Gene editing methods and materials for adult onset leukoencephalopathy

Abstract

Described herein are compositions and methods for highly efficient and specific base-editing to correct the most common mutation associated with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), CSF1R

Description

[0001] GENE EDITING METHODS AND MATERIALS FOR ADULT ONSET LEUKOENCEPHALOPATHY

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 546,914, filed on November 1, 2023. The entire contents of the foregoing are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named 29539-0806WOl_SL_ST26.xml. The XML file, created on October 31, 2024, is 31,890 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with Government support under Grant Nos. CA281401, HL142494, NS065743, TR002823, and NS134784 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] Described herein are compositions and methods for highly efficient and specific base-editing to correct the most common mutation associated with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), CSF1R c.2381 T>C, p.I794T.

[0010] BACKGROUND

[0011] Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rapidly progressive and invariably fatal neurodegenerative disorder for which there is currently no cure.1Clinical manifestations include progressive cognitive decline, neuropsychiatric dysfunction, motor and gait disturbances, and seizures, culminating rapidly in premature death within a lew years of symptom onset.2It is the most common adult-onset inherited white matter disease of the central nervous system (CNS), and approximately 10.000-25.000 patients in the United States alone carry this diagnosis.2 4 Patients with ALSP have autosomal dominant mutations in the colony stimulating factor 1 receptor (CSFJR) gene, which encodes a tyrosine kinase receptor expressed primarily in microglial cells of the CNS and myeloid lineage cells of the hematopoietic system.4Microglia are innate immune cells that have essential roles in CNS development and homeostasis. Aberrant microglial activation in ALSP and other neuro-inflammatory disorders triggers trafficking of hematopoietic myeloid cells to the CNS. where they differentiate into macrophage-derived microglia-like (MDMi) cells that can therapeutically modulate the neuro-inflammatory environment.

[0012] SUMMARY

[0013] Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is the most common adult-onset genetic neurodegenerative disorder caused by mutations in the human CSFJR gene. There are currently no approved therapies for ALSP, and the disease is rapidly and invariably fatal. Described herein are genome editing approaches to correct mutations in the CSF1R gene. The precise correction of CSFJR mutations via ex vivo or in vivo editing can provide a durable long-lasting treatment for ALSP patients.

[0014] Provided herein are guide RNAs (gRNAs) comprising a spacer sequence listed in any of Tables 3-5. Also provided herein are compositions comprising the gRNAs.

[0015] Additionally, provided herein are compositions comprising a recombinant SpCas9 cytosine base editor (BE) protein and a gRNA targeting CSF1R, preferably as a ribonucleoprotein complex, preferably wherein the gRNA comprises a sequence shown in any of Tables 3-5, optionally wherein:

[0016] (a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0017] (b) the gRNA targets the BE to CSF R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-LWRYEK, SpCas9-MQKSER, SpCas9-LWKFEG, SpCas9(MQKSER+LllllR), SpCas9(LWKFEG+A1285K+Ll 1 HR),

[0018] SpCas9(MQKSER+L 1111 R+ A 1322R+A61 R): or

[0019] (c) the gRNA targets the BE to CSFJR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations. In some embodiments, the recombinant SpCas9 cytosine base editor protein and gRNA targeting CSF1R are in a carrier, e.g., a lipid nanoparticle (LNP), liposome, exosome, or virus-like particle.

[0020] Further provided herein are compositions comprising a nucleic acid sequence encoding an SpCas9 cytosine base editor protein, and a gRNA targeting CSF1R, preferably wherein the gRNA comprises a sequence shown in any of Tables 3-5, optionally wherein:

[0021] (d) the gRNA targets the BE to CSF1R mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0022] (e) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG. SpCas9-LWRYEK, SpCas9-MQKSER, SpCas9-LWKFEG, SpCas9(MQKSER+Lll llR), SpCas9(LWKFEG+A1285K+Ll 111R), SpCas9(MQKSER+LllllR+A1322R+A61R); or

[0023] (f) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations.

[0024] Also provided herein are compositions comprising a nucleic acid sequence encoding an SpCas9 cytosine base editor protein and a nucleic acid sequence encoding a gRNA targeting CSF1R, preferably wherein the gRNA comprises a sequence shown in any of Tables 3-5, optionally wherein:

[0025] (g) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0026] (h) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-LWRYEK, SpCas9-MQKSER, SpCas9-LWKFEG, SpCas9(MQKSER+Lll llR), SpCas9(LWKFEG+A1285K+Ll 1 HR), SpCas9(MQKSER+LllllR+A1322R+A61R); or

[0027] (i) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations.

[0028] In some embodiments, the nucleic acid sequences encoding the base editor protein and / or gRNA comprise RNA, optionally mRNA, circRNA, or synthetic RNA; or are in an expression vector, e.g., a viral vector or a plasmid, and / or in a carrier, e.g., a lipid nanoparticle (LNP). liposome, exosome, or virus-like particle.

[0029] In some embodiments, the nucleic acid sequence encoding an SpCas9 cytosine base editor protein is an mRNA and the gRNA is a synthetic RNA.

[0030] In some embodiments, the compositions further comprise a carrier, e.g., a lipid nanoparticle (LNP), nanoparticle (NP), liposome, exosome, or virus-like particle.

[0031] Further provided herein are methods of editing a cell comprising a mutation in an allele of CSFJR, the method comprising contacting the cell with or expressing in the cell an SpCas9 cytosine base editor protein and a gRNA targeting CSFJR as described herien, preferably wherein the gRNA comprises a sequence shown in any of Tables 3-5, optionally wherein:

[0032] (j) the gRNA targets the BE to CSFJR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0033] (k) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG. SpCas9-LWRYEK, SpCas9-MQKSER, SpCas9-LWKFEG, SpCas9(MQKSER+Lll llR). SpCas9(LWKFEG+A1285K+Ll 111R), SpCas9(MQKSER+Llll lR+A1322R+A61R); or

[0034] (l) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations.

[0035] In some embodiments, the methods comprise contacting the cell with or expressing in the cell a composition as described herein.

[0036] In some embodiments, the methods comprise contacting the cell with mRNA encoding the SpCas9 cytosine base editor protein and a synthetic gRNA targeting CSFJR.

[0037] As used herein, “contacting a cell” includes introducing the compositions into the cell and / or into the nucleus of the cell using methods known in the art.

[0038] Further, provided herein are isolated cells from a subject who has a disease caused by a mutation in CSFJR, wherein the mutation has been corrected by contacting the cell with or expressing in the cell an SpCas9 cytosine base editor protein and a gRNA targeting CSFJR, preferably a gRNA that comprises a spacer sequence shown in any of Tables 3-5, optionally wherein: (m) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0039] (n) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-LWRYEK, SpCas9-MQKSER, SpCas9-LWKFEG, SpCas9(MQKSER+LllllR), SpCas9(LWKFEG+A1285K+Ll 111R),

[0040] SpCas9(MQKSER+Ll 111R+A1322R+A61R); or

[0041] (o) the gRNA targets the BE to CSF1R mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations..

[0042] In some embodiments, the isolated cell is a hematopoietic stem cell (HSC).

[0043] Additionally, provided herein are methods of treating a subject who has a disease caused by a mutation in CSF1R. The methods comprise administering to the subject a therapeutically effective amount of an isolated autologous cell, wherein the mutation in the cell has been corrected by contacting the cell with or expressing in the cell an SpCas9 cytosine base editor protein and a gRNA targeting CSFJR, preferably a gRNA comprising a spacer sequence shown in any of Tables 3-5, optionally wherein:

[0044] (p) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0045] (q) the gRNA targets the BE to CSF1R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-LWRYEK, SpCas9-MQKSER, SpCas9-LWKTEG, SpCas9(MQKSER+LllllR), SpCas9(LWKFEG+A1285K+Ll 111R),

[0046] SpCas9(MQKSER+L 1111 R+A 1322R+A61 R); or

[0047] (r) the gRNA targets the BE to CSF 1R mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations In some embodiments, the cell is a hematopoietic stem cell (HSC).

[0048] Also provided herein are methods of treating a subject who has a disease caused by a mutation in CSF1R comprising administering to the subj ect a therapeutically effective amount of a nucleic acid encoding an SpCas9 cytosine base editor protein and a gRNA targeting CSFJR. preferably a gRNA comprising a spacer sequence shown in any of Tables 3-5, optionally wherein:

[0049] (s) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or

[0050] (t) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG. SpCas9-LWRYEK, SpCas9-MQKSER. SpCas9-LWKFEG. SpCas9(MQKSER+Ll ll lR), SpCas9(LWKFEG+Al 285K+L111 1R), SpCas9(MQKSER+Llll lR+A1322R+A61R); or

[0051] (u) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations.

[0052] Additionally provided herein is the use of the compositions described herein for treating a subject who has a disease caused by a mutation in CSFJR, e.g., using a method described herein.

[0053] In some embodiments, the nucleic acid sequences encoding the base editor protein and / or gRNA are in a vector, e g., a viral vector or a plasmid, and / or in a carrier, e.g., a lipid nanoparticle (LNP), nanoparticle (NP), liposome, exosome, or virus-like particle.

[0054] In some embodiments, the nucleic acid sequence encoding the base editor protein is an mRNA or circRNA, and the method comprises administering the mRNA and gRNA in an LNP.

[0055] In some embodiments, the recombinant SpCas9 cytosine base editor protein and gRNA targeting CSFJR are administered in a carrier, e.g.. a lipid nanoparticle (LNP), liposome, exosome, or virus-like particle.

[0056] In some embodiments of the methods and compositions described herein, the cytosine base editor comprises a cytosine deaminase domain selected from the group consisting of cytosine deaminase domains from APOBEC1, APOBEC3A, APOBEC3G, AID / CDA1, BE3, BE4max, evoAPOBEC 1 -BE4max. Anc BE4max, FERNY-BE4max, evoFERNY-BE4max, CDAl-BE4max, evoCDAl-BE4max, or engineered TadA-based CBEs, optionally TadCBEs (optionally TadCBEd), TadDEs, CBE-Ts, CABE-Ts, Td-CBEs, Td-CGBEs, Sdd CBEs (optionally Sddl, Sdd,3, Sdd6, or Sdd7), or CBE6 (optionally CBE6b or CBE6a). In some embodiments of the methods and compositions described herein, the cytosine base editor comprises a fidelity -enhancing mutations selected from SpCas9- HF1 (N497A / R661 A / Q695A / Q926A), SpCas9-HiFi (R691A), eSpCas9(1.0) (K810A / K1003A / R1060A), and eSpCas9(l.l) (K848A / K1003A / R1060A).

[0057] Also provided herein are gRNAs listed in tables 3, 4, and 5, which are specific to CSF7A-I794T. and the use of C-to-T base editors (CBEs) with each of the gRNAs listed herein. The CBEs will be comprised of: a. Cas9 enzymes: i. SpCas9 (wild-type) and various engineered forms that read new PAMs, including, but not limited to: SpRY, SpG, SpCas9-VRER, SpCas9-VRQR, SpCas9- D1135E, SpCas9-MQKSER, SpCas9-LWKFEG, activity-enhanced versions of SpCas9 PAM vanants, SpCas9-NG, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, etc., and b. Cystoine deaminase domains i. cytosine deaminase domains include APOBEC1, APOBEC3A, APOBEC3G, A1D / CDA1, the cytosine deaminase domains from engineered CBEs (e g. BE4max, evoAPOBEC 1 -BE4max, Anc BE4max, FERNY-BE4max, evoFERNY-BE4max, CDAl-BE4max, evoCDAl-BE4max, the cytosine deamianse domains from engineered TadA-based CBEs (e.g. TadCBEs or TadDEs (Neugebauer et al.. Nature Biotecnology, 2023). CBETs or CABE-Ts (Lam et al., Nature Biotechnology, 2023), Td-CBEs or Td-CGBEs (Chen et al., Nature Biotechnology, 2023), etc.), etc.

[0058] In preferred embodiments, the combinations comprise: a. gRNA C6 using WT SpCas9 and various CBE configurations listed herein; or b. gRNA C5 using SpG, SpCas9-MQKSER, or SpCas9-LWKFEG and various CBE configurations listed herein.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0060] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0061] DESCRIPTION OF DRAWINGS

[0062] FIG. 1. Schematics of base editors. Cytosine base editors (CBEs) predominantly mediate C-to-T edits (left panel). Adenine base editors (ABEs) predominantly mediate A-to-G edits (right panel). CBEs are typically composed of a cytosine deaminase (e.g. rat APOBEC1, rAPOl, etc.) linked to SpCas9 nickase (D10A) or other nickase Cas enzymes, and fused to one or more uracil DNA glycosylase inhibitors (UGI). Adenine base editor (ABE) are typically composed of an engineered adenine deaminase domain (TadA*) linked to SpCas9 nickase (D10A) or other nickase Cas enzymes.

[0063] FIG. 2. Exemplary base editor target sites for the CSF1R-I794T mutation. Target sites encoding a protospacer and PAM are shown, with the position of the target cytosine (C) base indicated (where numbering is from the PAM-distal end of the 20 nt spacer), and the 3 nt PAM indicated. C-to-T editing of the target base by a CBE will correct the I794T mutation. Exemplary' target sites for gRNAs C4, C5, C6, and C7 are illustrated; additional gRNAs have been designed to place the target cytosine in different positions of the spacer.

[0064] FIG. 3. Editing efficiencies in the HEK293T reporter cell line for various CBEs and C4 gRNA combinations targeting the CSF1R I794T mutation delivered by transfection. On-target cytosine (C4A) and bystander cytosine (C 11, C14, Cl 8) edits were quantified after transfection of plasmid DNA encoding TadCBE6b-SpRY, miniSdd7-SpRY, and BE4-CBE-SpRY base editors paired with the C4 gRNA. HEK293T cells in the control group were untransfected.

[0065] FIG. 4. Editing efficiencies in the HEK293T reporter cell line for various CBEs and C5 gRNA combinations targeting the CSF1R I794T mutation delivered by transfection. On-target cytosine (C5A) and bystander cytosine (C 12, C15, C19) edits were quantified after transfection of plasmid DNA encoding miniSdd- SpG, TadCBE6b-SpG, and BE4max-SpG base editors paired with the C5 gRNA. HEK293T cells in the control group were untransfected. FIG. 5. Editing efficiencies in the HEK293T reporter cell line for various CBEs and C6 gRNA combinations targeting the CSF1R I794T mutation delivered by transfection. On-target cytosine (C6A) and bystander cytosine (C l, C13, C16, C20) edits were quantified after transfection of plasmid DNA encoding CBE4max-SpCas9, TadA-CBEd-SpCas9, Tad-CBEmax-SpCas9, miniSdd7-SpCas9, and TadCBE6b-SpCas9 base editors paired with the C6 gRNA. HEK293T cells in the control group were untransfected.

[0066] FIG. 6. Editing efficiencies in the HEK293T reporter cell line for various CBEs and C7 gRNA combinations targeting the CSF1R I794T mutation delivered by transfection. On-target cytosine (C7A) and bystander cytosine (Cl, C2, C14, C17) edits were quantified after transfection of plasmid DNA encoding miniSdd7-SpRY, TadA-CBE6b-SpRY, and BE4-CBE-SpRY base editors paired with the C7 gRNA. HEK293T cells in the control group were untransfected.

[0067] FIG. 7. Editing efficiencies in ALSP patient-derived primary fibroblasts for various CBE and C6 gRNA combinations targeting the CSF1R I794T mutation delivered by transfection. On-target cytosine (C6A) and bystander cytosine (Cl, C13, C16, C20) edits were quantified after transfection of plasmid DNA encoding TadA-CBEd-SpCas9, BE4max-SpCas9, and Tad-CBEmax-SpCas9 base editors paired with the C6 gRNA. Fibroblasts in the control group were transfected with plasmid DNA encoding ABE9-SpCas9 adenine base editor and a non-targeting gRNA. Prior to sequencing analysis, fibroblasts were enriched for GFP+ transfected cells by flow cytometry and recovered for seven days post-enrichment.

[0068] FIG. 8. Editing efficiencies in ALSP patient-derived primary fibroblasts for various CBE and gRNA combinations targeting the CSF1R I794T mutation delivered by lentivirus-mediated transduction. On-target cytosine (C6A) and bystander cytosine (Cl, C13, C16, C20) edits were quantified after transduction of recombinant lentivirus expressing TadA-CBEd-SpCas9 or BE4max-SpCas9 base editors paired with the C6 gRNA, or CBE4max-SpG base editor paired with the C5 gRNA. Prior to sequencing analysis, fibroblasts were enriched for GFP+ transfected cells by flow cytometry and recovered for seven days post-enrichment.

[0069] FIG. 9. Editing efficiencies in ALSP patient-derived primary lymphoblasts for the TadA-CBEd-SpCas9 and C6 gRNA combination targeting the CSF1R I794T mutation delivered by transfection. On-target cytosine (C6A) and bystander cytosine (Cl, Cl 3, Cl 6, C20) edits were quantified after transfection of plasmid DNA encoding TadA-CBEd-SpCas9 base editor and C6 gRNA using transfection programs created by Lonza for the 4D-Nucleofector X unit (DS-120, DZ- 150, FA-150, DS-150, EH-116, EH-120, EN-150, EH-156, EH-198, ER-150). Prior to sequencing analysis, lymphoblasts were expanded for seven days, but were not enriched for GFP+ transfected cells.

[0070] DETAILED DESCRIPTION

[0071] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the only available treatment for ALSP? It harnesses the crosstalk between the hematopoietic myeloid cells and CNS microglia by providing a source of healthy donor-derived monocytes and macrophages that can therapeutically modulate the neuro-inflammatory environment. However, access to allo-HSCT is significantly limited by lack of available donors. Further, therapeutic efficacy is significantly limited by graft versus host disease and chronic immunosuppression, which increase transplant-associated mortality and morbidity.6’7Hematopoietic myeloid cells originate from hematopoietic stem cells (HSCs), which have been successfully targeted in gene-based therapy clinical trials for other inherited neurodegenerative disorders with aberrant microglial activation, such as X-linked adrenoleukodystrophy and metachromatic leukodystrophy.89Importantly, HSCs are easily accessible from patients via mobilization and apheresis for ex vivo genome editing, with emerging in vivo approaches being recently described by Breda et al.10and others.

[0072] The present methods include the use of base editing agents to correct the CSF1R-I794T mutations in hematopoietic stem cells (HSCs), preferably patient- derived HSCs. The methods can also include the use of expression vectors for in vitro or in vivo transfection and expression of a Cas9 base editor and suitable guide RNAs targeting CSF1R in cells, e.g., HSCs. Alternatively, or in addition, the methods can include the in vitro or in vivo use of purified Cas9 base editor proteins complexed with suitable guide RNAs targeting CSF1R in cells, e.g., HSCs.

[0073] Described herein are compositions and methods for highly efficient and specific base-editing to correct the most common ALSP mutation, CSF1R c.2381 T>C. CAS9 Base Editing Agents

[0074] Base editing is a CRISPR-based genome editing approach that could potentially correct pathogenic point mutations and precisely edit DNA sequences without intentionally inducing DNA double strand breaks"14(FIG. 1). Base editors allow for the targeted deamination of cytosines and adenines that are exposed on ssDNA by RNA-guided CRISPR-Cas proteins. There are two prototypical types of base editors (BEs). Cytosine base editors (CBEs) (FIG. 1A) predominantly mediate C-to-T edits, and are typically composed of a cytosine deaminase domain (e g., rat APOBEC 1 (rAPOl), AID, and engineered forms of these deaminases, etc.) linked to Streptococcus pyogenes Cas9 (SpCas9) nickase (with a D10A mutation) or other nickase Cas enzymes, and fused to one or more uracil DNA glycosylase inhibitors (UGI) to improve editing efficiency and C-to-T purit1 1 12 14 22. Adenine base editors (ABEs) (FIG. IB) predominantly mediate A-to-G edits and are ty pically composed of an engineered adenine deaminase domain linked to SpCas9 nickase (D10A) or other nickase Cas enzymesl2 22'25. Other types of base editors have been described that enable transversion mutations26,27. These and other classes of BEs can be utilized to install precise genetic edits (for example, to correct specific patient mutations).

[0075] Cytosine base editors (CBEs), such as BE3 or BE4max, catalyze the conversion of target C*G base pairs to T«A, while adenine base editors (ABEs), such as ABE7.10, ABEmax, or ABE8, convert target A»T base pairs to G»C. The Cas9 base editors (e.g., adenine base editors) used herein are fusion proteins comprising an adenosine deaminase domain and a Cas9 DNA binding domain.

[0076] Base editing with canonical base editors requires the presence of a PAM located approximately 15±2 base pairs from the target nucleotide(s). See, e.g., Komor, A.C. et al.. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity, Sci Adv 3 (2017); Rees, H.A. et al., Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery, Nat. Commun. 8, 15790 (2017); US2018 / 0073012, US2017 / 0121693. W02017 / 070633, US2015 / 0166980, U.S. Patent No. 9,840,699; and U.S. Patent No. 10,077,453. Split ABEs can also be used.

[0077] Like other CRISPR-Cas enzy mes, BEs are directed to their target site by user- specifiable programming of ~20 nucleotides (nts) on the 5' end of a guide RNA (gRNA). The gRNA pairs with the Cas enzyme, enabling the Cas enzyme to search the genome for regions complementary to the 5’ ~20 nt spacer region of the gRNA (to match the protospacer sequence of the target site). The 5’ spacer region of the gRNA can be of various lengths and compositions. For base editing with SpCas9 enzymes, gRNAs with 20 nt spacers are ty pically utilized. The gRNA spacer bases ty pically perfectly match the protospacer of the target site when using chemically synthesized gRNAs when delivered by lipid nanoparticle or other non-viral methods. When the gRNA is delivered from a plasmid or viral vector (e.g. AAV), to enhance gRNA transcription from the polIII U6 promoter, in certain cases the 5’ base of the spacer is mismatched to a guanine (G) nucleotide. Alternatively, an additional 5' G base can be appended to the 5’ end of the gRNA spacer, making the spacer 21 nts long. The Cas enzyme must also recognize a short sequence through protein:DNA interactions, called a protospacer-adjacent motif (PAM). The preferred PAM for wild-type SpCas9 is NGG28 30. where N is any nucleotide. Other versions of SpCas9 with altered or relaxed PAM preferences could be used, pennitting the Cas enzyme to target additional sequences.

[0078] Exemplary cytosine deaminase domains include APOBEC111, APOBEC3A40, APOBEC3G41, AID / CDA112, the cytosine deaminase domains from engineered CBEs (e.g. BE3, BE4max15, evoAPOBECl-BE4max21, AncBE4max15, FERNY-BE4max21, evoFERNY-BE4max21, CDAl-BE4max21, evoCDAl-BE4max), the cytosine deaminase domains from engineered TadA-based CBEs (e.g. TadCBEs or TadDEs18, CBE-Ts or CABE-Ts20, Td-CBEs or Td-CGBEs22, etc ), Sdd CBEs17, CBE6 enzy mes19, etc.

[0079] Exemplary SpCas9 enzyme variants that can access alternate PAMs include SpCas9-VRQR, -VQR, -EQR, or VRER30,31, SpG or SpRY32, SpCas9-NG33, SpCas9- NRRH, SpCas9-NRCH, or SpCas9-NRTH34, activity-enhanced and / or P AM-altered versions of SpCas9 variants enzy mes (see, e.g., WO2021151085, WO2021151073, WO2016141224, and W02019040650), including variants with mutations at DI 135, SI 136, G1218. E1219. R1335. and / or T1337 including NGCT PAM enzymes MQKSER, LWRYEK, LWKFEG, and LWKYQS. The sequences of the Cas9s are known in the art; see, e.g., Walton et al., Science. 2020 Apr 17;368(6488):290-296; Kleinstiver et al., Nature. 2015 Jul 23; 523(7561): 481-485; WO 2021151085; WO 2016 / 141224; US 9,512,446: US-2014-0295557; WO 2014 / 204578; and WO 2014 / 144761. See also tables B and C. The methods can also include the use of the other previously described variants of the SpCas9 platform (e.g., truncated gRNAs (Tsai et al., Nat Biotechnol 33, 187-197 (2015); Fu et al., Nat Biotechnol 32, 279-284 (2014)), and nickase mutations (Mali et al., Nat Biotechnol 31, 833-838 (2013); Ran et al., Cell 154, 1380-1389 (2013)).

[0080] Preferably, to correct the c.2381 T>C mutation, a gRNA comprising a spacer sequence as listed in Tables 3-5. or a gRNA, base editor, or combination of gRNA and base editor (comprised of a deaminase domain and wild-type SpCas9 or an engineered PAM variant derivative, as shown in Table A, is used, optionally a variant that also includes one or more additional mutations that improve activity such as high fidelity (e.g., as shown in Tables B and C).

[0081] Table A. Exemplary SpCas9 enzymes compatible with each gRNA. Preferably, the CRISPR editing complex is specific, i.e.. induces genomic alterations preferentially at the target site, and does not induce alterations at other sites, or only rarely induces alterations at other sites.

[0082] The variant proteins can also include one or more mutations that increase activity, reduce off- target effects (fidelity-enhancing mutations), and / or alter protospacer adjacent motif (PAM) or target adjacent motif (TAM) specificity (Tables B and C). In some embodiments, the fidelity-enhancing mutations comprise SpCas9- HF1 (N497A / R661 A / Q695A / Q926A), SpCas9-HiFi (R691A), eSpCas9(1.0) (K810A / K1003A / R1060A), or eSpCas9(l. l) (K848A / K1003A / R1060A). Table B: List of Exemplary High Fidelity and / or PAM-relaxed RGN Variants

[0083] * predicted based on UniRule annotation on the UniProt database. Table C. List of Exemplary SpCas9 Activity-Altering Mutations

[0084] Nucleic Acids Encoding a CRISP R Gene Editing Complex

[0085] The present methods can include the delivery of nucleic acids encoding a CRISPR gene editing complex. The gene editing complex includes a Cas9 base editing enzyme and one or more guide RNAs directing the editing enzyme to target CSF1R.

[0086] Guide RNAs directing the editing enzyme to CSF1R

[0087] The gene editing complex also includes guide RNAs directing the editing enzyme to a mutation in CSFIR, i.e., comprising a sequence that is complementary to the sequence of a nucleic acid encoding a mutant CSF1R, and that include a PAM sequence that is targetable by the co-administered Cas9 editing enzyme.

[0088] Exemplary sequences for human CSF1R can be found in GenBank, e.g., at NG 012303.2, range 31819..65078 (RelSeqGene genomic sequence). Exemplary’ sequences of gRNAs targeting the mutations are provided below, e.g., in Tables 3-5. CSF JR is also known as colony stimulating factor 1 receptor. The sequences provided herein are exemplary, and additional gRNAs targeting the sequences identified herein could be designed and utilized. The guide RNAs can be produced in a cell or subject directly, e.g., by expression from a coding sequence, or can be delivered as a synthetic RNA, e.g., with a nucleic acid (e.g., mRNA or circRNA) encoding the base editor or as part of a RNP with a BE protein. Synthetic gRNAs can include one or more modifications, e.g., as described in Hendel et al., Nature Biotechnology 33:985-989 (2015); Yin et al., Nature Biotechnology 35: 1179-1187 (2017); Finn et al., Cell Rep. 2018 Feb 27;22(9):2227-2235; and Chen et al. Adv Drug Deliv Rev. 2021 Jan: 168:246-258. Optionally, the gRNAs comprise modifications including incorporation of phosphorothioate backbone and / or 2’0 methylation.

[0089] Delivering Cas9 base editing agents

[0090] The methods include the delivery of Cas9 base editing agents as described herein to cells or subjects, e.g., as nucleic acids or as recombinant proteins. As used herein “contacting a cell” with a composition as described herein can include any known methods for introducing the nucleic acids or proteins into the cell, e.g., into the nucleus of the cell.

[0091] Nucleic Acids

[0092] Nucleic acids comprising expression constructs or RNA (e.g., mRNA or circRNA) encoding one or both of guide RNAs and / or Cas9 base editing enzymes can be used in the present methods, including any composition capable of effectively delivering the gene to cells in vivo. Approaches include insertion of the coding sequence in viral vectors, including recombinant retroviruses, adenovirus, adeno- associated virus (AAV), lentivirus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered naked or with the help of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as electroporation or direct injection of the gene construct or CaPC>4 precipitation carried out in vivo. RNA can be delivered naked or encapsulated within or mixed with lipids such as liposomes, e.g., lipid nanoparticles (LNPs).

[0093] One approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing a nucleic acid, e.g., a cDNA. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid. In some embodiments, nucleic acids encoding a CRISPR base editing complex (e.g., a Cas9 BE or gRNA) are entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins). These delivery vehicles can also be used to deliver Cas9 BE protein / gRNA complexes (e.g., RNPs) as described below.

[0094] In clinical settings, the nucleic acids encoding a CRISPR base editing complex can be introduced into a subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells will occur predominantly from specificity of transfection, provided by the gene delivery vehicle, cell-type or tissuetype expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery of the nucleic acids encoding a CRISPR base editing complex is more limited, with introduction into the subject being quite localized. In some embodiments, a controlled-release hydrogel comprising the nucleic acids encoding a CRISPR gene editing complex is administered to provide a steady dose of the nucleic acids encoding a CRISPR gene editing complex over time.

[0095] A pharmaceutical preparation of the nucleic acids encoding a CRISPR gene editing complex can consist essentially of the gene delivery system (e.g., nucleic acids such as viral vector(s)) in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g.. retroviral vectors, the pharmaceutical preparation can comprise one or more cells, which produce the gene delivery system.

[0096] In some embodiments, the methods include ex vivo electroporation of BE mRNA and synthetic gRNA into patient derived HSCs, followed by introduction of the HSCs back into the patient.

[0097] Alternatively, the methods can include in vivo delivery to a patient of BE mRNA and synthetic gRNA via engineered LNPs / NPs that are HSC-specific, e.g., as described by Breda et al.10and others. Recombinant Protein-RNA Complexes

[0098] The Cas9 BE can be delivered as a purified protein (e.g., a recombinantly produced purified protein, prefolded and optionally complexed with the gRNA, in a ribonucleoprotein complex (RNP)). Purified Cas9 proteins can be produced using methods known in the art, e.g., expressed in prokary otic or eukary otic cells and purified using standard methodology. See, e.g., Liang et al., Journal of Biotechnology 208:44-53 (2015); Kim et al., Genome Res. 2014 Jun; 24(6): 1012-1019. Efficiency of protein delivery can be enhanced, e.g., using electroporation (see, e g., Wang et al.. Journal of Genetics and Genomics 43(5): 319-327 (2016)); cationic or lipophilic carriers (see, e.g., Yu et al., Biotechnol Lett. 2016; 38: 919-929; Zuris et al., Nat Biotechnol. 33(l):73-80 (2015)); or even lentiviral packaging particles (see, e.g., Choi et al., Gene Therapy 23, 627-633 (2016)).

[0099] Pharmaceutical Compositions

[0100] The methods described herein can include the administration of pharmaceutical compositions and formulations comprising nucleic acids encoding base editors and gRNAs or recombinant base editor protein / gRNA RNP complexes targeting CSF1R mutations as described herein, or cells that have been edited ex vivo to correct a CSF 1R mutation using a method described herein.

[0101] In some embodiments, the compositions are formulated with a pharmaceutically acceptable carrier, such as a lipid nanoparticle (LNP), liposome, exosome, virus-like particle, or a variant thereof (see, e.g., Zhou and Yao, Mol Biomed 4, 10 (2023). doi.org / 10.1186 / s43556-023-00115-5; Banskota et al., Cell. 2022 Jan 20; 185(2): 250-265. el6; US 11020470; Raguram et al.. Cell. Volume 185, Issue 15, 21 July 2022, Pages 2806-2827; WO2020252455; W02022020800; WO2024108001; WO2024107959 and WO2024107983). The LNPs can comprise various lipids, including dioleoylphosphatidylethanolamine (DOPE); 1,2-distearoyl- sn-glycero-3 -phosphocholine (DSPC); l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC); l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC); l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC); and l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). See also Jung et al., Theranostics. 2022; 12(17): 7509-7531.

[0102] The pharmaceutical compositions and formulations can be administered parenterally, e.g., by intravenous administration. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see. e.g., Remington: The Science and Practice of Pharmacy, 21st ed„ 2005.

[0103] Methods of Treatment

[0104] The Cas9 BEs described herein targeting CSF 1R mutations can be administered for therapeutic treatment of subjects. In some embodiments, the compositions (e.g., nucleic acids or protein / gRNA complexes) are administered to a subject who has a disorder caused by a CSF1R mutation, e.g., adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), in an amount sufficient to cure, alleviate or partially arrest (e.g.. slow or stop progression or development of) the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount. Diagnosis of ALSP can be made using methods known in the art, including genetic and non-genetic testing.2Table A provides sets of exemplary combinations of BEs and gRNA, but others can also be used, e.g., using gRNAs comprising spacer sequences listed in Tables 3-5. In some embodiments, a therapeutically effective amount is sufficient to reduce development or expansion of white matter lesions in the frontal region, corpus callosum and corticospinal tracts of the brain, as w ell as recognize enlarged ventricles due to cerebral atrophy2, or to reduce or delay development of symptoms of ALSP including personality changes, impaired cognition, memory derangement, bouts of depression or motor dysfunction, such as muscle weakness, impaired gait, slow movement, rigidity, and tremor.2

[0105] The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient’s physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration. The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents’ rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo- Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51 :337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.

[0106] Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g.. effect on tumor size or growth), and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

[0107] In alternative embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0108] The methods and compositions described herein can also be used for cell therapy. For example, hematopoietic stem cells from the patient are collected, optionally expanded or cultured, corrected using a method described herein, and reinfused as an intravenous infusion, a process referred to as cell therapy. The patient can receive chemotherapy busulfan or other chemotherapeutic agents to precondition and suppress the bone marrow before hematopoietic stem cell transplantation. Corrected stem cells engraft and grow into functional blood cells.

[0109] Exemplary sequences

[0110] In some embodiments, the sequence of a protein or nucleic acid used in a composition or method described herein is at least 80%. 85%. 90%. 95%. 97%. 98%. or 99% identical to a sequence set forth herein. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0111] The comparison of sequences and detennination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0112] In some embodiments, the sequence of a protein or nucleic acid used in a composition or method described herein has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions as compared to a sequence set forth herein. In some embodiments, the substitutions are conservative substitutions. EXAMPLES

[0113] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0114] Methods

[0115] The following materials and methods were used in the Example below. gRNA and CBE plasmids

[0116] Oligonucleotides for the top and bottom DNA strands encoding 5’ extensions for cloning along with: (1) 20 nt spacers that perfectly matched the protospacer of the target site (Table 3; typically used for direct gRNA deliver}' where the gRNA does not need to be transcribed from a DNA template), (2) 20 nt spacers where all gRNA spacers encode a 5’ G base (in cases where the native 5' base is an A, C, or T, the base is mismatched to a G; Table 4), or (3) 21 nt spacer that encode one additional 5' G (Table 5) were designed and ordered. Expression plasmids for human U6 promoter-driven gRNAs were generated by annealing and ligating duplexed top and bottom strand oligonucleotides corresponding to spacer sequences into BsmBI- digested pUC19-U6-BsmBI_cassette-SpCas9_sgRNA (BPK1520; Addgene plasmid 65777). Cloned plasmids were sequence verified by Sanger sequencing.

[0117] CBE plasmids were cloned via isothermal assembly (Table 6).

[0118] Generation of HEK 293T cell lines harbouring the CSF1R I794T sequence

[0119] For base editing experiments, a HEK 293T reporter cell line encoding CSF1R I794T was generated via lentiviral vector transduction. Briefly, 181 nt of the CSF1R I794T sequence (90 nt flanking on each site of the mutation) was cloned into a lentiviral backbone via isothermal assembly. Lentiviral vectors encoding the reporter sequence were produced in ~ 3.5 million HEK 293T cells seeded in 10mm plates ~ 24 hours earlier, via transfection with 2840 ng of a packaging plasmid (psPAX2; Addgene plasmid 12260), 964 ng of an envelope plasmid (pMD2.G; Addgene plasmid 12259), as well as 5170 ng of the reporter plasmid. Six hours posttransfection, human cells were washed twice with 6mL of pre-w armed Phosphate- Buffered Saline (PBS) and incubated with fresh culture media for 48-72 hours. Lentiviral vectors were isolated through differential centrifugation by collecting conditioned media, centrifuging at 300 g for 10 min, followed by a 10 min spin at 2,000 g to remove detached cells and cell debris. Lentiviral vectors were concentrated through ultracentrifugation at 70,000 g, and the vector pellet was resuspended using iced PBS. To generate the CSF1R I794T containing reporter cell line, low passage HEK 293T cells were stably transduced with 2 different MOIs of virus. Low MOI was transduced with 100 uL and high MOI condition transduced with 300 uL of lentiviral vectors and further selected with puromycin (1 pg / mL) over two passages.

[0120] Cell culture and transfections

[0121] Human HEK 293T cells (American Type Culture Collection; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS (HI-FBS) and 1% penicillin-streptomycin. Samples of supernatant media from cell culture experiments were analyzed monthly for the presence of mycoplasma using PCR.

[0122] Fibroblasts and lymphoblasts w ere derived from skin biopsies and blood draws, respectively, obtained from patients with ALSP (Table 1). Written informed consent was obtained from each patient under the institutional ethics review board at the Massachusetts General Hospital (protocol # 2007P002248) for each skin biopsy. Fibroblasts were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% HI-FBS and 1% penicillin / streptomycin. Lymphoblasts were cultured in Roswell Park Memorial Institute Medium (RPMI)-1640 medium supplemented with 10% HI-FBS and 1% penicillin / streptomycin. After transfection of plasmid DNA encoding genome editors and gRNA, the media was modified to contain 20% HI-FBS for recovery. Fibroblasts and lymphoblasts were transfected using the Nucleofector system (Lonza). Transfections were performed with a nontargeting gRNA to establish a ‘'control” line and naive cells were untreated. Approximately 72 hours after transfection, GFP+ fibroblasts were sorted (MGB HSCI CRM Flow Cytometry7Core; BD FACS Arialll cell sorter) and seeded into a pooled GFP+ population to grow7for an additional 7 days. Two additional passages were performed to expand the sorted cells, which were then used to extract gDNA as described above at passages 3, 4, 5, and 6 post-transfections (with 2-3 days of cell growth per passage). Lymphoblasts were not sorted for the GFP+ transfected population due to intolerance and poor survival post-sorting. Transfected and non- transfected lymphoblasts were cultured for an additional 7 days prior to extraction of gDNA for sequencing analysis.

[0123] For HEK 293T human cell experiments, transfections were performed 20 hours following seeding of 2x104HEK 293T cells per well in 96-well plates. Transfections containing 70 ng of ABE expression plasmid and 30 ng gRNA expression plasmid mixed with 0.72 pL of TransIT-X2 (Mirus) in a total volume of 15 pL Opti-MEM (Thermo Fisher Scientific), incubated for 15 minutes at room temperature, and distributed across the seeded HEK 293T cells. Experiments were halted after 72 hours and genomic DNA (gDNA) was collected by discarding the media, resuspending the cells in 100 pL of quick lysis buffer (20 mM Hepes pH 7.5, 100 mM KC1, 5 mM MgCh. 5% glycerol, 25 mM DTT. 0.1% Triton X-100, and 60 ng / pL Proteinase K (New England Biolabs; NEB)), heating the lysate for 6 minutes at 65 °C, heating at 98 °C for 2 minutes, and then storing at -20 °C.

[0124] Assessment of genome editor activities in human cells

[0125] The efficiency of genome modification by CBEs was determined by nextgeneration sequencing using a 2-step PCR-based Illumina library construction method, similar to as previously described32. Briefly, genomic loci were amplified using approximately 50-100 ng of gDNA, Q5 High-fidelity DNA Polymerase (NEB), and PCR-1 primers (Table 7) with cycling conditions of 1 cycle at 98 °C for 2 min; 35 cycles of 98 °C for 10 sec, 58 °C for 10 sec. 72 °C for 20 sec; and 1 cycle of 72 °C for 1 min. PCR products were purified using paramagnetic beads prepared as previously described4243. Approximately 20 ng of purified PCR-1 products were used as template for a second round of PCR (PCR-2) to add barcodes and Illumina adapter sequences using Q5 and standard Illumina PCR primers and cycling conditions of 1 cycle at 98 °C for 2 min; 10 cycles at 98 °C for 10 sec, 65 °C for 30 sec, 72 °C 30 sec; and 1 cycle at 72 °C for 5 min. PCR products were purified prior to quantification via capillary7electrophoresis (Qiagen QIAxcel), normalization, and pooling. Final libraries were quantified by qPCR using the KAPA Library Quantification Kit (Complete kit; Universal) (Roche) and sequenced on a MiSeq sequencer using a 300- cycle v2 kit (Illumina).

[0126] On-target genome editing activities were determined from sequencing data using CRISPResso2 (ref.44) in pooled mode with custom input parameters: - min reads to use region 100 -quantification window size 10 - quantification window center -10 — base editor output — min frequency alleles around cut to plot 0.05.

[0127] Example 1. Base Editors (BEs) for correction of pathogenic mutations in the human CSF1R gene

[0128] Here we explored the use of BEs to correct pathogenic mutations in the human CSFJR gene. In an initial approach, we designed several gRNA target sites to explore CBE-mediated correction of the most common CSF1R mutation (p.Ile794Thr; p.I794T; c.2381 T>C), which placed the target cytosine in positions 2 through 10 of the target site (counting from the PAM distal end of the spacer; Table 2; FIG. 2). The gRNAs were designed with various sequence configurations, including: (1) 20 nt spacers that perfectly matched the protospacer of the target site (Table 3; typically used for direct delivery' of chemically modified gRNAs where the gRNA does not need to be transcribed from a DNA template, or when the 5' base of the gRNA spacer naturally encodes a guanine base), (2) 20 nt spacers where all gRNA spacers encode a 5’ G base (in cases where the native 5’ base is an A, C, or T, the base is mismatched to a G; Table 4), or (3) 21 nt spacer that encode one additional 5’ G (Table 5). Oligonucleotides encoding the spacer sequences in Table 4 and Table 5 were cloned into a prototypical SpCas9 expression plasmid encoding a U6 promoter and the conventional SpCas9 gRNA scaffold followed by a transcriptional terminator (7xT sequence).

[0129] The gRNAs were paired with various CBE constructs (Table 6), including those bearing variant cytosine deaminase domains (to augment the efficiency and purity of editing) or variant nSpCas9(D10A) domains bearing different PAM preferences. CBE constructs were designed and cloned by generating combinations of the cytosine deaminase domains and SpCas9 enzy me variants.

[0130] To investigate CBE-mediated correction of the CSF1R I794T mutation, we first established a lentiviral vector-based HEK 293T cell line carrying the CSFJR I794T mutation. This reporter cell line was generated by cloning 181 nt of the CSF1R I794T sequence (90 nt on each side of the mutation) into a lentiviral packaging plasmid, producing the lentiviral vector, and transducing HEK 293T cells to incorporate this synthetic sequence into the genome. Using CSF1R I794T cell line, we tested CBEs paired with 4 different gRNAs targeted to the I794T mutation including sites harboring NCT (C4 gRNA, FIG. 3), NCG (C5 gRNA, FIG. 4), NGG (C6 gRNA, FIG. 5), or NTG (C7 gRNA, FIG. 6) PAMs that position the target cytosine in positions C4-C7, respectively (FIG. 2). The CBEs (Table 6) were comprised of wild type (WT) or engineered cytosine deaminase domains coupled with the wildtype SpCas9 enzyme that targets NGG PAMs (for gRNA C6), SpG for targeting NGN PAMs (for gRNA C5), and the near-PAMless SpRY variant that targets NRN>NYN PAMs (for gRNAs C4 and C7).

[0131] On-target editing efficiencies for correcting the I794T mutation in the HEK 293T reporter cell line varied substantially depending on the CBE and gRNA used. The highest levels of on-target correction were observed for CBE4max-SpCas9 and miniSdd7-SpCas9 CBEs paired with the C6 gRNA, reaching -50% reversion of the I794T causative mutation (FIG. 5). We also observed substantial on-target correction (-35-40%) when using TadA-CBEd-WT or CBE6b-WT with gRNA C6 (FIG. 5). In all cases, we observed some bystander editing of nearby cytosine bases that were all silent mutations, including editing at Cl (p.A792A), C13 (p.D796D), C16 (p.F797F), and C20 (p.L799L); the degree and nature of bystander editing varied based on the combination of CBE and gRNA used (FIG. 5). Minimal editing was observed with TadCBEmax-WT and gRNA C6 (FIG. 5).

[0132] With gRNAs C5 and C7, we also observed substantial on-target editing with SpG- and SpRY -based CBEs, respectively (FIGs. 4 and 6, respectively). Using Sdd7- SpG, CBE6-SpG, and BE4max-SpG with gRNA C5 resulted in >20% on-target correction, with Sdd7-SpG reaching -33% (FIG. 4). Minimal bystander editing was observed and all bystander edits were silent. For gRNA C7, both CBE6b-SpRY and BE4max-SpRY resulted in -35% correction (FIG. 6) and mostly silent bystander edits, with the exception of a Cl bystander edit that causes p.A792G. In contrast, all CBEs paired with the C4 gRNA showed minimal on-target editing efficiency (-1%) (FIG. 3)

[0133] Since combinations of CBEs with the SpCas9 WT enzyme variant paired with C6 gRNA mostly corrected the I794T mutation, we next tested these combinations in primary patient-derived fibroblasts (FIG. 7). Transfection of plasmid DNA encoding the CAG promoter and BE4max-WT paired with U6-C6 gRNA achieved -80% on- target editing efficiency for the targeted p.I794T mutation, whereas plasmid DNA encoding a CMV promoter expressing TadA-CBEd-WT and U6-gRNA C6 achieved -90% on-target correction (FIG. 7). When using lentiviral vector encoding a CMV promoter expressing BE4max-WT and U6-C6 gRNA, we observed lower levels of correction at -25%, suggesting that the TadA-CBEd-WT construct is a more potent editor under these conditions (FIG. 7). The results with pCMV-TadA-CBEd-WT and gRNA C6 restores normal CSF1R allele frequency from 50% in the heterozygous state to 95% after base editing. This is a level of therapeutic correction that is comparable to allogeneic EISCT with full donor chimerism. Importantly, there was not detectable bystander editing for the BE4max + C6 gRNA combination and minimal bystander editing for the TadA-CBEd + C6 gRNA combination. These data provide important proof-of-concept that the I794T mutation is correctable using genome editing approaches employing cytosine base editors when delivered to patient-derived fibroblasts.

[0134] We also explored lentiviral vector-mediated delivery of different CBEs and C6 gRNA into patient-derived fibroblasts to compare the on-target and bystander editing efficiencies between lentivirus-mediated delivery7and plasmids transfection (FIG. 8). Transfection results in short exposure to a high dose of base editor and gRNA while lentivirus transduction results in longer exposure to the CBE, which, in theory, can increase on-target and bystander editing. While transduction of lentiviral vectors encoding BE4max-WT and C6 gRNA increased on-target editing efficiency to >90%, we also observed increased bystander editing at C16, which produces a silent mutation (FIG. 8). Interestingly, transduction of recombinant lentivirus encoding TadA-CBEd-SpCas9 and C6 gRNA did not increase on-target C-to-T editing compared to transfection of DNA plasmid encoding this base editor and gRNA combination (-65% editing in transduced fibroblasts and -90% editing in transfected fibroblasts). Similar to the data derived from the HEK293T reporter cell line (FIGs. 3-6), on-target editing efficiency targeting the I794T mutation was significantly lower in fibroblasts transduced with the CBE4max-SpG base editor and C5 gRNA combination. These data reveal that different CBE and gRNA combinations and different modalities of genome editor delivery can produce dramatically different editing efficiencies when targeted to the same mutation, and that multiple CBE and gRNA combinations need to be assayed to determine the optimal combination.

[0135] We next assessed editing efficiency targeting the I794T mutation in ALSP patient-derived primary7lymphoblasts, which are more difficult to maintain in culture and transfect, but significantly closer in lineage to HSCs than primary fibroblasts. When transfecting primary lymphoblasts we observed substantial toxicity, and after screening multiple different transfection programs, eliminating the post-transfection enrichment for GFP+ transfected lymphoblasts, and optimizing the transfection protocol, the TadA-CBEd-WT CBE in combination with gRNA C6 achieved up to 16% on-target editing to correct the I794T mutation. There was near-zero bystander editing and no nonsynonymous bystander editing (Figure 9). These data provide important proof-of-concept that the CSF1R I794T mutation can be effectively targeted and corrected in ALSP patient-derived lymphoblasts. Future optimization of this electroporation protocol to reduce toxicity7to cells should result in higher levels of editing (e.g. by electroporating CBE mRNA and synthetic gRNA instead of DNA plasmids).

[0136] Together, these results reveal that specific combinations of CBEs and gRNAs can result in high levels of on-target correction of CSFJR I794T, supporting a genome editing-based treatment for ALSP patients.

[0137] Table 1. Demographics of ALSP patients from the clinical database at our institution and the respective pathogenic CSF1R variant(s) they cany7. The last column lists possible genome editing techniques with the theoretical capability for correcting each mutation through genome editing approaches, with the potential for becoming clinically approved precision-based genome editing treatment approaches in the future.

[0138] Table 2. Target sites for base editor-mediated correction of CSF1R-I794T.

[0139] Table 3. gRNA spacer sequences for base editor-mediated correction of CSF1R- I794T using 20 nt perfectly matched spacers. Table 4. gRNA spacer sequences for base editor-mediated correction of CSF1R- I794T using 20 nt spacers with 5’ guanine bases.

[0140] Table 5 gRNA spacer sequences for base editor-mediated correction of CSF1R- I794T using 21 nt spacers that harbor +1 5’G bases.

[0141] Table 6. Plasmids

[0142] Table 7. Next-generation PCR-1 primers.

[0143] References

[0144] 1. Rademakers, R. et al. Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids. Nat Genet 44, 200-205 (201 1).

[0145] 2. Papapetropoulos, S. et al. Adult-Onset Leukoencephalopathy With Axonal Spheroids and Pigmented Glia: Review of Clinical Manifestations as Foundations for Therapeutic Development. Front. Neurol. 12. (2022).

[0146] 3. Papapetropoulos, S. et al. Clinical presentation and diagnosis of adultonset leukoencephalopathy with axonal spheroids and pigmented glia: a literature analysis of case studies. Front. Neurol. 15, (2024). 4. Hu, B. et al. Insights Into the Role of CSF1R in the Central Nervous

[0147] System and Neurological Disorders. Front Aging Neurosci 13, 789834 (2021).

[0148] 5. Dulski, J. et al. Hematopoietic Stem Cell Transplantation in CSF1R- Related Leukoencephalopathy: Retrospective Study on Predictors of Outcomes. Pharmaceutics 14, 2778 (2022). 6. Penack, O. et al. How much has allogeneic stem cell transplant-related mortality improved since the 1980s? A retrospective analysis from the EBMT. Blood Adv 4, 6283-6290 (2020).

[0149] 7. Musolino, P. L. et al. Hematopoietic stem cell transplantation in the leukodystrophies: a systematic review of the literature. Neuropediatrics 45, 169-174 (2014).

[0150] 8. Eichler. F. S. et al. Disease Stabilization Following Treatment with elivaldogene autotemcel (eli-cel, Lenti-D) Gene Therapy for the Treatment of Cerebral Adrenoleukodystrophy: Interim Results from Phase 2 / 3 (ALD-102) and Phase 3 (ALD-104) Studies (2064). Neurology 96, (2021).

[0151] 9. Fumagalli, F. et al. Lentiviral haematopoietic stem-cell gene therapy for early -onset metachromatic leukodystrophy: long-term results from a non- randomised, open-label, phase 1 / 2 trial and expanded access. Lancet 399, 372-383 (2022).

[0152] 10. Breda, L. et al. In vivo hematopoietic stem cell modification by mRNA delivery. Science 381, 436-443 (2023).

[0153] 11. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016).

[0154] 12. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, (2016).

[0155] 13. Gaudelli, N. M. et al. Programmable base editing of A«T to G«C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017).

[0156] 14. Rees, H. A. & Liu, D. R. Base editing: precision chemistry’ on the genome and transcriptome of living cells. Nature Reviews Genetics 19, 770-788 (2018).

[0157] 15. Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nature Biotechnology 36, 843- 846 (2018).

[0158] 16. Komor, A. C., Badran, A. H. & Liu, D. R. Editing the Genome Without Double-Stranded DNA Breaks. ACS Chem. Biol. 13, 383-388 (2018).

[0159] 17. Huang, J. et al. Discovery' of deaminase functions by structure-based protein clustering. Cell 186, 3182-3195. el4 (2023). 18. Neugebauer, M. E. et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat Biotechnol 1-13 (2022) doi: 10.1038 / s41587-022-01533-6. Nat Biotechnol. 2023 May;41(5):673-685;

[0160] 19. Zhang, E., Neugebauer, M. E., Krasnow, N. A. & Liu, D. R. Phage- assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference. Nat Commun 15, 1697 (2024).

[0161] 20. Lam, D. K. et al. Improved cytosine base editors generated from TadA variants. Nat Biotechnol 1-12 (2023) doi: 10.1038 / s41587-022-01 11 -9.

[0162] 21. Thuronyi, B. W. et al. Continuous evolution of base editors with expanded target compatibility' and improved activity. Nature Biotechnology 37, 1070- 1079 (2019).

[0163] 22. Chen, L. et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nat Biotechnol 1-10 (2022) doi: 10.1038 / s41587-022-01532-7.

[0164] 23. Gaudelli, N. M. et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat Biotechnol 38, 892-900 (2020).

[0165] 24. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nature Biotechnology 38, 883- 891 (2020).

[0166] 25. Lapinaite. A. et al. DNA capture by a CRISPR-Cas9-guided adenine base editor. Science 369, 566-571 (2020).

[0167] 26. Tong, H. et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat Biotechnol 1-5 (2023) doi: 10.1038 / s41587-022-01595-6.

[0168] 27. Chen, L. et al. Adenine transversion editors enable precise, efficient A»T-to-C»G base editing in mammalian cells and embryos. Nat Biotechnol 1-13 (2023) doi:10. 1038 / s41587-023-01821-9.

[0169] 28. Jinek, M. et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816-821 (2012).

[0170] 29. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA- guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31, 233-239 (2013). 30. Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523. 481-485 (2015).

[0171] 31. Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495 (2016).

[0172] 32. Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368, 290-296 (2020).

[0173] 33. Nishimasu, H. et al. Engineered CR1SPR-Cas9 nuclease with expanded targeting space. Science 361, 1259-1262 (2018).

[0174] 34. Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat Biotechnol 38, 471-481 (2020).

[0175] 35. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019).

[0176] 36. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nature Biotechnology 38, 824-844 (2020).

[0177] 37. Ferreira da Silva, J. et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. Nat Biotechnol 1-13 (2024) doi : 10. 1038 / s41587-024-02324-x.

[0178] 38. Liu. B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat Biotechnol 1-7 (2023) doi: 10.1038 / s41587-023-01947-w.

[0179] 39. Nguyen, L. T. et al. Efficient Genome Editing with Chimeric Oligonucleotide-Directed Editing. 2024.07.09.602710 Preprint at https: / / d01.0rg / l 0. 1101 / 2024.07.09.602710 (2024).

[0180] 40. Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol 36, 977-982 (2018).

[0181] 41. Lee, S. et al. Single C-to-T substitution using engineered APOBEC3G- nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Science Advances 6, eabal773 (2020).

[0182] 42. Kleinstiver, B. P. et al. Engineered CRISPR-Casl2a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nature Biotechnology 37, 276-282 (2019). 43. Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939-946 (2012).

[0183] 44. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nature Biotechnology 37, 224-226 (2019). OTHER EMBODIMENTS

[0184] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A guide RNA (gRNA) comprising a spacer sequence listed in any of Tables 3-5.

2. A composition comprising a recombinant SpCas9 cytosine base editor (BE) protein and a gRNA targeting CSFJR, preferably as a ribonucleoprotein complex, preferably wherein the gRNA comprises a spacer sequence shown in any of Tables 3-5, optionally wherein:(a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-MQKSER, SpCas9(MQKSER+LllllR). orSpCas9(MQKSER+Ll l 11R+A1322R+A61R); or(c) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity -enhancing mutations.

3. The composition of claim 2, wherein the recombinant SpCas9 cytosine base editor protein and gRNA targeting CSF JR are in a carrier, e.g., a lipid nanoparticle (LNP), liposome, exosome, or virus-like particle.

4. A composition comprising a nucleic acid sequence encoding an SpCas9 cytosine base editor protein, and a gRNA targeting CSFJR, preferably wherein the gRNA comprises a spacer sequence show n in any of Tables 3-5, optionally w herein:(a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-MQKSER, SpCas9(MQKSER+LllllR). orSpCas9(MQKSER+L 1111 R+ A 1322R+A61 R); or(c) the gRNA targets the BE to CSFJR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity -enhancing mutations.

5. A composition comprising a nucleic acid sequence encoding an SpCas9 cytosine base editor protein and a nucleic acid sequence encoding a gRNA targeting CSFJR, preferably wherein the gRNA compnses a spacer sequence shown in any of Tables 3-5, optionally wherein:(a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF R mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-MQKSER, SpCas9(MQKSER+Llll lR), or SpCas9(MQKSER+LllllR+A1322R+A61R); or(c) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity -enhancing mutations.

6. The composition of claims 4 or 5, wherein the nucleic acid sequences encoding the base editor protein and / or gRNA comprise RNA, optionally mRNA, circRNA, or synthetic RNA; or are in an expression vector, e.g., a viral vector or a plasmid, and / or in a carrier, e.g., a lipid nanoparticle (LNP), liposome, exosome, or viruslike particle.

7. The composition of claim 4, wherein the nucleic acid sequence encoding an SpCas9 cytosine base editor protein is an mRNA and the gRNA is a synthetic RNA.

8. The composition of any of claims 2-7, further comprising a carrier.

9. The composition of claim 8, wherein the carrier comprises a lipid nanoparticle (LNP), nanoparticle (NP), liposome, exosome, or virus-like particle.

10. A method of editing a cell comprising a mutation in an allele of CSF JR, the method comprising contacting the cell with or expressing in the cell an SpCas9 cytosine base editor protein and a gRNA targeting CSF JR, preferably wherein the gRNA comprises a spacer sequence shown in any of Tables 3-5, optionally wherein:(a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG. SpCas9-MQKSER, SpCas9(MQKSER+L 1111 R), SpCas9(MQKSER+Ll 111 R+Al 322R+A61 R); or(c) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity -enhancing mutations.

11. The method of claim 10, comprising contacting the cell with or expressing in the cell the composition of any of claims 2-9.

12. The method of claim 10, comprising contacting the cell with mRNA encoding the SpCas9 cytosine base editor protein and a synthetic gRNA targeting CSF1R.

13. An isolated cell from a subject who has a disease caused by a mutation in CSF JR, wherein the mutation has been corrected by contacting the cell with or expressing in the cell an SpCas9 cytosine base editor protein and a gRNA targeting CSF JR, preferably wherein the gRNA comprises a spacer sequence shown in any of Tables 3-5. optionally wherein:(a) the gRNA targets the BE to CSFJR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG. SpCas9-MQKSER, SpCas9(MQKSER+L 11 HR). SpCas9(MQKSER+Ll 111 R+Al 322R+A61 R); or(c) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity -enhancing mutations..

14. The isolated cell of claim 13, wherein the cell is a hematopoietic stem cell (HSC).

15. A method of treating a subject who has a disease caused by a mutation in CSFJR, the method comprising administering to the subject a therapeutically effective amount of an isolated autologous cell, wherein the mutation in the cell has been corrected by contacting the cell with or expressing in the cell an SpCas9 cytosinebase editor protein and a gRNA targeting CSF JR, preferably wherein the gRNA comprises a spacer sequence shown in any of Tables 3-5, optionally wherein:(a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG. SpCas9-MQKSER, SpCas9(MQKSER+Ll 1 HR). SpCas9(MQKSER+Ll 111R+A1322R+A61R); or(c) the gRNA targets the BE to CSF R mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity -enhancing mutations16. The method of claim 15, wherein the cell is a hematopoietic stem cell (HSC).

17. A method of treating a subject who has a disease caused by a mutation in CSF / R. the method comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding an SpCas9 cytosine base editor protein and a gRNA targeting CSF JR, preferably wherein the gRNA comprises a spacer sequence shown in any of Tables 3-5, optionally wherein:(a) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C6, and the BE comprises WT SpCas9; or(b) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C5 or C9, and the BE comprises SpG, SpCas9-MQKSER, SpCas9(MQKSER+L 1111 R). SpCas9(MQKSER+L 1111 R+ Al 322R+ A61 R); or(c) the gRNA targets the BE to CSF JR mutation c.2381 T>C and comprises spacer sequence C7, and the BE comprises SpRY; or optionally wherein the BE comprises one or more fidelity-enhancing mutations.

18. The method of claim 17, wherein the nucleic acid sequences encoding the base editor protein and / or gRNA are in a vector, e.g., a viral vector or a plasmid, and / or in a carrier, e.g., a lipid nanoparticle (LNP), nanoparticle (NP), liposome, exosome, or virus-like particle.

19. The method of claim 18, wherein the nucleic acid sequence encoding the base editor protein is an mRNA or circRNA, and the method comprises administering the mRNA and gRNA in an LNP.

20. The method of claim 17, wherein the recombinant SpCas9 cytosine base editor protein and gRNA targeting CSF1R are administered in a carrier, e.g.. a lipid nanoparticle (LNP), liposome, exosome, or virus-like particle.

21. Any of claims 2-20, wherein the cytosine base editor comprises a cytosine deaminase domain selected from the group consisting of cytosine deaminase domains from APOBEC1, APOBEC3A. APOBEC3G. A1D / CDA1, BE3, BE4max. evoAPOBECl-BE4max, Anc BE4max, FERNY-BE4max, evoFERNY-BE4max, CDAl-BE4max, evoCDAl-BE4max, or engineered TadA-based CBEs, optionally TadCBEs (optionally TadCBEd). TadDEs, CBE-Ts, CABE-Ts, Td-CBEs, Td- CGBEs, Sdd CBEs (optionally Sddl, Sdd.

3. Sdd6, or Sdd7), or CBE6 (optionally CBE6b or CBE6a).

22. Any of claims 2-20, wherein the cytosine base editor comprises a fidelityenhancing mutations selected from SpCas9-HFl (N497A / R661A / Q695A / Q926A), SpCas9-HiFi (R691A), eSpCas9(1.0) (K810A / K1003A / R1060A), and eSpCas9(l.l) (K848A / K1003A / R1060A).

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