Methods and compositions for editing a CD40LG gene

Abstract

Embodiments of the methods and compositions provided herein relate to polynucleotides and vectors for modifying a CD40LG gene in a cell genome. Some embodiments include the use of such polynucleotides and vectors to introduce a nucleic acid encoding a CD40L polypeptide operably linked to an endogenous CD40LG promoter.

Description

SCRI.586WO PATENT METHODS AND COMPOSITIONS FOR EDITING A CD40LG GENE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Prov. App. No. 63 / 675,035 filed July 24, 2024 entitled “METHODS AND COMPOSITIONS FOR EDITING A CD40LG GENE”, which is incorporated by reference herein in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SCRI586WOSEQLIST.XML, created July 1, 2025, which is approximately 35,165 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. FIELD OF THE INVENTION

[0003] Embodiments of the methods and compositions provided herein relate to polynucleotides and vectors for modifying a CD40LG gene in a cell genome. Some embodiments include the use of such polynucleotides and vectors to introduce a nucleic acid encoding a CD40L polypeptide operably linked to an endogenous CD40LG promoter. BACKGROUND OF THE INVENTION

[0004] X-linked hyper IgM syndrome (XHIM) is a primary immune disorder caused by inactivating mutations in the CD40LG gene which encodes CD40 ligand (CD40L). Mainly expressed on the surface of activated CD4 T cells, CD40L triggers CD40-dependent activation of B cells and other antigen presenting cells.1,2In CD40-dependent B cell activation, the CD40:CD40L interaction promotes germinal center formation, class switch recombination, somatic hypermutation, and formation of plasma and memory B cells.3In XHIM patients, CD40L deficiency results in failure to generate protective class-switched, pathogen-specific, high-titer antibodies leading to high susceptibility to recurrent and opportunistic infections4and reduced long-term survival.5Allogeneic hematopoietic stem cell transplant offers long- term therapeutic benefit, but risks graft rejection, infection, and requires matched donoravailability.5Therefore, there is a need for additional therapies to treat, inhibit, or ameliorate XHIM. SUMMARY OF THE INVENTION

[0005] Some embodiments of the methods and compositions provided herein include a polynucleotide for modifying a human CD40LG gene in a cell genome, comprising:a 5 homology arm; a CD40L coding sequence; a polyadenylation signal; and a 3 homologyarm. Some embodiments also include a woodchuck hepatitis virus posttranscriptional regulatory element 3 (WPRE3) located between the CD40L coding sequence and the polyadenylation signal. In some embodiments, the encoded CD40L polypeptide binds CD40.

[0006] In some embodiments, the CD40L coding sequence comprises a cDNA. In some embodiments, the CD40L coding sequence comprises a codon-diverged cDNA. In some embodiments, the CD40L coding sequence comprises a nucleotide sequence: (i) having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:19; (ii) encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; or (iii) encoding an amino acid sequence set forth in SEQ ID NO:24 having 0-26 conservative substitutions.

[0007] In some embodiments, the WPRE3 comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:20.

[0008] In some embodiments, the 5 homology arm lacks a nucleotide sequencecapable of specifically hybridizing to the nucleotide sequence set forth in SEQ ID NO:23. Insome embodiments, the 5 homology arm comprises or consists of sequences 5 of the firstcoding exon of the CD40LG gene, and / or comprises or consists of sequences 5 of proteincoding sequences of the first coding exon of the CD40LG gene. In some embodiments, the 5homology arm comprises or consists of sequences 5 of the first coding exon of the CD40LGgene and a portion of the first coding exon, and / or comprises or consists of sequences 5 ofprotein coding sequences of the first coding exon of the CD40LG gene and a portion of thefirst coding exon. In some embodiments, the 5 homology arm comprises a sequence having atleast 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotidesequence set forth in SEQ ID NO:18. In some embodiments, the 5 homology arm comprisesa CD40LG promoter or portion thereof. In some embodiments, the 5 homology arm has alength in a range from about 500 nucleotides to about 1500 nucleotides; optionally, in a range from about 750 nucleotides to about 1250 nucleotides.

[0009] In some embodiments, the 3 homology arm comprises or consists of a wild-type CD40L genomic sequence. In some embodiments, the 3 homology arm comprisessequences 3 of the first coding exon of the CD40LG gene. In some embodiments, the 3homology arm comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:22. In someembodiments, the 3 homology arm has a length in a range from about 500 nucleotides to about1500 nucleotides; optionally, in a range from about 750 nucleotides to about 1250 nucleotides.

[0010] In some embodiments, the polyadenylation signal comprises an SV40 polyA signal. In some embodiments, the polyadenylation signal comprises a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:21.

[0011] In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded.

[0012] Some embodiments of the methods and compositions provided herein include a vector comprising any one of the polynucleotides provided herein. In some embodiments, the vector is an adeno- associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector

[0013] Some embodiments of the methods and compositions provided herein include a method of genetically modifying a CD40LG gene in a cell genome, comprising: introducing into a cell any one of the polynucleotides or vectors provided herein.

[0014] Some embodiments also include introducing into the cell a nuclease or nucleic acid encoding the nuclease, wherein the nuclease is capable of specifically cleaving a first coding exon of a CD40LG gene. In some embodiments, the nuclease is a Cas nuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), or a meganuclease; optionally, wherein the nuclease is the Cas nuclease or derivative thereof such as a mutant Cas nuclease; optionally, wherein the nuclease is a Cas9 nuclease.

[0015] Some embodiments also include introducing into the cell a guide RNA (gRNA). In some embodiments, the gRNA comprises a sequence capable of specifically hybridizing to a nucleotide sequence of a first coding exon of the CD40LG gene. In some embodiments, the gRNA comprises a sequence having at least 70%, 80%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:23.

[0016] Some embodiments also include contacting the cell with a DNA-dependent protein kinase (DNA-PK) inhibitor. In some embodiments, the DNA-PK inhibitor is selected from AZD7648, NU7441, NU7026, KU-0060648, LY3023414, CC-115, VX-984 or M3814. In some embodiments, the DNA-PK inhibitor comprises AZD7648.

[0017] Some embodiments of the methods and compositions provided herein include a cell prepared with any one of the methods provided herein. Some embodiments of the methods and compositions provided herein include a cell comprising any one of the polynucleotides or vectors provided herein. In some embodiments, the cell is a T cell, a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), and / or a CD34+ cell. In some embodiments, the cell comprises a single X chromosome.

[0018] Some embodiments of the methods and compositions provided herein include a pharmaceutical composition comprising any one of the cells provided herein and a pharmaceutically acceptable excipient.

[0019] Some embodiments of the methods and compositions provided herein include a system for modifying a human CD40LG gene in a cell genome, comprising: any one of the polynucleotides provided herein; and a nuclease or nucleic acid encoding the nuclease, wherein the nuclease is capable of specifically cleaving a first coding exon of a CD40LG gene.

[0020] In some embodiments, the nuclease is a Cas nuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), or a meganuclease; optionally, wherein the nuclease is the Cas nuclease or derivative thereof such as a mutant Cas nuclease; optionally, wherein the nuclease is a Cas9 nuclease.

[0021] Some embodiments also include a guide RNA (gRNA). In some embodiments, the gRNA comprises a sequence capable of specifically hybridizing to a nucleotide sequence of a first coding exon of the CD40LG gene. In some embodiments, the gRNA comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:23.

[0022] Some embodiments also include a cell. In some embodiments, the cell is a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), a T cell, and / or a CD34+ cell.

[0023] Some embodiments also include a DNA-dependent protein kinase (DNA- PK) inhibitor. In some embodiments, the DNA-PK inhibitor is selected from AZD7648, NU7441, NU7026, KU-0060648, LY3023414, CC-115, VX-984 or M3814. In some embodiments, the DNA-PK inhibitor comprises AZD7648.

[0024] Some embodiments of the methods and compositions provided herein include a method of treating, ameliorating or inhibiting a disorder in a subject, comprising administering to the subject any one of the cells provided herein. In some embodiments, the cell is autologous to the subject. In some embodiments, the cell is allogeneic to the subject. In some embodiments, the disorder comprises X-linked hyper IgM syndrome (XHIM). In some embodiments, the subject is male. In some embodiments, the subject is human.

[0025] Some embodiments of the methods and compositions provided herein include any one of the cells provided herein for treating, ameliorating or inhibiting a disorder in a subject; optionally, wherein the disorder comprises X-linked hyper IgM syndrome (XHIM).

[0026] Some embodiments of the methods and compositions provided herein include any one of the cells provided herein for for use in the preparation of a medicament for treating, ameliorating or inhibiting a disorder in a subject; optionally, wherein the disorder comprises X-linked hyper IgM syndrome (XHIM). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIGs.1, and 2A-2G relate to inhibition of DNA-PKcs enhanced AAV-based HDR editing efficiency in CD34+HSPCs.

[0028] FIG.1 depicts a schematic for CRISPR-Cas9 targeting of CD40LG exon 1 (E1). Above E1, the sgRNA sequence (“CTT … AAA”) and location in relation to the start codon (‘ATG”) and PAM sequence (“GGT”) is highlighted. Below E1, schematic of MND.GFP HDR donor template containing 1 kB homology arms flanking a constitutive MND promoter-driven GFP cassette followed by WPRE and synthetic polyadenylation (pA) regulatory elements.

[0029] FIG. 2A depicts a study to characterize HSPC phenotype and HDR efficiency with rAAV6+RNP-based HDR editing and inhibition of DNA-PKcs.

[0030] FIG. 2B depicts a graph of viability of HSPCs at 24h after indicated treatments, n=2-4 CD34+ donors from 2-3 independent experiments.

[0031] FIG. 2C depicts a representative flow cytometry analysis of HSPC phenotype 24h after indicated treatments.

[0032] FIG. 2D depicts graphs for proportion of CD34+(left), CD34+CD38- (middle), and CD34+CD38-CD90+(right) HSPCs as percentage of total cells 24h after editing, editing in combination with iDNA-PKcs, or electroporation only. n=3 CD34+donors from 3 independent experiments.

[0033] FIG. 2E depicts graphs for proportion of GFP+cells in CD34+(left), CD34+CD38- (middle), or CD34+CD38-CD90+(right) HSPC populations at 24h after editing. n=3 CD34+ donors from 3 independent experiments.

[0034] FIG.2F depicts a representative flow cytometry analysis to quantify HDR. HDR was determined by the proportion of GFPhighevents, using AAV-only treated cells as a negative control, 5 days after editing.

[0035] FIG. 2G depicts a graph for HDR editing efficiency measured by GFPhigh. n= 4 CD34+donors from 3 independent experiments. In FIG.2B, FIG.2D, FIG.2E, FIG.2G, each different shaded symbol represents a unique CD34+donor. AZD7648 (0.1 or 0.3μM) and rAAV (3.75-7.7 × 105GC / cell) were delivered at D2 as indicated.

[0036] FIGs. 3A-3E relate to CD40L-edited HSPCs durably engrafted with comparable differentiation capacity to untreated HSPCs.

[0037] FIG.3A depicts a schematic for xenotransplantation studies to assess long- term engraftment and differentiation capacity of untreated control, edited, or edited + iDNA- PKcs CD34+ HSPCs.24h after editing, HSPCs were transplanted to pre-conditioned NBSGW recipients. Some cells were retained to assess HDR efficiency of input cells. Animals were bled at weeks 10,12,14, and 16 to assess engraftment kinetics and frequency of GFP+ cells in the periphery. At 16 weeks, BM and spleens are harvested to assess engraftment and %GFP+.

[0038] FIG. 3B depicts graphs for mean peripheral blood engraftment (left, % hCD45+) and proportion of edited cells (right, %GFP+ from hCD45+ gate), over time post- transplant. Bars representSEM.

[0039] FIG. 3C depicts a representative FACS analysis of BM from mice transplanted with untreated control or edited + iDNA-PKcs HSPCs. Gating: hCD45 vs mCD45 gated from Live / Single cells, and CD19 and CD33 from hCD45+. GFP from hCD45+, CD33+, and CD19+ gates is displayed with shades matching parental gate.

[0040] FIG. 3D depicts graphs for an analysis of BM 16 weeks after transplant of indicated treatment groups. Engraftment (%hCD45+), proportion of B (CD19+) and myeloid lineage cells (CD33+), and proportion of edited cells in each subset is depicted; each donor indicated by unique shade. Statistical significance assessed by linear regression model. Mean + / - SD.

[0041] FIG. 3D depicts graphs for an analysis of spleen at 16 weeks. Statistical significance assessed by linear regression model. Mean + / - SD. For B-E: n=5 CD34+ donors, 6 independent experiments (dark gray and black are same donor repeated). Input %HDR (%GFPhighcells assessed at Day 5 using an aliquot of the edited cell population cultured in vitro) AAV+RNP: 52.2, 23.4 (light gray), 38.8 (dark gray), 34.6. Input %HDR AAV+RNP+iDNA-PKcs: 66.2, 36.4 (light gray), 47.0 (dark gray), 43.5. Input %HDR not recovered due to contamination: black, red. AZD7648 (0.1-0.3μM) and rAAV6 (3.75-7.7 × 105GC / cell) were delivered at D2.

[0042] FIGs.4A-4F relate to DNA-PK inhibition enhanced long-term engraftment of HDR-edited HSPCs.

[0043] FIG. 4A depicts a representative flow cytometry analysis of BM HSPC compartment at 16 weeks post-transplant using untreated control or edited + iDNA-PKcs HSPCs. Cells were first gated on Live / Single / hCD45+. CD34 vs CD38 is plotted to identify bulk CD34+ HSPCs and HSC-enriched CD34+CD38- HSPCs. CD90 is gated from CD34+CD38- to further define a subpopulation of HSC-enriched HSPCs.

[0044] FIG.4B depicts graphs for proportion of HSPC populations defined in FIG. 4A and recovered from the BM of transplanted mice.

[0045] FIG. 4C depicts a representative FACS analysis of GFP+cells in HSPC populations defined in FIG.4A.

[0046] FIG. 4D depicts graphs for proportion of GFP+HDR-edited HSPCs in populations defined in FIG.4A and FIG.4C, and recovered from the BM of edited or edited + iDNA-PKcs HSPC-transplanted mice. For FIGs. 4A-D, n=5 CD34+ donors, 6 independentexperiments. For FIG. 4B and FIG. 4D, statistical significance was assessed using a linear regression model. Bars represent mean + / - SD.

[0047] FIG. 4E depicts a graph for equal numbers of BM cells harvested from transplanted animals in (FIGs. 4A-4D) which were pooled within treatment groups and transplanted into secondary NSG-SGM3 recipient mice. After 12 weeks, BM was harvested, and engraftment was quantified by %hCD45+.

[0048] FIG. 4F depicts a graph for proportion of HDR edited hCD45+ cells recovered from BM of secondary transplant recipients. For FIGs.4E-4F, n= 3 CD34+ donors, 3 independent experiments. Unique donors are indicated by same shading scheme as FIG.3.

[0049] FIGs. 5A-5I. Long-term engraftment and T-lineage differentiation of CD40L cDNA-edited HSPCs.

[0050] FIG. 5A depicts a schematic for therapeutic CD40L cDNA HDR donor template. A codon-diverged CD40L coding sequence (“cDNA”) followed by WPRE3 and SV40USE polyadenylation (pA) elements flanked by 1kB homology arms is inserted at E1 using the same sgRNA and homology arms described in FIG. 2A. Expression of the CD40L cDNA cassette is driven by the endogenous CD40LG promoter.

[0051] FIG. 5B depicts a graph for viability measured 48h after treatment with increasing doses of rAAV6 CD40L cDNA with or without RNP treatment. n=2 CD34+ donors, 1 independent experiment.

[0052] FIG.5C depicts a graph for mean + / - SEM, wherein HDR was measured by ddPCR 5 days after treatment with increasing doses of rAAV6 CD40L cDNA with RNP treatment, or maximal dose or rAAV6 CD40L cDNA without RNP. n=2 CD34+ donors, 4 independent experiments. Mean + / - SEM.

[0053] FIG. 5D depicts graphs for xenotransplantation studies, which were conducted as described in FIG. 3A utilizing the rAAV6 CD40L cDNA donor cassette + / - iDNA-PKcs. After 16 weeks, gDNA was extracted from harvested BM and spleens and the proportion of HDR-edited cells present in the BM (left) and spleen (right) was quantified by ddPCR. Statistical significance assessed by linear regression model. MeanSD.

[0054] FIG. 5E depicts graphs for engraftment (%hCD45+), and proportion of HSPCs (%CD34+), B cells (%CD19+) and myeloid cells (%CD33+) present in the BM ofmice transplanted with edited or edited + iDNA-PKcs HSPCs. Statistical significance assessed by linear regression model. Mean + / - SD.

[0055] FIG. 5F depicts graphs for engraftment (%hCD45+), and proportion of B cells (%CD19+) and myeloid cells (%CD33+) present in the spleens of mice transplanted with edited or edited + iDNA-PKcs HSPCs. Statistical significance assessed by linear regression model. Mean + / - SD. For FIGs.5D-5F, n=3 CD34+ donors (gold, light gray, dark gray / black), 4 independent experiments (dark gray and black are same donor repeated). AZD7648 (0.3μM) and rAAV (6300 GC / cell) were delivered at D2. Input HDR, which was determined by ddPCR at Day 5 using an aliquot of the edited cell population cultured in vitro. AAV+RNP: 58.9 (gold), 30.6 (light gray), 23.0 (dark gray), lost from contamination (black). Input HDR AAV+RNP+iDNA-PKcs 64.9 (gold), 44.3 (light gray), 30.0 (dark gray), lost from contamination (black).

[0056] FIG.5G depicts a FACS analysis for CD34+ cells, which were isolated from BM harvested from animals in FIG. 5D and FIG. 5E and differentiated ex vivo in artificial thymic organoid (ATO) cultures for 6-8 weeks. In one study, edited samples were pooled between iDNA-PKcs groups (+ and – pooled) due to low CD34+ yield. In the next study, only iDNA-PKcs-treated edited HSPCs were isolated and used to initiate ATO cultures. T-lineage differentiation was characterized by FACS analysis. Cells were first gated by hCD45+CD34- CD14 / CD19 / CD56-. CD3+TCRab+ cells are indicated in FACS plots.

[0057] FIG.5H depicts a graph for proportion of CD5+CD7+ T-lineage committed (pre-T-1) cells present at 6-8 weeks after initiation of ATO cultures with CD34+ HSPCs isolated from the BM of untreated or edited recipient animals. Statistical significance assessed by linear regression model. Mean + / - SD.

[0058] FIG.5I depicts a graph for HDR frequency in pooled input cells (D0) or in individual ATO cultures. Statistical significance assessed by linear regression model. Mean SD. For FIGs. 5G-5I, n= 2 CD34+ donors, 2 independent experiments.

[0059] FIGs. 6A-6E relate to ex vivo stimulation of T cells derived from in vivo differentiation of CD40L-edited HSPCs in NSG-SGM3 mice.

[0060] FIG. 6A depicts a schematic of rAAV6 CD40L.GFP donor template consisting of a codon-diverged CD40L cDNA 2A-linked to a GFP reporter followed byWPRE3 and SV40 polyA sequences with flanking by 1 kB homology arms. Expression of the knock-in cassette is driven by the endogenous CD40L promoter.

[0061] FIG.6B depicts a representative flow cytometry analysis of splenic cells in a NSG-SGM3 recipient mouse at 16 wks post-transplant of CD40L.GFP + iDNA-PKcs-edited HSPCs.

[0062] FIG. 6C depicts graphs for human cell engraftment (%hCD45+; left panel) and T cells (%CD3+; middle panel) quantified by flow, and HDR quantified by ddPCR (right panel) in BM and spleen. Symbols indicate individual animals Bars represent mean + / - SEM.

[0063] FIG. 6D depicts flow cytometry plots showing CD40L and GFP co- expression 3.5 hours after PMA and ionomycin stimulation of splenocytes harvested from an CD40L.GFP + iDNA-PKcs-edited HSPC recipient animal.

[0064] FIG.6E depicts a graph for CD40L MFI in GFP+and GFP- quadrants from FIG. 6D measured at 0, 1, 3.5, 24, and 48 hours after PMA and ionomycin stimulation. For FIGs.6B-6E, representative data from 1 of 2 experiments. AZD7648 (0.3μM) and rAAV (6690 GC / cell) were delivered at D2.

[0065] FIGs. 7A-7D relate to establishment of CRISPR Cas9-based HDR editing at CD40L locus in CD34+ HSPCs.

[0066] FIG. 7A depicts a schematic for human CD40LG locus showing nuclease recognition site relative to exons (boxes) and translated coding sequence (taller boxes); below is AAV donor template with MND.GFP reporter; grey shading shows location of CD40LG homology.

[0067] FIG. 7B depicts a schematic for an experimental timeline: day 0 is designated as editing (electroporation with Cas9 RNP or TALEN mRNA) followed by culturing in media containing AAV donor template.

[0068] FIG. 7C depicts representative flow cytometry plots for mock, AAV only (MOI=1000) and AAV (MOI=1000) and TALEN (50 g / ml) or RNP (80 g / ml) conditions showing gating for viability (SSC vs. FSC) and GFP expression within the live cell gate (SSC vs. GFP) on the indicated day post-editing.

[0069] FIG.7D depicts graphs for mean ± SEM cell viability (left) and percentage of GFP+ edited cells (right) as determined by flow cytometry 2 and 5 days post-editing,respectively (N=9 replicates with 4 unique donors). P value was determined using unpaired t- test. *p < 0.05.

[0070] FIG. 8 relates to confirmation of on-target integration of MND.GFP reporter. FIG. 8 depicts a graph showing average editing rates using MND.GFP reporter template in CD34+ donors as determined by ddPCR vs. flow cytometry for GFP. Data are presented as mean ± SEM (N=5 replicates with 2 unique donors).

[0071] FIGs. 9A-9C relate to the incorporation of UM171 and SR1 within the in vitro CD34+ HSPC culturing protocol.

[0072] FIG.9A depicts a graph for viability (measured by FSC-A vs. SSC-A) 48h after mock-treatment (electroporation), AAV-treatment, or AAV+RNP editing of HSPCs cultured in the presence or absence of UM171 (35nM) and SR1 (1uM).

[0073] FIG.9B depicts a graph for fold-change in the percentage of HSC-enriched CD34+CD38-CD90+CD133+ HSPCs in cultures 48h after editing reagent treatment. For each editing condition, the frequency of HSC-enriched HSPCs in the UM171 / SR1 group is normalized to the same treatment without UM171 / SR1.

[0074] FIG. 9C depicts a graph for fold-change in HDR efficiency with UM171 and SR1 treatment 5 days after editing (a normalized to the same treatment without UM171 / SR1). For FIGs. 9A-9C, n=2-4 CD34+ donors, 2-3 independent experiments Bars represent mean + / - SEM.

[0075] FIG. 10 relates to a peripheral blood FACS analysis. FIG. 10 depicts a FACS analysis with a gating scheme for peripheral blood FACS analysis at 10, 12, 14, and 16 weeks post-transplant.

[0076] FIGs. 11 and 12 relate to indel profiling of rAAV6+RNP-based editing outcomes with or without DNA-PKcs addition.

[0077] FIG.11 depicts a graph for Indel frequencies following HDR editing.

[0078] FIG.12 depicts a graph for Indel frequencies following HDR editing. FIG. 11 and FIG. 12 are representative of n = 2 CD34+ donors with analysis performed in 2 independent experiments using HDR edited cells from 2 of the 3 donors shown in FIGs. 5A- 5I. Indel frequencies following HDR editing using rAAV6+RNP vs. rAAV6+RNP with AZD7648 were evaluated using ICE analysis (see Methods). Indels ±2 bp were categorized as NHEJ, while indels ±3 bp were defined as MMEJ. Histograms represent the percentagesof WT, NHEJ, and MMEJ events for each treatment group. A portion of each HDR edited cell population was retained and cultured in vitro for 5 days prior to extraction of gDNA.

[0079] FIG. 13 relates to peripheral blood engraftment of CD40L cDNA-edited HSPCs. FIG. 14 depicts a graph for percentage of hCD45+ cells in peripheral blood of mice transplanted with CD40L cDNA-edited or CD40L cDNA + iDNA-PKcs-edited HSPCs. n=3 CD34+ donors, 4 independent experiments Points represent mean + / - SEM.

[0080] FIG. 14 depicts a schematic representation of AAV donor templates described herein for GFP.CD40L and CD40L and GFP expression cassettes. #1368 - CD40LG[GFP.CD40L.WPRE]) comprising the codon diverged CD40L CDS, WPRE element, Syn.pA and 1 kb 5’ homology arm. #3359 - CD40LG[GFP.T2A.CD40L.cDNA.WPRE3] comprising a cis-linked GFP, codon diverged CD40L CDS, WPRE truncated element (WPRE3), SV40 polyA and 1 kb 5’ homology arm. #3367 - CD40LG[CD40L.cDNA.WPRE3.SV40 PolyA] comprising a codon diverged CD40L CDS, WPRE3 element, SV40 poly A, and optimized 5’ homology arm. #3377 – CD40LG[MND.GFP.WPRE.syn.pA] comprising MND.GFP, WPRE, Syn.PA and optimized 5’ homology arm. #1416 - CD40LG[CD40L.cDNA.WPRE3], described in US 2021 / 0324381 A1 (sequence #15), comprising a codon optimized CD40L CDS, WPRE3, SV40 poly A and 5’ homology arm (unoptimized).

[0081] FIG.15 depicts a schematic of an experimental timeline for gene editing in human T cells and downstream analysis.

[0082] FIG. 16A depicts a FACS analysis of GFP transgene expression following gene editing in healthy donor T cells (donor R003697). Shown are mock transduced cells, RNP only and RNP + AAV (#3367, #1368, #3359 and #3377) day 3 following editing. The corresponding editing rate as measured by ddPCR on day 14 is listed below the flow plots.

[0083] FIG. 16B depicts a FACS analysis for kinetics of CD40L expression in edited healthy donor T cells: Time course of CD40L surface expression on healthy CD4+ T cells edited using indicated donor templates. Flow was performed at 0, 0.5, 2, 3, 8, 24, and 54 hours following PMA / Ionomycin activation. Shown are the flow plots for CD154 / GFP expression for unstimulated and PMA / Ionomycin stimulated cells. X-axis corresponds to increasing GFP signal. Rows correspond to time flow performed in an increasing order with 0 hours the upper most row, as shown for ‘mock’.

[0084] FIG. 16C depicts graphs for a time course of CD40L surface expression on healthy CD4+ T cells edited using indicated donor templates. Shown are the percent CD40L+ expression over time (left) and average MFI (right) taken from flow plots in FIG.16B. Results show that T cells edited using donor templates #3367 and #3359 (both containing WPRE3) had similar CD40L expression levels and kinetics as endogenous CD40L (Mock edited). In contrast, T cells edited using donor template #1368 (containing WPRE) had higher CD40L MFI and slightly increased % CD40L expression.

[0085] FIG. 16D depicts graphs for kinetics of CD40L expression (edited vs unedited internal control). MFI of CD40L expression in T cells unedited and edited with #1368 - CD40L WPRE (left) and #3359 - CD40L WPRE3 (right) are shown. #3359-edited (GFP+) have similar CD40L kinetics as endogenous CD40L (GFP-) T cells. In contrast #1368-edited GPF+ cells, express elevated levels of CD40L for longer duration compared to endogenous CD40L (GFP-) T cells.

[0086] FIG 17A depicts a FACS analysis for GFP transgene expression following gene editing in healthy donor T cells (donor R0003797). Shown are mock transduced cells, RNP only and RNP + AAV (#3367, #1359, and #3377) day 3 following editing. The corresponding editing rate, as measured by ddPCR on day 8, is shown in the upper right coordinate of the flow plot.

[0087] FIG. 17B depicts a FACS analysis for kinetics of CD40L expression in edited healthy donor T cells (donor R0003797): Time course of CD40L surface expression on healthy CD4+ T cells edited using indicated donor templates. Flow was performed at 0, 0.5, 2, 3, 8, 24, and 54 hours following PMA / Ionomycin activation. Shown are the flow plots for CD154 / GFP expression for unstimulated and PMA / Ionomycin stimulated cells. There was no GFP expression in cells treated with AAV in the absence of RNP. Rows correspond to time flow performed in an increasing order with 0.5 hours the upper most row, as shown for ‘mock’.

[0088] FIG. 17C depicts graphs for a time course of CD40L surface expression on healthy CD4+ T cells edited using indicated donor templates. Shown are the percent CD40L+ expression over time (left) and average MFI (right) taken from flow plots in FIG.17B. Results showed that T cells edited using donor templates #3367 and #3359 (both containing WPRE3) have similar CD40L expression levels and kinetics as endogenous CD40L (Mock edited).

[0089] FIG. 17D depicts a graph for kinetics of CD40L expression (edited vs unedited internal control). MFI of CD40L expression in T cells unedited and edited with #3359 (CD40L WPRE3) is shown. Results show that #3359-edited (GFP+) have similar CD40L kinetics as endogenous CD40L (GFP-) T cells.

[0090] FIG. 17E depicts graphs for a comparison of CD40L kinetics in 2 healthy donors R00357 and R0003797, edited with #3359 (from FIG.16D and FIG.17D respectively).

[0091] FIG. 18A depicts a schematic for therapeutic CD40L cDNA HDR donor template (#3367). A codon-diverged CD40L coding sequence (“cDNA”) followed by WPRE3 and SV40USE polyadenylation (pA) elements flanked by optimized 1 kB homology arms was inserted at E1. Above E1, the sgRNA sequence and location in relation to the start codon and PAM sequence is highlighted of the CD40L cDNA cassette is driven by the endogenous CD40LG promoter.

[0092] FIG. 18B depicts a graph for viability measured 48h after treatment with increasing doses of rAAV6 CD40L cDNA (#3367) with or without RNP treatment. n=2 CD34+ donors, 1 independent experiment.

[0093] FIG. 18C depicts a graph for HDR measured by ddPCR 5 days after treatment with increasing doses of rAAV6 CD40L cDNA (#3367) with RNP treatment, or maximal dose or rAAV6 CD40L cDNA without RNP. n=2 CD34+ donors, 4 independent experiments.

[0094] FIG.18D depicts a schematic for xenotransplantation studies to assess long- term engraftment and differentiation capacity of untreated control, edited, or edited + iDNA- PKcs CD34+ HSPCs.24h after editing, HSPCs were transplanted to pre-conditioned NBSGW recipients. Some cells were retained to assess HDR efficiency of input cells. Animals were bled at weeks 10,12,14, and 16 to assess engraftment kinetics. At 16 weeks, BM and spleens were harvested to assess the proportion of HDR-edited cells as measured by ddPCR, engraftment in a variety of cell subsets, and for differentiated ex vivo in artificial thymic organoid (ATO) cultures.

[0095] FIG.18E depicts a graph for percentage of hCD45+ cells in peripheral blood of mice transplanted with CD40L cDNA-edited or CD40L cDNA + iDNA-PKcs-edited HSPCs. n=3 CD34+ donors, 4 independent experiments. Points represent mean+ / - SEM.

[0096] FIG. 18F depicts graphs for xenotransplantation studies which were conducted utilizing the rAAV6 CD40L cDNA (#3367) donor cassette + / - iDNA-PKcs. After 16 weeks, gDNA was extracted from harvested BM and spleen cells and the proportion of HDR-edited cells present in the BM (left) and spleen (right) was quantified by ddPCR.

[0097] FIG. 18G depicts graphs for engraftment (%hCD45+), and proportion of HSPCs (%CD34+), B cells (%CD19+) and myeloid cells (%CD33+) present in the BM of mice transplanted with edited or edited + iDNA-PKcs HSPCs.

[0098] FIG.18H depicts graphs for engraftment (%hCD45+), and proportion of B cells (%CD19+) and myeloid cells (%CD33+) present in the spleens of mice transplanted with edited or edited + iDNA-PKcs HSPCs. For FIGs.18F-18H, n=3 CD34+ donors, 4 independent experiments. AZD7648 (0.3mM) and rAAV (6300 GC / cell) were delivered at D2. Input HDR AAV+RNP: 58.9, 30.6, 23.0, lost from contamination. Input HDR AAV+RNP+iDNA-PKcs 64.9, 44.3, 30.0, lost from contamination.

[0099] FIG. 18I depicts a FACS analysis for CD34+ cells were isolated from BM harvested from animals in (FIG. 18F) and (FIG. 18G) and differentiated ex vivo in artificial thymic organoid (ATO) cultures for 6-8 weeks. In one study, edited samples were pooled between iDNA-PKcs groups (+ and – pooled) due to low CD34+ yield. In the next study, only iDNA-PKcs-treated edited HSPCs were isolated and used to initiate ATO cultures. T-lineage differentiation was characterized by FACS analysis. Cells were first gated by hCD45+CD34- CD14 / CD19 / CD56-. CD3+TCRab+ cells are indicated in FACS plots. For FIGs. 18G-18I, n= 2 CD34+ donors, 2 independent experiments. Statistical significance was assessed using a linear regression model. Bars represent mean + / - SEM.

[0100] FIG.18J depicts a graph for proportion of CD5+CD7+ T-lineage committed (pre-T-1) cells present at 6-8 weeks after initiation of ATO cultures with CD34+ HSPCs isolated from the BM of untreated or edited recipient animals.

[0101] FIG. 18K depicts a graph for HDR frequency in pooled input cells (D0) or in individual ATO cultures.

[0102] FIG. 19A depicts a schematic of rAAV6 CD40L.GFP donor template (#3359) consisting of a codon-diverged CD40L cDNA T2A-linked to a GFP reporter followed by WPRE3 and SV40 polyA sequences with flanking by 1kB homology arms. Expression of the knock-in cassette is driven by the endogenous CD40L promoter.

[0103] FIG. 19B depicts a representative flow cytometry analysis of splenic cells in a NSG-SGM3 recipient mouse at 16 weeks post-transplant of CD40L.GFP + iDNA-PKcs- edited HSPCs.

[0104] FIG.19C depicts graphs for human cell engraftment (%hCD45+; left panel) and T cells (%CD3+; middle panel) quantified by flow, and HDR quantified by ddPCR (right panel) in BM and spleen. Symbols indicate individual animals.

[0105] FIG. 19D depicts flow cytometry plots showing CD40L and GFP co- expression 3.5 hrs after PMA and ionomycin stimulation of splenocytes harvested from an CD40L.GFP + iDNA-PKcs-edited HSPC recipient animal.

[0106] FIG. 19E depicts a graph for CD40L MFI in GFP+ and GFP- quadrants from (FIG. 19D) measured at 0, 1, 3.5, 24, and 48 hrs after PMA and ionomycin stimulation. For FIGs. 19B-19E, representative data from 1 of 2 experiments. AZD7648 (0.3mM) and rAAV (6690 GC / cell) were delivered at D2. DETAILED DESCRIPTION

[0107] Some embodiments of the methods and compositions provided herein relate to polynucleotides and vectors for modifying a CD40LG gene in a cell genome. Some embodiments include the use of such polynucleotides and vectors to introduce a nucleic acid encoding a CD40L polypeptide operably linked to an endogenous CD40LG promoter.

[0108] Some embodiments include polynucleotides comprising an WPRE3 element which includes a truncated WPRE containing the and elements of WPRE; a SV40poly A signal, instead of a synthetic poly A (Syn.pA); and optimized 5 homology arm whichlacked mutations designed to eliminate TALEN binding, thus increasing homologous stretchwithin the 5 homology arm. Unexpectedly, the changes in at least the WPRE and polyAelements had a surprisingly large impact on results observed compared to prior constructs. For example, it had been believed that additional elements or alternative elements would have been required for appropriate levels and patterns of expression in loci modified with the construct, such as inclusion of intronic sequences and / or use of an alternative poly A signal. However, overall it was surprising that these modifications achieve at least: endogenous levels of surface expression (based on Flow staining) and endogenous kinetics of expression. In contrast, priorart constructs had higher basal expression and prolonged / sustained expression both of which could be a problem for clinical application.

[0109] Targeted gene editing to restore CD40L expression via homology directed repair (HDR) in CD34+hematopoietic stem and progenitor cells (HSPCs) provides for a potential long-term therapy for X-linked hyper-immunoglobulin M syndrome (XHIM). However, clinical translation of HSPC editing is limited by inefficient long-term engraftment of HDR edited HSPCs. As disclosed herein, this issue was addressed by employing a small molecule inhibitor of DNA-PKcs, AZD7648, to bias DNA repair mechanisms to facilitate HDR upon CRISPR SpCas9-based gene editing. Using AZD7648 treatment and a clinically relevant HSPC source, mobilized peripheral blood CD34+ cells with ~60% HDR efficiency at the CD40LG locus and enhanced engraftment of HDR-edited HSPCs in primary and secondary xenotransplants was achieved. Specifically, a 1.6-fold increase of HDR-edited long-term HSPCs in primary transplant recipients without disturbing chimerism levels or differentiation capacity was obtained. As CD40L is primarily expressed in T cells, T cell differentiation from HDR-edited HSPCs in vivo and in artificial thymic organoid cultures, and endogenously regulated CD40L expression following activation of in vivo derived CD4+T cells was demonstrated. These combined findings evidence HDR editing at the CD40LG locus at clinically beneficial levels. More broadly, these data support using DNA-PKcs inhibition with AZD7648 as a simple and efficacious addition to HSPC editing platforms.

[0110] As XHIM is a monogenic disorder, previous studies have aimed to identify approaches to restore expression of wildtype CD40L protein in XHIM T cells or hematopoietic stem and progenitor cells (HSPCs). CD40L expression achieved via retroviral delivery of a CD40L cDNA expression cassette in CD40L - / - mice resulted in most animals developing lymphoproliferative disorders, highlighting the importance of maintaining endogenous transcriptional control of CD40L expression.6XHIM-causing mutations have been identified throughout the CD40L gene and lack a dominant mutational hotspot.7Therefore, precise correction by homology directed repair (HDR)-based editing with short oligonucleotide donor cassettes8or, alternatively, via base- or prime-editing approaches9,10would require development of multiple patient-specific designs, not feasible for broad clinical application. In contrast, HDR-based gene editing to insert CD40L cDNA downstream of the CD40LG promoter elements allows for restoration of endogenous functional gene expression and hasbeen described in both human T cells and HSPCs.11-13In female carriers of XHIM, skewed random X-chromosome inactivation suggests as little as 10% HDR in HSPCs might be sufficient for therapeutic benefit, assuming efficient engraftment and transgene expression and function.14

[0111] Due to the simultaneous self-renewal and multilineage differentiation capacity of long-term hematopoietic stem cells (LT-HSCs), efficient HDR editing in LT-HSCs provides long-term therapeutic benefit in XHIM. However, while efficient ex vivo HDR editing in CD34+HSPCs has been reported for multiple blood and immune disorders, xenotransplantation studies consistently demonstrate inefficient long-term engraftment of HDR edited cells.15-20Further, in comparison to fetal or neonatal HSPC sources (fetal liver or cord blood), the engraftment deficit of HDR edited HPSCs is most evident using clinically relevant, mobilized peripheral blood (mPB) HSPCs.21

[0112] HDR in LT-HSCs is directly related to cell-cycle dependent DNA repair mechanisms. In the context of genome editing and delivery of homologous DNA donor template, repair of the nuclease-mediated double stranded DNA break (DSB) occurs via two competing pathways, non-homologous end joining (NHEJ) vs. HDR. NHEJ is rapid and cell cycle independent, whereas HDR is a slower repair pathway that occurs predominantly in S / G2.22,23Consistent with these observations, engraftment enriched, CD34+CD38- HSPCs are quiescent (G0) and biased toward NHEJ repair.24

[0113] NHEJ repair is mediated by Ku70 / Ku80 recruitment to the DSB, followed by catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) to facilitate DNA end- processing and ligation.25DNA-PKcs inhibitors can enhance HDR by inhibiting NHEJ.26,27Notably, a recently identified DNA-PKcs inhibitor with superior selectivity, AZD7648,28has been shown to enhance HDR efficiency in vitro in cell lines, primary cells and umbilical cord blood-derived HSPCs.29-31

[0114] Efficient rAAV6-based HDR editing in primary T cells as a potential therapeutic strategy for XHIM has been described.11Importantly, the expression kinetics of CD40L in gene-edited T cells partially rescued CD40L expression and function in XHIM T cells. While promising as a T cell therapy, translation of this approach to HSPCs would provide potential curative treatment for XHIM. Embodiments disclosed herein include efficient and sustained HDR editing at the CD40LG locus using optimized CD40L cDNAAAV donor templates and clinically relevant mPB HSPCs. The addition of AZD7648 to editing protocols resulted in significantly enhanced HDR and superior long-term engraftment of HDR-edited HSPCs. In addition, CD40LG-edited HSPCs differentiated into T cells in vivo.

[0115] Importantly, in contrast to previous data where CD40L expression in HDR edited T cells was higher and more prolonged in comparison to endogenous CD40L (see e.g., Hubbard, N., et al. (2016). Blood 127, 2513-2522), the optimized CD40L HDR cassette provided herein mediated lineage restricted and endogenously regulated, CD40L expression in both primary T cell and T cells isolated in vivo from differentiated HDR-edited HSPCs.

[0116] Certain embodiments of the methods and compositions provided herein may include aspects disclosed in US 2021 / 0324381; WO 2024112944; and WO 2024112945 which are each expressly incorporated by reference in their entireties. Certain polynucleotides, polypeptides and vectors

[0117] Some embodiments of the methods and compositions provided herein include polynucleotides for modifying a human CD40LG gene in a cell genome. In some embodiments, the polynucleotides can be substrates for homologous recombination. Somesuch embodiments include, in a 5 to 3 order: a 5 homology arm; a CD40L coding sequence;a polyadenylation signal; and a 3 homology arm. Some embodiments also include awoodchuck hepatitis virus posttranscriptional regulatory element 3 (WPRE3) located between the CD40L coding sequence and the polyadenylation signal. In some embodiments, the encoded CD40L polypeptide binds CD40. In some embodiments, the polynucleotide is single- stranded. In some embodiments, the polynucleotide is double-stranded. In some suchembodiments, the 5 homology arm and the 3 homology arm comprise portions of theCD40LG gene.

[0118] In some embodiments, the CD40L coding sequence comprises a cDNA. In some embodiments, the CD40L coding sequence comprises a codon-diverged cDNA. In some embodiments, the CD40L coding sequence comprises a nucleotide sequence: (i) having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:19; (ii) encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acidsequence set forth in SEQ ID NO:24; or (iii) encoding an amino acid sequence set forth in SEQ ID NO:24 having 0-26 conservative substitutions.

[0119] Some embodiments include a nucleotide sequence encoding an amino acid sequence of CD40L. In some embodiments, the amino acid sequence comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:19. In some embodiments, the amino acid sequence includes one or more conservative substitutions. As is well-known in the art, a “conservative substitution” of an amino acid or a “conservative substitution variant” of a polypeptide refers to an amino acid substitution which maintains: (1) the secondary structure of the polypeptide; (2) the charge or hydrophobicity of the amino acid; and (3) the bulkiness of the side chain or any one or more of these characteristics. In one embodiment, the conservative amino acid substitution can be with an amino acid analog. Illustratively, the well-known terminologies “hydrophilic residues” relate to serine or threonine. “Hydrophobic residues” refer to leucine, isoleucine, phenylalanine, valine or alanine, or the like. “Positively charged residues” relate to lysine, arginine, ornithine, or histidine. “Negatively charged residues” refer to aspartic acid or glutamic acid. Residues having “bulky side chains” refer to phenylalanine, tryptophan or tyrosine, or the like. An exemplary list of conservative amino acid substitutions is given in TABLE 1. TABLE 1 Amino acid Example substitution Alanine D-Ala, Gly, Aib, -Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine D-GIn, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, GIn, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, -Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, IleAmino acid Example substitution Lysine D-Lys, Arg, D-Arg, Orn, D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0120] In some embodiments, the WPRE3 comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:20.

[0121] In some embodiments, the 5 homology arm lacks a nucleotide sequencecapable of specifically hybridizing to the nucleotide sequence set forth in SEQ ID NO:23. Insome embodiments, the 5 homology arm comprises or consists of sequences 5 of the firstcoding exon of the CD40LG gene, and / or 5 of protein coding sequences of the first codingexon of the CD40LG gene. In some embodiments, the 5 homology arm comprises or consistsof sequences 5 of the first coding exon of the CD40LG gene and a portion of the first codingexon, and / or 5 of protein coding sequences of the first coding exon of the CD40LG gene anda portion of the first coding exon. In some embodiments, the 5 homology arm comprises asequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the nucleotide sequence set forth in SEQ ID NO:18. In some embodiments, the 5homology arm comprises a CD40LG promoter or portion thereof. In some embodiments, the5 homology arm has a length in a range from about 500 nucleotides to about 1500 nucleotides;optionally, in a range from about 750 nucleotides to about 1250 nucleotides.

[0122] In some embodiments, the 3 homology arm comprises or consists of a wild-type CD40L genomic sequence. In some embodiments, the 3 homology arm comprisessequences 3 of the first coding exon of the CD40LG gene. In some embodiments, the 3homology arm comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:22. In someembodiments, the 3 homology arm has a length in a range from about 500 nucleotides to about1500 nucleotides; optionally, in a range from about 750 nucleotides to about 1250 nucleotides.

[0123] In some embodiments, the polyadenylation signal comprises an SV40 polyA signal. In some embodiments, the polyadenylation signal comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:21.

[0124] Some embodiments of the methods and compositions provided herein include vectors. In some embodiments, a vector comprises any one of the polynucleotides provided herein. In some embodiments, the vector is an adeno- associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV6 vector

[0125] Vectors suitable for use with embodiments described herein include viral vectors derived from adenovirus, adeno- associated virus (AAV), herpes simplex virus (HSV), retrovirus, lentivirus, self-amplifying single-strand RNA (ssRNA) viruses such as alphavirus (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis, Ml), and flavivirus (e.g., Kunjin virus, West Nile virus, Dengue virus), rhabdovirus (e.g., rabies, vesicular stomatitis virus), measles virus, Newcastle Disease virus (NDV) or poxivirus as described by, for example, Lundstrom (2019, Diseases, 6: 42). In some embodiments, the vector is an adeno- associated virus (AAV) vector. Exemplary AAV vectors, which can be used with embodiments described herein, include, without limitation, those derived from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, or using synthetic or modified AAV capsid proteins such as those optimized for efficient in vivo transduction. A recombinant AAV vector describes replication-defective virus that includes an AAV capsid shell encapsidating an AAV genome. Typically, one or more of the wild-type AAV genes have been deleted from the genome in whole or part, preferably the rep and / or cap genes.

[0126] TABLE 2 lists nucleic acid sequences useful with the methods and compositions provided herein. TABLE 2 Feature SEQ ID NO Nucleotide sequence 5HASEQ ID TGCTTAACGTGTTTCAAATTTCTTCCATGCACATCTT (optimized) NO:18 TATTAGATCTTCACAGCAACCTACAGGATAAGCAAG ACAGGTGCAAGTGCCTCCTTTGGGTATGAGGAAACTFeature SEQ ID NO Nucleotide sequence GAGGTCTAAAGAGATGAAGTGATTTGCCCAAGGCTC ATAGCAATTTATTGGTAGAGCAAAGACTAGAATTCA GATCTCTTAACTGCAGCCTATTTTCCCTATTCTGAAC TGTTACATCAGCATCAACAATTATCTAATGGATTGGA ACAGTGTACACAGGCAGCTTAGCTACGTCAAGTCAC GATTTTTACTTTAACTTCAATTCCAGAGTCTTGGCCT GATTTCCCTCAAGACCCTACTTATCTTTGCCTTTGCA AAATTTATTTTTCTTGCATTATCTTTCCAGCTAAATTT TATTTAATAACCATCAGCATGCTTTTTTTGCTTTATGC CATGTAGACTTGACCTGAAAACCTGCCAGGCTTTCA TTGAGTTTAGTGATTAAAGAAGTAAAGTTCTGAGAA GCAATTAGTTGATGGGACACCAGTCATAAAATCAAT CCAAACTTTTGTTGACATGTGTTTCTTTCTCCATATAC CAGGTTCCCGCTTCGTATTAGTAAGATTGAAATTGAA ATAAGTCTATTGCTGGTGGATGAATTTGTCACTTTCC TTGAAACTGGTGAACCCAAAAAGTTAGACAGTGATA GGAAAATACTGCCATTGTCTGTTAAGAAGTCTATGA CATTTCAAGGCAAGAATGAATATATGGAAGAAGAAA CTTGTTTCTTCTTTACTTACAAAAAGGAAAGCCTGG AAGTGAATGATATGGGTATAATTAAAAAAAAAAAAA AAAACAAAAAACCTTTACGTAACGTTTTTGCTGGGA GAGAAGACTACGAAGCACATTTTCCAGGAAGTGTG GGCTGCAACGATTGTGCGCTCTTAACTAATCCTGAG TAAGGTGGCCACTTTGACAGTCTTCTCATGCTGCCT CTGCCACCTTCTCTGGCAGAGGATACCATTTCAACTT TAACACAGC CD40L SEQ ID ATGATTGAGACTTATAATCAGACCTCTCCTCGCAGCG NO:19 CGGCAACGGGGCTGCCGATCTCTATGAAGATCTTCA TGTACCTTCTGACTGTCTTCCTCATTACACAAATGAT AGGATCTGCCTTGTTTGCAGTCTACTTGCACCGCCG ACTGGATAAAATCGAGGACGAGCGAAATCTGCACG AAGACTTCGTGTTTATGAAGACCATTCAGCGGTGCA ATACAGGCGAACGATCCCTGAGCCTGTTGAACTGCG AAGAAATCAAGAGTCAATTCGAGGGGTTCGTCAAG GACATCATGCTCAACAAGGAAGAGACTAAAAAGGA GAATTCTTTTGAGATGCAGAAGGGGGACCAAAACC CCCAGATTGCCGCCCACGTGATCAGCGAAGCCTCCA GTAAGACGACCAGTGTTCTGCAATGGGCCGAGAAG GGATATTACACGATGTCCAATAACCTGGTCACACTTG AGAACGGAAAGCAACTCACTGTTAAGAGGCAGGGC CTGTATTACATCTACGCCCAGGTAACGTTTTGCTCGA ACCGCGAGGCATCCAGCCAGGCTCCGTTTATCGCTT CCCTGTGTCTCAAAAGCCCTGGCAGGTTTGAACGCA TTCTCCTTAGGGCCGCTAATACGCACAGCTCTGCGAFeature SEQ ID NO Nucleotide sequence AGCCCTGCGGTCAACAGTCGATCCATCTGGGTGGCG TTTTCGAGCTTCAGCCGGGAGCCAGTGTCTTCGTCA ACGTGACAGATCCCTCCCAGGTCTCACATGGGACCG GGTTTACCAGCTTCGGACTGCTGAAGTTGTGA WPRE3 SEQ ID GATAATCAACCTCTGGATTACAAAATTTGTGAAAGAT NO:20 TGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA TGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTAT TGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATA AATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCG CCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGC TGTTGGGCACTGACAATTCCGTGG SV40 SEQ ID TTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGT polyA NO:21 AACCATTCTAGCTTTATTTGTGAAATTTGTGATGCTAT TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAA GTTAACAACAACAATTGCATTCATTTTATGTTTCAGG TTCAGGGGGAGATGTGGGAGGTTTTTTAAAGC3 HA SEQ IDATGATCGAAACATACAACCAAACTTCTCCCCGATCT NO:22 GCGGCCACTGGACTGCCCATCAGCATGAAAATTTTT ATGTATTTACTTACTGTTTTTCTTATCACCCAGATGAT TGGGTCAGCACTTTTTGCTGTGTATCTTCATAGAAGG TTGGACAAGGTAAGATGAACCACAAGCCTTTATTAA CTAAATTTGGGGTCCTTACTAATTCATAGGTTGGTTC TACCCAAATGATGGATGATGGTAGAAACCAAATAGA AGAATGGTCTTGTGGCATAATGTTTGTTGCCTAGTCA ATGAAGTCTCATATTCTTGTCTCTGGTTAGGATCTTG GGATCTGGAGTCAGACTGCCTGGGTTCAAATCTTGG CTCTGCCCATACCATCTCTGTTATCCTGGGGCAAGTG CCTCAGTTTCCACATCTGAGAAATGGGGATGGTATT GGTGTCCATTTCATAGATTAAGTGAGTTTAGCCTTGT AAAAAGCTTAGGAGGGGGTCTGATACATAGTAAGCA CTATGTACGCACTAGCTATAATTATTTGCTAAAGTTCT GCTTTAAAAGTAAGCTATTTTTTTATGGAGACAGCTT TTTTCTTTTAAATTTCCAGCTAGGCAAGAAGAGCGT CAATTTGATCTAAAATTTCATAATGCTTCAGATTAAC ATAGACATGGATAAGTCCCAGAATTTGCAGTCTTTTA GTAAAAGTAGCATTTTCTGTGTAATTCTTCACAAGCA CTGATTGTAGTTGCAGGATGCTCAGTCTCCCTCTGA GATGTTTTACATTTTTAAATGGTTAGACTTGCAGGAA CAAAAGAGCAGAGTAACTTAGTAGGCTGTTTTGCAT TCTTAGGAAAAGAAAACCATCAGGACTTATTTTGTT TTCATGTATTTTTTCACTTCCACTGAGGAGTATAATTG GCTGGTGTTGACAAAATACCAATCATAGATGTAAAGFeature SEQ ID NO Nucleotide sequence GAGAAAGTTGATTAGTTTTCTGGCTGTTCCTAAAATT CTGGATGCAG gRNA SEQ ID AAAGTTGAAATGGTATCTTCTGGC NO:23 CD40L SEQ ID MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGS NO:24 ALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGE RSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNN LVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPF IASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGG VFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Certain methods for modifying a CD40LG gene

[0127] Some embodiments of the methods and compositions provided herein include methods of genetically modifying a CD40LG gene in a cell genome. Some such embodiments include introducing into a cell any one of the polynucleotides provided herein or any one of the vectors provided herein. Some embodiments also include introducing into the cell a nuclease or nucleic acid encoding the nuclease, wherein the nuclease is capable of specifically cleaving a first coding exon of a CD40LG gene. In some embodiments, the nuclease is a Cas nuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), or a meganuclease; optionally, wherein the nuclease is the Cas nuclease or derivative thereof such as a mutant Cas nuclease; optionally, wherein the nuclease is a Cas9 nuclease. Some embodiments also include introducing into the cell a guide RNA (gRNA). In some embodiments, the gRNA comprises a sequence capable of specifically hybridizing to a nucleotide sequence of a first coding exon of the CD40LG gene. In some embodiments, the gRNA comprises a sequence having at least 70%, 80%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:23.

[0128] Methods and tools for the design of TALENs and ZFNs are also useful for some of the embodiments described herein and illustrative examples of such include CHOPCHOP, TALE-NT (Doyle, et al., 2012, Nucleic Acids Research, 4O(W1): W117-W122) and ZF Tools (Mandell and Barbas, 2006, Nucleic Acids Research, 34(W1): W516-W523).

[0129] Some embodiments also include contacting the cell with a DNA-dependent protein kinase (DNA-PK) inhibitor. Some embodiments also include contacting the cell witha DNA-dependent protein kinase (DNA-PK) inhibitor. In some embodiments, the DNA-PK inhibitor is selected from AZD7648, NU7441, NU7026, KU-0060648, LY3023414, CC-115, VX-984 or M3814. In some embodiments, the DNA-PK inhibitor comprises AZD7648. See e.g., Smith, M. C. et al. Characterization of LY3023414, a novel PI3K / mTOR dual inhibitor eliciting transient target modulation to impede tumor growth. Mol. Cancer Ther. 15, 2344– 2356 (2016); Tsuji, T. et al. CC-115, a dual inhibitor of mTOR kinase and DNA-PK, blocks DNA damage repair pathways and selectively inhibits ATM-deficient cell growth in vitro. Oncotarget 8, 74688–74702 (2017); Boucher, D. et al. Abstract 3716: potent radiation enhancement with VX-984, a selective DNA-PKcs inhibitor for the treatment of NSCLC. Cancer Res.76, 3716 (2016); Fuchss, T. et al. Abstract 4198: highly potent and selective DNA- PK inhibitor M3814 with sustainable anti-tumor activity in combination with radiotherapy. Cancer Res.77, 4198 (2017); Zenke, F. T. et al. Abstract 1658: M3814, a novel investigational DNA-PK inhibitor: enhancing the effect of fractionated radiotherapy leading to complete regression of tumors in mice. Cancer Res.76, 1658 (2016); Willmore, E., et al., A novel DNA- dependent protein kinase inhibitor, NU7026, potentiates the cytotoxicity of topoisomerase II poisons used in the treatment of leukemia. Blood. 2004 Jun 15;103(12):4659-65; and Matsumoto, Y. Development and Evolution of DNA-Dependent Protein Kinase Inhibitors toward Cancer Therapy. Int J Mol Sci. 2022 Apr 12;23(8):4264, which are each incorporated by reference in its entirety.

[0130] The repair of DSBs is mediated, at least in part, by DNA-dependent protein kinase (DNA-PK), a complex that consists of the KU heterodimers (KU70 and KU80) and the DNA-dependent protein kinase catalytic subunit DNA-PKcs. DNA-PK is also associated with cellular processes, such as modulation of chromatin structure, telomere maintenance and transcriptional regulation. In view of the importance of DNA-PK activity in the repair of DSB, small molecule inhibitors have been developed for the treatment of, e.g., cancer. First- generation DNA-PK inhibitors, such as NU7441, NU7026 and KU-0060648, while effective for inhibiting DNA-PK, have limited selectivity against PI3K and PIKK members, e.g., mTOR and PI3Ky. Second-generation DNA-PK inhibitors, such as VX-984, M3814 and AZD7648 have improved selectivity against secondary targets, such as ATM, ATR, mTOR, and PI3K isoforms (i.e., PI3Ka, PI3KP and PI3K5), see, e.g., Fok et al., 2019, Nature Communications, 10: 5065. Second-generation DNA-PK inhibitors have a 50-fold or greater selectivity forDNA-PKcs against six or more of the kinases selected from: ATM, ATR, mTOR, PI3Ka, PI3KP, PI3K5, and PI3Ky. Any one or more of the aforementioned DNA-PK inhibitors are useful with embodiments described herein. Certain cells and pharmaceutical compositions

[0131] Some embodiments of the methods and compositions provided herein include cells prepared by any one or more of the methods described herein entailing genetically modifying a CD40LG gene in a cell genome. In some embodiments, the cell is a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), T cell, or a CD34+ cell. In some embodiments, the cell comprises a single X chromosome.

[0132] As used herein the terms "hematopoietic stem cell" or "HSC" refer to multipotent cells capable of differentiating into all of the cell types of the hematopoietic system, including, but not limited to, granulocytes, monocytes, erythrocytes, megakaryocytes, lymphocytes, dendritic cells; or self-renewal activity, i.e., the ability to divide and generate at least one daughter cell with the identical (e.g., self-renewing) characteristics of the parent cell.

[0133] Some embodiments of the methods and compositions provided herein include cells comprising any one of the polynucleotides provided herein or any one of the vectors provided herein. In some embodiments, the cell is a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), T cell, and / or a CD34+ cell. In some embodiments, the cell comprises a single X chromosome.

[0134] Some embodiments of the methods and compositions provided herein include pharmaceutical compositions comprising any one of the cells provided herein and a pharmaceutically acceptable excipient.

[0135] The term "pharmaceutical composition" as used herein refers to a composition that is in a form that allows the biological activity of the active ingredient (e.g., the cell of the present disclosure) to be effective, and that does not contain additional ingredients that have unacceptable toxicity to the subject to which the composition is to be administered.

[0136] In an embodiment, the pharmaceutical composition comprises the cell (e.g., a population of cells) in a number sufficient to administer a dosage of 104to 109cells / kg body weight per dose. Accordingly, the pharmaceutical composition may comprise the cell (e.g., apopulation of cells) in a number sufficient to administer a dosage of 104, 105, 106, 107, 108or 109cells / kg body weight per dose.

[0137] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers or solutes to render the composition isotonic with the blood of the intended recipient, aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, anti-fungal or anti-bacterial agents, isotonic or absorption agents. Certain therapeutic methods

[0138] Some embodiments of the methods and compositions provided herein include methods of treating, ameliorating or inhibiting a disorder in a subject or use of a composition described herein as a medicament. Some such embodiments include administering to the subject any one of the cells provided herein, such as a cell modified by a method provided herein. In some embodiments, the cell is autologous to the subject. In some embodiments, the cell is allogeneic to the subject. In some embodiments, the disorder comprises X-linked hyper IgM syndrome (XHIM). In some embodiments, the subject is male. In some embodiments, the subject is human.

[0139] The therapeutic regimen for the treatment, inhibition, or prevention of a disease or disorder can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the type, stage and molecular characteristics of the disease or disorder in addition to the age, weight and general health of the subject. Another determinative factor may be the risk of developing recurrent disease. For instance, for a subject identified as being at high risk or higher risk or developing recurrent disease, a more aggressive therapeutic regimen may be prescribed as compared to a subject who is deemed at a low or lower risk of developing recurrent disease. Similarly, for a subject identified as having a more advanced stage of disease or disorder, a more aggressive therapeutic regimen may be prescribed as compared to a subject that has a less advanced stage of the disease or disorder.

[0140] The term “subject” as used herein refers to any mammal, including livestock and other farm animals (e.g., cattle, goats, sheep, horses, pigs or chickens), performance animals (e.g., racehorses), companion animals (e.g., cats or dogs), laboratory test animals orhumans. In an embodiment, the subject is a human. In an embodiment, the subject is an adult. In another embodiment, the subject is a child.

[0141] The terms “treat”, "treating", “treatment” and the like are used interchangeably herein to mean relieving, reducing, alleviating, ameliorating or otherwise inhibiting the severity and / or progression of a disease or disorder, or a symptom thereof, in a subject. It is to be understood that the terms “treat”, "treating", “treatment” and the like, as used herein, do not imply that a subject is treated until clinical symptoms of the disease or disorder have been eliminated or are no longer evident. Said treatment may also reduce the severity of the disease or disorder by preventing progression or alleviating the symptoms associated with the disease or disorder.

[0142] The terms “prevent”, “preventing”, “prevention” and the like are used interchangeably herein to mean inhibit, hinder, retard, reduce or otherwise delay the development of a disease or disorder and / or progression of the disease or disorder, or a symptom thereof, in a subject. In the context of the present disclosure, the term “prevent” and variations thereof does not necessarily imply the complete prevention of the specified event. Rather, the prevention may be to an extent, and / or for a time, sufficient to produce the desired effect. Prevention may be inhibition, retardation, reduction or otherwise hindrance of the event, activity or function. Such preventative effects may be in magnitude and / or be temporal in nature.

[0143] The term "autologous" as used herein refers to any material derived from the same subject to whom it is later to be administered into the subject in accordance with the methods disclosed herein. Accordingly, in certain embodiments, cells isolated from the subject may be modified according to the method of the present disclosure and cultured ex vivo for a time and under conditions suitable for the integration of the donor template, before being reinfused back into the subject in accordance with the method of treatment or inhibition as set forth in this disclosure.

[0144] The term "allogenic" as used herein refers to any material derived from a different animal of the same species as the subject to whom the material is administered.Certain systems and kits

[0145] Some embodiments of the methods and compositions provided herein include systems or kits for modifying a human CD40LG gene in a cell genome. Some embodiments include any one of the polynucleotides provided herein, and a nuclease or nucleic acid encoding the nuclease, wherein the nuclease is capable of specifically cleaving a first coding exon of a CD40LG gene.

[0146] In some embodiments, the nuclease is a Cas nuclease, a mutant Cas nuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), or a meganuclease; optionally, wherein the nuclease is the Cas nuclease or mutant Cas nuclease; optionally, wherein the nuclease is a Cas9 nuclease or a mutant Cas nuclease derived from a Cas molecule.

[0147] Some embodiments also include a guide RNA (gRNA). In some embodiments, the gRNA comprises a sequence capable of specifically hybridizing to a nucleotide sequence of a first coding exon of the CD40LG gene. In some embodiments, the gRNA comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:23.

[0148] Some embodiments also include a cell. In some embodiments, the cell is, a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), a T cell, or a CD34+ cell.

[0149] Some embodiments also include a DNA-dependent protein kinase (DNA- PK) inhibitor. Some embodiments also include contacting the cell with a DNA-dependent protein kinase (DNA-PK) inhibitor. In some embodiments, the DNA-PK inhibitor is selected from AZD7648, NU7441, NU7026, KU-0060648, LY3023414, CC-115, VX-984 or M3814. In some embodiments, the DNA-PK inhibitor comprises AZD7648.EXAMPLES Example 1— DNA-PK inhibition enhances gene editing efficiency in hematopoietic stem and progenitor cells for CRISPR-based treatment or inhibition of X-linked hyper IgM syndrome Establishment and optimization of SpCas9-based editing at CD40LG in mobilized peripheral blood (mPB) CD34+HSPCs

[0150] Efficient editing of the CD40LG locus in human primary T cells with rAAV6 donor template and TALEN nuclease has been previously reported.11To establish andcompare this approach with a CRISPR-SpCas9 editing strategy, an sgRNA targeting the 5UTR of CD40LG and a rAAV6 donor template to deliver a fluorescent reporter cassette were designed. The rAAV donor template utilized a constitutively active MND promoter driving GFP expression followed by WPRE and synthetic poly-adenylation signal elements flanked by 1 kb homology arms (FIG.7A).

[0151] As the SpCas9 and TALEN target sites were separated by only 15 bp, an identical MND.GFP AAV donor vector for both nucleases, designed to avoid recutting of the repaired DNA locus by either nuclease was initially employed. TALEN and SpCas9 RNP (SpCas9 protein + CD40LG sgRNA) were tested, in parallel, in mPB CD34+HSPCs following the timeline outlined in FIG. 7B. At matched rAAV6 multiplicity of infection (MOI), SpCas9 outperformed TALEN in preserving HSPC viability while enabling superior HDR efficiency (FIG. 7C, FIG.7D). HDR rates quantified by FACS matched genomic HDR rates quantified by in-out ddPCR (FIG. 8). After identifying SpCas9 as the preferable nuclease, an optimized MND.GFP AAV donor without previous mutations designed to eliminate TALEN binding wasgenerated, thus increasing the homologous stretch within the 5 homology arm. This donorresulted in enhanced HDR efficiencies and was used in all subsequent studies.

[0152] Next, the incorporation of the small molecule drugs, SR1 and UM171, previously reported to support survival and possible ex vivo expansion of phenotypically primitive HSPCs32,33into our HSPC culturing protocol was assessed. Culturing in the presence of SR1 and UM171 trended toward higher cell viability at 48 hours post-editing in mock- treated, AAV-treated, and AAV+RNP-treated conditions relative to the matched condition cultured without SR1 and UM171 (FIG. 9A). Based upon flow cytometry analysis, roughly twice the proportion of phenotypically primitive, HSC-enriched, CD34+CD38-CD90+CD133+cells were present in culture 24h after editing with SR1 and UM171 treatment with similarfold-expansion independent of treatment with AAV or AAV + RNP (FIG. 9B). Similar HDR efficiencies were observed at 1E3 or 2.5E3 MOI of AAV with SR1 and UM171 treatment (FIG.9C). iDNA-PKcs enables enhanced HDR editing at CD40LG locus in CD34+HSPCs

[0153] The effect of iDNA-PKcs in rAAV-based HDR editing at the CD40LG locus was determined. The RNP targeting CD40L Exon 1 (FIG. 7A; sequence shown schematically in FIG. 1) and a rAAV6 GFP donor template were utilized to mediate HDR in mPB HPSCs using the experimental approach shown in FIG. 2A. Analysis of cell viability at 24h post-editing indicated minor toxicity associated with rAAV6 treatment that increased slightly with RNP co-delivery (FIG. 2B). Notably, no difference in viability was observed in the presence vs. absence of iDNA-PKcs treatment (FIG.2B). FACS analysis of HSPC surface markers 24h post-editing showed similar proportions of bulk CD34+HSPCs, CD34+CD38- “engraftment enriched” HSPCs24,39and CD34+CD38-CD90+HSC-enriched cells40across treatments (FIG. 2C, FIG. 2D). Comparison of rAAV6 transduction, as quantified by %GFP+cells in HSPC subsets at Day 1 post-editing (as identified in FIG. 2D), revealed similar GFP expression levels independent of iDNA-PKcs treatment (FIG. 2E). Importantly, HDR rates, based on proportion of GFPhighcells at Day 5 post-editing, were significantly increased with iDNA-PKcs treatment (FIG. 2F, FIG. 2G; 3 experiments, 4 independent donors). Of note, to optimally account for the variability of starting HDR frequencies across independent experiments and HSPC donors in this and most subsequent experiments, a linear regression statistical model was utilized to determine the significance of the effect of iDNA-PKcs treatment on HDR.

[0154] In addition to characterizing cell viability and HSPC phenotype, cells were removed 24h after editing and plated in methylcellulose to assess the colony formation potential. To define CFU input cell phenotype, some cells were retained in culture to assess viability, proportion of CD34+and CD34+CD38-, and GFP (HDR) within CD34+and CD34+CD38 populations at day 5 post-editing. After 15 days, colonies were enumerated and phenotypically defined. It was found that the individual editing components; electroporation (mock-edited), RNP only, AAV only and iDNA-PKcs impacted total colony formation relative to the untreated control. The combination of RNP+AAV and RNP+AAV+iDNA-PKcs furtherlimited total colony formation, with a ~46% and 56% loss in the RNP+AAV and RNP+AAV+iDNA-PKcs groups compared to untreated control, respectively. No statistical difference in colony formation using RNP+AAV vs. RNP+AAV+iDNA-PKcs (p-value 0.256) was observed. No skewing of colony-types between groups was observed. To specifically test if iDNA-PKcs impacts the colony-forming potential of HDR-edited cells, GFP+colonies were counted to assess %HDR. It was found that the increased GFP+population observed at day 5 post-editing in the RNP+AAV+iDNA-PKcs group was maintained at day 15 based on %GFP+colonies.

[0155] Overall, these results demonstrated that iDNA-PKcs treatment significantly increased HDR efficiency in mPB CD34+HSPCs and resulted in minimal additional disturbances in HSPC phenotype. Superior multilineage engraftment of HDR edited cells with iDNA-PKcs

[0156] To determine the impact of treatment with iDNA-PKcs on the engraftment of HDR-edited mPB HPSCs, a series of xenotransplantation studies were conducted (FIG.3A). Edited (MND.GFP rAAV6 + RNP), edited + iDNA-PKcs-treated, or untreated control mPB CD34+HSPCs were transplanted (at 24h post-editing) into adult NBSGW recipient animals, an immune deficient strain that supports high levels of human hematopoietic chimerism and multilineage differentiation.21,41

[0157] Peripheral blood was analyzed for engraftment (%hCD45+) and proportion of edited cells (%GFP+; FIG. 10), revealing engraftment across all cohorts (FIG. 3B left). A trend of higher GFP+, HDR edited cells was observed across the duration of the studies with inclusion of iDNA-PKcs (FIG.3B right). After 16 weeks, bone marrow (BM) and spleens were harvested and analyzed to determine human cell engraftment (%hCD45+), differentiation to B cell (%CD19+) vs myeloid lineage (%CD33+), and presence of edited cells in each population (FIG.3C). Analysis of recipient BM (FIG.3D) and spleen (FIG.3E) showed engraftment and multilineage differentiation in all animals (left 3 panels, respectively) and a significantly increased proportion of HDR edited cells in iDNA-PKcs treated recipients in both compartments across all lineages (right 3 panels, respectively).

[0158] Together, these observations indicated that mPB CD34+HSPCs edited at the CD40LG locus can engraft and differentiate in vivo at levels similar to untreated HSPCs.While a limited deficit in BM engraftment with AAV+RNP treatment (mean: 66.5%) relative to untreated control mPB CD34 HSPCs was observed (mean: 72.2%), treatment with iDNA- PKcs had no additional impact (mean: 65.5%). Importantly, these studies demonstrated that the improved HDR efficiency observed in vitro with iDNA-PKcs (FIG. 2G) was sustained following engraftment in vivo (FIGs. 3C-3E). Sustained, long-term engraftment of CD40LG HDR edited HSPCs

[0159] To understand the phenotype and HDR editing frequency of HSPCs engrafted in the BM of recipient mice, an HSPC-specific immunophenotyping panel was utilized (FIG. 4A). Consistent with slightly reduced bulk (hCD45+) engraftment in AAV- treated conditions, a reduced frequency of total CD34+HSPCs in the AAV+RNP and AAV+RNP+iDNA-PKcs recipient animals was observed (FIG. 4B). Treatment with iDNA- PKcs had no additional impact on the proportion of CD34+cells compared to HDR editing with AAV+RNP alone. In contrast to bulk CD34+cells, no differences in the proportion of more- primitive, CD34+CD38- and CD34+CD38-CD90+HSPC populations was observed (FIG.4B). Strikingly, analysis of GFP in CD34+, CD34+CD38-, and CD34+CD38-CD90+populations each showed a significant increase in the proportion of HDR edited HSPCs with iDNA-PKcs treatment across all donor groups (FIG. 4C, FIG.4D).

[0160] Next, to stringently characterize the long-term engraftment and self-renewal capacity of HSPCs recovered from NBSGW recipients, secondary transplant studies were conducted utilizing NSG-SGM3 as recipient mice (previously reported to support superior secondary engraftment compared to NSG recipients).42Upon sacrifice at week 16, BM from NBSGW primary recipient animals (shown in FIGs. 4A-4D) was pooled across groups and transplanted into NSG-SGM3 mice (BM from 4 primary recipients were pooled and transplanted into 2-3 secondary recipients). After 12-14 weeks, BM was harvested from secondary recipients and analyzed for human engraftment (FIG. 4E) and the proportion of HDR edited cells (FIG.4F). Comparable levels of engraftment (averaging ~1%) were observed for all groups (untreated, edited, and edited+iDNA-PKcs). Notably, an average of 0.97% vs. 6.9% HDR edited cells were recovered from AAV+RNP and AAV+RNP+iDNA-PKcs recipients, respectively. Greater than 1% of HDR edited cells were present in 2 / 6 AAV+RNP vs. 3 / 7 AAV+RNP+iDNA-PKcs recipients. Notably, in 2 of 6 AAV+RNP+iDNA-PKcsrecipients, but none of the AAV+RNP recipients, the proportion of HDR edited cells recovered was equal to or greater than the average initial proportion of HDR edited HSPCs (e.g., within the pooled BM isolated from primary NBSGW recipients). These variable data likely reflected a limited number of HSPCs engrafting in secondary recipients, leading to stochastic variation in HDR. However, recovery of HDR edited cells from secondary recipients suggested HDR editing in long-lived HSCs. Together, this characterization of primary and secondary xenotransplantation outcomes provided evidence that iDNA-PKcs supported enhanced HDR editing in primitive, long-term engrafting HSPCs. Long-term engraftment and T-lineage differentiation of CD40L cDNA-edited HSPCs

[0161] Moving toward a therapeutic platform for XHIM, a rAAV6 donor template consisting of a codon diverged CD40L coding sequence (CDS) (identical to coding sequences in11) and WPRE3 and synthetic polyA elements was generated (FIG. 5A). Following HDR, expression of the codon diverged CD40L CDS was designed to be driven by the endogenous CD40LG promoter, with the goal of achieving physiological regulation. Using this “cDNA” donor template, in vitro conditions were identified for co-delivery of RNP and rAAV6 donor in mPB CD34+HSPCs that preserved viability (FIG. 5B) while maximizing HDR efficiency (FIG.5C).

[0162] To determine the long-term engraftment capacity of the mPB CD34+HSPCs edited with this therapeutic donor, xenotransplantation studies were performed in NGSGW recipient mice (FIGs. 5D-5F). A similar experimental approach to that detailed in FIG. 3A was utilized and incorporated HDR editing with or without iDNA-PKcs treatment. Four experiments were performed using 3 CD34+HPSC donors with input HDR rates of 23- 59%. In parallel, the impact of editing in association with iDNA-PKcs was assessed on DNA repair outcomes not resulting in HDR. HDR editing using RNP+AAV in association with iDNA-PKcs resulted in increased rates of MMEJ and a concomitant reduction in NHEJ (FIG. 11, FIG.12).

[0163] Following adoptive transfer, similar levels of human cell engraftment within the peripheral blood were observed in cDNA-edited vs. cDNA-edited + iDNA-PKcs treated recipients (FIG. 13); levels comparable to engraftment observed with GFP-edited recipients (FIG. 3B). Following sacrifice at 16 weeks, HDR in engrafted human cells wasassessed using ddPCR (FIG. 5D). In comparison with cDNA-editing alone, treatment with iDNA-PKcs resulted in significantly higher average levels of long-term engrafted HDR edited cells in both BM and spleen (BM AAV+RNP: 7.7%; BM AAV+RNP+iDNA-PKcs: 11.9%; SP AAV+RNP: 11.5%; SP AAV+RNP+iDNA-PKcs: 16.2%). For all CD34 donors evaluated, similar human cell engraftment and lineage distribution in BM (FIG.5E) and spleen (FIG.5F) was observed.

[0164] A constraint of the adult NBSGW humanized model is lack of thymic differentiation of human T cells following transplant of mPB CD34 HSPCs.21Therefore, as an alternative means to assess T cells generated from HDR edited HPSCs, an artificial thymic organoid (ATO) system43was utilized to assess T-lineage differentiation of BM CD34+ HSPCs isolated from long-term engrafted, NBSGW recipient mice. Human CD34+cells were enriched from BM pools from recipient mice (2 independent transplant experiments) and used to initiate ATO cultures. Following 6-8 weeks in culture, T cell differentiation was characterized by immunophenotyping (FIG. 5G, FIG. 5H). T lineage cells across a range of differentiation stages, including abundant CD5+CD7+ pre-T1 cells as well as DP and limited numbers of SP T cells, were present in cultures initiated using either untreated or CD40L cDNA-edited HSPCs (FIG. 5G, upper vs lower panels). To determine whether HDR edited HSPCs contributed to the generation of T lineage cells, %HDR was quantified by ddPCR in ATOs (after 6-8 weeks) and compared to % HDR of input CD34+ HSPCs isolated from long- term engrafted, cDNA-edited, recipient animal BM (FIG. 5I). While variable across the studies, the proportion of HDR edited T cells was similar to the proportion of edited cells within the input HSPC population. These findings indicated that long-term engrafted HSPCs recovered from xenotransplantation studies retain T-lineage differentiation capacity and that HDR editing at the CD40LG locus did not impair their differentiation capability. T cells derived in vivo from CD40LG-edited HSPCs exhibit regulated CD40L expression

[0165] Characterization of CD40L cDNA expression is useful to infer the functionality and safety of CD4 T cells expressing a candidate therapeutic HDR cassette. Moving toward a HSPC editing therapy and to allow for direct tracking of HDR edited, CD40L expressing T cells, an alternative AAV donor cassette containing a GFP.T2A.CD40L cDNA expression cassette was generated (FIG. 6A). This cis-linked GFP donor design was utilizedto permit flow-based tracking of naïve T cells derived from HDR-edited HPSCs in vivo. mPB CD34+HSPCs were edited in association with iDNA-PKcs treatment using this new donor as detailed in FIG.3A. In comparison with the NSG or NBSGW strains, the NSG-SGM3 mouse strain has previously been reported to support more efficient T cell differentiation from engrafted human HPSCs.42Therefore, to facilitate generation of T cells in vivo from HDR- edited adult mPB HPSCs, transplant studies using this strain were performed. Busulfan conditioned, adult NSG-SGM3 recipient mice were transplanted with unedited or edited HPSCs and recipient mice were evaluated at 16 weeks post-transplant. Similar levels of human chimerism were observed in recipients transplanted with unedited vs. edited HSPCs (FIG.6B, FIG.6C). As expected, based on previous studies, overall human cell chimerism was lower in this strain (FIG.6B, FIG. 6C) compared to NBSGW recipients shown in FIG.3, FIG. 4, FIG. 5. Variable proportions of CD3+ T cells were present in both the BM and spleen across both cohorts. Notably, following long-term engraftment, HDR rates in both BM (44.3%) and spleen (52.3%) were similar to the HDR level of input HSPCs (63.4%) (FIG. 6C) demonstrating sustained engraftment of CD40L HDR edited, iDNA-PKcs treated, adult mPB HPSCs in this alternative model.

[0166] Consistent with intracellular, endogenously regulated, CD40L expression that occurs in naïve human T cells, flow cytometry of a CD40L cDNA.GFP + iDNA-PKcs treated recipient revealed T cell-specific, baseline cis-linked GFP expression driven off the integrated HDR cassette (FIG. 6B). Next, to determine whether HDR-edited T cells exhibit activation-dependent surface CD40L expression, a modified activation protocol was used.11Transgenic CD40L cDNA expression kinetics were characterized by stimulating with PMA and ionomycin to activate surface CD40L expression and tracked expression based on co- expression of GFP and CD40L (CD154) surface staining. As T cell numbers were limited, total spleen cells were activated and stained for surface CD40L expression. Activation led to a CD40L+GFP+ double positive population. Comparing CD40L expression in GFP+ vs. GFP- populations revealed a similar level of CD40L surface expression (based upon CD154 MFI) and comparable expression kinetics consistent with endogenous CD40L expression in response to T cell activation (FIG. 6D, FIG. 6E). Together, these data demonstrated sustained engraftment of CD40L HDR edited mPB HPSCs, differentiation of edited HPSC into naïve Tcells in vivo, and endogenously regulated CD40L surface expression following ex vivo activation of T cells derived from HDR edited mPB HPSCs. Discussion

[0167] HDR-based gene editing to precisely insert a therapeutic expression cassette in HSPCs has emerged as a potential future therapeutic strategy for monogenic blood and immune disorders.19,20,44Here, utilizing a high specificity, small molecule inhibitor of DNA- PKcs, AZD7648, in association with co-delivery of CRISPR-SpCas9 RNP and a rAAV6 homology template, an efficient HDR-based HSPC gene editing approach to treat XHIM syndrome was established. Using clinically relevant, mPB CD34+HSPCs, precise insertion of a therapeutic CD40L cDNA cassette at the CD40LG locus and long-term engraftment of CD40L-edited HSPCs in vivo, at predicted therapeutically beneficial levels was achieved. Importantly, this approach permitted T cell differentiation in vivo and in vitro and led to endogenous promoter-regulated CD40L expression kinetics.

[0168] Previously, efficient HDR editing at the CD40LG locus in primary T cells using TALEN and rAAV6 has been reported.11By targeting CD40LG exon 1, the upstream promoter and intronic regulatory elements are conserved, enabling regulated expression of the rAAV6-delivered DNA donor cassette. Embodiments disclosed herein include an sgRNA proximal to the previously reported TALEN site and compared CD40LG editing efficiency and viability in mPB CD34+HSPCs. At matched rAAV6 doses, superior HDR editing efficiency and higher cell viability with SpCas9 nuclease was found. Next, HSPC culturing parameters were optimized through addition of small molecules SR1 and UM17132,33that increased the proportion of primitive CD34+CD38-CD90+CD133+HSPCs. Optimization of HSPC culturing and nuclease choice yielded roughly 40% HDR in vitro using a therapeutic CD40L cDNA cassette. In previous work,12a similar strategy was used to introduce a CD40L cDNA leading to efficient HDR in HSPCs and XHIM patient-derived T cells in vitro. However, transplant of HDR-edited HSPCs yielded limited long-term persistence of HDR edited cells in vivo; comparable to levels reported here in the absence of iDNA-PKcs. The authors speculated that long-term engraftment may be driven by a quiescent fraction of HSPCs resistant to HDR. An alternative HDR strategy has also been used previously to target intron 1 of the CD40LG gene for HDR editing;13an approach designed to avoid unregulatedexpression from off-target integration but not capable of addressing mutations within exon 1. Using cord blood CD34+HSPCs, the authors reported efficient HDR and engraftment of HDR edited cells at higher levels when editing was performed in association with inhibition of p53 via GSE56 mRNA;45,46observations that highlight the potential clinical benefit of transiently targeting the DNA damage response.47Combinatorial inhibition of DNA-PKcs and p53 (via GSE56 mRNA or via alternative approaches) in mPB CD34+HSPCs could be considered in future studies.

[0169] While efficient HDR has now been reported for many genetic loci in vitro, reduced in vivo engraftment of HDR edited HSPCs remains a major hurdle to clinical translation.15-17,47,48This challenge is especially important using clinically relevant HSPCs including mPB CD34+cells. Engraftment-enriched CD34+CD38- HSPCs are more quiescent relative to bulk CD34+HSPCs and skew towards NHEJ repair, rather than HDR, in the context of gene editing.24Small molecule inhibitors of DNA-PKcs, an enzyme critical in facilitating NHEJ repair, have been used to enhance HDR in cell lines and primary cell populations.27,29-31,49Disclosed herein it has been demonstrated that AZD7648, a highly selective inhibitor of DNA-PKcs,28potently inhibits NHEJ allowing for enhanced HDR rates in HSPCs with both ssODN and rAAV6 DNA donor templates. Up to 60% HDR was achieved at the CD40LG locus with rAAV6 DNA donors with sustained benefit in CFU assays. Most notably, using a series of xenotransplantation studies, iDNA-PKcs co-delivery improved the in vivo recovery of long-term repopulating, HDR edited, HSPCs. Analysis of BM from recipient NBSGW animals at 16 weeks showed ~70% higher levels of HDR-edited CD34+CD38-CD90+HSPCs following editing in the presence of iDNA-PKcs. Consistent with findings disclosed herein, Selvaraj et al., identified AZD7648 to be superior to alternative DNA-PKcs inhibitors in enhancing the HDR:INDEL ratio in cord blood CD34+HSPCs in vitro using an rAAV6 / RNP editing strategy, findings that correlated with an increased proportion of HDR-edited CFU colonies.29While the authors did not perform xenotransplantation studies, they speculated that AZD7648 might increase engraftment of gene-targeted HSPCs. This speculation is validated by our in vivo findings.

[0170] Inhibition of 53BP1 in CD34+HSPCs has been evaluated as an alternative approach to limit NHEJ and promote HDR.50,51Transient delivery of i53 mRNA52led to an ~50% increase in HDR efficiency at the CYBB locus, and combination of i53 and GSE56mRNAs resulted in 76% greater HDR efficiency at the MAGT1 locus and an ~50% increase HDR edited cells recovered in NSG-SGM3 recipients. In contrast to AZD7648 small molecule delivery, mRNA delivery requires generation of high-purity, non-immunostimulatory products to limit innate RNA sensing. Interestingly, across multiple groups, maximal mean HDR efficiency with rAAV6 templates reach ~60-70% in CD34+HSPCs. Of note, combinatorialinhibition of DNA-PKcs and Pol mediated further HDR enhancement by simultaneouslytargeting NHEJ and theta-mediated end joining;30however, this approach has not been assessed for in vivo efficacy using HPSCs. Inhibition of theta-mediated end joining has also been reported to reduce the frequency of large deletions occurring after DSB generation.53Overall, targeted inhibition of NHEJ provides a key tool for enhancing HSPC HDR editing, a conclusion strongly supported by our combined in vivo findings.

[0171] While targeting DNA repair mechanisms can partially address the deficit in engrafted HDR-edited CD34+HSPCs, other mechanisms likely also limit the clinical translation of rAAV6-based HSPC editing. As disclosed herein, modest evidence for rAAV6- associated toxicity in vivo was observed in comparison to mice transplanted with control edited HSPCs. rAAV6 exposure triggers a p53-dependent DNA damage response (DDR) in HSPCs that can be partially mitigated via transient p53 inhibition.45,46,50,54While rAAV likely directly mediates a transient DDR, AAV ITR fragments trapped at on- and off-target sites DSBs may also impact the p53-dependent DDR.54Interestingly, DNA-PK has been implicated in a STING-independent DNA sensing pathway.55The proportional increase in HDR observed in vivo suggests that AZD7648, in addition to promoting HDR, may play a role in limiting toxicity in HSPCs sensitive to rAAV6-assocaited toxicities. Future work is required to determine whether a combination of AZD7648 and p53 inhibition, or targeting other innate signaling pathways can additionally mitigate impacts of rAAV6 exposure. Potential candidate small molecule inhibitors may include those tested in primary T cells in association with a dsDNA donor template.56

[0172] While rAAV6 remains the primary method for delivering large DNA donors for HDR editing in HSPCs, HDR mediated by alternative DNA templates might be similarly potentiated with AZD7648. Consistent with this concept, we observed a ~50% increase in HDR rates in edited mPB CD34+HSPCs using AZD7648 in association with ssODNs. While relatively inefficient in CD34+ HSPCs, single- or double-stranded DNA donors may alsoexhibit improved efficiency in combination with AZD7648. Consistent with this concept, combined use of inhibitors of DNA-PKcs, HDAC1 / 2, and CDC7 facilitated knock-in of non- viral, DNA donors in primary T cells.57Further, IDLV-based editing can achieve HDR efficiencies comparable to rAAV654and might be similarly facilitated with AZD7648 delivery.

[0173] Achieving physiological rescue of CD40L expression is critical for successful XHIM therapy, as unregulated CD40L expression results in lymphoproliferative disease.6The CD40L cDNA cis-linked GFP cassette utilized in embodiments disclosed herein mediated T-cell specific expression and activation-dependent CD40L surface expression by T cells differentiated from CD40L-edited mPB HSPCs in vivo. This design was similar to previous work in primary T cells.11However, here the donor cassette was altered by placing the GFP and codon-diverged CD40L cDNA bicistronic elements downstream of the DNA cut site and utilized a truncated WPRE3 element. In T cells, derived in vivo from mPB CD34+HSPC-transplant recipients, cis-linked GFP was specifically expressed in unstimulated CD3+cells, consistent with endogenous, lineage-specific, CD40L promoter activity leading to intracellular loading of CD40L containing vesicles. Further, physiological, T cell activation dependent, CD40L surface expression was demonstrated herein. In a similar study,13CD40L surface expression was shown following stimulation of CD4+T cells recovered from mice transplanted using HDR edited cord blood CD34+HSPCs. These combined findings support the safety and function of our therapeutic CD40L cDNA donor, confirming T lymphocyte differentiation in vivo and physiological ex vivo activation following editing at the CD40LG locus and long-term engraftment of mPB CD34+HSPCs.

[0174] Importantly, embodiments disclosed herein focused on HDR editing and transplantation of clinically relevant, mPB CD34+HSPCs into adult recipient animals to most accurately model future therapeutic strategies. One challenge with existing xenotransplantation models (particularly in using IV delivery of adult HSPCs in adult recipient mice) is the limited ability to achieve sustained engraftment of primitive HSPCs in parallel with efficient T cell differentiation. While robust T cell development is observed following intrahepatic transplant in neonatal recipients,50,58T cell development following transplantation of mPB CD34+HSPCs into adult immune deficient animals is more limited.21Conversely, while alternative models, including NSG-SGM3 mice, permit more efficient T cell development, HSC engraftment progressively declines over time.42As an overall approach to assess both CD34+HSPCengraftment and T cell differentiation, a combinational approach of NBSGW and NSG-SGM3 models, primary and secondary transplants, and in vitro ATO studies using CD34+HSPCs recovered post-transplant were used. In the NSG-SGM3 model, T lineage-specific GFP expression was observed consistent with endogenously regulated expression. Notably, higher levels of HDR edited cells were recovered in the NSG-SGM3 model; levels nearly equivalent to input HDR rates. This observation is consistent with other work50,59and likely reflects expression of human cytokines that promote lineage committed, HDR-edited human progenitors, rather than more primitive CD34+CD38- HSPCs. To stringently characterize editing in long-term engrafted HSPCs, secondary transplant studies, transplanting BM from primary NBSGW recipients into secondary recipient NSG-SGM3 animals were performed. This approach was based upon previous findings42indicating superior secondary engraftment in NSG-SGM3 mice. GFP+edited cells were recovered in secondary recipients showing HDR editing in long-term engrafted HSPCs. Despite the limited human chimerism and small number of secondary recipients studied, our findings suggest that iDNA-PKcs may facilitate increased long-term engraftment of HDR edited HSPCs. Moving forward, it will also be important to assess the clonality of HSPCs engrafted in vivo46to further assess the safety of proposed editing strategies for XHIM.

[0175] In summary, embodiments disclosed herein demonstrated a SpCas9-based HDR editing strategy to deliver functional CD40L cDNA to the CD40LG locus. Inclusion of the specific DNA-PKcs inhibitor, AZD7648, led to improved levels of long-term engrafted, HDR edited, mPB HSPCs approaching levels predicted to provide clinical benefit in HIGM14and generated T cells with endogenous promoter-regulated CD40L expression. Materials and Methods Nuclease reagents

[0176] CD40LG TALENs were identical to those previously described in Hubbard et al.,11except that they were cloned into a pEVL backbone rather than a pUC57 backbone, which was linearized using BsaI. TALEN mRNAs were transcribed from the linearizedplasmids and capped (5 7-methylguanylate cap with cap-1 structure) in vitro using T7mScript mRNA Production System (CellScript Madison, WI, USA) following the manufacturer’s instructions and as previously described.60,61Final purification was performedusing NucleoSpin RNA clean-up (Machery Nagel, Germany). TALENs were electroporated at 50 μg / mL for each TALEN. SpCas9RNPs targeting CD40LG or B2M were generated using Alt-R V3 SpCas9 (Integrated DNA Technologies, Coralville, IA, USA) and the following sgRNAs: CD40L (SEQ ID NO:01); B2M (SEQ ID NO:02). SpCas9 RNPs were made by incubating the SpCas9 protein with sgRNA synthesized by IDT at 2:1 sgRNA:SpCas9 ratio for 10-15 min at 37°C. SpCas9 RNP was electroporated at a concentration of 150 μg / mL. For all other experiments, SpCas9 protein was incubated with sgRNAs from Synthego (Redwood City, CA, USA) at 1:1.2 molar ratio for 15 min at room temperature, and the resulting SpCas9 RNP was electroporated at a concentration of 80 μg / mL reaction volume. The following ssODN donor template for B2M (IDT) was included in the RNP complexing reaction: (SEQ ID NO:03). The following TABLE 3 lists oligonucleotides useful with embodiments provided herein. TABLE 3 Oligonucleotide SEQ ID NO Nucleotide sequence sgRNA, CD40L SEQ ID NO:01 AAAGUUGAAAUGGUAUCUUC sgRNA, B2M SEQ ID NO:02 CGAUAAGCGUCAGAGCGCCG SEQ ID NO:03 AAAGAGCGGAAGAGAAACCCTCC ssODN donor template for CCCAACCTCGGCTCGAGCGCTCTG B2M ACGCTTATCGACGCCCTAAACTTT GTCCCGACC For ddPCR Forward SEQ ID NO:04 ACCTGTCAGCTCCTTTCC detection of Reverse SEQ ID NO:05 GGTCCAGATCCTAAGAGAGG CD40L MND.GFPPSEQ ID NO:06HDRrobe TCAATCCAGCGGACCTTCCTFor ddPCR Forward SEQ ID NO:07 AGTGTCTTCGTCAACGTGACAG detection of Reverse SEQ ID NO:08 TCCCTGATAAAGTGCAATCATCC CD40L cDNA and SEQ ID NO:09 GFP.T2A Probe TCCTGGTTAGTTCTTGCCAC HDR For ddPCR Forward SEQ ID NO:10 ACTCTGCAGGTTCTATTTGC detection of Reverse SEQ ID NO:11 AATGATCTGAGGAGGGAAGG Beta Actin (ActB) Probe SEQ ID NO:12 ATCAAGGTGGGTGTCTTTCCOligonucleotide SEQ ID NO Nucleotide sequence B2MSEQ ID NO:13For forwardGCGTGAGTCTCTCCTACCCTCamplifying and B2MSEQ ID NO:14uencing reveCCTAGACGAAGTCCACAGCTCseq rse B2M B2MSEQ ID NO:15sequencingCTCTGGTCCTTCCTCTCCCGFor indel CD40LGSEQ ID NO:16profiling of forwardCGTAACGTTTTTGCTGGGAGAGrAAV6+RN SEQ ID NO:17 P based CD40LG HDR at reverse GCAAAAAGTGCTGACCCAATCA CD40LG rAAV6 design and production

[0177] Plasmids were adapted from those previously described in Hubbard et al.11To generate the pAAV.MND.GFP.WPRE construct, the MND modified retroviral promoter was inserted into the previously described pAAVCD40LG[GFP.WPRE] plasmid. The full CD40L.cDNA.WPRE3.SV40PolyA was synthesized (GeneArt) and cloned into a pAAV backbone with 1kB CD40LG homology arms by Infusion cloning (Clontech). The pAAV.CD40LG [GFP.2A.CD40L cDNA.WPRE3]11was cloned into a pAAV backbone with 1 kB CD40LG homology arms. AAV6 vector stocks were produced and titered as previously described.62HSPC culture and editing

[0178] mPB CD34+HSPCs (CD34-purified peripheral blood stem cells apheresed from G-CSF-mobilized adult male donors) were purchased from Fred Hutchinson Cancer Research Center (Seattle, WA, USA) or Charles River Laboratories (Wilmington, MA, USA). For most experiments, cells were thawed and cultured at a concentration of 0.25 × 106cells / mL in HSPC growth media: serum-free expansion media II (SFEMII) (STEMCELL Technologies, Vancouver, CA) supplemented with TPO, SCF, FLT3-L, IL-6 (100 ng / mL each, Preprotech, Waltham, MA, USA) and 1 μM SR1 (STEMCELL Technologies) and 35 nM UM171 (ApexBio, Houston, TX, USA). However, experiments in FIG.7 and FIG.8 used a cell density of 1 × 106cells / mL and serum-free stem cell growth media (SCGM; CellGenix, Sartorius,Germany) in place of SFEMII; experiments in FIG. 7 did not include SR1 or UM171 in the growth media.

[0179] For the experiments shown in FIG.7 and FIG. 8, cells were cultured for 48 hours at 37oC, resuspended in Neon Buffer T (Thermo Fisher Scientific), then electroporated with either TALEN mRNA or SpCas9 RNP (1400 V, 20ms, one pulse) using a Neon Transfection System (Thermo Fisher Scientific). Immediately after electroporation, cells were plated at a concentration of 5 × 105cells / mL in fresh HSPC growth media with rAAV6 added 1-5 × 105GC (viral genome copies) / cell. 24 h after editing, AAV-containing media was removed and replaced with fresh media.

[0180] In all other experiments, cells were cultured for 48 h at 37oC, resuspended in P3 Buffer (Lonza, Switzerland) then electroporated with the 4D-Nucleofector system (Lonza) using CM149 or DZ100 electroporation protocols. Cells were rested for 5-10 min post- electroporation, then plated at a concentration of 1 × 106cells / mL. rAAV6 was added at 1.5% of the final culture volume (unless otherwise noted) and multiplicity of infection [genome copies (GC) / cell] was determined based on viral titer. AZD7648 in DMSO (Selleck Chemicals, Houston, TX, USA) was diluted in SFEMII prior to addition to rAAV6-containing media at concentration of 0.3 μM unless otherwise noted. Twenty-four hours after editing, AAV or AZD7648-containing media was removed and replaced with fresh media and analyzed by flow cytometry to assess viability, %GFP+, and HSPC phenotype using the following antibodies: CD34, CD38, CD90, CD133. Viability was determined using BD Via-Probe Red Nucleic Acid Stain (BD Sciences). Otherwise, viability was determined by FSC vs. SSC. Five days after editing, cells were analyzed by flow cytometry to assess viability and %GFPhighto determine HDR. CFU assays

[0181] Twenty-four hours after editing, cells were counted and processed for CFU assays. Viability and cell number were determined on the CytoflexS (Beckman Coulter) with DAPI used as a viability dye and the recording set to volume to determine cells / mL. Thereafter, the viable cells / mL concentration was used to plate an equal number of cells per condition to establish the impact of each individual gene editing component and the combinations thereof on colony formation.

[0182] Viable cells from each condition were seeded at 250 cells per well in 6-well SmartDish™ (StemCell Technologies) in MethoCult™ H4034 Optimum (StemCell Technologies) according to the manufacturer’s instructions and incubated at 37°C for 14 days. Plates were imaged using a STEMVision™ (StemCell Technologies) instrument and dark and brightfield images were processed for CFU determination using the STEMVision™ Analyser software using the 14-day EPO huPB program. Colony assignments (BFU-E, CFU-G / GM / GM and CFU-GEMM) by the software were further interrogated by eye using the ColonyMarker software (StemCell Technologies). Colony counts are depicted as stacked histograms, with each stack representing the different colony assignments. The cumulative stack represents the total colony count per condition for each well evaluated. To determine impact on colony type, each colony type was normalized to total colony counts and represented as a percentage of the total for each treatment group.

[0183] GFP-expressing colonies were acquired using a Cytation 5 imager (Biotek, Agilent Technologies, Santa Clara, CA, USA). Images were processed using the Gen 5 Secure Image Prime software (BioTek, Agilent Technologies). The following settings were used for image acquisition: Temperature set to 37°C; brightfield conditions set to Illumination: 10, Integration time: 5 and Gain: 3.3 and GFP conditions set to Illumination: 10, Integration time: 5 and Gain: 22.8. Images were acquired in montage mode with 11 X 11 (columns X rows) images to cover the entire 6-well. Tile overlap was set to auto for stitching. After acquisition, images were processed for stitching using the linear blend method with a reduction of the total image size to 20% of the original image. GFP+ colonies were counted using ImageJ multipoint tool (ImageJ 1.53t, National Institutes of Health, USA). The %GFP+ was calculated as the numbers of GFP+ colonies divided by total colonies (as assessed on the STEMVision) × 100. Xenotransplantation

[0184] NBSGW and NSG-SGM3 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) and maintained in a pathogen-free facility at Seattle Children’s Research Institute. All animal studies were approved by Seattle Children’s Research Institute’s Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For primary transplants in NBSGW mice, 6-10-week-old female mice were treated with 12. 5mg / kg busulfan 24 h prior to cell transplant. Twenty-four hours after editing, 1.5-2 × 106cells were delivered retro-orbitally (equal doses across treatment groups). Nonmanipulated cells were transferred as control. Peripheral blood was collected 10-, 12-, 14-, and 16-weeks post- transplant and analyzed by flow cytometry. After 16 weeks post-transplant, the mice were euthanized. Leukocytes collected from the BM and spleen were used for immunophenotyping and to extract gDNA for molecular analyses. To assess engraftment and differentiation, cells were analyzed by flow cytometry after labeling with antibodies against human antigens CD45, CD33 and CD19, as well as murine CD45 (all antibodies recognize the human antigen unless specified). LT-HSCs were phenotypically detected by staining with CD34, CD38, and CD90 antibodies.

[0185] For primary transplant into the NSG-SGM3 strain, 8-10 week-old female mice were treated with 25 mg / kg Busulfan 24 h prior to cell transplant.24 h after editing, 2 × 106cells were delivered retro-orbitally. Mice were euthanized at 14-weeks post-transplant and assessed as above; splenocytes were also stimulated in culture with PMA and ionomycin (below).

[0186] In some cases, BM cells from the NBSGW primary transplanted mice were serially passaged into NSG-SGM3 mice (secondary transplant recipients). Here, 6-8 week-old female NSG-SGM3 mice were treated with 25 mg / kg Busulfan 24 h prior to cell transplant. On the day of secondary transplant, BM from NBSGW primary xenotransplant recipients was extracted and pooled within treatment groups. 2 × 106BM cells were transplanted retro- orbitally to recipient NSG-SGM3 mice. After 12-14 weeks, BM was harvested and immunophenotyping performed to assess the presence of the xenograft and HDR edited cells. ATO cultures

[0187] Artificial thymic organoid (ATO) cultures were performed following the protocol described by Montel-Hagen et al.43Cultures were initiated with 5000 CD34+ HSPCs isolated with a CD34 microbead kit (Miltenyi Biotec, Germany) from pooled BM of recipient NBSGW mice. Input cells were analyzed by FACS to confirm purity and ddPCR was performed on gDNA extracted to quantify input HDR rate. Cultures were maintained for 6-8 weeks. After 6-8 weeks, cultures were harvested for gDNA extraction and ddPCR analysis toquantify HDR or FACS analysis to assess differentiation using the following antibodies:hCD45, CD34, CD14, CD56, CD19, CD1a, CD7, CD3, TCR ß, CD4, and CD8.PMA Ionomycin stimulation of NSG-SGM3 splenocytes

[0188] Splenocytes isolated from primary-transplanted NSG-SGM3 mice were cultured at 1-2 × 106cells / mL and stimulated with 50 ng / mL PMA and 1 μg / mL ionomycin (Sigma Aldrich, St Louis, MO, USA). After stimulation, cells were washed in PBS, then stained with the following antibodies: CD45, CD33, CD19, CD4, CD8, CD154 (CD40L) and Live / Dead-Near IR (ThermoFisher Scientific). Quantification of HDR by ddPCR

[0189] Genomic DNA was isolated from in vitro cultured HSPCs or BM and splenic leukocytes using DNeasy Blood and Tissue kit (Qiagen, Germany). To quantify HDR editing, “in-out” ddPCR was performed with the forward primer binding within the knock-in cassette and reverse primer binding outside the region of homology. A control amplicon of similar size (1.3 kB) was generated for the ActB gene. Probes for both amplicons were labeled with FAM and amplified in separate ddPCR reactions in duplicate. The PCR reaction included 900 nM each primer (IDT), 250 nM probe (IDT), 50 ng gDNA, and ddPCR Supermix for Probes without dUTP (Bio-Rad). Droplets were created using a QX200 Droplet Generator (Bio-Rad). Droplets were analyzed using the QX200 Droplet Digital PCR System (Bio-Rad) and analyzed using Quantasoft (Bio-Rad). As CD40LG is on the X chromosome and all CD34 donors were male, editing rates were calculated as a ratio of copies / μL from CD40LG / ActB multiplied by two. Sequencing analysis of B2M locus

[0190] Genomic DNA (gDNA) was extracted from CD34+HSPCs 48 hrs after editing using QuickExtract DNA Extraction Solution (Biosearch Technologies UK). The concentration of the resulting gDNA sample was measured using the Qubit 3.0 Fluorometer (Invitrogen, MA, USA). PCR amplification of the edited region was performed using primers flanking the B2M target region. After amplification, the PCR product was visualized by electrophoresis on the E-Gel Power Snap System (Invitrogen) in accordance with themanufacturer's instructions to confirm the presence of the expected ~ 500 bp bands. gDNA samples with detectable bands were subjected to Sanger sequencing (Genewiz, Azenta Life Sciences). Sequencing was performed using the instructions for "Difficult Templates" (Genewiz, Azenta, Life Sciences). Deconvolution of the trace data was analyzed for InDel frequency and or insertional frequency using inference of CRISPR edits, or ICE analysis (Synthego). Editing efficiency rates (i.e., percentage of the pool with non-wild type sequence) were determined by comparing the edited trace to a control (B2M) trace. Indels were counted and graphed as WT (0), NHEJ (+ / -1, + / -2) or MMEJ (-3 ; 3). Analysis of indels and integration by ICE was also used to infer the dominant DNA repair pathway. HDR was inferred from template integration, MMEJ dominance inferred from large deletions (i.e., 3 bp deletion) and NHEJ dominance inferred from small indels (i.e., 3 bp indel). Sequencing analysis of CD40L locus

[0191] Genomic DNA (gDNA) was extracted from in vitro cultured CD34+HSPCs 5 days after HDR editing using DNeasy Blood and Tissue kit (Qiagen, Germany). PCR amplification of the edited region was performed using primers flanking the CD40L target region. After amplification, PCR products were visualized by electrophoresis by Gel Doc XR+ (Bio-Rad) in accordance with the manufacturer's instructions to confirm the presence of the expected ~300 bp (non-HDR repair) and ~ 1.8kB (HDR) bands. The ~ 300bp band was extracted with QIAquick Gel Extraction kit (Qiagen, Germany) and subjected to Sanger sequencing (Genewiz, Azenta Life Sciences). Deconvolution of the trace data was analyzed for InDel frequency using inference of CRISPR edits (ICE) analysis (Synthego). Indels were counted and graphed as WT (0), NHEJ (+ / -1, + / -2) or MMEJ (-3 ; 3). Flow cytometry

[0192] Flow cytometry analysis was done with an LSR II (BD Biosciences) or MACSquant Analyzer (Miltenyi Biotec) and analyzed with FlowJo V.10.8.1 (BD Biosciences).Statistical Analysis

[0193] GraphPad Prism V 9.1.1 (GraphPad Software, CA, USA) was used to plot graphs and for statistical analyses. For HDR, NHEJ and AAV dose curves, a 4-parameter logistic (4PL) non-linear regression model was used to determine the IC50 (NHEJ) and EC50 (HDR and AAV).

[0194] Relationships between treatment (Untreated, AAV, AAV+RNP, AAV+RNP+iDNA-PKcs) and experimental readouts were assessed using a linear regression model in the following Figures: FIG.2B, FIGs. 2D-2G, FIG. 3D, FIG. 3E; FIG. 4B, FIG. 4D, FIG. 4E; FIGs. 5D-5I. Readouts (continuous variables) were considered as dependent variables, whereas treatment was considered as an independent variable (categorical variable). Since the results are clustered by donor and study, the model used incorporated a random intercept to account for variations in baseline level of the readouts between the different clusters formed by donors and studies (i.e., all donor and study combinations). Additionally, a robust estimate of the variance was incorporated to account for potential between-treatment heteroscedasticity. For FIG. 2I and FIG. 4F, a Fixed intercept model was used because it was not possible to fit random intercept models for the corresponding readouts. The cell type (BFU- E, CFU-G / M / GM or CFU-GEMM) was added to the model as a fixed effect in interaction with the treatment effect. Example 2—Preparation and analysis of CD40L vectors CD40L expression and kinetics in CD4+ T cells: Comparing donor templates containing WPRE vs WPRE3

[0195] Previous studies in T cells demonstrated that cells edited with CD40LG [GFP.CD40L.WPRE] (#1368 in FIG. 14) demonstrated a consistently higher baseline percentage of CD40L+ and CD40L MFI compared to a CD40LG [GFP.CD40L.3’UTR] and nonedited healthy donors. Due to the relative overexpression of CD40L on the cell surface using the WPRE construct, compared with endogenous CD40L expression in healthy control, an optimized therapeutic donor template that would result in CD40L expression and kinetics comparable to endogenous CD40L expression was developed. Two new donor templates #3359 and #3367 were generated and compared to #1368, in CD4 T cells (AAV donor template schematics shown in FIG. 14). Both new CD40L cDNA constructs (#3367 and 3359) utilizeda truncated version of WPRE, WPRE3, containing only the and elements of WPRE and aSV40 poly A instead of Syn.pA. In addition, #3367 also comprised optimized 5 homologyarm which lacked mutations designed to eliminate TALEN binding, thus increasinghomologous stretch within the 5 homology arm.

[0196] FIG. 14 depicts schematic representations of AAV donor templates. The vectors included expression cassettes for an eGFP reporter gene linked to a CD40L cDNA (GFP.CD40L), a CD40L cDNA, or a GFP. Vector #1368 - CD40LG[GFP.CD40L.WPRE])comprised a 1 kb 5 homology arm (HA), an eGFP reporter linked to a codon diverged CD40LCDS, a WPRE element, a synthetic poly A (Syn.pA) and a 3 HA. Vector #3359 - CD40LG[GFP.T2A.CD40L.cDNA.WPRE3] comprised a 1 kb 5 HA, an eGFP reporter linked to acodon diverged CD40L CDS, a truncated WPRE element (WPRE3), a SV40 poly A (SV40)and a 3 HA. Vector #3367 - CD40LG [CD40L.cDNA.WPRE3.SV40 PolyA] comprised anoptimized 5 HA, a codon diverged CD40L CDS, a WPRE3, a SV40 poly A and a 3 HA.Vector #3377 – CD40LG[MND.GFP.WPRE.syn.pA] comprised an optimized 5 HA, an MNDpromoter, an eGFP, a WPRE element, a Syn.pA and a 3 HA. Vector #1416 - CD40LG[CD40L.cDNA.WPRE3] comprised a 1 kb 5 HA, a codon optimized CD40L CDS, WPRE3,SV40 poly A, and a 3 HA. Aspects of the expression vectors are summarized in TABLE 4.TABLE 2 lists nucleotide sequences for # 3367. TABLE 4 Construct: 1368 3367 3359 3377 1416 donor CD40LG[GF CD40LG[C CD40LG[GF 40L.W D40L.c MND.GFP. CD40LG[C template P.CD DNA P.T2A.CD40 WPRE.syn.p D40L.cDNA cassette: PRE] .WPRE3.SV L.cDNA.WP 40PolyA] RE3] A .WPRE3] 1 kb - 1 kb - optimized optimized 5' HA: 1 kb (guide 1 kb (guide 1 kb specific specific mutation) mutation) 3' HA: 1 kb 1 kb 1 kb 1 kb 1 kb CD40L codon codon codon d divergeddivergedNcodon CDS: divergeAdiverged WPRE: WPRE WPRE3 WPRE3 WPRE WPRE3 GFP: GFP NA GFP MND.GFP NAConstruct: 1368 3367 3359 3377 1416 poly A: Syn.pA SV40 SV40 Syn.pA SV40 Comparison of donor templates with WPRE3 vs WPRE

[0197] To evaluate the CD40L expression and kinetics in T cells, CD4+ T cells were edited using the new donors / vectors as detailed in FIG.15. GFP expression was assessed 3 days post editing and HDR at 14 days post editing (FIG. 16A) with average editing rates (percentage of GFP) of 39%, 32% and 35% with #1368, #3359 and #3377 (control MND.GFP template) respectively and HDR ranging from 44.1% to 55.8% via ddPCR. The expression of CD40L on the cell surface was characterized following activation with PMA and ionomycin (P / I) (FIG.16B, FIG.16C). Donor template #1368 (WPRE) editing resulted in more sustained CD40L expression with a higher MFI compared to #3367 and 3359 (both containing WPRE3) which performed identically to the expression pattern of endogenous CD40L (mock edited cells). Comparing CD40L expression of GFP+ vs GFP- populations demonstrated similar CD40L MFI for #3359 edited T cells (CD40L.WPRE3). In contrast in #1368 edited T cells (CD40L.WPRE), GFP+ (edited cells) exhibited an increased MFI relative to the GFP- unedited population. Independent CD40L-editing study using a second healthy donor confirmed that donor templates containing WPRE3 had CD40L kinetics similar to endogenous CD40L expression

[0198] To confirm CD40L expression levels after editing with WPRE3 expression templates, the study was repeated with #3367 and #3359 donor templates in a different healthy donor resulting in comparable results (FIGs.17A-17E). Combined, this data demonstrated that CD40L surface expression and kinetics using #3367 and #3359, containing the WPRE3, closely resembled the expression pattern and kinetics of endogenous CD40L in multiple donors. Compared to #1368 which contained the full length WPRE element, these data indicated that WPRE3 may be superior in this setting to optimally mimic endogenous CD40L expression.Long-term engraftment and T-lineage differentiation of CD40L cDNA-edited HSPCs

[0199] Moving toward a therapeutic platform for XHIM, a rAAV6 donor template was generated consisting of a codon diverged CD40L coding sequence (CDS) and WPRE3 followed by SV40 poly A elements (FIG. 14: donor template #3367, and FIG. 18A). This donor template had several key features that differed from a prior cCD40L donor template(#1368), including a truncated WPRE (WPRE3), an optimized 5 homology arm, SV40 polyAinstead of Syn.pA and no GFP.

[0200] Following HDR, expression of the codon diverged CD40L CDS was designed to be driven by the endogenous CD40LG promoter, with the goal of achieving physiological regulation. Using this “cDNA” donor template, in vitro conditions for co- delivery of RNP and rAAV6 donor in mPB CD34+ HSPCs were identified that preserved viability (FIG.18B) while maximizing HDR efficiency (FIG. 18C).

[0201] To determine the long-term engraftment capacity of mobilized peripheral blood (mPB) CD34+ HSPCs edited with this therapeutic donor, xenotransplantation studies were performed in NBSGW recipient mice as detailed in FIG. 18D and incorporated HDR editing with or without iDNA-PKcs treatment. Similar levels of human cell engraftment within the peripheral blood were observed in cDNA-edited vs. cDNA-edited + iDNA-PKcs treated recipients (FIG. 18E). Following sacrifice at 16 weeks, HDR levels in engrafted human cells was assessed using ddPCR. In comparison with cDNA-editing alone, treatment with iDNA- PKcs resulted in a significantly higher average levels of long-term engrafted HDR edited cells in both BM and spleen (BM AAV+RNP: 7.7%; BM AAV+RNP+iDNA-PKcs: 11.9%; SP AAV+RNP: 11.5%; SP AAV+RNP+iDNA-PKcs: 16.2%; FIG. 18F). For all CD34 donors evaluated, similar human cell engraftment and lineage distribution in BM (FIG.18G) and spleen (FIG. 18H) were observed. Importantly, the mean % HDR in BM for cDNA-edited + iDNA-PKcs treated recipients exceeded the hypothesized threshold of ~10% HDR required for therapeutic benefit, assuming efficient transgene expression and function.

[0202] A constraint of the adult NBSGW humanized model is lack of thymic differentiation of human T cells following transplant of mPB CD34 HSPCs. Therefore, as an alternative means to assess T cells generated from HDR edited HPSCs, an artificial thymic organoid (ATO) system was utilized to assess T-lineage differentiation of BM CD34+ HSPCs isolated from long-term engrafted, NBSGW recipient mice. Human CD34+ cells wereenriched from BM pools from recipient mice (2 independent transplant studies experiments) and used to initiate ATO cultures. Following 6-8 weeks in culture, T cell differentiation was characterized by immunophenotyping (FIGs. 18I-18K). T lineage cells across a range of differentiation stages, including CD5+CD7+ pre-T1 cells, DP, and SP T cells, were present in cultures initiated using both untreated and CD40L cDNA-edited HSPCs (FIG. 18I, upper vslower panels). Mature TCR + cells and overall cellularity were also similar in both groups(FIG. 18I, FIG.18J). To determine whether HDR edited HSPCs contributed to the generation of T lineage cells, %HDR was quantified by ddPCR in ATOs (after 6-8 weeks) and compared to % HDR of input CD34+ HSPCs isolated from long-term engrafted, cDNA-edited, recipient animal BM (FIG.18K). While variable across the studies, the proportion of HDR edited T cells was similar to the proportion of edited cells within the input HSPC population. These findings indicated that long-term engrafted HSPCs recovered from xenotransplantation studies retained T-lineage differentiation capacity and that HDR editing at the CD40LG locus did not impair their differentiation capability. T cells derived in vivo from CD40LG-edited HSPCs exhibit regulated CD40L expression

[0203] Characterization of CD40L cDNA expression is useful to infer the functionality and safety of CD4 T cells expressing a candidate therapeutic HDR cassette. Moving toward a HSPC editing therapy and to allow for direct tracking of HDR edited, CD40L expressing T cells, an alternative AAV donor template was generated containing a GFP.T2A.CD40L cDNA expression cassette (FIG. 14: #3359, and FIG.19A). This cis-linked GFP donor design was utilized to permit flow-based tracking of naïve T cells derived from HDR-edited HPSCs in vivo. mPB CD34+ HSPCs were edited in association with iDNA-PKcs treatment using this new donor as detailed in FIG. 18D. In comparison with the NSG or NBSGW strains, the NSG-SGM3 mouse strain has previously been reported to support more efficient T cell differentiation from engrafted human HPSCs. Therefore, to facilitate generation of T cells in vivo from HDR-edited adult mPB HPSCs, transplant studies using this strain was performed. Busulfan conditioned, adult NSG-SGM3 recipient mice were transplanted with unedited or edited HPSCs and recipient mice were evaluated at 16 weeks post-transplant. Similar levels of human chimerism were observed in recipients transplanted with unedited vs. edited HSPCs (FIG. 19B, FIG. 19C). As expected, based on previous studies, overall humancell chimerism was lower in this strain (FIG.19B, FIG.19C) compared to NBSGW recipients shown in FIG.18G and FIG.18H. Variable proportions of CD3+ T cells were present in both the BM and spleen across both cohorts. Notably, following long-term engraftment, HDR rates in both BM (44.3%) and spleen (52.3%) were similar to the HDR level of input HSPCs (63.4%) (FIG. 19C) demonstrating sustained engraftment of CD40L HDR edited, iDNA-PKcs treated, adult mPB HPSCs in this alternative model.

[0204] Consistent with intracellular, endogenously regulated, CD40L expression that occurs in naïve human T cells, flow cytometry of a CD40L cDNA.GFP + iDNA-PKcs treated recipient revealed T cell-specific, baseline cis-linked GFP expression driven off the integrated HDR cassette (FIG. 19B). Next, to determine whether HDR-edited T cells exhibit activation-dependent surface CD40L expression, a modified activation protocol was used (see Hubbard et al, 2016). Transgenic CD40L cDNA expression kinetics were characterized by stimulating with PMA and ionomycin to activate surface CD40L expression and tracked expression based on co-expression of GFP and CD154 surface staining. As T cell numbers were limited, total spleen cells were activated and stained for surface CD40L expression. Activation led to a CD40L+GFP+ double positive population. Comparing CD40L expression in GFP+ vs. GFP- populations revealed a similar level of CD40L surface expression (based upon CD154 MFI) and comparable expression kinetics consistent with endogenous CD40L expression in response to T cell activation (FIG. 19D, FIG. 19E). Together, these data demonstrated sustained engraftment of CD40L HDR edited mPB HPSCs, differentiation of edited HPSC into naïve T cells in vivo, and endogenously regulated CD40L surface expression following ex vivo activation of T cells derived from HDR edited mPB HPSCs.

[0205] The following references are each incorporated by reference herein in its entirety. References 1. Koguchi, Y., et al. (2007). Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner. Blood 110, 2520-2527. 2. van Kooten, C., and Banchereau, J. (2000). CD40-CD40 ligand. J Leukoc Biol 67, 2-17. 3. Elgueta, R., et al. (2009). Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunol Rev 229, 152-172.4. Notarangelo, L.D., and Hayward, A.R. (2000). X-linked immunodeficiency with hyper-IgM (XHIM). Clin Exp Immunol 120, 399-405. 5. de la Morena, M.T. et al. (2017). Long-term outcomes of 176 patients with X- linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation. J Allergy Clin Immunol 139, 1282-1292. 6. Brown, M.P., et al. (1998). Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice. Nat Med 4, 1253-1260. 7. Prasad, M.L., et al. (2005). Mutational screening of the CD40 ligand (CD40L) gene in patients with X linked hyper-IgM syndrome (XHIM) and determination of carrier status in female relatives. J Clin Pathol 58, 90-92. 8. DeWitt, M.A., et al. (2016). Selection-free genome editing of the sickle mutation in human adult hematopoietic stem / progenitor cells. Sci Transl Med 8, 360ra134. 9. McAuley, G.E., et al. (2023). Human T cell generation is restored in CD3delta severe combined immunodeficiency through adenine base editing. Cell 186, 1398-1416 e1323. 10. Anzalone, A.V., et al. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157. 11. Hubbard, N., et al. (2016). Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome. Blood 127, 2513-2522. 12. Kuo, C.Y., et al. (2018). Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome. Cell Rep 23, 2606-2616. 13. Vavassori, V., et al. (2021). Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome. EMBO Mol Med 13, e13545. 14. Hollenbaugh, D., et al. (1994). The random inactivation of the X chromosome carrying the defective gene responsible for X-linked hyper IgM syndrome (X-HIM) in female carriers of HIGM1. J Clin Invest 94, 616-622. 15. Hoban, M.D., et al. (2015). Correction of the sickle cell disease mutation in human hematopoietic stem / progenitor cells. Blood 125, 2597-2604. 16. De Ravin, S.S., et al. (2016). Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat Biotechnol 34, 424-429. 17. Dever, D.P., et al. (2016). CRISPR / Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature 539, 384-389. 18. De Ravin, S.S., et al. (2017). CRISPR-Cas9 gene repair of hematopoietic stem cells from patients with X-linked chronic granulomatous disease. Sci Transl Med 9, eaah3480. 19. Lee, B.C., et al. (2021). Understanding and overcoming adverse consequences of genome editing on hematopoietic stem and progenitor cells. Mol Ther 29, 3205-3218. 20. Allen, D., et al. (2023). Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders. Pharmaceutics 15, 1329. 21. Hess, N.J., et al (2020). Different Human Immune Lineage Compositions Are Generated in Non-Conditioned NBSGW Mice Depending on HSPC Source. Front Immunol 11, 573406.22. Hustedt, N., and Durocher, D. (2016). The control of DNA repair by the cell cycle. Nat Cell Biol 19, 1-9. 23. Nambiar, T.S., et al. (2022). CRISPR-based genome editing through the lens of DNA repair. Mol Cell 82, 348-388. 24. Shin, J.J., et al et al. (2020). Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells. Cell Rep 32, 108093. 25. Wu, Q., et al. (2019). Understanding the structure and role of DNA-PK in NHEJ: How X-ray diffraction and cryo-EM contribute in complementary ways. Prog Biophys Mol Biol 147, 26-32. 26. Robert, F., et al. (2015). Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing. Genome Med 7, 93. 27. Riesenberg, S., and Maricic, T. (2018). Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat Commun 9, 2164. 28. Fok, J.H.L., et al. (2019). AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity. Nat Commun 10, 5065. 29. Selvaraj, S., et al. (2023). High-efficiency transgene integration by homology- directed repair in human primary cells using DNA-PKcs inhibition. Nat Biotechnol 42, 731- 30. Wimberger, et al. (2023). Simultaneous inhibition of DNA-PK and Polimproves integration efficiency and precision of genome editing. Nat Commun 14, 4761. 31. Cloarec-Ung, et al. (2023). Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells. bioRxiv, 2023.2005.2026.542436. 32. Boitano, A.E., et al. (2010). Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science 329, 1345-1348. 33. Fares, I., et al. (2014). Cord blood expansion. Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal. Science 345, 1509-1512. 34. Turchiano, G., et al. (2021). Quantitative evaluation of chromosomal rearrangements in gene-edited human stem cells by CAST-Seq. Cell Stem Cell 28, 1136-1147 e1135. 35. Tatiossian, K.J., et al. (2021). Rational Selection of CRISPR-Cas9 Guide RNAs for Homology-Directed Genome Editing. Mol Ther 29, 1057-1069. 36. Modarai, S.R., et al. (2021). Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples. Gene Ther 28, 105-113. 37. Lessard, S., et al. (2017). Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci. Proc Natl Acad Sci U S A 114, E11257-E11266. 38. Conant, D., et al. (2022). Inference of CRISPR Edits from Sanger Trace Data. CRISPR J 5, 123-130. 39. Zonari, E., et al. (2017). Efficient Ex Vivo Engineering and Expansion of Highly Purified Human Hematopoietic Stem and Progenitor Cell Populations for Gene Therapy. Stem Cell Reports 8, 977-990.40. Radtke, S., et al. (2017). A distinct hematopoietic stem cell population for rapid multilineage engraftment in nonhuman primates. Sci Transl Med 9, eaan1145. 41. McIntosh, B.E., et al. (2015). Nonirradiated NOD,B6.SCID Il2rgamma- / - Kit(W41 / W41) (NBSGW) mice support multilineage engraftment of human hematopoietic cells. Stem Cell Reports 4, 171-180. 42. Wunderlich, M., et al. (2018). Improved multilineage human hematopoietic reconstitution and function in NSGS mice. PLoS One 13, e0209034. 43. Montel-Hagen, A., et al. (2022). Generation of Artificial Thymic Organoids from Human and Murine Hematopoietic Stem and Progenitor Cells. Curr Protoc 2, e403. 44. Koniali, L., et al. (2021). Therapy Development by Genome Editing of Hematopoietic Stem Cells. Cells 10, 1492. 45. Schiroli, et al. (2019). Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response. Cell Stem Cell 24, 551- 565 e558. 46. Ferrari, S., et al. (2020). Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking. Nat Biotechnol 38, 1298-1308. 47. Dorset, S.R., and Bak, R.O. (2023). The p53 challenge of hematopoietic stem cell gene editing. Mol Ther Methods Clin Dev 30, 83-89. 48. Maganti, H.B., et al. (2021). Persistence of CRISPR / Cas9 gene edited hematopoietic stem cells following transplantation: A systematic review and meta-analysis of preclinical studies. Stem Cells Transl Med 10, 996-1007. 49. Jayavaradhan, R., et al. (2019). A Versatile Tool for the Quantification of CRISPR / Cas9-Induced Genome Editing Events in Human Hematopoietic Cell Lines and Hematopoietic Stem / Progenitor Cells. J Mol Biol 431, 102-110. 50. Brault, J., et al. (2021). CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes. Blood 138, 2768-2780. 51. Sweeney, C.L., et al. (2021). Correction of X-CGD patient HSPCs by targeted CYBB cDNA insertion using CRISPR / Cas9 with 53BP1 inhibition for enhanced homology- directed repair. Gene Ther 28, 373-390. 52. Canny, M.D., et al. (2018). Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol 36, 95- 102. 53. Schimmel, J., et al. (2023). Modulating mutational outcomes and improving precise gene editing at CRISPR-Cas9-induced breaks by chemical inhibition of end-joining pathways. Cell Rep 42, 112019. 54. Ferrari, S., et al. (2022). Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell 29, 1428- 1444 e1429. 55. Burleigh, K., et al.. (2020). Human DNA-PK activates a STING-independent DNA sensing pathway. Sci Immunol 5, eaba4219. 56. Kath, J., et al. (2022). Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells. Mol Ther Methods Clin Dev 25, 311-330.57. Shy, B.R., et al. (2023). High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol 41, 521-531. 58. Goodwin, M., et al. (2020). CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells. Sci Adv 6, eaaz0571. 59. Brault, J., et al. (2022). CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells. Front Immunol 13, 1067417. 60. Romano Ibarra, G.S., et al. (2016). Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance. Mol Ther Nucleic Acids 5, e352. 61. Sather, B.D., et al. (2015). Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template. Sci Transl Med 7, 307ra156. 62. Pattabhi, S., et al. (2019). In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods. Mol Ther Nucleic Acids 17, 277-288. 63. Rhiel, M., et al. (2023). T-CAST: An optimized CAST-Seq pipeline for TALEN confirms superior safety and efficacy of obligate-heterodimeric scaffolds. Front Genome Ed 5, 1130736.

[0206] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

[0207] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0208] The above description discloses several methods and materials of the present invention. This invention is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the invention disclosed herein. Consequently, it is not intended that this invention be limited to the specific embodiments disclosed herein, but that it cover all modifications and alternatives coming within the true scope and spirit of the invention.

[0209] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extentpublications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide for modifying a human CD40LG gene in a cell genome,comprising, in a 5 to 3 order:a 5 homology arm;a CD40L coding sequence, optionally wherein the encoded CD40L polypeptide binds CD40; a polyadenylation signal; and a3 homology arm;optionally, further comprising a woodchuck hepatitis virus posttranscriptional regulatory element 3 (WPRE3) located between the CD40L coding sequence and the polyadenylation signal.

2. The polynucleotide of claim 1, wherein the CD40L coding sequence comprises a cDNA.

3. The polynucleotide of claim 1 or 2, wherein the CD40L coding sequence comprises a codon-diverged cDNA.

4. The polynucleotide of any one of claims 1-3, wherein the CD40L coding sequence comprises a nucleotide sequence: (i) having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:19; (ii) encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; or (iii) encoding an amino acid sequence set forth in SEQ ID NO:24 having 0-26 conservative substitutions.

5. The polynucleotide of any one of claims 1-4, wherein the WPRE3 comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:

20.

6. The polynucleotide of any one of claims 1-5, wherein the 5 homology armlacks a nucleotide sequence capable of specifically hybridizing to the nucleotide sequence set forth in SEQ ID NO:23.

7. The polynucleotide of any one of claims 1-6, wherein the 5 homology armcomprises or consists of sequences 5 of the first coding exon of the CD40LG gene, and / or ofsequences 5 of protein coding sequences of the first coding exon of the CD40LG gene.

8. The polynucleotide of any one of claims 1-7, wherein the 5 homology armcomprises or consists of sequences 5 of the first coding exon of the CD40LG gene and aportion of the first coding exon, and / or of sequences 5 of protein coding sequences of the firstcoding exon of the CD40LG gene and a portion of the first coding exon.

9. The polynucleotide of any one of claims 1-8, wherein the 5 homology armcomprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:

18.

10. The polynucleotide of any one of claims 1-9, wherein the 5 homology armcomprises a CD40LG promoter or portion thereof.

11. The polynucleotide of any one of claims 1-10, wherein the 5 homology armhas a length in a range from about 500 nucleotides to about 1500 nucleotides; optionally, in a range from about 750 nucleotides to about 1250 nucleotides.

12. The polynucleotide of any one of claims 1-11, wherein the 3 homology armcomprises or consists of a wild-type CD40L genomic sequence.

13. The polynucleotide of any one of claims 1-12, wherein the 3 homology armcomprises sequences 3 of the first coding exon of the CD40LG gene.

14. The polynucleotide of any one of claims 1-13, wherein the 3 homology armcomprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:

22.

15. The polynucleotide of any one of claims 1-14, wherein the 3 homology armhas a length in a range from about 500 nucleotides to about 1500 nucleotides; optionally, in a range from about 750 nucleotides to about 1250 nucleotides.

16. The polynucleotide of any one of claims 1-15, wherein the polyadenylation signal comprises an SV40 polyA signal.

17. The polynucleotide of any one of claims 1-16, wherein the polyadenylation signal comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:21.

18. The polynucleotide of any one of claims 1-17, wherein the polynucleotide is single-stranded.

19. The polynucleotide of any one of claims 1-18, wherein the polynucleotide is double-stranded.

20. A vector comprising the polynucleotide of any one of claims 1-19.

21. The vector of claim 20, wherein the vector is an adeno- associated virus (AAV) vector.

22. The vector of claim 21, wherein the AAV vector is an AAV6 vector 23. A method of genetically modifying a CD40LG gene in a cell genome, comprising: introducing into a cell the polynucleotide of any one of claims 1-19 or the vector of any one of claims 20-22; optionally, wherein the cell is a T cell, a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), and / or a CD34+ cell; optionally, wherein the cell is a HSPC; optionally, wherein the cell is a CD34+ cell.

24. The method of claim 23, further comprising introducing into the cell a nuclease or nucleic acid encoding the nuclease, wherein the nuclease is capable of specifically cleaving a first coding exon of a CD40LG gene.

25. The method of claim 24, wherein the nuclease is a Cas nuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), or a meganuclease; optionally, wherein the nuclease is the Cas nuclease or derivative thereof such as a mutant Cas nuclease; optionally, wherein the nuclease is a Cas9 nuclease.

26. The method of any one of claims 23-25, further comprising introducing into the cell a guide RNA (gRNA).

27. The method of claim 26, wherein the gRNA comprises a sequence capable of specifically hybridizing to a nucleotide sequence of a first coding exon of the CD40LG gene.

28. The method of claim 26 or 27, wherein the gRNA comprises a sequence having at least 70%, 80%, 90%, 95% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:

23.

29. The method of any one of claims 23-28, further comprising contacting the cell with a DNA-dependent protein kinase (DNA-PK) inhibitor.

30. The method of claim 29, wherein the DNA-PK inhibitor is selected from AZD7648, NU7441, NU7026, KU-0060648, LY3023414, CC-115, VX-984 or M3814; optionally, wherein the DNA-PK inhibitor comprises AZD7648.

31. A cell prepared with the method of any one of claim 23-30.

32. A cell comprising the polynucleotide of any one of claim 1-19 or the vector of any one of claims 20-22.

33. The cell of claim 31 or 32, wherein the cell is a T cell, a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), and / or a CD34+ cell; optionally, wherein the cell is a HSPC; optionally, wherein the cell is a CD34+ cell.

34. The cell of any one of claims 31-33, wherein the cell comprises a single X chromosome; and / or is derived from a male subject.

35. A pharmaceutical composition comprising the cell of any one of claims 31-34 and a pharmaceutically acceptable excipient.

36. A system for modifying a human CD40LG gene in a cell genome, comprising: the polynucleotide of any one of claims 1-19; and a nuclease or nucleic acid encoding the nuclease, wherein the nuclease is capable of specifically cleaving a first coding exon of a CD40LG gene.

37. The system of claim 36, wherein the nuclease is a Cas nuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), or a meganuclease; optionally, wherein the nuclease is the Cas nuclease or derivative thereof such as a mutant Cas nuclease; optionally, wherein the nuclease is a Cas9 nuclease.

38. The system of claim 36 or 37, further comprising a guide RNA (gRNA).

39. The system of claim 38, wherein the gRNA comprises a sequence capable of specifically hybridizing to a nucleotide sequence of a first coding exon of the CD40LG gene.

40. The system of claim 38 or 39, wherein the gRNA comprises a sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO:

23.

41. The system of any one of claims 36-40, further comprising a cell.

42. The system of claim 41, wherein the cell is, a stem cell, a hematopoietic stem cell, a hematopoietic stem and progenitor cell (HSPC), a T cell, and / or a CD34+ cell; optionally, wherein the cell is a HSPC; optionally, wherein the cell is a CD34+ cell.

43. The system of any one of claims 36-42, further comprising a DNA-dependent protein kinase (DNA-PK) inhibitor.

44. The system of claim 43, wherein the DNA-PK inhibitor is selected from AZD7648, NU7441, NU7026, KU-0060648, LY3023414, CC-115, VX-984 or M3814; optionally, wherein the DNA-PK inhibitor comprises AZD7648.

45. A method of treating, ameliorating or inhibiting a disorder in a subject, comprising administering to the subject the cell of any one of claims 31-34.

46. The method of claim 45, wherein the cell is autologous to the subject.

47. The method of claim 45, wherein the cell is allogeneic to the subject.

48. The method of any one of claims 45-47, wherein the disorder comprises X- linked hyper IgM syndrome (XHIM).

49. The method of any one of claims 45-48, wherein the subject is male.

50. The method of any one of claims 45-49, wherein the subject is human.

51. The cell of any one of claims 31-34 for treating, ameliorating or inhibiting a disorder in a subject; optionally, wherein the disorder comprises X-linked hyper IgM syndrome (XHIM).

52. The cell of any one of claims 31-34 for use in the preparation of a medicament for treating, ameliorating or inhibiting a disorder in a subject; optionally, wherein the disorder comprises X-linked hyper IgM syndrome (XHIM).

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