, wherein the first member is a SpyTag, and wherein the second homologous member is a KTag. In some embodiments, the specific binding pair is a SpyTag:KTag, wherein the first member is a KTag, and wherein the second homologous member is a SpyTag. In some embodiments, the specific binding pair is an isopeptide tag:pilin-C, wherein the first member is an isopeptide tag, and wherein the second homologous member is pilin-C or a portion thereof. In some embodiments, the specific binding pair is a SnoopTag:SnoopCatcher, and the first member is a SnoopTag and the second homologous member is a SnoopCatcher.

[0219] In some embodiments, the recombinant virus capsid protein described herein is derived from an adeno-associated virus (AAV) capsid gene, for example, a genetically modified capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant virus capsid protein is derived from an AAV2 capsid gene, an AAV6 capsid gene, an AAV1 capsid gene, or an AAV9 capsid gene. In some embodiments, the recombinant virus capsid protein is derived from an AAV2 capsid gene, for example, a genetically modified AAV2 VP1 capsid protein. In some embodiments, the recombinant virus capsid protein is derived from an AAV1 capsid gene, for example, a genetically modified AAV1 VP1 capsid protein. In some embodiments, the recombinant virus capsid protein is derived from an AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from an AAV6 capsid gene, for example, a genetically modified VP1 capsid protein of AAV6. In some embodiments, a heterologous epitope is inserted into I-453 of an AAV9 capsid protein.

[0220] Typically, the recombinant viral capsid protein described herein comprises a heterologous epitope inserted into the capsid protein and / or displayed by the capsid protein, so that the heterologous epitope reduces and / or eliminates the natural tropism of the capsid protein or the capsid comprising it. In some embodiments, the heterologous epitope is inserted into a capsid protein region associated with the natural tropism of the wild-type reference capsid protein, for example, a region of the capsid protein associated with a cell receptor. In some embodiments, the heterologous epitope is inserted into the knob domain of the Ad fiber protein and / or displayed by the knob domain. In some embodiments, the heterologous epitope is inserted into the HI loop of the Ad fiber protein and / or displayed by the HI loop. In some embodiments, the heterologous epitope is inserted after the amino acid position selected from the group consisting of: G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, Q585 of AAV6 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the heterologous epitope is inserted and / or displayed between amino acids N587 and R588 of the AAV2 VP1 capsid. Other suitable insertion sites identified by using AAV2 are well known in the art (Wu et al. (2000) J. Virol 74:8635-8647), including 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713 and 1-716. The recombinant viral capsid protein described herein can be an AAV2 capsid protein comprising a heterologous epitope inserted into a position selected from the group consisting of: I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, I-716, and combinations thereof. Other suitable insertion sites identified by using other AAV serotypes are well known, including I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), and I-585 (AAV5). In some embodiments, the recombinant viral capsid protein described herein can be an AAV2 capsid protein, which comprises a heterologous epitope inserted into a position selected from the group consisting of: I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

[0221] The nomenclature I-### used herein refers to the insertion site, wherein ### designates the amino acid number of the VP1 protein relative to the AAV capsid protein, however such insertion may be located directly within 5 amino acids of the N or C terminal end of a given amino acid, preferably 3, more preferably 2, and especially 1 amino acid of the N or C terminal end of the given amino acid sequence, preferably the C terminal end. In addition, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and mutations thereof) of the VP2 and VP3 capsid proteins encoded by the capsid gene can be easily identified by sequence alignment of the VP1, VP2 and VP3 proteins encoded by the reference AAV capsid gene.

[0222] Thus, insertion of the corresponding position of the encoding nucleic acid at one of these sites of the cap gene results in insertion of VP1, VP2 and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Thus, for example, for AAV2, according to this nomenclature, insertions between amino acids 1 and 138 are inserted only into VP1, insertions between 138 and 203 are inserted into VP1 and VP2, and insertions between 203 and the C-terminus are inserted into VP1, VP2 and VP3, and of course the same is true for insertion site 1-587. Thus, the present invention encompasses AAV structural genes having corresponding insertions in the VP1, VP2 and / or VP3 proteins.

[0223] In addition, due to the high conservation of at least the large segment and the large members of closely related family members, the corresponding insertion sites of AAVs other than the listed AAVs can be identified by performing amino acid alignments or by comparison of capsid structures. For exemplary alignments of different AAV capsid proteins, see, e.g., Rutledge et al. (1998) J. Virol 72:309-19 and U.S. Pat. No. 9,624,274, each of which is incorporated herein by reference in its entirety.

[0224] In some compositions comprising recombinant viral capsids disclosed herein, the recombinant viral capsid protein is an AAV2 capsid protein VP1 with a heterologous epitope inserted at the I587 site, wherein the heterologous epitope does not contain an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions comprising recombinant viral capsids disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T448 and N449, wherein the heterologous epitope does not contain c-myc. In some compositions comprising recombinant viral capsids disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at the I-447 site, wherein the heterologous epitope does not contain L14 or HA.

[0225] In some compositions comprising recombinant viral capsids, the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at the I587 site, wherein the heterologous epitope comprises an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions comprising recombinant viral capsids disclosed herein, the viral capsid is a VP1 capsid, the heterologous epitope comprises c-myc, and the heterologous epitope is inserted between T448 and N449, or between N587 and R588. In some compositions comprising recombinant viral capsids disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at the I-447 site, wherein the heterologous epitope comprises L14 or HA. In some compositions comprising recombinant viral capsids disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T448 and N449, wherein the heterologous epitope comprises c-myc. US Patent No. 9,624,274 describes I-453 of the AAV capsid protein as a suitable insertion site for heterologous epitopes.

[0226] In some embodiments, the insertion (display) of a heterologous epitope eliminates the natural tropism of the viral vector, for example, in the absence of a suitable binding molecule, the transduction of cells and / or target cells that naturally allow the infection of a wild-type reference viral vector is undetectable. In some embodiments, for example, compared to the transduction of cells that naturally allow the infection of a wild-type reference viral vector, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 10%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 20%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 30%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 40%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 50%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 60%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 70%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 80%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 90%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 95%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of a viral vector by at least 90%. In these embodiments, wherein the insertion (display) of a heterologous epitope does not eliminate the natural tropism of a recombinant viral capsid, the natural tropism of such a recombinant viral capsid can be eliminated by a second different mutation. For example, in one embodiment, the recombinant viral capsid protein described herein can be derived from an AAV9 capsid gene, comprise a heterologous epitope, and can further comprise a mutation, such as a W503A mutation. Other non-limiting examples of the second mutation include, for example, Y445F and V473D (for AAV1 or AAV6 capsid).

[0227] This de-targeting of the virus from its natural host cells is important, especially when the viral vector is intended to be administered systemically rather than locally or regionally, because the uptake of the viral vector by the natural host cells limits the effective dose of the viral vector. In the case of AAV2 and AAV6, HSPGs are reported to be the main receptors for viral uptake in a large number of cells, especially hepatocytes. For AAV2, HSPG binding activity depends on a set of 5 basic amino acids: R484, R487, R585, R588 and K532 (Kern et al. (2003) J Virol. 77 (20): 11072-81). Recently, it was reported that the amino acid substitution K531E of lysine to glutamic acid resulted in inhibition of the ability of AAV6 to bind heparin or HSPG (Wu et al., 2006) J. of Virology 80 (22): 11393-11397). Therefore, preferred point mutations are those that reduce the transduction activity of the viral vector to a given target cell by at least 50%, preferably at least 80%, and especially at least 95%, wherein the transduction activity is mediated by the natural receptor, or in the case where HSPG is the primary receptor, the binding of the viral vector to HSPG.

[0228] Therefore, other preferred mutations for HSPG binding to viral vectors are those that deplete or replace basic amino acids (such as R, K or H, preferably R or K) involved in HSPG binding of the corresponding virus with non-basic amino acids (such as A, D, G, Q, S and T, preferably A or amino acids present at corresponding positions in different but highly conserved AAV serotypes that lack such basic amino acids at that position). Thus, preferred amino acid substitutions are R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A or R588T (especially R585A and / or R588A) (for AAV2) and K531A or K531E (for AAV6). A particularly preferred embodiment of the present invention is a capsid protein mutant of AAV2 that additionally contains two point mutations R585A and R588A, because these two point mutations are sufficient to largely eliminate HSPG binding activity. These point mutations enable efficient de-targeting from cells expressing HSPG, which increases the specificity of the corresponding mutant virus for its new target cells for targeting purposes.

[0229] One embodiment of the present invention is a multimeric structure comprising a recombinant viral capsid protein of the present invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 recombinant viral capsid proteins comprising heterologous epitopes as described herein. They can form regular viral capsids (empty viral particles) or viral vectors (capsids encapsulating target nucleotides). The formation of a viral vector capable of packaging a viral genome is a highly preferred feature of using the recombinant viral capsids described herein as viral vectors.

[0230] In some embodiments, according to an indirect recombination method, a targeting ligand can be associated with (e.g., displayed by, operably linked to, bound to) a modified AAV capsid protein and the resulting AAV capsid, wherein the AAV capsid protein is modified to comprise a first member of a binding pair (e.g., a heterologous scaffold), and optionally wherein the first member of the binding pair is linked to (e.g., covalently or non-covalently bound to) a second homologous member of the binding pair (e.g., an adaptor), further optionally wherein the second homologous member of the binding pair is fused to the targeting ligand. Non-limiting and exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12: 248-265.

[0231] Thus, in some embodiments, modifications of the capsid protein described herein include those modifications that are typically produced by modifications at the genetic level (e.g., by modification of the cap gene), such as insertion of a first member of a binding pair (e.g., a protein:protein binding pair, a protein:nucleic acid binding pair), a detectable marker, etc. for display by the Cap protein.

[0232] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member forms a binding pair with a metal ion (e.g., Ni 2+ 、Co 2+ , Cu 2+ 、Zn 2+ , Fe 3+ In some embodiments, the first member is selected from the group consisting of: streptavidin, Strep II, HA, L14, 4C-RGD, LH and protein A.

[0233] In some embodiments, the binding pair comprises an enzyme:nucleic acid binding pair. In some embodiments, the first member comprises a HUH endonuclease or a HUH tag, and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises a HUH tag. See, e.g., US2021 / 018008, which is incorporated herein by reference in its entirety.

[0234] In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0235] In some embodiments, the first member and the second member of the peptide:peptide binding pair each comprise an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8:2004018 (1 of 22); Muik et al. (2017) Biomaterials 144:84, each of which is incorporated herein by reference in its entirety.

[0236] In some embodiments, the first member is a B cell epitope, eg, between about 1 amino acid and about 35 amino acids in length, and forms a binding pair with an antibody epitope, eg, an immunoglobulin variable domain.

[0237] In some embodiments, the capsid protein of the present invention comprises a protein containing a detectable marker: a first member of a protein binding pair, which can also be used for detection and / or separation of the Cap protein and / or as a protein: a first member of a protein binding pair. In some embodiments, the detectable marker serves as a first member of a protein binding pair for binding to a targeting ligand: a multispecific binding protein that can bind to a detectable marker and a target expressed by a target cell. In some embodiments, the Cap protein of the present invention comprises a protein containing c-myc, FLAG or HA: a first member of a protein binding pair. The use of a detectable marker as a first member of a protein binding pair is described in, for example, WO2019006043.

[0238] In some embodiments, the capsid protein comprises a protein: a first member of a protein binding pair, wherein the protein: the protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the peptide: peptide binding pair is covalently bound to the homologous second member of the peptide: peptide binding pair by an isopeptide bond, and optionally, wherein the homologous second member of the peptide: peptide binding pair is fused to a targeting ligand, which binds to a target expressed by a target cell. In some embodiments, the protein: protein binding pair can be selected from SpyTag: SpyCatcher, SpyTag002: SpyCatcher002, SpyTag003: SpyCatcher003, SpyTag: KTag, isopeptide tags: Pili protein-C and SnoopTag: SnoopCatche. In some embodiments, wherein the first member is SpyTag (or a biologically active portion or variant thereof), the protein (second homologous member) is SpyCatcher (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag (or a biologically active portion or variant thereof), the protein (second homologous member) is KTag (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is KTag (or a biologically active portion or variant thereof), the protein (second homologous member) is SpyTag (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SnoopTag (or a biologically active portion or variant thereof), the protein (second homologous member) is SnoopCatcher (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is an isopeptide tag (or a biologically active portion or variant thereof), the protein (second homologous member) is pilin-C (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag002 (or a biologically active portion or variant thereof), the protein (second homologous member) is SpyCatcher002 (or a biologically active portion or variant thereof). In some embodiments, wherein the first member is SpyTag003 (or a biologically active portion or variant thereof), the protein (second homologous member) is SpyCatcher003 (or a biologically active portion or variant thereof). In some embodiments, the Cap protein of the present invention comprises SpyTag, or a biologically active portion or variant thereof. The use of the first member of a protein:protein binding pair is described in WO2019006046 (incorporated herein in its entirety).

[0239] In some embodiments, the viral capsid comprising the modified viral capsid protein described herein is a chimeric capsid, for example, comprising at least two groups of VP1, VP2 and / or VP3 proteins, each of which is encoded by a different cap gene. The chimeric capsid herein generally refers to a chimera of a first viral capsid protein and a second corresponding viral capsid protein, wherein the first viral capsid protein is modified to include a first member of a binding pair, and the second corresponding viral capsid protein lacks the first member of the binding pair. With regard to chimeric capsids, the second viral capsid protein lacking the first member of the binding pair may be referred to as a reference capsid protein encoded by a reference cap gene. In some chimeric capsid embodiments, preferably, when the VP1, VP2 and / or VP3 capsid proteins modified with the first member of the protein: protein pair are not chimeric capsid proteins, except that the reference capsid protein lacks the first member of the binding pair, the VP1, VP2 and / or VP3 reference capsid proteins may include the same amino acid sequence as the viral VP1, VP2 and / or VP3 capsid proteins modified with the first member of the binding pair. In some chimeric capsid embodiments, the VP1, VP2 and / or VP3 reference capsid protein corresponds to a viral VP1, VP2 and / or VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP1 reference capsid protein corresponds to a viral VP1 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some chimeric capsid embodiments comprising a chimeric VP1, VP2 and / or VP3 capsid protein that has been further modified to comprise a first member of a binding pair, the reference protein may be a corresponding capsid protein from which a portion of the chimeric capsid protein forms a portion of the chimeric capsid protein. As a non-limiting example in some embodiments, a chimeric capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to comprise a first member of a binding pair may further comprise an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member as a reference capsid protein. Similarly, in some embodiments, a chimeric capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to comprise a first member of a binding pair may further comprise an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member as a reference capsid protein.In some embodiments, the chimeric capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to comprise a first member of a binding pair may further comprise an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member as a reference capsid protein. In some chimeric capsid embodiments, the reference capsid protein may be any capsid protein as long as it lacks the first member of the binding pair and is capable of forming a capsid with the first capsid protein modified with the first member of the binding pair.

[0240] Typically, chimeric particles can be produced by transfecting a mixture of modified Cap genes and reference Cap genes into production cells at a specified ratio. The ratio of protein subunits in the particle, such as the ratio of modified VP protein: unmodified VP protein, can, but does not necessarily, stoichiometrically reflect the ratio of at least two species of a cap gene encoding a first capsid protein modified with a first member of a binding pair to one or more reference cap genes (e.g., a modified cap gene transfected into a packaging cell: one or more reference cap genes). In some embodiments, the ratio of protein subunits in the particle does not stoichiometrically reflect the ratio of a modified cap gene: one or more reference cap genes transfected into a packaging cell.

[0241] In some chimeric viral particle embodiments, the protein subunit ratio is in the range of about 1:59 to about 59:1.

[0242] In some embodiments of non-chimeric viral particles, the protein subunit ratio can be 1:0, wherein each capsid protein of the non-chimeric viral particle is modified with the first member of the binding pair. In some embodiments of non-chimeric viral particles, the protein subunit ratio can be 0:1, wherein each capsid protein of the non-chimeric viral particle is not modified with the first member of the binding pair.

[0243] Due to the high conservation of at least the large segments and the large members of closely related family members, the corresponding insertion sites of AAVs other than the listed AAVs can be identified by performing an amino acid alignment or by comparing the capsid structures. For exemplary alignments of different AAV capsid proteins, see, for example, Rutledge et al. (1998) J. Virol 72: 309-19; Mietzsch et al. (2019) Viruses 11,362,1-34 and U.S. Patent No. 9,624,274, each of which is incorporated herein by reference in its entirety. For example, Mietzcsh et al. (2019) provide a band coverage from different parvoviruses in Figure 7, describing variable regions VR I to VR IX. Using such structural analysis and sequence analysis as described therein, a technician can determine which amino acids within the variable region correspond to the amino acid sequence of AAV that can accommodate the insertion of a targeting ligand, a first member of a binding pair, and / or a detectable marker, such as described herein.

[0244] Typically, the targeting ligand, the first member of the binding pair, and / or the detectable label can be inserted into the variable region or variable loop of the AAV capsid protein, the GH loop of the AAV capsid protein, or the like.

[0245] In some embodiments, the first member of the binding pair and / or the detectable label is inserted into the VP1 capsid protein of non-primate AAV after the amino acid position corresponding to the amino acid position selected from the group consisting of: G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the first member of the binding pair and / or the detectable label is inserted into the VP1 capsid protein of non-primate AAV between the amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid. Other suitable insertion sites for non-primate VP1 capsid proteins include those corresponding to 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713 and 1-716 of the VP1 capsid protein of AAV2 (Wu et al. (2000) J. Virol 74:8635-8647). The modified viral capsid protein described herein can be a non-primate capsid protein comprising a first member of a binding pair and / or a detectable label inserted into a position corresponding to a position of an AAV2 capsid protein selected from the group consisting of: 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Other suitable insertion sites for non-primate AAVs include those corresponding to I-587 or I-590 of AAV1, I-589 of AAV1, I-585 of AAV3, I-584 or I-585 of AAV4, and I-575 or I-585 of AAV5. In some embodiments, the modified viral capsid protein described herein can be a non-primate capsid protein comprising a targeting ligand, a first member of a binding pair, and / or a detectable label inserted at a position corresponding to a position selected from the group consisting of: I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

[0246] In some embodiments, the first member of the binding pair and / or the detectable label is inserted into the VP1 capsid protein of a non-primate AAV after the amino acid position corresponding to the amino acid position selected from the group consisting of: I444 of avian AAV capsid protein VP1, I580 of avian AAV capsid protein VP1, I573 of bearded dragon AAV capsid protein VP1, I436 of bearded dragon AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I437 of sea lion AAV capsid protein VP1, and I565 of sea lion AAV capsid protein VP1.

[0247] The nomenclature I-###, I#, etc. in this article refers to the insertion site (I), wherein ### designates the amino acid number of the VP1 protein relative to the AAV capsid protein, however, such insertion may be located directly within 5 amino acids of the N or C terminal end of a given amino acid, preferably 3, more preferably 2, and especially N or C terminal end of one amino acid in a sequence of 1 amino acid, preferably the C terminal end. In addition, the positions mentioned herein are relative to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and point mutations thereof) of the VP2 and VP3 capsid proteins encoded by the capsid gene can be easily identified by sequence alignment of the VP1, VP2 and VP3 proteins encoded by the appropriate AAV capsid gene.

[0248] Thus, insertion of the corresponding position of the encoding nucleic acid at one of these sites of the cap gene results in insertion of VP1, VP2 and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Thus, for example, for AAV2, according to this nomenclature, insertions between amino acids 1 and 138 are inserted only into VP1, insertions between 138 and 203 are inserted into VP1 and VP2, and insertions between 203 and the C-terminus are inserted into VP1, VP2 and VP3, and of course the same is true for insertion site 1-587. Thus, the present invention encompasses structural genes of AAV having corresponding insertions in the VP1, VP2 and / or VP3 proteins.

[0249] Also provided herein are nucleic acids encoding the VP3 capsid protein described herein. AAV capsid proteins can (but need not) be encoded by overlapping reading frames of the same gene with staggered start codons. In some embodiments, the nucleic acid encoding the VP3 capsid protein described herein does not encode the VP2 capsid protein or VP1 capsid protein of the present invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein described herein may also encode the VP2 capsid protein described herein, but does not encode the VP1 capsid of the present invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein described herein may also encode the VP2 capsid protein described herein and the VP1 capsid described herein.

[0250] In some embodiments, a viral capsid comprising a modified viral capsid protein containing a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprising a multispecific binding protein, etc.) is capable of infecting a specific cell, for example, with an enhanced ability compared to the ability of a control viral capsid to target and bind to a specific cell, the control viral capsid being identical to the modified viral capsid protein except that it lacks one or both of the first member and the second member of the binding pair, for example, comprising a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein that is bound to a first member and a second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid.

[0251] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprising a multispecific binding protein, etc.) is capable of infecting a specific cell, for example, with an enhanced ability to target and bind to a specific cell as compared to the ability of a control viral capsid, which is identical to the modified viral capsid protein except that it lacks one or both of the first member and the second member of the binding pair, for example, comprising a control capsid protein. In some embodiments, the viral particles of the invention (which comprise a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, and optionally comprise a first member and a second member of a binding pair (e.g., wherein the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.)) are better able to evade neutralization by pre-existing antibodies in serum isolated from a human patient than an appropriate control viral particle (e.g., comprising a viral capsid of an AAV serotype, a portion of which, e.g., as a portion of a viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, is contained in the viral capsid of the invention). In some embodiments, a viral particle of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate AAV, a distant AAV, or a combination thereof requires at least 2-fold more total IVIG or IgG to neutralize (e.g., 50% or more inhibition of infection) compared to an appropriate control viral particle, e.g., (e.g., the IC of the viral particle of the invention is 50 The value was at least 2-fold that of the control virus particles).

[0252] In some embodiments of the invention comprising a detectable marker, the targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to a detectable marker and (ii) a second binding domain that specifically binds to a receptor, which second domain may be conjugated to a bead surface (e.g., for purification) or expressed by a target cell. Thus, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to a detectable marker and (ii) a second binding domain that specifically binds to a receptor targets the viral particle. Such "targeting" or "direction" may include situations in which wild-type viral particles target several cells in a tissue and / or several organs in an organism, and such broad targeting of tissues or organs may be reduced to elimination by inserting a detectable marker, and such retargeting to more specific cells in a tissue or more specific organs in an organism is achieved with a multispecific binding molecule. Such retargeting or redirection may also include situations in which wild-type viral particles target tissues, such tissue targeting may be reduced to elimination by inserting a detectable marker, and such retargeting to completely different tissues may be achieved using a multispecific binding molecule. The antibody paratope described herein generally comprises at least a complementary determining region (CDR) that specifically recognizes a detectable marker, such as a CDR3 region of a heavy chain and / or light chain variable domain. In some embodiments, the multispecific binding molecule comprises an antibody (or portion thereof) containing an antibody paratope that specifically binds to a detectable marker. For example, the multispecific binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or the single domain light chain variable region comprises an antibody paratope that specifically binds to a detectable marker. In some embodiments, the multispecific binding molecule may comprise an Fv region, for example, the multispecific binding molecule may comprise an scFv, which comprises an antibody paratope that specifically binds to a detectable marker. In some embodiments, the multispecific binding molecule as described herein comprises an antibody paratope that specifically binds to c-myc.

[0253] Another embodiment of the present invention is the use of at least one modified viral capsid protein and / or nucleic acid encoding the protein, preferably at least one multimeric structure (eg, viral particle) for producing target nucleotides and for transferring target nucleotides to target cells.

[0254] In some embodiments, the viral particles described herein include components of a virus, such as a capsid, a glycoprotein, etc., selected from the group consisting of human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), murine stem cell virus (MSCV) or murine leukemia virus (MLV). In some embodiments, the viral particles described herein include HIV capsids, multiple HIV capsids and / or HIV capsids, for example, HIV viral particles and / or produced by HIV.

[0255] In some embodiments, in addition to the B cell or HSC targeting moiety, the viral particles described herein also display a fusogen. In some embodiments, the fusogen is a protein; for example, a viral protein (e.g., a vesicular virus protein [e.g., vesicular stomatitis virus G glycoprotein (VSVG)], an alpha virus protein [e.g., a Sindbis virus glycoprotein], an orthomyxovirus protein [e.g., an influenza HA protein], a paramyxovirus protein [e.g., a Nipah virus F protein or a measles virus F protein]); or a fragment, mutant or derivative thereof. In a specific embodiment, the fusogen is heterologous to the reference wild-type virus from which the particle is produced. In some embodiments, the fusogen is a mutant protein that does not bind to its natural ligand.

[0256] In some embodiments, the targeting moiety and the fusogen are contained in a fusion protein.

[0257] In some embodiments described herein, the viral particles include fusogens. Many different proteins and non-protein fusogens can be used. In some embodiments, the fusogen is a protein. In a specific embodiment, the fusogen is a viral protein. Non-limiting examples of useful viral fusogens include, for example, vesicular virus fusogens (e.g., vesicular stomatitis virus G glycoprotein (VSVG)), alphavirus fusogens (e.g., Sindbis virus glycoprotein), orthomyxovirus fusogens (e.g., influenza HA protein), paramyxovirus fusogens (e.g., Nipah virus F protein or measles virus F protein) and from dengue virus (DV), Lassa fever virus, tick-borne encephalitis virus, dengue virus, hepatitis B virus, rabies virus, Semliki Forest virus, Ross River virus, Ora virus, Borna disease virus, Hanta virus, SARS-CoV virus fusogens and various fragments, mutants and derivatives thereof. Other exemplary fusogen molecules and related methods are described, for example, in U.S. Patent Application Publications 2005 / 0238626 and 2007 / 0020238.

[0258] In a specific embodiment, the fusogen is heterologous to the virus from which the particle is produced.

[0259] There are two recognized classes of viral fusogens, both of which can be used as targeting moieties (DS Dimitrov, Nature Rev. Microbio. 2, 109 (2004)). Class I fusogens use a helical coiled-coil structure to trigger membrane fusion, while class II fusogens use 13 barrels to trigger fusion. In some embodiments, class I fusogens are used. In other embodiments, class II fusogens are used. In other embodiments, class I and class II fusogens are used. See, e.g., Skehel and Wiley, Annu. Rev. Biochem. 69, 531-569 (2000); Smit, J. et al. J. Virol. 73, 8476-8484 (1999), Morizono et al. J. Virol. 75, 8016-8020 (2005), Mukhopadhyay et al. (2005) Rev. Microbiol. 3, 13-22.

[0260] In some embodiments, a form of hemagglutinin (HA), a class I fusogen, from influenza A / fowl plague virus / Rostock / 34 (FPV) is used (Hatziioannou et al., J. Virol. 72, 5313 (1998)). In some embodiments, a form of FPV HA is used (Lin et al., Hum. Gene. Ther. 12, 323 (2001)). HA-mediated fusion is generally considered to be independent of receptor binding (Lavillette et al., Cosset, Curr. Opin. Biotech. 12, 461 (2001)).

[0261] In other embodiments, the Sindbis virus glycoprotein (class II fusogen) from the Alphaviridae family is used (Wang et al., J. Virol. 66, 4992 (1992); Mukhopadhyay et al., Nature Rev. Microbio. 3, 13 (2005), Morizono et al., Nature Med. 11, 346 (2005)).

[0262] In some embodiments, mutant fusogens are used that retain their fusogenic ability but have reduced or abolished binding capacity or specificity.The functional properties of mutant fusogens can be tested, for example, in cell culture or by determining their ability to stimulate an immune response in vivo without causing adverse side effects.

[0263] To select the most effective and non-toxic combination of targeting moiety and fusogen (wild-type or mutant), viral particles carrying these molecules can be tested for selectivity and / or their ability to facilitate penetration of target cell membranes.

[0264] In certain embodiments, the fusogenic molecule is the Sindbis virus envelope protein (SIN). Sindbis virus transfers its RNA into cells by low pH-mediated membrane fusion. SIN comprises five structural proteins: E1, E2, E3, 6K and capsid. E2 comprises a receptor binding sequence that allows wild-type SIN to bind, and E1 is known to comprise properties necessary for membrane fusion (Konoochik et al., Virology Journal 2011, 8: 304). E1, E2 and E3 are encoded by polyproteins, and their amino acid sequences are provided by, for example, accession numbers VHWVB, VHWVB2 and P03316: nucleic acid sequences are provided by, for example, accession numbers SVU90536 and V01403 (see also Rice & Strauss, Proc. Nat'l Acad. Sci USA 78: 2062-2066 (1981); and Strauss et al., Virology 133: 92-110 (1984)).

[0265] In certain embodiments, the Sindbis virus envelope protein is mutated (SINmu). In certain embodiments, the mutation reduces the natural tropism of the Sindbis virus. In certain embodiments, SINmu comprises SIN proteins E1, E2 and E3, wherein at least one of E1, E2 or E3 is mutated compared to the wild-type sequence. For example, one or more of E1, E2 or E3 proteins can be mutated at one or more amino acid positions. In addition, the combination of mutations in E1, E2 and E3 is included in the fusogens described herein, such as mutations in E1 and E2, or E2 and E3, or E3 and E1, or E1, E2 and E3. In certain embodiments, at least E2 is mutated.

[0266] In certain embodiments, SINmu comprises the following envelope protein mutations compared to the wild-type Sindbis virus envelope protein: (i) deletion of E3 amino acids 61-64; (ii) E2KE159-160AA; and (iii) E2 SLKQ68-71AAAA ("SLKQ" and "AAAA" are disclosed as SEQ ID NOs. 10-11, respectively). In other embodiments, SINmu further comprises the envelope protein mutation E1 AK226-227SG. Examples of SINmu can be found, for example, in U.S. Pat. No. 9,163,248; WO2011011584; Cronin et al., Curr Gene Ther. 2005Aug; 5(4): 387-398.

[0267] Envelopes of other Togaviridae, such as envelopes from the genus Alphavirus, such as Semliki Forest virus, Ross River virus, and Equine Encephalitis virus, can also be used to pseudotype the vectors described herein. Envelope protein sequences of such alphaviruses are known in the art.

[0268] In certain embodiments, the fusogen is a vesicular stomatitis virus (VSV) envelope protein. In certain embodiments, the fusogen is the G protein of VSV (VSV-G; Burns et al., Proc. Natl. Acad. Sci. USA 1993, Vol. 90, No. 17, pp. 1833-7) or a fragment, mutant, derivative or homologue thereof. VSV-G interacts with the phospholipid components of the cell (e.g., T cell) membrane to mediate viral entry by membrane fusion (Mastromarino et al., J Gen Virol. 1998, Vol. 68, No. 9, pp. 2359-69; Marsh et al., Adv Virus Res. 1989, Vol. 107, No. 36, pp. 107-51. Examples of VSV-G can be found in, for example, WO2008058752.

[0269] One embodiment of the present invention is a nucleic acid encoding a capsid protein as described above. The nucleic acid is preferably a vector comprising the claimed nucleic acid sequence. The nucleic acid (especially the vector) is necessary for recombinant expression of the capsid protein of the present invention.

[0270] Another embodiment of the present invention is the use of at least one recombinant viral capsid protein and / or nucleic acid encoding the same, preferably at least one multimeric structure (eg, viral vector) for production and use as a gene transfer vector.

[0271] Heterologous epitope

[0272] Typically, the recombinant viral capsid protein and / or the viral vector comprising the recombinant viral capsid comprises a heterologous epitope that enables, for example, retargeting of the viral vector by a binding molecule (e.g., an antibody). In some embodiments, the heterologous epitope is a B cell epitope, for example, having a length between about 1 amino acid and about 35 amino acids, and forms a binding pair with an antibody paratope (e.g., an immunoglobulin variable domain). In some embodiments, the heterologous epitope comprises an affinity tag.

[0273] A large number of tags are known in the prior art. (See, for example, Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins" Protein Expression and Purification 11:1-16, Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60:523-533 and references therein). Affinity tags include, but are not limited to, binding to immobilized divalent cations (e.g., Ni 2+ ), a polyhistidine tag (e.g., a His-6, His-8, or His-10 tag) that binds to immobilized avidin (e.g., on a polypeptide sequence that is biotinylated in vivo), a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S tag that binds to immobilized S protein, an antigen that binds to an immobilized antibody or a domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to corresponding antibodies), a FLASH tag (a high affinity tag coupled to a specific arsenic moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), a protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, a maltose binding protein (MBP) that binds to immobilized amylose, an albumin binding protein that binds to immobilized albumin, a chitin binding domain that binds to immobilized chitin, a calmodulin binding peptide that binds to immobilized calmodulin, and a cellulose binding domain that binds to immobilized cellulose. Another exemplary tag is a SNAP-tag available from Covalys (www.covalys.com). In some embodiments, a heterologous epitope disclosed herein comprises an affinity tag that is recognized only by an antibody paratope. In some embodiments, a heterologous epitope disclosed herein comprises an affinity tag that is recognized by an antibody paratope and other specific binding pairs.

[0274] In some embodiments, the heterologous epitope and / or affinity tag does not form a binding pair with an immunoglobulin constant domain. In some embodiments, the heterologous epitope and / or affinity tag does not bind to a metal ion (e.g., Ni 2+ 、Co 2+ , Cu 2+ 、Zn 2 +, Fe 3+ In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of: streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A.

[0275] In some embodiments, the affinity tag is selected from the group consisting of FLAG, HA, and c-myc (EQKLISEEDL (SEQ ID NO: 12)). In some embodiments, the heterologous epitope is c-myc.

[0276] In some embodiments, the recombinant viral capsid described herein comprises the amino acid sequence EQKLISEEDL (SEQ ID NO: 12) flanked and / or operably linked to at least 5 consecutive amino acids of the AAV VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein comprises the amino acid sequence EQKLISEEDL (SEQ ID NO: 12) flanked and / or operably linked to at least 5 consecutive amino acids of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein comprises EQKLISEEDL (SEQ ID NO: 12) inserted between N587 and R588 of the AAV2 VP1 capsid protein.

[0277] In some embodiments, the heterologous epitope comprises an affinity tag and one or more linkers. In some embodiments, the heterologous epitope comprises an affinity tag flanked by linkers, for example, the heterologous epitope comprises a first linker, an affinity tag, and a second linker from the N-terminus to the C-terminus. In some embodiments, the length of the first linker and the second linker is each independently at least one amino acid. In some embodiments, the first linker and the second linker are the same.

[0278] In general, the heterologous epitopes described herein, such as affinity tags alone or in combination with one or more linkers, are between about 5 amino acids and about 35 amino acids in length. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is at least 5 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 6 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 7 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 8 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 9 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 10 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 11 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 12 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 13 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 14 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 15 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 16 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 17 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 18 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 19 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 20 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 21 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 22 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 23 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 24 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 25 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 26 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 27 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 28 amino acids.In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 29 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 30 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 31 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 32 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 33 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 34 amino acids. In some embodiments, the length of the heterologous epitope (alone or in combination with one or more linkers) is 35 amino acids.

[0279] Retargeting part

[0280] In the absence of binding molecules, particularly binding molecules that specifically bind to surface molecules expressed by target cells (e.g., B cells or hematopoietic stem cells), the transduction capacity of the viral vectors described herein is reduced to disappearance. In some embodiments, the binding molecule comprises an antibody (or fragment thereof) containing an antibody paratope that specifically binds to a heterologous epitope. For example, the binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, wherein the single domain heavy chain variable region or the single domain light chain variable region comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the binding molecule may comprise an Fv region, for example, the binding molecule may comprise an scFv, which comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the binding molecules described herein comprise an antibody paratope that specifically binds to c-myc.

[0281] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in a given HCVR and / or LCVR amino acid sequence disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on the variability of the sequence, the Chothia definition is based on the position of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases can also be used to identify CDR sequences in antibodies.

[0282] In some embodiments, the binding molecule binds to a protein expressed on the surface of a cell, such as a cell surface protein on a hematopoietic cell (e.g., a B cell or hematopoietic stem cell (HSC)). There are a large number of cell surface proteins, such as cell surface receptors, which are suitable for being targeted by a retargeting ligand, and retargeting ligands, such as antibodies or portions thereof, are available for them. Such structures include, but are not limited to, B cell receptors and related proteins (e.g., CD19, CD20, CD22, CD34, CD38, CD40, CD22, CD79, CD180, B cell activating factor (BAFF), ASGR1, CD117, Sca1, etc.) and HSC receptors and related proteins (e.g., CD34, etc.). The recombinant viral capsid described herein allows specific infection of cell types by using a binding molecule comprising a retargeting ligand that binds to a surface antigen of a differentiated cell as a target of the viral vector complex.

[0283] The viral particles described herein may also comprise a second member of a specific binding pair that specifically forms a covalent bond with a first member of the specific binding pair inserted into / displayed by the recombinant viral capsid protein, wherein the second member is fused to the binding molecule.

[0284] In certain exemplary embodiments, the binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the case of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first antigen and a second antigen-binding domain, the CDR of the first antigen-binding domain can be named with the prefix "A1", and the CDR of the second antigen-binding domain can be named with the prefix "A2". Therefore, the CDR of the first antigen-binding domain may be referred to as A1-HCDR1, A1-HCDR2, and A1-HCDR3 herein; and the CDR of the second antigen-binding domain may be referred to as A2-HCDR1, A2-HCDR2, and A2-HCDR3 herein.

[0285] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form the bispecific antigen binding molecules of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain can be connected to a separate multimerization domain. The association of a multimerization domain and another multimerization domain promotes the association between the two antigen-binding domains, thereby forming a bispecific antigen binding molecule. As used herein, "multimerization domain" is any macromolecule, protein, polypeptide, peptide or amino acid with the ability to associate with the second multimerization domain of the same or similar structure or composition. For example, the multimerization domain can be a polypeptide comprising an immunoglobulin CH3 domain. The non-limiting example of a multimerization component is the Fc part of an immunoglobulin (comprising a CH2-CH3 domain), for example, the Fc domain of an IgG selected from isotypes IgG1, IgG2, IgG3 and IgG4, and any allotype in each isotype group.

[0286] The bispecific antigen binding molecules of the present invention generally comprise two multimerization domains, such as two Fc domains, each of which is an independent part of a single antibody heavy chain. The first and second multimerization domains can be the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerization domains can be different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0287] In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence of 1 to about 200 amino acids in length comprising at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue, or a short peptide containing cysteine. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0288] Any bispecific antibody form or technology can be used to prepare the bispecific antigen binding molecules of the present invention. For example, an antibody or fragment thereof with a first antigen binding specificity can be functionally connected (e.g., by chemical coupling, gene fusion, non-covalent association or otherwise) with one or more other molecular entities (such as another antibody or antibody fragment with a second antigen binding specificity) to produce a bispecific antigen binding molecule. Specific exemplary bispecific formats that can be used in the present description include, but are not limited to, for example, scFv-based bispecific formats or diabody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG and Mab2 bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein; see also Brinkmann and Konterman (2017) mAbs 9:182-212; each of which is incorporated by reference in its entirety).

[0289] The present invention also includes a bispecific antigen binding molecule comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the difference in at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds to protein A and the second Ig CH3 domain comprises a mutation that reduces or eliminates protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R, according to EU numbering). The second CH3 may also comprise a Y96F modification (according to IMGT; Y436F, according to EU). Other modifications that may be present in the second CH3 include: in the case of an IgG1 antibody, D16E, L18M, N44S, K52N, V57M and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M and V422I, according to EU); in the case of an IgG2 antibody, N44S, K52N and V82I (according to IMGT; N384S, K392N and V422I, according to EU); and in the case of an IgG4 antibody, Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I, according to EU); see, e.g., WO 2010 / 151792.

[0290] In certain embodiments, the Fc domain may be chimeric, incorporating Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include part or all of a CH2 sequence derived from a human IgG1, human IgG2, or human IgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgG1, human IgG2, or human IgG4. A chimeric Fc domain may also include a chimeric hinge region. For example, a chimeric hinge may include a combination of an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region and a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any antigen-binding molecule described herein includes from N-terminus to C-terminus: [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any antigen binding molecule described herein comprises, from N-terminus to C-terminus: [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any antigen binding molecule of the invention are described in PCT application WO2014 / 022540 (incorporated by reference in its entirety). Chimeric Fc domains and variants thereof having these general structural arrangements may have altered Fc receptor binding, which in turn affects the function of the Fc effector.

[0291] Liposomes, lipid nanoparticles and other delivery vehicles

[0292] In some embodiments, the first component and / or the second component of the systems described herein can be a lipid-based vehicle, such as a lipid nanoparticle (LNP), a liposome, a lipidoid, or a lipoplex.

[0293] In some embodiments, the first component and / or the second component of the system described herein can include liposomes or LNPs. Liposomes and LNPs are vesicles that include one or more lipid bilayers. In some embodiments, liposomes or LNPs include two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more proteins, polysaccharides or other molecules.

[0294] Lipid preparations can protect biomolecules from being degraded while improving their cellular uptake. Liposomes or LNPs are particles comprising multiple lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including single-layer and multi-layer vesicles, such as liposomes), dispersed phases in emulsions, micelles or internal phases in suspensions. Such liposomes or LNPs can be used to encapsulate one or more nucleic acids or proteins for delivery. Preparations containing cationic lipids can be used to deliver polyanions such as nucleic acids. Other lipids that may be included are neutral lipids (i.e., uncharged lipids or zwitterionic lipids), anionic lipids, auxiliary lipids for enhancing transfection, and stealth lipids that increase the time length that nanoparticles can exist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, auxiliary lipids, and stealth lipids are found in WO 2016 / 010840 A1 and WO2017 / 173054 A1, each of which is incorporated herein by reference in its entirety for all purposes. Exemplary lipid nanoparticles may include cationic lipids and one or more other components. In one example, another component can include auxiliary lipids such as cholesterol. In another example, other components can include auxiliary lipids such as cholesterol and neutral lipids such as distearoylphosphatidylcholine (DSPC). In another example, other components can include auxiliary lipids such as cholesterol, optional neutral lipids such as DSPC and stealth lipids such as S010, S024, S027, S031 or S033.

[0295] Liposomes are amphiphilic lipids that can form a bilayer in an aqueous environment to encapsulate an aqueous core. Polypeptides (e.g., Cas proteins) or polynucleotides (e.g., guide RNAs) can be incorporated into the aqueous core. These lipids can have anionic, cationic, or zwitterionic hydrophilic head groups. Liposomes can be formed from a single lipid or a lipid mixture. The mixture can include (1) a mixture of anionic lipids; (2) a mixture of cationic lipids; (3) a mixture of zwitterionic lipids; (4) a mixture of anionic lipids and cationic lipids; (5) a mixture of anionic lipids and zwitterionic lipids; (6) a mixture of zwitterionic lipids and cationic lipids; or (7) a mixture of anionic lipids, cationic lipids, and zwitterionic lipids. Similarly, the mixture can include saturated and unsaturated lipids. Exemplary phospholipids include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. Cationic lipids include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), dioleoyltrimethylammonium propane (DOTAP), 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids include dodecylphosphocholine, DPPC, and DOPC.

[0296] The liposome or LNP may comprise one or more or all of the following: (i) lipids for encapsulation and endosomal escape; (ii) neutral lipids for stabilization; (iii) auxiliary lipids for stabilization; and (iv) stealth lipids. See, e.g., Finn et al. (2018) Cell Rep. 22(9): 2227-2235 and WO 2017 / 173054 A1, each of which is incorporated herein by reference in its entirety for all purposes.

[0297] In some instances, liposome or LNP include cationic lipid. In some instances, liposome or LNP include 18 carbon -9,12- dienoic acid (9Z, 12Z) -3- ((4,4- bis (octyloxy) butyryl) oxygen base) -2- ((((3- (diethylamino) propoxy) carbonyl) oxygen base) methyl) propyl ester (also referred to as (9Z, 12Z) -18 carbon -9,12- dienoic acid 3- ((4,4- bis (octyloxy) butyryl) oxygen base) -2- ((((3- (diethylamino) propoxy) carbonyl) oxygen base) methyl) propyl ester) or another ionizable lipid. See, for example, WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340 and WO 2014 / 136086, for all purposes, each of these documents is incorporated herein by reference in its entirety. In some instances, the LNP comprises a molar ratio (N:P) of cationic lipid amine to RNA phosphate of about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. In some instances, the terms cationic and ionizable are interchangeable in the context of LNP lipids (e.g., where the ionizable lipid is cationic depending on the pH).

[0298] The lipid for encapsulation and endosomal escape can be a cationic lipid. The lipid can also be a biodegradable lipid, such as a biodegradable ionizable lipid. An example of a suitable lipid is lipid A or LP01, which is octadecane-9,12-dienoic acid (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z, 12Z)-octadecane-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester. See, e.g., Finn et al. (2018) Cell Rep. 22(9): 2227-2235 and WO 2017 / 173054 A1, each of which is incorporated herein by reference in its entirety for all purposes. Another example of a suitable lipid is lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), also known as ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Another example of a suitable lipid is lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadec-9,12-dienoate). Another example of a suitable lipid is lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octyl undecanoate. Other suitable lipids include 4-(dimethylamino)butyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester (also known as [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butyrate or Dlin-MC3-DMA (MC3))).

[0299] Other suitable cationic lipids include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N -trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). For example, cationic lipids having a positive charge at less than physiological pH include, but are not limited to, DODAP, DODMA, and DMDMA. In some embodiments, the cationic lipid comprises a C18 alkyl chain, an ether linkage between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, and DODMA. The cationic lipid may comprise an ether linkage and a pH titratable head group. Such lipids include, for example, DODMA. Other cationic lipids are described in U.S. Pat. Nos. 7,745,651, 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992, which are incorporated herein by reference.

[0300] In some embodiments, cationic lipids may include protonatable tertiary amine head groups. Such lipids are referred to herein as ionizable lipids. Ionizable lipids refer to lipid species comprising ionizable amine head groups and generally comprising pKa less than about 7. In an acidic pH environment, ionizable amine head groups are protonated so that ionizable lipids preferentially interact with negatively charged molecules (e.g., nucleic acids, such as recombinant polynucleotides described herein), thereby promoting the assembly and encapsulation of liposomes or LNPs. Therefore, in some embodiments, ionizable lipids can increase the loading of nucleic acids into liposomes or LNPs. In an environment where pH is greater than about 7 (e.g., 7.4 physiological pH), ionizable lipids include neutral charges. When particles comprising ionizable lipids enter the low pH environment of endosomes (e.g., pH <7), ionizable lipids are protonated again and associate with anionic endosomal membranes, promoting the release of the contents encapsulated by the particles.

[0301] In some embodiments, the liposomes or LNPs may include one or more non-cationic helper lipids. Exemplary helper lipids include (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DiPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), (1,2-dioleoyl-sn-glycero-3-phospho-(1′-racemic glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), ceramide, sphingomyelin, and cholesterol.

[0302] Some such lipids suitable for use in liposomes or LNPs described herein are biodegradable in vivo. Examples of biodegradable lipids include, but are not limited to, octadecane-9,12-dienoic acid (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-20(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z, 12Z)-octadecane-9,12-dienoate or another ionizable lipid. See, e.g., PCT Publication Nos. WO2017 / 173054, WO2015 / 095340, and WO2014 / 136086. In some embodiments, the terms cationic and ionizable are interchangeable in the context of liposomes or LNP lipids, for example, where an ionizable lipid is cationic depending on the pH.

[0303] Such lipids can be ionizable, depending on the pH of the medium in which they are located. For example, in slightly acidic media, lipids can be protonated and therefore carry a positive charge. On the contrary, in weakly alkaline media, such as blood having a pH of about 7.35, lipids may not be protonated and therefore have no charge. In some embodiments, lipids can be protonated at a pH of at least about 9, 9.5, or 10. The ability of such lipids to carry a charge is related to their intrinsic pKa. For example, lipids can independently have a pKa of about 5.8 to about 6.2.

[0304] The function of the neutral lipid is to stabilize and improve the processing of the liposomes or LNPs.Examples of suitable neutral lipids include various neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present invention include, but are not limited to, 5-heptadecanylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-diamidonoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl basic phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1 -palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diacetoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-diicosanoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and combinations thereof. For example, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

[0305] Helper lipids include lipids that enhance transfection. The mechanism by which helper lipids enhance transfection may include enhancing particle stability. In some cases, helper lipids can enhance membrane fusogenicity. Helper lipids include steroids, sterols, and alkylresorcinols. Examples of suitable helper lipids include cholesterol, 5-heptadecanylresorcinol, and cholesterol hemisuccinate. In one example, the helper lipid can be cholesterol or cholesterol hemisuccinate.

[0306] Stealth lipids include lipids that change the length of time that nanoparticles can exist in vivo. Stealth lipids can help the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids can modulate the pharmacokinetic properties of liposomes or LNPs. Suitable stealth lipids include lipids with a hydrophilic head group connected to the lipid moiety.

[0307] The hydrophilic head group of the stealth lipid may comprise, for example, a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinyl pyrrolidone), polyamino acids, and poly-N-(2-hydroxypropyl)methacrylamide. The term PEG refers to any polyethylene glycol or other polyalkylene ether polymer. In certain liposome or LNP formulations, the PEG is PEG-2K, also known as PEG 2000, which has an average molecular weight of approximately 2,000 Daltons. See, e.g., WO 2017 / 173054 A1, which is incorporated herein by reference in its entirety for all purposes.

[0308] The lipid portion of the stealth lipid may be derived from, for example, diacylglycerol or diacylglycerol amide, including those comprising a dialkylglycerol or dialkylglycerol amide group having an alkyl chain length independently comprising saturated or unsaturated carbon atoms from about C4 to about C40, wherein the chain may comprise one or more functional groups, such as amides or esters. The dialkylglycerol or dialkyl imidazole diamide group may also comprise one or more substituted alkyl groups.

[0309] As an example, the stealth lipid can be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilauroylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)carboxamido-3',6'-dioxooctanoyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-dioctylbenzyl-[ω]-methyl- ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000)(PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000](PEG2k-DSA). In a specific example, the stealth lipid can be PEG2k-DMG.

[0310] In some embodiments, the liposome or LNP may also include one or more PEG-modified lipids comprising a 5 kDa poly(ethylene glycol) chain covalently attached to a lipid comprising one or more C6-C20 alkyl groups. In some embodiments, the liposome or LNP also includes 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-amine). In some embodiments, the PEG-modified lipid accounts for about 0.1% to about 1% of the total lipid content in the lipid nanoparticle. In some embodiments, the PEG-modified lipid accounts for about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% of the total lipid content in the liposome or lipid nanoparticle.

[0311] In some embodiments, liposome as herein described or LNP can comprise the conjugated lipid that suppresses lipid particle aggregation.Such lipid conjugates include but are not limited to PEG-lipid conjugates, such as PEG (such as PEG-DAA conjugates) coupled to dialkoxypropyl, PEG (such as PEG-DAG conjugates) coupled to diacylglycerol, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamine and PEG (see, for example, U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (such as, POZ-DAA conjugates), polyamide oligomers (such as, ATTA-lipid conjugates) and mixtures thereof coupled to ceramide.PCT discloses other examples of POZ-lipid conjugates described in No. WO 2010 / 006282.PEG or POZ can be directly conjugated with lipid, or can be connected with lipid by linker moiety.Can use any linker moiety suitable for PEG or POZ to be coupled to lipid, including, for example, non-ester-containing linker moiety and ester-containing linker moiety. In certain embodiments, non-ester containing linker moieties are used, such as amides or carbamates.

[0312] Liposomes or LNPs may contain different respective molar ratios of the component lipids in the formulation. The mol-% of CCD lipids may be, for example, about 30 mol-% to about 60 mol-%. The mol-% of auxiliary lipids may be, for example, about 30 mol-% to about 60 mol-%. The mol-% of neutral lipids may be, for example, about 1 mol-% to about 20 mol-%. The mol-% of stealth lipids may be, for example, about 1 mol-% to about 10 mol-%.

[0313] Liposomes or LNPs can have different ratios between the positively charged amine groups (N) of the biodegradable lipids and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the formula N / P. For example, the N / P ratio can be from about 0.5 to about 100. The N / P ratio can also be from about 4 to about 6.

[0314] In some embodiments, the liposome or LNP may include a nuclease (e.g., CRISPR / Cas system, ZFN or TALEN), may include a polynucleotide molecule (e.g., guide RNA), may include a nucleic acid construct encoding a target polypeptide (e.g., an antibody or antigen binding fragment), or may include a nuclease (e.g., CRISPR / Cas system) and a nucleic acid construct encoding a target polypeptide (e.g., a donor template for gene editing). With regard to the CRISPR / Cas system, the liposome or LNP may include any form of Cas protein (e.g., protein, DNA or mRNA) and / or may include any form of guide RNA (e.g., DNA or RNA). In one example, the liposome or LNP includes a Cas protein in the form of mRNA (e.g., a modified RNA as described herein) and one or more guide RNAs in the form of RNA (e.g., a modified guide RNA as disclosed herein). As another example, the liposome or LNP may include a Cas protein in the form of a protein and one or more guide RNAs in the form of RNA. In one example, the guide RNA and the Cas protein are each introduced into the same LNP in the form of RNA by LNP-mediated delivery. As discussed in more detail elsewhere herein, one or more RNAs can be modified. For example, the guide RNA can be modified to include modifications of one or more stable ends at the 5' end and / or the 3' end. Such modifications may include, for example, one or more phosphorothioate linkages at the 5' end and / or the 3' end and / or one or more 2'-O-methyl modifications at the 5' end and / or the 3' end. As another example, Cas mRNA modifications may include substitution with pseudouridine (e.g., complete substitution with pseudouridine), 5' caps, and polyadenylation. As disclosed elsewhere herein, other modifications are also contemplated. Delivery by such methods can result in transient expression of Cas and / or transient presence of guide RNA, and biodegradable lipids improve clearance, improve tolerance, and reduce immunogenicity.

[0315] In some liposomes or LNPs, goods may include guide RNA or nucleic acid encoding guide RNA. In some liposomes or LNPs, goods may include mRNA encoding Cas nuclease (such as Cas9) and guide RNA or nucleic acid encoding guide RNA. In some liposomes or LNPs, goods may include nucleic acid constructs encoding target polypeptide (for example, antibody or Fab), as described elsewhere herein. In some liposomes or LNPs, goods may include mRNA encoding Cas nuclease (such as Cas9), guide RNA or nucleic acid encoding guide RNA, and nucleic acid constructs encoding target polypeptide (such as antibody or Fab). In some liposomes or LNPs, lipid components include amine lipids, such as biodegradable, ionizable lipids. In some cases, lipid components include biodegradable, ionizable lipids, cholesterol, DSPC and PEG-DMG. For example, Cas9 mRNA and gRNA can be delivered to cells and animals using a lipid formulation containing the ionizable lipid octadec-9,12-dienoic acid ((9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester (also known as (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester), cholesterol, DSPC and PEG2k-DMG.

[0316] In some liposomes or LNPs, the cargo may include Cas mRNA (e.g., Cas9 mRNA) and gRNA. Cas mRNA and gRNA may have different ratios. For example, the LNP formulation may include a ratio of Cas mRNA to gRNA nucleic acid of about 25:1 to about 1:25. Alternatively, the liposome or LNP formulation may include a ratio of Cas mRNA to gRNA nucleic acid of about 2:1 to about 1:2. In a specific example, the ratio of Cas mRNA to gRNA may be about 2:1.

[0317] In some liposomes or LNPs, goods may include nucleic acid constructs encoding target polypeptides (e.g., antibodies or Fabs) and gRNAs. Nucleic acid constructs encoding target polypeptides (e.g., antibodies or Fabs) and gRNAs may have different ratios. For example, liposomes or LNP formulations may include a ratio of nucleic acid constructs to gRNA nucleic acids from about 25:1 to about 1:25.

[0318] A specific example of a suitable LNP has a nitrogen-phosphorus (N / P) ratio of about 4.5 and contains a biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of about 45:44:9:2 (about 45: about 44: about 9: about 2). The biodegradable cationic lipid can be octadecane-9,12-dienoic acid (9Z, 12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z, 12Z)-octadecane-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester. See, e.g., Finn et al. (2018) Cell Rep. 22 (9): 2227-2235, which is incorporated herein by reference in its entirety for all purposes. The weight ratio of Cas9 mRNA to guide RNA may be about 1: 1 (about 1: 1). Another specific example of suitable LNPs includes Dlin-MC3-DMA (MC3), cholesterol, DSPC, and PEG-DMG in a molar ratio of about 50: 38.5: 10: 1.5 (about 50: about 38.5: about 10: about 1.5). The weight ratio of Cas9 mRNA to guide RNA is about 1: 2 (about 1: about 2). The weight ratio of Cas9 mRNA to guide RNA is about 1: 1 (about 1: about 1). The weight ratio of Cas9 mRNA to guide RNA is about 2: 1 (about 2: about 1).

[0319] Another specific example of a suitable LNP has a nitrogen-phosphorus (N / P) ratio of about 6 and contains a biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of about 50:38:9:3 (about 50: about 38: about 9: about 3). The biodegradable cationic lipid can be lipid A (octadec-9,12-dienoic acid (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl ester, also known as (9Z,12Z)-octadec-9,12-dienoic acid 3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)oxy)carbonyl)methyl)propyl ester). The weight ratio of Cas9 mRNA to guide RNA is about 1:2 (about 1: about 2). The weight ratio of Cas9 mRNA to guide RNA is about 1:1 (about 1: about 1). The weight ratio of Cas9 mRNA to guide RNA is about 2:1 (about 2: about 1).

[0320] Another specific example of a suitable LNP has a nitrogen to phosphorus (N / P) ratio of about 3 and comprises a cationic lipid, a structural lipid, cholesterol (e.g., cholesterol (sheep) (Avanti 700000)), and PEG2k-DMG (e.g., PEG-DMG 2000 (NOF America- GM-020 (DMG-PEG)). The structural lipid can be, for example, DSPC (e.g., DSPC (Avanti 850365)), SOPC, DOPC, or DOPE. The cationic / ionizable lipid can be, for example, Dlin-MC3-DMA (e.g., Dlin-MC3-DMA (Biofine International)). The weight ratio of Cas9 mRNA to guide RNA is about 1:2 (about 1: about 2). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1: about 1). The weight ratio of Cas9 mRNA to guide RNA can be about 2:1 (about 2:1).

[0321] Another specific example of suitable LNP comprises Dlin-MC3-DMA, DSPC, cholesterol and PEG lipid in a ratio of about 45:9:44:2 (about 45: about 9: about 44: about 2). Another specific example of suitable LNP comprises Dlin-MC3-DMA, DOPE, cholesterol and PEG lipid or PEG DMG in a ratio of about 50:10:39:1 (about 50: about 10: about 39: about 1). Another specific example of suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol and PEG lipid or PEG DMG in a ratio of about 55:10:32.5:2.5 (about 55: about 10: about 32.5: about 2.5). Another specific example of suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol and PEG2k-DMG in a ratio of about 50:10:38.5:1.5 (about 50: about 10: about 38.5: about 1.5). Another specific example of suitable LNP has Dlin-MC3-DMA, DSPC, cholesterol and PEG-DMG in a ratio of about 50:10:38.5:1.5 (about 50: about 10: about 38.5: about 1.5). The weight ratio of Cas9 mRNA to guide RNA can be about 1:2 (about 1: about 2). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1: about 1). The weight ratio of Cas9 mRNA to guide RNA can be about 2:1 (about 2: about 1).

[0322] Other examples of suitable LNPs can be found in, for example, WO 2019 / 067992, WO 2020 / 082042, US2020 / 0270617, WO 2020 / 082041, US2020 / 0268906, WO 2020 / 082046 (see, e.g., pages 85-86), and US2020 / 0289628, each of which is incorporated herein by reference in its entirety for all purposes.

[0323] Dynamic light scattering ("DLS") can be used to characterize the polydispersity index ("PDI") and size of liposomes and LNPs. In some embodiments, the PDI can be in the range of about 0.005 to about 0.75. In some embodiments, the PDI can be in the range of about 0.01 to about 0.5. In some embodiments, the PDI can be in the range of about 0.02 to about 0.4. In some embodiments, the PDI can be in the range of about 0.03 to about 0.35. In some embodiments, the PDI can be in the range of about 0.1 to about 0.35.

[0324] LNP disclosed herein may have a size of about 1nm to about 250nm. In some embodiments, LNP may have a size of about 10nm to about 200nm. In some embodiments, LNP may have a size of about 20nm to about 150nm. In some embodiments, LNP may have a size of about 50nm to about 150nm. In some embodiments, LNP may have a size of about 50nm to about 100nm. In some embodiments, LNP may have a size of about 50nm to about 120nm. In some embodiments, LNP may have a size of about 75nm to about 150nm. In some embodiments, LNP may have a size of about 30nm to about 200nm. In some embodiments, the average size (diameter) of fully formed nanoparticles is measured by dynamic light scattering on a Malvern Zetasizer (e.g., a nanoparticle sample may be diluted in phosphate buffered saline (PBS) so that the count rate is about 200-400kct, and the data may be expressed as a weighted average of intensity measurements).

[0325] In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency of about 50% to about 100%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency of about 50% to about 70%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency of about 70% to about 90%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency of about 90% to about 100%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency of about 75% to about 95%.

[0326] In addition to liposomes and LNPs, the first and / or second components of the systems described herein may also be in the form of other carriers for delivery of nucleic acids and / or protein molecules. Examples of other suitable carriers include, but are not limited to, lipid and liposome complexes, granules or polymer nanoparticles, inorganic nanoparticles, peptide carriers, nanoparticle mimics, nanotubes, conjugates, immunostimulatory complexes (ISCOMs), virus-like particles (VLPs), self-assembling proteins, or emulsion delivery systems such as cationic submicron oil-in-water emulsions.

[0327] Polymer microparticles or nanoparticles can also be used to encapsulate or adsorb polypeptides (e.g., Cas proteins) or polynucleotides (e.g., guide RNAs). The particles can be substantially nontoxic and biodegradable. The particles used to deliver polynucleotides (e.g., guide RNAs) can have an optimal size and zeta potential. For example, the diameter of the microparticles can be in the range of 0.02 μm to 8 μm. In the case where the composition has a population of micro- or nanoparticles having different diameters, at least 80%, 85%, 90%, or 95% of these particles ideally have a diameter in the range of 0.03 μm to 7 μm. The particles may also have a zeta potential between 40 mV and 100 mV to provide maximum adsorption of the particles to polynucleotides (e.g., guide RNAs).

[0328] Non-toxic and biodegradable polymers include, but are not limited to, poly(hydroxy acids), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanones, polyvalerolactones, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinylpyrrolidone or polyesteramides, one or more natural polymers such as polysaccharides, e.g., pullulan, alginates, inulin, and chitosan, and combinations thereof. In some embodiments, the particles are formed from poly(hydroxy acids), such as poly(lactide) (PLA), poly(g-glutamic acid) (g-PGA), poly(ethylene glycol) (PEG), polystyrene, copolymers of lactide and glycolide, such as poly(D,L-lactide-co-glycolide) (PLG), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers can include those having a lactide / glycolide molar ratio of, for example, 20:80 to 80:20, such as 25:75, 40:60, 45:55, 55:45, 60:40, 75:25. Useful PLG polymers include those having a molecular weight of, for example, between 5,000 Da and 200,000 Da, such as between 10,000 Da and 100,000 Da, between 20,000 Da and 70,000 Da, between 40,000 Da and 50,000 Da.

[0329] Polymer nanoparticles can also form hydrogel nanoparticles, hydrophilic three-dimensional polymer networks with advantageous properties including flexible mesh size, large surface area for multivalent conjugation, high water content, and high antigen loading capacity. Polymers such as poly (L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are suitable for forming hydrogel nanoparticles.

[0330] For example, inorganic nanoparticles can be calcium phosphate nanoparticles, silicon nanoparticles or gold nanoparticles. Inorganic nanoparticles usually have a rigid structure and comprise a core to which a shell or polypeptide or polynucleotide encapsulating a polypeptide or polynucleotide can be covalently attached. The core can comprise one or more atoms, such as gold (Au), silver (Ag), copper (Cu) atoms, Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd or Au / Ag / Cu / Pd or calcium phosphate (CaP).

[0331] Other molecules suitable for complexing with the polypeptides or polynucleotides of the invention include cationic molecules such as polyamidoamines, dendritic polylysine, polyethyleneimine or polypropyleneimine, polylysine, chitosan, DNA-gelatin polymers, DEAE dextran, dendrimers or polyethyleneimine (PEI).

[0332] In some embodiments, the polypeptide or polynucleotide of the present invention can be conjugated to nanoparticles. Nanoparticles that can be used to conjugate with antigens and / or antibodies of the present invention include, but are not limited to, chitosan shell nanoparticles, carbon nanotubes, PEGylated liposomes, poly (d, l-lactide-co-glycolide) / montmorillonite (PLGA / MMT) nanoparticles, poly (lactide-co-glycolide) (PLGA) nanoparticles, poly (malic acid)-based nanoparticles, and other inorganic nanoparticles (e.g., nanoparticles made of magnesium-aluminum layered double hydroxide and disuccinimidyl carbonate (DSC) and TiO2 nanoparticles).

[0333] Oil-in-water emulsions can also be used for delivering polypeptides or polynucleotides (e.g., mRNA) to a subject. Examples of oils that can be used to prepare emulsions include animal oils (e.g., fish oils) or vegetable oils (e.g., nuts, cereals, and seeds). The oil can be biodegradable and biocompatible. Exemplary oils include, but are not limited to, tocopherol and squalene, shark liver oil as branched unsaturated terpenoids, and combinations thereof. Terpenoids are branched oils biochemically synthesized in 5 carbon isoprene units.

[0334] The aqueous component of the emulsion can be water or water to which additional components have been added. For example, it can include salts that form buffers, such as citrate or phosphate, such as sodium salts. Exemplary buffers include borate buffers, citrate buffers, histidine buffers, phosphate buffers, Tris buffers, or succinate buffers.

[0335] In some embodiments, the oil-in-water emulsion includes one or more cationic molecules. For example, a cationic lipid may be included in the emulsion to provide a positively charged droplet surface to which a negatively charged polynucleotide (e.g., mRNA) may be attached. Exemplary cationic lipids include, but are not limited to: 1,2-dioleoyloxy-3-(trimethylammonium) propane (DOTAP), 1,2-dimyristoyl-3-trimethyl-ammonium propane (DMTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC cholesterol), dimethyldioctadecyl ammonium (DDA, e.g., bromide), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearyl trimethylammonium propane (DSTAP).Other useful cationic lipids include benzalkonium chloride (BAK), benzethonium chloride, cholesterol hemisuccinate choline ester, lipopolyamines (e.g., dioctadecylamido glycyl spermine (DOGS), dipalmitoylphosphatidylethanolamido spermine (DPPES)), ceramides, cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), cationic derivatives of cholesterol (e.g., cholesteryl-3β-oxysuccinylethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinylethylene-dimethylamine, cholesteryl-3β-carboxamidoethylenetrimethylammonium salt, and cholesteryl-3β-carboxamidoethylenedimethylamine ), N,N',N'--polyoxyethylene (10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium chloride, benzyltrimethylmethylammonium, hexadecyldimethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzalacetammonium, decamethylquaternary ammonium chloride, methyl mixed trialkylammonium chloride, methyl trioctyl ammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy)ethyl] -phenylmethanediamine chloride (DEBDA), (4'-trimethylammonium) cholesteryl butyrate, N-alkyl pyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), N-alkyl piperidinium salts, dicationic bolaform electrolytes (C12Me6; C12BU6), dialkylglycylphosphocholine, lysolecithin, L-α-dioleoylphosphatidylethanolamine, lipopoly-L (or D)-lysine (LPLL, LPDL), poly (L (or D)-lysine conjugated to N-glutarylphosphatidylethanolamine, dialkyldimethylammonium salts, [1-(2,3-dioleoyloxy)-propyl]-N,N,N-trioleylphosphatidylethanolamine Methylammonium chloride, trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (the acyl group may be dimyristoyl, dipalmitoyl, distearoyl or dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (the acyl group may be dimyristoyl, dipalmitoyl, distearoyl or dioleoyl), 1,2-dioleoyl-3-(4'-trimethyl-ammonio)butyryl-sn-glycerol, 1,2-dioleoyl 3-succinyl-sn-glycerol choline ester, didodecyl glutamate (CGluPhCnN) having a pendant amino group, and ditetradecyl glutamate (C14GluCnN+) having a pendant amino group.

[0336] In some embodiments, in addition to oil and cationic lipids, the emulsion may also include nonionic surfactants and / or zwitterionic surfactants. Examples of useful surfactants include, but are not limited to: polyoxyethylene sorbitan ester surfactants, such as polysorbate 20 and polysorbate 80; copolymers of ethylene oxide, propylene oxide and / or butylene oxide, linear block copolymers; phospholipids, such as phosphatidylcholine; polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol and oleyl alcohol; polyoxyethylene-9-lauryl ether; octoxynol; (octylphenoxy) polyethoxyethanol; and sorbitan esters.

[0337] In some embodiments, the polynucleotides described herein can be incorporated into polynucleotide complexes, such as, but not limited to, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconductor / metal nanoparticles), gels and hydrogels, polynucleotide complexes with cations and anions, microparticles, and any combination thereof.

[0338] In some embodiments, polynucleotide disclosed herein can be configured to self-assembled nanoparticles. As non-limiting examples, polynucleotides can be used to prepare nanoparticles, which can be used in the delivery system of polynucleotides (see, for example, PCT discloses No. WO2012 / 125987). In some embodiments, polynucleotide self-assembled nanoparticles can include the core and polymer shells of polynucleotides disclosed herein. The polymer shell can be any polymer described herein, and is known in the art. In another embodiment, the polymer shell can be used to protect the polynucleotides in the core.

[0339] In some embodiments, the self-assembled nanoparticles can be microsponges formed by long polymers of polynucleotide hairpins, which form crystalline "wrinkled" sheets before self-assembling into microsponges. These microsponges are densely packed sponge-like particles that can be used as efficient carriers and can deliver cargo to cells. The diameter of the microsponges can be 1 μm to 300 nm. The microsponges can be compounded with other agents known in the art to form larger microsponges. As a non-limiting example, microsponges can be compounded to form an outer layer to promote cellular uptake, such as polycationic polyethyleneimine (PEI). This complex can form particles with a diameter of 250 nm, which can remain stable at high temperatures (150° C.) (Grabow and Jaegar, Nature Materials 2012, 11: 269-269). In addition, these microsponges may be able to show an abnormal degree of protection against ribonuclease degradation. In one embodiment, polymer-based self-assembled nanoparticles, such as but not limited to microsponges, can be fully programmable nanoparticles. The geometry, size, and stoichiometry of the nanoparticles can be precisely controlled to produce optimal nanoparticles for delivery of cargo such as, but not limited to, polynucleotides.

[0340] In some embodiments, the polynucleotides disclosed herein can be formulated in inorganic nanoparticles (see U.S. Pat. No. 8,257,745). Inorganic nanoparticles may include, but are not limited to, water-swellable clay materials. As a non-limiting example, inorganic nanoparticles may include synthetic layered clays made from simple silicates (see U.S. Pat. Nos. 5,585,108 and 8,257,745).

[0341] In some embodiments, the polynucleotides disclosed herein can be formulated in water-dispersible nanoparticles comprising semiconductor or metallic materials (U.S. Patent Application Publication No. 2012 / 0228565; incorporated herein by reference in its entirety) or formed in magnetic nanoparticles (U.S. Patent Application Publication Nos. 2012 / 0265001 and 2012 / 0283503). The water-dispersible nanoparticles can be hydrophobic nanoparticles or hydrophilic nanoparticles.

[0342] In some embodiments, the polynucleotides disclosed herein can be encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. A hydrogel is a network of hydrophilic polymer chains, sometimes found as a colloidal gel with water as the dispersion medium. Hydrogels are highly hygroscopic (they can contain more than 99% water) natural or synthetic polymers. Hydrogels also have a degree of flexibility, very similar to natural tissues, because they contain large amounts of water. The hydrogels described herein can be used to encapsulate biocompatible, biodegradable and / or porous lipid nanoparticles.

[0343] As a non-limiting example, the hydrogel can be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel can be programmed to release one or more polynucleotides using polynucleotide hybridization. (Battig et al., J. Am. Chem. Society. 2012 134: 12410-12413). In some embodiments, the polynucleotides can be encapsulated in lipid nanoparticles, which can then be encapsulated in the hydrogel.

[0344] In some embodiments, the polynucleotides disclosed herein can be encapsulated in fibrin gel, fibrin hydrogel or fibrin glue. In another embodiment, polynucleotides can be prepared in lipid nanoparticles or rapidly eliminated lipid nanoparticles before being encapsulated in fibrin gel, fibrin hydrogel or fibrin glue. In another embodiment, polynucleotides can be formulated into liposome complexes before being encapsulated in fibrin gel, hydrogel or fibrin glue. Fibrin gel, hydrogel and glue comprise two components, fibrinogen solution and calcium-rich thrombin solution (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010.148:49-55; Kidd et al. Journal of Controlled Release 2012.157:80-85). The component concentrations of fibrin gel, hydrogel and / or glue can be changed to change the properties, network mesh size and / or degradation characteristics of gel, hydrogel and / or glue, such as but not limited to changing the release characteristics of fibrin gel, hydrogel and / or glue. (See, e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148:49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128). This feature may be advantageous when used to deliver the polynucleotides disclosed herein. (See, e.g., Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128).

[0345] In some embodiments, the polynucleotides disclosed herein may include cations or anions. In one embodiment, the formulation comprises a metal cation, such as but not limited to Zn 2+ , Ca 2+ , Cu2+ Mg 2+ As a non-limiting example, a formulation may include a polymer and a polynucleotide complexed with a metal cation (see US Pat. Nos. 6,265,389 and 6,555,525).

[0346] In some embodiments, the polynucleotides may be formulated in nanoparticles and / or microparticles. These nanoparticles and / or microparticles may be molded into any size, shape, and chemical properties. For example, the nanoparticles and / or microparticles may be prepared using LIQUIDA TECHNOLOGIES (Morrisville, NC). The invention is manufactured by using the technology (see, for example, International Publication No. WO2007 / 024323).

[0347] In some embodiments, the polynucleotides disclosed herein can be formulated in NanoJackets and NanoLiposomes by Keystone Nano (State College, Pa.). NanoJackets are made of compounds naturally present in the human body, including calcium and phosphate, and may also include a small amount of silicate. The size range of NanoJackets can be 5nm to 50nm, and can be used to deliver hydrophilic and hydrophobic compounds, such as but not limited to polynucleotides, primary constructs and / or polynucleotides. Nanoliposomes are made of lipids (such as but not limited to naturally occurring lipids in the body). The size of nanoliposomes can be in the range of 60nm to 80nm, and can be used to deliver hydrophilic and hydrophobic compounds, such as but not limited to polynucleotides, primary constructs and / or polynucleotides. On the one hand, the polynucleotides disclosed herein are formulated in nanoliposomes (such as but not limited to ceramide nanoliposomes).

[0348] Gene editing system

[0349] In one aspect, the systems or compositions described herein are capable of introducing a gene editing system (e.g., a CRISPR / Cas system) into a target cell (e.g., a B cell or HSC). The system comprises a gene editing molecule or a polynucleotide molecule containing a sequence encoding the gene editing molecule in one component.

[0350] In some embodiments, at least one component of the systems described herein may further comprise a guide RNA (gRNA) molecule or a sequence encoding the gRNA molecule.

[0351] In one embodiment, the system or composition of the present invention comprises a recombinant viral particle containing a gene editing molecule and a second recombinant viral particle containing a guide RNA (gRNA) and a sequence encoding an antibody or a fragment thereof. In certain embodiments, the gene editing molecule is a functional fragment or derivative thereof.

[0352] "Gene editing molecules" are molecules (e.g., proteins or polynucleotide molecules encoding such proteins (e.g., mRNA)) used to modify a target genomic locus (i.e., a target) in a cell (e.g., a eukaryotic cell, a mammalian cell, a human cell, or a non-human cell). Such modifications include, but are not limited to, the destruction, deletion, repair, mutation, addition, change, or modification of the gene sequence at the target locus in the gene. Examples of gene editing molecules include, but are not limited to, endonucleases. Endonucleases are enzymes that cut phosphodiester bonds within polynucleotide chains, but they only break internal phosphodiester bonds. Examples of gene editing endonucleases that can be used in the compositions and methods of the present invention include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, restriction endonucleases, recombinases, and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) proteins.

[0353] A. Cas fusion molecules

[0354] The methods and compositions disclosed herein can utilize clustered regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems or components of such systems to modify the genome within a cell. The CRISPR / Cas system includes transcripts and other elements that participate in Cas gene expression or guide Cas gene activity. The CRISPR / Cas system can be, for example, a type I, type II, or type III system. Alternatively, the CRISPR / Cas system can be a type V system (e.g., subtype VA or subtype VB). The methods and compositions disclosed herein can use the CRISPR / Cas system by using a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-directed cutting of nucleic acids.

[0355] The CRISPR / Cas systems used in the compositions and methods disclosed herein can be non-naturally occurring. "Non-naturally occurring" systems include anything that refers to something involving human hand, such as being altered or mutated from their naturally occurring state, at least substantially free of at least one other component with which they are naturally associated in nature, or one or more components of the system associated with at least one other component with which they are non-naturally associated. For example, some CRISPR / Cas systems employ non-naturally occurring CRISPR complexes that include gRNA and Cas proteins that do not naturally occur together, employ non-naturally occurring Cas proteins, or employ non-naturally occurring gRNAs.

[0356] (i) Cas molecules

[0357] "Cas molecules", "Cas proteins" or "Cas nucleases" useful in the compositions and methods of the present invention generally include at least one RNA recognition or binding domain that can interact with a guide RNA (gRNA, described in more detail below). Cas proteins may also include nuclease domains (e.g., DNA enzyme or RNA enzyme domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. The nuclease domain has catalytic activity for nucleic acid cleavage, which includes the breaking of covalent bonds of nucleic acid molecules. The cleavage can produce blunt ends or staggered ends, and it can be single-stranded or double-stranded. For example, wild-type Cas9 proteins generally produce blunt-ended cleavage products. Alternatively, wild-type Cpf1 proteins (e.g., FnCpf1) can produce cleavage products with 5 nucleotide 5' overhangs, and the cleavage occurs after the 18th base pair of the PAM sequence on the non-target strand and after the 23rd base on the target strand. The Cas protein can have full cleavage activity to generate double-strand breaks (e.g., double-strand breaks with blunt ends) at the target genomic locus, or it can be a nickase that generates single-strand breaks at the target genomic locus.

[0358] Examples of Cas proteins that can be used in the compositions and methods of the present invention include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csb1, Csb2, Csb3, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified forms thereof.

[0359] An exemplary Cas protein is a Cas9 protein or a Cas9 protein derived from a type II CRISPR / Cas system. The Cas9 protein is from a type II CRISPR / Cas system and generally shares four key motifs with a conserved structure. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is a HNH motif. Exemplary Cas9 proteins are from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiral, Streptomyces viridochromogene, Streptomyces viridochromogene, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum), Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivoran, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudi, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilu, Pelotomaculumthermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum), a species of the genus Marinobacter sp., Nitrosococcus halophilu, Nitrosococcus watsoni, Pseudoalteromonas halophilus, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, a species of the genus Nostoc sp., Arthrospira maxima, Arthrospira platensis, a species of the genus Arthrospira sp., a species of the genus Lyngbya sp., Microcoleus chthonoplastes, a species of the genus Oscillatoria sp.), Petrotogamobilis, Thermosipho africanus, Acaryochloris marina, Neisseria meningitidis, or Campylobacter jejuni. Other examples of Cas9 family members are described in WO 2014 / 131833 (incorporated herein in its entirety by reference for all purposes). Cas9 (SpCas9) from Streptococcus pyogenes (assigned SwissProt accession number Q99ZW2) is an exemplary cas9 protein. Cas9 (SaCas9) from Staphylococcus aureus (assigned UniProt accession number J7RUA5) is another exemplary Cas9 protein. Cas9 (assigned UniProt accession number Q0P897) from Campylobacter jejuni (CjCas9) is another exemplary Cas9 protein. See, e.g., Kim et al. (2017) Nat. Comm. 8:14500, which is incorporated herein by reference in its entirety for all purposes. SaCas9 is smaller than SpCas9, and CjCas9 is smaller than both SaCas9 and SpCas9. .

[0360] Another example of a Cas protein is Cpf1 (CRISPR from Prevotella and Francisella 1) protein. Cpf1 is a large protein (about 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9 and a counterpart to the characteristic arginine-rich cluster of Cas9. However, Cpf1 lacks the HNH nuclease domain present in the Cas9 protein, and the RuvC-like domain is continuous in the Cpf1 sequence, in contrast to Cas9, which contains a long insert including the HNH domain. See, e.g., Zetsche et al. (2015) Cell 163(3):759-771, incorporated herein by reference in its entirety for all purposes. Exemplary Cpf1 proteins are from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020 MA2020), Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae.Cpf1 from Francisella novicida U112 (FnCpf1; assigned UniProt accession number A0Q7Q2) is an exemplary Cpf1 protein.

[0361] The Cas protein can be a wild-type protein (i.e., a naturally occurring protein), a modified Cas protein (i.e., a Cas protein variant), or a fragment of a wild-type or modified Cas protein. In terms of the catalytic activity of a wild-type or modified Cas protein, the Cas protein can also be an active variant or fragment. In terms of catalytic activity, the active variant or fragment may comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a wild-type or modified Cas protein or a portion thereof, wherein the active variant retains the ability to cut at the desired cleavage site, and thus retains nick induction or double-strand break induction activity. The determination of nick induction or double-strand break induction activity is known, and the overall activity and specificity of the Cas protein on a DNA substrate containing a cleavage site are generally measured.

[0362] An example of a modified Cas protein is a modified SpCas9-HF1 protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9 with changes designed to reduce nonspecific DNA contacts (N497A / R661A / Q695A / Q926A). See, e.g., Kleinstiver et al. (2016) Nature 529(7587):490-495, which is incorporated herein by reference in its entirety for all purposes. Another example of a modified Cas protein is a modified eSpCas9 variant (K848A / K1003A / R1060A) designed to reduce off-target effects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88, which is incorporated herein by reference in its entirety for all purposes. Other SpCas9 variants include K855A and K810A / K1003A / R1060A.

[0363] The Cas protein can be modified to increase or decrease one or more of nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. The Cas protein can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or the Cas protein can be truncated to remove domains that are not essential to the function of the protein or to optimize (e.g., enhance or reduce) the activity of the Cas protein.

[0364] The Cas protein may include at least one nuclease domain, such as a DNA enzyme domain. For example, the wild-type Cpf1 protein generally includes a RuvC-like domain, which cuts two strands of the target DNA, possibly in a dimer configuration. The Cas protein may also include at least two nuclease domains, such as a DNA enzyme domain. For example, the wild-type Cas9 protein generally includes a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC and HNH domains can each cut different strands of double-stranded DNA, thereby producing double-strand breaks in the DNA. See, for example, Jinek et al. (2012) Science 337: 816-821, which is incorporated herein by reference in its entirety for all purposes.

[0365] One or more nuclease domains may be deleted or mutated so that they are no longer functional or have reduced nuclease activity. For example, if one of the nuclease domains in the Cas9 protein is deleted or mutated, the resulting Cas9 protein may be referred to as a nickase, and may produce single-strand breaks at the guide RNA recognition sequence in double-stranded DNA, but may not produce double-strand breaks (i.e., it may cut complementary or non-complementary strands, but may not cut both). If both nuclease domains are deleted or mutated, the ability of the resulting Cas protein (e.g., Cas9) to cut both strands of double-stranded DNA will be reduced (e.g., nuclease-ineffective or nuclease-inactivated Cas protein, or catalytically dead Cas protein (dCas)). An example of a mutation that converts Cas9 to a nickase is a D10A (aspartic acid at position 10 of Cas9 becomes alanine) mutation in the RuvC domain of Cas9 from Streptococcus pyogenes. Similarly, H939A (histidine at amino acid position 839 becomes alanine), H840A (histidine at amino acid position 840 becomes alanine) or N863A (asparagine at amino acid position N863 becomes alanine) in the HNH domain of Cas9 from Streptococcus pyogenes can convert Cas9 into a nickase. Other examples of mutations that convert Cas9 into a nickase include corresponding mutations to Cas9 from Streptococcus thermophilus. See, for example, Sapranauskas et al. (2011) Nucleic Acids Research 39:9275-9282 and WO 2013 / 141680, each of which is incorporated herein by reference in its entirety for all purposes. Such mutations can be produced using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or full gene synthesis. Examples of other mutations producing nickases can be found in, for example, WO 2013 / 176772 and WO2013 / 142578, each of which is incorporated herein by reference in its entirety for all purposes. If all nuclease domains in the Cas protein are deleted or mutated (for example, both nuclease domains in the Cas9 protein are deleted or mutated), the ability of the resulting Cas protein (for example, Cas9) to cut two chains of double-stranded DNA will be reduced (for example, nuclease is invalid or nuclease inactivated Cas protein). A specific example is a D10A / H840A Streptococcus pyogenes Cas9 double mutant or a corresponding double mutant from another species of Cas9 when optimally compared with Streptococcus pyogenes Cas9. Another specific example is a D10A / N863A Streptococcus pyogenes Cas9 double mutant or a corresponding double mutant from another species of Cas9 when optimally compared with Streptococcus pyogenes Cas9.

[0366] Examples of inactivating mutations in the catalytic domain of the Staphylococcus aureus Cas9 protein are also known. For example, the Staphylococcus aureus Cas9 enzyme (SaCas9) may include a substitution at position N580 (e.g., N580A substitution) and a substitution at position D10 (e.g., D10A substitution) to produce a nuclease-inactive Cas protein. See, for example, WO 2016 / 106236, which is incorporated herein by reference in its entirety for all purposes.

[0367] Examples of inactivating mutations in the catalytic domain of Cpf1 proteins are also known. With reference to Cpf1 proteins from Francisella novicida U112 (FnCpf1), Acidaminococcus species BV3L6 (AsCpf1), Lachnospiraceae bacteria ND2006 (LbCpf1) and Moraxella bovis 237 (MbCpf1 Cpf1), such mutations may include mutations at positions 908, 993 or 1263 of AsCpf1 or corresponding positions in Cpf1 orthologs, or positions 832, 925, 947 or 1180 of LbCpf1 or corresponding positions in Cpf1 orthologs. Such mutations may include, for example, one or more of the D908A, E993A, and D1263A mutations of AsCpf1 or corresponding mutations in Cpf1 orthologs or the D832A, E925A, D947A, and D1180A mutations of LbCpf1 or corresponding mutations in Cpf1 orthologs. See, e.g., US2016 / 0208243, which is incorporated herein by reference in its entirety for all purposes.

[0368] The Cas fusion protein can also be tethered to the labeled nucleic acid. Such tethering (i.e., physical connection) can be achieved through covalent interactions or non-covalent interactions, and the tethering can be direct (e.g., by direct fusion or chemical conjugation, which can be achieved by modification of cysteine ​​or lysine residues on the protein or modification of inteins), or can be achieved through one or more intervening linkers or adapter molecules such as streptavidin or aptamers. See, e.g., Pierce et al. (2005) Mini Rev. Med. Chem. 5(1):41-55; Duckworth et al. (2007) Angew. Chem. Int. Ed. Engl. 46(46):8819-8822; Schaeffer and Dixon (2009) Australian J. Chem. 62(10):1328-1332; Goodman et al. (2009) Chembiochem. 10(9):1551-1557; and Khatwani et al. (2012) Bioorg. Med. Chem. 20(14):4532-4539, each of which is incorporated herein by reference in its entirety for all purposes. Non-covalent strategies for synthesizing protein-nucleic acid conjugates include biotin-streptavidin and nickel-histidine methods. Covalent protein-nucleic acid conjugates can be synthesized by connecting appropriately functionalized nucleic acids and proteins using a variety of chemical methods. Some of these chemical methods involve direct attachment of oligonucleotides to amino acid residues (e.g., lysine amines or cysteine ​​thiols) on the surface of proteins, while other more complex schemes require post-translational modifications of proteins or involve catalytic or reactive protein domains. Methods for covalently attaching proteins to nucleic acids may include, for example, chemical crosslinking of oligonucleotides to protein lysine or cysteine ​​residues, expressed protein-connections, chemoenzymatic methods, and the use of photoaptamers. The labeled nucleic acid can be tethered to the C-terminus, N-terminus, or internal region of the Cas protein. Preferably, the labeled nucleic acid can be tethered to the C-terminus or N-terminus of the Cas protein. Similarly, the Cas protein can be tethered to the 5' end, 3' end, or internal region of the labeled nucleic acid. That is, the labeled nucleic acid can be tethered in any direction and polarity. Preferably, the Cas protein is tethered to the 5' end or 3' end of the labeled nucleic acid.

[0369] In some embodiments, the nucleic acid encoding the Cas protein of the present invention or its functional fragment or derivative can be codon optimized so as to be efficiently translated into protein in a specific cell or organism. For example, the nucleic acid encoding the Cas protein or its functional fragment or derivative can be modified to replace codons with higher usage frequencies in bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells or any other host (e.g., packaging) and / or target cells of interest. When a fusion RNA encoding a Cas protein or its functional fragment or derivative is introduced into a cell, the Cas protein or its functional fragment or derivative can be expressed transiently or conditionally in the cell.

[0370] In certain embodiments, the Cas molecule is a Cas9 molecule, or a functional fragment or derivative thereof. In certain embodiments, Cas9 can be a wild-type Cas9, a Cas9 nicking enzyme, a dead Cas9 (dCas9), a split Cas9, and a Cas9 fusion protein. In certain embodiments, Cas9 is Streptococcus pyogenes or Staphylococcus aureus Cas9. In certain embodiments, the sequence of Cas9mRNA is codon optimized for expression in eukaryotic cells.

[0371] Optionally, Cas mRNA can be codon optimized so that it can be efficiently translated into Cas protein or its functional fragment or derivative in a specific cell or organism. For example, compared with a naturally occurring polynucleotide sequence, the nucleic acid sequence encoding the Cas protein or its functional fragment or derivative can be modified to replace codons with higher frequencies in bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells or any other host (e.g., packaging) and / or target cells of interest.

[0372] In certain embodiments, the Cas protein is Cas9 or a functional fragment or derivative thereof. In certain embodiments, Cas9 is selected from the group consisting of wild-type Cas9, Cas9 nicking enzyme, dead Cas9 (dCas9), split Cas9, inducible Cas9 and Cas9 fusion protein. In certain embodiments, Cas9 is Streptococcus pyogenes or Staphylococcus aureus Cas9. In certain embodiments, the sequence of Cas9 mRNA is codon optimized for expression in eukaryotic cells.

[0373] Cas protein or its functional fragment or derivative can also be operably connected to other heterologous polypeptides as fusion protein.For example, Cas protein or its functional fragment or derivative can be fused with cleavage domain, epigenetic modification domain, transcriptional activation domain or transcriptional transcription repressor domain.See WO 2014 / 089290, which is incorporated herein by reference in its entirety for all purposes.Examples of transcriptional activation domains include herpes simplex virus VP16 activation domain, VP64 (which is a tetramer derivative of VP16), NFκB p65 activation domain, p53 activation domain 1 and 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain and NFAT (nuclear factor of activated T cells) activation domain. Other examples include activation domains from Oct1, Oct-2A, SP1, AP-2, CTF1, P300, CBP, PCAF, SRC1, PvALF, ERF-2, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7, ARF-8, CPRF1, CPRF4, MYC-RP / GP, TRAB1PC4, and HSF1. See, for example, US2016 / 0237456, EP3045537, and WO 2011 / 145121, each of which is incorporated by reference in its entirety for all purposes. In some cases, a transcriptional activation system comprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1 can be used. The guide RNA in such a system can be designed with an aptamer sequence attached to the sgRNA tetraloop and stem-loop 2 designed to bind to the dimerized MS2 phage coat protein. See, e.g., Konermann et al. (2015) Nature 517(7536):583-588, which is incorporated herein by reference in its entirety for all purposes. Examples of transcriptional repressor domains include inducible cAMP early repressor (ICER) domains, Kruppel-associated box A (KRAB-A) repressor domains, YY1 glycine-rich repressor domains, Sp1-like repressors, E (spl) repressors, IκB repressors, and MeCP2. Other examples include transcriptional repressor domains from A / B, KOX, TGF-β-inducible early gene (TIEG), v-erbA, SID, SID4X, MBD2, MBD3, DNMT1, DNMG3A, DNMT3B, Rb, ROM2, see, for example, EP3045537 and WO 2011 / 145121, each of which is incorporated by reference in its entirety for all purposes. Cas protein can also be fused to heterologous polypeptides to provide enhanced or reduced stability. Fusion domains or heterologous polypeptides can be located at the N-terminus, C-terminus or internal of Cas protein or its functional fragment or derivative.

[0374] As an example, the Cas protein or its functional fragment or derivative can be fused to one or more heterologous polypeptides providing subcellular localization. Such heterologous polypeptides can include, for example, one or more nuclear localization signals (NLS) (such as SV40 NLS and / or α-importin NLS targeting the nucleus), mitochondrial localization signals targeting mitochondria, endoplasmic reticulum retention signals, etc. See, for example, Lange et al. (2007) J. Biol. Chem. 282: 5101-5105, which is incorporated herein by reference in its entirety for all purposes. Such subcellular localization signals may be located at the N-terminus, C-terminus, or anywhere at the Cas protein or its functional fragment or derivative. The NLS may comprise a stretch of basic amino acids and may be a single-part sequence or a two-part sequence. Optionally, the Cas protein or its functional fragment or derivative comprises two or more NLSs, including an NLS at the N-terminus (e.g., an importin NLS) and / or an NLS at the C-terminus (e.g., SV40 NLS).

[0375] The Cas protein or its functional fragment or derivative can also be operably linked to a heterologous polypeptide for tracking or purification, such as a fluorescent protein, a purification tag or an epitope tag. Examples of fluorescent proteins include green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, lemon yellow, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed monomer , HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) and any other suitable fluorescent protein. Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly (NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP) and calmodulin.

[0376] Cas fusion proteins can be produced using conventional molecular biology techniques, such as those described above or in He et al. (supra). Alternatively, Cas fusion proteins can be prepared by various other methods.

[0377] In certain embodiments, the nucleic acid encoding the Cas fusion protein comprises a regulatory element, including, for example, a promoter, an enhancer, or a transcriptional repressor binding element. Exemplary expression control sequences are known in the art and are described in, for example, Goeddel, (1990) Gene Expression Technology: Methods in Enzymology, Vol. 185, AAcademic Press, San Diego, Calif. (incorporated herein in its entirety by reference for all purposes).

[0378] B. Transcription activator-like effector nucleases, zinc finger nucleases, meganucleases, and restriction endonucleases

[0379] In certain embodiments, the gene editing molecule can be a zinc finger nuclease (ZFns), a transcription activator-like effector nuclease (TALEN), a meganuclease and / or a restriction endonuclease. The fusion RNA and fusion protein molecules using these gene editing molecules or their functional fragments or derivatives for the compositions and methods of the present invention can be prepared in the same manner and structure as disclosed above for Cas molecules or their functional fragments or derivatives.

[0380] Transcription activator-like effector nucleases (TALENs) are restriction endonucleases that can be engineered to target sequences that cut DNA. They are made by fusing the TAL effector DNA binding domain with a DNA cleavage domain (a nuclease that cuts DNA chains). TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genomes of prokaryotes or eukaryotes. TAL effector nucleases are produced by fusing natural or engineered transcription activator-like (TAL) effectors or their functional parts with the catalytic domain of a nuclease (e.g., FokI). The unique modular TAL effector DNA binding domain allows the design of proteins with potential for any given DNA recognition specificity. Therefore, the DNA binding domain of the TAL effector nuclease can be engineered to recognize specific DNA target sites, thereby being used to produce double-strand breaks on the desired target sequence. See WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. doi: 10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29:143-148; all of which are incorporated herein by reference in their entirety for all purposes.

[0381] Examples of suitable TAL nucleases and methods of making suitable TAL nucleases are disclosed, for example, in U.S. Patent Application Nos. 2011 / 0239315A1, 2011 / 0269234A1, 2011 / 0145940A1, 2003 / 0232410A1, 2005 / 0208489A1, 2005 / 0026157A1, 2005 / 0064474A1, 2006 / 0188987A1, and 2006 / 0063231A1 (each of which is hereby incorporated by reference in its entirety for all purposes). In various embodiments, a TAL effector nuclease is engineered that cuts in or near a target nucleic acid sequence, for example, in a genomic locus of interest, wherein the target nucleic acid sequence is at or near a sequence to be modified by a targeting vector. TAL nucleases suitable for use in the various methods and compositions provided herein include those specifically designed to bind at or near a target nucleic acid sequence to be modified by a targeting vector.

[0382] In one embodiment, each monomer of TALEN comprises 12-25 TAL repeats, wherein each TAL repeat binds to a 1bp subsite. In certain embodiments, the gene editing molecule is a chimeric protein comprising a TAL repeat-based DNA binding domain operably linked to an independent nuclease. In certain embodiments, the independent nuclease is a FokI nuclease. In one embodiment, the gene editing molecule comprises a first TAL repeat-based DNA binding domain and a second TAL repeat-based DNA binding domain, wherein each of the first and second TAL repeat-based DNA binding domains is operably linked to the FokI nuclease, wherein the first and second TAL repeat-based DNA binding domains recognize two adjacent target DNA sequences separated by about 6bp to about 40bp cleavage sites in each chain of the target DNA sequence, and wherein the FokI nuclease dimerizes and produces a double-stranded break at the target sequence.

[0383] In certain embodiments, the gene editing molecule comprises a first TAL repeat sequence-based DNA binding domain and a second TAL repeat sequence-based DNA binding domain, wherein each of the first and second TAL repeat sequence-based DNA binding domains is operably linked to a FokI nuclease, wherein the first and second TAL repeat sequence-based DNA binding domains recognize two adjacent target DNA sequences separated by a 5bp or 6bp cleavage site in each strand of the target DNA sequence, and wherein the FokI nuclease dimerizes and produces a double-stranded break.

[0384] The gene editing molecules used in the various methods and compositions disclosed herein may also include zinc finger nucleases (ZFNs). Zinc finger nucleases (ZFNs) are a class of engineered DNA binding proteins that assist in targeted editing of the genome by generating double-strand breaks (DSBs) in the DNA at the target location. ZFNs contain two functional domains: i) a DNA binding domain, which contains a series of two-finger module chains (each recognizing a unique hexamer (6bp) sequence of DNA-two-finger modules stitched together to form zinc finger proteins, each with a specificity of ≥24bp) and ii) a DNA cleavage domain, which contains the nuclease domain of FokⅠ. When the DNA binding and cleavage domains are fused together, a pair of highly specific "genomic scissors" is produced.

[0385] In certain embodiments, each monomer of the ZFN comprises 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain binds to a 3bp subsite. In other embodiments, the ZFN is a chimeric protein comprising a zinc finger-based DNA binding domain operably connected to an independent nuclease. In certain embodiments, the independent endonuclease is a FokI endonuclease. In certain embodiments, the gene editing molecule comprises a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably connected to a FokI nuclease, wherein the first and second ZFNs recognize two adjacent target DNA sequences separated by a cleavage site of about 6bp to about 40bp or a cleavage site of about 5bp to about 6bp in each chain of the target DNA sequence, and wherein the FokI nuclease dimerizes and produces a double-strand break. See, e.g., US20060246567, US20080182332, US20020081614, US20030021776, WO / 2002 / 057308A2, US20130123484, US20100291048, and WO / 2011 / 017293A2, each of which is herein incorporated by reference in its entirety for all purposes.

[0386] In certain embodiments of the compositions and methods provided herein, the gene editing molecule comprises: (a) a chimeric protein comprising a zinc finger-based DNA binding domain fused to a FokI endonuclease; or (b) a chimeric protein comprising a transcription activator-like effector nuclease (TALEN) fused to a FokI endonuclease.

[0387] In another embodiment, the gene editing molecule is a meganuclease. Based on the conserved sequence motifs, meganucleases are divided into four families, namely LAGLIDADG, GIY-YIG, HNH and His-Cys box families. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. HE enzymes are known for their long recognition sites and for allowing some sequence polymorphisms in their DNA substrates. Meganuclease domains, structures and functions are known, see, e.g., Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55:1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764. In some examples, naturally occurring variants and / or engineered derivative meganucleases are used. Methods for modifying kinetics, cofactor interactions, expression, optimal conditions and / or recognition site specificity and screening for activity are known, see, e.g., Epinat et al., (2003) Nucleic Acids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154; WO2005105989; WO2003078619; WO2006097854; WO2006097853; WO2006097784 and WO2004031346.

[0388] Any meganuclease can be used in this article, including but not limited to I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-Aural, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII or any active variant or fragment thereof.

[0389] In one embodiment, the meganuclease recognizes a double-stranded DNA sequence of 12 to 40 base pairs. In one embodiment, the meganuclease recognizes a fully matched target sequence in the genome. In one embodiment, the meganuclease is a homing nuclease. In one embodiment, the homing nuclease is a LAGLIDADG family of homing nucleases. In one embodiment, the LAGLIDADG family of homing nucleases is selected from I-SceI, I-CreI and I-Dmol.

[0390] Gene editing molecules may also include restriction endonucleases, including type I, type II, type III and type IV endonucleases. Type I and type III restriction endonucleases recognize specific recognition sites, but are usually cut at variable positions away from the nuclease binding site, which may be hundreds of base pairs away from the cutting site (recognition site). In type II systems, restriction activity is independent of any methylase activity, and cutting usually occurs at a specific site within or near the binding site. Most type II enzymes cut palindromic sequences, but type IIa enzymes recognize non-palindromic recognition sites and cut outside the recognition site, type IIb enzymes cut sequences twice, and both sites are outside the recognition site, and type IIs enzymes recognize asymmetric recognition sites and cut at a determined distance of about 1-20 nucleotides from the recognition site on one side. Type IV restriction enzymes target methylated DNA. For example, restriction enzymes are further described and classified in the REBASE database (web page at rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-20), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al., (2002) in Mobile DNA II, pp. 761-783, ed. Craigie et al., (ASM Press, Washington, DC).

[0391] ZFN and TALEN introduce DSB in the target genomic sequence and activate non-homologous end joining (NHEJ)-mediated DNA repair, which produces mutant alleles containing insertion or deletion of nucleic acid sequence on the target genomic locus, thereby causing destruction of the target genomic locus in the cell. If a repair template is provided, DSB also stimulates homology-directed repair (HDR) by homologous recombination. HDR can lead to perfect repair, i.e., restore the original sequence of the break site, or it can be used to guide designed modifications, such as deletion, insertion or replacement of the sequence at the double-strand break site.

[0392] C. Guide RNA

[0393] In one aspect, the systems described herein comprise a guide RNA (gRNA).

[0394] "Guide RNA" or "gRNA" is an RNA molecule that binds to a Cas protein (e.g., Cas9 protein) or a functional fragment or derivative thereof, and targets the Cas protein to a specific location within the target DNA. A guide RNA may comprise two segments: a "DNA targeting segment" and a "protein binding segment". A "segment" includes a portion or region of a molecule, such as a continuous nucleotide fragment in an RNA. Some gRNAs, such as the gRNA of Cas9, may comprise two independent RNA molecules: an "activation RNA" (e.g., tracrRNA) and a "target RNA" (e.g., CRISPR RNA or crRNA). Other gRNAs are single RNA molecules (single RNA polynucleotides), also referred to as "single-molecule gRNAs", "single guide RNAs" or "sgRNAs". See, for example, WO 2013 / 176772, WO 2014 / 065596, WO2014 / 089290, WO 2014 / 093622, WO 2014 / 099750, WO 2013 / 142578, and WO 2014 / 131833, each of which is incorporated by reference in its entirety for all purposes. For example, for Cas9, a single guide RNA may include a crRNA fused to tracrRNA (e.g., through a linker). For example, for Cpf1, only crRNA is required to achieve binding to a target sequence. The terms "guide RNA" and "gRNA" include bimolecular (i.e., module) gRNA and single molecule gRNA.

[0395] Exemplary two-molecule gRNAs include a crRNA-like ("CRISPR RNA" or "target-RNA" or "crRNA" or "crRNA repeats") molecule and a corresponding TRAC RNA-like ("trans-acting CRISPR RNA" or "activating RNA" or "tracrRNA") molecule. The crRNA contains the DNA targeting segment (single strand) of the gRNA and a stretch of nucleotides that forms half of the dsRNA duplex of the protein binding segment of the gRNA.

[0396] The corresponding tracrRNA (activating RNA) contains a segment of nucleotides that forms the other half of the dsRNA duplex of the protein binding segment of the gRNA. A segment of nucleotides of crRNA is complementary and hybridized with a segment of nucleotides of tracrRNA to form a dsRNA duplex of the protein binding domain of the gRNA. Therefore, it can be said that each crRNA has a corresponding tracrRNA.

[0397] In a system where both crRNA and tracrRNA are needed, crRNA and corresponding tracrRNA hybridize to form gRNA. In a system where only crRNA is needed, crRNA can be gRNA. CrRNA also provides a single-stranded DNA targeting segment that hybridizes with the guide RNA recognition sequence. If used for intracellular modification, the exact sequence of a given crRNA or tracrRNA molecule can be designed to be specific to the species in which the RNA molecule will be used. See, e.g., Mali et al. (2013) Science 339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al. (2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol. 31:233-239; and Cong et al. (2013) Science 339:819-823, each of which is incorporated herein by reference in its entirety for all purposes.

[0398] The DNA targeting segment (crRNA) of a given gRNA comprises a nucleotide sequence complementary to a sequence in the target DNA (i.e., a guide RNA recognition sequence). The DNA targeting segment of gRNA interacts with the target DNA in a sequence-specific manner by hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the DNA targeting segment can vary, and determines the position at which the gRNA interacts with the target DNA in the target DNA. The DNA targeting segment of the gRNA of the present invention can be modified to hybridize with any desired sequence in the target DNA. Naturally occurring crRNAs are different according to the CRISPR / Cas system and organism, but generally comprise a targeting segment having a length between 21 nucleotides and 72 nucleotides, flanked by two forward repeats (DR) having a length between 21 nucleotides and 46 nucleotides (see, e.g., WO 2014 / 131833, incorporated herein by reference in its entirety for all purposes). In the case of Streptococcus pyogenes, the DR is 36 nucleotides in length and the targeting segment is 30 nucleotides in length. The DR at the 3' end is complementary and hybridized with the corresponding tracrRNA, which in turn binds to the Cas protein.

[0399] The length of the DNA targeting segment may be at least about 12 nucleotides, at least about 15 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, or at least about 40 nucleotides. Such DNA targeting segments may be about 12 nucleotides to about 100 nucleotides, about 12 nucleotides to about 80 nucleotides, about 12 nucleotides to about 50 nucleotides, about 12 nucleotides to about 40 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 25 nucleotides, or about 12 nucleotides to about 20 nucleotides in length. For example, the DNA targeting region fragment may be about 15 nucleotides to about 25 nucleotides (e.g., about 17 nucleotides to about 20 nucleotides, or about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, or about 20 nucleotides). See, e.g., US2016 / 0024523, which is incorporated herein by reference in its entirety for all purposes. For Cas9 from Streptococcus pyogenes, a typical DNA targeting segment is between 16 and 20 nucleotides in length, or between 17 and 20 nucleotides in length. For Cas9 from Staphylococcus aureus, a typical DNA targeting segment is between 21 and 23 nucleotides in length. For Cpf1, a typical DNA targeting segment is at least 16 nucleotides or at least 18 nucleotides in length.

[0400] TRAC RNA can be in any form (e.g., full-length tracrRNA or active partial tracrRNA) and have different lengths. They may include primary transcripts or processed forms. For example, tracrRNA (as a part of a single guide RNA or as an independent molecule as a part of a two-molecule gRNA) may include all or part of a wild-type tracrRNA sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracrRNA sequence) or consist of it. Examples of wild-type tracrRNA sequences from Streptococcus pyogenes include forms of 171 nucleotides, 89 nucleotides, 75 nucleotides, and 65 nucleotides. See, e.g., Deltcheva et al. (2011) Nature 471: 602-607; WO 2014 / 093661, each of which is incorporated herein by reference in its entirety for all purposes. Examples of tracrRNA in a single guide RNA (sgRNA) include tracrRNA segments found in the +48, ​​+54, +67, and +85 forms of sgRNA, where "+n" indicates that up to +n nucleotides of wild-type tracrRNA are included in the sgRNA. See US 8,697,359, which is incorporated herein by reference in its entirety for all purposes.

[0401] The percentage of complementarity between the DNA targeting sequence and the guide RNA recognition sequence within the target DNA can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%). The percentage of complementarity between the DNA targeting sequence and the guide RNA recognition sequence within the target DNA can be at least 60% over about 20 consecutive nucleotides. For example, the percentage of complementarity between the DNA targeting sequence and the guide RNA recognition sequence within the target DNA is 100% over 14 consecutive nucleotides at the 5' end of the guide RNA recognition sequence within the complementary strand of the target DNA, and as low as 0% over the rest. In this case, the length of the DNA targeting sequence can be considered to be 14 nucleotides. As another example, the percentage of complementarity between the DNA targeting sequence and the guide RNA recognition sequence within the target DNA is 100% over 7 consecutive nucleotides at the 5' end of the guide RNA recognition sequence within the complementary strand of the target DNA, and as low as 0% over the rest. In this case, the length of the DNA targeting sequence can be considered to be 7 nucleotides. In some guide RNAs, at least 17 nucleotides within the DNA-target sequence are complementary to the target DNA. For example, the DNA-targeting sequence can be 20 nucleotides in length and can contain 1, 2, or 3 mismatches with the target DNA (guide RNA recognition sequence). Preferably, the mismatch is not adjacent to the protospacer adjacent motif (PAM) sequence (e.g., the mismatch is located at the 5' end of the DNA targeting sequence, or the mismatch is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs away from the PAM sequence).

[0402] The protein binding segment of the gRNA may include two nucleotide fragments that are complementary to each other. The complementary nucleotides of the protein binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein binding segment of the gRNA of the present invention interacts with the Cas protein or its functional fragment or derivative, and the gRNA guides the bound Cas protein or its functional fragment or derivative to a specific nucleic acid sequence in the target DNA through the DNA targeting segment.

[0403] The guide RNA may include modifications or sequences that provide additional desired characteristics (e.g., modified or modulated stability; subcellular targeting; tracking with fluorescent markers; binding sites for proteins or protein complexes; etc.). Examples of such modifications include, for example, a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (i.e., a 3' poly(A) tail); a riboswitch sequence (e.g., allowing a protein and / or protein complex to regulate stability and / or regulate accessibility); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin); a modification or sequence that targets the RNA to a subcellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, etc.); and combinations thereof. Other examples of modification include engineered stem-loop duplex structures, engineered bulge regions, engineered hairpins 3' of stem-loop duplex structures, or any combination thereof. See, for example, US2015 / 0376586, which is incorporated herein by reference in its entirety for all purposes. The bulge region can be an unpaired nucleic acid region in a duplex consisting of a crRNA-like region and a minimum tracrRNA-like region. On one side of the duplex, the bulge may include an unpaired 5'-XXXY-3', wherein X is any purine, and Y may be a nucleotide that can form a wobble pair with a nucleotide on the opposite chain, and an unpaired nucleotide region is included on the other side of the duplex.

[0404] In some cases, a transcriptional activation system comprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1 can be used. The guide RNA in such a system can be designed with aptamer sequences attached to the sgRNA tetraloop and stem-loop 2 designed to bind to the dimerizing MS2 phage coat protein. See, e.g., Konermann et al. (2015) Nature 517(7536):583-588, which is incorporated herein by reference in its entirety for all purposes.

[0405] The guide RNA can be provided in any form. For example, the gRNA can be provided in the form of RNA, or as two molecules (independent crRNA and tracrRNA) or as one molecule (sgRNA). The gRNA can also be provided in the form of a DNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or a separate RNA molecule (e.g., a separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA can be provided as a DNA molecule, or as separate DNA molecules encoding crRNA and tracrRNA, respectively.

[0406] When gRNA is provided in the form of DNA, gRNA can be expressed transiently, conditionally or constitutively in cells. The DNA encoding gRNA can be stably integrated into the genome of the cell and operably connected to a promoter active in the cell. Alternatively, the DNA encoding gRNA can be operably connected to a promoter in an expression construct. For example, the DNA encoding gRNA can be present in a vector comprising a heterologous nucleic acid. Promoters that can be used for such expression constructs include, for example, eukaryotic cells, human cells, non-human cells, mammalian cells, non-human mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, rabbit cells, pluripotent cells, embryonic stem (ES) cells, adult stem cells, developmentally restricted progenitor cells, induced pluripotent stem (iPS) cells, or one or more active promoters in a single cell stage embryo. Such promoters can be, for example, conditional promoters, inducible promoters, constitutive promoters, or tissue-specific promoters. Such promoters can also be, for example, bidirectional promoters. In certain embodiments, the RNA Pol III promoter can be operably linked to a gRNA sequence (if included in a lentiviral vector) to control the expression of the sequence. RNA Pol III promoters are often used to express small RNAs, such as small interfering RNA (siRNA) / short hairpin RNA (shRNA) and guide RNA sequences used in CRISPR-Cas9 systems. Examples of RNA Pol III promoters that can be used in the present invention include, but are not limited to, human U6 promoters, rat U6 polymerase III promoters or mouse U6 polymerase III promoters and H1 promoters, which are described in, for example, Goomer and Kunkel, Nucl. Acids Res., 20 (18): 4903-4912 (1992) and Myslinski et al., Nucleic Acids Res., 29 (12): 2502-9 (2001).

[0407] D. Guide RNA recognition sequence

[0408] The term "guide RNA recognition sequence" includes a nucleic acid sequence present in the target DNA, and the DNA targeting segment of the gRNA will bind to the nucleic acid sequence as long as there are sufficient binding conditions. For example, the gRNA recognition sequence includes a sequence to which the gRNA is designed to be complementary, wherein the hybridization between the guide RNA recognition sequence and the DNA targeting sequence promotes the formation of the CRISPR complex. It is not necessarily required to be completely complementary, as long as there is enough complementarity to cause hybridization and promote the formation of the CRISPR complex. The guide RNA recognition sequence also includes the cleavage site of the Cas protein, which will be described in more detail below. The gRNA recognition sequence may include any polynucleotide that may be located, for example, in the nucleus or cytoplasm of a cell, or in an organelle of the cell such as a mitochondria or chloroplast.

[0409] The gRNA recognition sequence in the target DNA can be targeted by Cas protein or gRNA (i.e., combined with or hybridized or complementary thereto). Suitable DNA / RNA binding conditions include physiological conditions that are generally present in cells. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001), for all purposes, incorporated herein by reference in its entirety). The chain of the target DNA that is complementary and hybridized with Cas protein or gRNA can be referred to as a "complementary chain", and the chain of the target DNA that is complementary to the "complementary chain" (and therefore not complementary to the Cas protein or gRNA) can be referred to as a "non-complementary chain" or "template chain".

[0410] Cas protein can cut nucleic acid at a site within or outside the nucleic acid sequence that will be bound to the DNA targeting segment of gRNA present in the target DNA. "Cut site" includes the position in the nucleic acid where the Cas protein produces a single-strand break or a double-strand break. For example, the formation of a CRISPR complex (comprising a gRNA that hybridizes with a guide RNA recognition sequence and is compounded with a Cas protein) can cause one or two chains to be cut in or near the nucleic acid sequence that will be bound to the DNA targeting segment of gRNA present in the target DNA (for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). If the cleavage site is outside the nucleic acid sequence that will be bound to the DNA targeting segment of gRNA, the cleavage site is still considered to be within the "guide RNA recognition sequence". The cleavage site can be only on one strand of the nucleic acid or on two strands. The cleavage site can be at the same position on the two strands of the nucleic acid (producing a flat end), or it can be at different positions on each strand (producing staggered ends (i.e., overhangs)). For example, staggered ends can be produced by using two Cas proteins, each of which produces single-strand breaks at different cleavage sites on different chains, thereby producing double-strand breaks. For example, a first nickase can produce single-strand breaks on the first strand of double-stranded DNA (dsDNA), and a second nickase can produce single-strand breaks on the second strand of dsDNA, thereby producing an overhang sequence. In some cases, the guide RNA recognition sequence of the nickase on the first strand is separated from the guide RNA recognition sequence of the nickase on the second strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500 or 1,000 base pairs.

[0411] Site-specific binding and cleavage of the target DNA by the Cas protein can occur in the target DNA at a position determined by (i) base pairing complementarity between the gRNA and the target DNA and (ii) a short motif called a protospacer adjacent motif (PAM). The PAM can be located on the flank of the guide RNA recognition sequence. Optionally, the 3' end of the guide RNA recognition sequence can be flanked by the PAM. Alternatively, the 5' end of the guide RNA recognition sequence can be flanked by the PAM. For example, the cleavage site of the Cas protein can be about 1 to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some cases (e.g., when using Cas9 from Streptococcus pyogenes or a closely related Cas9), the PAM sequence of the non-complementary chain can be 5'-N1GG-3', where N1 is any DNA nucleotide and is immediately adjacent to the 3' of the guide RNA recognition sequence of the non-complementary chain of the target DNA. In this way, the PAM sequence of the complementary strand will be 5'-CCN2-3', where N2 is any DNA nucleotide and is immediately 5' of the guide RNA recognition sequence of the complementary strand of the target DNA. In some such cases, N1 and N2 can be complementary, and the N1-N2 base pair can be any base pair (e.g., N1=C, N2=G; N1=G, N2=C; N1=A, N2=T; or N1=T, N2=A). In the case of Cas9 from Staphylococcus aureus, the PAM can be NNGRRT or NNGRR, where N can be A, G, C or T, and R can be G or A. In the case of Cas9 from Campylobacter jejuni, the PAM can be, for example, NNNNACAC or NNNNRYAC, where N can be A, G, C or T, and R can be G or A. In some cases (e.g., for FnCpf1), the PAM sequence can be upstream of the 5' end and have the sequence 5'-TTN-3'.

[0412] Examples of gRNA recognition sequences include DNA sequences complementary to the DNA targeting segment of the gRNA, or such DNA sequences other than the PAM sequence. For example, the target motif can be a DNA sequence of 20 nucleotides just before the NGG motif recognized by the Cas9 protein (such as, G N G 19 NGG or N 20 NGG). See, for example, WO 2014 / 165825, which is incorporated herein by reference in its entirety for all purposes. The guanine at the 5' end can promote transcription by RNA polymerase in cells. Other examples of guide RNA recognition sequences may include two guanine nucleotides at the 5' end (e.g., GGN 20NGG) to promote efficient transcription of T7 polymerase in vitro. See, for example, WO 2014 / 065596, which is incorporated herein by reference in its entirety for all purposes. The length of other guide RNA recognition sequences may be between 4 nucleotides and 22 nucleotides, including 5'G or GG and 3'GG or NGG. However, the length of other guide RNA recognition sequences may be between 14 nucleotides and 20 nucleotides.

[0413] In various embodiments, the gRNA is complementary to a sequence at an IgH locus, a J chain locus, or an Igκ locus in a target cell (e.g., a B cell or HSC). In some embodiments, the gRNA is complementary to a sequence at a J chain locus. In one embodiment, the gRNA is complementary to a sequence in the 4th exon of the J chain locus. In one embodiment, the gRNA is complementary to a sequence in the first intron of the J chain locus.

[0414] As non-limiting examples, the nucleotide sequence encoding antibody, Fab and antibody-like molecule (e.g., single-chain antibody-like molecule) can be inserted into the IgH locus. The expressed antibody, Fab and antibody-like molecule (e.g., single-chain antibody-like molecule) can be used as the surface BCR that can be converted into the secretory antibody form according to natural BCR. As another non-limiting example, the sequence encoding antibody, Fab or antibody-like protein (e.g., single-chain antibody-like molecule) and non-antibody protein can be inserted into J chain and / or Igκ locus, and expressed as secretory product.

[0415] E. Repair template

[0416] In one aspect, the systems described herein include a sequence corresponding to a repair template.

[0417] As used herein, the terms "repair template", "RT", "recombination template", "donor nucleic acid molecule" or "donor polynucleotide" are used interchangeably and refer to a DNA fragment that is desired to be integrated at a target locus. In certain embodiments, the repair template comprises one or more target polynucleotides. In other embodiments, the repair template may comprise one or more expression cassettes. A given expression cassette may comprise a target polynucleotide, a polynucleotide encoding a selectable marker and / or a reporter gene, and various regulatory components that affect expression.

[0418] In certain embodiments, the repair template may comprise a segment of genomic DNA, a cDNA, a regulatory region, or any portion or combination thereof. In certain embodiments, the repair template may comprise a nucleic acid from a eukaryotic organism, a mammal, a human, a non-human mammal, a rodent, a rat, a non-rat rodent, a mouse, a hamster, a rabbit, a pig, a cow, a deer, a sheep, a goat, a chicken, a cat, a dog, a ferret, a primate (e.g., a marmoset, a rhesus monkey), a domesticated mammal, or an agricultural mammal, or any other organism of interest.

[0419] In certain embodiments, the repair template comprises a knock-in allele of at least one exon of an endogenous gene. In certain embodiments, the repair template comprises a knock-in allele of an intact endogenous gene (ie, a "gene exchange knock-in").

[0420] In other embodiments, the repair template comprises a conditional allele. In certain embodiments, the conditional allele is a multifunctional allele, as described in US2011 / 0104799 (which is incorporated by reference in its entirety). In certain embodiments, the conditional allele comprises: (a) an actuating sequence in a sense orientation relative to target gene transcription, and a drug selection cassette in a sense or antisense orientation; (b) a target nucleotide sequence (NSI) in an antisense orientation and a conditional by inversion module (COIN, which utilizes exon-splitting introns and a reversible gene trap-like module; see, e.g., US2011 / 0104799, which is incorporated herein by reference in ...

Claims

1. A system for producing an antibody or an antigen-binding fragment thereof in a subject, the system comprising: a) a first component comprising a polynucleotide molecule, wherein the polynucleotide molecule comprises a sequence encoding the antibody or antigen-binding fragment thereof; and b) A second component, which comprises a gene editing molecule or a polynucleotide molecule containing a sequence encoding the gene editing molecule.

2. The system of claim 1, wherein administration of the first component and the second component to the subject results in integration of sequences encoding the antibody or antigen-binding fragment thereof into DNA of B cells and / or hematopoietic stem cells (HSCs) of the subject, thereby resulting in production of the antibody or antigen-binding fragment in the subject.

3. A system according to claim 1, wherein the ex vivo administration of the first component and the second component to B cells and / or hematopoietic stem cells (HSCs) results in the integration of sequences encoding the antibody or its antigen-binding fragment into the DNA of the cells to produce modified B cells or modified HSCs, thereby resulting in the production of the antibody or its antigen-binding fragment in the subject after administration of the modified B cells or HSCs to the subject.

4. The system of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof binds to an antigen associated with a disease or disorder.

5. The system of claim 4, wherein the disease or disorder is an infection, cancer, an autoimmune disease, a cardiovascular disease, a musculoskeletal disorder, or a neurodegenerative disease.

6. The system of claim 5, wherein the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

7. The system of any one of claims 4-6, wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor-associated antigen (TAA).

8. The system of any one of claims 1-7, wherein the gene editing molecule is a Cas nuclease.

9. The system of claim 8, wherein the Cas nuclease is a Cas9 nuclease.

10. The system of any one of claims 1-9, wherein the first or second component further comprises a guide RNA (gRNA) molecule or a sequence encoding the gRNA molecule.

11. The system according to claim 10, wherein the first component comprises the polynucleotide molecule, which comprises a sequence encoding the antibody or antigen-binding fragment thereof and a sequence encoding the gRNA.

12. The system of claim 10, wherein the first component comprises (i) a first polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof; and (ii) a second polynucleotide molecule comprising a sequence encoding the gRNA.

13. The system of claim 10, wherein the first component comprises (i) a first polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof; and (ii) the gRNA molecule.

14. The system of claim 10, wherein the second component comprises the gRNA molecule or a sequence encoding the gRNA molecule.

15. The system of any one of claims 10-14, wherein the gRNA is complementary to a sequence at the IgH locus, the J chain locus, or the Igκ locus.

16. The system of claim 15, wherein the gRNA is complementary to a sequence in exon 4 of the J chain locus.

17. The system of claim 15, wherein the gRNA is complementary to a sequence in the first intron of the J chain locus.

18. The system of any one of claims 1-17, wherein the sequence encoding the antibody or antigen-binding fragment thereof comprises a sequence encoding a light chain variable region and optionally a light chain constant region of the antibody.

19. The system of any one of claims 1-18, wherein the sequence encoding the antibody or antigen-binding fragment thereof comprises a sequence encoding a heavy chain variable region of the antibody.

20. The system of any one of claims 1-20, wherein the sequence encoding the antibody or antigen-binding fragment thereof is integrated at the IgH locus in the genomic region downstream of the last J gene but upstream of the Eμ enhancer.

21. The system of any one of claims 1-20, wherein integration of the sequence encoding the antibody or antigen-binding fragment thereof into the DNA of a B cell or HSC results in disruption of the kappa light chain constant region.

22. The system of any one of claims 1-21, wherein the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a 5' IgH homology region, a splice acceptor, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a splice donor sequence, and a 3' IgH homology region, wherein the heavy chain sequence and the light chain sequence can be placed in either order.

23. The system of any one of claims 1-21, wherein the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a 5' J chain exon 4 homology region, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence having a 5' furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a sequence encoding the heavy chain constant region of the antibody, and a 3' J chain exon 4 homology region, wherein the heavy chain sequence and the light chain sequence can be placed in either order.

24. The system of any one of claims 1-23, wherein the sequence encoding the antibody or antigen-binding fragment thereof does not comprise a promoter sequence.

25. The system of claim 24, wherein: When the sequence encoding the antibody or antigen-binding fragment thereof is integrated into the DNA of the B cell or HSC, the sequence is under the transcriptional control of the endogenous heavy chain promoter in the B cell or HSC.

26. The system of claim 24, wherein: When the sequence encoding the antibody or antigen-binding fragment thereof is integrated into the DNA of the B cell or HSC, the sequence is under the transcriptional control of the endogenous J chain promoter in the B cell or HSC.

27. The system of any one of claims 1-23, wherein the sequence encoding the antibody or antigen-binding fragment thereof comprises a promoter sequence.

28. The system of claim 27, wherein the promoter is a B cell-specific promoter or a HSC-specific promoter.

29. The system of claim 27, wherein the promoter is the Hg38-mCP promoter.

30. The system of claim 27, wherein the promoter is the spleen focus forming virus (SFFV) promoter or a fragment thereof.

31. The system according to any one of claims 1-30, wherein the first component and / or the second component are independently selected from viral vectors, virus-like particles (VLPs), lipid nanoparticles (LNPs), liposomes, and ribonucleoprotein (RNP) complexes.

32. The system of claim 31, wherein the first component and the second component are both viral vectors.

33. The system of claim 32, wherein the viral vectors are derived from the same viral species.

34. The system of claim 32, wherein the viral vectors are derived from different viral species.

35. The system of any one of claims 31-34, wherein the viral vector is an adeno-associated viral (AAV) vector.

36. The system of claim 35, wherein the AAV vector is derived from AAV1, AAV2, AAV6, AAV9 or AAV9.PHP.

37. The system of claim 35 or 36, wherein the AAV vector capsid comprises one or more mutations, wherein the one or more mutations eliminate the natural tropism of the AAV vector.

38. The system of any one of claims 31-34, wherein the viral vector is a retroviral vector.

39. The system of claim 38, wherein the retroviral vector is a lentiviral vector.

40. The system of any one of claims 31-39, wherein the viral vector further comprises a targeting moiety.

41. The system of claim 40, wherein the viral vector is an AAV vector and the targeting moiety is inserted into, or covalently or non-covalently attached to, a protein that forms the viral capsid.

42. The system of claim 41, wherein the targeting moiety is attached to the viral capsid via a first member and a second member of a binding pair, wherein the first member and the second member form an isopeptide bond.

43. The system of claim 40, wherein the viral vector is a lentiviral vector and the targeting moiety is covalently or non-covalently attached to a fusogen.

44. The system of any one of claims 40-43, wherein the targeting moiety is a targeting antibody or antigen-binding fragment thereof.

45. The system of claim 44, wherein the targeting antibody or antigen-binding fragment thereof binds CD5, CD19, CD20, CD22, CD34, CD38, CD40, CD117, CD79, CD180, B cell receptor (BCR), B cell activating factor (BAFF), or Sca-1.

46. ​​The system of any one of claims 1-45, wherein the subject is a human.

47. The system of any one of claims 1-45, wherein the subject is an experimental animal.

48. A modified B cell or a modified hematopoietic stem cell (HSC) comprising the system of any one of claims 1-47.

49. A pharmaceutical composition comprising the system according to any one of claims 1-47 and a pharmaceutically acceptable carrier or excipient.

50. A kit comprising (i) the system according to any one of claims 1-47 and optionally (ii) a container and / or instructions for use.

51. A method for generating a modified B cell or modified hematopoietic stem cell (HSC) that produces an antibody or an antigen-binding fragment thereof, the method comprising transducing a B cell or HSC ex vivo with an effective amount of the system according to any one of claims 1-47, wherein the first component and the second component of the system are administered to the cell simultaneously or sequentially in any order, and wherein the administration of the first component and the second component results in the integration of the sequence encoding the antibody or the antigen-binding fragment thereof into the DNA of the cell, wherein the cell becomes a modified cell.

52. The method of claim 51, wherein the first component and the second component of the system are administered to the cell simultaneously as two separate compositions.

53. The method of claim 51, wherein the first component and the second component of the system are administered to the cell simultaneously as a composition.

54. The method of any one of claims 51 to 53, wherein the B cells or HSCs are present in a heterogeneous cell population during transduction.

55. The method of any one of claims 51-53, wherein the B cells have been isolated from spleen, peritoneum or peripheral blood.

56. The method of any one of claims 51-55, wherein the B cell is a primary B cell.

57. The method of any one of claims 51-55, wherein the B cell is a B2 B cell.

58. The method of any one of claims 51-55, wherein the B cell is a B1 B cell.

59. The method of any one of claims 51-58, wherein the B cells are cultured under stimulating conditions before and / or after the transduction.

60. The method of claim 59, wherein the stimulatory conditions promote B cell activation without differentiation.

61. The method of claim 59 or claim 60, wherein the B cells are cultured in the presence of an agonist for CD40 and / or an agonist for CD180 before and / or after the transduction.

62. The method of claim 61, wherein the CD40 agonist is CD40L or an anti-CD40 antibody.

63. The method of claim 61 or claim 62, wherein the agonist of CD180 is an anti-CD180 antibody.

64. The method of claim 63, wherein the B cells are cultured in the presence of CD40L and / or anti-CD180 antibodies before and / or after the transduction.

65. The method of claim 64, wherein the B cells are cultured in the presence of CD40L and anti-CD180 antibodies before and / or after the transduction.

66. The method of claim 64 or claim 65, wherein the B cells are cultured in the presence of about 20 ng / ml or less CD40L and / or about 100 ng / ml or less anti-CD180 antibody before and / or after the transduction.

67. The method of claim 66, wherein the B cells are cultured in the presence of about 20 ng / ml CD40L and about 20 ng / ml anti-CD180 antibody before and / or after the transduction.

68. The method of any one of claims 64-67, wherein the B cells are cultured in the presence of CD40L and / or anti-CD180 antibodies for 4 days or less prior to the transduction.

69. The method of claim 68, wherein the B cells are cultured in the presence of CD40L and / or anti-CD180 antibodies for about 2 days prior to the transduction.

70. The method of any one of claims 51-69, further comprising culturing the modified B cells or modified HSCs under differentiation conditions to promote differentiation of the modified B cells or modified HSCs into modified plasma cells.

71. The method of any one of claims 51-70, further comprising introducing the modified B cell or the modified HSC or the modified plasma cell into a subject.

72. The method of claim 71, wherein the modified cells are introduced into the subject intraperitoneally.

73. The method of claim 71 or claim 72, wherein the subject has been depleted of CD20+ cells prior to introducing the modified cells.

74. The method of any one of claims 71-73, wherein after the modified cells are introduced into the subject, the modified cells are expanded in vivo by administering to the subject an antigen that is recognized by an antibody or antigen-binding fragment thereof produced by the modified cells.

75. The method of any one of claims 71-74, wherein the subject is autologous to the modified cells.

76. The method of any one of claims 71-74, wherein the subject is allogeneic to the modified cells.

77. The method of any one of claims 71-76, wherein the subject is a human.

78. The method of any one of claims 71-76, wherein the subject is an experimental animal.

79. A modified B cell or a modified hematopoietic stem cell (HSC) produced by the method of any one of claims 51-69.

80. Modified plasma cells produced by the method of claim 70.

81. A method for producing an antibody or an antigen-binding fragment thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of the system according to any one of claims 1-47, wherein the first component and the second component of the system are administered simultaneously or sequentially in any order, and wherein administration of the first component and the second component to the subject results in the integration of the sequence encoding the antibody or antigen-binding fragment thereof into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), resulting in the production of the antibody or antigen-binding fragment thereof in the subject.

82. The method of claim 81, wherein the first component and the second component of the system are administered to the subject simultaneously as two separate compositions.

83. The method of claim 81, wherein the first component and the second component of the system are administered to the subject simultaneously as a composition.

84. The method of any one of claims 81-83, wherein the first component and / or the second component of the system is administered intraperitoneally to the subject.

85. The method of any one of claims 81-84, wherein the method further comprises administering to the subject an effective amount of an agonist of CD40 and / or an agonist of CD180 prior to administering the system to the subject.

86. The method of claim 85, wherein the CD40 agonist is CD40L or an anti-CD40 antibody.

87. The method of claim 85 or claim 86, wherein the agonist of CD180 is an anti-CD180 antibody.

88. The method of claim 87, wherein the method comprises administering to the subject an effective amount of an anti-CD180 antibody and / or an anti-CD40 antibody prior to administering the system to the subject.

89. The method of claim 88, wherein the method comprises administering to the subject an effective amount of an anti-CD180 antibody and an anti-CD40 antibody prior to administering the system to the subject.

90. The method of claim 88 or claim 89, wherein the method comprises administering to the subject about 8.5 mg / kg or less of an anti-CD180 antibody and / or about 1.8 mg / kg or less of an anti-CD40 antibody prior to administering the system to the subject.

91. The method of any one of claims 88-90, wherein the method comprises administering about 0.4 mg / kg of the anti-CD180 antibody to the subject.

92. The method of any one of claims 88-91, wherein the method comprises administering the anti-CD180 antibody and / or anti-CD40 antibody to the subject about 7 days or less prior to administering the system to the subject.

93. The method of claim 92, wherein the method comprises administering an anti-CD180 antibody and / or an anti-CD40 antibody to the subject about 2-3 days prior to administering the system to the subject.

94. The method of any one of claims 81-93, wherein the method further comprises administering to the subject an effective amount of an antigen recognized by the antibody or antigen-binding fragment thereof, wherein the antigen is administered before and / or after administration of the first component and / or the second component of the system.

95. The method of claim 94, wherein the antigen has a low affinity for the antibody or antigen-binding fragment thereof.

96. The method of claim 94, wherein the antigen has a high affinity for the antibody or antigen-binding fragment thereof.

97. A method according to any one of claims 94-96, wherein the method comprises administering to the subject an effective amount of a first antigen, and administering to the subject an effective amount of a second antigen; wherein the first antigen has a low affinity for the antibody or antigen-binding fragment thereof, and wherein the first antigen is administered before administration of the first component and the second component of the system; wherein the second antigen has a high affinity for the antibody or antigen-binding fragment thereof, and wherein the second antigen is administered after administration of the first component and the second component of the system.

98. The method of any one of claims 81-97, wherein the subject is a human.

99. The method of any one of claims 81-97, wherein the subject is an experimental animal.

100. A method for treating or reducing the likelihood of a disease or condition in a subject in need thereof, the method comprising practicing the method of any one of claims 71-78 or the method of any one of claims 81-99, wherein the method results in the production of an effective amount of the antibody or antigen-binding fragment thereof in the subject.

101. The method of claim 100, wherein the disease or disorder is an infection, cancer, an autoimmune disease, a cardiovascular disease, a musculoskeletal disorder, or a neurodegenerative disease.

102. The method of claim 101, wherein the infection is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

103. The method of any one of claims 100-102, wherein the subject is a human.

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