Systems and methods for treating hyper igm syndrome

Abstract

To provide genome editing systems and related methods which allow for treatment of Hyper IgM Syndrome, a group of disorders characterized by defective CD40 signaling.SOLUTION: Provided herein is an isolated oligonucleotide donor template which comprises, from 5' to 3', A1-N-UTR-pA-A2, where A1 is a homology arm that is substantially identical to a first homology arm of a target CD40L nucleic acid; N is a cargo comprising one or more of exons 2-5 of a CD40L gene; UTR is a CD40L 3' untranslated region (UTR); pA is a polyA tail; and A2 is a homology arm that is substantially identical to a second homology arm of the target CD40L nucleic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 576,277, filed October 24, 2017, U.S. Provisional Patent Application No. 62 / 664,800, filed April 30, 2018, U.S. Provisional Patent Application No. 62 / 673,039, filed May 17, 2018, U.S. Provisional Patent Application No. 62 / 690,284, filed June 26, 2018, and U.S. Provisional Patent Application No. 62 / 746,900, filed October 17, 2018, the entire contents of each of which are expressly incorporated herein by reference.

[0002] Field The present disclosure relates to CRISPR / Cas-related methods and compositions for treating Hyper-IgM syndrome, methods for editing CD40L target nucleic acid sequences and modulating expression of CD40L target nucleic acid sequences, and applications thereof. [Background technology]

[0003] CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) evolved in bacteria and archaea as an adaptive immune system to defend against viral attacks. After exposure to a virus, a short segment of viral DNA is integrated into the CRISPR locus. From the part of the CRISPR locus that contains the viral sequence, RNA is transcribed. The RNA, which contains a sequence that is complementary to the viral genome, mediates the targeting of Cas9 protein to the target sequence in the viral genome. Cas9 protein then cleaves the viral target, thereby silencing the viral target.

[0004] Recently, the CRISPR / Cas system has been applied for genome editing in eukaryotic cells. The introduction of site-specific double-strand breaks (DSBs) allows modification of the target sequence through endogenous DNA repair mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR).

[0005] Hyper-IgM syndrome (HIGM) is an immunoglobulin deficiency characterized by normal or elevated serum IgM levels and reduced or absent levels of other serum immunoglobulins, resulting in susceptibility to infections. Most cases of HIGM are X-linked and are caused by mutations in a gene on the X chromosome that encodes a protein (CD40 ligand, "CD40L") on the surface of activated helper T cells. Mutations in the CD40L gene ("CD40LG") in subjects with hyper-IgM syndrome include missense mutations, nonsense mutations, splice site mutations, deletions and insertions, and are well known to those skilled in the art. See, for example, Seyama et al., (1998) Blood, 92(7):2421-2434. In the presence of cytokines, normal CD40 ligand interacts with B cells, thus signaling them to switch from producing IgM to producing IgA, IgG and / or IgE. In X-linked hyper-IgM syndrome, T cells lack functional CD40 ligand and are unable to signal B cells to switch. Thus, B cells in HIGM subjects produce only IgM, and IgM levels may be normal or elevated. Summary of the Invention [Problem to be solved by the invention]

[0006] Treatment of HIGM syndrome usually includes immunoglobulin replacement therapy, long-term antibiotic and antifungal prophylaxis, and G-CSF treatment for neutropenia. Hematopoietic stem cell transplantation is also preferred if an HLA-matched sibling donor is available. However, current treatments are ineffective, as approximately 80% of HIGM patients die before the age of 30. Thus, there remains a continuing unmet need for the development of novel therapeutic strategies and products to treat HIGM. [Means for solving the problem]

[0007] An adoptive immunotherapy approach based on gene-modified autologous T cells may be sufficient to induce the development of protective immunity in recipient HIGM1 patients, and perhaps even the production of some long-lived memory B cells. However, multiple administrations of edited T cells may be required to reconstitute a long-lasting, broad T cell repertoire that can mediate efficient T cell help in response to a wide range of pathogens. Furthermore, although CD40LG has important functions in CD4 T cells (Th0, Th1 and Th2), this molecule is also expressed in several other hematopoietic cell types, such as activated B cells, platelets, NK cells, monocytes, basophils and eosinophils. Thus, the present disclosure surprisingly extends gene editing strategies from the modification of T cells to the modification of autologous hematopoietic stem / progenitor cells (HSPCs), which provides a much broader and longer-lasting therapeutic benefit.

[0008] The present disclosure is based, at least in part, on the discovery that CD40 ligand (CD40L) edited cells surprisingly produce functionally active CD40L protein and can be used to restore B cell class switching function for the treatment of hyper-IgM syndrome. In particular, when one or more of exons 2-5 of the CD40L gene are edited in cells, e.g., T cells or hematopoietic stem cells (HSCs), using a genome editing system, the edited cells demonstrate high levels of CD40L gene editing and high levels of expression of functional exogenous CD40L. Specifically, the CD40L-edited T cells restored B cell class switching capability by increasing the levels of IgG produced while decreasing the levels of IgM. Furthermore, mice that received hematopoietic stem cells containing at least 10% CD40L-edited cells produced higher levels of IgG following vaccination, demonstrating the therapeutic benefit of CD40L-edited hematopoietic stem cells for use in the treatment of hyper-IgM syndrome.

[0009] The genome editing system disclosed herein is designed such that exon 1 of the CD40L gene is not edited, avoiding the possibility of random integration or off-target events. Specifically, editing of exon 1 can lead to loss of promoter control of the locus, affecting expression of the CD40L gene and leading to lymphoproliferative disorders, especially in the context of hematopoietic stem cell transplantation.

[0010] Thus, in one aspect, disclosed herein is an isolated oligonucleotide donor template comprising, from 5' to 3', A1-N-UTR-pA-A2, where A1 is a homologous arm that is substantially identical to a first homologous arm of a target CD40L nucleic acid; N is a cargo comprising one or more of exons 2-5 of the CD40L gene; UTR is the 3' untranslated region (UTR) of CD40L; pA is the polyA tail; and A2 is a homologous arm that is substantially identical to a second homologous arm of the target CD40L nucleic acid.

[0011] In one aspect, disclosed herein is an isolated oligonucleotide donor template comprising, from 5' to 3', A1-S1-N-UTR-pA-S2-A2, where A1 is a homologous arm substantially identical to a first homologous arm of a target CD40L nucleic acid; S1 is a first stuffer, N is a cargo comprising one or more of exons 2-5 of the CD40L gene; UTR is the 3' untranslated region (UTR) of CD40L; pA is a polyA tail; S2 is a second stuffer, and A2 is a homologous arm substantially identical to a second homologous arm of a target CD40L nucleic acid.

[0012] In one aspect, disclosed herein is an isolated oligonucleotide donor template comprising, from 5' to 3', A1-S1-N-UTR-pA-R-S2-A2, where A1 is a homologous arm substantially identical to a first homologous arm of a target CD40L nucleic acid; S1 is a first stuffer, N is a cargo comprising one or more of exons 2-5 of the CD40L gene; UTR is the 3' untranslated region (UTR) of CD40L; pA is a polyA tail; R is a reporter; S2 is a second stuffer, and A2 is a homologous arm substantially identical to a second homologous arm of a target CD40L nucleic acid.

[0013] In one aspect, disclosed herein is an isolated oligonucleotide donor template comprising, from 5' to 3', A1-N-UTR-pA-R-A2, where A1 is a homologous arm substantially identical to a first homologous arm of a target CD40L nucleic acid; N is a cargo comprising one or more of exons 2-5 of the CD40L gene; UTR is the 3' untranslated region (UTR) of CD40L; pA is a polyA tail; R is a reporter; and A2 is a homologous arm substantially identical to a second homologous arm of the target CD40L nucleic acid.

[0014] In any of the aspects described herein, the cargo may comprise exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or exons 4 and 5 of the CD40L gene. In one embodiment, the cargo comprises a codon-optimized CD40L sequence. For example, the cargo may comprise a codon-optimized version of CD40L exon 2, CD40L exon 3, CD40L exon 4, CD40L exon 5, or combinations thereof, such as a codon-optimized exon 2-5 sequence, a codon-optimized exon 2-4 sequence; a codon-optimized exon 2 and 3 sequence; a codon-optimized exon 3-5 sequence; a codon-optimized exon 3 and 4 sequence; or a codon-optimized exon 4 and 5 sequence. An exemplary codon-optimized version of exons 2-5 of CD40L is provided in SEQ ID NO:48.

[0015] In one embodiment, the CD40L gene is a wild-type CD40L sequence. In one embodiment, the target CD40L nucleic acid comprises a mutation. In one embodiment, the oligonucleotide donor template further comprises a sequence encoding a reporter. In one embodiment, the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase. In one embodiment, the reporter may be a marker that can be detected in living cells using methods standard in the art, such as a surface antigen detectable by an antibody, a peptide that catalyzes or otherwise facilitates a chemical reaction that produces an optically detectable product, such as luciferase. In one embodiment, the reporter is a selection marker. In an exemplary embodiment, the selection marker is a low affinity nerve growth factor receptor (NGFR). In other embodiments, the selectable marker may be a drug resistance protein (e.g., neomycin or puromycin resistance, a mutant version of the MGMT gene), a truncated version of a cell surface protein (e.g., CD19, EGFR), a gene that confers a selective growth and / or engraftment advantage following in vivo transplantation of the edited cells (e.g., CXCR4, CD47, IL2 receptor), or a fluorescent reporter protein (e.g., green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc., or a combination thereof. In one embodiment, the isolated oligonucleotide donor template is a single-stranded oligonucleotide donor template. In one embodiment, the isolated oligonucleotide donor template is a double-stranded oligonucleotide donor template.

[0016] In one aspect, disclosed herein is a composition comprising an isolated oligonucleotide donor template and, optionally, a pharma- ceutically acceptable carrier.

[0017] In one aspect, disclosed herein is a vector comprising an isolated oligonucleotide donor template.In one embodiment, the vector is an AAV vector, a lentivirus, a naked DNA vector, or a lipid nanoparticle.In one embodiment, the AAV vector is an AAV6 vector.

[0018] In one aspect, disclosed herein is an isolated gRNA molecule comprising any one of SEQ ID NOs: 18-25. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 18. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 19. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 20. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 21. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 22. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 23. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 24. In one embodiment, the isolated gRNA molecule comprises SEQ ID NO: 25.

[0019] In one aspect, a genome editing system is disclosed herein, comprising an isolated oligonucleotide donor template. In one embodiment, the genome editing system further comprises an RNA-guided nuclease and at least one gRNA molecule. In one embodiment, the genome editing system further comprises an isolated gRNA molecule disclosed herein.

[0020] In one aspect, disclosed herein is a method of modifying a cell comprising contacting the cell with a genome editing system disclosed herein, thereby modifying the cell.

[0021] In one aspect, disclosed herein is a kit comprising a genome editing system.

[0022] In one aspect, disclosed herein are isolated oligonucleotide donor templates, compositions, vectors, isolated gRNA molecules, gene editing systems, methods or kits for use in medicine.

[0023] In one aspect, disclosed herein is a method of modifying a cell comprising forming at least one single- or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an modified CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm, thereby modifying the cell.

[0024] In one aspect, disclosed herein is a method of modifying a cell comprising forming at least one single or double stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an modified CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm substantially identical to the first homologous arm, a first stuffer, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, a second stuffer, and a second donor homologous arm substantially identical to the second homologous arm.

[0025] In one aspect, disclosed herein is a method of modifying a cell comprising forming at least one single or double stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an modified CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm substantially identical to the first homologous arm, a first stuffer, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, a reporter, a second stuffer, and a second donor homologous arm substantially identical to the second homologous arm, thereby modifying the cell.

[0026] In one aspect, disclosed herein is a method of modifying a cell, the method comprising forming at least one single- or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an modified CD40L nucleic acid, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, a reporter, and a second donor homologous arm substantially identical to the second homologous arm.

[0027] In one embodiment, the cargo comprises all or part of: exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or exons 4 and 5 of the CD40L gene.

[0028] In one embodiment, the CD40L gene is a wild-type CD40L sequence. In one embodiment, the target CD40L nucleic acid comprises a mutation.

[0029] In one embodiment, the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase. In one embodiment, the reporter may be a marker that can be detected in living cells using standard methods in the art, such as a surface antigen detectable by an antibody, a peptide that catalyzes or otherwise promotes a chemical reaction that produces an optically detectable product, such as luciferase. In another embodiment, the reporter is a selectable marker. In an exemplary embodiment, the reporter is NGFR.

[0030] In one embodiment, the step of forming at least one single-stranded or double-stranded break comprises contacting the cell with an RNA-guided nuclease. In one embodiment, the RNA-guided nuclease is a class 2 clustered regularly interspersed repeat (CRISPR)-associated nuclease. In one embodiment, the RNA-guided nuclease is selected from the group consisting of wild-type Cas9, Cas9 nickase, wild-type Cpf1, and Cpf1 nickase.

[0031] In one embodiment, contacting the RNA-guided nuclease with the cell comprises introducing a ribonucleoprotein (RNP) complex comprising the RNA-guided nuclease and a guide RNA (gRNA) into the cell. In one embodiment, recombining an exogenous oligonucleotide donor template into the target CD40L nucleic acid by homologous recombination comprises introducing the exogenous oligonucleotide donor template into the cell. In one embodiment, the introducing comprises electroporation of the cell in the presence of the RNP complex and / or the exogenous oligonucleotide donor template.

[0032] In one aspect, a method of modifying a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site is provided, comprising contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, the exogenous oligonucleotide donor template comprising a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' to 5' end of the gRNA molecule, and a second homologous arm 3' to the cleavage site. Disclosed herein are methods for modifying a target CD40L nucleic acid in a cell, the method comprising the steps of: a gRNA molecule and an RNA-guided nuclease molecule interacting with a target CD40L nucleic acid, the target CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm, resulting in a cleavage event at or near the cleavage site, and the cleavage event being repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid.

[0033] In one aspect, a method of modifying a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site is provided, comprising contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a first stuffer, a cargo comprising one or more of exons 2-5 of the CD40L gene, a second homologous arm 3' to the cleavage site, and a gRNA molecule comprising at least one gRNA molecule. Disclosed herein are methods of modifying a target CD40L nucleic acid in a cell, the method comprising the steps of: a gRNA molecule and an RNA-guided nuclease molecule interacting with a target CD40L nucleic acid, the target CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, a second stuffer, and a second donor homologous arm that is substantially identical to the second homologous arm, resulting in a cleavage event at or near the cleavage site, and the cleavage event being repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid.

[0034] In one aspect, a method of modifying a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site is provided, comprising contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a first stuffer, a cargo comprising one or more of exons 2-5 of the CD40L gene, a second homologous arm 3' to the cleavage site, and a gRNA molecule comprising at least one gRNA molecule. Disclosed herein are methods of modifying a target CD40L nucleic acid in a cell, the method comprising the steps of: a gRNA molecule and an RNA-guided nuclease molecule interacting with a target CD40L nucleic acid, the target CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, a reporter, a second stuffer, and a second donor homologous arm that is substantially identical to the second homologous arm, resulting in a cleavage event at or near the cleavage site, and the cleavage event being repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid.

[0035] In one aspect, a method of modifying a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site is provided, comprising contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, the exogenous oligonucleotide donor template comprising a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' to 5' end of the gRNA molecule, and a second homologous arm 3' to the cleavage site. Disclosed herein are methods for modifying a target CD40L nucleic acid in a cell, the method comprising the steps of: a gRNA molecule and an RNA-guided nuclease molecule interacting with a target CD40L nucleic acid, the target CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, a reporter, and a second donor homologous arm that is substantially identical to the second homologous arm, resulting in a cleavage event at or near the cleavage site, and the cleavage event being repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid.

[0036] In one embodiment, the method further comprises (d) contacting the cell with a second gRNA molecule, where the second gRNA molecule and the RNA-guided nuclease molecule interact with the target nucleic acid resulting in a second cleavage event at or near the cleavage site, and where the second cleavage event is repaired by at least one DNA repair pathway.

[0037] In one embodiment, the cargo comprises all or part of: exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or exons 4 and 5 of the CD40L gene.

[0038] In one embodiment, the CD40L gene is a wild-type CD40L sequence. In one embodiment, the target CD40L nucleic acid comprises a mutation.

[0039] In one embodiment, the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase. In one embodiment, the reporter may be a marker that can be detected in living cells using methods standard in the art, such as a surface antigen detectable by an antibody, a peptide that catalyzes or otherwise promotes a chemical reaction that produces an optically detectable product, such as luciferase. In another embodiment, the reporter is a selectable marker. In an exemplary embodiment, the reporter is NGFR.

[0040] In one embodiment, the modified CD40L nucleic acid comprises, from 5' to 3', a first donor homologous arm, cargo, 3' UTR, polyA tail, and a second donor homologous arm. In one embodiment, the modified CD40L nucleic acid comprises, from 5' to 3', a first donor homologous arm, first stuffer, cargo, 3' UTR, polyA tail, second stuffer, and a second donor homologous arm. In one embodiment, the modified CD40L nucleic acid comprises, from 5' to 3', a first donor homologous arm, first stuffer, cargo, 3' UTR, polyA tail, reporter, second stuffer, and a second donor homologous arm. In one embodiment, the modified CD40L nucleic acid comprises, from 5' to 3', a first donor homologous arm, cargo, 3' UTR, polyA tail, reporter, second stuffer, and a second donor homologous arm.

[0041] In one embodiment, the cell is first contacted with at least one gRNA molecule and an RNA-guided nuclease molecule, and then the cell is contacted with an exogenous oligonucleotide donor template. In one embodiment, the cell is contacted with at least one gRNA molecule, an RNA-guided nuclease molecule, and an exogenous oligonucleotide donor template simultaneously.

[0042] In one embodiment, the exogenous oligonucleotide donor template is a single-stranded oligonucleotide donor template. In one embodiment, the exogenous oligonucleotide donor template is a double-stranded oligonucleotide donor template.

[0043] In one embodiment, the exogenous oligonucleotide donor template is present in a vector. In one embodiment, the vector is a viral vector. In one embodiment, the viral vector is an AAV vector or a lentiviral vector.

[0044] In one embodiment, the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template. In one embodiment, the cleavage event, or both the cleavage event and the second cleavage event, are repaired by genetic correction.

[0045] In one embodiment, the altered CD40L nucleic acid comprises a sequence that differs from the sequence of the target CD40L nucleic acid.

[0046] In one embodiment, the gRNA molecule is a gRNA nucleic acid and the RNA-guided nuclease molecule is an RNA-guided nuclease protein. In one embodiment, the gRNA molecule is a gRNA nucleic acid and the RNA-guided nuclease molecule is an RNA-guided nuclease nucleic acid. In one embodiment, the cell is contacted with the gRNA molecule and the RNA-guided nuclease molecule as a preformed complex. In one embodiment, the RNA-guided nuclease is selected from the group consisting of wild-type Cas9, Cas9 nickase, wild-type Cpf1 and Cpf1 nickase.

[0047] In one embodiment, the cell is a eukaryotic cell. In one embodiment, the eukaryotic cell is a human cell. In one embodiment, the cell is a T cell. In one embodiment, the cell is a hematopoietic stem cell (HSC).

[0048] In one embodiment, the cell is derived from a subject suffering from hyper-IgM syndrome. In one embodiment, the cell is derived from a subject having at least one mutation at the cleavage site. In one embodiment, the method further comprises isolating the cell from the subject before the forming step or the contacting step. In one embodiment, the method further comprises introducing the cell into the subject after the recombination step or after the cleavage event is repaired by at least one DNA repair pathway.

[0049] In one embodiment, the forming and recombining, or contacting, steps are performed in vitro. In one embodiment, the forming and recombining, or contacting, steps are performed ex vivo. In one embodiment, the forming and recombining, or contacting, steps are performed in vivo.

[0050] In one embodiment, the recipient subject is conditioned prior to administration of the edited cells to the subject. Conditioning may include lymphodepletion of the subject. For example, in one embodiment, prior to receipt of the edited cells, the subject is conditioned with chemotherapy that causes lymphodepletion. In one embodiment, the subject's T cells are depleted by conditioning. Agents suitable for conditioning the subject include agents that induce lymphodepletion, e.g., depletion of T cells. In one embodiment, the conditioning agent is cyclophosphamide. In some embodiments, the method may optionally include administering to the subject a conditioning agent, e.g., a conditioning agent that causes lymphodepletion. In one embodiment, the conditioning agent is a chemotherapeutic agent, e.g., cyclophosphamide, fludarabine, busulfan, treosulfan, or a combination thereof. In one embodiment, the conditioning agent is a polyclonal or monoclonal depleting antibody, e.g., anti-thymocyte globulin (ATG), anti-CD3, anti-CD4, anti-CD52, anti-CD2, anti-TCRαβ, anti-IL2Rα, or a combination thereof. In another embodiment, the subject is not conditioned prior to administration of the edited cells to the subject.

[0051] In one embodiment, the cells are stimulated prior to administration to a subject, hi one embodiment, the cells are stimulated using a cytokine, such as IL-7, IL-15, IL-2, or a combination thereof.

[0052] In one embodiment, class switching is restored in the subject. In one embodiment, the level of IgM is reduced in the subject. In one embodiment, the level of IgG is increased in the subject.

[0053] In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 10% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 15% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 20% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 30% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 40% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 50% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 60% of the cells of the cell population. In one embodiment, the cell is a cell population and the DNA repair pathway repairs the target CD40L nucleic acid resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 75% of the cells of the cell population.

[0054] In one aspect, disclosed herein are cells altered by the methods disclosed herein. In one aspect, disclosed herein are cell populations altered by the methods disclosed herein.

[0055] In one embodiment, the cells are hematopoietic stem cells (HSCs), and at least about 10% of the cells in a population of HSCs contain an altered CD40L nucleic acid that is a functional CD40L allele, hi one embodiment, the cells are T cells.

[0056] In one aspect, disclosed herein is a pharmaceutical composition comprising the cell or cell population.

[0057] In one aspect, disclosed herein is a method of treating a subject having hyper-IgM syndrome by altering a target CD40L in a cell, the method comprising: forming at least one single- or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm substantially identical to the second homologous arm, thereby treating a subject having hyper-IgM syndrome.

[0058] In one aspect, a method of treating a subject with hyper-IgM syndrome by altering a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site is provided, comprising contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' to 5' end of the cleavage site, and a second homologous arm 3' to the cleavage site. Disclosed herein is a method of treating a subject having hyper-IgM syndrome, comprising: a target CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm, wherein a gRNA molecule and an RNA-guided nuclease molecule interact with a target CD40L nucleic acid resulting in a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid.

[0059] In one aspect, disclosed herein is a method of restoring B cell class switching in a subject by altering a target CD40L in a cell, the method comprising: forming at least one single- or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm substantially identical to the second homologous arm, thereby restoring B cell class switching in the subject.

[0060] In one aspect, a method of restoring B cell class switching in a subject by modifying a target CD40L nucleic acid in a cell to comprise a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site comprises contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' to 5' end of the cleavage site, and a second homologous arm 3' to the cleavage site. Disclosed herein is a method of targeting a CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm, wherein a gRNA molecule and an RNA-guided nuclease molecule interact with a target CD40L nucleic acid resulting in a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid, thereby restoring B cell class switching in a subject.

[0061] In one aspect, disclosed herein is a method of reducing levels of IgM in a subject by altering a target CD40L in a cell, the method comprising: forming at least one single- or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm substantially identical to the second homologous arm, thereby reducing levels of IgM in the subject.

[0062] In one aspect, a method of reducing levels of IgM in a subject by altering a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site comprises contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' to 5' end of the cleavage site, and a second homologous arm 3' to the cleavage site. Disclosed herein is a method of reducing the level of IgM in a subject, comprising: a target CD40L nucleic acid comprising a CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm; a gRNA molecule and an RNA-guided nuclease molecule interacting with a target CD40L nucleic acid resulting in a cleavage event at or near the cleavage site; and the cleavage event being repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid, thereby reducing the level of IgM in a subject.

[0063] In one aspect, disclosed herein is a method of increasing levels of IgG in a subject by altering a target CD40L in a cell, the method comprising: forming at least one single- or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, wherein the target CD40L nucleic acid comprises a first homologous arm 5' of the cleavage site and a second homologous arm 3' of the cleavage site; and recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, the exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm substantially identical to the second homologous arm, thereby increasing levels of IgG in the subject.

[0064] In one aspect, a method of increasing a level of IgG in a subject by altering a target CD40L nucleic acid in a cell comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site comprises contacting the cell with (a) at least one gRNA molecule, (b) an RNA-guided nuclease molecule, and (c) an exogenous oligonucleotide donor template, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' to 5' end of the cleavage site, and a second homologous arm 3' to the cleavage site. Disclosed herein are methods for increasing levels of IgG in a subject, comprising: a CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm, wherein a gRNA molecule and an RNA-guided nuclease molecule interact with a target CD40L nucleic acid, resulting in a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid, thereby increasing the level of IgG in a subject.

[0065] In one aspect, disclosed herein is a cell population characterized in that at least 10% of the cells of the population comprise at least one copy of an engineered CD40L sequence. In one embodiment, at least 15% of the cells of the population comprise at least one copy of an engineered CD40L sequence. In one embodiment, at least 20% of the cells of the population comprise at least one copy of an engineered CD40L sequence. In one embodiment, at least 30% of the cells of the population comprise at least one copy of an engineered CD40L sequence. In one embodiment, at least 40% of the cells of the population comprise at least one copy of an engineered CD40L sequence. In one embodiment, at least 50% of the cells of the population comprise at least one copy of an engineered CD40L sequence. In one embodiment, at least 60% of the cells of the population comprise at least one copy of an engineered CD40L sequence.

[0066] In one embodiment, at least one copy is inserted into an intron of the endogenous CD40L gene, hi one embodiment, the cell population or progeny of the cell population are capable of restoring class switching in a subject suffering from hyper-IgM syndrome.

[0067] In one aspect, disclosed herein is an isolated cell population, wherein at least about 5% to about 100% of the cells of the cell population express a functional CD40L allele, and wherein the cell population is isolated from a subject previously diagnosed with hyper-IgM syndrome. In one embodiment, at least about 6% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 7% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 8% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 9% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 10% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 15% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 20% of the cells of the cell population express a functional CD40L allele. In one embodiment, at least about 25% of the cells in the cell population express a functional CD40L allele.

[0068] The accompanying drawings provide illustrative and schematic, rather than comprehensive, examples of certain aspects and embodiments of the present disclosure. The drawings are not limited or bound to any particular theory or model, and are not necessarily scaled. Without limiting the above, nucleic acids and polypeptides can be represented as linear sequences or as schematic two- or three-dimensional structures; these depictions are illustrative and are not limited or bound to any particular model or theory regarding their structure. [Brief description of the drawings]

[0069] [Figure 1A-D]

[0023] Figure 1A depicts an exemplary CD40L donor template configuration for gene editing. Figure 1A depicts a construct comprising homologous arms, CD40L cDNA, GFP, and a stuffer sequence. Figure 1B depicts a construct comprising homologous arms, CD40L cDNA, and GFP, but without a stuffer sequence. Figure 1C depicts a construct comprising homologous arms, CD40L cDNA, and a stuffer sequence, but without GFP. Figure 1D depicts a construct comprising homologous arms and CD40L cDNA, but without GFP and without a stuffer sequence. [Figure 2A]Figure 2 shows that the genome editing system described herein resulted in high levels of CD40LG gene editing. Specifically, Figure 2A is a schematic diagram of an exemplary construct for CD40L gene editing. Figure 2B shows the targeted location in CD40LG intron 1 of 8 S. pyogenes and 30 S. aureus gRNAs screened for cleavage efficacy of CD40LG in primary human T cells. Figure 2C shows the editing percentage including insertion / deletion rate for the exemplary gRNA from Figure 2B delivered with nuclease as RNP complex to primary human T cells. Figure 2D shows the experimental procedure of CD40L gene editing in T lymphocytes. Figure 2E shows the percentage of GFP-positive cells in the T cell population after gene editing. T stem memory cells (TSCM) are defined as CD62L+CD45RA+, T central memory (TCM) as CD62L+CD45RA-, T effector memory (TEM) as CD62L-CD45RA-, and terminal effector (TEMRA) as CD62L-CD45RA+. Figure 2F shows representative plots of T cells expressing CD45RA and CD62 by FACS analysis after 7 days (left panel) and 20 days (right panel) of treatment. Figure 2G shows the T cell culture composition after 7 and 19 days (left panel) and 20 days (right panel) of treatment as measured by FACS analysis. Figure 2H shows a schematic protocol for gene editing in T cells using donor templates provided by AAV6 or IDLV. Figure 2I shows the results of HDR in various T cell subpopulations (assessed as the percentage of GFP+ cells). Figure 2J shows the levels of gene editing in HD and patient cells. [Figure 2B] This is a continuation of Figure 2A. [Figure 2C-D] This is a continuation of Figure 2B. [Figure 2E-F] Continuation of Figures 2C-D. [Figure 2G] Continuation of Figures 2E-F. [Figure 2H-I] Continuation of Figure 2G. [Figure 2J] This is a continuation of Figure 2H-I. [Figure 3A-C] FIG. 3 depicts targeted integration and CD40L expression after editing with three exemplary gRNAs (gRNA A, D, and G). FIG. 3A depicts the location of exemplary gRNAs within CD40LG intron 1. FIG. 3B depicts the experimental procedure and timeline for the experiments presented in FIGS. 3D-3E. FIG. 3C provides the rate of targeted integration (% of GFP+ cells) after treatment with the indicated RNP complexes and AAV6 donor templates. FIG. 3D depicts CD40L expression in the GFP-positive cell population as determined by FACS analysis. FIG. 3E depicts the percentage of CD40L+ cells at various time points after restimulation. [Fig. 3D-E] Continuation of Figures 3A-C. [Figure 4A-C] FIG. 4 shows that edited CD40L preserves physiological regulation. Specifically, FIG. 4A and 4B show the constructs and experimental procedures used for CD40L gene editing and expression in T lymphocytes. FIG. 4C and 4E show the kinetics of CD40L expression in edited T cells after induction of CD40L surface expression by polyclonal stimulation (PMA / ionomycin). FIG. 4D shows representative plots showing expression of CD40L in activated CD4+ and CD8+ cells. FIG. 4F shows representative plots showing expression of CD40L in stimulated CD4+ cells. [Fig. 4D-F] Continuation of Figures 4A-C. [Figure 5A-B] Figure 5 shows that edited CD40L preserves physiological regulation. Specifically, Figures 5A and 5B show the constructs and experimental procedures used for CD40L gene editing and expression in T lymphocytes. Figure 5C shows the expression of CD40L and CD8 in wild-type and edited T cells. Figure 5D shows the kinetics of CD40L expression in edited T cells. Figure 5E shows the effect of AAV6 on gene editing efficiency. [Figure 5C] This is a continuation of Figures 5A-B. [Figure 5D-E] This is a continuation of Figure 5C. [Figure 6A]FIG. 6 demonstrates that gene editing produces high levels of CD40L gene editing (FIG. 6A) and high levels of exogenous CD40L expression (FIG. 6B) using different transgene configurations (wild-type cDNA (no recoding) or codon-optimized (recoded) sequences; endogenous polyadenylation signal or viral SV40 polyadenylation signal). [Figure 6B] This is a continuation of Figure 6A. [Figure 7A-B] Figure 7 depicts the results of an experiment evaluating the specificity of an exemplary gRNA targeting CD40LG. Figure 7A presents Guide-Seq analysis of gRNA A and gRNA D. Figures 7B and 7E validate the on-target and off-target editing observed with gRNA A using Amplicon-Seq analysis. Figures 7C-7D present the results of a Digenome analysis to identify potential off-target sites for gRNA A and gRNA D. Figure 7F represents a summary of the results of an Amplicon-Seq analysis for the off-target cleavage sites identified for gRNA A and D. [Figure 7C-E] This is a continuation of Figures 7A-B. [Figure 7F] Continuation of Figures 7C-E. [Figure 8] Figure 1 shows that CD40L-edited T cells restored B cell class switching capacity in an in vitro co-culture assay. The histograms show the percentage of B cells that expressed IgG on their surface after 5 days of co-culture with wild-type T cells (unedited), T cells with an edited CD40L gene (GFP+), T cells treated for gene editing but negative for integration of the corrective transgene (GFP-), or T cells in which the endogenous CD40L gene was knocked out by integration of a reporter cassette. Cultures with B cells alone and with B cells plus soluble CD40L protein were used as negative and positive controls, respectively. All T cells were tested without any stimulation (resting) or after stimulation with anti-CD3-CD28 beads (beads) or PMA / ionomycin. [Figure 9]FIG. 13 depicts reduced levels of IgM and increased levels of IgG in culture medium of B cells co-cultured with T cells harboring an edited CD40L locus. [Figure 10] FIG. 1 depicts baseline levels of targeted integration at four gRNA cleavage sites using an AAV6 donor template containing the following cargo: stuffer sequence, CD40LG cDNA / 3' UTR / polyA, and a GFP expression cassette. The predicted cleavage sites were flanked by short homology arms. Differences in baseline levels of targeted integration between gRNAs were detected based on GFP expression. [Figure 11] Figure 10 compares the levels of targeted integration obtained using donor templates with various homology arm lengths. AAV6 donor templates were prepared as in Figure 10, with the homology arms extended to 500 base pairs on each side of the cargo sequence. Increasing the length of the homology arms increased the levels of targeted integration for both gRNA cleavage sites tested. [Figure 12A-B] Figure 12 depicts the phenotype of transferred T cells at different time points post-injection. Figure 12A shows the percentages of CD8 and CD4 cells measured in the circulating wild-type T cell population. Figure 12B shows the percentages of effector (CD44+CD62L-), central memory (TCM, CD44+CD62L+), naive (CD44-CD62L+), and early effector (CD44-CD62L-) T cells measured in the CD8 and CD4 fractions of circulating wild-type T cells. [Figure 13]Figure 1 depicts adoptive T cell therapy in CD40L KO mice using wild type T cells transplanted with or without cyclophosphamide preconditioning regimen. The graph on the left represents the percentage of recipient and donor T cells over time in mice treated with or without the preconditioning regimen. The dot plots on the right represent the amount of antigen-specific IgG in the serum of transplanted animals 14 days before and 28 days after immunization of transplanted mice with trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) antigen. HIGM1 mice were used as negative controls. Mice transplanted with a full dose of wild type hematopoietic stem cells (HSCT) were used as positive controls. [Figure 14A-B] Figure 14 shows the response to vaccination of HIGM1 mice transplanted with wild-type T cells. Figure 14A shows the amount of antigen-specific IgG response in the serum of HIGM1 mice transplanted with or without different dose adjustments, before and after two boosts with TNP-KLH antigen. Figure 14B shows a correlation graph between serum levels of TNP / KLH-specific IgG and the amount of circulating wild-type CD4 T cells (percentages on top, absolute numbers on bottom). [Fig. 14C-D] Figure 14 depicts adoptive T cell therapy in CD40L KO mice using wild type T cells transferred with or without anti-leukocyte (ALS) or anti-CD4 antibody preconditioning regimen. Figures 14C and 14E depict the percentage of recipient and donor T cells over time in mice treated with or without preconditioning regimen. The level of specific IgG response to vaccination correlates with the conditioning dose administered (Figure 14D). [Figure 14E] Continuation of Figures 14C-D. [Figure 15A] Figure 15 depicts immunohistochemical analysis of GC B cells in spleen sections of adoptively transferred mice from Figure 14 after TNP-KLH immunization. Figure 15A depicts immunohistochemical staining analysis of GC B cells in spleen sections, with peanut agglutinin in brown and hematoxylin counterstain in blue. Figure 15B depicts a histogram quantifying the immunohistochemical analysis shown in Figure 15A. [Figure 15B]This is a continuation of Figure 15A. [Figure 16A] Figure 16 depicts hematopoietic stem cell therapy in a mouse model of hyper-IgM syndrome. As shown in Figure 16A, different doses of CD40LG wild-type hematopoietic stem cells harvested from wild-type C57BL / 6 mice were transplanted in different ratios together with CD40LG negative HSPCs harvested from HIGM1 mice in recipient HIGM1 mice prior to immunization with thymus-dependent antigen (TNP-KLH). Dot plots represent the amount of TNP-KLH-specific IgG in the serum of transplanted animals 14 days before or after, and 28 days before or after, immunization of transplanted mice with TNP-KLH antigen. Figure 16B depicts a second experiment in which different percentages of wild-type HSPCs and CD40LG negative HSPCs were transplanted into CD40L mice as described for Figure 16A. Mice were immunized with TNP-KLH and then with ovalbumin. Dot plots represent the amount of TNP-specific IgG (left panel) and OVA-specific IgG (right panel) before and after immunization. Figure 16C shows donor T cell engraftment in peripheral blood measured at different time points after transplantation. [Figure 16B] This is a continuation of Figure 16A. [Figure 16C] This is a continuation of Figure 16B. [Figure 17A-D]Figure 17 depicts high polyclonal CD40L targeting in both healthy donor (HD) and patient derived T cells. CD4 T cells were collected from different healthy donors (HD, n=4) or patients with HIGM1 induced mutations in exon 3 (patient) stimulated in vitro and treated with AAV6 at 25 pmol RNP A (Sp) and MOI: 5*10^4 vg / cell. Figure 17A shows the percentage of GFP+ edited cells measured in different T cell subpopulations after 17 days of treatment. Figure 17B shows the culture composition measured by cytofluorometric analysis (GFP marking). Figure 17C compares gene editing efficiency measured by ddPCR molecular analysis (left bar) and by cytofluorometric analysis (GFP by FACS, right bar). Figure 17D shows TCR diversity in untreated patient cells (grey / right panel) and edited patient cells (black / left panel) assessed by 5 different multiplex PCR reactions identifying 21 families of TCR-B. [Figure 18A-B] Figure 18 shows that targeted integration of a correction construct restored expression and physiological regulation of CD40L. Figure 18A shows representative flow cytometry dot plots depicting CD40L and GFP expression after 8 hours of PMA / ionomycin (P / I) stimulation in HD and patient cells from Figure 17. Figure 18B shows the time course of CD40L surface expression as relative fluorescence intensity (RFI, calculated as a ratio to TO) and percentage, measured 0, 3, 8, 24 and 48 hours after P / I activation. [Figure 19A-B] Figure 19 depicts the ability of CD40L-edited T cells to restore B cell proliferation in co-culture assays. Figure 19A shows a schematic of the T and B cell co-culture assay protocol. Figures 19B and 19C depict flow cytometry plots and histograms, respectively, showing the percentage of proliferating B cells in co-culture with resting bead-activated (1:1 ratio) or PMA / ionomycin-stimulated T cells (untreated (UT) or edited (GFP+)). Negative control: B cells only; positive control: B cells + soluble CD40L. [Figure 19C] This is a continuation of Figures 19A-B. [Figure 20A]Ability of CD40L-edited T cells to restore B cell class switching in co-culture assays. Figures 20A and 20B show Elispot images and histograms, respectively, showing the percentage of IgG-switched B cells in co-culture with resting, bead-activated (1:1 ratio) or PMA / ionomycin-stimulated T cells (naive (UT) or edited (GFP+)). Negative control: B cells only; Positive control: B cells + soluble CD40L. [Figure 20B] This is a continuation of Figure 20A. [Figure 21A-D] FIG. 21A shows that the genome editing system described herein resulted in high levels of CD40LG gene editing in human hematopoietic stem / progenitor cells (HSPCs). FIG. 21A is a schematic of the donor construct used to edit CD34+ cells. FIG. 21B, FIG. 21C, and FIG. 21E show the percentage of targeted integration in bulk treated CD34 cells and the indicated sorted subpopulations, respectively, as measured by digital droplet PCR analysis performed on the 5′ vector to the genome junction. FIG. 21D shows the culture composition of treated and untreated (UT) cells. FIG. 21F shows the percentage of hCD45+ cells (left panel) and the percentage of HDR cells (right panel) in the grafts, measured at different time points after transplantation. [Fig. 21E-F] Continuation of Figures 21A-D. [Figure 22A-C]FIG. 22 depicts the selection strategy developed to enrich for CD40LG-edited T cells. FIG. 22A depicts a donor DNA construct with a modified CD40LG gene, an internal ribosome entry site (IRES) sequence, and a selection gene (e.g., delta-LNGFR). FIG. 22B depicts a schematic of the targeted integration protocol, showing different time points at which expression of the selection gene was assessed by cytofluorometric analysis. FIG. 22C depicts reporter gene expression at the indicated time points (days 6, 13, and 20) in CD4 and CD8 T cells treated as described in FIG. 22B. FIG. 22D depicts the kinetics of CD40L expression after restimulation of T cells treated for gene editing. FIG. 22E depicts the expression levels (mean fluorescence intensity, MFI) of the CD40L gene (left) and the percentage of CD40L+ cells in untreated NGFR+ and NGFR- T cell populations. Figure 22F depicts the ability of CD40L-edited T cells to restore B cell class switching in co-culture assays, showing the percentage of IgG-switched B cells in co-culture with resting, bead-activated (1:1 ratio) or PMA / ionomycin-stimulated T cells (naive (UT) or edited (NGFP+)). Negative control: B cells only; Positive control: B cells + soluble CD40L. [Fig. 22D-E] Continuation of Figures 22A-C. [Figure 22F] This is a continuation of Figure 22D-E. [Figure 23A-C] Figure 23 shows the selection of CD40LG edited cells using NGFR selection gene. Figure 23A shows the percentage of NGFR+ cells obtained after gene editing measured among different T cell subpopulations (from left to right: terminal effector (TEMRA), CD45RA+ CD62L-; effector memory (TEM), CD62L- CD45RA-; central memory (TCM), CD45RA-CD62L+; and T stem memory cells (TSCM), CD45RA+CD62L+). Figure 23B shows the percentage of T cell subpopulations measured before and after NGFR selection. Figure 23C shows the percentage of CD40LG gene editing measured by molecular analysis (ddPCR) or NGFR expression before and after NGFR selection. [Figure 24A]Figure 24 depicts engraftment of CD40LG edited cells following xenotransplantation in NSG mice. Figure 24A depicts the percentage of human cells (hCD45+ cells; top left and bottom left and right panels) and the percentage of NGFR+ cells (top right panel) within the human grafts measured at different time points following transplantation. Figure 24B depicts the proportion of T cell subpopulations (terminal effector (TEMRA), CD45RA+ CD62L-; effector memory (TEM), CD62L- CD45RA-; central memory (TCM), CD45RA-CD62L+; and T stem memory cells (TSCM), CD45RA+CD62L+) of engrafted T cells measured 4, 8, and 16 weeks after transplantation. Figure 24C depicts the level of CD40LG expression and the percentage of CD40LG+ cells recovered from NSG mice. [Figure 24B] This is a continuation of Figure 24A. [Figure 24C] This is a continuation of Figure 24B. [Figure 25A-C] Figure 25 depicts an experiment demonstrating that HSPC editing is transferable to the CRISPR / Cas platform. Figure 25A presents a protocol for editing CD34+ cells obtained from umbilical cord blood. Figure 25B depicts the targeted integration efficiency (expressed as a percentage of GFP+ cells) in various cell populations obtained through gene editing using ZFN or RNP. Figure 25C depicts the culture composition after gene editing using ZFN or RNP compared to untreated cells (UT). Figure 25D depicts the fold induction of interferon (IFN)-responsive genes RIG1, IRF7, and OAS1 following treatment with RNP, ZFN, Cas9 alone, or RNP-mod compared to untreated cells (UT). [Figure 25D] Continuation of Figures 25A-C. [Figure 26A-B]Figure 26 depicts further experiments demonstrating that HSPC editing is transferable to the CRISPR / Cas platform. Figure 26A presents a protocol for editing CD34+ cells from cord blood (CB), bone marrow (BM) or mobile peripheral blood (MPB). Figure 26B depicts the targeted integration efficiency (expressed as a percentage of GFP+ cells) in various cell populations obtained using the indicated editing procedure. Figure 26C depicts the culture composition after editing. Figure 26D depicts the expression of IFN-responsive genes RIG1, IRF7 and OAS1 in edited cells. [Fig. 26C-D] Continuation of Figures 26A-B. [Figure 27A-B] Figure 27 depicts data showing that human HSPCs from various sources can be efficiently edited using various donor vehicles and nuclease configurations. Figure 27A depicts the percentage of edited (GFP+) cell subpopulations using ZFNs or RNPs with IDLV or AAV6 donor templates. Figure 27B depicts the percentage of edited (GFP+) cell subpopulations using CD34+ cells derived from umbilical cord blood, bone marrow, and mobilized peripheral blood (mPB). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Definitions and Abbreviations Unless otherwise stated, each of the following terms has the meaning associated with it in this section.

[0071] The indefinite articles "a" and "an" refer to at least one of the associated noun and are used interchangeably with the terms "at least one" and "one or more." For example, "a module" means at least one module or one or more modules.

[0072] The conjunctions "or" and "and / or" are used interchangeably as a non-exclusive logical or.

[0073] "Alt-HDR", "alternative homology-guided repair" or "alternative HDR" are used interchangeably to refer to the process of repairing DNA damage using homologous nucleic acid (e.g., endogenous homologous sequences, e.g., sister chromatids, or exogenous nucleic acid, e.g., template nucleic acid). Alt-HDR differs from canonical HDR in that the process utilizes a different pathway than canonical HDR, and can be inhibited by canonical HDR mediators, RAD51 and BRCA2. Alt-HDR is also distinguished by the involvement of a single-stranded or nicked homologous nucleic acid template, whereas canonical HDR generally involves a double-stranded homologous template.

[0074] "Canonical HDR", "canonical homology-induced repair" or "cHDR" refers to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, e.g., a sister chromatid, or an exogenous nucleic acid, e.g., a template nucleic acid). Canonical HDR generally operates when there is substantial excision at the double-stranded break, forming at least one single-stranded portion of DNA. In normal cells, cHDR generally involves a series of steps, such as recognition of the break, stabilization of the break, excision, stabilization of single-stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation. This process requires RAD51 and BRCA2, and the homologous nucleic acid is generally double-stranded.

[0075] Unless otherwise specified, the term "HDR" as used herein encompasses both regular HDR and Alt-HDR.

[0076] As used herein, the term "cleavage event" refers to a cut in a nucleic acid molecule. The cleavage event may be a single-stranded or double-stranded cleavage event. A single-stranded cleavage event may result in a 5' overhang or a 3' overhang. A double-stranded cleavage event may result in a blunt end, two 5' overhangs or two 3' overhangs.

[0077] The term "cleavage site" as used herein with respect to a site on a target nucleic acid sequence, e.g., a target CD40L nucleic acid sequence, refers to a target location where a double-stranded break occurs between two nucleotide residues of the target nucleic acid, or a target location within a few nucleotide residues of the target nucleic acid where two single-stranded breaks occur, mediated by an RNA-guided nuclease-dependent process. The cleavage site may be, for example, a target location for a blunt double-stranded break. Alternatively, the cleavage site may be, for example, a target site within a few nucleotide residues of the target nucleic acid for two single-stranded breaks or nicks that form a double-stranded break and are separated by, for example, about 10 base pairs. The closer of the double-stranded break or the two single-stranded nicks in the pair is ideally within 0-500 bp from the target location (e.g., 450 or less, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target location). When dual nickases are used, the two nicks in the pair are within 25-55 bp of each other (e.g., 25-50, 25-45, 25-40, 25-35, 25-30, 50-55, 45-55, 40-55, 35-55, 30-55, 30-50, 35-50, 40-50, 45-50, 35-45 or 40-45 bp) and are no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).

[0078] The phrase "consisting essentially of" means that the recited species is the predominant species, but other species may be present in minor amounts, or in amounts that do not affect the structure, function, or behavior of the subject composition. For example, a composition that consists essentially of a particular species will typically contain 90%, 95%, 96% or more of that species.

[0079] "Domain" is used to describe a segment of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional properties.

[0080] As used herein, the term "endogenous" gene, "endogenous" nucleic acid or "endogenous" homologous region refers to a naturally occurring gene, nucleic acid or genetic region that is in its natural location in the genome of a cell, e.g., a chromosome or a plasmid. In contrast, the term "exogenous" gene or "exogenous" nucleic acid refers to a gene, nucleic acid or genetic region that is not naturally occurring in the cell, but that is introduced into the cell during the methods of the invention. An exogenous gene or exogenous nucleic acid may be homologous or identical to an endogenous gene or endogenous nucleic acid.

[0081] The term "exon" refers to the coding region of a gene that contains the information required to code for a protein. In eukaryotes, genes are composed of coding exons interspersed with non-coding introns. During RNA splicing, the introns are removed and the exons are covalently joined to each other to form a functional mature messenger RNA (mRNA) that can be translated into a protein. The term exon refers to both the DNA sequences in a gene and the corresponding sequences in the RNA transcript.

[0082] "Gene conversion" refers to the alteration of a DNA sequence by the integration of an endogenous homologous sequence (e.g., a homologous sequence in a gene array). "Gene correction" refers to the alteration of a DNA sequence by the integration of an exogenous homologous sequence, e.g., an exogenous single-stranded or double-stranded donor template DNA. Gene conversion and gene correction are the products of repair of DNA double-strand breaks by HDR pathways, such as those described below.

[0083] The terms "homology" or "identity", used interchangeably herein, refer to the sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being the more strict comparison. The phrases "percent identity or homology" and "% identity or homology" refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. The identity of two or more sequences can be anywhere from 0 to 100% or any value therebetween. Identity can be determined by comparing positions in each sequence, which can be aligned for comparison to a reference sequence. If a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. The degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. The degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. The degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[0084] Calculation of homology or sequence identity (these terms are used interchangeably herein) between two sequences is performed as follows: The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences.

[0085] As used herein, the term "homology arms" refers to one or more regions that are homologous to the target sequence to be cleaved, e.g., a region of DNA within or near (e.g., adjacent or proximal to) the cleavage site, e.g., a target nucleic acid.

[0086] "Indel" is an insertion and / or deletion in a nucleic acid sequence. Indels can be the product of repair of DNA double-strand breaks, such as the double-strand breaks formed by the genome editing system of the present disclosure. Indels are most commonly formed when the break is repaired by an "error-prone" repair pathway, such as the NHEJ pathway described below.

[0087] Indels, gene conversions, gene corrections, and other genome editing results are typically assessed by sequencing (most commonly by "next generation" or "sequencing by synthesis" methods, but Sanger sequencing can also be used) and quantified by the relative frequency of numerical changes (e.g., ±1, ±2 or more bases) at the site of interest among all sequencing reads. DNA samples for sequencing can be prepared by a variety of methods known in the art, including polymerase chain reaction (PCR), capture of DNA ends generated by double-strand breaks as in the GUIDEseq process described in Tsai et al. (Nat. Biotechnol. 34(5): 483 (2016), incorporated herein by reference), or amplification of the site of interest by other means known in the art. Genome editing results can also be assessed by in situ hybridization methods, such as the FiberComb™ system commercialized by Genomic Vision (Bagneux, France), and any other suitable methods known in the art.

[0088] The term "intron" refers to long stretches of non-coding DNA found between the exons (or coding regions) of genes. Introns usually start and end with specific series of nucleotides and are spliced ​​out before the RNA molecule is translated into a protein. These sequences act as boundaries between introns and exons and are known as splice sites. The recognition of the boundaries between coding and non-coding DNA is important for the formation of functional genes.

[0089] The term "isolated gene" or "isolated nucleic acid" refers to a gene or nucleic acid that is not associated with naturally associated components that accompany it in its natural state by reason of its origin or source of derivation; is substantially free of other proteins from the same species; is expressed by cells of a different species; or is not naturally occurring. Thus, a nucleic acid that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived is "isolated" from its naturally associated components. Nucleic acids can also be rendered substantially free of naturally associated components by isolation, using protein purification techniques that are well known in the art.

[0090] In the nucleotide sequences presented herein, conventional IUPAC notation is used as shown in Table 1 below (see also Cornish-Bowden A, Nucleic Acids Res. 1985 May 10; 13(9):3021-30, which is incorporated herein by reference). However, it should be noted that where the sequence can be encoded by DNA or RNA, for example in the gRNA targeting domain, "T" stands for "thymine or uracil."

[0091] JPEG2025011076000001.jpg10456

[0092] A "kit" refers to any collection of two or more components that together constitute a functional unit that can be used for a specific purpose. By way of example (and not limitation), a kit according to this disclosure may include a guide RNA that is complexed or can form a complex with an RNA-guided nuclease, together with (e.g., suspended or suspendable in) a pharma- ceutically acceptable carrier. The kit can be used, for example, to introduce the complex into a cell or subject, for the purpose of causing a desired genomic alteration in such cell or subject. The components of the kit can be packaged together, or they can be packaged separately. A kit according to this disclosure also optionally includes a Instructions for Use (DFU), for example, that describes the use of the kit according to the method of this disclosure. The DFU can be physically packaged with the kit, or it can be made available to a user of the kit, for example, by electronic means.

[0093] "Non-homologous end joining" or "NHEJ" refers to ligation-mediated repair and / or non-template-mediated repair, including canonical NHEJ (cNHEJ) and alternative NHEJ (altNHEJ), which in turn includes microhomology-mediated end joining (MMEJ), single-strand annealing (SSA) and synthesis-dependent microhomology-mediated end joining (SD-MMEJ).

[0094] The terms "poly(A)", "poly(A) tail", and "polyadenylation" refer to the addition of a poly(A) tail to messenger RNA. A poly(A) tail is a long chain of adenine nucleotides that is added to a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule. Shortly after a gene in a eukaryotic cell is transcribed, the new RNA molecule undergoes several modifications known as RNA processing. These modifications alter both ends of the primary RNA transcript to produce a mature mRNA molecule. Processing of the 3' end adds a poly(A) tail to the RNA molecule.

[0095] The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" refer to a series of nucleotide bases (also called "nucleotides") in DNA and RNA, meaning any chain of two or more nucleotides. Polynucleotides, nucleotide sequences, nucleic acids, etc. may be single-stranded or double-stranded chimeric mixtures or derivatives or modified versions thereof. They can be modified, for example, in the base moiety, sugar moiety or phosphate backbone to improve the stability of the molecule, its hybridization parameters, etc. Nucleotide sequences generally carry genetic information, including but not limited to information used by cellular machinery to make proteins and enzymes. These terms include double-stranded or single-stranded genomic DNA, RNA, any synthetic and genetically engineered polynucleotides, and sense and antisense polynucleotides. These terms also include nucleic acids containing modified bases. As used herein, "CD40L" nucleic acid refers to a series of nucleotide bases that encodes the CD40L protein.

[0096] "Prevent," "preventing," and "prevention" refer to the prevention of disease in a mammal, such as a human, and include (a) avoiding or eliminating the disease; (b) affecting predisposition to the disease; or (c) arresting or delaying the onset of at least one symptom of the disease.

[0097] The terms "protein," "peptide," and "polypeptide" are used interchangeably to refer to a continuous chain of amino acids linked together through peptide bonds. These terms include individual proteins, groups or complexes of proteins linked together, as well as fragments or portions, variants, derivatives, and analogs of such proteins. Peptide sequences are presented herein using conventional notation, beginning with the amino or N-terminus at the left and proceeding to the carboxyl or C-terminus at the right. Standard one-letter or three-letter abbreviations can be used.

[0098] When used in reference to modification of a molecule (eg, a nucleic acid or protein), "substitution" or "substituted" does not require a process restriction but merely indicates that a replacement entity is present.

[0099] The term "stuffer sequence" as used herein refers to a non-homologous or random nucleic acid sequence selected to promote targeted integration of a donor template into a target site and subsequent amplification of an amplicon that includes the stuffer sequence. The presence of a stuffer sequence also prevents promotion of integration of the donor template into another site. Stuffer sequences are described in more detail herein.

[0100] "Subject" means a human or non-human animal. A human subject may be of any age (e.g., a child, a young adult, or an adult), may suffer from a disease, or may require genetic modification. Alternatively, the subject may be an animal, which term includes, but is not limited to, mammals, birds, fish, reptiles, amphibians, and more particularly, non-human primates, rodents (e.g., mice, rats, hamsters, etc.), rabbits, guinea pigs, dogs, cats, and the like. In certain embodiments of this disclosure, the subject is a livestock animal, such as a cow, horse, sheep, or goat. In certain embodiments, the subject is a poultry animal.

[0101] As used herein, the term "substantially" refers to a qualitative condition exhibiting the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the art of biology understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0102] The term "substantially identical" as used herein with respect to two nucleic acid sequences, in some embodiments, refers to at least 95% sequence identity between a contiguous stretch of at least 20 nucleotides of each of the two nucleic acid sequences. For example, a first nucleic acid sequence is substantially identical to a second nucleic acid sequence if the first nucleic acid sequence has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity over a contiguous stretch of at least 20 nucleotides of the second nucleic acid sequence. In some embodiments, a first nucleic acid sequence is substantially identical to a second nucleic acid sequence if the first nucleic acid sequence has at least 95% identity over the entire length of the second nucleic acid sequence. In some embodiments, the term "substantially identical" is used in connection with a priming site and refers to the ability of the priming site to support priming by the same PCR primer during an amplification reaction. In some embodiments, the term "substantially identical" is used to describe the relationship between a homologous arm of a donor template and a homologous arm of a target nucleic acid, and refers to a nucleic acid identity between the homologous arms that allows for efficient recombination of the donor template with the target nucleic acid, to tolerate some degree of polymorphism, e.g., to eliminate PAM or protospacer sequences at the recombined locus.

[0103] As used herein, a "target CD40L nucleic acid" refers to a CD40L nucleic acid, e.g., present in a chromosome, that is the target of modification by an RNA-guided nuclease-dependent process using the genome editing system disclosed herein. The target CD40L nucleic acid can, for example, contain a mutation. In some embodiments, the mutation leads to loss of B-cell class switching due to defective CD40 signaling.

[0104] As used herein, a "target location" refers to a site on a target nucleic acid (e.g., a chromosome) that is modified by an RNA-guided nuclease-dependent process. In one embodiment, a target location may be a site between two nucleotides, e.g., adjacent nucleotides, on a target nucleic acid. A target location may include one or more nucleotides that are altered, e.g., modified, by a template nucleic acid. In one embodiment, a target location is within a "target sequence" (e.g., a sequence to which a gRNA binds). In one embodiment, a target location is upstream or downstream of a target sequence (e.g., a sequence to which a gRNA binds).

[0105] "Treat", "treating" and "treatment" refer to the treatment of a disease in a subject (e.g., a human subject), including one or more of: inhibiting the disease, i.e., arresting or preventing its development or progression; relieving the disease, i.e., causing regression of the disease state; alleviating one or more symptoms of the disease; and curing the disease.

[0106] The term "untranslated region" or "UTR" refers to either one of the two non-protein coding sections of an mRNA. The 5' untranslated region (5' UTR), also known as the leader sequence or leader RNA, is the region of an mRNA immediately upstream of the start codon. The 3' untranslated region (3' UTR) refers to the section of an mRNA immediately following the translation stop codon.

[0107] The term "variant" refers to an entity, e.g., a polypeptide, polynucleotide, or small molecule, that exhibits significant structural identity with a reference entity, but differs structurally from the reference entity in the presence or level of one or more chemical moieties compared to the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is appropriately considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity.

[0108] overview Provided herein are genome editing systems, donor templates, gRNA molecules and related methods that enable the treatment of Hyper-IgM Syndrome, a group of disorders characterized by defective CD40 signaling. The compositions and methods described herein rely on the use of donor templates that contain one or more of exons 2-5 of CD40L to functionally restore proper CD40 signaling and class switch recombination.

[0109] Genome editing system The term "genome editing system" refers to any system that has RNA-guided DNA editing activity. The genome editing system of the present disclosure includes at least two components adapted from the naturally occurring CRISPR system: guide RNA (gRNA) and RNA-guided nuclease. These two components bind to a specific nucleic acid sequence and form a complex that can edit the nucleic acid sequence or the DNA around it, for example, by forming one or more of single-strand breaks (SSBs or nicks), double-strand breaks (DSBs) and / or point mutations.

[0110] Naturally occurring CRISPR systems are evolutionarily organized into two classes and five types (Makarova et al. Nat Rev Microbiol. 2011 Jun; 9(6): 467-477 (Makarova), incorporated herein by reference), and although the genome editing system of the present disclosure can apply components of any type or class of naturally occurring CRISPR systems, the embodiments presented herein apply generally from class 2 and type II or V CRISPR systems. Class 2 systems, including types II and V, are characterized by a relatively large multi-domain RNA-guided nuclease protein (e.g., Cas9 or Cpf1) and one or more guide RNAs (e.g., crRNA and, optionally, tracrRNA) that form a ribonucleoprotein (RNP) complex that binds (i.e., targets) and cleaves a specific locus complementary to the targeting (or spacer) sequence of the crRNA. The genome editing system according to the present disclosure similarly targets and edits cellular DNA sequences, but is significantly different from naturally occurring CRISPR systems. For example, the unimolecular guide RNAs described herein do not occur in nature, and both the guide RNAs and the RNA-guided nucleases according to this disclosure can incorporate any number of non-naturally occurring modifications.

[0111] The genome editing system can be implemented (e.g., administered or delivered to a cell or subject) in a variety of ways, and different implementations may be suitable for different applications. For example, in certain embodiments, the genome editing system is implemented as a protein / RNA complex (ribonucleoprotein or RNP), which may be included in a pharmaceutical composition, optionally including a pharmaceutically acceptable carrier and / or encapsulant, such as lipid or polymer microparticles or nanoparticles, micelles, liposomes, etc. In certain embodiments, the genome editing system is implemented as one or more nucleic acids encoding the above-mentioned RNA-guided nuclease and guide RNA components (optionally together with one or more additional components); in certain embodiments, the genome editing system is implemented as one or more vectors, such as viral vectors, such as adeno-associated viruses, that contain such nucleic acids; in certain embodiments, the genome editing system is implemented as any combination of the above. Additional or modified implementations that operate according to the principles set forth herein will be apparent to those skilled in the art and are within the scope of this disclosure.

[0112] It should be noted that the genome editing system of the present disclosure can target a single specific nucleotide sequence, or can target two or more specific nucleotide sequences through the use of two or more guide RNAs, and can edit in parallel. Throughout this disclosure, the use of multiple gRNAs is referred to as "multiplexing" and can be used to target multiple unrelated target sequences of interest, or to form multiple SSBs or DSBs within a single target domain, and in some cases, to generate specific edits within such target domains. For example, International Patent Application Publication No. 2015 / 138510 by Maeder et al. (Maeder), incorporated herein by reference, describes a genome editing system for correcting a point mutation (C.2991+1655A to G) in the human CEP290 gene, which results in the formation of a cryptic splice site that then reduces or eliminates the function of the gene. Maeder's genome editing system utilizes two guide RNAs that target sequences on either side (i.e., adjacent) of the point mutation, forming DSBs adjacent to the mutation. This, in turn, facilitates deletion of the intervening sequence containing the mutation, thereby eliminating the cryptic splice site and restoring normal gene function.

[0113] As another example, WO 2016 / 073990 by Cotta-Ramusino et al. ("Cotta-Ramusino"), incorporated herein by reference, describes a genome editing system that utilizes two gRNAs in combination with a Cas9 nickase (a Cas9 that forms a single-stranded nick, such as S. pyogenes D10A), a configuration referred to as a "dual nickase system." The Cotta-Ramusino dual nickase system is configured to form two nicks on opposite strands of a sequence of interest offset by one or more nucleotides, and the nicks join to form a double-stranded break with an overhang (5' in the case of Cotta-Ramusino, but a 3' overhang is also possible). The overhang can then promote homology-guided repair events in some cases. As yet another example, WO 2015 / 070083 by Palestrant et al. ("Palestrant", incorporated herein by reference) describes a gRNA (referred to as a "controller RNA") that targets a nucleotide sequence encoding Cas9, which may be included in a genome editing system that includes one or more additional gRNAs to allow for transient expression of Cas9 that may otherwise be constitutively expressed, for example in some virally transduced cells. These multiplexing applications are illustrative and not limiting, and one of skill in the art will appreciate that other applications of multiplexing are generally compatible with the genome editing systems described herein.

[0114] Genome editing systems can, in some cases, form double-strand breaks that are repaired by cellular DNA double-strand break mechanisms, such as NHEJ or HDR. These mechanisms are described throughout the literature, for example, by Davis & Maizels, PNAS, 111(10):E924-932, March 11, 2014 (Davis) (describing Alt-HDR); Frit et al. DNA Repair 17(2014) 81-97 (Frit) (describing Alt-NHEJ); and Iyama and Wilson III, DNA Repair (Amst.) 2013-Aug; 12(8): 620-636 (Iyama) (describing canonical HDR and NHEJ pathways in general).

[0115] Where a genome editing system operates by forming DSBs, such systems optionally include one or more components that promote or encourage a particular mode of double-stranded break repair or a particular repair outcome. For example, Cotta-Ramusino also describes a genome editing system in which a single-stranded oligonucleotide "donor template" is added; the donor template can be incorporated into the target region of cellular DNA that is cut by the genome editing system, resulting in an alteration to the target sequence.

[0116] In certain embodiments, the genome editing system modifies the target sequence or modifies the expression of a gene at or near the target sequence without causing single- or double-strand breaks. For example, the genome editing system may include an RNA-guided nuclease fused to a functional domain that acts on DNA and thereby modifies the target sequence or its expression. As an example, the RNA-guided nuclease can be connected (e.g., fused) to a cytidine deaminase functional domain and can operate by generating a targeted C to A substitution. Exemplary nuclease / deaminase fusions are described in Komor et al. Nature 533, 420-424 (19 May 2016) ("Komor"), which is incorporated by reference. Alternatively, genome editing systems can utilize cleavage-inactivating (i.e., "deactivated") nucleases, such as deactivated Cas9 (dCas9), which can operate by forming a stable complex at one or more targeted regions of cellular DNA, thereby interfering with functions involving the targeted regions, including, but not limited to, mRNA transcription, chromatin remodeling, etc.

[0117] Guide RNA (gRNA) molecules The terms "guide RNA" and "gRNA" refer to any nucleic acid that facilitates the specific binding (or "targeting") of an RNA-guided nuclease, such as Cas9 or Cpf1, to a target sequence within a cell, such as a CD40L sequence. gRNAs can be unimolecular (comprising a single RNA molecule, alternatively referred to as chimeric) or modular (comprising more than one, and typically two, separate RNA molecules, such as crRNA and tracrRNA, which are typically linked to each other, e.g., by duplexing). gRNAs and their component parts are described throughout the literature, e.g., in Briner et al. (Molecular Cell 56(2), 333-339, October 23, 2014 (Briner)), and Cotta-Ramusino, which are incorporated by reference.

[0118] In bacteria and archaea, type II CRISPR systems generally include an RNA-guided nuclease protein, e.g., Cas9, a CRISPR RNA (crRNA) that includes a 5' region that is complementary to an exogenous sequence, and a trans-activating crRNA (tracrRNA) that includes a 5' region that is complementary to and forms a duplex with the 3' region of the crRNA. Without being bound by any theory, it is believed that this duplex promotes the formation of the Cas9 / gRNA complex and is necessary for its activity. As type II CRISPR systems are adapted for use in gene editing, in one non-limiting example, it has been discovered that the crRNA and tracrRNA can be linked in a single unimolecular or chimeric guide RNA by a four nucleotide (e.g., GAAA), "tetraloop" or "linker" sequence that bridges the complementary regions of the crRNA (at its 3' end) and the tracrRNA (at its 5' end). (Mali et al. Science. 2013 Feb 15; 339(6121): 823-826 ("Mali"); Jiang et al. Nat Biotechnol. 2013 Mar; 31(3): 233-239 ("Jiang"); and Jinek et al., 2012 Science Aug. 17; 337(6096): 816-821 ("Jinek"), all of which are incorporated herein by reference).

[0119] Guide RNAs, whether unimolecular or modular, contain a "targeting domain" that is fully or partially complementary to a target sequence in the genome of a cell where editing is desired, such as a target domain in the CD40L sequence. Targeting domains are referred to by various names in the literature, including, but not limited to, "guide sequence" (Hsu et al., Nat Biotechnol. 2013 Sep; 31(9): 827-832, ("Hsu"), incorporated herein by reference), "complementarity region" (Cotta-Ramusino), "spacer" (Briner), and collectively "crRNA" (Jiang). Regardless of the name given to them, targeting domains are generally 10-30 nucleotides in length, and in certain embodiments 16-24 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length), and are located at or near the 5' end in the case of Cas9 gRNA, and at or near the 3' end in the case of Cpf1 gRNA.

[0120] In addition to the targeting domain, gRNAs typically (but not necessarily, as discussed below) contain multiple domains that can affect the formation or activity of the gRNA / Cas9 complex. For example, as noted above, the duplex (also called the repeat:anti-repeat duplex) formed by the first and second complementarity domains of gRNA can interact with the recognition (REC) lobe of Cas9 and mediate the formation of the Cas9 / gRNA complex. (Nishimasu et al., Cell 156, 935-949, February 27, 2014 (Nishimasu 2014) and Nishimasu et al., Cell 162, 1113-1126, August 27, 2015 (Nishimasu 2015), both incorporated herein by reference). It should be noted that the first and / or second complementarity domains may contain one or more polyA tracts, which RNA polymerase can recognize as termination signals. Thus, the sequences of the first and second complementarity domains are optionally modified to eliminate these tracts and facilitate complete in vitro transcription of the gRNA, for example, through the use of AG exchanges as described in Briner, or through the use of AU exchanges. These and other similar modifications to the first and second complementarity domains are within the scope of this disclosure.

[0121] Along with the first and second complementarity domains, Cas9 gRNAs typically contain two or more additional duplex regions that are involved in nuclease activity in vivo, but not necessarily in vitro. (Nishimasu 2015). The first stem-loop one near the 3' portion of the second complementarity domain is variously called the "proximal domain", (Cotta-Ramusino) "stem-loop 1" (Nishimasu 2014 and 2015) and "nexus" (Briner). One or more additional stem-loop structures are typically present near the 3' end of the gRNA, the number of which varies by species: S. pyogenes gRNAs typically contain two 3' stem-loops (four stem-loops in total, including the repeat:anti-repeat duplex), while S. aureus and other species have only one (three stem-loop structures in total). A description of the conserved stem-loop structures (and gRNA structures, more generally) organized by species is provided in Briner.

[0122] While the foregoing description has focused on gRNAs for use with Cas9, it should be understood that other RNA-guided nucleases have been discovered or invented (or will be discovered or invented in the future) that utilize gRNAs that differ in some respects from those described in this section. For example, Cpf1 ("CRISPR from Prevotella and Franciscella 1") is a recently discovered RNA-guided nuclease that does not require tracrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771 October 22, 2015 (Zetsche I), incorporated herein by reference). gRNAs for use in the Cpf1 genome editing system generally include a targeting domain and a complementarity domain (alternatively referred to as a "handle"). It should also be noted that in gRNAs for use with Cpf1, the targeting domain is typically located at or near the 3' end rather than the 5' end as described above in relation to the Cas9 gRNA (the handle is at or near the 5' end of the Cpf1 gRNA).

[0123] Those skilled in the art will understand that although there may be structural differences between gRNAs from different prokaryotic species, or between Cpf1 and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be loosely defined by their targeting domain sequences, and those skilled in the art will understand that a given targeting domain sequence can be incorporated into any suitable gRNA, including monomolecular or chimeric gRNAs, or into gRNAs that contain one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Therefore, for efficient use of the presentation in this disclosure, gRNAs are described only with respect to their targeting domain sequences.

[0124] More generally, those skilled in the art will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. For this reason, unless otherwise stated, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, not just gRNAs that are compatible with a particular species of Cas9 or Cpf1. By way of example, the term gRNA may, in certain embodiments, include gRNAs for use with any RNA-guided nuclease present in class 2 CRISPR systems, such as type II or type V or CRISPR systems, or RNA-guided nucleases derived or adapted therefrom.

[0125] gRNA design Methods for target sequence selection and validation and off-target analysis have been previously described, for example, in Mali; Hsu; Fu et al., 2014 Nat Biotechnol. 32(3): 279-84, Heigwer et al., 2014 Nat methods 11(2):122-3; Bae et al. (2014) Bioinformatics 30(10): 1473-5; and Xiao A et al. (2014) Bioinformatics 30(8): 1180-1182. Each of these references is incorporated herein by reference. As a non-limiting example, gRNA design can include the use of software tools to optimize the selection of potential target sequences corresponding to the user's target sequence, for example to minimize total off-target activity across the genome. Off-target activity is not limited to cleavage, but cleavage efficiency at each off-target sequence can be predicted, for example, using empirically derived metric schemes. These and other guide selection methods are described in detail in Maeder and Cotta-Ramusino.

[0126] gRNA modification The activity, stability or other characteristics of gRNA can be altered through the incorporation of certain modifications. As an example, transiently expressed or delivered nucleic acids may be susceptible to degradation, for example, by cellular nucleases. Thus, the gRNAs described herein can contain one or more modified nucleosides or nucleotides that introduce stability against nucleases. Without wishing to be bound by theory, it is also believed that certain modified gRNAs described herein, when introduced into cells, can exhibit reduced innate immune responses. Those skilled in the art will be aware of certain cellular responses that are commonly observed in cells, e.g., mammalian cells, in response to exogenous nucleic acids, particularly those of viral or bacterial origin. Such responses, which may include cytokine expression and release and induction of cell death, can be reduced or completely eliminated by the modifications presented herein.

[0127] Certain exemplary modifications discussed in this section may be included anywhere in the gRNA sequence, including, but not limited to, at or near the 5' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5' end), and / or at or near the 3' end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 3' end). In some cases, modifications are located within functional motifs, such as the repeat-anti-repeat duplex of a Cas9 gRNA, the stem-loop structure of a Cas9 or Cpf1 gRNA, and / or the targeting domain of the gRNA.

[0128] As an example, the 5' end of the gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., G(5')ppp(5')G cap analog, m7G(5')ppp(5')G cap analog, or 3'-O-Me-m7G(5')ppp(5')G anti-reverse cap analog (ARCA)) as shown below:

[0129] [ka] A cap or cap analog may be included either during chemical synthesis or during in vitro transcription of the gRNA.

[0130] Along similar lines, the 5' end of the gRNA may lack a 5' triphosphate group. For example, in vitro transcribed gRNA can be phosphatase treated (e.g., using calf intestinal alkaline phosphatase) to remove the 5' triphosphate group.

[0131] Another common modification involves the addition of multiple (e.g., 1-10, 10-20, or 25-200) adenine (A) residues, called a polyA tract, at the 3' end of the gRNA. PolyA tracts can be added to gRNAs during chemical synthesis after in vitro transcription using a polyadenosine polymerase (e.g., E. coli poly(A) polymerase) or in vivo by a polyadenylation sequence as described by Maeder.

[0132] It should be noted that the modifications described herein can be combined in any suitable manner, for example, gRNAs, whether in vivo transcribed from a DNA vector or in vitro transcribed, can include one or both of a 5' cap structure or cap analog and a 3' polyA tract.

[0133] Guide RNAs can be modified with a 3'-terminal U-ribose. For example, the two terminal hydroxyl groups of U-ribose can be oxidized to aldehyde groups, with simultaneous opening of the ribose ring to give the modified nucleoside, as shown below:

[0134] [ka] Here, "U" can be unmodified or modified uridine.

[0135] The 3' terminal U ribose can be modified with a 2'3' cyclic phosphate as shown below:

[0136] [ka] Here, "U" can be unmodified or modified uridine.

[0137] Guide RNAs can contain 3' nucleotides that can be stabilized against degradation, for example, by incorporating one or more of the modified nucleotides described herein. In certain embodiments, uridine can be replaced with modified uridines, such as 5-(2-amino)propyluridine and 5-bromouridine, or any of the modified uridines described herein; adenosine and guanosine can be replaced with modified adenosines and guanosines, for example with modifications at position 8, such as 8-bromoguanosine, or any of the modified adenosines or guanosines described herein.

[0138] In certain embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., where the 2'-OH group is replaced with a group selected from H, -OR, -R (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), halo, -SH, -SR (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), amino (where amino can be, e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid); or cyano (-CN). In certain embodiments, the phosphate backbone can be modified, e.g., with a phosphothioate (PhTx) group, as described herein. In certain embodiments, one or more of the nucleotides of the gRNA may each independently be a modified or unmodified nucleotide, including, but not limited to, a 2'-sugar modification, such as a 2'-O-methyl, 2'-O-methoxyethyl, or 2'-fluoro modification, such as 2'-F or 2'-O-methyl, adenosine (A), 2'-F or 2'-O-methyl, cytidine (C), 2'-F or 2'-O-methyl, uridine (U), 2'-F or 2'-O-methyl, thymidine (T), 2'-F or 2'-O-methyl, guanosine (G), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof.

[0139] Guide RNAs can also include "locked" nucleic acids (LNAs) in which the 2'-OH group can be connected to the 4' carbon of the same ribose sugar, for example, by a C1-6 alkylene or C1-6 heteroalkylene bridge. Any suitable moiety can be used to provide such a bridge, such as methylene, propylene, ether or amino bridges; O-amino (where amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino); and aminoalkoxy or O(CH2). n -amino (wherein amino may be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine or polyamino).

[0140] In certain embodiments, gRNAs may include modified nucleotides that are polycyclic (e.g., tricyclic; and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, where the ribose is replaced with a glycol unit linked to a phosphodiester bond), or threose nucleic acids (TNAs, where the ribose is replaced with α-L-threofuranosyl-(3'→2')).

[0141] Generally, gRNAs contain the sugar group ribose, which is a five-membered ring with oxygen. Exemplary modified gRNAs may include, but are not limited to, substitution of oxygen in ribose (e.g., by sulfur (S), selenium (Se), or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a cyclobutane or oxetane four-membered ring); ring expansion of ribose (e.g., to form a 6- or 7-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have a phosphoramidate backbone). Most sugar analogue modifications are localized at the 2' position, but other sites, including the 4' position, are amenable to modification. In certain embodiments, the gRNA comprises a 4'-S, 4'-Se, or 4'-C-aminomethyl-2'-O-Me modification.

[0142] In certain embodiments, deazanucleotides, such as 7-deazaadenosine, can be incorporated into the gRNA. In certain embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, can be incorporated into the gRNA. In certain embodiments, one or more or all of the nucleotides in the gRNA are deoxynucleotides.

[0143] CD40L donor template Donor templates, e.g., oligonucleotide donor templates, according to this disclosure can be implemented in any suitable manner, including, but not limited to, single-stranded or double-stranded DNA, linear or circular, naked or contained within a vector, and / or covalently or non-covalently linked to a guide RNA (e.g., by direct hybridization or splint hybridization). Some preferred donor template designs are depicted diagrammatically in FIG.

[0144] In some embodiments, the donor template is a ssODN. When a linear ssODN is used, it can be configured to (i) anneal to the nicked strand of the target nucleic acid, (ii) anneal to the intact strand of the target nucleic acid, (iii) anneal to the positive strand of the target nucleic acid, and / or (iv) anneal to the negative strand of the target nucleic acid. The ssODN can have any suitable length, for example, about 150-200 nucleotides (e.g., 150, 160, 170, 180, 190 or 200 nucleotides) or less. In other embodiments, the ssODN has a length of about 100 to 3000 nucleotides, for example, about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 nucleotides. In other embodiments, the ssODN has a length of at least 100 to 3000 nucleotides, e.g., at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 nucleotides. In other embodiments, the ssODN has a length of 100 or less to 3000 nucleotides, for example, 100 or less, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 nucleotides.

[0145] In other embodiments, the donor template is a dsODN. In one embodiment, the donor template comprises a first strand. In another embodiment, the donor template comprises a first strand and a second strand. The dsODN can have any suitable length, for example, about 150-200 base pairs (e.g., 150, 160, 170, 180, 190, or 200 base pairs) or less. In other embodiments, the dsODN has a length of about 100 to 3000 base pairs, for example, about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 base pairs. In other embodiments, the dsODN has a length of at least 100 to 3000 base pairs, for example, at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 base pairs. In other embodiments, the dsODN has a length of 100 or less to 3000 base pairs, for example, 100 or less, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000 base pairs.

[0146] In some embodiments, the donor template is an exogenous oligonucleotide, e.g., an oligonucleotide that does not naturally occur in the cell.

[0147] It should be noted that the donor template may be contained in a nucleic acid vector, such as a viral genome or a circular double-stranded DNA, such as a plasmid. In some embodiments, the donor template may be a dog-bone DNA (see, for example, U.S. Pat. No. 9,499,847). The nucleic acid vector containing the donor template may contain other coding or non-coding elements. For example, the donor template nucleic acid may be delivered as part of a viral genome (e.g., an AAV or lentiviral genome) that contains certain genomic backbone elements (e.g., in the case of an AAV genome, an inverted terminal repeat sequence) and optionally contains additional sequences encoding gRNAs and / or RNA-guided nucleases. In certain embodiments, the donor template may be adjacent to or adjacent to a target site recognized by one or more gRNAs to promote the formation of a free DSB at one or both ends of the donor template that can participate in the repair of the corresponding SSB or DSB formed in the cellular DNA using the same gRNA. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino. In one embodiment, the donor template is in an AAV6 vector. In another embodiment, the donor template is in an IDLV vector.

[0148] A. homologous arm The donor template, whether single-stranded or double-stranded, typically contains one or more regions that are homologous to the target sequence to be cleaved, e.g., a region of DNA within or near (e.g., adjacent or proximal to) the cleavage site, e.g., the target nucleic acid, CD40L sequence. These regions of homology are referred to herein as "homology arms" and are illustrated diagrammatically in FIG. 1.

[0149] The homologous arms of the donor template described herein may be of any suitable length, provided that such length is sufficient to allow efficient resolution of the cut site on the targeting nucleic acid by the DNA repair process that requires the donor template. In some embodiments, when it is desired to amplify the homologous arms, for example by PCR, the homologous arms are of such a length that the amplification can be carried out. In some embodiments, when it is desired to sequence the homologous arms, the homologous arms are of such a length that the sequencing can be carried out. In some embodiments, when it is desired to quantitatively evaluate the amplicons, the homologous arms are of such a length that similar times of amplification of each amplicon are achieved, for example by having similar G / C content, amplification temperature, etc. In some embodiments, the homologous arms are double-stranded. In some embodiments, the double-stranded homologous arms are single-stranded.

[0150] In some embodiments, the 5' homologous arm is between 150-250 nucleotides in length. In some embodiments, the 5' homologous arm is 700 nucleotides or less in length. In some embodiments, the 5' homologous arm is 650 nucleotides or less in length. In some embodiments, the 5' homologous arm is 600 nucleotides or less in length. In some embodiments, the 5' homologous arm is 550 nucleotides or less in length. In some embodiments, the 5' homologous arm is 500 nucleotides or less in length. In some embodiments, the 5' homologous arm is 400 nucleotides or less in length. In some embodiments, the 5' homologous arm is 300 nucleotides or less in length. In some embodiments, the 5' homologous arm is 250 nucleotides or less in length. In some embodiments, the 5' homologous arm is 200 nucleotides or less in length. In some embodiments, the 5' homologous arm is 150 nucleotides or less in length. In some embodiments, the 5' homologous arm is less than 100 nucleotides in length. In some embodiments, the 5' homology arm is 50 nucleotides or less in length. In some embodiments, the 5' homology arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the 5' homology arm is 40 nucleotides in length. In some embodiments, the 3' homology arm is 250 nucleotides or less in length. In other embodiments, the 5' homology arm is at least 50 nucleotides in length. In some embodiments, the 5' homologous arm is at least 100 nucleotides in length. In some embodiments, the 5' homologous arm is at least 150 nucleotides in length. In some embodiments, the 5' homologous arm is at least 200 nucleotides in length. In some embodiments, the 5' homologous arm is at least 250 nucleotides in length. In some embodiments, the 5' homologous arm is at least 300 nucleotides in length. In some embodiments, the 5' homologous arm is at least 350 nucleotides in length. In some embodiments, the 5' homologous arm is at least 40 nucleotides in length. 0 nucleotides in length. In some embodiments, the 5' homologous arm is at least 450 nucleotides in length. In some embodiments, the 5' homologous arm is at least 500 nucleotides in length. In some embodiments, the 5' homologous arm is at least 550 nucleotides in length. In some embodiments, the 5' homologous arm is at least 600 nucleotides in length. In some embodiments, the 5' homologous arm is at least 650 nucleotides in length. In some embodiments, the 5' homologous arm is at least 700 nucleotides in length. In one embodiment, the 5' homologous arm is between 200 and 700 nucleotides in length. In one embodiment, the 5' homologous arm is between 200 and 300 nucleotides in length. In one embodiment, the 5' homologous arm is between 200 and 400 nucleotides in length. In one embodiment, the 5' homologous arm is between 200 and 500 nucleotides in length. In one embodiment, the 5' homologous arm is between 400 and 700 nucleotides in length. In one embodiment, the 5' homology arm is between 500 and 700 nucleotides in length. In one embodiment, the 5' homology arm is about 500 nucleotides in length.

[0151] In some embodiments, the 3' homologous arms are between 150-250 nucleotides in length. In some embodiments, the 3' homologous arms are 700 nucleotides in length or less. In some embodiments, the 3' homologous arms are 650 nucleotides in length or less. In some embodiments, the 3' homologous arms are 600 nucleotides in length or less. In some embodiments, the 3' homologous arms are 550 nucleotides in length or less. In some embodiments, the 3' homologous arms are 500 nucleotides in length or less. In some embodiments, the 3' homologous arms are 400 nucleotides in length or less. In some embodiments, the 3' homologous arms are 300 nucleotides in length or less. In some embodiments, the 3' homologous arms are 200 nucleotides in length or less. In some embodiments, the 3' homologous arms are 150 nucleotides in length or less. In some embodiments, the 3' homologous arms are 100 nucleotides in length or less. In some embodiments, the 3' homologous arms are 50 nucleotides in length or less. In some embodiments, the 3' homology arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the 3' homology arm is 40 nucleotides in length. In other embodiments, the 3' homology arm is at least 50 nucleotides in length. In some embodiments, the 3' homology arm is at least 100 nucleotides in length. In some embodiments, the 3' homology arm is at least 150 nucleotides in length. In some embodiments, the 3' homologous arm is at least 200 nucleotides in length. In some embodiments, the 3' homologous arm is at least 250 nucleotides in length. In some embodiments, the 3' homologous arm is at least 300 nucleotides in length. In some embodiments, the 3' homologous arm is at least 350 nucleotides in length. In some embodiments, the 3' homologous arm is at least 400 nucleotides in length. In some embodiments, the 3' homologous arm is at least 450 nucleotides in length. In some embodiments, the 3' homologous arm is at least In some embodiments, the 3' homologous arm is at least 500 nucleotides in length. In some embodiments, the 3' homologous arm is at least 550 nucleotides in length. In some embodiments, the 3' homologous arm is at least 600 nucleotides in length. In some embodiments, the 3' homologous arm is at least 650 nucleotides in length. In some embodiments, the 3' homologous arm is at least 700 nucleotides in length. In one embodiment, the 3' homologous arm is between 200 and 700 nucleotides in length. In one embodiment, the 3' homologous arm is between 200 and 300 nucleotides in length. In one embodiment, the 3' homologous arm is between 200 and 400 nucleotides in length. In one embodiment, the 3' homologous arm is between 200 and 500 nucleotides in length. In one embodiment, the 3' homologous arm is between 400 and 700 nucleotides in length. In one embodiment, the 3' homologous arm is between 500 and 700 nucleotides in length. In one embodiment, the 3' homologous arm is about 500 nucleotides in length.

[0152] In some embodiments, the 5' homologous arms are between 150 and 250 base pairs in length. In some embodiments, the 5' homologous arms are 700 base pairs or less in length. In some embodiments, the 5' homologous arms are 650 base pairs or less in length. In some embodiments, the 5' homologous arms are 600 base pairs or less in length. In some embodiments, the 5' homologous arms are 550 base pairs or less in length. In some embodiments, the 5' homologous arms are 500 base pairs or less in length. In some embodiments, the 5' homologous arms are 400 base pairs or less in length. In some embodiments, the 5' homologous arms are 300 base pairs or less in length. In some embodiments, the 5' homologous arms are 250 base pairs or less in length. In some embodiments, the 5' homologous arms are 200 base pairs or less in length. In some embodiments, the 5' homologous arms are 150 base pairs or less in length. In some embodiments, the 5' homologous arms are less than 100 base pairs in length. In some embodiments, the 5' homologous arm is 50 base pairs or less in length. In some embodiments, the 5' homologous arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs in length. In some embodiments, the 5' homologous arm is 40 base pairs in length. In other embodiments, the 5' homologous arm is at least 50 base pairs in length. In some embodiments, the 5' homologous arm is at least 100 base pairs in length. In some embodiments, the 5' homologous arm is at least 150 base pairs in length. In some embodiments, the 5' homologous arm is at least 200 base pairs in length. In some embodiments, the 5' homologous arm is at least 250 base pairs in length. In some embodiments, the 5' homologous arm is at least 300 base pairs in length. In some embodiments, the 5' homologous arm is at least 350 base pairs in length. In some embodiments, the 5' homologous arm is at least 400 base pairs in length. In some embodiments, the 5' homologous arm is at least 450 base pairs in length. In some embodiments, the 5' homologous arm is at least 500 base pairs in length. In some embodiments, the 5' homologous arm is at least 600 base pairs in length. is at least 550 base pairs. In some embodiments, the 5' homologous arm is at least 600 base pairs in length. In some embodiments, the 5' homologous arm is at least 650 base pairs in length. In some embodiments, the 5' homologous arm is at least 700 base pairs in length. In one embodiment, the 5' homologous arm is between 200 and 700 base pairs in length. In one embodiment, the 5' homologous arm is between 200 and 300 base pairs in length. In one embodiment, the 5' homologous arm is between 200 and 400 base pairs in length. In one embodiment, the 5' homologous arm is between 200 and 500 base pairs in length. In one embodiment, the 5' homologous arm is between 400 and 700 base pairs in length. In one embodiment, the 5' homologous arm is between 500 and 700 base pairs in length. In one embodiment, the 5' homologous arm is about 500 base pairs in length.

[0153] In some embodiments, the 3' homologous arms are between 150 and 250 base pairs in length. In some embodiments, the 3' homologous arms are 700 base pairs or less in length. In some embodiments, the 3' homologous arms are 650 base pairs or less in length. In some embodiments, the 3' homologous arms are 600 base pairs or less in length. In some embodiments, the 3' homologous arms are 550 base pairs or less in length. In some embodiments, the 3' homologous arms are 500 base pairs or less in length. In some embodiments, the 3' homologous arms are 400 base pairs or less in length. In some embodiments, the 3' homologous arms are 300 base pairs or less in length. In some embodiments, the 3' homologous arms are 250 base pairs or less in length. In some embodiments, the 3' homologous arms are 200 base pairs or less in length. In some embodiments, the 3' homologous arms are 150 base pairs or less in length. In some embodiments, the 3' homologous arms are 100 base pairs or less in length. In some embodiments, the 3' homologous arm is 50 base pairs or less in length. In some embodiments, the 3' homologous arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 base pairs in length. In some embodiments, the 3' homologous arm is 40 base pairs in length. In other embodiments, the 3' homologous arm is at least 50 base pairs in length. In some embodiments, the 3' homologous arm is at least 100 base pairs in length. In some embodiments, the 3' homologous arm is at least 150 base pairs in length. In some embodiments, the 3' homologous arm is at least 200 base pairs in length. In some embodiments, the 3' homologous arm is at least 250 base pairs in length. In some embodiments, the 3' homologous arm is at least 300 base pairs in length. In some embodiments, the 3' homologous arm is at least 350 base pairs in length. In some embodiments, the 3' homologous arm is at least 400 base pairs in length. In some embodiments, the 3' homologous arm is at least 450 base pairs in length. In some embodiments, the 3' homologous arm is at least 500 base pairs in length. In some embodiments, the 3' homologous arm is at least 600 base pairs in length. is at least 550 base pairs. In some embodiments, the 3' homologous arm is at least 600 base pairs in length. In some embodiments, the 3' homologous arm is at least 650 base pairs in length. In some embodiments, the 3' homologous arm is at least 700 base pairs in length. In one embodiment, the 3' homologous arm is between 200 and 700 base pairs in length. In one embodiment, the 3' homologous arm is between 200 and 300 base pairs in length. In one embodiment, the 3' homologous arm is between 200 and 400 base pairs in length. In one embodiment, the 3' homologous arm is between 200 and 500 base pairs in length. In one embodiment, the 3' homologous arm is between 400 and 700 base pairs in length. In one embodiment, the 3' homologous arm is between 500 and 700 base pairs in length. In one embodiment, the 3' homologous arm is about 500 base pairs in length.

[0154] The 5' and 3' homologous arms may be the same length or may be different in length. In some embodiments, the 5' and 3' homologous arms are amplified to allow for quantitative assessment of gene editing events, such as targeted integration, in a target nucleic acid, such as CD40L. In some embodiments, quantitative assessment of gene editing events can rely on amplification of both the 5' and 3' junctions of the targeted integration site by amplifying all or part of the homologous arms using a single PCR primer pair in a single amplification reaction. Thus, the lengths of the 5' and 3' homologous arms may be different, but if desired, the length of each homologous arm should be amplifiable (e.g., using PCR). Furthermore, if amplification of both the 5' and the difference in length between the 5' and 3' homologous arms in a single PCR reaction is desired, the difference in length between the 5' and 3' homologous arms should allow PCR amplification using a single PCR primer pair.

[0155] In some embodiments, the lengths of the 5' and 3' homology arms do not differ by more than 75 nucleotides. Thus, in some embodiments, when the lengths of the 5' and 3' homology arms differ, the difference in length between the homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides or base pairs. In some embodiments, the 5' and 3' homology arms differ in length by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 nucleotides. In some embodiments, the difference in length between the 5' and 3' homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pairs. In some embodiments, the 5' and 3' homology arms differ in length by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 base pairs.

[0156] Whatever format is used, the donor template can be designed to avoid undesired sequences, in certain embodiments, one or both homology arms can be truncated to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.

[0157] B. Cargo - CD40L exon The donor template of the gene editing system described herein comprises a cargo (e.g., a CD40L gene-corrected cargo). The cargo disclosed herein may comprise one or more of exons 2-5 of the CD40L nucleic acid. The cargo may be of any length necessary to achieve the desired result. For example, the cargo sequence may be less than 2500 base pairs or less than 2500 nucleotides in length. If the donor template is delivered using a delivery vehicle with a size limit (e.g., a viral delivery vehicle, such as an adeno-associated virus (AAV) or a herpes simplex virus (HSV) delivery vehicle), one of skill in the art will readily ascertain that the size of the donor template containing the cargo should not exceed the size limit of the delivery system.

[0158] In some embodiments, the cargo comprises a replacement sequence. In one embodiment, the replacement sequence is a wild-type CD40L sequence. In one embodiment, the replacement sequence is a codon-optimized CD40L sequence, which will increase translation of the CD40L protein. In one embodiment, the wild-type CD40L sequence does not contain a mutation that would render CD40L non-functional or affect its expression and / or splicing.

[0159] In some embodiments, the cargo comprises all or a portion of an exon of the gene sequence CD40L, for example all or a portion of one or more of exons 2, 3, 4, and 5 of CD40L. In some embodiments, the cargo comprises all or a portion of exon 2 of CD40L. In some embodiments, the cargo comprises all or a portion of exon 3 of CD40L. In some embodiments, the cargo comprises all or a portion of exon 4 of CD40L. In some embodiments, the cargo comprises all or a portion of exon 5 of CD40L. In some embodiments, the cargo comprises all or a portion of exons 2-5 of CD40L. In some embodiments, the cargo comprises all or a portion of exons 2 and 3 of CD40L. In some embodiments, the cargo comprises all or a portion of exons 2-4 of CD40L. In some embodiments, the cargo comprises all or a portion of exons 3 and 4 of CD40L. In some embodiments, the cargo comprises all or a portion of exons 3-5 of CD40L. In some embodiments, the cargo comprises all or part of exons 3 and 5 of CD40L. In some embodiments, the cargo comprises all or part of exons 4 and 5 of CD40L. In some embodiments, the cargo does not comprise exon 1 of CD40L. In preferred embodiments, the cargo comprises a splice acceptor (SA) signal before CD40L.

[0160] The sequence and exon and intron boundaries of the CD40L gene are well known to those of skill in the art. In one embodiment, the CD40L mRNA sequence is described at least in NM_000074, the entire contents of which are expressly incorporated herein by reference. In one embodiment, the CD40L RefSeq gene sequence for the X chromosome is described at least in NG_007280.1, the entire contents of which are expressly incorporated herein by reference. As described herein, in some embodiments, a codon-optimized version of one or more exons of CD40L can be used in the donor template. An exemplary codon-optimized version of exons 2-5 of CD40L is provided in SEQ ID NO:48.

[0161] JPEG2025011076000005.jpg177170JPEG2025011076000006.jpg247170JPEG2025011076000007.jpg247170JPEG2025011076000008.jpg234170

[0162] In some embodiments, the cargo comprises an intron of a gene sequence. In some embodiments, the cargo comprises a cDNA sequence. In some embodiments, the cargo comprises a transcriptional regulatory element. In some embodiments, the cargo comprises a reverse complement of a replacement sequence, an exon of a gene sequence, an intron of a gene sequence, a cDNA sequence, or a transcriptional regulatory element. In some embodiments, the cargo comprises a portion of a replacement sequence, an exon of a gene sequence, an intron of a gene sequence, a cDNA sequence, or a transcriptional regulatory element.

[0163] The replacement sequence in the donor template has been described in other publications, including Cotta-Ramusino et al. The replacement sequence may be of any suitable length (including 0 nucleotides, where the desired repair result is a deletion), and generally includes one, two, three or more sequence modifications compared to the naturally occurring sequence in the cell in which editing is desired. One common sequence modification includes modification of the naturally occurring sequence to repair a mutation associated with the disease or condition that is desired to be treated. Another common sequence modification includes modification of one or more sequences that are complementary to or code for the PAM sequence of an RNA-guided nuclease or the targeting domain of the gRNA used to generate SSB or DSB, to reduce or eliminate repeated cleavage of the target site after the replacement sequence is integrated into the target site.

[0164] In some embodiments, the oligonucleotide donor template may further comprise a sequence encoding a reporter. In one embodiment, the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase. In one embodiment, the reporter may be a marker that can be detected in living cells using methods standard in the art, such as a surface antigen detectable by an antibody, a peptide that catalyzes or otherwise promotes a chemical reaction that produces an optically detectable product, such as luciferase. In one embodiment, the reporter is a selection marker. In an exemplary embodiment, the selection marker is a low affinity nerve growth factor receptor (NGFR). In other embodiments, the selectable marker may be a drug resistance protein (e.g., neomycin or puromycin resistance, a mutant version of the MGMT gene), a truncated version of a cell surface protein (e.g., CD19, EGFR), a gene that confers a selective growth and / or engraftment advantage following in vivo transplantation of the edited cells (e.g., CXCR4, CD47, IL2 receptor), or a fluorescent reporter protein (e.g., green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc., or a combination thereof.

[0165] C.Stuffer In some embodiments, the donor template may optionally include one or more stuffer sequences. In general, the stuffer sequence is a non-homologous or random nucleic acid sequence selected to (a) promote (or not inhibit) the targeted integration of the donor template of the present disclosure into the target site and subsequent amplification of the amplicon containing the stuffer sequence by certain methods of this disclosure, but (b) avoid promoting the integration of the donor template into another site. The stuffer sequence can be placed, for example, between the homologous arm and the CD40L exon sequence to adjust the size of the amplicon generated when the donor template sequence is integrated into the target site. Such size adjustment can be used, by way of example, to balance the size of the amplicon generated by integrated and unintegrated target sites, and thus the efficiency with which each amplicon is generated in a single PCR reaction; this can facilitate quantitative assessment of the rate of targeted integration based on the relative abundance of the two amplicons in the reaction mixture.

[0166] To facilitate targeted integration and amplification, stuffer sequences can be selected to minimize the formation of secondary structures that may interfere with resolution of the cleavage site by DNA repair mechanisms (e.g., through homologous recombination) or that may interfere with amplification.

[0167] In one embodiment, the first stuffer is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155 , at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475 or at least 500 nucleotides.In another embodiment, the second stuffer is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155 , at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475 or at least 500 nucleotides.

[0168] It is preferred that the stuffer sequence does not interfere with the resolution of the cleavage site of the target nucleic acid.Therefore, the stuffer sequence should have a minimum sequence identity with the nucleic acid sequence of the cleavage site of the target nucleic acid.In some embodiments, the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% identical to any nucleic acid sequence within 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 nucleotides from the cleavage site of the target nucleic acid. In some embodiments, the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% identical to any nucleic acid sequence within 500, 450, 400, 350, 300, 250, 200, 150, 100, 50 base pairs of the cleavage site of the target nucleic acid.

[0169] To avoid off-target molecular recombination events, it is preferred that the stuffer sequence has minimal homology with nucleic acid sequences in the genome of the target cell. In some embodiments, the stuffer sequence has minimal sequence identity with nucleic acids in the genome of the target cell. In some embodiments, the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% identical (measured in base pairs or nucleotides) to any nucleic acid sequence of the same length in the genome of the target cell. In some embodiments, a 20 base pair stretch of the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% identical to any stretch of at least 20 base pairs of nucleic acid in the genome of the target cell. In some embodiments, a 20 nucleotide stretch of the stuffer sequence is less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% identical to any nucleic acid stretch of at least 20 nucleotides in the target cell genome.

[0170] In some embodiments, the stuffer sequence has minimal sequence identity with a nucleic acid sequence in the donor template (e.g., a nucleic acid sequence of the cargo or a nucleic acid sequence of a priming site present in the donor template). In some embodiments, the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10% identical (measured in base pairs or nucleotides) to any nucleic acid sequence of the same length in the donor template. In some embodiments, a 20 base pair stretch of the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10% identical to any 20 base pair stretch of nucleic acid in the donor template. In some embodiments, a 20 nucleotide stretch of the stuffer sequence is less than 80%, 70%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20% or 10% identical to any 20 nucleotide stretch of nucleic acid in the donor template.

[0171] Stuffer sequences are described in further detail in U.S. Provisional Application No. 62 / 532,509, filed July 14, 2017, the entire contents of which are expressly incorporated herein by reference.

[0172] RNA-guided nucleases RNA-guided nucleases according to the present disclosure include, but are not limited to, naturally occurring class 2 CRISPR nucleases, such as Cas9 and Cpf1, as well as other nucleases derived or obtained therefrom. In terms of function, an RNA-guided nuclease is defined as a nuclease that (a) interacts with (e.g., forms a complex with) a gRNA; and (b) binds to and optionally cleaves or modifies a target region of DNA together with the gRNA, which comprises (i) a sequence complementary to the targeting domain of the gRNA, and optionally (ii) an additional sequence, referred to as a "protospacer adjacent motif" or "PAM", as described in more detail below. As illustrated by the examples below, RNA-guided nucleases can be broadly defined by their PAM specificity and cleavage activity, although there can be variation between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Those skilled in the art will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease with a particular PAM specificity and / or cleavage activity. For this reason, unless otherwise indicated, it should be understood that the term RNA-guided nuclease is generic and not limited to any particular type (e.g., Cas9 vs. Cpf1), species (e.g., S. pyogenes vs. S. aureus) or variant (e.g., full-length vs. truncated or split; naturally occurring vs. engineered PAM specificity, etc.) of RNA-guided nuclease.

[0173] The PAM sequence gets its name from its sequential relationship to a "protospacer" sequence that is complementary to the gRNA targeting domain (or "spacer"). Together with the protospacer sequence, the PAM sequence defines a target region or sequence for a specific RNA-guided nuclease / gRNA combination.

[0174] Different RNA-guided nucleases may require different sequential relationships between the PAM and the protospacer. In general, Cas9s recognizes the PAM sequence 3' to the protospacer, as visualized on the bottom or non-complementary strand: JPEG2025011076000009.jpg13119

[0175] Cpf1, on the other hand, generally recognizes PAM sequences 5' to the protospacer, as visualized on the bottom or non-complementary strand: JPEG2025011076000010.jpg13119

[0176] In addition to recognizing specific sequential orientations of PAM and protospacer, RNA-guided nucleases can also recognize specific PAM sequences. S. aureus Cas9, for example, recognizes the PAM sequence NNGRRT or NNGRRV, where the N residue is immediately 3' to the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes the PAM sequence NGG. F. novicida Cpf1 recognizes the PAM sequence TTN. PAM sequences have been identified for various RNA-guided nucleases, and a strategy for identifying novel PAM sequences is described in Shmakov et al., 2015, Molecular Cell 60, 385-397, November 5, 2015. It should also be noted that an engineered RNA-guided nuclease can have a PAM specificity that differs from that of the reference molecule (e.g., in the case of an engineered RNA-guided nuclease, the reference molecule may be a naturally occurring variant from which the RNA-guided nuclease is derived, or a naturally occurring variant that has the greatest amino acid sequence homology to the engineered RNA-guided nuclease).

[0177] In addition to their PAM specificity, RNA-guided nucleases can be characterized by their DNA cleavage activity: naturally occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered mutants have been generated that generate only SSBs (discussed above) (Ran & Hsu, et al., Cell 154(6), 1380-1389, September 12, 2013 (Ran), incorporated herein by reference) or do not cleave at all.

[0178] Cas9 The crystal structures of S. pyogenes Cas9 (Jinek 2014) and S. aureus Cas9 in complex with single guide RNA and target DNA have been determined (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

[0179] Naturally occurring Cas9 protein contains two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each contains specific structural and / or functional domains. The REC lobe contains an arginine-rich bridge helix (BH) domain and at least one REC domain (e.g., a REC1 domain and, optionally, a REC2 domain). The REC lobe does not share structural similarity with other known proteins, indicating that it is a unique functional domain. Without wishing to be bound by any theory, mutational analysis suggests specific functional roles for the BH and REC domains: the BH domain appears to play a role in gRNA:DNA recognition, and the REC domain is thought to interact with the repeat:anti-repeat duplex of the gRNA and mediate the formation of the Cas9 / gRNA complex.

[0180] The NUC lobe contains a RuvC domain, an HNH domain, and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity with members of the retroviral integrase superfamily and cleaves the non-complementary (i.e., bottom) strand of the target nucleic acid. It can be formed from two or more split RuvC motifs (e.g., RuvCI, RuvCII, and RuvCIII in S. pyogenes and S. aureus). Meanwhile, the HNH domain is structurally similar to the HNN endonuclease motif and cleaves the complementary (i.e., top) strand of the target nucleic acid. As the name suggests, the PI domain contributes to PAM specificity.

[0181] While certain functions of Cas9 are associated with (but not necessarily entirely determined by) the specific domains shown above, these and other functions can be mediated or influenced by other Cas9 domains, or by multiple domains on either lobe. For example, as described in Nishimasu 2014, in S. pyogenes Cas9, the repeat:anti-repeat duplex of the gRNA fits into a groove between the REC and NUC lobes, and nucleotides in the duplex interact with amino acids in the BH, PI, and REC domains. Some nucleotides in the first stem-loop structure also interact with amino acids in multiple domains (PI, BH, and REC1), as do some nucleotides in the second and third stem-loops (RuvC and PI domains).

[0182] Cpf1 The crystal structure of Cpf1 from Acidaminococcus species complexed with crRNA and a double-stranded (ds) DNA target containing a TTTN PAM sequence has been solved by Yamano et al. (Yamano et al. (Cell. 2016 May 5; 165(4): 949-962 (Yamano), incorporated herein by reference). Like Cas9, Cpf1 has two lobes: the REC (recognition) lobe and the NUC (nuclease) lobe. The REC lobe contains the REC1 and REC2 domains, which lack similarity to any known protein structure. Meanwhile, the NUC lobe contains three RuvC domains (RuvC-I, -II, and -III) and a BH domain. However, in contrast to Cas9, Cpf1 The REC lobe lacks the HNH domain and contains other domains that also lack similarity to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED-I, -II and -III) and a nuclease (Nuc) domain.

[0183] Although Cas9 and Cpf1 share similarities in structure and function, it should be recognized that certain Cpf1 activities are mediated by structural domains that are not similar to any Cas9 domain. For example, cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which is sequentially and spatially distinct from the HNH domain of Cas9. Furthermore, the non-targeting portion (handle) of the Cpf1 gRNA adopts a pseudoknot structure rather than the stem-loop structure formed by the repeat:anti-repeat duplex in the Cas9 gRNA.

[0184] Engineering RNA-guided nucleases While the RNA-guided nucleases described above have activities and properties that can be useful in a variety of applications, those of skill in the art will appreciate that RNA-guided nucleases can also be modified in certain cases to alter cleavage activity, PAM specificity, or other structural or functional features.

[0185] Turning first to modifications that change cleavage activity, mutations that reduce or eliminate the activity of domains in the NUC lobe are described above. Exemplary mutations that can be made in the RuvC domain, Cas9 HNH domain, or Cpf1 Nuc domain are described in Ran and Yamano, and Cotta-Ramusino. In general, mutations that reduce or eliminate activity in one of the two nuclease domains result in an RNA-guided nuclease with nickase activity, but it should be noted that the type of nickase activity differs depending on which domain is inactivated. As an example, inactivation of the RuvC domain of Cas9 results in a nickase that cuts the complementary or top strand. On the other hand, inactivation of the Cas9 HNH domain results in a nickase that cuts the bottom or non-complementary strand.

[0186] Modifications of PAM specificity compared to naturally occurring Cas9 reference molecules have been described by Kleinstiver et al. for both S. pyogenes (Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5 (Kleinstiver I)) and S. aureus (Kleinstiver et al., Nat Biotechnol. 2015 Dec;33(12):1293-1298 (Kleinstiver II)). Kleinstiver et al. also describe modifications that improve the targeting fidelity of Cas9 (Nature, 2016 Jan 28;529, 490-495 (Kleinstiver III)). Each of these references is incorporated herein by reference.

[0187] As described by Zetsche et al. (Nat Biotechnol. 2015 Feb;33(2):139-42 (Zetsche II), incorporated by reference) and Fine et al. (Sci. Rep. 2015 Jul 1;5:10777 (Fine), incorporated by reference), RNA-guided nucleases have been split into two or more parts.

[0188] In certain embodiments, the RNA-guided nuclease can be size-optimized or truncated, for example, through one or more deletions that reduce the size of the nuclease while still retaining gRNA binding, target and PAM recognition, and cleavage activity. In certain embodiments, the RNA-guided nuclease is covalently or non-covalently linked to another polypeptide, nucleotide, or other structure, optionally by a linker. Exemplary linked nucleases and linkers are described by Guilinger et al., Nature Biotechnology 32, 577-582 (2014), which is incorporated herein by reference for all purposes.

[0189] RNA-guided nuclease also optionally comprises a tag, for example, but not limited to, a nuclear localization signal to facilitate the transport of RNA-guided nuclease protein into the nucleus.In certain embodiments, RNA-guided nuclease can incorporate C-terminal and / or N-terminal nuclear localization signals.Nuclear localization sequences are known in the art and are described in Maeder and others.

[0190] The above list of modifications is exemplary in nature, and those skilled in the art will appreciate in light of the present disclosure that other modifications may be possible or desirable in certain applications. For the sake of brevity, therefore, the exemplary systems, methods and compositions of the present disclosure are presented with respect to specific RNA-guided nucleases, but it should be understood that the RNA-guided nucleases used can be modified in ways that do not change their principles of operation. Such modifications are within the scope of the present disclosure.

[0191] Nucleic acid encoding an RNA-guided nuclease Provided herein are nucleic acids encoding RNA-guided nucleases, such as Cas9, Cpf1, or functional fragments thereof. Exemplary nucleic acids encoding RNA-guided nucleases have been previously described (see, e.g., Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

[0192] In some cases, the nucleic acid encoding the RNA-guided nuclease may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule may be chemically modified. In certain embodiments, the mRNA encoding the RNA-guided nuclease has one or more (e.g., all) of the following characteristics: capping; polyadenylation; and 5-methylcytidine and / or pseudouridine substitution.

[0193] The synthetic nucleic acid sequence can also be codon-optimized, e.g., at least one uncommon or less common codon is replaced with a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., as described herein. An example of a codon-optimized Cas9 coding sequence is provided in Cotta-Ramusino.

[0194] Additionally, or alternatively, the nucleic acid encoding the RNA-guided nuclease may contain a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.

[0195] Functional analysis of candidate molecules Candidate RNA-guided nucleases, gRNAs and their complexes can be evaluated by standard methods known in the art. See, for example, Cotta-Ramusino. The stability of RNP complexes can be evaluated by differential scanning fluorimetry as described below.

[0196] Differential Scanning Fluorescence (DSF) The thermostability of a ribonucleoprotein (RNP) complex containing a gRNA and an RNA-guided nuclease can be measured through DSF. The DSF technique measures the thermostability of a protein, which can increase under favorable conditions, such as the addition of a binding RNA molecule, e.g., gRNA.

[0197] The DSF assay can be performed by any suitable protocol and can be used in any suitable context, including, but not limited to, (a) testing different conditions (e.g., different stoichiometric ratios of gRNA:RNA-guided nuclease protein, different buffers, etc.) to identify optimal conditions for RNP formation; and (b) testing modifications (e.g., chemical modifications, sequence changes, etc.) of the RNA-guided nuclease and / or gRNA to identify modifications that improve RNP formation or stability. One readout of the DSF assay is a shift in the melting temperature of the RNP complex; a relatively high shift suggests that the RNP complex is more stable (and therefore may have greater activity or better formation kinetics, disassembly kinetics, or another functional characteristic) compared to a reference RNP complex characterized by a lower shift. When the DSF assay is deployed as a screening tool, a threshold melting temperature shift can be specified such that the output is one or more RNPs with a melting temperature shift equal to or greater than the threshold. For example, the threshold may be 5-10° C. (e.g., 5°, 6°, 7°, 8°, 9°, 10°) or higher, and the output may be one or more RNPs characterized by a melting point shift above the threshold.

[0198] Two non-limiting examples of DSF assay conditions are provided below:

[0199] To determine the best solution to form RNP complexes, a fixed concentration of Cas9 (e.g., 2 μM) in water + 10×SYPRO Orange® (Life Technologies cat#S-6650) is dispensed into a 384-well plate. Equimolar amounts of gRNA diluted in solutions with various pH and salt are then added. After 10 minutes of incubation at room temperature and a brief centrifugation to remove any air bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler is used with Bio-Rad CFX Manager software to run a gradient from 20°C to 90°C, increasing the temperature by 1°C every 10 seconds.

[0200] The second assay consists of mixing various concentrations of gRNA with a fixed concentration (e.g., 2 μM) of Cas9 in the optimal buffer from assay 1 above and incubating in a 384-well plate (e.g., 10 min at room temperature). An equal volume of optimal buffer + 10x SYPRO Orange® (Life Technologies cat#S-6650) is added and the plate is sealed with Microseal® B adhesive (MSB-1001). Following a brief centrifugation to remove any air bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler is used with Bio-Rad CFX Manager software to run a gradient from 20°C to 90°C, increasing the temperature by 1°C every 10 seconds.

[0201] Genome editing strategies In various embodiments of the present disclosure, the above-described genome editing system is used to generate (i.e., modify) edits in targeted regions of DNA in or obtained from cells. Various strategies are described herein to generate specific edits, and these strategies are generally described in terms of the desired repair outcome, the number and arrangement of individual edits (e.g., SSBs or DSBs), and the target sites of such edits.

[0202] Genome editing strategies involving the formation of SSBs or DSBs are characterized by repair outcomes including: (a) deletion of all or part of the targeted region; (b) insertion into or replacement of all or part of the targeted region; or (c) disruption of all or part of the targeted region. This grouping is not intended to be limiting or binding to any particular theory or model, but is provided merely for the purposes of efficient use of the presentation. Those skilled in the art will understand that the results listed are not mutually exclusive, and that some repairs may lead to other outcomes. Description of a particular editing strategy or method should not be understood as requiring a particular repair outcome unless otherwise specified.

[0203] Replacement of the targeted region generally involves replacing all or part of the existing sequence in the targeted region with a homologous sequence, for example through gene correction or gene conversion, two repair outcomes mediated by the HDR pathway. As described in more detail below, HDR is facilitated using a donor template, which may be single-stranded or double-stranded. The single-stranded or double-stranded templates may be exogenous, in which case they facilitate gene correction, or they may be endogenous (e.g., a homologous sequence in the cellular genome), and facilitate gene conversion. As described, for example, by Richardson et al. (Nature Biotechnology 34, 339-344 (2016), (Richardson), incorporated by reference), the exogenous template may have an asymmetric overhang (i.e., the portion of the template that is complementary to the site of the DSB may be offset in the 3' or 5' direction rather than being centered in the donor template). If the template is single-stranded, it may correspond to the complementary (top) or non-complementary (bottom) strand of the targeted region.

[0204] As described by Ran and Cotta-Ramusino, gene conversion and gene correction are in some cases facilitated by the formation of one or more nicks at or around the targeted region. In some cases, a double nickase strategy is used to create two offset SSBs that in turn form a single DSB with an overhang (e.g., a 5' overhang).

[0205] The interruption and / or deletion of all or part of the target sequence can be achieved by various repair outcomes.As one example, the sequence can be deleted by simultaneously generating two or more DSBs adjacent to the target region, which is then excised when the DSB is repaired, as described in Maeder for LCA10 mutation.As another example, the sequence can be interrupted by the formation of a double-stranded break with a single-stranded overhang, followed by the deletion generated by exonuclease degradative processing of the overhang before repair.

[0206] One specific subset of target sequence interruptions is mediated by the formation of indels in the target sequence, where the repair outcome is generally mediated by the NHEJ pathway (including Alt-NHEJ). NHEJ is called the "error-prone" repair pathway because of its association with indel mutations. However, in some cases, DSBs are repaired by NHEJ without altering the sequence around them (so-called "complete" or "scarless" repair); this generally requires that the two ends of the DSB are completely ligated. On the other hand, indels are thought to result from enzymatic processing of free DNA ends before they are ligated, adding and / or removing nucleotides from one or both strands of one or both free ends.

[0207] Since the enzymatic processing of free DSB ends can be stochastic in nature, indel mutations tend to vary along a distribution and can be influenced by various factors including the specific target site, the cell type used, the genome editing strategy used, etc. Even so, it is possible to draw limited generalizations about indel formation: deletions formed by repair of a single DSB are most commonly in the range of 1-50 bp, but can exceed 100-200 bp. Insertions formed by repair of a single DSB tend to be shorter, often containing a short duplication of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases the inserted sequence was often derived from other regions of the genome or from plasmid DNA present in the cell.

[0208] Indel mutations, and genome editing systems configured to generate indels, are useful for interrupting target sequences, for example, when the generation of a specific final sequence is not required and / or frameshift mutations are acceptable. They can also be useful in situations where a specific sequence is preferred, as long as a certain sequence desired at a given site tends to preferentially result from the repair of an SSB or DSB. Indel mutations are also useful tools for evaluating or screening the activity of specific genome editing systems and their components. In these and other situations, indels can be characterized by (a) their relative and absolute frequency in the genome of cells contacted with the genome editing system, and (b) the distribution of numerical differences compared to the unedited sequence, e.g., ±1, ±2, ±3, etc. As an example, in a lead discovery situation, multiple gRNAs can be screened to identify the gRNA that most efficiently promotes cleavage at the target site based on the indel readout under controlled conditions. Guides that generate indels at or above a threshold frequency or generate a specific distribution of indels can be selected for further research and development. The frequency and distribution of indels can also be useful as a readout to evaluate different genome editing system implementations or formulations and delivery methods, for example, by keeping the gRNA constant and varying certain other reaction conditions or delivery methods.

[0209] Multiple Strategies Although the above exemplary strategies focus on the repair outcome mediated by a single DSB, the genome editing system according to this disclosure can also be used to generate two or more DSBs at the same locus or at different loci. Strategies for editing involving the formation of multiple DSBs or SSBs are described, for example, in Cotta-Ramusino.

[0210] Donor Mold Design Donor template design is described in detail in the literature, for example in Cotta-Ramusino. DNA oligomer donor templates (oligodeoxynucleotides or ODNs), which can be single-stranded (ssODN) or double-stranded (dsODN), can be used to promote HDR-based repair of DSBs, and are particularly useful for introducing changes into target DNA sequences, inserting new sequences into target sequences, or completely replacing target sequences.

[0211] Whether single-stranded or double-stranded, the donor template will generally contain a region that is homologous to a region of DNA within or near (e.g., adjacent or proximal to) the target sequence to be cleaved. These regions of homology are referred to herein as "homology arms" and are illustrated diagrammatically below: [5' homologous arm]-[substitution sequence]-[3' homologous arm].

[0212] The homology arms can have any suitable length (including 0 nucleotides when only one homology arm is used), and the 3' and 5' homology arms can have the same length or can be different in length. The selection of the appropriate homology arm length can be influenced by various factors, such as the desire to avoid homology or microhomology with certain sequences, such as Alu repeat sequences or other very common elements. For example, the 5' homology arm can be shortened to avoid sequence repeat elements. In other embodiments, the 3' homology arm can be shortened to avoid sequence repeat elements. In some embodiments, both the 5' and 3' homology arms can be shortened to avoid the inclusion of certain sequence repeat elements. Furthermore, some homology arm designs can improve editing efficiency or increase the frequency of the desired repair outcome. For example, Richardson et al. Nature Biotechnology 34, 339-344 (2016) (Richardson), incorporated by reference, found that the relative asymmetry of the 3' and 5' homology arms of a single-stranded donor template affects the rate and / or outcome of repair.

[0213] The replacement sequence in the donor template has been described in other publications, including Cotta-Ramusino et al. The replacement sequence may be of any suitable length (including 0 nucleotides, in the case where the desired repair result is a deletion), and generally comprises one, two, three or more sequence modifications compared to the naturally occurring sequence in the cell in which editing is desired. One common sequence modification comprises alteration of the naturally occurring sequence to repair the mutation associated with the disease or condition that is desired to be treated. Another common sequence modification comprises alteration of one or more sequences that are complementary to or code for the PAM sequence of an RNA-guided nuclease or the targeting domain of the gRNA used to generate SSB or DSB, to reduce or eliminate repeated cleavage of the target site after the replacement sequence is integrated into the target site.

[0214] When a linear ssODN is used, it can be configured to (i) anneal to a nicked strand of a target nucleic acid, (ii) anneal to an intact strand of a target nucleic acid, (iii) anneal to a plus strand of a target nucleic acid, and / or (iv) anneal to a minus strand of a target nucleic acid. The ssODN can have any suitable length, for example, about, or at least, 150-200 nucleotides (e.g., 150, 160, 170, 180, 190, or 200 nucleotides) or less.

[0215] It should be noted that the template nucleic acid may be a nucleic acid vector, such as a viral genome or a circular double-stranded DNA, such as a plasmid. The nucleic acid vector containing the donor template may contain other coding or non-coding elements. For example, the template nucleic acid can be delivered as part of a viral genome (e.g., AAV or lentiviral genome), which contains certain genomic backbone elements (e.g., in the case of the AAV genome, inverted terminal repeat sequences) and optionally contains additional sequences encoding gRNAs and / or RNA-guided nucleases. In certain embodiments, the donor template can be adjacent to or adjacent to a target site recognized by one or more gRNAs to promote the formation of free DSBs at one or both ends of the donor template, which can participate in the repair of the corresponding SSBs or DSBs formed in cellular DNA using the same gRNA. Exemplary nucleic acid vectors suitable for use as donor templates are described in Cotta-Ramusino.

[0216] Whatever format is used, the template nucleic acid can be designed to avoid undesirable sequences, in certain embodiments, one or both homology arms can be truncated to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.

[0217] In addition to the homologous arms, the donor template may include additional elements, such as a stuffer sequence and / or a reporter sequence, such as a GFP reporter (see FIG. 1) or a NGFR sequence (see FIG. 22). In some embodiments, the donor template includes a first homologous arm, a first stuffer sequence, a cargo sequence, such as one or more of exons 2-5 of CD40L, a 3' UTR sequence, a polyA sequence, a reporter sequence, such as a GFP reporter sequence or a NGFR sequence, a second stuffer sequence, and a second homologous arm. In another embodiment, the donor template includes a first homologous arm, a cargo sequence, such as one or more of exons 2-5 of CD40L, a 3' UTR sequence, a polyA sequence, a reporter sequence, such as a GFP reporter sequence or a NGFR sequence, and a second homologous arm. In another embodiment, the donor template comprises a first homologous arm, a first stuffer sequence, a cargo sequence, e.g., one or more of exons 2-5 of CD40L, a 3' UTR sequence, a polyA sequence, a second stuffer sequence, and a second homologous arm. In another embodiment, the donor template comprises a first homologous arm, a cargo sequence, e.g., one or more of exons 2-5 of CD40L, a 3' UTR sequence, a polyA sequence, and a second homologous arm.

[0218] target cell Genome editing systems according to this disclosure can be used to engineer or modify cells, for example to edit or modify a target nucleic acid. In various embodiments, the engineering can occur in vivo or ex vivo.

[0219] Various cell types can be engineered or modified by embodiments of this disclosure, and in some cases, such as in vivo applications, multiple cell types are modified or engineered, for example, by delivering a genome editing system according to this disclosure to multiple cell types. However, in other cases, it may be desirable to restrict the engineering or modification to a specific cell type. For example, in some cases, it may be desirable to edit cells with limited differentiation capacity or terminally differentiated cells, such as photoreceptor cells in the case of Maeder, where modification of the genotype is expected to result in a change in the cell phenotype. However, in other cases, it may be desirable to edit less differentiated, multipotent or pluripotent stem or progenitor cells. By way of example, the cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs), or other stem or progenitor cell types that differentiate into cell types relevant to a given application or indication.

[0220] For the treatment of hyper-IgM syndrome, the target cell may be a hematopoietic stem cell (HSC) or a population of HSC. Alternatively, the target cell may be a T cell or a population of T cells.

[0221] As a natural consequence, the cells that are altered or engineered can be dividing or non-dividing cells, depending on the cell type targeted and / or the desired editing outcome.

[0222] If the cells are manipulated or altered ex vivo, the cells can be used immediately (e.g., administered to a subject) or they can be maintained or stored for later use. One of skill in the art will appreciate that the cells can be maintained in culture or stored (e.g., frozen in liquid nitrogen) using any suitable method known in the art.

[0223] Treatment Method The genome editing system according to this disclosure can be used to treat Hyper-IgM syndrome (HIGM) and related disorders. HIGM is an immunoglobulin deficiency characterized by normal or elevated serum IgM levels and reduced or absent levels of other serum immunoglobulins, resulting in susceptibility to bacterial infections. HIGM syndrome patients are unable to switch from producing IgM type antibodies to IgG, IgA and / or IgE type antibodies. As a result, subjects with this disease have reduced levels of IgG and IgA in the blood, but normal or elevated levels of IgM. These different types of antibodies perform different functions and are all important in fighting infections. Normally, B lymphocytes can produce IgM antibodies by themselves, but to switch from IgM to IgG, IgA or IgE, they require bidirectional assistance from T lymphocytes. HIGM results from various genetic defects that affect this interaction between T lymphocytes and B lymphocytes.

[0224] The most common form of HIGM syndrome results from a defect or deficiency in a protein found on the surface of activated T lymphocytes. The affected protein is called CD40 ligand (CD40L) because it binds or ligates to a protein on B lymphocytes called CD40. CD40 ligand is made by a gene on the X chromosome. Thus, this primary immunodeficiency disease is inherited as an X-linked recessive trait.

[0225] As a result of the deficiency of CD40 ligand, T lymphocytes in X-linked hyper IgM (XHIGM) patients are unable to instruct B lymphocytes to switch immunoglobulin production from IgM to IgG, IgA, and / or IgE. Because CD40 ligand is also important for other functions performed by T lymphocytes, X-linked hyper IgM syndrome (XHIM) patients have defective cell-mediated immunity and are susceptible to many types of infections, especially opportunistic infections, and also some types of cancer. Patients with this form may also have severe neutropenia and often present with Pneumocystis jirovecii pneumonia during childhood. Because the defective CD40 ligand signaling does not activate B cells, lymphoid tissue is very small.

[0226] The diagnosis of hyper-IgM syndrome is presumed based on clinical criteria. Serum Ig levels are measured; normal or elevated serum IgM levels and reduced or absent levels of other immunoglobulins support the diagnosis. Flow cytometric testing of CD40 ligand expression on the surface of T cells should be performed. When possible, the diagnosis is confirmed by genetic testing. If women considering pregnancy have a family history of CD40 ligand deficiency, prenatal genetic testing can be offered to them. Genetic testing of other relatives is not routinely performed. Other laboratory findings include reduced numbers of memory B cells (CD27) and the absence of class-switched memory B cells (IgD-CD27).

[0227] Treatments shown to help prevent recurrent infections associated with hyper-IgM syndrome include prophylactic antibiotics and / or infusions with plasma-derived antibodies (immunoglobulins). Additionally, although steroid therapy is often effective in treating neutropenia, autoimmune disorders in children with hyper-IgM syndrome can present a difficult treatment dilemma, as the use of steroid drugs often suppresses an already weakened immune system. In some cases, nonsteroidal anti-inflammatory drugs can be helpful in controlling autoimmune-like symptoms, avoiding the use of corticosteroids.

[0228] The data presented herein provide evidence that when T cells and hematopoietic stem cells are edited to correct CD40L expression and function using the genome editing system described herein, the edited T cells regain the ability to B cell class switching. Similarly, mice receiving hematopoietic stem cells containing 10% CD40L edited cells have significantly higher levels of IgG, suggesting that hematopoietic stem cells with at least 10% CD40L editing demonstrate great therapeutic potential for the treatment of hyper IgM syndrome. Thus, the genome editing system described herein provides an important new treatment for subjects with hyper IgM syndrome.

[0229] In one embodiment, at least 5% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 6% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 7% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 8% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 9% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 10% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 15% of the edited cells in the cell population are sufficient to functionally restore class cell switching. In another embodiment, at least 20% of the edited cells in the cell population are sufficient to functionally restore class cell switching.

[0230] In some embodiments, it may be desirable to lymphodeplete a subject with hyper-IgM syndrome prior to administration of the edited cells. Thus, in one embodiment, the subject is conditioned prior to administration of the edited cells. Conditioning may include lymphodepletion of the subject. For example, in one embodiment, prior to receipt of the edited cells, the subject is conditioned with chemotherapy that causes lymphodepletion. In one embodiment, the subject's T cells are depleted by conditioning. Agents suitable for conditioning the subject include agents that induce lymphodepletion, e.g., depletion of T cells. Agents that induce lymphodepletion, i.e., conditioning agents, are known in the art. In one embodiment, the conditioning agent is a chemotherapeutic agent. In one embodiment, the conditioning agent is cyclophosphamide. In some embodiments, the method may optionally include administering to the subject a conditioning agent, e.g., a conditioning agent that causes lymphodepletion. In one embodiment, the conditioning agent is a chemotherapeutic agent. In one embodiment, the conditioning agent is cyclophosphamide.

[0231] In another embodiment, the edited cells are administered to the subject without lymphodepletion, in this embodiment, the subject is not conditioned prior to administration of the edited cells.

[0232] In one embodiment, the cells are stimulated prior to administration to a subject. In one embodiment, the cells are stimulated using a cytokine, e.g., a stimulatory cytokine. For example, the cells can be stimulated with IL-7, IL-15, IL-2, or a combination thereof.

[0233] Implementation of genome editing systems: delivery, formulation and routes of administration As mentioned above, the genome editing system of this disclosure can be implemented in any suitable manner, which means that the components of such a system, including but not limited to RNA-guided nuclease, gRNA and optional donor template nucleic acid, can be delivered, formulated or administered in any suitable form or combination of forms that result in transduction, expression or introduction of the genome editing system and / or cause the desired repair result in a cell, tissue or subject. Tables 5 and 6 show some non-limiting examples of genome editing system implementations. However, those skilled in the art will understand that these lists are not comprehensive and that other implementations are possible. With particular reference to Table 5, this table lists some exemplary implementations of a genome editing system that includes a single gRNA and an optional donor template. However, the genome editing system according to this disclosure can incorporate other components such as multiple gRNAs, multiple RNA-guided nucleases, and proteins, and various implementations will be apparent to those skilled in the art based on the principles illustrated in the table. In the table, [N / A] indicates that the genome editing system does not include the indicated component.

[0234] JPEG2025011076000011.jpg213161

[0235] Table 6 summarizes various delivery methods for the components of the genome editing system described herein. Again, the list is illustrative rather than limiting.

[0236] JPEG2025011076000012.jpg161160

[0237] Nucleic acid-based delivery of genome editing systems Nucleic acids encoding various elements of the genome editing system according to the present disclosure can be administered to a subject or delivered to a cell by methods known in the art or as described herein. For example, DNA encoding an RNA-guided nuclease and / or encoding a gRNA, as well as a donor template nucleic acid, can be delivered, for example, by a vector (e.g., a viral or non-viral vector), a non-vector-based method (e.g., using naked DNA or DNA complexes), or a combination thereof.

[0238] Nucleic acids encoding the genome editing system or its components can be delivered directly to cells as naked DNA or RNA, for example by transfection or electroporation, or can be conjugated to molecules (e.g., N-acetylgalactosamine) that facilitate uptake by target cells (e.g., red blood cells, HSCs). Nucleic acid vectors, such as those summarized in Table 6, can also be used.

[0239] The nucleic acid vector may include one or more sequences encoding genome editing system components, such as an RNA-guided nuclease, a gRNA, and / or a donor template. The vector may also include a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, or mitochondrial localization) associated with (e.g., inserted or fused to) the protein-encoding sequence. As an example, the nucleic acid vector may include a Cas9 coding sequence that includes one or more nuclear localization sequences (e.g., a nuclear localization sequence from SV40).

[0240] The nucleic acid vector can also include any suitable number of regulatory / control elements, such as promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, or internal ribosome entry sites (IRES), which are well known in the art and described in Cotta-Ramusino.

[0241] Nucleic acid vectors according to this disclosure include recombinant viral vectors. Exemplary viral vectors are shown in Table 6, and further suitable viral vectors and their use and production are described in Cotta-Ramusino. Other viral vectors known in the art can also be used. In addition, viral particles can be used to deliver genome editing system components in the form of nucleic acids and / or peptides. For example, "empty" viral particles can be assembled to contain any suitable cargo. Viral vectors and viral particles can also be engineered to incorporate targeting ligands to alter target tissue specificity.

[0242] In addition to viral vectors, non-viral vectors can be used to deliver the nucleic acid encoding the genome editing system according to the present disclosure. One important category of non-viral nucleic acid vectors is nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art and are summarized in Cotta-Ramusino. Any suitable nanoparticle design can be used to deliver the genome editing system components or nucleic acid encoding such components. For example, in certain embodiments of this disclosure, organic (e.g., lipid and / or polymer) nanoparticles may be suitable for use as delivery vehicles. Exemplary lipids for use in nanoparticle formulations and / or gene transfer are shown in Table 7, and Table 8 lists exemplary polymers for use in gene transfer and / or nanoparticle formulations.

[0243] JPEG2025011076000013.jpg228152JPEG2025011076000014.jpg80152

[0244] JPEG2025011076000015.jpg177107

[0245] Non-viral vectors optionally include targeting modifications to improve uptake and / or selectively target certain cell types. These targeting modifications may include, for example, cell-specific antigens, monoclonal antibodies, single-chain antibodies, aptamers, polymers, sugars (e.g., N-acetylgalactosamine (GalNAc)), and cell-penetrating peptides. Such vectors further optionally use fusogenic and endosome-destabilizing peptides / polymers, undergo acid-induced conformational changes (e.g., to accelerate endosomal escape of the cargo), and / or incorporate stimuli-cleavable polymers, for example, for release into cellular compartments. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.

[0246] In certain embodiments, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of the genome editing system, such as the RNA-guided nuclease component and / or gRNA component described herein, are delivered. In certain embodiments, the nucleic acid molecule is delivered simultaneously with one or more of the components of the genome editing system. In certain embodiments, the nucleic acid molecule is delivered before or after one or more of the components of the genome editing system are delivered (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks). In certain embodiments, the nucleic acid molecule is delivered by a different means than one or more of the components of the genome editing system, such as the RNA-guided nuclease component and / or gRNA component. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, such as an integration-defective lentivirus, and the RNA-guided nuclease molecule component and / or the gRNA component can be delivered by electroporation, for example, to reduce the toxicity caused by the nucleic acid (e.g., DNA). In certain embodiments, the nucleic acid molecule encodes a therapeutic protein, such as a protein described herein. In certain embodiments, the nucleic acid molecule encodes an RNA molecule, such as an RNA molecule described herein.

[0247] Delivery of RNPs and / or RNAs encoding genome editing system components RNP (complex of gRNA and RNA-guided nuclease) and / or RNA encoding the RNA-guided nuclease and / or gRNA can be delivered to cells or administered to a subject by methods known in the art, some of which are described in Cotta-Ramusino. In vitro, RNA encoding the RNA-guided nuclease and / or gRNA can be delivered by, for example, microinjection, electroporation, temporary cell compression or squeezing (see, for example, Lee 2012). Lipid-mediated transfection, peptide-mediated delivery, GalNAc or other conjugate-mediated delivery and combinations thereof can also be used for in vitro and in vivo delivery.

[0248] In vitro, delivery via electroporation involves mixing cells with RNA encoding the RNA-guided nuclease and / or gRNA in a cartridge, chamber, or cuvette, with or without a donor template nucleic acid molecule, and applying one or more electrical impulses of defined duration and amplitude. Systems and protocols for electroporation are known in the art, and any suitable electroporation tool and / or protocol can be used in connection with the various embodiments of this disclosure.

[0249] Route of administration Genome editing systems or cells modified or engineered using these systems can be administered to a subject by any suitable mode or route, whether local or systemic. Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intramedullary, intraarterial, intramuscular, intradermal, subcutaneous, intranasal, and intraperitoneal routes. Components administered systemically can be modified or formulated to target, for example, HSCs, hematopoietic stem / progenitor cells, or erythroid precursor or progenitor cells.

[0250] Examples of localized modes of administration include intramedullary injection into bone trabeculae or intrafemoral injection into the medullary space, and injection into the portal vein. In certain embodiments, a significantly smaller amount of a component (compared to a systemic approach) may be effective when administered locally (e.g., directly into the bone marrow) compared to when administered systemically (e.g., intravenously). A localized mode of administration may reduce or eliminate the occurrence of potential toxic side effects that may occur when a therapeutically effective amount of a component is administered systemically.

[0251] Dosing can be provided as periodic bolus doses (e.g., intravenously) or as continuous infusion from an internal or external reservoir (e.g., from an intravenous bag or implantable pump). The components can be administered locally, for example, by continuous release from a sustained drug delivery device.

[0252] In addition, the components can be formulated to allow release over an extended period of time. The release system can have a matrix of biodegradable materials or materials that release the incorporated components by diffusion. The components can be distributed homogeneously or heterogeneously within the release system. A variety of release systems can be useful, and the selection of the appropriate system will depend on the rate of release required for a particular application. Non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents, such as, but not limited to, calcium carbonate and sugars (e.g., trehalose). The release system can be natural or synthetic. However, synthetic release systems are preferred, as they generally produce more reliable, more reproducible, and more defined release profiles. The release system material can be selected such that components with different molecular weights are released by diffusion or degradation of the material.

[0253] Representative synthetic, biodegradable polymers include, for example: polyamides, such as poly(amino acids) and poly(peptides); polyesters, such as poly(lactic acid), poly(glycolic acid), poly(lactic acid-glycolic acid copolymers), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and their chemical derivatives (substitutions, addition of chemical groups, e.g., alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely performed by one of ordinary skill in the art), copolymers, and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers, such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates, such as methyl, ethyl, other alkyl, hydroxyethyl methacrylates, acrylic and methacrylic acid, and other vinyl polymers, such as poly(vinyl alcohol), poly(vinyl pyrrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives, such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and the various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitution, addition of chemical groups, e.g., alkyl, alkylene, hydroxylation, oxidation, and other modifications routinely performed by one of ordinary skill in the art), copolymers, and mixtures thereof.

[0254] Poly(lactide-glycolide copolymer) microspheres may also be used. Generally, the microspheres are composed of polymers of lactic acid and glycolic acid and are constructed to form hollow spheres. The spheres may be approximately 15-30 microns in diameter and may be loaded with the components described herein.

[0255] Multimodal or differential delivery of ingredients From this disclosure, those skilled in the art will recognize in light of this disclosure that different components of the genome editing systems disclosed herein can be delivered together or separately and simultaneously or separately. Separate and / or non-simultaneous delivery of genome editing system components can be particularly desirable to provide temporal or spatial control over the function of the genome editing system and limit certain effects caused by its activity.

[0256] As used herein, different or differential modes refer to modes of delivery that result in different pharmacodynamic or pharmacokinetic properties for the component molecules of interest, such as RNA-guided nuclease molecules, gRNAs, template nucleic acids, or payloads. For example, the modes of delivery may result in different tissue distribution, different half-lives, or different temporal distribution, for example, in selected compartments, tissues, or organs.

[0257] For example, some modes of delivery, such as delivery by nucleic acid vectors that persist in the cell or cell progeny by self-replication or insertion into cellular nucleic acid, result in more sustained expression and presence of the component. Examples include viral, such as AAV or lentiviral delivery.

[0258] For example, components of the genome editing system, such as the RNA-guided nuclease and gRNA, may be delivered by different modes in terms of the half-life or persistence of the delivered components obtained in the body or in a particular compartment, tissue or organ. In certain embodiments, the gRNA may be delivered by such a mode. The RNA-guided nuclease molecule component may be delivered by a mode that results in low persistence or low exposure to the body or a particular compartment, tissue or organ.

[0259] More generally, in certain embodiments, a first mode of delivery is used to deliver a first component, and a second mode of delivery is used to deliver a second component. The first mode of delivery provides a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, for example, the distribution, persistence, or exposure of the component, or the nucleic acid encoding the component, in the body, compartment, tissue, or organ. The second mode of delivery provides a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, for example, the distribution, persistence, or exposure of the component, or the nucleic acid encoding the component, in the body, compartment, tissue, or organ.

[0260] In certain embodiments, a first pharmacodynamic or pharmacokinetic property, eg, distribution, persistence or exposure, is more defined than a second pharmacodynamic or pharmacokinetic property.

[0261] In certain embodiments, the first mode of delivery is selected to optimize, eg, minimize, a pharmacodynamic or pharmacokinetic property, eg, distribution, duration, or exposure.

[0262] In certain embodiments, the second mode of delivery is selected to optimize, eg, maximize, a pharmacodynamic or pharmacokinetic property, eg, distribution, duration, or exposure.

[0263] In certain embodiments, the first mode of delivery involves the use of a relatively persistent element, such as a nucleic acid, such as a plasmid or a viral vector, such as an AAV or lentivirus, such that the product transcribed therefrom is relatively persistent.

[0264] In certain embodiments, the second mode of delivery comprises a relatively transient element, such as RNA or a protein.

[0265] In certain embodiments, the first component comprises a gRNA and the delivery mode is relatively sustained, for example, the gRNA is transcribed from a plasmid or a viral vector, such as an AAV or lentivirus. The transcription of these genes has little physiological impact, since the genes do not code for protein products and the gRNA cannot act alone. The second component, the RNA-guided nuclease molecule, is delivered in a transient manner, for example as an mRNA or protein, ensuring that the complete RNA-guided nuclease molecule / gRNA complex is present and active for only a short period of time.

[0266] Furthermore, the components may be delivered in different molecular forms or in different delivery vectors that complement each other to enhance safety and tissue specificity.

[0267] The use of differential delivery modes can enhance performance, safety and / or efficacy, for example reducing the likelihood of eventual off-target modification. Because bacterially derived Cas enzyme-derived peptides are presented on the surface of cells by MHC molecules, delivery of immunogenic components, such as Cas9 molecules, can reduce immunogenicity by a less sustained manner. A two-component delivery system can alleviate these drawbacks.

[0268] Differential delivery modes can be used to deliver components to different but overlapping target regions. Formation active complexes are minimized outside the overlap of target regions. Thus, in certain embodiments, a first component, e.g., gRNA, is delivered by a first delivery mode that results in a first spatial, e.g., tissue distribution. A second component, e.g., RNA-guided nuclease molecule, is delivered by a second delivery mode that results in a second spatial, e.g., tissue distribution. In certain embodiments, the first mode comprises a first element selected from liposomes, nanoparticles, e.g., polymer nanoparticles, and nucleic acids, e.g., viral vectors. The second mode comprises a second element selected from the group. In certain embodiments, the first mode of delivery comprises a first targeting element, e.g., a cell-specific receptor or antibody, and the second mode of delivery does not comprise that element. In certain embodiments, the second mode of delivery comprises a second targeting element, e.g., a second cell-specific receptor or a second antibody.

[0269] When the RNA-guided nuclease molecule is delivered in a viral delivery vector, liposome, or polymer nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, even when it may be desirable to only target a single tissue. A two-component delivery system can solve this problem and enhance tissue specificity. When the gRNA and the RNA-guided nuclease molecule are packaged in separate delivery vehicles with unique but overlapping tissue affinities, a fully functional complex is formed only in tissues that are targeted by both vectors.

[0270] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications, and figures cited throughout this application are hereby incorporated by reference. EXAMPLES

[0271] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0272] [Example 1] Gene editing of CD40L on T lymphocytes Targeted integration experiments were performed in T cells using wild-type S. pyogenes Cas9 ribonucleoprotein (RNP) targeted to the CD40L locus. The gRNA sequences are shown in Table 9. The CD40L donor sequence is shown in Table 10. Figure 2A shows a schematic diagram of the CD40L locus after targeted integration. Eight S. pyogenes and 30 S. aureus gRNAs targeted to CD40LG exon 1 were screened for editing efficiency in combination with S. pyogenes or S. aureus wild-type Cas9 in human primary T cells. The location of the gRNA in CD40LG exon 1 is illustrated in Figure 2B. Approximately 90%-95% of CD40LG mutations were corrected in S. pyogenes and S. aureus, restoring both expression and function of the gene. Figure 2C shows the insertion / deletion rate of each gRNA measured in T cells. To determine the level and type of editing at the multiple gRNA sites identified in CD40LG intron 1, ribonucleoproteins (RNPs) were synthesized from human male primary CD4 +T cells were transduced with 2.27 μM RNP at a gRNA:Cas9 ratio of 2:1 using the Lonza nucleofection system. The cells were then expanded in T cell media for 4 days before genomic DNA was isolated. Genomic DNA (gDNA) was then PCR amplified using primers surrounding the cleavage site of interest, sequenced, and analyzed for insertions and deletions at the cleavage site. Over 70% overall editing was achieved with each gRNA presented in Figure 2C, although the ratio of insertions to deletions varied. The results depicted in Figure 2C represent at least two independent samples. The percentage of editing achieved with each RNP is also presented in Table 9.

[0273] The gRNAs that exhibited high editing efficiency were used in targeted integration experiments in combination with AAV6 vectors containing the CD40LG donor template as illustrated in Figure 2A. As shown in Figure 2D, AAV6 was added after electroporation of RNPs on day 3, and GFP fluorescence was measured by FACS 20 days after the experiment. The percentage of GFP-positive cells in the T cell population after gene editing is shown in Figure 2E. T stem memory cells (TSCM) are defined as CD62L+CD45RA+, T central memory (TCM) as CD62L+CD45RA-, T effector memory (TEM) as CD62L-CD45RA-, and terminal effector (TEMRA) as CD62LCD45RA+. The combination of CRISPR / Cas9 and AAV6 enabled high levels of CD40L gene editing in multiple T cell populations. Representative plots of T cells expressing CD45RA and CD62L at days 7 (left panel) and 20 (right panel) after treatment are shown in FIG. 2F. T cell culture composition at days 7 and 19 (left panel) and 20 (right panel) after treatment is shown in FIG. 2G. The data show that there is no skewing of T cell subpopulations after gene editing at the CD40L locus compared to unedited ("cells only") controls. Targeted integration experiments were also performed in combination with IDLV vectors containing CD40LG donor templates, as shown in FIG. 2H. The levels of HDR (assessed by determining the percentage of GFP+ cells) within various T cell lineages are shown in FIG. 2I. FIG. 2J illustrates that similar and high levels of gene editing are observed in both HD and patient cells.

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[0275] JPEG2025011076000018.jpg145165JPEG2025011076000019.jpg244165JPEG2025011076000020.jpg244165JPEG2025011076000021.jpg244165JPEG202 5011076000022.jpg244165JPEG2025011076000023.jpg245166JPEG2025011 076000024.jpg245166JPEG2025011076000025.jpg245166JPEG20250110760 00026.jpg245166JPEG2025011076000027.jpg245166JPEG2025011076000028.jpg245166JPEG2025011076000029.jpg245166JPEG2025011076000030.j pg244165JPEG2025011076000031.jpg244165JPEG2025011076000032.jpg244165JPEG2025011076000033.jpg244165JPEG2025011076000034.jpg149165

[0276] CD40L expression after targeted integration and editing by three RNP complexes containing representative gRNAs (gRNA A, D, and G) was determined. The location of CD40LG, intron 1, targeted by these gRNAs is illustrated in FIG. 3A. The experimental procedure is outlined in FIG. 3B. The rate of targeted integration (shown as a percentage of GFP+ cells) after treatment with RNP complexes and AAV6 donor templates is presented in FIG. 3C. The level of targeted integration obtained with gRNA A was comparable to that obtained with gRNA D and higher than that obtained with gRNA G. FIG. 3D illustrates CD40L expression in the GFP-positive cell population as determined by FACS analysis. FIG. 3E illustrates the percentage of CD40L+ cells at various time points after restimulation. Compared to integration of the corrected repair template in the middle of intron 1 using gRNA A or gRNA D, integration of the corrected repair template at the 3' end of intron 1 using gRNA G did not change CD40L expression.

[0277] To test whether gene editing of CD40L preserves physiological regulation, the kinetics of CD40L expression was determined. As shown in Figure 4B, after RNP electroporation and donor DNA addition, the kinetics of CD40L expression was monitored over a 48-hour time series starting on day 15 after induction of CD40L expression with PMA / ionomycin. Figures 4C and 4E illustrate the kinetics of CD40L expression in edited CD4+ T cells after PMA / ionomycin treatment. Expression of CD40L in activated CD4+ and CD8+ cells is shown in Figures 4D and 4F. This data indicates that normal CD40L expression was restored by gene editing.

[0278] To confirm these results, another experiment was performed. Human T cells were electroporated with RNP and transduced with AAV6 vector carrying the corrected donor 15 minutes later. FACS analysis was performed 6 days after RNP electroporation and the kinetics of CD40L expression was determined at day 15 (Figure 5B). As shown in Figures 5C and 5D, editing of CD40LG allows normal expression of CD40L protein and preserves the physiological regulation of CD40L. Figures 5C and 5D illustrate the kinetics of CD40L expression in CD4+ T cells edited with wild-type or codon-optimized CD40LG ("co") donor after PMA / ionomycin treatment. CD40L expression in activated CD4+ and CD8+ cells is shown in Figure 5C. The data show that the codon-optimized CD40LG cDNA allows better restoration of CD40L expression. In addition, gene editing efficiency improved when higher concentrations of AAV6 donor were added to the cells (Figure 5E). Modified CD40L expression peaks at a lower level than endogenous CD40L, but they both exhibit a sharp decline in expression after approximately 6 hours (data not shown). Similarly, gene editing efficiency was improved by optimizing the dose of RNP and AAV6. Figures 6A and 6B demonstrate that the methods described herein generate high levels of CD40L gene editing (Figure 6A) and high levels of exogenous CD40L expression (Figure 6B). It was observed that AAV inhibits T cell expansion after electroporation depending on its MOI (data not shown).

[0279] The specificity of gRNA A and gRNA D was evaluated using Guide-Seq and Amplicon-Seq (Amp-Seq) analysis. Guide-Seq analysis was performed to evaluate the number of on-target and off-target editing events that occurred after editing with RNPs containing gRNA A or gRNA D, and the results are shown in Figure 7A. Guide-Seq was performed in male T cells after cell activation and expansion. Cells were nucleofected with RNPs containing gRNA A or D and short double-stranded oligos (Nat. Biotech. 2015, 33: 187-197). gDNA was isolated, sheared, and adapters for PCR amplification were added before PCR amplification. Sequences flanking the Guide-Seq oligos were aligned to the genome to find the location of the double-stranded oligo insertion. The results of the Guide-Seq analysis are summarized in Table 11.

[0280] JPEG2025011076000035.jpg34157

[0281] As shown in Figure 7B, Amp-Seq analysis was performed to verify the off-target editing events observed in gRNA A. Amp-Seq was performed using PCR amplification around the off-target cleavage sites found from Guide-Seq analysis, followed by high-throughput sequencing and computer alignment to determine whether there were editing events at the cleavage sites. One off-target cleavage site was identified for gRNA A by Guide-Seq. This cleavage site was on chromosome 8, approximately 300 kb from another gene. As shown in Figure 7B, off-target cleavage was reduced to background levels using higher fidelity Cas9 (eCas9). Target editing events for gRNA A are illustrated in Figure 7B and Figure 7E. No detectable off-target cleavage sites were observed in gRNA D. Digenome analysis revealed a significantly higher number of potential in vitro cleavage sites for gRNA A (205) compared to gRNA D (10) (Figures 7C-7D). As shown above, gRNA A and gRNA D exhibited high efficiency of editing and targeted integration combined with a high level of specificity. CD40L expression from the integrated cargo was comparable to endogenous levels of CD40L. Figure 7F illustrates a summary of the results of Amplicon-Seq analysis for the off-target cleavage sites identified for gRNA A and D.

[0282] [Example 2] Edited CD40L cells are functionally active and restore B cell class switch recombination To determine whether function was restored in CD40L-edited cells, B cells were co-cultured with edited T cells and surface expression of IgG on B cells was determined. Gene editing of CD40L in T cells was performed as described above. B cells were co-cultured with wild-type T cells (unedited), T cells edited and selected for targeted integration of the modified CD40L gene (GFP+), T cells treated for gene editing but negative for integration of the modified CD40L gene (GFP-), and T cells in which the endogenous CD40L gene had been knocked out by integration of a reporter cassette (CD40L KO). (i) B cells alone and (ii) cultures containing soluble CD40L protein (sCD40L) in addition to B cells were used as negative and positive controls, respectively. T cells were tested without stimulation (resting), after stimulation with anti-CD3 / CD28 beads (beads), or after stimulation with PMA / ionomycin (PMA / Iono). After 5 days of co-culture, FACS analysis was performed. As shown in FIG. 8, the edited T cells were able to induce B cell class switching in the co-culture assay. Furthermore, IgM levels were significantly decreased and IgG levels were significantly increased in the medium of B cells co-cultured with T cells after gene editing (FIG. 9). Taken together, the data reported in FIG. 8 and FIG. 9 demonstrate that the edited T cells express functional CD40L and are capable of providing contact-dependent signals to B cells that induced class switching in vitro.

[0283] [Example 3] Targeted integration at the CD40L locus Baseline levels of targeted integration at the four gRNA cleavage sites from Example 1 were determined: gRNA A (S. pyogenes), gRNA C (S. pyogenes), gRNA D (S. aureus) and gRNA G (S. aureus). AAV6 donor templates were designed according to the depiction in Figure 1A such that all donors contained the same cargo (stuffer sequence, CD40LG cDNA / 3' UTR / polyA and GFP expression cassette) within short homology arms designed to flank the predicted cleavage site. The homology arm lengths in each construct are provided in Table 12.

[0284] JPEG2025011076000036.jpg3562

[0285] Human male primary CD4 + T cells were grown and nucleofected with the indicated gRNAs as described above. Cells were transferred to fresh culture medium and AAV6 particles were added to the cultures 15 min later at either a multiplicity of infection (MOI) of 5e4 or 1e5. Cells were expanded for 7 days and then assessed for targeted integration based on GFP expression using flow cytometry analysis. At this time point after nucleofection, there was very little detectable expression of GFP from the non-integrated AAV donor (data not shown), indicating that cells with high levels of GFP expression had undergone targeted integration. As shown in FIG. 10, differences between gRNAs were found in the baseline levels of targeted integration based on GFP expression (gRNA A approx. 35%, gRNA C approx. 15%, gRNA D approx. 28%, and gRNA G approx. 17%). No dose-dependent reduction in targeted integration was observed by reducing the amount of AAV6 donor given to the cells. Results are representative of at least two independent samples.

[0286] To increase the baseline level of targeted integration, the effect of lengthening the homology arms was examined. AAV6 donor templates with long homology arms (500 bp on each side of the cargo) were designed for the top gRNA site based on the targeted integration determined in Figure 10 (gRNA A and gRNA D). Targeted integration experiments were performed in human male primary CD4+ T cells as described above. Cells were fed AAV6 donor templates with either short HA (same as AAV6 donor used in Figure 10) or long HA. GFP expression was determined by flow cytometry 7 days after nucleofection and is plotted in Figure 11. Long HA improved the level of targeted integration for both gRNA cleavage sites. Targeted integration at the gRNA A site increased by about 35% to about 44%, and targeted integration at the gRNA D site increased by about 28% to about 42%.

[0287] [Example 4] Adoptive T cell therapy in mouse models Mice were pretreated or conditioned with cyclophosphamide on day 0 to deplete recipient cells and free up lymphocytic niches, allowing better engraftment of transplanted cells. Wild-type T cells were injected on day 2, and their engraftment was measured longitudinally over time. The CD8 / CD4 ratio remained stable in the unconditioned group, but increased significantly in conditioned mice (see FIG. 12A). During that time, the majority of circulating donor CD8 and CD4 T cells expressed T central memory and effector memory phenotypes, respectively (FIG. 12B). TNP / KLH vaccination was introduced on day 21, followed by serological assays on days 35 and 50. As shown in FIG. 13, conditioning with cyclophosphamide resulted in higher levels of TNP / KLH-specific IgG in mice. The level of specific IgG response to vaccination correlates with the conditioning dose administered (FIG. 14A) and the amount of circulating wild-type CD4 T cells (percentage and absolute numbers, FIG. 14B). However, the conditioning step is not absolutely necessary, since unconditioned mice also showed increased IgG levels in response to vaccination (Figures 13 and 14A). In a second experiment, mice were pretreated or conditioned with anti-leukocyte (ALS) or anti-CD4 antibodies on day 0. Figures 14C and 14E illustrate the percentage of recipient and donor T cells over time in mice treated with or without the preconditioning regimen. The level of specific IgG response to vaccination correlates with the conditioning dose administered (Figure 14D). This data provides evidence that lymphodepletion conditioning could also be performed by an immunobiological strategy. Adoptive T cell transfer also restored the presence of germinal centers in the spleens of transplanted mice (Figures 15A and 15B).

[0288] [Example 5] Hematopoietic stem cell therapy in mouse models CD40L negative mice were treated with CD40L wild type HSPCs harvested from 6-8 week old wild type C57B / 6 mice and mixed with CD40LG negative cells harvested from HIGM1 mice at different percentages (100%, 10%, 1% and 0%). Recipient mice were then immunized with TNP-KLH and the levels of TNP-KLH specific IgG were measured. As shown in Figure 16A, mice receiving hematopoietic stem cells containing 10% and 100% CD40L wild type cells had very high levels of IgG, suggesting that hematopoietic stem cells containing at least 10% CD40L edited cells show therapeutic efficacy in treating diseases, such as hyper-IgM syndrome.

[0289] In the second experiment, mice were challenged with two antigens, TNP-KLH and ovalbumin (OVA), as shown in FIG. 16B. Different percentages of wild-type HSPCs were transplanted into CD40LG-negative mice on day 0. Mice were immunized with TNP-KLH at weeks 12 and 15 after transplantation, and subsequently immunized with OVA at weeks 20 and 23 after transplantation. TNP-specific IgG responses were assessed (i) before immunization with TNP-KLH (pre-challenge), (ii) 14 days after initial immunization with TNP-KLH (day 14), and (iii) 28 days after initial immunization with TNP-KLH (post-boost, day 28). OVA-specific IgG responses were assessed (i) before immunization with OVA (pre-challenge), (ii) 14 days after initial immunization with OVA (day 14), and (iii) 28 days after initial immunization with OVA (post-boost, day 28). The results show that recovery of at least 10% of CD40LG positive HSPCs allows partial restoration of immune responses to different T cell-dependent vaccinations. Figure 16C illustrates donor T cell engraftment in peripheral blood.

[0290] [Example 6] Proof-of-concept of CD40LG correction on T cells obtained from HIGM1 patients To confirm the correction of the HIGM1 causative mutation by targeted integration of the corrected donor template, gene editing was performed on T cells harvested from a male subject with a genotyped inactivating mutation (c.334 G > T) on exon 3 of the CD40L gene. CD4 + T cells were grown and nucleofected with RNP (gRNA A) and 15 min later transduced with AAV6 vector carrying the modified donor template depicted in Figure 2A at an MOI of 5e4. Targeted integration was measured by cytofluorimetric analysis scoring the percentage of GFP+ cells within the following CD4 T cell subpopulations: T stem cell memory (TSCM, CD45RA+ CD62L+), central memory (CM, CD45RA-CD62L+), effector memory (EM, CD45RA-CD62L-) and effector memory RA (TEMRA, CD45RA+CD62L-; Figures 17A, 17B). Targeted insertion was verified by digital droplet PCR analysis quantifying the 5' vector-to-genome junction, thereby confirming a similar amount of homology-directed repair as measured by GFP expression (Figure 17C). Polyclonal T cell targeting was confirmed by quantification of T cell receptor diversity by TCR-beta spectratyping (Figure 17D). Integration of the correction construct restored expression and physiological regulation of CD40L in edited patient T cells after PMA / ionomycin stimulation (Figure 18). To assess the functionality of the edited cells, sorted GFP+ cells were tested in a TB cell co-culture assay (Figure 19A) and confirmed the restoration of T cell-dependent signals to B cells inducing B cell proliferation (Figure 19B,C) and the production of class-switched IgG (Figure 20).

[0291] [Example 7] Selection of CD40LG-edited cells before transplantation In addition to the methods described above, the correction construct used to repair the CD40LG mutation can be equipped with a selection gene to enrich for edited cells prior to transplantation. Coupling the corrected CD40LG cDNA to a selection gene (e.g., nerve growth factor receptor [NGFR]) followed by an internal ribosome entry site (IRES) sequence (Figure 22A) allows targeted integration to occur and expression of the selection gene only in cells that concomitantly express the CD40LG gene. Although CD40LG requires strong stimulation to be exposed on the surface of T lymphocytes, basal levels of expression from the CD40LG promoter are sufficient to surface express the NGFR selection gene in edited cells, beginning 6 days after gene editing, followed by antibody selection on day 13 (Figures 22B and 22C). Following PMA / ionomycin stimulation of treated cells, the CD40LG promoter rapidly increased expression of both CD40L and NGFR proteins (Figure 22D). However, the kinetics of CD40L surface expression is comparable to that measured in mock (unedited, UT) T cells (Figure 22E). Using an NGFR expression cassette, we obtained physiological expression of the CD40LG gene in edited cells, both in terms of the level of expression and the percentage of CD40L+ cells (Figure 22E). Figure 22F illustrates the ability of CD40L-edited T cells to restore B cell class switching in co-culture assays, showing the percentage of IgG-switched B cells in co-culture with resting, bead-activated (1:1 ratio) or PMA / ionomycin-stimulated T cells (naive [UT], or edited [NGFR+]). The targeted integration efficiency of the corrected donor containing the selection cassette (Figure 23A) was similar to that measured with the original corrected donor template (Figure 6A). Targeted cells expressing CD40LG could be enriched by NGFR selection without any skewing in the T cell culture composition (Figure 23B), and molecular analysis confirmed the presence of targeted integration in NGFR+ cells (Figure 23C). NSG mice were transplanted with edited CD4+ T cells that were either selected or not for NGFR expression. Levels of engraftment were comparable to those observed in mice transplanted with unedited T cells, and the proportion of edited cells remained stable over time (Figure 24A). These data demonstrate that the CD40LG editing procedure and NGFR selection do not impair the repopulating capacity of treated T cells, which are generally capable of engrafting long-term and physiologically differentiating into effector cells after engraftment (Figure 24B). Figure 24C shows that edited CD40LG is expressed by edited T cells recovered from NSG mice.

[0292] The selection strategies described herein may be utilized to increase the percentage and absolute numbers of therapeutically relevant corrected T cells that can be adoptively transferred in HIGM patients while minimizing the number of unmodified T cells cultured in vitro that do not confer any therapeutic benefit.

[0293] [Example 8] Gene editing of CD40L in human hematopoietic stem precursor cells Adoptive immunotherapy approaches based on gene-modified autologous T cells may be sufficient to induce the development of protective immunity in recipient HIGM1 patients and perhaps even the production of some long-lived memory B cells. However, multiple administrations of edited T cells may be required to reconstitute a long-lasting, broad T cell repertoire that can mediate efficient T cell help in response to a wide range of pathogens. Furthermore, although CD40LG has critical functions on CD4 T cells (Th0, Th1 and Th2), this molecule is also expressed in several other hematopoietic cell types, such as activated B cells, platelets, NK cells, monocytes, basophils and eosinophils. Therefore, it will be useful to extend gene editing strategies from T cell modification to autologous hematopoietic stem / progenitor cell (HSPC) modification, which may provide broader and longer-lasting therapeutic benefits.

[0294] To demonstrate the transferability of the described gene correction strategy for human HSPCs, we first adapted a previously reported ZFN-based gene editing protocol to the CRISPR / Cas9 system. Briefly, as shown in FIG. 25A, CD34+ cells were isolated from umbilical cord blood and pre-stimulated with SR1+dmPGE2 for 2 days. Cells were infected with IDLV donor on day 2 and electroporated on day 3 with ribonucleoprotein (RNP) complexes composed of in vitro transcribed gRNA or a modified version of in vitro transcribed gRNA (gRNAmod: HPLC purified, ARCA capped, 5' phosphatase) and assembled Cas9 protein. Then on day 7, cells were FACS analyzed for the expression of GFP and cell surface markers. The targeted integration efficiency achieved by the two different RNPs and reference ZFN in the indicated cell populations is shown in FIG. 25B. The culture composition after editing with ZFN and RNP is shown in FIG. 25C compared to untreated cells. The induction of interferon (IFN) responsive genes RIG1, IRF7 and OAS1 in edited cells is shown in Figure 25D. These results demonstrate that delivery of modified RNPs allows levels of homology-mediated editing in human HSPCs comparable to our historical, gold standard ZFN protocol, without inducing a deleterious IFN response and without affecting the phenotype of treated cells.

[0295] Additional experiments were performed using CD34+ cells obtained from umbilical cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB). Briefly, as shown in FIG. 26A, cells were pre-stimulated with SR1, dmPGE2 and UM171 for 3 days, at which time the cells were electroporated with RNP complexes composed of Cas9 protein assembled with single or two-component synthetic gRNA and infected with AAV6 donor. Then on day 6, cells were FACS sorted for expression of GFP and cell surface markers. Targeted integration efficiency (expressed as percentage of GFP+ cells) is shown in FIG. 26B. Cell culture composition after editing is shown in FIG. 26C. Expression of IFN-responsive genes is shown in FIG. 26D. These results confirmed that homology-mediated editing in human HSPCs can be achieved without inducing adverse IFN responses and without affecting the phenotype of treated cells, and using AAV6 or RNP assembled with synthetic gRNA as a delivery vehicle for donor DNA.

[0296] As shown in Figure 27, human HSPCs from diverse sources can be efficiently edited using a variety of donor vehicles and nuclease configurations. The indicated cell types were edited using either ZFNs or RNPs with IDLV or AAV6 donor templates. The percentage of edited (GFP+) cells in each subpopulation is shown in Figure 27A. The percentage of edited (GFP+) cell populations obtained from cord blood, bone marrow, and mobilized peripheral blood (mPB) is shown in Figure 27B.

[0297] To confirm that the CD40L gene correction strategy is feasible in human HSPCs, targeted integration of the corrected donor was performed in umbilical cord blood CD34+ cells harvested from a healthy male donor. HSPCs were grown, nucleofected with different RNP (gRNA A) doses, and transduced 15 min later with different doses of the AAV6 vector carrying the corrected donor (MOI: 1e4, 5e4, and 1e5 vg / cell) (Figure 21A). Targeted integration was measured by analytical digital droplet PCR quantifying the 5' vector-genome junction. To confirm editing in all the different HSPC cell subpopulations, treated cells were sorted using surface markers that previously identified more primitive (CD34+CD133+CD90+), early (CD34+CD133+CD90-) and committed (CD34+CD133-) precursors, as well as differentiated cells (CD34-). High levels of targeted integration were measured in both bulk and different sorted populations (FIGS. 21B, C, and E) without modifying the overall culture composition (FIG. 21D). Levels of engraftment were comparable to those observed in mice transferred with unedited T cells, and the proportion of edited cells remained stable over time (FIG. 21F).

[0298] INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0299] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the following claims.

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Claims

1. An isolated oligonucleotide donor template comprising, from 5' to 3', A1-N-UTR-pA-A2, A1 is a homologous arm that is substantially identical to the first homologous arm of a target CD40L nucleic acid; N is a cargo containing one or more of exons 2 to 5 of the CD40L gene; UTR is the 3′ untranslated region (UTR) of CD40L; pA is the polyA tail; A2 is a homologous arm that is substantially identical to a second homologous arm of the target CD40L nucleic acid; An isolated oligonucleotide donor template.

2. From 5' to 3', it contains A1-S1-N-UTR-pA-S2-A2, S1 is the first stuffer and S2 is the second stuffer, 2. The isolated oligonucleotide donor template of claim 1.

3. 3. The isolated oligonucleotide donor template of claim 2, comprising from 5' to 3' A1-S1-N-UTR-pA-R-S2-A2, wherein R is a reporter.

4. 2. The isolated oligonucleotide donor template of claim 1, comprising from 5' to 3' A1-N-UTR-pA-R-A2, wherein R is a reporter.

5. the cargo is selected from exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or Exons 4 and 5 of the CD40L gene 5. The isolated oligonucleotide donor template of claim 1, comprising:

6. 6. The isolated oligonucleotide donor template of any one of claims 1 to 5, wherein the CD40L gene is a wild-type CD40L sequence.

7. 7. The isolated oligonucleotide donor template of any one of claims 1 to 6, wherein the target CD40L nucleic acid comprises a mutation.

8. 8. The isolated oligonucleotide donor template of any one of claims 1 to 7, wherein the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase.

9. 9. The isolated oligonucleotide donor template of any one of claims 1 to 8, which is a single-stranded oligonucleotide donor template or a double-stranded oligonucleotide donor template.

10. A composition comprising the isolated oligonucleotide donor template of any one of claims 1 to 9, and optionally a pharma- ceutically acceptable carrier.

11. A vector comprising the isolated oligonucleotide donor template of any one of claims 1 to 9.

12. The vector of claim 11, wherein the vector is an AAV vector, a lentivirus, a naked DNA vector or a lipid nanoparticle.

13. An isolated gRNA molecule comprising any one of SEQ ID NOs: 18-25.

14. A genome editing system comprising an isolated oligonucleotide donor template according to any one of claims 1 to 9.

15. The genome editing system of claim 14, further comprising an RNA-guided nuclease and at least one gRNA molecule.

16. The genome editing system of claim 14, further comprising the isolated gRNA molecule of claim 13.

17. 17. A method of modifying a cell, comprising contacting the cell with a genome editing system described in any one of claims 14 to 16, thereby modifying the cell.

18. A kit comprising the genome editing system described in any one of claims 14 to 16.

19. The isolated oligonucleotide donor template of any one of claims 1 to 9, the composition of claim 10, the vector of any one of claims 11 to 12, the isolated gRNA molecule of claim 13, the gene editing system of any one of claims 14 to 16, the method of claim 17 or the kit of claim 18 for use in medicine.

20. 1. A method of modifying a cell, comprising: creating at least one single-stranded or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, the target CD40L nucleic acid comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site; recombining an exogenous oligonucleotide donor template with said target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, said exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to said first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm substantially identical to said second homologous arm; Including, thereby modifying said cells.

21. 21. The method of claim 20, wherein the exogenous oligonucleotide donor template comprises the first donor homologous arm that is substantially identical to the first homologous arm, a first stuffer, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3' CD40L untranslated region (UTR), the polyA tail, a second stuffer, and the second donor homologous arm that is substantially identical to the second homologous arm.

22. 22. The method of claim 21, wherein the exogenous oligonucleotide donor template comprises the first donor homologous arm that is substantially identical to the first homologous arm, the first stuffer, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3' CD40L untranslated region (UTR), the polyA tail, a reporter, the second stuffer, and the second donor homologous arm that is substantially identical to the second homologous arm.

23. 21. The method of claim 20, wherein the exogenous oligonucleotide donor template comprises the first donor homologous arm that is substantially identical to the first homologous arm, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3' CD40L untranslated region (UTR), the polyA tail, a reporter, and the second donor homologous arm that is substantially identical to the second homologous arm.

24. 24. The method of any one of claims 20 to 23, wherein the cargo comprises exon 2 of the CD40L gene; exon 3 of the CD40L gene; exon 4 of the CD40L gene; exon 5 of the CD40L gene; exons 2-5 of the CD40L gene; exons 2-4 of the CD40L gene; exons 2 and 3 of the CD40L gene; exons 3-5 of the CD40L gene; exons 3 and 4 of the CD40L gene; or exons 4 and 5 of the CD40L gene.

25. The method of any one of claims 20 to 24, wherein the CD40L gene is a wild-type CD40L sequence.

26. The method of any one of claims 20 to 25, wherein the target CD40L nucleic acid comprises a mutation.

27. 24. The method of claim 22 or 23, wherein the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase.

28. 28. The method of any one of claims 20 to 27, wherein the step of forming at least one single-stranded or double-stranded break comprises contacting the cell with an RNA-guided nuclease.

29. 29. The method of claim 28, wherein the RNA-guided nuclease is a class 2 clustered regularly interspersed repeat (CRISPR)-associated nuclease.

30. 30. The method of claim 29, wherein the RNA-guided nuclease is selected from the group consisting of wild-type Cas9, Cas9 nickase, wild-type Cpf1, and Cpf1 nickase.

31. 31. The method of any one of claims 28 to 30, wherein contacting the cell with the RNA-guided nuclease comprises introducing into the cell a ribonucleoprotein (RNP) complex comprising the RNA-guided nuclease and a guide RNA (gRNA).

32. 32. The method of any one of claims 28 to 31, wherein the step of recombining the exogenous oligonucleotide donor template into the target CD40L nucleic acid by homologous recombination comprises introducing the exogenous oligonucleotide donor template into the cell.

33. 33. The method of claim 31 or claim 32, wherein the step of introducing comprises electroporation of the cells in the presence of the RNP complex and / or the exogenous oligonucleotide donor template.

34. 1. A method of modifying a target CD40L nucleic acid in a cell, the target CD40L nucleic acid comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site, the method comprising: The cells, (a) at least one gRNA molecule; (b) an RNA-guided nuclease molecule, and (c) Exogenous oligonucleotide donor template wherein the exogenous oligonucleotide donor template comprises a first donor homology arm substantially identical to the first homology arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homology arm substantially identical to the second homology arm; wherein the gRNA molecule and the RNA-guided nuclease molecule interact with the target CD40L nucleic acid to effect a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid; Thereby altering said target CD40L nucleic acid in said cell.

35. The cells, 35. The method of Claim 34, further comprising the step of: (d) contacting with a second gRNA molecule, wherein said second gRNA molecule and said RNA-guided nuclease molecule interact with the target nucleic acid resulting in a second cleavage event at or near the cleavage site, and wherein said second cleavage event is repaired by at least one of said DNA repair pathways.

36. 36. The method of claim 34 or claim 35, wherein the exogenous oligonucleotide donor template comprises the first donor homologous arm that is substantially identical to the first homologous arm, a first stuffer, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3' CD40L untranslated region (UTR), the polyA tail, a second stuffer, and the second donor homologous arm that is substantially identical to the second homologous arm.

37. 37. The method of claim 36, wherein the exogenous oligonucleotide donor template comprises the first donor homologous arm that is substantially identical to the first homologous arm, the first stuffer, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3' CD40L untranslated region (UTR), the polyA tail, a reporter, the second stuffer, and the second donor homologous arm that is substantially identical to the second homologous arm.

38. 36. The method of claim 34 or claim 35, wherein the exogenous oligonucleotide donor template comprises the first donor homologous arm that is substantially identical to the first homologous arm, the cargo comprising one or more of exons 2-5 of the CD40L gene, the 3' CD40L untranslated region (UTR), the polyA tail, a reporter, and the second donor homologous arm that is substantially identical to the second homologous arm.

39. The cargo is Exon 2 of the CD40L gene; Exon 3 of the CD40L gene; Exon 4 of the CD40L gene; Exon 5 of the CD40L gene; Exons 2 to 5 of the CD40L gene; Exons 2 to 4 of the CD40L gene; Exons 2 and 3 of the CD40L gene; Exons 3 to 5 of the CD40L gene; Exons 3 and 4 of the CD40L gene; or Exons 4 and 5 of the CD40L gene The method according to any one of claims 34 to 38, comprising:

40. The method of any one of claims 34 to 39, wherein the CD40L gene is a wild-type CD40L sequence.

41. The method of any one of claims 34 to 40, wherein the target CD40L nucleic acid comprises a mutation.

42. 39. The method of claim 37 or 38, wherein the reporter is green fluorescent protein (GFP), yellow fluorescent protein (YFB), DS-Red or luciferase.

43. 43. The method of any one of claims 20 to 42, wherein the altered CD40L nucleic acid comprises, from 5' to 3', the first donor homologous arm, the cargo, the 3' UTR, the polyA tail and the second donor homologous arm.

44. 37. The method of any one of claims 20-36, wherein the altered CD40L nucleic acid comprises, from 5' to 3', the first donor homologous arm, the first stuffer, the cargo, the 3' UTR, the polyA tail, the second stuffer, and the second donor homologous arm.

45. 38. The method of any one of claims 20-37, wherein the altered CD40L nucleic acid comprises, from 5' to 3', the first donor homologous arm, the first stuffer, the cargo, the 3' UTR, the polyA tail, the reporter, the second stuffer, and the second donor homologous arm.

46. 39. The method of any one of claims 20, 34, 35 or 38, wherein the altered CD40L nucleic acid comprises, from 5' to 3', the first donor homologous arm, the cargo, the 3' UTR, the polyA tail, the reporter, and the second donor homologous arm.

47. 47. The method of any one of claims 34 to 46, wherein the cell is first contacted with at least one of the gRNA molecule and the RNA-guided nuclease molecule, and subsequently the cell is contacted with the exogenous oligonucleotide donor template.

48. 47. The method of any one of claims 34 to 46, wherein the cell is contacted with at least one of the gRNA molecule, the RNA-guided nuclease molecule and the exogenous oligonucleotide donor template simultaneously.

49. The method of any one of claims 34 to 48, wherein the exogenous oligonucleotide donor template is present in a vector.

50. 50. The method of claim 49, wherein the vector is a viral vector.

51. 51. The method of claim 50, wherein the viral vector is an AAV vector or a lentiviral vector.

52. 52. The method of any one of claims 34-51, wherein the DNA repair pathway repairs the target CD40L nucleic acid, resulting in targeted integration of the exogenous oligonucleotide donor template.

53. 53. The method of any one of claims 34 to 52, wherein the cleavage event, or both the cleavage event and the second cleavage event, are repaired by genetic correction.

54. The method of any one of claims 34 to 53, wherein the altered CD40L nucleic acid comprises a sequence that differs from the sequence of the target CD40L nucleic acid.

55. 55. The method of any one of claims 34 to 54, wherein said gRNA molecule is a gRNA nucleic acid and said RNA-guided nuclease molecule is a RNA-guided nuclease protein.

56. 55. The method of any one of claims 34 to 54, wherein said gRNA molecule is a gRNA nucleic acid and said RNA-guided nuclease molecule is a RNA-guided nuclease nucleic acid.

57. 55. The method of any one of claims 34 to 54, wherein the cell is contacted with the gRNA molecule and the RNA-guided nuclease molecule as a preformed complex.

58. 58. The method of any one of claims 34 to 57, wherein the RNA-guided nuclease is selected from the group consisting of wild-type Cas9, Cas9 nickase, wild-type Cpf1 and Cpf1 nickase.

59. The method of any one of claims 20 to 58, wherein the cell is a eukaryotic cell.

60. 60. The method of claim 59, wherein the eukaryotic cell is a human cell.

61. The method of any one of claims 20 to 60, wherein the cell is a T cell.

62. The method of any one of claims 20 to 60, wherein the cells are hematopoietic stem cells (HSCs).

63. The method of any one of claims 20 to 62, wherein the cells are derived from a subject suffering from Hyper-IgM Syndrome.

64. 64. The method of any one of claims 20 to 63, wherein the cell is derived from a subject having at least one mutation at the cleavage site.

65. 65. The method of any one of Claims 63 or 64, further comprising the step of isolating said cells from said subject prior to said forming or said contacting steps.

66. 66. The method of any one of claims 20 to 65, further comprising introducing said cells into a subject after said recombination step or after said cleavage event has been repaired by at least one said DNA repair pathway.

67. 67. The method of any one of claims 20 to 66, wherein the forming and recombining steps, or the contacting steps, are carried out in vitro.

68. 67. The method of any one of claims 20 to 66, wherein the forming and recombining steps, or the contacting steps, are carried out ex vivo.

69. 67. The method of any one of claims 20 to 66, wherein the forming and recombining steps, or the contacting steps, are carried out in vivo.

70. 67. The method of claim 66, wherein the subject is conditioned prior to the introduction of the cells.

71. 71. The method of claim 70, wherein the subject is conditioned with cyclophosphamide.

72. The method of claim 66, wherein the subject is not conditioned prior to the introduction of the cells.

73. 67. The method of claim 66, wherein class switching is restored in the subject.

74. 67. The method of claim 66, wherein the level of IgM is reduced in the subject.

75. 67. The method of claim 66, wherein the level of IgG is increased in the subject.

76. 76. The method of any one of claims 20-75, wherein the cell is a cell population and the DNA repair pathway repairs the targeted CD40L nucleic acid, resulting in targeted integration of the exogenous oligonucleotide donor template in at least about 10% of cells of the cell population.

77. 77. A cell or cell population modified by the method of any one of claims 20 to 76.

78. The cell population of Claim 77, wherein the cells are hematopoietic stem cells (HSCs) and at least about 10% of the cells in the population of HSCs comprise an altered CD40L nucleic acid that is a functional CD40L allele.

79. 79. A pharmaceutical composition comprising a cell or cell population of claim 77 or claim 78.

80. 1. A method of treating a subject with hyper-IgM syndrome by altering target CD40L in a cell, comprising: creating at least one single-stranded or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, the target CD40L nucleic acid comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site; recombining an exogenous oligonucleotide donor template with said target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, said exogenous oligonucleotide donor template comprising a first donor homologous arm substantially identical to said first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm substantially identical to said second homologous arm; Including, Thereby, the subject having hyper-IgM syndrome is treated.

81. 1. A method of treating a subject with hyper-IgM syndrome by altering a target CD40L nucleic acid in a cell to include a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site, comprising: The cells, (a) at least one gRNA molecule; (b) an RNA-guided nuclease molecule, and (c) Exogenous oligonucleotide donor template wherein the exogenous oligonucleotide donor template comprises a first donor homology arm substantially identical to the first homology arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homology arm substantially identical to the second homology arm; wherein the gRNA molecule and the RNA-guided nuclease molecule interact with the target CD40L nucleic acid to effect a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid; Thereby, the subject having hyper-IgM syndrome is treated.

82. 1. A method of restoring B cell class switching in a subject by altering targeting CD40L in cells, comprising: creating at least one single-stranded or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, the target CD40L nucleic acid comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site; recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm; Including, Thereby restoring B cell class switching in said subject.

83. 1. A method of restoring B cell class switching in a subject by altering a target CD40L nucleic acid in a cell to include a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site, comprising: The cells, (a) at least one gRNA molecule; (b) an RNA-guided nuclease molecule, and (c) Exogenous oligonucleotide donor template wherein the exogenous oligonucleotide donor template comprises a first donor homology arm substantially identical to the first homology arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homology arm substantially identical to the second homology arm; wherein the gRNA molecule and the RNA-guided nuclease molecule interact with the target CD40L nucleic acid to effect a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid; Thereby restoring B cell class switching in said subject.

84. 1. A method of reducing levels of IgM in a subject by altering target CD40L in a cell, comprising: creating at least one single-stranded or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, the target CD40L nucleic acid comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site; recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm; Including, Thereby reducing the level of IgM in said subject.

85. 1. A method of reducing levels of IgM in a subject by altering a target CD40L nucleic acid in a cell to include a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site, comprising: The cells, (a) at least one gRNA molecule; (b) an RNA-guided nuclease molecule, and (c) Exogenous oligonucleotide donor template wherein the exogenous oligonucleotide donor template comprises a first donor homology arm substantially identical to the first homology arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homology arm substantially identical to the second homology arm; wherein the gRNA molecule and the RNA-guided nuclease molecule interact with the target CD40L nucleic acid to effect a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid; Thereby reducing the level of IgM in said subject.

86. 1. A method for increasing levels of IgG in a subject by altering target CD40L in cells, comprising: creating at least one single-stranded or double-stranded break in a target CD40L nucleic acid of the cell at a cleavage site, the target CD40L nucleic acid comprising a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site; recombining an exogenous oligonucleotide donor template with the target CD40L nucleic acid by homologous recombination to generate an altered CD40L nucleic acid, wherein the exogenous oligonucleotide donor template comprises a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm; Including, Thereby increasing the level of IgG in the subject.

87. 1. A method of increasing levels of IgG in a subject by altering a target CD40L nucleic acid in a cell to include a first homologous arm 5' to the cleavage site and a second homologous arm 3' to the cleavage site, comprising: The cells, (a) at least one gRNA molecule; (b) an RNA-guided nuclease molecule, and (c) Exogenous oligonucleotide donor template wherein the exogenous oligonucleotide donor template comprises a first donor homology arm substantially identical to the first homology arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homology arm substantially identical to the second homology arm; wherein the gRNA molecule and the RNA-guided nuclease molecule interact with the target CD40L nucleic acid to effect a cleavage event at or near the cleavage site, and the cleavage event is repaired by at least one DNA repair pathway to generate an altered CD40L nucleic acid; Thereby increasing the level of IgG in the subject.

88. A cell population, characterized in that at least 30% of the cells in said population contain at least one copy of an engineered CD40L sequence.

89. The cell population of claim 88, wherein at least one of said copies is inserted in an intron of the endogenous CD40L gene.

90. 89. The cell population of claim 88, wherein said cell population or a progeny of said cell population is capable of restoring class switching in a subject suffering from hyper-IgM syndrome.

91. An isolated cell population, wherein at least about 5% to about 100% of the cells of said cell population express a functional CD40L allele, said cell population being isolated from a subject previously diagnosed with hyper-IgM syndrome.

92. (a) at least one gRNA molecule; (b) an RNA-guided nuclease molecule, and (c) Oligonucleotide donor template 1. A composition comprising: said oligonucleotide donor template comprising a first donor homologous arm that is substantially identical to the first homologous arm, a cargo comprising one or more of exons 2-5 of the CD40L gene, a 3' CD40L untranslated region (UTR), a polyA tail, and a second donor homologous arm that is substantially identical to the second homologous arm.

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