Compositions and Methods for Reducing Complement Activation

Abstract

Compositions and methods for reducing complement activation by introducing one or more modifications into intracellular complement component 3 (C3) polynucleotides. In certain embodiments, the disclosure features a base editor system (e.g., a fusion protein or complex comprising a programmable DNA-binding protein, a nucleic acid base editor, and a gRNA) for modifying a C3 polynucleotide, wherein the modification is associated with reduced expression and / or reduced activity of the C3 polypeptide encoded by the polynucleotide.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority of U.S. Provisional Application No. 63 / 352,547, filed on June 15, 2022, the entire content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in XML format, the entire content of which is incorporated herein by reference. The sequence listing XML file created on June 14, 2023, is named 180802 - 055102PCT_SL.xml and has a size of 1,364,888 bytes.

Background Art

[0003] The complement system is an important part of the innate immune system and is involved in the removal of microorganisms and cell debris, as well as the activation of inflammation and various immune pathways. Although over - activation of the complement system or inappropriate targeting of its own cells can cause diseases, inhibition of complement activity has been successfully and safely shown to provide therapeutic benefits to patients suffering from an over - active complement system. Therefore, improved methods for reducing complement activation in such patients.

Summary of the Invention

[0004] As described below, the present disclosure features compositions and methods for reducing complement activation by introducing one or more modifications into intracellular complement component 3 (C3) polynucleotides. In certain embodiments, the present disclosure features a base editor system (e.g., a fusion protein or complex comprising a programmable DNA - binding protein, a nucleic acid base editor, and a gRNA) for modifying the C3 polynucleotide, and the modification is associated with reduced expression of the C3 polypeptide encoded by the polynucleotide and / or reduced activity. Non - limiting examples of the modification include base editing.

[0005] In one aspect, the present disclosure features a method of modifying a nucleobase of a complement component 3 (C3) polynucleotide. The method comprises contacting the C3 polynucleotide with one or more guide RNAs, or one or more polynucleotides encoding one or more guide RNAs, and a base editor containing a nucleic acid programmable DNA-binding protein (napDNAbp) domain and a deaminase domain, or a base editor system containing one or more polynucleotides encoding a base editor, thereby modifying the nucleobase of the C3 polynucleotide. The method includes (a), (b), (c), (d), and / or (e). In (a), the one or more guide RNAs target the base editor to effect a modification of a nucleobase of the C3 polynucleotide that disrupts a splice site within the C3 polynucleotide. In (b), the deaminase domain is a TadA variant (TadA*) containing a combination of modifications listed in Tables 5A, 5B, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6E, 6F, or 7, where the TadA* is not TadA*7.9 or TadA*7.10, and / or the TadA* variant is a TadA*8.8, TadA*8.17, or TadA*8.20 variant that includes one or more of the amino acid modifications V82T, Y147T, and Q154S. In (c), the one or more guide RNAs contain a nucleic acid sequence containing at least 10 to 23 consecutive nucleotides of a spacer nucleic acid sequence listed in any one of Tables 1A, 1B, 1C, 1D, 1E, 1F, and 2. In (d), the one or more guide RNAs target the base editor to the following reference sequence: C3 amino acid sequence

[0006] In another aspect, the disclosure features a method of modifying a nucleobase of a complement component 3 (C3) polynucleotide. The method involves contacting the C3 polynucleotide with one or more guide RNAs, or one or more polynucleotides encoding one or more guide RNAs, and a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, thereby modifying the nucleobase of the complement component 3 (C3) polynucleotide. The method further includes (a) and / or (b). In (a), the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, TadA*8.20_V82T_Y147T_Q154S, rAPOBEC1, and ppAPOBEC. In (b), the one or more guide RNAs contain a spacer corresponding to a guide polynucleotide selected from one or more of gRNA661, gRNA662, gRNA676, gRNA695, gRNA696, gRNA701, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA1793, gRNA1798, gRNA3342, gRNA3343, and gRNA3345.

[0007] In another aspect, the present disclosure features a method of treating a disease or disorder in a subject in need of treatment for a disease or disorder associated with inappropriate activation of the complement system. The method comprises modifying the nucleobases of a complement component 3 (C3) polynucleotide in a subject by administering to the subject one or more guide RNAs, or one or more polynucleotides encoding one or more RNAs, and a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding a base editor, thereby treating the disease or disorder. The method includes (a), (b), (c), (d), and / or (e). In (a), the one or more guide RNAs target the base editor to effect modification of the nucleobases of the C3 polynucleotide that disrupts a splice site within the C3 polynucleotide. In (b), the deaminase domain is a TadA variant (TadA*) containing a combination of modifications listed in Tables 5A, 5B, 5C, 5D, 5E, 6A, 6B, 6C, 6D, 6E, 6F, or 7, and the TadA* is not TadA*7.9 or TadA*7.10. In (c), the one or more guide RNAs contain a nucleic acid sequence containing at least 10 to 23 consecutive nucleotides of a spacer nucleic acid sequence listed in any one of Tables 1A, 1B, 1C, 1D, 1E, 1F, and 2. In (d), the one or more guide RNAs target the base editor to the following reference sequences: C3 amino acid sequence

[0008] In another aspect, the present disclosure features a method of treating a disease or disorder in a subject in need of treatment for an inappropriate activation of the complement system. The method comprises modifying the nucleobases of a complement component 3 (C3) polynucleotide in the subject by administering to the subject one or more guide RNAs, or one or more polynucleotides encoding one or more guide RNAs, and a base editor containing a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, thereby treating the disease or disorder. The method further comprises (a) and (b). In (a), the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, TadA*8.20_V82T_Y147T_Q154S, rAPOBEC1, and ppAPOBEC. In (b), the one or more guide RNAs contain a spacer corresponding to a guide polynucleotide selected from one or more of gRNA661, gRNA662, gRNA676, gRNA695, gRNA696, gRNA701, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA1793, gRNA1798, gRNA3342, gRNA3343, and gRNA3345.

[0009] In another aspect, the present disclosure features a cell produced by the method of any aspect of the present disclosure, or an embodiment thereof.

[0010] In another aspect, the present disclosure features a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding a base editor, and one or more guide polynucleotides, or a base editor system comprising one or more polynucleotides encoding one or more guide polynucleotides, wherein the one or more guide polynucleotides contain at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleic acid bases of the spacers listed in Tables 1A, 1B, 1C, 1D, 1E, 1F, or 2.

[0011] In another aspect, the present disclosure features a polynucleotide or set of polynucleotides encoding a base editor of any aspect of the present disclosure, or an embodiment thereof, a base editor system, or a component thereof.

[0012] In another aspect, the present disclosure features a kit comprising a base editor system or base editor of any aspect of the present disclosure, and / or one or more polynucleotides encoding the same or a component thereof, or an embodiment thereof.

[0013] In another aspect, the present disclosure features a lipid nanoparticle comprising a base editor system or base editor of any aspect of the present disclosure, and / or one or more polynucleotides encoding the same or a component thereof, or an embodiment thereof.

[0014] In another aspect, the present disclosure features a kit containing a base editor or a base editor system containing one or more polynucleotides encoding a base editor, wherein the base editor contains a nucleic acid programmable DNA binding protein domain (napDNAbp), a deaminase domain, and one or more guide polynucleotides, or one or more polynucleotides encoding one or more guide polynucleotides, and the one or more guide polynucleotides contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleic acid bases of the spacers listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2.

[0015] In another aspect, the present disclosure features a pharmaceutical composition containing an effective amount of a base editor system of any aspect of the present disclosure or an embodiment thereof.

[0016] In another aspect, the present disclosure features a pharmaceutical composition containing a base editor or a base editor system containing one or more polynucleotides encoding a base editor, wherein the base editor contains a nucleic acid programmable DNA binding protein domain (napDNAbp), a deaminase domain, and one or more guide polynucleotides, or one or more polynucleotides encoding one or more guide polynucleotides, and the one or more guide polynucleotides contain at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleic acid bases of the spacers listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2.

[0017] In another aspect, the present disclosure features a guide polynucleotide containing a spacer sequence listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 2.

[0018] In another aspect, the present disclosure features a base editor comprising a nucleic acid programmable DNA binding protein (napDNAbp) domain and a deaminase domain, or one or more polynucleotides encoding the base editor, and one or more guide polynucleotides, or a base editor system comprising one or more polynucleotides encoding one or more guide polynucleotides, a) the guide polynucleotide contains a spacer corresponding to gRNA661, the napDNAbp domain is SpCas9 or SpCas9 having amino acid modifications A1283D and E1250K, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.8 having amino acid modification V82T, TadA*8.8 having amino acid modifications V82T, Y147T, and Q154S, TadA*8.20 having amino acid modification V82T, and TadA*8.20 having amino acid modifications V82T, Y147T, and Q154S, or b) the guide polynucleotide contains a spacer corresponding to gRNA676, the napDNAbp domain is SpCas9, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.8 having amino acid modifications V82T, Y147T, and Q154S, TadA*8.20 having amino acid modification V82T, and TadA*8.20 having amino acid modifications V82T, Y147T, and Q154S, or c) the guide polynucleotide contains a spacer corresponding to gRNA696, the napDNAbp domain is SpCas9 or SpCas9 having amino acid modifications A1283D and E1250K, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.20 having amino acid modification V82T, and TadA*8.20 having amino acid modifications V82T, Y147T, and Q154S, or d) The guide polynucleotide contains a spacer corresponding to gRNA715, the napDNAbp domain is SpCas9 or SpCas9-VRQR, and the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, and TadA*8.20 having the amino acid modification V82T, or e) The guide polynucleotide contains a spacer corresponding to gRNA821, the napDNAbp is SpCas9, the deaminase domain is a cytidine deaminase domain, and the base editor further contains two uracil glycosylase inhibitors, or f) The guide polynucleotide contains a spacer corresponding to gRNA827, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8, or g) The guide polynucleotide contains a spacer corresponding to gRNA828, the napDNAbp is h) SpCas9, and the deaminase domain is TadA*8.8, or h) The guide polynucleotide contains a spacer corresponding to gRNA829, the napDNAbp is SpCas9, and the deaminase domain is TadA*8.8, or i) The guide polynucleotide contains a spacer corresponding to gRNA837, the napDNAbp is SpCas9-VRQR, and the deaminase domain is TadA*8.8, or j) The guide polynucleotide contains a spacer corresponding to gRNA838, the napDNAbp is SpCas9-VRQR, and the deaminase domain is TadA*8.8, or k) The guide polynucleotide contains a spacer corresponding to gRNA3342, the napDNAbp is SpCas9, and the deaminase is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.20, TadA*8.8 having amino acid modifications V82T, Y147T, and Q154S, TadA*8.17 having amino acid modification V82T, TadA*8.17 having amino acid modifications V82T, Y147T, and Q154S, TadA*8.20 having amino acid modification V82T, and TadA*8.20 having amino acid modifications V82T, Y147T, and Q154S, or l) The guide polynucleotide contains a spacer corresponding to gRNA3343, and the napDNAbp is i) SpCas9-MQKFRAER, ii) SpCas9-MQKFRAER having a combination of amino acid modifications I322V, S409I, E427G, R654L, R753G, R1114G, and R1337K, iii) SpCas9-MQKFRAER having a combination of amino acid modifications I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D, and E1250K, and iv) selected from one or more of SpCas9-MQKFRAER having a combination of amino acid modifications I322V, S409I, E427G, R654L, R753G, and R1114G, the deaminase domain is TadA*8.20 or TadA*7.10, or m) The guide polynucleotide contains a spacer corresponding to gRNA3345, the napDNAbp domain is SaCas9-KHH, the deaminase domain is a cytidine deaminase domain, and the base editor further contains two uracil glycosylase inhibitors.

[0019] In another aspect, d is characterized by a method for modifying the nucleobases of a complement component 3 (C3) polynucleotide. This method includes contacting the C3 polynucleotide with a base editor system of any aspect of the present disclosure or an embodiment thereof.

[0020] In another aspect, the present disclosure is characterized by a method of treating a disease or disorder in a subject in need of treatment for an inappropriate activation of the complement system. This method includes modifying the C3 polynucleotide in the subject by administering to the subject a base editor system of any aspect of the present disclosure or an embodiment thereof.

[0021] In any aspect of the present disclosure, or an embodiment thereof, the splice site corresponds to any one of the protospacers listed in Table 1A, 1B, or 1C.

[0022] In any aspect of the present disclosure, or an embodiment thereof, the base editor is selected from one or more of ABE8.8, ABE8.13, ABE8.17, ABE8.20, ABE8.8_V82T, ABE8.8_V82T_Y147T_Q154S, ABE8.17_V82T, ABE8.17_V82T_Y147T_Q154S, ABE8.20_V82T, ABE8.20_V82T_Y147T_Q154S, BE4, and those base editors listed in Table 1A, 1B, 1C, 1D, 1E, 1F, 2, and 28 - 15.

[0023] In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides target the base editor to the following reference sequences: C3 amino acid sequence

[0024] In any aspect of the present disclosure, or an embodiment thereof, the editing rate for the base editor system exceeds 35%.

[0025] In any aspect of the present disclosure, or an embodiment thereof, the guide RNA or guide polynucleotide contains a spacer that contains or consists of about 19 to about 23 nucleotides. In any aspect of the present disclosure, or an embodiment thereof, the spacer contains or consists of 21 nucleotides.

[0026] In any aspect of the present disclosure, or an embodiment thereof, the modification of the nucleobase results in a modification to the encoded amino acid residue, and the modification disrupts opsonization by C3. In any aspect of the present disclosure, or an embodiment thereof, the modification of the nucleobase disrupts the splicing of the C3 transcript. In any aspect of the present disclosure, or an embodiment thereof, the modification of the nucleobase results in a modification to the encoded amino acid residue, and the modification disrupts the cleavage of the C3 polypeptide by the C3 convertase.

[0027] In any aspect of the present disclosure, or an embodiment thereof, the C3 polynucleotide is intracellular. In an embodiment, the cell is a mammalian cell. In an embodiment, the cell is a primate cell. In an embodiment, the primate is a human. In an embodiment, the cell is a retinal cell or other cell of the eye, a cell of the CNS, or a hepatocyte.

[0028] In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides target a base editor to effect a modification of the nucleobase of the C3 polynucleotide that disrupts a splice site in the C3 polynucleotide, and the splice site is selected from one or more of those splice sites corresponding to any of the protospacers listed in Table 1A, 1B, 1C, 1D, 1E, 1F, or 1G.

[0029] In any aspect of the present disclosure, or an embodiment thereof, the editing rate exceeds 50%.

[0030] In any aspect of the present disclosure, or an embodiment thereof, the C3 activity and / or expression is reduced by at least about 50% compared to a control without modification. In any aspect of the present disclosure, or an embodiment thereof, the C3 activity and / or expression is reduced by at least about 60% compared to an unmodified control cell or subject.

[0031] In any aspect of the present disclosure, or an embodiment thereof, inappropriate activation of the complement system is associated with increased levels of one or more of inflammation, the presence of autoantibodies, neurodegeneration, and microthrombosis. In any aspect of the present disclosure, or an embodiment thereof, inappropriate activation of the complement system is associated with damage to the central nervous system (CNS), eyes, gastrointestinal system, pulmonary system, musculoskeletal system, circulatory system, integumentary system, blood cells, thyroid, kidneys, joints, gastrointestinal system, or transplanted organs.In any aspect of the present disclosure, or an embodiment thereof, the disease or disorder is selected from one or more of acute antibody-related rejection, age-related macular degeneration, allergic bronchopulmonary aspergillosis, allergic neuritis, allergic rhinitis, amyotrophic lateral sclerosis, anaphylaxis, and episcleritis, atopic dermatitis, atypical hemolytic uremic syndrome (aHUS), autoimmune hemolytic anemia, Behçet's disease, bronchiolitis, C3 glomerulopathy, central nervous system (CNS) inflammatory disorder, choroidal neovascularization (CNV), choroiditis, chronic allograft vasculopathy, chronic hepatitis, chronic myositis, chronic pain, chronic pancreatitis, chronic urticaria, Churg-Strauss syndrome, conjunctivitis, cyclitis, demyelinating disease, dermatitis, dermatomyositis, diabetic retinopathy, encephalitis, eosinophilic pneumonia, geographic atrophy, giant cell arteritis, glaucoma, glomerulonephritis, graft or transplant rejection or disorder, HELLP syndrome, Henoch-Schönlein purpura, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), IgA nephropathy (IgAN), inflammatory bowel disease, inflammatory joint condition, inflammatory skin disease, infusion reaction, interstitial pneumonia, iridocyclitis, iritis, ischemia / reperfusion injury, Kawasaki disease, keratitis, lupus nephritis, membranoproliferative glomerulonephritis (MPGN), meningitis, microscopic polyangiitis, myasthenia gravis, myocarditis, nasal polyps, neuromyelitis optica, neuropathic pain, ocular inflammation, osteoarthritis, pancreatitis, panniculitis, paroxysmal nocturnal hemoglobinuria (PNH), pars planitis, pemphigoid, pemphigus, polyarteritis nodosa, polymyositis, primary membranous nephropathy, proliferative vitreoretinopathy, proteinuria, psoriasis, pulmonary fibrosis, renal disease, respiratory distress syndrome, retinal neovascularization (RNV), retinopathy of prematurity, rheumatoid arthritis (RA), rhinosinusitis, sarcoid, sarcoidosis, episcleritis, scleroderma, scleroderma-dermatomyositis, sclerosis, Sjögren's syndrome, systemic lupus erythematosus, systemic scleroderma, Takayasu arteritis, thyroiditis, thyroidoisis, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis.

[0032] In any aspect of the present disclosure, or an embodiment thereof, the administration is local administration. In an embodiment, the local administration is administration to the eye, cerebrospinal fluid, or liver.

[0033] In any aspect of the present disclosure, or an embodiment thereof, the C3 polynucleotide contacts two or more guide RNAs or guide polynucleotides, and each guide RNA or guide - polynucleotide binds to a different position within the C3 polynucleotide.

[0034] In any aspect of the present disclosure, or an embodiment thereof, the deaminase is adenosine deaminase or cytidine deaminase. In an embodiment, adenosine deaminase converts a target A●T within the C3 polynucleotide to G●C. In an embodiment, cytidine deaminase converts a target C●G within the C3 polynucleotide to T●A.

[0035] In any aspect of the present disclosure, or an embodiment thereof, the modification of the nucleobase results in a premature stop codon.

[0036] In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain contains a Cas9, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, or Cas12j / CasΦ polynucleotide or a functional portion thereof. In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain contains a Cas9 polynucleotide having endonuclease activity on both strands of a double-stranded DNA molecule or a functional portion thereof. In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain contains inactive Cas9 (dCas9) or Cas9 nickase (nCas9). In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain is a modified Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), modified Streptococcus pyogenes Cas9 (SpCas9), or a variant thereof. In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain contains a variant of SpCas9 having modified protospacer adjacent motif (PAM) specificity.

[0037] In any aspect of the present disclosure, or an embodiment thereof, the cytidine deaminase domain is an APOBEC deaminase domain or a derivative thereof. In any aspect of the present disclosure, or an embodiment thereof, the adenosine deaminase domain is a TadA deaminase domain.

[0038] In any aspect of the present disclosure, or an embodiment thereof, the guide RNA or guide polynucleotide contains a nucleic acid analog. In any aspect of the present disclosure, or an embodiment thereof, the guide RNA or guide polynucleotide contains one or more of 2'-OMe and phosphorothioate.

[0039] In any aspect of the present disclosure, or an embodiment thereof, the base editor further contains one or more uracil glycosylase inhibitors (UGIs). In any aspect of the present disclosure, or an embodiment thereof, the base editor further contains one or more nuclear localization sequences (NLSs).

[0040] In any aspect of the present disclosure, or an embodiment thereof, napDNAbp is a nuclease-inactive variant or a nickase variant.

[0041] In any aspect of the present disclosure, or an embodiment thereof, the deaminase domain can deaminate cytosine or adenine in DNA. In any aspect of the present disclosure, or an embodiment thereof, the deaminase domain is a cytidine deaminase domain. In an embodiment, the cytidine deaminase is an APOBEC deaminase or a derivative thereof. In any aspect of the present disclosure, or an embodiment thereof, the deaminase domain is an adenosine deaminase domain. In an embodiment, the adenosine deaminase is a TadA*8 variant. In an embodiment, the adenosine deaminase is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In any aspect of the present disclosure, or an embodiment thereof, the deaminase is an adenosine deaminase selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, and TadA*8.20_V82T_Y147T_Q154S. In any aspect of the present disclosure, or an embodiment thereof, the deaminase domain is a monomer or a heterodimer.In any aspect of the present disclosure, or an embodiment thereof, the deaminase domain is selected from one or more of TadA*8.8, TadA*8.13, TadA*8.17, TadA*8.20, TadA*8.8_V82T, TadA*8.8_V82T_Y147T_Q154S, TadA*8.17_V82T, TadA*8.17_V82T_Y147T_Q154S, TadA*8.20_V82T, and TadA*8.20_V82T_Y147T_Q154S, rAPOBEC1, and ppAPOBEC.

[0042] In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides contain a spacer corresponding to a guide polynucleotide selected from one or more of gRNA676, gRNA661, gRNA715, gRNA821, gRNA837, gRNA838, gRNA827, gRNA828, gRNA829, gRNA3342, gRNA3343, and gRNA3345.

[0043] In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain contains a Cas9 variant. In any aspect of the present disclosure, or an embodiment thereof, the Cas9 variant contains one or more of the amino acid modifications A1283D and E1250K relative to the SpCas9 reference amino acid sequence. In any aspect of the present disclosure, or an embodiment thereof, the Cas9 variant is a combination of the following amino acid modifications relative to the following spCas9 reference amino acid sequence: I322V, S409I, E427G, R654L, R753G, and R1114G; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, and R1337K; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, and A1283D; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R220A, and R221A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A, and Q768A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, Q1136Y, R1337K, A1283D, R765A, Q768A, K772A, and K775A; I322V, S409I, E427G, R654L, R753G, R1114G, M1135L, R1337K, A1283D, and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, A1283D, and E1250K; I322V, S409I, E427G, R654L, R753G, R1114G, Q1136Y, and R1337K; and one or more of I322V, S409I, E427G, R654L, R753G, R1114G, and R1337K. In any aspect of the present disclosure, or an embodiment thereof, the Cas9 variant is SaCas9-KHH, SpCas9-MQKFRAER, or SpCas9-VRQR.

[0044] In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides contain nucleic acid analogs. In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides contain one or more of 2'-OMe and phosphorothioate. In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides have the following nucleotide sequences: Terminal-modified SpCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsmUsmUsmU (SEQ ID NO: 943), Terminal-modified SaCas9 guide polynucleotide: mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 944), H M01: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 855), H M07: mNsmNsmNsmNmNmNmNmNmNmNNNNNNNNNNNmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 856), NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsmU (SEQ ID NO: 857), LONG EST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 859), NLS + LONG ST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 860), or LONGEST + G OLD: contains one of the following: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 861). The term "mN" indicates a 2'-OMe modification of nucleotide "N", and "Ns" indicates that nucleotide "N" is linked to the next nucleotide by phosphorothioate (PS). In any aspect of the present disclosure, or an embodiment thereof, one or more or one or more guide RNAs or guide polynucleotides are covalently attached at the 5' end to a peptide having the following amino acid sequence: CKRTADGSEFESPKKKRKV (SEQ ID NO: 858).

[0045] In any aspect of the present disclosure, or an embodiment thereof, the base editor contains a linker peptide between the deaminase domain and the napDNAbp domain, and the linker peptide contains the amino acid sequence KGPKPKKEESEK (SEQ ID NO: 940).

[0046] In any aspect of the present disclosure, or an embodiment thereof, one or more guide RNAs or guide polynucleotides target the base editor to effect a modification of a nucleic acid base at the start codon of a C3 polynucleotide.

[0047] In any aspect of the present disclosure, or an embodiment thereof, the method includes administering to a subject a lipid nanoparticle containing one or more guide RNAs or guide polynucleotides and an mRNA molecule encoding a base editor.

[0048] In any aspect of the present disclosure, or an embodiment thereof, the method further includes administering to the subject one or more guide RNAs or guide polynucleotides, or one or more polynucleotides encoding one or more guide RNAs or guide polynucleotides, and a base editor, or one or more polynucleotides encoding a base editor, a second time. The second administration is about or at least about 1 month, 6 months, or 1 year after the first administration.

[0049] In any aspect of the present disclosure, or an embodiment thereof, the napDNAbp domain is a nickase. In any aspect of the present disclosure, or an embodiment thereof, the polynucleotide encoding the base editor is codon-optimized. In any aspect of the present disclosure, or an embodiment thereof, the kit further contains written instructions regarding the use of the kit in the treatment of a disease or disorder associated with inappropriate activation of the complement system in a subject. In any aspect provided herein, or an embodiment thereof, the method is not a process for modifying the genetic identity of the human germline.

[0050] Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide one of ordinary skill in the art with general definitions of many of the terms used in this disclosure. Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings set forth below unless otherwise specified.

[0051] The term "complement component 3 (C3) polypeptide" means a C3 protein that is capable of activating the complement system and has at least about 85% amino acid sequence identity with Ensembl accession number ENSP00000245907, or a fragment thereof, provided below. >ENSG00000125730:ENST00000245907.11 peptide:ENSP00000245907 pep:protein_coding

[0052] The "complement component 3 (C3) polynucleotide" means a nucleic acid molecule encoding the C3 polypeptide, as well as introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, the C3 polynucleotide is a genomic sequence, cDNA, mRNA, or gene related to and / or required for C3 expression. Exemplary C3 nucleotide sequences from Homo sapiens are provided below (see Ensembl accession numbers ENST00000245907.11 and ENSG00000125730):

[0053] >ENSG00000125730:ENST00000245907.11 cds:protein_coding

[0054] >19 dna: Chromosome Chromosome: GRCh38: 19: 6677704: 6730562: -1 (The exon sequences are in bold) TIFF2025524415000001.tif 177152 TIFF2025524415000002.tif 216149 TIFF2025524415000003.tif 217148 TIFF2025524415000004.tif 223150 TIFF2025524415000005.tif 216145 TIFF2025524415000006.tif 215149 TIFF2025524415000007.tif 215147 TIFF2025524415000008.tif 223150 TIFF2025524415000009.tif 217151 TIFF2025524415000010.tif 218152 TIFF2025524415000011.tif 217146 TIFF2025524415000012.tif 219148 TIFF2025524415000013.tif 222153 TIFF2025524415000014.tif 223149 TIFF2025524415000015.tif 221150 TIFF2025524415000016.tif 222152 TIFF2025524415000017.tif 219147 TIFF2025524415000018.tif 218149 TIFF2025524415000019.tif 221149 TIFF2025524415000020.tif 228151 TIFF2025524415000021.tif 217151 TIFF2025524415000022.tif 222150 TIFF2025524415000023.tif 224149 TIFF2025524415000024.tif 226151 TIFF2025524415000025.tif 222151 TIFF2025524415000026.tif 81159

[0055] "Adenine" or "9H-purin-6-amine" has the molecular formula C5H5N5 and the structure [Chemical formula] It has and means a purine nucleobase corresponding to CAS number 73 - 24 - 5.

[0056] "Adenosine" or "4 - amino - 1 - [(2R,3R,4S,5R) - 3,4 - dihydroxy - 5 - (hydroxymethyl)oxolan - 2 - yl]pyrimidin - 2(1H) - one" is bonded to ribose sugar via a glycosidic bond and has the structure [Chemical formula] It has and means an adenine molecule corresponding to CAS number 65 - 46 - 3. Its molecular formula is C 10 H 13 N5O4.

[0057] "Adenosine deaminase" or "adenine deaminase" means a polypeptide or a fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., modified adenosine deaminases, evolved adenosine deaminases) can be derived from any organism (e.g., eukaryote, prokaryote), including, but not limited to, algae, bacteria, fungi, plants, invertebrates (e.g., insects), and vertebrates (e.g., amphibians, mammals). In some embodiments, the adenosine deaminase is an adenosine deaminase variant having one or more modifications and can deaminate both adenine and cytosine in a target polynucleotide (e.g., DNA, RNA) and may be referred to as a "dual deaminase." Non-limiting examples of dual deaminases include those described in PCT / US22 / 22050. In some embodiments, the target polynucleotide is single-stranded or double-stranded. In some embodiments, the adenosine deaminase variant can deaminate both adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant can deaminate both adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variant can deaminate both adenine and cytosine in RNA. In embodiments, the adenosine deaminase variant is selected from those described in PCT / US2020 / 018192, PCT / US2020 / 049975, PCT / US2017 / 045381, and PCT / US2020 / 028568, the entire contents of which are hereby incorporated by reference in their entirety for all purposes.

[0058] "Adenosine deaminase activity" means catalyzing the deamination of adenine or adenosine in a polynucleotide to guanine. In some embodiments, the adenosine deaminase variants provided herein maintain adenosine deaminase activity (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)).

[0059] "Adenosine base editor (ABE)" means a base editor that includes adenosine deaminase.

[0060] "Adenosine base editor (ABE) polynucleotide" means a polynucleotide that encodes an ABE. "Adenosine base editor 8 (ABE8) polypeptide" or "ABE8" means a base editor as defined herein that includes an adenosine deaminase or adenosine deaminase variant, wherein the adenosine deaminase or adenosine deaminase variant includes one or more of the modifications listed in Table 5B, one of the combinations of modifications listed in Table 5B, or modifications at one or more of the amino acid positions listed in Table 5B, such modifications being relative to the following reference sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 1), or corresponding positions in another adenosine deaminase. In embodiments, ABE8 includes a modification at amino acid 82 and / or 166 of SEQ ID NO: 1. In some embodiments, ABE8 includes additional modifications as described herein relative to the reference sequence.

[0061] The "Adenosine Base Editor 8 (ABE8) polynucleotide" means a polynucleotide encoding the ABE8 polypeptide.

[0062] "Administering" is referred to herein as providing one or more of the compositions described herein to a patient or subject.

[0063] "Administering" is referred to herein as providing one or more of the compositions described herein to a patient or subject. By way of example, but not limitation, administration of a composition (e.g., injection) can be performed by intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection. One or more such routes can be used. Parenteral administration can be performed, for example, by bolus injection or by gradually perfusing over time. In some embodiments, parenteral administration includes infusion or injection into the vasculature, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, and intrasternal. Alternatively or concurrently, administration can be performed by an oral route.

[0064] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof. "Modification" means a change in the level, structure, or activity of an analyte, gene, or polypeptide detected by standard methods known in the art, such as the methods described herein. As used herein, modification includes a change in the expression level (e.g., increase or decrease). In embodiments, the increase or decrease in the expression level is 10%, 25%, 40%, 50% or more. In some embodiments, modification includes insertion, deletion, or substitution of a nucleobase or amino acid (e.g., by genetic manipulation).

[0065] "Remission" means reducing, suppressing, attenuating, decreasing, arresting, or stabilizing the onset or progression of a disease.

[0066] "Analog" means a molecule having similar functional or structural features but not being identical. For example, a polypeptide analog has specific biochemical modifications that enhance the function of the analog relative to the corresponding natural polypeptide while retaining the biological activity of the natural polypeptide. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog, for example, without modifying ligand binding. An analog may contain non-natural amino acids.

[0067] "Base editor (BE)" or "nucleic acid base editor polypeptide (NBE)" means an agent that binds to a polynucleotide and has nucleic acid base modification activity. In various embodiments, a base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9 or Cpf1) in combination with a nucleic acid base modification polypeptide (e.g., a deaminase) and a guide polynucleotide (e.g., guide RNA (gRNA)). Representative nucleic acid and protein sequences of base editors include sequences having about or at least about 85% sequence identity to any of the base editor sequences provided in the Sequence Listing, e.g., sequences corresponding to SEQ ID NOs: 2-11.

[0068] "Base editing activity" means acting to chemically modify a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, e.g., conversion of a target C●G to T●A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, e.g., conversion of A●T to G●C. The term "base editor system" refers to an intermolecular complex for editing nucleic acid bases of a target nucleotide sequence. In various embodiments, a base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating a nucleic acid base in a target nucleotide sequence, and (2) one or more guide polynucleotides (e.g., guide RNA) combined with the polynucleotide programmable nucleotide binding domain. In various embodiments, a base editor (BE) system comprises a nucleic acid base editor domain selected from adenosine deaminase or cytidine deaminase, and a domain having nucleic acid sequence-specific binding activity. In some embodiments, a base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleic acid bases in a target nucleotide sequence, and (2) one or more guide RNAs combined with the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE), or a cytidine or cytosine base editor (CBE). In some embodiments, a base editor system (e.g., a base editor system comprising cytidine deaminase) comprises a uracil glycosylase inhibitor or other agent or peptide (e.g., a uracil stabilizing protein, e.g., as provided in WO2022015969, the disclosure of which is hereby incorporated by reference in its entirety for all purposes) that inhibits the inosine base excision repair system.

[0069] "BE3" refers to a base editor that includes a cytidine deaminase domain, a nucleic acid programmable DNA binding protein domain (napDNAbp), and a single uracil glycosylase inhibitor domain. In some embodiments, the npDNAbp is a SpCas9(D10A) nickase domain.

[0070] "BE4" refers to a base editor that includes a cytidine deaminase domain, a nucleic acid programmable DNA binding protein domain (napDNAbp), and two uracil glycosylase inhibitor domains. In some embodiments, the npDNAbp is a SpCas9(D10A) nickase domain.

[0071] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease that includes a Cas9 protein or a fragment thereof (e.g., a protein that includes an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). The Cas9 nuclease may also be referred to as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease.

[0072] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that has common properties. A functional way to define the common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Schulz, G.E. and Schirmer, R.H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such an analysis, groups of amino acids can be defined when the amino acids within the group are preferentially exchanged with each other, and thus the effects on the overall protein structure are most similar to each other (Schulz, G.E. and Schirmer, R.H., supra). Non-limiting examples of conservative mutations include amino acid substitutions, such as the substitution of arginine with lysine so as to maintain a positive charge, and vice versa, the substitution of aspartic acid with glutamic acid so as to maintain a negative charge, and vice versa, the substitution of threonine with serine so as to maintain a free -OH, and the substitution of asparagine with glutamine so as to maintain a free -NH2.

[0073] As used interchangeably herein, the term "coding sequence" or "protein coding sequence" refers to a segment of a polynucleotide that encodes a protein. The coding sequence may also be referred to as an open reading frame. A region or sequence is bounded proximally at the 5' end by a start codon and proximally at the 3' end by a stop codon. Stop codons useful in the base editors described herein include: TAG, TAA, and TGA.

[0074] "Complex" means a combination of two or more molecules whose interaction depends on intermolecular forces. Non-limiting examples of intermolecular forces include covalent interactions and non-covalent interactions. Non-limiting examples of non-covalent interactions include hydrogen bonds, ionic bonds, halogen bonds, hydrophobic bonds, van der Waals interactions (e.g., dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces), and π effects. In one embodiment, the complex comprises a polypeptide, a polynucleotide, or a combination of one or more polypeptides and one or more polynucleotides. In one embodiment, the complex comprises one or more polypeptides that associate to form a base editor (e.g., a nucleic acid programmable DNA binding protein such as Cas9, and a base editor comprising a deaminase) and a polynucleotide (e.g., a guide RNA). In one embodiment, the complex is held together by hydrogen bonds. It should be understood that one or more components of the base editor (e.g., a deaminase, or a nucleic acid programmable DNA binding protein) can associate covalently or non-covalently. As an example, the base editor can comprise a deaminase covalently bound to a nucleic acid programmable DNA binding protein (e.g., by a peptide bond). Alternatively, the base editor can comprise a deaminase and a nucleic acid programmable DNA binding protein that associate non-covalently (e.g., when one or more components of the base editor are supplied in trans and associate directly or through another molecule such as a protein or nucleic acid). In one embodiment, one or more components of the complex are held together by hydrogen bonds.

[0075] "Cytosine" or "4-aminopyrimidin-2(1H)-one" means a purine nucleobase having the molecular formula C4H5N3O and the structure

Chemical formula

[0076] "Cytidine" is attached to a ribose sugar via a glycosidic bond and has the structure [Chem.] has, and means a cytosine molecule corresponding to the CAS number 65-46-3. Its molecular formula is C9H 13 N3O5.

[0077] "Cytidine base editor (CBE)" means a base editor containing cytidine deaminase.

[0078] "Cytidine base editor (CBE) polynucleotide" means a polynucleotide encoding CBE.

[0079] As used herein, the term "cytidine deaminase" or "cytosine deaminase" means a polypeptide or fragment thereof that can deaminate cytidine or cytosine. In embodiments, the cytidine or cytosine is present in a polynucleotide. In one embodiment, the cytidine deaminase converts cytosine to uracil or 5-methylcytosine to thymine. The terms "cytidine deaminase" and "cytosine deaminase" are used interchangeably throughout this application. Petromyzon marinus cytosine deaminase 1 (PmCDA1) (SEQ ID NOs: 13-14), activation-induced cytidine deaminase (AICDA) (SEQ ID NOs: 15-21), and APOBEC (SEQ ID NOs: 12-61) are exemplary cytidine deaminases. Further exemplary cytidine deaminase (CDA) sequences are shown in the Sequence Listing as SEQ ID NOs: 62-189. Non-limiting examples of cytidine deaminases include those described in PCT / US20 / 16288, PCT / US2018 / 021878, 180802-021804 / PCT, PCT / US2018 / 048969, and PCT / US2016 / 058344. "Cytosine deaminase activity" means catalyzing the deamination of cytosine or cytidine. In one embodiment, a polypeptide having cytosine deaminase activity converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil (i.e., C to U) or 5-methylcytosine to thymine (i.e., 5mC to T). In some embodiments, the cytosine deaminases provided herein have high cytosine deaminase activity (e.g., at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more) relative to a reference cytosine deaminase.

[0080] As used herein, the terms "deaminase" or "deaminase domain" refer to a protein or fragment thereof that catalyzes a deamination reaction.

[0081] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence modifications in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an indel is detected.

[0082] "Detectable label" means a composition that enables the detection of a molecule of interest when linked to the molecule of interest, through spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (e.g., enzymes commonly used in enzyme-linked immunosorbent assay (ELISA)), biotin, digoxigenin, or haptens.

[0083] "Disease" means any pathological condition or disorder that impairs or interferes with the normal function of cells, tissues, or organs. Exemplary diseases include diseases that can be treated and that result in a reduction in the activity and / or expression of complement 3 polypeptide in cells, involving the introduction of a modification into the complement component 3 polynucleotide in the cells. In some cases, the disease is a disease associated with inappropriate activation of the complement system in a subject. Non-limiting examples of diseases associated with inappropriate activation of the complement system include blood disorders, transplantation or graft rejection, inflammatory diseases or disorders, eye diseases or disorders, kidney diseases or disorders, heart disorders, respiratory diseases or disorders, autoimmune disorders, inflammatory bowel diseases or disorders, arthritis, neurodegenerative diseases or disorders, musculoskeletal diseases or disorders associated with inflammation, disorders affecting the integumentary system, diseases or disorders affecting the central nervous system, diseases or disorders affecting the circulatory system, diseases or disorders affecting the gastrointestinal system, diseases or disorders affecting the thyroid, chronic pain, allergies, and lung diseases. Further non-limiting examples of diseases associated with inappropriate activation of the complement system include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), HELLP syndrome, autoimmune hemolytic anemia, transplant rejection, ischemia / reperfusion injury, transplant injury, hyperacute rejection, graft rejection or disorder, acute antibody-mediated rejection reaction, chronic inflammation, chronic allograft vasculopathy, chronic rejection of transplantation or graft, age-related macular degeneration (e.g., wet or dry age-related macular degeneration), diabetic retinopathy, glaucoma, uveitis, autoimmune diseases, myasthenia gravis, neuromyelitis optica (NMO), kidney diseases, membranoproliferative glomerulonephritis (MPGN) (e.g., MPGNType I, II, or III), IgA nephropathy (IgAN), primary membranous nephropathy, C3 glomerulopathy, proteinuria, neurodegenerative diseases, neuropathic pain, rhinosinusitis, nasal polyps, cancer, sepsis, respiratory distress syndrome, anaphylaxis, infusion reactions, respiratory diseases or disorders (e.g., asthma or chronic obstructive pulmonary disease (COPD), or idiopathic pulmonary fibrosis, or asthma), Th2-related disorders (e.g., disorders associated with high levels or hyperactivation of the Th2 subtype of CD4+ helper T cells), disorders associated with high levels or inappropriate activation of the Th17 subtype of CD4+ helper T cells, inflammatory bowel diseases (e.g., Crohn's disease or ulcerative colitis), inflammatory skin diseases, chronic inflammatory diseases, psoriasis, atopic dermatitis, systemic sclerosis, scleroderma, Behcet's disease, dermatomyositis, polymyositis, multiple sclerosis (MS), dermatitis, meningitis, encephalitis, uveitis, osteoarthritis, lupus nephritis, rheumatoid arthritis (RA), Sjögren's syndrome (Sjoren’ssyndrome), vasculitis, central nervous system (CNS) inflammatory disorders, chronic hepatitis, chronic pancreatitis, glomerulonephritis, sarcoidosis, thyroid disorders, pathological immune responses to tissue / organ transplantation, bronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), periodontitis, gingivitis, disorders associated with excessive or inappropriate activation of IgE-producing cells, neuromyelitis optica, pemphigoid, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener's granulomatosis, bronchiolitis obliterans, allergic rhinitis, inflammatory joint conditions (e.g., arthritis such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthritis Reiter's syndrome, or gout), dermatomyositis, polymyositis, chronic myositis, pemphigus, systemic lupus erythematosus, dermatomyositis, scleroderma, scleroderma dermatomyositis, Sjögren's syndrome, chronic urticaria, demyelinating diseases, amyotrophic lateral sclerosis, chronic pain, stroke, allergic neuritis, Huntington's disease, Alzheimer's disease, Parkinson's disease, cardiovascular diseases, polyarteritis nodosa, Wegener's granulomatosis, giant cell arteritis, Churg-Strauss syndrome, microscopic polyangiitis, Henoch-Schönlein purpura, Takayasu arteritis, Kawasaki disease, Behçet's disease, ulcerative colitis, thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease, postpartum thyroiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis, glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis), panniculitis, eye disorders, choroidal neovascularization (CNV), retinal neovascularization (RNV), eye inflammation, retinopathy of prematurity, proliferative vitreoretinopathy, uveitis, keratitis, conjunctivitis, and scleritis, geographic atrophy, conjunctivitis, keratitis, scleritis, iritis, iridocyclitis, cyclitis, pars planitis, choroiditis, persistent asthma, and allergic asthma.

[0084] "Dual editing activity" or "dual deaminase activity" means having adenosine deaminase and cytidine deaminase activities. In one embodiment, a base editor having dual editing activity has both A→G and C→T activities, and the two activities are approximately equal to each other or within the range of about 10% or 20%. In another embodiment, the dual editor has an A→G activity that is about 10% or 20% or less greater than the C→T activity. In another embodiment, the dual editor has an A→G activity that is about 10% or 20% or less less than the C→T activity. In some embodiments, the adenosine deaminase variant has mainly cytidine deaminase activity and little or no adenosine deaminase activity. In some embodiments, the adenosine deaminase variant has cytidine deaminase activity and no significant or detectable adenosine deaminase activity.

[0085] "Effective amount" means the amount of a drug or active compound, such as a base editor described herein, required to alleviate the symptoms of a disease in an untreated patient or an individual without the disease, i.e., a healthy individual, or is an amount of a drug or active compound sufficient to induce a desired biological response. The effective amount of the active compound(s) used to practice the present disclosure for the therapeutic treatment of a disease will vary depending on the mode of administration, age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amounts and dosing regimen. Such amounts are referred to as "effective" amounts. In one embodiment, the effective amount is the amount of a base editor of the present disclosure sufficient to introduce a modification in a gene of interest in a cell (e.g., an in vitro or in vivo cell). In one embodiment, the effective amount is the amount of a base editor required to achieve a therapeutic effect. Such a therapeutic effect need not be sufficient to modify pathogenic genes in all cells of the subject, tissue, or organ, and may be sufficient to modify pathogenic genes in about 1%, 5%, 10%, 25%, 50%, 75%, or more of the cells present in the subject, tissue, or organ. In one embodiment, the effective amount is sufficient to alleviate one or more symptoms of the disease.

[0086] A "fragment" means a portion of a polypeptide or nucleic acid molecule. This portion contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. The fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. In some embodiments, the fragment is a functional fragment. A "guide polynucleotide" means a polynucleotide or polynucleotide complex that is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule.

[0087] "Hybridization" means hydrogen bonding, which may be Watson-Crick type, Hoogsteen type, or reverse Hoogsteen type hydrogen bonding between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

[0088] "Inappropriate activation" in the context of C3 means any increase in complement activation associated with a disease or disorder. In one embodiment, inappropriate activation is activation that increases or rises locally (e.g., within an organ or tissue such as the central nervous system or the eye) or systemically as compared to a healthy reference (e.g., a healthy subject). In some cases, "inappropriate activation" is activation associated with chronic (e.g., persisting for more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks) inflammation in a subject. In some instances, inappropriate activation is activation that is directed against and / or results in unwanted damage to the subject's tissue, cells, or organs. In embodiments, diseases or disorders associated with inappropriate activation of the complement system can be treated by any of the methods or compositions provided herein for reducing or eliminating the expression and / or activity of the C3 polypeptide. In one embodiment, complement activation is detected by measuring the levels of C3 polypeptide and / or cleaved C3 polypeptide (e.g., C3a fragment or C3b fragment), and inappropriate activation can be determined as a high level of C3 polypeptide and / or cleaved C3 polypeptide as compared to a healthy reference subject.

[0089] "Increase" means a positive modification of at least 10%, 25%, 50%, 75% or 100%, or about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, or about 100-fold.

[0090] The terms "inhibitor of base repair", "base repair inhibitor", "IBR" or their grammatical equivalents refer to a protein capable of inhibiting the activity of a nucleic acid repair enzyme, e.g., a base excision repair enzyme.

[0091] "Intein" is a protein fragment that can excise itself and ligate the remaining fragments (exteins) by peptide bonds in a process known as protein splicing.

[0092] The terms "isolated", "purified", or "biologically pure" refer to materials that contain, to varying degrees, fewer of the components that are normally associated with them as found in their natural state. "Isolating" indicates the degree of separation from the original source or surroundings. "Purifying" indicates a higher degree of separation than isolation. A "purified" or "biologically pure" protein contains few enough other substances that no impurity substantially affects the protein's biological properties or causes other adverse events. That is, a nucleic acid or peptide of the present disclosure is purified if, when produced by recombinant DNA technology, it substantially lacks cellular, viral, or culture medium substances, or if chemically synthesized, it substantially lacks chemical precursors or other chemicals. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein yields essentially one band in an electrophoretic gel. For example, in the case of a protein that can be subject to modifications such as phosphorylation or glycosylation, different modifications can yield different isolated proteins that can be purified separately.

[0093] "Isolated polynucleotide" means a nucleic acid molecule that does not contain the genes adjacent to the gene in the natural genome of the organism from which the nucleic acid molecule of the present disclosure is derived. Thus, this term includes, for example, a recombinant DNA incorporated into a vector, an autonomously replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote, or a recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA, or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion). Further, this term includes an RNA molecule transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0094] "Isolated polypeptide" means a polypeptide of the present disclosure that is separated from the components naturally associated therewith. Generally, a polypeptide is isolated when it is at least 60% by weight and does not contain proteins and naturally occurring organic molecules naturally associated therewith. In embodiments, the preparation is at least 75% by weight, at least 90% by weight, or at least 99% by weight of the polypeptide of the present disclosure. The isolated polypeptides of the present disclosure can be obtained, for example, by extraction from natural sources, by expression of recombinant nucleic acids encoding such polypeptides, or by chemically synthesizing the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0095] As used herein, the term "linker" refers to a molecule that connects two moieties. In one embodiment, the term "linker" refers to a covalent linker (e.g., a covalent bond) or a non-covalent linker.

[0096] As used herein, "marker" means any protein or polynucleotide having an alteration in expression, level, structure, or activity associated with a disease or disorder. In embodiments, the disease or disorder is associated with inappropriate activation of the complement system. In some cases, the marker is a C3 polynucleotide or polypeptide.

[0097] As used herein, the term "mutation" refers to a substitution of a residue within a sequence, e.g., by another residue of a nucleic acid or amino acid sequence, or a deletion or insertion of one or more residues within the sequence. Mutations are typically described herein by identifying the original residue, followed by identifying the position of the residue within the sequence, and then identifying the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art and are provided, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[0098] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to a compound containing a nucleobase and an acidic moiety, such as a nucleoside, nucleotide, or polymer of nucleotides. Usually, polymeric nucleic acids, such as nucleic acid molecules containing three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester bonds. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide strand containing three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids can be natural, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other natural nucleic acid molecule. On the other hand, nucleic acid molecules can be non-natural molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or may contain non-natural nucleotides or nucleosides. Further, "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, such as analogs having a backbone other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally purified or chemically synthesized. If desired, for example, in the case of chemically synthesized molecules, nucleic acids include nucleoside analogs, such as those having chemically modified bases or sugars, and backbone modifications. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise specified.In some embodiments, the nucleic acid is a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), a nucleotide analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), a chemically modified base, a biologically modified base (e.g., a methylated base), an intercalated base, a modified sugar (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or a modified phosphate group (e.g., phosphorothioate and 5'-N-phosphoramidite linkages), or comprises them.

[0099] The term "nuclear localization sequence", "nuclear localization signal", or "NLS" refers to an amino acid sequence that promotes the import of a protein into the cell nucleus. Nuclear localization sequences are known in the art and are described, for example, in International PCT Application PCT / EP2000 / 011690, filed November 23, 2000 by Plank et al. (published May 31, 2001 as WO / 2001 / 038547), the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is, for example, an optimized NLS as described in Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO: 190), KRPAATKKAGQAKKKK (SEQ ID NO: 191), KKTELQTTNAENKTKKL (SEQ ID NO: 192), KRGINDRNFWRGENGRKTR (SEQ ID NO: 193), RKSGKIAAIVVKRPRK (SEQ ID NO: 194), PKKKRKV (SEQ ID NO: 195), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 196), PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328), or RKSGKIAAIVVKRPRKPKKKRKV (SEQ ID NO: 329).

[0100] The terms "nucleobase", "nitrogenous base", or "base" are used interchangeably herein and refer to nitrogen-containing biological compounds that form nucleosides, which are components of nucleotides. The ability of nucleobases to form base pairs and stack on top of each other is directly related to the long-chain helix structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), are referred to as the major or standard ones. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA can also contain other (non-primary) bases that are modified. Non-limiting exemplary modified nucleobases include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydroxymethylcytosine. Both hypoxanthine and xanthine can be generated via deamination (substituting an amine group with a carbonyl group) due to the presence of mutagens. Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can result from the deamination of cytosine. A "nucleoside" consists of a nucleobase and a pentose sugar (either ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthosine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C), and pseudouridine (Ψ). A "nucleotide" consists of a nucleobase, a pentose sugar (either ribose or deoxyribose), and at least one phosphate group.Non-limiting examples of modified nucleobases and / or non-limiting examples of chemical modifications that a modified nucleobase may include are as follows: pseudouridine, 5-methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-O-methylthioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), constrained ethyl (S-cEt), 2'-O-methyl ("M"), 2'-O-methyl-3'-phosphorothioate ("MS"), 2'-O-methyl-3'-thiophosphonoacetate ("MSP"), 5-methoxyuridine, phosphorothioate, and N1-methylpseudouridine.

[0101] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" may be used interchangeably with "polynucleotide programmable nucleotide binding domain", and can refer to a protein that associates with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or guide polynucleotide (e.g., gRNA) that directs the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can associate with a guide RNA that guides the Cas9 protein to a specific DNA sequence complementary to the guide RNA. In some embodiments, the napDNAbp is a Cas9 domain, such as nuclease-active Cas9, Cas9 nickase (nCas9), or nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid programmable DNA binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ (Cas12j / Casphi).Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, Cpf1, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector proteins, type V Cas effector proteins, type VI Cas effector proteins, CARF, DinG, their homologs, or their modified or engineered versions. Other nucleic acid programmable DNA-binding proteins are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, for example, Makarova et al., “Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?” CRISPR J. 2018 Oct; 1:325-336. doi:10.1089 / crispr.2018.0033, Yan et al., “Functionally diverse type V CRISPR-Cas systems” Science. 2019 Jan 4; 363(6422):88-91. doi:10.1126 / science.aav7271 (the entire content of each is incorporated herein by reference).Exemplary nucleic acid programmable DNA binding proteins and nucleic acid sequences encoding nucleic acid programmable DNA binding proteins are provided in the sequence listing as SEQ ID NOs: 197-245, 254-260, and 378.

[0102] As used herein, the terms "nucleobase editing domain" or "nucleobase editing protein" refer to a protein or enzyme that can catalyze nucleobase modifications in RNA or DNA, such as deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine), and deamination of adenine (or adenosine) to hypoxanthine (or inosine), as well as non-template nucleotide addition and insertion. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase, or a cytidine deaminase or cytosine deaminase).

[0103] As used herein, "obtaining" in "obtaining an agent" includes synthesizing, purchasing, or otherwise obtaining the agent.

[0104] "Subject" or "patient" means a mammal, including but not limited to, a human or non-human mammal. In embodiments, the mammal is a cow, horse, dog, sheep, rabbit, rodent, non-human primate, or cat. In one embodiment, "patient" refers to a mammalian subject that has a higher than average likelihood of developing a disease or disorder. Exemplary patients can be human, non-human primate, cat, dog, pig, cow, cat, horse, camel, llama, goat, sheep, rodent (e.g., mouse, rabbit, rat, or guinea pig), and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0105] As used herein, the term "patient in need thereof" or "subject in need thereof" refers to a patient diagnosed with a disease or disorder, a patient at risk of or having the disease or disorder, a patient determined in advance to have the disease or disorder, or a patient suspected of having the disease or disorder.

[0106] The terms "pathogenic variant", "pathogenic mutation", "etiologic variant", "etiologic mutation", "harmful mutation" or "predisposing mutation" refer to a genetic modification or mutation associated with a disease or disorder, or a genetic modification or mutation that increases an individual's susceptibility or predisposition to a particular disease or disorder. In some embodiments, a pathogenic variant comprises at least one wild-type amino acid substituted by at least one pathogenic amino acid within a protein encoded by a gene. In some embodiments, a pathogenic variant is within a termination region (e.g., a stop codon). In some embodiments, a pathogenic variant is within a non-coding region (e.g., an intron, a promoter, etc.).

[0107] The term "Pongo pygmaeus (orangutan) APOBEC (ppAPOBEC) polypeptide" means a cytidine deaminase polypeptide having at least about 85% amino acid sequence identity with an exemplary ppAPOBEC polypeptide sequence provided below, or a fragment thereof having cytidine deaminase activity. Exemplary ppAPOBEC polypeptide sequence: MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMDQRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR (SEQ ID NO: 941).

[0108] "Pongo pygmaeus (orangutan) APOBEC (ppAPOBEC) polynucleotide" means a nucleic acid molecule encoding the ppAPOBEC polypeptide, as well as introns, exons, 3' untranslated regions, 5' untranslated regions, and regulatory sequences associated with its expression, or fragments thereof. In embodiments, the ppAPOBEC polynucleotide is a genomic sequence, cDNA, mRNA, or gene associated with and / or required for ppAPOBEC expression. Exemplary ppAPOBEC nucleotides are provided below. Exemplary ppAPOBEC Polynucleotide Sequence: ATGACCTCTGAGAAGGGCCCTAGCACAGGCGACCCCACCCTGCGGCGGAGAATCGAGAGCTGGGAGTTCGACGTGTTCTACGACCCTAGAGAACTGAGAAAGGAAACCTGCCTGCTGTACGAGATCAAGTGGGGCATGAGCAGAAAGATCTGGCGGAGCTCTGGCAAGAACACCACCAACCACGTGGAAGTGAATTTCATCAAGAAGTTCACCAGCGAGAGAAGGTTCCACAGCAGCATCAGCTGCAGCATCACCTGGTTCCTGAGCTGGTCCCCTTGCTGGGAATGCAGCCAGGCCATCAGAGAGTTCCTGAGCCAACACCCCGGAGTGACACTGGTGATCTACGTGGCCAGACTGTTCTGGCACATGGACCAGAGAAACAGACAGGGCCTGAGAGATCTGGTCAACAGCGGCGTGACTATCCAGATCATGCGGGCCAGCGAGTACTACCACTGTTGGCGGAACTTCGTGAACTACCCCCCCGGCGATGAGGCCCACTGGCCTCAGTACCCTCCTCTGTGGATGATGCTGTACGCCCTGGAACTGCACTGCATCATCCTGTCTCTGCCTCCATGTCTGAAGATCTCTAGAAGATGGCAGAACCACCTGGCCTTCTTCAGACTGCACCTGCAGAATTGCCACTACCAGACCATCCCCCCCCACATCCTGCTGGCTACAGGCCTGATCCACCCTTCTGTGACCTGGAGA (SEQ ID NO: 942).

[0109] The terms “protein,” “peptide,” “polypeptide,” and their grammatical equivalents are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. A protein, peptide, or polypeptide can be natural, recombinant, synthetic, or any combination thereof.

[0110] As used herein, the term "fusion protein" refers to a hybrid polypeptide that contains protein domains derived from at least two different proteins.

[0111] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that is not naturally occurring but is the product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule contains an amino acid sequence or nucleotide sequence that includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 mutations as compared to any natural sequence.

[0112] "Decrease" means a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0113] "Reference" means standard or control conditions. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments, the reference is an untreated cell that is not subjected to the test conditions or is subjected to a placebo or saline, medium, buffer, and / or a control vector that does not carry the polynucleotide of interest. In an embodiment, the reference is a healthy subject or cell without inappropriate activation of the complement system. In some cases, the reference is an unedited or untreated cell (e.g., hepatocyte), tissue (e.g., a component of the central nervous system or an organ such as the liver, eye), and / or subject.

[0114] A "reference array" is a defined array used as a basis for array comparison. The reference array may be a subset or the entirety of a particular array, such as a segment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. In the case of a polypeptide, the length of the reference polypeptide sequence is generally at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. In the case of a nucleic acid, the length of the reference nucleic acid sequence is generally at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer therebetween or therearound. In some embodiments, the reference array is the wild-type sequence of the protein of interest. In other embodiments, the reference array is a polynucleotide sequence encoding a wild-type protein.

[0115] The terms "RNA programmable nuclease" and "RNA-guided nuclease" refer to a nuclease that forms a complex with one or more RNAs that are not the target of cleavage (e.g., binds or associates with it). In some embodiments, an RNA programmable nuclease, when in complex with RNA, may be referred to as a nuclease-RNA complex. Usually, the bound RNA(s) is / are called guide RNA (gRNA). In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, e.g., Cas9 from Streptococcus pyogenes (Csnl) (e.g., SEQ ID NO: 197), Cas9 from Neisseria meningitidis (NmeCas9, SEQ ID NO: 208), Nme2Cas9 (SEQ ID NO: 209), Streptococcus constellatus (ScoCas9), or a derivative thereof (e.g., a sequence having at least about 85% sequence identity to Cas9 such as Nme2Cas9 or spCas9).

[0116] Amino acids can generally be classified into classes according to the following general side-chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basic: His, Lys, Arg, (5) Residues affecting chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0117] In some embodiments, conservative substitutions may involve exchanging one member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve exchanging one member of one of these classes for another class.

[0118] The term "single nucleotide polymorphism (SNP)" refers to a variation of a single nucleotide that occurs at a specific position within the genome, and each variation is present within a population to a certain extent (e.g., > 1%). SNPs can be included in the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some embodiments, SNPs within the coding sequence do not necessarily change the amino acid sequence of the resulting protein due to the degeneracy of the genetic code. There are two types of SNPs in the coding region: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. Non-synonymous SNPs are of two types: missense and nonsense. SNPs that are not in the protein-coding region can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNA. Gene expression affected by this type of SNP is referred to as eSNP (expression SNP) and can be upstream or downstream of the gene. A single nucleotide variant (SNV) is a variation in a single nucleotide without a frequency limit and can occur in somatic cells. Somatic single nucleotide variations can also be called single nucleotide changes. By "specifically binds" is meant that in a sample, e.g., a biological sample, a nucleic acid molecule, polypeptide, polypeptide / polynucleotide complex, compound, or molecule recognizes and binds to a polypeptide and / or nucleic acid molecule of the disclosure, but does not substantially recognize or substantially bind to other molecules.

[0119] "Substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence. In one embodiment, the reference sequence is a wild-type amino acid or nucleic acid sequence. In another embodiment, the reference sequence is any one of the amino acid or nucleic acid sequences described herein. In one embodiment, such a sequence is at least about 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or even 99.99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0120] Sequence identity is typically measured using sequence analysis software (e.g., the BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs in the sequence analysis software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0121] Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule that encodes a polypeptide of the present disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will typically be able to hybridize to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule that encodes a polypeptide of the present disclosure or a functional fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial identity. A polynucleotide having "substantial identity" to an endogenous sequence will typically be able to hybridize to at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means that a pair forms a double-stranded molecule between complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof under various stringency conditions. (See, for example, Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399, Kimmel, A.R. (1987) Methods Enzymol.152:507).

[0122] "Split" means being divided into two or more fragments.

[0123] "Split polypeptide" or "split protein" refers to a protein provided as an N-terminal fragment and a C-terminal fragment that are translated as two separate polypeptides from a nucleotide sequence(s). The polypeptides corresponding to the N-terminal and C-terminal portions of the split protein may, in some embodiments, be spliced to form a "reconstituted" protein. In embodiments, the split polypeptide is a nucleic acid programmable DNA binding protein (e.g., Cas9) or a base editor.

[0124] The term "target site" refers to a sequence within a nucleic acid molecule to be modified. In embodiments, the modification is deamination of a base. The deaminase can be a cytidine or adenosine deaminase. A fusion protein or base editing complex comprising a deaminase can include a dCas9-adenosine deaminase fusion protein, a Cas12b-adenosine deaminase fusion, or a base editor disclosed herein.

[0125] As used herein, the terms "treat," "treating," "treatment," etc. refer to reducing or alleviating a disorder and / or an associated symptom, or obtaining a desired pharmacological and / or physiological effect. It will be understood that treating a disorder or condition does not necessarily require, although not precluding, that the disorder, condition, or an associated symptom be completely eliminated. In some embodiments, the effect is therapeutic, i.e., the effect, without limitation, reduces, decreases, suppresses, alleviates, mitigates, attenuates, or cures a disease and / or a deleterious symptom caused by the disease, in part or in whole. In some embodiments, the effect is prophylactic, i.e., the effect defends against or prevents the occurrence or recurrence of a disease or condition. For this purpose, the methods disclosed herein include administering a therapeutically effective amount of a composition as described herein.

[0126] "Uracil glycosylase inhibitor" or "UGI" means an agent that inhibits the uracil excision repair system. A base editor comprising a cytidine deaminase converts cytosine to uracil, which is then converted to thymine via DNA replication or repair. In various embodiments, uracil DNA glycosylase (UGI) prevents base excision repair that would convert U back to C. In some cases, contacting a cell and / or polynucleotide with UGI and a base editor prevents base excision repair that would convert U back to C. Exemplary UGIs include the following amino acid sequences. >splP14739IUNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 231).

[0127] In some embodiments, the agent that inhibits the uracil excision repair system is uracil-stabilizing protein (USP). See, for example, WO2022015969A1, which is incorporated herein by reference.

[0128] The ranges provided herein are understood to be shorthand for all values within the range. For example, a range of 1 to 50 includes any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0129] As used herein, the term "vector" refers to a means for introducing a nucleic acid molecule into a cell and resulting in a transformed cell. Vectors include plasmids, transposons, phages, viruses, liposomes, lipid nanoparticles, and episomes.

[0130] The recitation of a list of chemical groups in any definition of a variable herein includes the definition of that variable as any single group, or combination of the recited groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment, or in combination with any other embodiment or part thereof.

[0131] All terms are intended to be understood as would be understood by one of ordinary skill in the art. 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 disclosure pertains.

[0132] For the purposes of this application, the use of the singular includes the plural unless otherwise indicated. It should be noted that as used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise specified. Further, the term "including", as well as the use of other forms such as "include", "includes", and "included", is not limiting.

[0133] As used in this specification and the claims (if any), the words "comprising" (and any form thereof, such as "comprise" and "comprises"), "having" (and any form thereof, such as "have" and "has"), "including" (and any form thereof, such as "includes" and "include"), or "containing" (and any form thereof, such as "contains" and "contain") are inclusive or open-ended. This phrase indicates the presence of a particular element, feature, component, and / or method step, but does not exclude the presence of other elements, features, components, and / or method steps. Any embodiment specified as "comprising" a particular component(s) or element(s) may also, in some embodiments, be contemplated as "consisting of" or "consisting essentially of" the particular component(s) or element(s). Any embodiment discussed herein is contemplated to be implemented with respect to any method or composition of the present disclosure, and vice versa. Further, the methods of the present disclosure can be achieved using the compositions of the present disclosure.

[0134] The terms "about" or "approximately" mean within the range of error tolerances for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined (i.e., the limitations of the measurement system).

[0135] References herein to "some embodiments", "an embodiment", "one embodiment" or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments.

Brief Description of the Drawings

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[0137] Base editors, endonucleases, and guide RNAs (gRNAs) for use in editing, modifying, or altering a target polynucleotide are provided herein. In certain embodiments, the base editors or endonucleases of the disclosure modify a complement component 3 (C3) polynucleotide. In certain embodiments, the base editors of the disclosure introduce a stop codon, missense mutation, or indel (e.g., an insertion or deletion (indel) of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides) modification into the C3 polynucleotide or disrupt a splice site in the C3 polynucleotide. The modification is associated with a reduction in the activity or level of intracellular C3 polypeptide and / or polynucleotide.

[0138] The present invention of the disclosure is at least partially based on the discovery that the complement system can be activated via several different pathways, each of which requires protein C3 for complement pathway amplification and function. The invention is further at least partially based on the discovery that base editing (e.g., disruption of splice acceptors or donors, or introduction of stop codons, missense mutations, or indel modifications) can be used to reduce the expression (e.g., inappropriate activation) of intracellular C3 polypeptide associated with dysregulated complement system. In particular, reduction of the activity and / or expression of C3 polypeptide in a subject diagnosed with a disease or disorder associated with overactivation of the complement system can be an effective treatment strategy. This reduction of activity and / or expression can be achieved using any of the base editing systems and / or endonucleases and methods provided herein. Accordingly, the disclosure features compositions and methods for editing C3 polynucleotides. Editing of C3 polynucleotides is related to reduction of the expression and / or activity of C3 polypeptide in the cells, tissues, and / or body fluids of a subject, as well as symptoms associated with overactivation or otherwise pathogenic activation of the complement system in the subject.

[0139] Accordingly, as described in the examples provided herein, base editor systems were successfully developed to disrupt complement system activity by functional disruption of C3 at the gene level. C3 disruption was performed using two separate approaches: 1) silencing of the C3 gene, and 2) generation of mutations that disrupt specific C3 functions (e.g., opsonization or cleavage by C3 convertases).

[0140] In embodiments, the methods of the present disclosure include disrupting splicing of C3 polynucleotide transcripts. For example, the base editors or base editor systems provided herein can be used to edit nucleic acid bases in a splice acceptor located 5' of an exon of a C3 polynucleotide. In some embodiments, the target sequence is a splice acceptor within an intron of an intron sequence adjacent to an exon of a C3 polynucleotide and is related to a change in the splice acceptor compared to the wild-type splice acceptor. In some embodiments, deamination of an A or C nucleic acid base within the splice acceptor results in disruption of splicing of the mRNA transcript during or after transcription. In some embodiments, the subject has, or is at risk of developing, a dysregulated and / or over-activated complement system and any associated disease or disorder.

[0141] In some cases, the methods of the present disclosure include modifying a C3 polynucleotide to introduce an amino acid modification into the encoded C3 polypeptide. In embodiments, the amino acid modification disrupts cleavage of the C3 polypeptide by a C3 convertase to yield a C3b fragment. In embodiments, the C3 convertase cleaves the C3 polypeptide between residues R748 and S749. In some cases, the amino acid modification disrupts opsonization. In embodiments, the amino acid modification disrupts opsonization by the C3b fragment. In embodiments, the modified amino acid is at position C1010, Q1013, E1128, and / or H1126. In some cases, the modified amino acid is R748.

[0142] In some cases, the methods of the present disclosure include modifying a C3 polynucleotide to introduce a stop codon, indel, or missense mutation associated with a reduction in the level or activity of the C3 polynucleotide and / or polypeptide. The modification can be effected by a base editor system or by an endonuclease (e.g., Cas9) such as those described herein.

[0143] In some embodiments, the present disclosure provides a base editor or nuclease that efficiently generates intended mutations, such as point mutations or indels in a nucleic acid molecule (e.g., a nucleic acid within a subject's genome), without generating a significant number of unintended mutations, such as unintended point mutations. In some embodiments, the intended mutations are mutations generated by a specific base editor (e.g., an adenosine base editor or a cytidine base editor) or endonuclease (e.g., Cas9) that binds to a guide polynucleotide (e.g., gRNA) specifically designed to generate the intended mutations. In some embodiments, the intended mutations are point mutations from adenine (A) to guanine (G) within the non-coding region of a gene. In some embodiments, the intended mutations are point mutations from cytosine (C) to thymine (T) within the non-coding region of a gene. In some embodiments, the intended mutations are mutations of a splice acceptor at the 5' of the non-coding region of an exon of a gene associated with a disease or disorder. In some cases, the intended mutations are indel mutations. In some embodiments, the intended mutations are point mutations from adenine (A) to guanine (G) within a splice acceptor within the 5' non-coding region of an exon of a gene associated with a disease or disorder. In some embodiments, the intended mutations are missense mutations. The intended mutations can include the introduction of a stop codon into the polynucleotide sequence. In some embodiments, the intended mutations are mutations that disrupt the normal splicing of the full-length transcript of a gene, such as a change from A to G within a splice acceptor within the non-coding region located 5' of an exon of a disease-causing gene or a gene associated with a disease. In some embodiments, the intended mutations are mutations within a splice acceptor that disrupt the splicing of a gene transcript and result in an alternative transcript that encodes a truncated and / or non-functional protein product.

[0144] In some embodiments, any of the base editors or endonucleases provided herein can generate a ratio of intended mutations to unintended mutations (e.g., intended point mutations: unintended point mutations) greater than 1:1. In some embodiments, any of the base editors provided herein has a ratio of intended mutations to unintended mutations (e.g., intended point mutations: unintended point mutations) of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 500:1, or at least 1000:1, or more, and can generate such a ratio.

[0145] In some embodiments, using the methods provided herein to edit multiple nucleic acid base pairs in one or more genes results in the formation of at least one intended mutation. In some embodiments, the formation of at least one intended mutation is within the 5' splice acceptor of an exon of a disease-related gene, resulting in disruption of splicing of the mRNA transcript of the disease-related gene. It should be understood that multiplex editing can be achieved using any of the methods or combinations of methods provided herein.

[0146] The present disclosure provides methods for treating a subject diagnosed with a dysregulated and / or over-activated complement system or any disease or disorder associated therewith. For example, in some embodiments, a subject having or prone to developing a dysregulated and / or over-activated complement system is administered an effective amount of a nucleic acid base editor (e.g., an adenosine deaminase base editor or a cytidine deaminase base editor) or an endonuclease to effect a modification of a C3 polynucleotide sequence.

[0147] The Complement System and C3 The complement is a system consisting of a number of plasma and cell-bound proteins that plays important roles in both innate and adaptive immunity. The proteins of the complement system act in a series of enzymatic cascades through various protein interactions and cleavage events.

[0148] The complement system plays an important role in defending the body against infectious agents. The complement system contains more than 30 serum and cell proteins involved in three major pathways known as the classical, alternative, and lectin pathways. The classical pathway is typically initiated by the binding of a complex of antigen and IgM or IgG antibody to C1 (although certain other activating factors can also initiate the pathway). Activated C1 cleaves C4 and C2, producing C4a and C4b in addition to C2a and C2b. C4b and C2a bind to form a C3 convertase, which cleaves C3 at a defined cleavage site to form C3a and C3b. The binding of C3b to the C3 convertase produces a C5 convertase that cleaves C5 into C5a and C5b. C3a, C4a, and C5a are anaphylatoxins that mediate multiple responses in the acute inflammatory response. C3a and C5a are also chemotactic factors that attract immune system cells such as neutrophils. Further details regarding C3 are provided in Ricklin, et al. “Complement component C3 - The ‘Swiss Army Knife’ of innate immunity and host defense.” Immunol Rev. 2016 Nov; 274(1):33 - 58, and Janssen, et al., “Structures of complement component C3 provide insights into the function and evolution of immunity.” Nature. 2005 Sep 22; 437(7058):505 - 11, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0149] Alternative pathways are typically initiated and amplified by microbial surfaces and various complex polysaccharides. In this pathway, the spontaneous hydrolysis of C3 to C3(H2O) that occurs at low levels results in the binding of factor B that is cleaved by factor D, generating a fluid-phase C3 convertase that activates complement by cleaving C3 into C3a and C3b. C3b binds to targets such as cell surfaces, forms a complex with factor B, and is later cleaved by factor D, resulting in a C3 convertase. The surface-bound C3 convertase is cleaved, additional C3 molecules are activated, resulting in rapid C3b deposition proximal to the activation site, resulting in the formation of additional C3 convertases, and then additional C3b is generated. This process results in a cycle of C3 cleavage and C3 convertase formation that significantly amplifies the response. The cleavage of C3 and the binding of another molecule of C3b to the C3 convertase results in a C5 convertase. The C3 and C5 convertases of this pathway are controlled by the cell molecules CR1, DAF, MCP, CD59, and fH. The mode of action of these proteins involves either, or both, decay-accelerating activity (i.e., the ability to dissociate the convertase), the ability to function as a cofactor in the degradation of C3b or C4b by factor I. Normally, the presence of complement regulatory proteins on cell surfaces prevents the occurrence of significant complement activation on those surfaces.

[0150] The C5 convertase produced in both pathways cleaves C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzes the polymerization of C9 to form the C5b-9 membrane attack complex (MAC), also known as the terminal complement complex (TCC). The MAC inserts itself into the target cell membrane and causes cell lysis. Small amounts of MAC on cell membranes can have various consequences other than cell death. If the TCC is not inserted into the membrane, it can circulate in the blood as soluble sC5b-9 (sC5b-9). The level of sC5b-9 in the blood can serve as an indicator of complement activation.

[0151] The lectin complement pathway can be initiated by the binding of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASP) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein that localizes to the intermediate region between the endoplasmic reticulum and the Golgi. The MBL-2 gene encodes a soluble mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, resulting in the above C3 convertase.

[0152] Diseases and / or disorders associated with increased inappropriate activation of the complement system Inappropriate activation of the complement system can result in various diseases and / or disorders in a subject. For example, inappropriate activation of the complement system in a subject can cause, inter alia, increased inflammation, the presence of autoantibodies, neurodegeneration, and damage to cells that cause microthrombosis. Inappropriate activation of the complement system can be associated with damage to the central nervous system (CNS), eyes, blood cells (e.g., red blood cells, white blood cells, and platelets), as well as transplanted organs, and damage to other organs or tissues associated with the presence of microemboli. Thus, effective treatment for such diseases and / or disorders can involve modifying the C3 nucleotide sequence to reduce and / or eliminate the expression and / or activity of the C3 polypeptide in the subject, thereby reducing the activation of the complement system in organs, cells, and / or tissues. In embodiments, the organ or tissue is the eye, kidney, central nervous system component, heart, or thyroid.

[0153] Non-limiting examples of diseases associated with inappropriate activation of the complement system include blood disorders, transplant or graft rejection, inflammatory diseases or disorders, eye diseases or disorders, kidney diseases or disorders, heart disorders, respiratory / lung diseases or disorders, autoimmune disorders, inflammatory bowel diseases or disorders, arthritis, neurodegenerative diseases or disorders, musculoskeletal diseases or disorders associated with inflammation, disorders affecting the integumentary system, diseases or disorders affecting the central nervous system, diseases or disorders affecting the circulatory system, diseases or disorders affecting the gastrointestinal system, diseases or disorders affecting the thyroid, chronic pain, allergies, and lung diseases. Further non-limiting examples of diseases associated with inappropriate activation of the complement system include acute antibody-mediated rejection, age-related macular degeneration (e.g., wet or dry age-related macular degeneration), allergic asthma, allergic bronchopulmonary aspergillosis, allergic neuritis, allergic rhinitis, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, atopic dermatitis, atypical hemolytic uremic syndrome (aHUS), autoimmune diseases, autoimmune hemolytic anemia, Bechet's disease, Behcet'sdisease), bronchiolitis, obliterative bronchiolitis, C3 glomerulopathy, cancer, central nervous system (CNS) inflammatory disorders, choroidal neovascularization (CNV), choroiditis, chronic allograft vasculopathy, chronic hepatitis, chronic inflammation, chronic inflammatory diseases, chronic myositis, chronic pain, chronic pancreatitis, chronic rejection of transplantation or graft, chronic urticaria, Churg-Strauss syndrome, conjunctivitis, cyclitis, demyelinating diseases, dermatitis, dermatomyositis, diabetic retinopathy, cardiovascular diseases, disorders associated with excessive or inappropriate activation of IgE-producing cells, disorders associated with high levels or inappropriate activation of Th17 subtype of CD4+ helper T cells, encephalitis, eosinophilic pneumonia, eye disorders, geographic atrophy, giant cell arteritis, gingivitis, glaucoma, glomerulonephritis, glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis), graft rejection or disorders, HELLP syndrome, Henoch-Schönlein purpura, hepatitis (e.g., hepatitis C), Huntington's disease, hyperacute rejection reaction, hypersensitivity pneumonia, idiopathic pulmonary fibrosis (IPF), IgA nephropathy (IgAN), inflammatory bowel diseases (e.g., Crohn's disease or ulcerative colitis), inflammatory joint conditions (e.g., arthritis such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthropathy Reiter's syndrome, or gout), inflammatory skin diseases, infusion reactions, interstitial pneumonia, iridocyclitis, iritis, ischemia / reperfusion injury, Kawasaki disease, keratitis, lupus nephritis, membranoproliferative glomerulonephritis (MPGN) (e.g., MPGNType I, II, or III), meningitis, microscopic polyangiitis, multiple sclerosis (MS), myasthenia gravis, myocarditis, nasal polyps, neurodegenerative diseases, neuromyelitis optica, neuromyelitis optica (NMO), neuropathic pain, eye inflammation, osteoarthritis, pancreatitis, panniculitis, Parkinson's disease, paroxysmal nocturnal hemoglobinuria (PNH), uveitis, pathological immune responses to tissue / organ transplantation, pemphigoid, pemphigus, periodontitis, persistent asthma, polyarteritis nodosa, polymyositis, primary membranous nephropathy, proliferative vitreoretinopathy, proteinuria, psoriasis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury, kidney diseases, respiratory diseases or disorders (e.g., asthma or chronic obstructive pulmonary disease (COPD), or idiopathic pulmonary fibrosis, or asthma), respiratory distress syndrome, retinal neovascularization (RNV), retinopathy of prematurity, rheumatoid arthritis (RA), rhinosinusitis, sarcoid, sarcoidosis, episcleritis, scleroderma, scleroderma-skin myositis, sclerosis, sepsis, Sjogren's syndrome, Schellen's syndrome, stroke, systemic lupus erythematosus, systemic scleroderma, Takayasu arteritis, Th2-related disorders (e.g., disorders associated with high levels or hyperactivation of Th2 subtypes of CD4+ helper T cells), thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease, or postpartum thyroiditis), thyroid disorders, transplantation injury, transplant rejection, ulcerative colitis, uveitis, vasculitis, and Wegener's granulomatosis. In embodiments, the methods of the present disclosure include reducing complement-mediated hemolysis in a subject. Further non-limiting examples of diseases include Creutzfeldt-Jakob disease, Pick's disease, mild cognitive impairment, fibromyalgia, frontotemporal dementia, dementia with Lewy bodies, multiple system atrophy, chronic inflammation, demyelinating polyneuropathy, Guillain-Barré syndrome, multifocal motor neuropathy, non-alcoholic fatty liver disease (NAFLD), e.g., non-alcoholic steatohepatitis (NASH), and Stargardt macular degeneration.

[0154] The methods and compositions of the present disclosure are suitable for use in the treatment of any of the above diseases or disorders associated with inappropriate activation of the complement system. In various instances, the methods include introducing a modification into a C3 polynucleotide that results in a reduction in the expression and / or activity of the C3 polypeptide in cells.

[0155] Editing of target gene Exemplary spacer sequences suitable for use in guide RNAs that can be used to generate the polynucleotide edits described herein (e.g., missense mutations, introduction of stop codons, splice site disrupting mutations, etc.) are listed in Tables 1A - 1F, and 2 below. To produce a polynucleotide edit, a cell (e.g., a cell in or derived from a subject) is contacted with one or more guide RNAs containing one or more of the spacer sequences listed in Tables 1A - 1F, or 2, or fragments thereof, and a complex containing a nucleobase editor polypeptide or nucleic acid programmable DNA binding protein (napDNAbp) and one or more deaminases having cytidine deaminase and / or adenosine deaminase activity (e.g., a "dual deaminase" having cytidine and adenosine deaminase activity). In embodiments, the base editor and / or endonuclease is introduced into the cell using a polynucleotide sequence (e.g., mRNA) encoding the base editor and / or endonuclease. Tables 1A - 1F, and 2 list representative guide RNA spacer sequences that can be used in combination with the indicated base editors. Using a guide RNA containing a spacer sequence listed in Tables 1A - 1F, and 2, the target sequences listed in Tables 1A - 1F, and 2 can be targeted to effect the edits listed in Tables 1A - 1F, and 2. In some embodiments, the gRNA comprises nucleotide analogs. In some cases, the gRNA is added directly to the cell. These nucleotide analogs can inhibit the degradation of the gRNA by cellular processes. Tables 1A - 1F, and 2 provide target sequences for use with the gRNA. Further exemplary spacer sequences suitable for use in gRNA sequences for use in the methods provided herein include any of the spacers provided in Tables 1A - 1F, and 2, and any fragment of any of the spacers provided in Tables 1A - 1F, and 2 modified to include extensions or truncations at the 3' and / or 5' ends (s).In embodiments, the spacer arrays of Tables 1A - 1F, and 2 can be modified to include an extension or truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3' and / or 5' end(s).

[0156] In various cases, it can be advantageous for the spacer array to include 5' and / or 3' "G" nucleotides. In some cases, for example, any of the spacer arrays or guide polynucleotides provided herein include or further include a 5' "G", and in some embodiments, the 5' "G" is or is not complementary to the target sequence. In some embodiments, a 5' "G" is added to a spacer array that does not yet contain a 5' "G". For example, when a guide RNA is expressed under the control of a U6 promoter, it can be advantageous for the guide RNA to include a 5' terminal "G" because the U6 promoter prefers a "G" at the transcription start site (see Cong, L. et al. “Multiplex genome engineering using CRISPR / Cas systems. Science 339:819 - 823 (2013) doi:10.1126 / science.1231143). In some cases, a 5' terminal "G" is added to a guide polynucleotide expressed under the control of a promoter, but optionally, it is not added to the guide polynucleotide when the guide polynucleotide is not expressed under the control of a promoter or at that time.

[0157] In some embodiments, the guide polynucleotides of the present disclosure contain a scaffold having one of the following chemical modification schemes, where "N" represents any nucleotide, "mN" represents a 2'-OMe modification of nucleotide "N", and "Ns" indicates that nucleotide "N" is linked to the next nucleotide by phosphorothioate (PS). End - modified SpCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUsmUsmUsmU (SEQ ID NO: 943) Terminal-modified SaCas9 guide polynucleotide mNsmNsmNsNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUsmUsmUsmU (SEQ ID NO: 944) HM01: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 855) HM07: mNsmNsmNsmNmNmNmNmNmNmNNNNNNNNNNNmGUUUUAGmAmGmCmUmAmGmAmAmAmUmAmGmCmAmAGUUmAAmAAmUAmAmGmGmCmUmAGUmCmCGUUAmUmCAAmCmUmUmGmAmAmAmAmAmGmUmGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 856) NLS (bpsv40): mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCmUsmUsmUsmU-NHC6-CrossL-ac-CKRTADGSEFESPKKKRKV (SEQ ID NOS: 857 and 858) LONGEST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGUGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 859) NLS+LONGEST: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU-NHC5-CrossL-CKRTADGSEFESPKKKRKV (SEQ ID NO: 860 and 858) LONGEST+GOLD: mNsmNsmNsNNNNNNNNNNNNNNNNNGUUUUAGAmGmCmCmGmGmCmGmGmAmAmAmCmGmCmCmGmGmCAAGUUAAAAUAAGGCUAGUCCGUUAmUmCAAmCmUmUGGACUUCGGUCCmAmAmGUGGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmUsmUsmUsmU (SEQ ID NO: 861)

[0158] Exemplary guide RNA sequences are provided in Tables 1A - 1F and 2 below.

Table 1A-1

Table 1A-2

Table 1A-3

Table 1A-4

Table 1A-5

Table 1A-6

Table 1A-7

Table 1A-8

Table 1A-9

Table 1A-10

[0159]

Table 1B

[0160]

Table 1C-1

Table 1C-2

[0161]

Table 1D

[0162]

Table 1E

[0163]

Table 1F-1

Table 1F-2

[0164]

Table 2-1

Table 2-2

[0165] Nucleobase editor Nucleobase editors that edit, modify or alter the target nucleotide sequence of a polynucleotide are useful in the methods and compositions described herein. The nucleobase editors described herein typically include a polynucleotide programmable nucleotide binding domain and a nucleobase editing domain (e.g., adenosine deaminase, cytidine deaminase, or dual deaminase). The polynucleotide programmable nucleotide binding domain can specifically bind to a target polynucleotide sequence when bound to a bound guide polynucleotide (e.g., gRNA), thereby localizing the base editor to the target nucleic acid sequence to be edited.

[0166] Polynucleotide programmable nucleotide binding domain The polynucleotide programmable nucleotide binding domain binds to a polynucleotide (e.g., RNA, DNA). The polynucleotide programmable nucleotide binding domain of a base editor can itself include one or more domains (e.g., one or more nuclease domains). In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain includes an endonuclease or exonuclease.

[0167] Disclosed herein are base editors that include a polynucleotide programmable nucleotide binding domain that includes all or a portion (e.g., a functional portion) of a CRISPR protein (i.e., a base editor that includes all or a portion (e.g., a functional protein) of a CRISPR protein (e.g., a Cas protein) as a domain, also referred to as the "CRISPR protein-derived domain" of the base editor). The CRISPR protein-derived domain incorporated into the base editor can be modified compared to the wild-type or native version of the CRISPR protein. The CRISPR protein-derived domain can include one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to the wild-type or native version of the CRISPR protein.

[0168] Cas proteins that can be used in this specification include Class 1 and Class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Cas12a / Cpf1, Cas12b / C2c1 (e.g., SEQ ID NO: 232), Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ, CARF, DinG, their homologs, or modified versions thereof. CRISPR enzymes can direct cleavage of one or both strands at a target sequence, e.g., within the target sequence and / or within the complementary sequence of the target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0169] A vector encoding a CRISPR enzyme that has been mutagenized relative to a corresponding wild-type enzyme can be used such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. A Cas protein (e.g., Cas9, Cas12) or Cas domain (e.g., Cas9, Cas12) can refer to a polypeptide or domain having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity and / or sequence homology to an exemplary wild-type Cas polypeptide or Cas domain. Cas (e.g., Cas9, Cas12) can refer to a wild-type or modified form of a Cas protein that can include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0170] In some embodiments, the CRISPR protein-derived domain of the base editor can comprise all or a part (e.g., a functional part) of Cas9 from Corynebacterium ulcerans (NCBI reference: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI reference: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI reference: NC_021284.1); Prevotella intermedia (NCBI reference: NC_017861.1); Spiroplasma taiwanense (NCBI reference: NC_021846.1); Streptococcus iniae (NCBI reference: NC_021314.1); Belliella baltica (NCBI reference: NC_018010.1); Psychroflexus torquis (NCBI reference: NC_018721.1); Streptococcus thermophilus (NCBI reference: YP_820832.1); Listeria innocua (NCBI reference: NP_472073.1); Campylobacter jejuni (NCBI reference: YP_002344900.1); Neisseria meningitides (NCBI reference: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0171] Some aspects of the present disclosure provide high-fidelity Cas9 domains. High-fidelity Cas9 domains are known in the art and are described, for example, in Kleinstiver, B.P., et al. “High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.” Nature 529, 490-495(2016) and Slaymaker, I.M., et al. “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015), the entire contents of each of which are incorporated herein by reference. An exemplary high-fidelity Cas9 domain is shown in the Sequence Listing as SEQ ID NO: 233.

[0172] In some embodiments, any of the Cas9 fusion proteins or complexes provided herein comprises one or more of the D10A, N497X, R661X, Q695X, and / or Q926X mutations, or corresponding mutations in any of the amino acid sequences provided herein, where X is any amino acid.

[0173] Typically, a Cas9 protein, e.g., Cas9 from S. pyogenes (spCas9), requires a “protospacer adjacent motif (PAM)” or PAM-like motif, which is a DNA sequence of 2 - 6 base pairs immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The presence of the NGG PAM sequence is required to bind to a specific nucleic acid region, where “N” in “NGG” is adenosine (A), thymidine (T), or cytosine (C), and G is guanosine. In some embodiments, any of the fusion proteins or complexes provided herein may include a Cas9 domain capable of binding to a nucleotide sequence that does not contain a canonical (e.g., NGG) PAM sequence. Cas9 domains that bind to non-canonical PAM sequences are described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver, B.P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523,481-485 (2015), and Kleinstiver, B.P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015) (the entire contents of each are incorporated herein by reference).

[0174] In some embodiments, napDNAbp is a circular permutant (e.g., SEQ ID NO: 238).

[0175] In some embodiments, the polynucleotide programmable nucleotide binding domain comprises a nickase domain. As used herein, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain that includes a nuclease domain capable of cleaving only one strand of a double-stranded nucleic acid molecule (e.g., DNA). For example, where the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the Cas9-derived nickase domain may comprise a D10A mutation and a histidine at position 840. In another example, the Cas9-derived nickase domain comprises an H840A mutation, while the amino acid residue at position 10 remains as D.

[0176] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., (in the case of SEQ ID NO: 201) Cas9 is a nickase and is referred to as the "nCas9" protein. The Cas9 nickase can be a Cas9 protein that can cleave only one strand of a double-stranded nucleic acid molecule (e.g., a double-stranded DNA molecule). In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the Cas9 nickases provided herein. Further suitable Cas9 nickases will be apparent to those skilled in the art based on the present disclosure and the knowledge in the art and are within the scope of the present disclosure.

[0177] Also provided herein are base editors that include a catalytically inactive (i.e., unable to cleave the target polynucleotide sequence) polynucleotide programmable nucleotide binding domain. For example, in the case of a base editor that includes a Cas9 domain, Cas9 can include both the D10A mutation and the H840A mutation. In further embodiments, the catalytically inactive polynucleotide programmable nucleotide binding domain includes point mutations (e.g., D10A or H840A), and deletions of all or a portion (e.g., the functional portion) of the nuclease domain. The dCas9 domain is known in the art; see, for example, Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5):1173-83. The entire content thereof is incorporated herein by reference.

[0178] The term “protospacer adjacent motif (PAM)” or PAM-like motif refers to a 2- to 6-base pair DNA sequence immediately following the DNA sequence targeted by a nucleic acid programmable DNA binding protein. In some embodiments, the PAM can be a 5’ PAM (i.e., a PAM located upstream of the 5’ end of the protospacer). In other embodiments, the PAM can be a 3’ PAM (i.e., a PAM located downstream of the 5’ end of the protospacer). The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNNRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAAC. Y is a pyrimidine, N is any nucleotide base, and W is A or T.

[0179] The base editors provided herein can include domains derived from CRISPR proteins, which can bind to nucleotide sequences containing canonical or non-canonical protospacer adjacent motif (PAM) sequences.

[0180] In some embodiments, for example, as described in R.T. Walton et al., 2020, Science, 10.1126 / science.aba8853 (2020) (the entire content of which is incorporated herein by reference), the PAM is either an "NRN" PAM (where "N" in "NRN" is adenine (A), thymine (T), guanine (G), or cytosine (C), and R is adenine (A) or guanine (G)), or the PAM is a "NYN" PAM (where "N" in "NYN" is adenine (A), thymine (T), guanine (G), or cytosine (C), and Y is cytosine (C) or thymine (T)).

[0181] Several PAM variants are described in Table 3 below. [Table 3]

[0182] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM Cas9 variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R of spCas9 (SEQ ID NO: 197) (collectively referred to as "MQKFRAER"), or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from D1135V, G1218R, R1335Q, and T1337R of spCas9 (SEQ ID NO: 197) (collectively referred to as VRQR), or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from D1135V, G1218R, R1335E, and T1337R of spCas9 (SEQ ID NO: 197) (collectively referred to as VRER), or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from E782K, N968K, and R1015H of saCas9 (SEQ ID NO: 218) (collectively referred to as KHH). In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R of spCas9 (SEQ ID NO: 197) (collectively referred to as "MQKSER"), or corresponding mutations in another Cas9. In some embodiments, the Cas9 variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219S, R1335E, and T1337R of spCas9 (SEQ ID NO: 197) (collectively referred to as "MQKSER"), or corresponding mutations in another Cas9.

[0183] In some embodiments, the CRISPR protein-derived domain of the base editor comprises all or a part (e.g., a functional part) of a Cas9 protein having a canonical PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor can use a non-canonical PAM sequence. Such sequences are described in the art and will be apparent to those skilled in the art. For example, Cas9 domains that bind to non-canonical PAM sequences are described in Kleinstiver, B.P., et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015), and Kleinstiver, B.P., et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition” Nature Biotechnology 33, 1293-1298 (2015), R.T. Walton et al. “Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants” Science 10.1126 / science.aba8853(2020), Hu et al. “Evolved Cas9 variants with broad PAM compatibility and high DNA specificity,” Nature, 2018 Apr.5, 556 (7699), 57-63, Miller et al., “Continuous evolution of SpCas9 variants compatible with non-G PAMs” Nat.Biotechnol., 2020 Apr; 38(4):471-481, the entire contents of which are incorporated herein by reference.

[0184] A fusion protein or complex comprising NapDNAbp and cytidine deaminase and / or adenosine deaminase Some aspects of the present disclosure provide a fusion protein or complex comprising a Cas9 domain or other nucleic acid programmable DNA binding protein (e.g., Cas12) and one or more cytidine deaminases, adenosine deaminases, or cytidine adenosine deaminase domains. It should be understood that the Cas9 domain can be either the Cas9 domain or Cas9 protein provided herein (e.g., dCas9 or nCas9). In some embodiments, any of the Cas9 domains or Cas9 proteins provided herein (e.g., dCas9 or nCas9) can be fused to any of the cytidine deaminases and / or adenosine deaminases provided herein. The domains of the base editors disclosed herein can be arranged in any order.

[0185] In some embodiments, a fusion protein or complex comprising a cytidine deaminase or adenosine deaminase and a napDNAbp (e.g., a Cas9 or Cas12 domain) does not contain a linker sequence. In some embodiments, the linker is present between the cytidine deaminase or adenosine deaminase and the napDNAbp. In some embodiments, the cytidine deaminase or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein. For example, in some embodiments, the cytidine deaminase or adenosine deaminase and the napDNAbp are fused via any of the linkers provided herein.

[0186] It should be understood that the fusion proteins or complexes of the present disclosure may include one or more additional features. For example, in some embodiments, the fusion protein or complex may include an inhibitor, a cytoplasmic localization sequence, a transport sequence (e.g., a nuclear export sequence), or other localization sequences, as well as sequence tags useful for solubilization, purification, or detection of the fusion protein or complex. Suitable protein tags provided herein include, but are not limited to, biotin carboxylase carrier protein (BCCP) tag, myc tag, calmodulin tag, FLAG tag, hemagglutinin (HA) tag, polyhistidine tag also called histidine tag or His tag, maltose binding protein (MBP) tag, nus tag, glutathione-S-transferase (GST) tag, green fluorescent protein (GFP) tag, thioredoxin tag, S tag, soft tag (e.g., soft tag 1, soft tag 3), streptag, biotin ligase tag, FlAsH tag, V5 tag, and SBP tag. Further suitable sequences will be apparent to those skilled in the art. In some embodiments, the fusion protein or complex includes one or more His tags.

[0187] Exemplary but non-limiting fusion proteins are described in International PCT Applications No. PCT / US2017 / 045381, No. PCT / US2019 / 044935, and No. PCT / US2020 / 016288 (each of which is incorporated herein by reference in its entirety).

[0188] Fusion proteins or complexes with internal insertions Provided herein are fusion proteins or complexes comprising a nucleic acid programmable nucleic acid binding protein, e.g., a heterologous polypeptide fused to napDNAbp. The heterologous polypeptide can be fused to napDNAbp at the C-terminus of napDNAbp, at the N-terminus of napDNAbp, or inserted at an internal position of napDNAbp. In some embodiments, the heterologous polypeptide is a deaminase (e.g., cytidine of adenosine deaminase) or a functional fragment thereof. For example, the fusion protein can comprise a deaminase adjacent to the N-terminal and C-terminal fragments of a Cas9 or Cas12 (e.g., Cas12b / C2c1) polypeptide.

[0189] The deaminase can be a circular permutant deaminase. In some embodiments, the deaminase is a circular permutant TadA that is circularly substituted at amino acid residue 116, 136, or 65 numbered in the TadA reference sequence.

[0190] The fusion protein or complex can comprise multiple deaminases. The fusion protein or complex can comprise, for example, 1, 2, 3, 4, 5, or more deaminases. The deaminases in the fusion protein or complex can be adenosine deaminases, cytidine deaminases, or combinations thereof.

[0191] In some embodiments, the napDNAbp within the fusion protein or complex contains a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide can be a variant Cas9 polypeptide. The Cas9 polypeptide can be a circularly substituted Cas9 protein.

[0192] A heterologous polypeptide (e.g., a deaminase) can be inserted into a napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b / C2c1)) at a suitable position such that, for example, the ability of the napDNAbp to bind to the target polynucleotide and the guide nucleic acid is retained. A deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase (dual deaminase)) can be inserted into the napDNAbp without impairing the function of the deaminase (e.g., base editing activity) or the napDNAbp (e.g., the ability to bind to the target nucleic acid and the guide nucleic acid).

[0193] In some embodiments, a deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted into a region of the Cas9 polypeptide that includes a B factor higher than the average B factor (e.g., a higher B factor compared to the entire protein or a protein domain including a disordered region). Positions of the Cas9 polypeptide that include a B factor higher than the average can include, for example, residues numbered 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 1247, and 1248 in the Cas9 reference sequence described above. Regions of the Cas9 polypeptide that include a B factor higher than the average B factor can include, for example, residues numbered 792 - 872, 792 - 906, and 2 - 791 in the Cas9 reference sequence described above.

[0194] In some embodiments, a heterologous polypeptide (e.g., a deaminase) is inserted into the flexible loop of the Cas9 polypeptide. The flexible loop portion can be selected from the group consisting of amino acid residues numbered 530-537, 569-570, 686-691, 943-947, 1002-1025, 1052-1077, 1232-1247, or 1298-1300 in the above Cas9 reference sequence, or the corresponding amino acid residues of another Cas9 polypeptide. The flexible loop portion can be selected from the group consisting of amino acid residues numbered 1-529, 538-568, 580-685, 692-942, 948-1001, 1026-1051, 1078-1231, or 1248-1297 in the above Cas9 reference sequence, or the corresponding amino acid residues of another Cas9 polypeptide.

[0195] A heterologous polypeptide (e.g., adenine deaminase) can be inserted into the Cas9 polypeptide region corresponding to amino acid residues numbered 1017-1069, 1242-1247, 1052-1056, 1060-1077, 1002-1003, 943-947, 530-537, 568-579, 686-691, 1242-1247, 1298-1300, 1066-1077, 1052-1056, or 1060-1077 in the above Cas9 reference sequence, or the corresponding amino acid residues of another Cas9 polypeptide. A heterologous polypeptide (e.g., adenine deaminase) can be inserted in place of the deletion region of the Cas9 polypeptide. The deletion region can correspond to the N-terminal portion or the C-terminal portion of the Cas9 polypeptide. Exemplary internal fusion base editors are provided in Table 4A below.

[0196]

Table 4A

[0197] A heterologous polypeptide (e.g., a deaminase) can be inserted into the structure or functional domain of a Cas9 polypeptide. A heterologous polypeptide (e.g., a deaminase) can be inserted between two structural or functional domains of a Cas9 polypeptide. A heterologous polypeptide (e.g., a deaminase) can be inserted, for example, after deleting a domain from a Cas9 polypeptide, in place of the structural or functional domain of the Cas9 polypeptide. The structural or functional domain of a Cas9 polypeptide can include, for example, RuvCI, RuvCII, RuvCIII, Rec1, Rec2, PI, or HNH.

[0198] The fusion protein can include a linker between the deaminase and the napDNAbp polypeptide. The linker can be a peptide or non-peptide linker. For example, the linker can be XTEN, (GGGS) n (SEQ ID NO: 246), SGGSSGGS (SEQ ID NO: 330), (GGGGS) n (SEQ ID NO: 247), (G) n , (EAAAK)n (SEQ ID NO: 248), (GGS) n , SGSETPGTSESATPES (SEQ ID NO: 249). In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein includes a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the N-terminal and C-terminal fragments of napDNAbp are connected to the deaminase by a linker. In some embodiments, the N-terminal and C-terminal fragments are linked to the deaminase domain without a linker. In some embodiments, the fusion protein includes a linker between the N-terminal Cas9 fragment and the deaminase, but does not include a linker between the C-terminal Cas9 fragment and the deaminase. In some embodiments, the fusion protein includes a linker between the C-terminal Cas9 fragment and the deaminase, but does not include a linker between the N-terminal Cas9 fragment and the deaminase.

[0199] In some embodiments, the napDNAbp in the fusion protein or complex is a Cas12 polypeptide, such as Cas12b / C2c1 or a functional fragment thereof, and can associate with a nucleic acid (e.g., gRNA) that directs Cas12 to a specific nucleic acid sequence. The Cas12 polypeptide can be a variant Cas12 polypeptide. In other embodiments, the N-terminal fragment or C-terminal fragment of the Cas12 polypeptide comprises a nucleic acid programmable DNA binding domain or an RuvC domain. In other embodiments, the fusion protein comprises a linker between the Cas12 polypeptide and the catalytic domain. In other embodiments, the amino acid sequence of the linker is GGSGGS (SEQ ID NO: 250) or GSSGSETPGTSESATPESSG (SEQ ID NO: 251). In other embodiments, the linker is a rigid linker. In other embodiments of aspects of the present disclosure, the linker is encoded by GGAGGCTCTGGAGGAAGC (SEQ ID NO: 252) or GGCTCTTCTGGATCTGAAACACCTGGCACAAGCGAGAGCGCCACCCCTGAGAGCTCTGGC (SEQ ID NO: 253).

[0200] In other embodiments, the fusion protein or complex comprises a nuclear localization signal (e.g., a bipartite nuclear localization signal). In other embodiments, the amino acid sequence of the nuclear localization signal is MAPKKKRKVGIHGVPAA (SEQ ID NO: 261). In other embodiments of aspects of the present disclosure, the nuclear localization signal is encoded by the following sequence: ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC (SEQ ID NO: 262). In other embodiments, the Cas12b polypeptide comprises a mutation that silences the catalytic activity of the RuvC domain. In other embodiments, the Cas12b polypeptide comprises D574A, D829A, and / or D952A mutations.

[0201] In some embodiments, the fusion protein or complex comprises a napDNAbp domain (e.g., a domain derived from Cas12) and has an internal fusion nucleic acid base editing domain (e.g., all or part (e.g., a functional part) of a deaminase domain (e.g., an adenosine deaminase domain)). In some embodiments, the napDNAbp is Cas12b. In some embodiments, the base editor comprises a BhCas12b domain and has an internal fusion TadA*8 domain inserted at the locus provided in Table 4B below.

[0202]

Table 4B

[0203] In some embodiments, the base editor system described herein is an ABE in which TadA is inserted into Cas9. The polypeptide sequences of related ABEs having TadA inserted into Cas9 are provided as SEQ ID NOs: 263-308 in the accompanying Sequence Listing.

[0204] Exemplary but non-limiting fusion proteins are described in International PCT Application No. PCT / US2020 / 016285 and U.S. Provisional Application Nos. 62 / 852,228 and 62 / 852,224 (the contents of which are incorporated herein by reference in their entirety).

[0205] Editing from A to G In some embodiments, the base editors described herein include an adenosine deaminase domain. Such an adenosine deaminase domain of a base editor can promote the editing of an adenine (A) nucleobase to a guanine (G) nucleobase by deaminating A to form inosine (I) which exhibits the base pairing properties of G. In some embodiments, the base editor for A to G further includes an inhibitor of base excision repair of inosine bases, such as a uracil glycosylase inhibitor (UGI) domain or a catalytically inactive inosine-specific nuclease. Without wishing to be bound by any particular theory, the UGI domain or catalytically inactive inosine-specific nuclease can inhibit or prevent base excision repair of deaminated adenosine residues (e.g., inosine), which can improve the activity or efficiency of the base editor.

[0206] Base editors containing adenosine deaminase can act on any polynucleotide including DNA, RNA, and DNA-RNA hybrids. In one embodiment, the adenosine deaminase domain of the base editor includes all or a part (e.g., a functional part) of ADAT that contains one or more mutations that enable ADAT to deaminate a target A in DNA. For example, the base editor can include all or a part (e.g., a functional part) of ADAT from Escherichia coli (EcTadA) that contains one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I156F, or corresponding mutations in another adenosine deaminase. Exemplary ADAT homologous polypeptide sequences are provided as SEQ ID NOs: 1 and 309-315 in the Sequence Listing.

[0207] Adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, adenosine deaminase is derived from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, adenine deaminase is a native adenosine deaminase that contains one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). Corresponding residues in any homologous protein can be identified, for example, by sequence alignment and determination of homologous residues. Mutations in any native adenosine deaminase corresponding to any of the mutations described herein (e.g., any of the mutations identified in ecTadA) can be generated accordingly.

[0208] In some embodiments, the adenosine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described for the adenosine deaminases provided herein. It should be understood that the adenosine deaminases provided herein may contain one or more mutations (e.g., any of the mutations provided herein). The present disclosure provides any deaminase domain having a particular percent identity, in addition to any of the mutations described herein or combinations thereof. In some embodiments, the adenosine deaminase comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations as compared to a reference sequence or any of the adenosine deaminases provided herein.

[0209] It should be understood that any of the mutations provided herein (e.g., based on a TadA reference sequence such as TadA*7.10 (SEQ ID NO: 1)) can be introduced into other adenosine deaminases, such as E. coli TadA (ecTadA), S. aureus TadA (saTadA), or other adenosine deaminases (e.g., bacterial adenosine deaminases). In some embodiments, the TadA reference sequence is TadA*7.10 (SEQ ID NO: 1). It will be apparent to one of skill in the art that additional deaminases can be similarly aligned to identify homologous amino acid residues and mutated as provided herein. Thus, any of the mutations identified in the TadA reference sequence can be made in other adenosine deaminases (e.g., ecTadA) that have homologous amino acid residues. It should also be understood that any of the mutations provided herein can be made in the TadA reference sequence or another adenosine deaminase, individually or in any combination.

[0210] In some embodiments, the adenosine deaminase comprises a modification or series of modifications selected from those listed in Tables 5A-5E below:

[0211] [Table 5A-1] [Table 5A-2]

[0212] [Table 5B]

[0213] [Table 5C-1] [Table 5C-2]

[0214] In some embodiments, the adenosine deaminase has M1I, S2A, S2E, V4D, V4E, V4M, F6S, H8E, H8Y, E9Y, M12S, R13H, R13I, R13Y, T17L, T17S, L18A, L18E, A19N, R21N, K20K, K20R, R21A, G22P, W23D, R23H, W23G, W23Q, W23L, W23R, D24E, D24G, E25F, E25M, E25D, E25A, E25G, E25R, E25V, E25S, E25Y, R26D, R26E, R26G, R26N, R26Q, R26C, R26L, R26K, R26W, E27V, E27D, P29V, V30G, L34S, L34V, L36H, H36L, H36N, N37N, N37T, N37S, N38G, N38R, W45A, W45L, W45N, N46N, R46W, R46F, R46Q, R46M, R47A, R47Q, R47F, R47K, R47P, R47W, R47M, P48T, P48L, P48A, P48I, P48S, I49G, I49H, I49V, I49F, I49H, G50L, R51H, R51L, R51N, L51W, R51Y, H52D, H52Y, D53P, P54C, P54T, A55H, T55A, A56E, A56S, E59A, E59G, E59I, E59Q, E59W, M61A, M61I, M61L, M61V, L63S, L63V, Q65V, G66C, G67D, G67L, G67V, L68Q, M70H, M70Q, L84F, M70V, M%70L, E70A, M70V, Q71M, Q71N, Q71L, Q71R, N72A, N72K, N72S, N72D, N72Y, Y73G, Y73I, Y73K, Y73R, Y73S, R74A, R74Q, R74G, R74K, R74L, R74N, I76D, I76F, I76I, I76N, I76T, I76Y, D77G, A78I, T79M, L80M, L80Y, V82A, V82S, V82G, V82T, L84E, L84F, L84Y, E85K, E85G, E85P, E85S, S87C, S87L, S87V, V88A, V88M, C90S, A91A, A91G, A91S, A91V, A91T, G92T, A93I, M94A, M94V, M94L, M94I, M94H, in the TadA reference sequence (e.g., TadA*7.10, ecTadA, or TadA8e).I95S, I95G, I95L, I95H, I95V, H96A, H96L, H96R, H96S, S97C, S97G, S97I, S97M, S97R, S97S, R98K, R98I, R98N, R98Q, G100R, G100V, R101V, R101R, V102A, V102F, V102I, V102V, D103A, F104G, D104N, F104V, F104I, F104L, A106T, V106Q, V106F, V106W, V106M, A106A, A106Q, A106F, A106G, A106W, A106M, A106V, A106R, R107C, R107G, R107P, R107K, R107A, R107N, R107W, R107H, R107S, D108N, D108F, D108G, D108V, D108A, D108Y, D108H, D108I, D108K, D108L, D108M, D108Q, N108Q, N108F, N108W, N108M, N108K, D108K, D108F, D108M, D108Q, D108R, D108W, D108S, A109H, A109K, A109R, A109S, A109T, A109V, K110G, K110H, K110I, K110R, K110T, T111A, T111G, T111H, T111R, G112A, A114G, A114H, A114V, G115S, L117M, L117N, L117V, M118D, M118G, M118K, M118N, M118V, D119L, D119N, D119S, D119V, V120H, V120L, H122H, H122N, H122P, H122R, H122S, H122Y, H123C, H123G, H123P, H123V, H123Y, Y123H, P124G, P124I, P124L, P124W, G125H, G125I, G125A, G125M, G125K, M126D, M126H, M126K, M126I, M126N, M126O, M126S, M126Y, N127H, N127S, N127D, N127K, N127R, H128R, R129H, R129Q, R129V, R129I, R129E, R129V, I132I, I132F, T133V, T133E, T133G, T133K, E134A, E134E, E134G, E134I, G135G, G135V, I136G, I136L, I136T, l137AOne or more of the mutations of l137D, l137E, L137M, l137S, A138D, A138E, A138G, S138A, A138N, A138S, A138T, A138V, A138Y, D139E, D139I, D139C, D139L, D139M, E140A, E140C, E140L, E140R, A142N, A142D, A142G, A142A, A142L, A142S, A142T, A142N, A142S, A142V, A143D, A143E, A143G,, A143D, A143G, A143E, A143L, A143W, A143M, A143S, A143Q, A143R, C146R, S146A, S146C, S146D, S146F, S146R, S146T, D147D, D147L, D147F, D147G, D147Y, Y147T, Y147R, Y147D, D147R, F148L, F148F, F148R, F148Y, F149C, F149M, F149R, F149Y, M151F, M151P, M151R, M151V, R152C, R152F, R152H, R152P, R152R, R153C, R153Q, R153R, R153V, Q154E, Q154H, Q154M, Q154R, Q154L, Q154S, Q154V, E155F, E155G, E155I, E155K, E155P, E155V, E155D, I156A, I156F, I156D, I156K, I156N, I156R, I156Y, E157A, E157F, E157I, E157P, E157T, E157V, N157K, K157N, K157R, A158Q, A158K, A158V, Q159F, Q159K, Q159L, Q159N, K160A, K160S, K160E, K160K, K160N, K161I, K161A, K161N, K161Q, K161S, K161T, A162D, A162Q, R162H, R162P, A162S, Q163G, Q163H, Q163N, Q163R, S164I, S164R, S164Y, S165A, S165D, S165I, S165T, S165Y, T166D, T166K, T166I, T166N, T166P, T166R, D167S and / or D167N, and any alternative mutations at the corresponding positions, or for the TadA reference sequence, R26, W23, E27, H36, R47,Any substitution from P48, R51, H52, R74, I76, V82, V88, M94, I95, H96, A106, D108, A109, K110, T111, A114, D119, H122, H123, M126, N127, A142, S146, D147, F149, R152, Q154, E155, I156, E157, K161, T166, and / or D167, or substitutions of 2 to 50 amino acids in the TadA reference sequence that can be selected from W23R, E27D, H36L, R47K, P48A, R51H, R51L, I76F, I76Y, V82S, Al06V, D108G, A109S, K110R, T111H, A114V, D119N, H122R, H122N, H123Y, M126I, N127K, S146C, D147R, R152P, Q154R, E155V, 1156F, K157N, K161N, T166I, and Dl67N, or one or more corresponding mutations in another adenosine deaminase. Additional mutations are described in U.S. Patent Application Publication No. 2022 / 0307003A1 and International Patent Application Publication Nos. WO2023 / 288304A2 and WO2023 / 034959A2, the disclosures of which are hereby incorporated by reference in their entirety for all purposes.

[0215] In embodiments, variants of TadA*7.10 include one or more modifications selected from any of these modifications provided herein.

[0216] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from Staphylococcus aureus (S.aureus) TadA, Bacillus subtilis (B.subtilis) TadA, Salmonella typhimurium (S.typhimurium) TadA, Shewanella putrefaciens (S.putrefaciens) TadA, Haemophilus influenzae F3031 (H.influenzae) TadA, Caulobacter crescents (C.crescentus) TadA, Geobacter sulfurreducens (G.sulfurreducens) TadA, or TadA*7.10.

[0217] In some embodiments, TadA*8 is a variant shown in Table 5D. Table 5D shows the numbers of specific amino acid positions in the TadA amino acid sequence and the amino acids present at those positions in TadA-7.10 adenosine deaminase. Table 5D also shows the amino acid changes of TadA variants relative to TadA-7.10 after phage-assisted non-sustained evolution (PANCE) and phage-assisted continuous evolution (PACE) described in M. Richter et al., 2020, Nature Biotechnology, doi.org / 10.1038 / s41587-020-0453-z (the entire content of which is incorporated herein by reference). In some embodiments, TadA*8 is TadA*8a, TadA*8b, TadA*8c, TadA*8d, or TadA*8e. In some embodiments, TadA*8 is TadA*8e. In one embodiment, the adenosine deaminase is TadA*8 comprising or consisting essentially of SEQ ID NO: 316 or a fragment thereof having adenosine deaminase activity.

[0218]

Table 5D

[0219] In some embodiments, the TadA variant is the variant shown in Table 5E. Table 5E shows the specific amino acid position numbers in the TadA amino acid sequence and the amino acids present at these positions in TadA*7.10 adenosine deaminase. In some embodiments, the TadA variant is MSP605, MSP680, MSP823, MSP824, MSP825, MSP827, MSP828, or MSP829. In some embodiments, the TadA variant is MSP828. In some embodiments, the TadA variant is MSP829.

[0220]

Table 5E

[0221] In certain embodiments, the fusion protein or complex comprises a single (e.g., provided as a monomer) TadA* (e.g., TadA*8 or TadA*9). Throughout the present disclosure, an adenosine deaminase base editor comprising a single TadA* domain is denoted using the terms ABEm or ABE#m, where "#" is an identification number (e.g., ABE8.20m), and where "m" denotes "monomer". In some embodiments, TadA* is linked to a Cas9 nickase. In some embodiments, the fusion protein or complex of the present disclosure comprises wild-type TadA (TadA(wt)) linked to TadA* as a heterodimer. Throughout the present disclosure, an adenosine deaminase base editor comprising a single TadA* domain and a TadA(wt) domain is denoted using the terms ABEd or ABE#d, where "#" is an identification number (e.g., ABE8.20d), and where "d" denotes "dimer". In other embodiments, the fusion protein or complex of the present disclosure comprises TadA*7.10 linked to TadA* as a heterodimer. In some embodiments, the base editor is ABE8 comprising a TadA* variant monomer. In some embodiments, the base editor is ABE comprising a heterodimer of TadA* and TadA(wt). In some embodiments, the base editor is ABE comprising a heterodimer of TadA* and TadA*7.10. In some embodiments, the base editor is ABE comprising a heterodimer of TadA*. In some embodiments, TadA* is selected from Tables 5A - 5E.

[0222] In some embodiments, the adenosine deaminase is expressed as a monomer. In other embodiments, the adenosine deaminase is expressed as a heterodimer. In some embodiments, the deaminase or other polypeptide sequence lacks methionine when included, for example, as a component of a fusion protein. This may change the numbering of positions. However, one of ordinary skill in the art will understand that such corresponding mutations refer to the same mutation.

[0223] Any of the mutations provided herein, and any additional mutations (e.g., based on the ecTadA amino acid sequence), can be introduced into any other adenosine deaminase. Any of the mutations provided herein can be made individually or in any combination in the TadA reference sequence or another adenosine deaminase (e.g., ecTadA).

[0224] Details of nucleic acid base editing proteins from A to G are described in International PCT Application No. PCT / US2017 / 045381 (WO2018 / 027078) and 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), the entire contents of which are incorporated herein by reference.

[0225] Editing from C to T In some embodiments, the base editors disclosed herein include a fusion protein or complex comprising a cytidine deaminase, which can deaminate the target cytidine (C) base of a polynucleotide to generate uridine (U) having the base - pairing properties of thymine. In some embodiments, for example, when the polynucleotide is double - stranded (e.g., DNA), the uridine base is then replaced with a thymidine base (e.g., by the cell's repair machinery), resulting in a transition from C:G to T:A. In other embodiments, deamination of nucleic acid from C to U by a base editor may not be accompanied by replacement of U with T.

[0226] Target C deamination of a polynucleotide to generate U is a non-limiting example of a type of base editing and can be performed by the base editors described herein. In another example, a base editor that includes a cytidine deaminase domain can mediate the conversion of a cytosine (C) base to a guanine (G) base. For example, the U of a polynucleotide produced by deamination of cytidine by the cytidine deaminase domain of a base editor can be removed from the polynucleotide by a base excision repair mechanism (e.g., by a uracil DNA glycosylase (UDG) domain), producing an abasic site. The nucleobase opposite the abasic site can then be replaced with another base (e.g., C), for example, by a translesion polymerase (e.g., by a base repair mechanism). Although it is typical for the nucleobase opposite the abasic site to be replaced with C, other substitutions (e.g., A, G, or T) can also occur.

[0227] Thus, in some embodiments, the base editors described herein include a deamination domain (e.g., a cytidine deaminase domain) that can deaminate a target C of a polynucleotide to U. Further, as described below, the base editor can, in some embodiments, include additional domains that facilitate the conversion of U resulting from deamination to T or the conversion of U to G. For example, a base editor that includes a cytidine deaminase domain can further include a uracil glycosylase inhibitor (UGI) domain that mediates the substitution of U by T, completing a C-to-T base editing event. In another example, the base editor can include a uracil-stabilizing protein as described herein. In another example, because a translesion polymerase can facilitate the incorporation of C opposite an abasic site (i.e., resulting in the incorporation of G at the abasic site and completing a C-to-G base editing event), a translesion polymerase can be incorporated into the base editor to improve the efficiency of C-to-G base editing.

[0228] Base editors that include cytidine deaminase as a domain can deaminate target C of any polynucleotide, including DNA, RNA, and DNA-RNA hybrids.

[0229] In some embodiments, the cytidine deaminase of the base editor includes all or part (e.g., the functional part) of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is a family of evolutionarily conserved cytidine deaminases. Members of this family are C-to-U editing enzymes. The N-terminal domain of APOBEC-like proteins is the catalytic domain, and the C-terminal domain is the pseudocatalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for the deamination of cytidine. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D (currently referred to as "APOBEC3E"), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine) deaminase.

[0230] According to aspects of the present disclosure, other exemplary deaminases that can be fused to Cas9 are provided below. In embodiments, the deaminase is activation-induced deaminase (AID). It should be understood that in some embodiments, the active domain of each sequence, e.g., the domain without a localization signal (no nuclear localization sequence, no nuclear export signal, cytoplasmic localization signal) can be used.

[0231] Some aspects of the present disclosure are based on the recognition that modulating the catalytic activity of the deaminase domain of any of the fusion proteins or complexes described herein, for example, by creating point mutations in the deaminase domain, can affect the processability of the fusion protein (e.g., base editor) or complex. For example, mutations that reduce but do not eliminate the catalytic activity of the deaminase domain in a base editing fusion protein or complex can lower the likelihood that the deaminase domain will catalyze the deamination of residues adjacent to the target residue, thereby narrowing the window of deamination. The ability to narrow the window of deamination can prevent unwanted deamination of residues adjacent to a particular target residue and can reduce or prevent off-target effects.

[0232] In some embodiments, the APOBEC deaminase incorporated into the base editor can include one or more mutations selected from the group consisting of H121R, H122R, R126A, R126E, R118A, W90A, W90Y, and R132E of rAPOBEC1; D316R, D317R, R320A, R320E, R313A, W285A, W285Y, and R326E of hAPOBEC3G, as well as any alternative mutations at corresponding positions, or one or more corresponding mutations in another APOBEC deaminase.

[0233] Several modified cytidine deaminases are commercially available, including but not limited to SaBE3, SaKKH-BE3, VQR-BE3, EQR-BE3, VRER-BE3, YE1-BE3, EE-BE3, YE2-BE3, and YEE-BE3, which are available from Addgene (plasmids 85169, 85170, 85171, 85172, 85173, 85174, 85175, 85176, 85177). In some embodiments, the deaminase incorporated into the base editor includes all or a portion (e.g., a functional portion) of the APOBEC1 deaminase.

[0234] In some embodiments, the fusion protein or complex of the present disclosure comprises one or more cytidine deaminase domains. In some embodiments, the cytidine deaminase provided herein can deaminate cytosine or 5-methylcytosine to uracil or thymine. In some embodiments, the cytidine deaminase provided herein can deaminate cytosine of DNA. The cytidine deaminase can be derived from any suitable organism. In some embodiments, the cytidine deaminase is a naturally occurring cytidine deaminase comprising one or more mutations corresponding to any of the mutations provided herein. One of ordinary skill in the art will be able to identify the corresponding residues of any homologous protein, for example, by sequence alignment and determination of homologous residues. Thus, one of ordinary skill in the art will be able to generate mutations in any natural cytidine deaminase corresponding to any of the mutations described herein. In some embodiments, the cytidine deaminase is of prokaryotic origin. In some embodiments, the cytidine deaminase is of bacterial origin. In some embodiments, the cytidine deaminase is of mammalian (e.g., human) origin.

[0235] In some embodiments, the cytidine deaminase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of the amino acid sequences described herein. It should be understood that the cytidine deaminase provided herein can comprise one or more mutations (e.g., any of the mutations provided herein). Some embodiments provide a polynucleotide molecule encoding any of the previous embodiments or the cytidine deaminase nucleic acid base editor polypeptide described herein. In some embodiments, the polynucleotide is codon-optimized.

[0236] In embodiments, the fusion protein of the present disclosure comprises two or more nucleic acid editing domains.

[0237] Details of cytosine to thymine nucleobase editing proteins are described in International PCT Application No. PCT / US2016 / 058344 (WO2017 / 070632) and Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016) (the entire contents of which are incorporated herein by reference).

[0238] Cytidine adenosine base editor (CABE) In some embodiments, the base editors described herein include adenosine deaminase variants having increased cytidine deaminase activity. Such base editors may be referred to as “cytidine adenosine base editors (CABE)” or “cytosine base editors derived from TadA*(CBE-Ts)”, and their corresponding deaminase domains are “TadA* that acts on DNA cytosine (T ADC) can be referred to as a "domain". In some cases, the adenosine deaminase variant has both adenine and cytosine deaminase activities (i.e., is a dual deaminase). In some embodiments, the adenosine deaminase variant deaminates adenine and cytosine in DNA. In some embodiments, the adenosine deaminase variant deaminates adenine and cytosine in single-stranded DNA. In some embodiments, the adenosine deaminase variant deaminates adenine and cytosine in RNA. In some embodiments, the adenosine deaminase variant predominantly deaminates cytosine in DNA and / or RNA (e.g., more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all deaminations catalyzed by the adenosine deaminase variant, or the number of cytosine deaminations catalyzed by the variant is about or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, 100-fold, 500-fold, or 1,000-fold greater than the number of adenine deaminations catalyzed by the variant). In some embodiments, the adenosine deaminase variant has approximately equal cytosine and adenosine deaminase activities (e.g., the two activities are within about 10% or 20% of each other). In some embodiments, the adenosine deaminase variant predominantly has cytosine deaminase activity and little to no adenosine deaminase activity, if any. In some embodiments, the adenosine deaminase variant has cytosine deaminase activity and no significant or detectable adenosine deaminase activity. In some embodiments, the target polynucleotide is present in cells in vitro or in vivo. In some embodiments, the cells are bacterial, yeast, fungal, insect, plant, or mammalian cells.

[0239] In some embodiments, CABE comprises a bacterial TadA deaminase variant (e.g., ecTadA). In some embodiments, CABE comprises a truncated TadA deaminase variant. In some embodiments, CABE comprises a fragment of a TadA deaminase variant. In some embodiments, CABE comprises the TadA*8.20 variant.

[0240] In some embodiments, the adenosine deaminase variant of the disclosure is a TadA adenosine deaminase comprising one or more modifications that increase cytidine deaminase activity (e.g., increase by at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more) while maintaining adenosine deaminase activity (e.g., at least about 30%, 40%, 50% or more of the activity of a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19)). In some cases, the adenosine deaminase variant comprises one or more modifications that increase cytidine deaminase activity (e.g., increase by at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more) relative to the activity of a reference adenosine deaminase and comprises undetectable adenosine deaminase activity or adenosine deaminase activity that is less than 30%, 20%, 10%, or 5% of the activity of the reference adenosine deaminase. In some embodiments, the reference adenosine deaminase is TadA*8.20 or TadA*8.19.

[0241] In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising two or more modifications at amino acid positions selected from the group consisting of 2, 4, 6, 8, 13, 17, 23, 27, 29, 30, 47, 48, 49, 67, 76, 77, 82, 84, 96, 100, 107, 112, 114, 115, 118, 119, 122, 127, 142, 143, 147, 149, 158, 159, 162, 165, 166, and 167 of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 1, or corresponding modifications in another deaminase. I

[0242] In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising one or more modifications selected from the group consisting of S2H, V4K, V4S, V4T, V4Y, F6G, F6H, F6Y, H8Q, R13G, T17A, T17W, R23Q, E27C, E27G, E27H, E27K, E27Q, E27S, E27G, P29A, P29G, P29K, V30F, V30I, R47G, R47S, A48G, I49K, I49M, I49N, I49Q, I49T, G67W, I76H, I76R, I76W, Y76H, Y76R, Y76W, F84A, F84M, H96N, G100A, G100K, T111H, G112H, A114C, G115M, M118L, H122G, H122R, H122T, N127I, N127K, N127P, A142E, R147H, A158V, Q159S, A162C, A162N, A162Q, and S165P of an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identity to SEQ ID NO: 1, or corresponding modifications in another deaminase.

[0243] In some embodiments, the adenosine deaminase variant is an adenosine deaminase comprising an amino acid modification or combination of amino acid modifications selected from those listed in any of Tables 6A - 6F.

[0244] The residue identities of exemplary adenosine deaminase variants capable of deaminating adenine and / or cytidine in target polynucleotides (e.g., DNA) are provided in Tables 6A-6F below. Further examples of adenosine deaminase variants include the following variants of 1.17 (see Table 6A): 1.17+E27H; 1.17+E27K; 1.17+E27S; 1.17+E27S+I49K; 1.17+E27G; 1.17+I49N; 1.17+E27G+I49N; and 1.17+E27Q. In some embodiments, any of the amino acid modifications provided herein are substituted with conservative amino acids. Additional mutations known in the art can be added to any of the adenosine deaminase variants provided herein.

[0245] In some embodiments, base editor systems comprising a CABE provided herein have at least about 30%, 40%, 50%, 60%, 70% or more C to T editing activity in a target polynucleotide (e.g., DNA). In some embodiments, base editor systems comprising a CABE provided herein have increased C to T base editing activity (e.g., at least about 30-fold, 40-fold, 50-fold, 60-fold, 70-fold or more increased) relative to a reference base editor system comprising a reference adenosine deaminase (e.g., TadA*8.20 or TadA*8.19).

[0246] [Table 6A-1] [Table 6A-2]

[0247] [Table 6B-1] [Table 6B-2]

Table 6B-3

Table 6B-4

Table 6B-5

Table 6B-6

Table 6B-7

[0248]

Table 6C-1

Table 6C-2

[0249]

Table 6D-1

Table 6D-2

[0250]

Table 6E

[0251]

Table 6F

[0252] Guide polynucleotide A polynucleotide programmable nucleotide binding domain, when combined with a bound guide polynucleotide (e.g., gRNA), specifically binds to a target polynucleotide sequence (i.e., through complementary base pairing between the bases of the bound guide nucleic acid and the bases of the target polynucleotide sequence), thereby localizing a base editor to the target nucleic acid sequence where editing is desired. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0253] In one embodiment, the guide polynucleotide described herein can be RNA or DNA. In one embodiment, the guide polynucleotide is a gRNA.

[0254] In some embodiments, the guide polynucleotide is at least one single guide RNA (“sgRNA” or “gRNA”). In some embodiments, the guide polynucleotide comprises two or more individual polynucleotides that can interact with each other, for example, through complementary base pairing (e.g., dual guide polynucleotide, dual gRNA). For example, the guide polynucleotide may comprise a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), or may comprise one or more trans-activating CRISPR RNAs (tracrRNAs).

[0255] The guide polynucleotide can include natural or non-natural (or unnatural) nucleotides (e.g., peptide nucleic acids or nucleotide analogs). In some cases, the target region of the guide nucleic acid sequence (e.g., the spacer) can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0256] In some embodiments, the methods described herein can utilize engineered Cas proteins. Guide RNAs (gRNAs) are short synthetic RNAs composed of a scaffold sequence necessary for Cas binding and a user-defined ~20 nucleotide spacer that defines the genomic target to be modified. Exemplary gRNA scaffold sequences are shown in the Sequence Listing as SEQ ID NOs: 317-327 and 425. Thus, one of skill in the art can change the genomic target of Cas protein specificity, and the target sequence of the gRNA is partially determined by how specific it is for the genomic target compared to the rest of the genome. In embodiments, the spacer is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. The spacer of the gRNA can be 19, 20, or 21 nucleotides in length, or about 19, 20, or 21 nucleotides in length.

[0257] The gRNA or guide polynucleotide can target any exon or intron of a gene target. In some embodiments, the composition comprises multiple gRNAs that all target the same exon or multiple gRNAs that target different exons. Exons and / or introns of a gene can be targeted. The gRNA or guide polynucleotide can target a nucleic acid sequence of about 20 nucleotides or less than about 20 nucleotides (e.g., at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 nucleotides), or any number from about 1 to 100 (e.g., 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides). The target nucleic acid sequence can be 20 bases immediately 5' of the first nucleotide of the PAM, or about 20 bases. The gRNA can target a nucleic acid sequence. The target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides.

[0258] The guide polynucleotide can include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs.

[0259] In some embodiments, the base editor system can include multiple guide polynucleotides (e.g., gRNAs). For example, the gRNAs can target one or more target loci included in the base editor system (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs). The multiple gRNA sequences can be arranged in series and separated by direct repeats.

[0260] Modified polynucleotide To enhance expression, stability and / or genome / base editing efficiency and / or to reduce potential toxicity, the base editor coding sequence (e.g., mRNA) and / or guide polynucleotide (e.g., gRNA) can be modified to include one or more modified nucleotides and / or chemical modifications, e.g., using pseudouridine, 5-methyl-cytosine, 2'-O-methyl-3'-phosphonoacetate, 2'-O-methylthioPACE (MSP), 2'-O-methyl-PACE (MP), 2'-fluoro RNA (2'-F-RNA), locked ethyl (S-cEt), 2'-O-methyl ("M"), 2'-O-methyl-3'-phosphorothioate ("MS"), 2'-O-methyl-3'-thiophosphonoacetate ("MSP"), 5-methoxyuridine, phosphorothioate and N1-methylpseudouridine. Chemically protected gRNAs can enhance stability and editing efficiency in vivo and ex vivo. Methods of using chemically modified mRNAs and guide RNAs are known in the art, e.g., as described by Jiang et al., Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). doi.org / 10.1038 / s41467-020-15892-8, Callum et al., N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells, Nucleic Acids Research, Volume 48, Issue 6, 06 April 2020, Page e35, and Andries et al., Journal of Controlled Release, Volume 217, 10 November 2015, Pages 337-344, each of which is incorporated herein by reference in its entirety.

[0261] In some embodiments, the guide polynucleotide comprises one or more modified nucleotides at the 5' end and / or 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, or four or more modified nucleotides at the 5' end and / or 3' end of the guide. In some embodiments, the guide polynucleotide comprises two, three, or four or more modified nucleotides at the 5' end and / or 3' end of the guide.

[0262] In some embodiments, the guide comprises at least about 50% to 75% modified nucleotides. In some embodiments, the guide comprises at least about 85% or more modified nucleotides. In some embodiments, at least about 1 to 5 nucleotides at the 5' end of the gRNA are modified, and at least about 1 to 5 nucleotides at the 3' end of the gRNA are modified. In some embodiments, at least about 3 to 5 consecutive nucleotides at each of the 5' and 3' ends of the gRNA are modified. In some embodiments, at least about 20% of the nucleotides present within the direct repeat or the anti-direct repeat are modified. In some embodiments, at least about 50% of the nucleotides present within the direct repeat or the anti-direct repeat are modified. In some embodiments, at least about 50 to 75% of the nucleotides present within the direct repeat or the anti-direct repeat are modified. In some embodiments, at least about 100 of the nucleotides present within the direct repeat or the anti-direct repeat are modified. In some embodiments, at least about 20% or more of the nucleotides present within the hairpin present within the gRNA scaffold are modified. In some embodiments, at least about 50% or more of the nucleotides present within the hairpin present within the gRNA scaffold are modified. In some embodiments, the guide comprises a variable length spacer. In some embodiments, the guide comprises a 20-40 nucleotide spacer. In some embodiments, the guide comprises a spacer comprising at least about 20 to 25 nucleotides or at least about 30 to 35 nucleotides. In some embodiments, the spacer comprises modified nucleotides. In some embodiments, the guide comprises two or more of the following: ● that at least about 1 to 5 nucleotides at the 5' end of the gRNA are modified and at least about 1 to 5 nucleotides at the 3' end of the gRNA are modified, ● that at least about 20% of the nucleotides present within the direct repeat or the anti-direct repeat are modified, ●At least about 50-75% of the nucleotides present within the direct repeat or anti-direct repeat are modified. ●At least about 20% or more of the nucleotides present within the hairpin present within the gRNA scaffold are modified. ●Variable length spacer, and ●A spacer comprising modified nucleotides.

[0263] In embodiments, the gRNA contains a number of modified nucleotides and / or chemical modifications (“heavy mod”). Such heavy modifications can increase base editing by about 2-fold in vivo or in vitro. In embodiments, the gRNA comprises 2'-O-methyl or phosphorothioate modifications. In one embodiment, the gRNA comprises 2'-O-methyl and phosphorothioate modifications. In one embodiment, the modification increases base editing by at least about 2-fold.

[0264] The guide polynucleotide can include one or more modifications to provide a nucleic acid having new or enhanced features. The guide polynucleotide can include a nucleic acid affinity tag. The guide polynucleotide can include synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0265] The gRNA or guide polynucleotide can also be modified by 5'-adenylate, 5'-guanosine-triphosphate cap, 5'-N7-methylguanosine-triphosphate cap, 5'-triphosphate cap, 3'-phosphate, 3'-thiophosphate, 5'-phosphate, 5'-thiophosphate, cis-syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, d spacer, PC spacer, r spacer, spacer 18, spacer 9, 3'-3' modification, 2'-O-methylthioPACE (MSP), 2'-O-methyl-PACE (MP) and constrained ethyl (S-cEt), 5'-5' modification, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'-DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-(-7), QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methylribonucleoside analog, sugar-modified analog, wobble / universal base, fluorescent dye label, 2'-fluoroRNA, 2'-O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0266] Note: There seems to be a potential error in the original text where "QSY-(-7)" is written. It's left as is in the translation following the instruction to preserve all 7 - digit tags exactly.In some cases, the gRNA, which is RNA enhanced with phosphorothioate, can inhibit RNase A, RNase T1, bovine serum nuclease, or any combination thereof. These properties can enable the use of PS-RNA gRNAs in applications where they are likely to be exposed to nucleases in vivo or in vitro. For example, phosphorothioate (PS) linkages can be introduced between the last 3 to 5 nucleotides at the 5' or 3' end of the gRNA, which can inhibit exonuclease degradation. In some cases, phosphorothioate linkages can be added throughout the gRNA to reduce attack by endonucleases. Fusion protein or complex containing a nuclear localization sequence (NLS)

[0267] In some embodiments, the fusion proteins or complexes provided herein further comprise one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLSs). In one embodiment, a bipartite NLS is used. In some embodiments, the NLS comprises an amino acid sequence that facilitates the import of a protein containing the NLS into the cell nucleus (e.g., by nuclear transport). In some embodiments, the NLS is fused to the N-terminus or C-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus or N-terminus of the nCas9 domain or dCas9 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of the Cas12 domain. In some embodiments, the NLS is fused to the N-terminus or C-terminus of cytidine or adenosine deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises the amino acid sequence of any one of the NLS sequences provided or referenced herein. Additional nuclear localization sequences are known in the art and will be apparent to those of skill in the art. For example, the NLS sequences are described in Plank et al., PCT / EP2000 / 011690 (the contents of which are hereby incorporated by reference for the disclosure of exemplary nuclear localization sequences).

[0268] In some embodiments, the NLS is present within a linker or adjacent to a linker, as described herein, for example. A bipartite NLS contains two basic amino acid clusters separated by a relatively short spacer sequence (thus, bipartite - two parts, while a monopartite NLS is not). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 191), is a prototype of a ubiquitous bipartite signal and consists of two clusters of basic amino acids separated by a spacer of about 10 amino acids. Exemplary bipartite NLS sequences are as follows: PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 328).

[0269] In some embodiments, any of the fusion proteins or complexes provided herein comprises an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 - 29 of SEQ ID NO: 328). In some embodiments, any of the adenosine base editors provided herein, e.g., ABE variant A, ABE variant B, ABE variant C, ABE variant D, ABE variant E, ABE variant F, ABE variant G, ABE variant H, ABE variant I, ABE variant J, ABE variant K, or ABE variant D, comprises an NLS comprising the amino acid sequence EGADKRTADGSEFESPKKKRKV (amino acids 8 - 29 of SEQ ID NO: 328). In some embodiments, the NLS is in the C-terminal portion of the adenosine base editor. In some embodiments, the NLS is at the C-terminus of the adenosine base editor.

[0270] Additional domain The base editors described herein can include any domain that helps facilitate the editing, modification, or alteration of the nucleic acid bases of a polynucleotide. In some embodiments, the base editor comprises a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a nucleic acid base editing domain (e.g., a deaminase domain), and one or more additional domains. In some embodiments, the additional domain can be a factor that promotes the enzymatic or catalytic function of the base editor, the binding function of the base editor, or an inhibitor of a cellular mechanism (e.g., an enzyme) that can interfere with the desired base editing outcome. In some embodiments, the base editor comprises a nuclease, nickase, recombinase, deaminase, methyltransferase, methylase, acetylase, acetyltransferase, transcriptional activator, or transcriptional repressor domain.

[0271] In some embodiments, the base editor comprises a uracil glycosylase inhibitor (UGI) domain. In some embodiments, the cellular DNA repair response to the presence of U:G heteroduplex DNA can cause a decrease in nucleic acid base editing efficiency in the cell. In such embodiments, uracil DNA glycosylase (UDG) can catalyze the removal of U from the cellular DNA, initiate base excision repair (BER), and can primarily result in the reversion from a U:G pair to a C:G pair. In such embodiments, BER can be inhibited by a base editor comprising one or more domains that bind to single strands, block the edited base, inhibit UGI, inhibit BER, protect the edited base, and / or promote repair of the unedited strand. Accordingly, the present disclosure contemplates a base editor fusion protein or complex comprising a UGI domain and / or a uracil stabilizing protein (USP) domain.

[0272] Base editor system Systems, compositions, and methods are provided herein for editing nucleic acid bases using a base editor system. In some embodiments, the base editor system comprises: (1) a base editor (BE) comprising a polynucleotide programmable nucleotide binding domain and a nucleic acid base editing domain (e.g., a deaminase domain) for editing nucleic acid bases; and (2) a guide polynucleotide (e.g., guide RNA) in combination with the polynucleotide programmable nucleotide binding domain. In some embodiments, the base editor system is a cytidine base editor (CBE) or an adenosine base editor (ABE). In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA or RNA binding domain. In some embodiments, the nucleic acid base editing domain is a deaminase domain. In some embodiments, the deaminase domain can be a cytidine deaminase or a cytosine deaminase. In some embodiments, the deaminase domain can be an adenine deaminase or an adenosine deaminase. In some embodiments, the adenosine base editor can deaminate adenine in DNA. In some embodiments, the base editor can deaminate cytidine in DNA.

[0273] The use of the base editor system provided herein involves (a) contacting a target nucleotide sequence of a target polynucleotide (e.g., double-stranded or single-stranded DNA or RNA) with a base editor system comprising a nucleic acid base editor (e.g., an adenosine base editor or a cytidine base editor) and a guide polynucleotide (e.g., gRNA), wherein the target nucleotide sequence comprises a target nucleobase pair, the contacting step; (b) inducing strand separation of the target region; (c) converting a first nucleobase of the target nucleobase pair in a single strand of the target region to a second nucleobase; and (d) cleaving one or fewer strands of the target region, wherein a third nucleobase complementary to the first nucleobase is replaced by a fourth nucleobase complementary to the second nucleobase. It should be understood that in some embodiments, step (b) is omitted. In some embodiments, the target nucleobase pair is a plurality of nucleobase pairs in one or more genes. In some embodiments, the base editor system provided by the present invention can multiplex-edit a plurality of nucleobase pairs in one or more genes. In some embodiments, the plurality of nucleobase pairs are located in the same gene. In some embodiments, the plurality of nucleobase pairs are located in one or more genes, and at least one gene is located at a different locus.

[0274] The components of a base editor system (e.g., a deaminase domain, a guide RNA, and / or a polynucleotide programmable nucleotide binding domain) can associate with each other covalently or non-covalently. For example, in some embodiments, the deaminase domain can be targeted to a target nucleotide sequence by a polynucleotide programmable nucleotide binding domain, and optionally, the polynucleotide programmable nucleotide binding domain forms a complex with a polynucleotide (e.g., a guide RNA). In some embodiments, the polynucleotide programmable nucleotide binding domain can be fused or linked to the deaminase domain. In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to a target nucleotide sequence by non-covalently interacting with or associating with the deaminase domain. For example, in some embodiments, a nucleic acid base editing component (e.g., a deaminase component) includes an additional heterologous moiety or domain, and the additional heterologous moiety or domain can interact with, associate with, or form a complex with a corresponding heterologous moiety, antigen, or domain that is part of the polynucleotide programmable nucleotide binding domain and / or a guide polynucleotide (e.g., a guide RNA) that forms a complex therewith. In some embodiments, the polynucleotide programmable nucleotide binding domain and / or a guide polynucleotide (e.g., a guide RNA) that forms a complex therewith includes an additional heterologous moiety or domain, and the additional heterologous moiety or domain can interact with, associate with, or form a complex with a corresponding heterologous moiety, antigen, or domain that is part of the nucleic acid base editing domain (e.g., a deaminase component). In some embodiments, the additional heterologous moiety may be able to bind to, interact with, associate with, or form a complex with a polypeptide. In some embodiments, the additional heterologous moiety may be able to bind to, interact with, associate with, or form a complex with a polynucleotide.or may be able to form a complex with a polynucleotide. In some embodiments, the additional heterologous moiety may be able to bind to the guide polynucleotide. In some embodiments, the additional heterologous moiety may be able to bind to a polypeptide linker. In some embodiments, the additional heterologous moiety may be capable of binding to a polynucleotide linker. The additional heterologous moiety can be a protein domain. In some embodiments, the additional heterologous moiety is a polypeptide, e.g., the 22-amino acid RNA-binding domain (N22p) of lambda bacteriophage antiterminator protein N, the 2G12 IgG homodimer domain, ABI, an antibody (e.g., an antibody that binds to a component of the base editor system or a heterologous moiety thereof) or a fragment thereof (e.g., the heavy chain domain 2 (CH2) of IgM (MHD2) or IgE (EHD2), the immunoglobulin Fc region, the heavy chain domain 3 (CH3) of IgG or IgA, the heavy chain domain 4 (CH4) of IgM or IgE, Fab, Fab2, minibody, and / or ZIP antibody), the barnase-barstar dimer domain, the Bcl-xL domain, the calcineurin A (CAN) domain, the cardiac phospholamban transmembrane pentamer domain, the collagen domain, the Com RNA-binding protein domain (e.g., the SfMu Com coat protein domain and the SfMu Com binding protein domain), the cyclophilin-Fas fusion protein (CyP-Fas) domain, the Fab domain, the Fe domain, the fibritin foldon domain, the FK506-binding protein (FKBP) domain, the FKBP-binding domain (FRB) domain of mTOR, the foldon domain, the fragment X domain, the GAI domain, the GID1 domain, the glycophorin A transmembrane domain, the GyrB domain, the Halo tag, the HIV Gp41 trimerization domain, the C-terminal dimer domain of HPV45 oncoprotein E7, a hydrophobic polypeptide, the K homology (KH) domain, the Ku protein domain (e.g., the Ku heterodimer), the leucine zipper, the LOV domain, the mitochondrial antiviral signaling protein CARD filament domain, the MS2 coat protein domain (MCP), a non-natural RNA aptamer ligand that binds to the corresponding RNA motif / aptamer,It includes a parathyroid hormone dimerization domain, a PP7 coat protein (PCP) domain, a PSD95-Dlgl-zo-1 (PDZ) domain, a PYL domain, a SNAP tag, a SpyCatcher moiety, a SpyTag moiety, a streptavidin domain, a streptavidin-binding protein domain, a streptavidin-binding protein (SBP) domain, a telomerase Sm7 protein domain (e.g., Sm7 homoheptamer or monomeric Sm-like protein), and / or fragments thereof. In embodiments, additional heterologous moieties are polynucleotides (e.g., RNA motifs), such as MS2 phage operator stem-loops (e.g., MS2, MS2 C-5 variant or MS2 F-5 variant), non-natural RNA motifs, PP7 operator stem-loops, SfMu phate Com stem-loops, sterile alpha motifs, telomerase Ku-binding motifs, telomerase Sm7-binding motifs and / or fragments thereof. Non-limiting examples of additional heterologous moieties include polypeptides or fragments thereof having at least about 85% sequence identity to any one or more of SEQ ID NOs: 380, 382, 384, 386-388. Non-limiting examples of additional heterologous moieties include polynucleotides or fragments thereof having at least about 85% sequence identity to any one or more of SEQ ID NOs: 379, 381, 383, 385.,

[0275] In some cases, the components of the base editing system associate with each other through the interaction of leucine zipper domains (e.g., SEQ ID NOs: 387 and 388). In some cases, the components of the base editing system associate with each other through polypeptide domains (e.g., FokI domain), and the polypeptide domains associate to form a protein complex, and the protein complex contains about 1, 2 (i.e., dimerization), 3, 4, 5, 6, 7, 8, 9, 10 polypeptide domain units, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 polypeptide domain units or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or fewer polypeptide domain units, and optionally, the polypeptide domains may include modifications that reduce or eliminate their activity.

[0276] In some cases, the components of the base editing system associate with each other through the interaction of multimeric antibodies or fragments thereof (e.g., IgG; IgD; IgA; IgM; IgE; heavy chain domain CH2 of IgM (MHD2) or IgE (EHD2); immunoglobulin Fc region; heavy chain domain CH3 of IgG or IgA; heavy chain domain CH4 of IgM or IgE; Fab; and Fab2). In some cases, the antibody is a dimer, trimer or tetramer. In embodiments, the dimeric antibody binds to a polypeptide or polynucleotide component of the base editing system.

[0277] In some cases, the components of the base editing system associate with each other through the interaction of polynucleotide-binding protein domain(s) and polynucleotide(s). In some cases, the components of the base editing system associate with each other through the interaction of one or more polynucleotide-binding protein domains and a polynucleotide, and the polynucleotides are self-complementary and / or complementary to each other, whereby the complementary binding of the polynucleotides to each other results in the association of their corresponding bound polynucleotide-binding protein domain(s).

[0278] In some cases, the components of the base editing system associate with each other through the interaction of a polypeptide domain(s) and a small molecule(s) (e.g., a dimer-inducing compound (CID) also known as a “dimerizer”). Non-limiting examples of CIDs are those disclosed in Amara, et al., “A versatile synthetic dimerizer for the regulation of protein-protein interactions,” PNAS, 94:10618-10623 (1997) and Voss, et al. “Chemically induced dimerization:reversible and spatiotemporal control of protein function in cells,” Current Opinion in Chemical Biology, 28:^{}194-201 (2015), the disclosures of each of which are hereby incorporated by reference in their entirety for all purposes. In some embodiments, the base editor inhibits base excision repair (BER) of the edited strand. In some embodiments, the base editor protects or binds the unedited strand. In some embodiments, the base editor comprises UGI activity or USP activity. In some embodiments, the base editor comprises a catalytically inactive inosine-specific nuclease.

[0279] The base editors of the present disclosure can include any domain, feature, or amino acid sequence that facilitates editing of a target polynucleotide sequence. For example, in some embodiments, the base editor comprises a nuclear localization sequence (NLS). In some embodiments, the NLS of the base editor is located between the deaminase domain and the polynucleotide programmable nucleotide binding domain. In some embodiments, the NLS of the base editor is located at the C-terminus of the polynucleotide programmable nucleotide binding domain.

[0280] The protein domains included in the fusion protein can be heterologous functional domains. Non-limiting examples of protein domains that can be included in the fusion protein include deaminase domains (e.g., cytidine deaminase and / or adenosine deaminase), uracil glycosylase inhibitor (UGI) domains, epitope tags, and reporter gene sequences.

[0281] In some embodiments, an adenosine base editor (ABE) can deaminate adenine in DNA. In some embodiments, ABE is generated by replacing the APOBEC1 component of BE3 with native or engineered E. coli TadA, human ADAR2, mouse ADA, or human ADAT2. In some embodiments, ABE includes an evolved TadA variant. In some embodiments, the base editor is ABE8.1 that includes or consists essentially of the following sequence or a fragment thereof having adenosine deaminase activity: SEQ ID NO: 331. Other ABE8 sequences are shown in the accompanying sequence listing (SEQ ID NOS: 332-354).

[0282] In some embodiments, the base editor includes an adenosine deaminase variant having an amino acid sequence that includes modifications relative to the ABE7*10 reference sequence as described herein. When used in Table 7, the term "monomer" refers to the monomeric form of TadA*7.10 that includes the described modifications. When used in Table 7, the term "heterodimer" refers to a specific wild-type E. coli TadA adenosine deaminase fused to TadA*7.10 that includes the described modifications.

[0283]

Table 7

[0284] In some embodiments, the base editor includes a domain that includes all or a portion (e.g., a functional portion) of a uracil glycosylase inhibitor (UGI) or uracil-stabilizing protein (USP) domain.

[0285] Linker In certain embodiments, a linker may be used to join either a peptide or a peptide domain of the present disclosure. The linker may be as simple as a covalent bond or a polymeric linker that is several atoms in length. In certain embodiments, the linker is a polypeptide or amino acid-based. In other embodiments, the linker is not peptidomimetic. In certain embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).

[0286] In some embodiments, any of the fusion proteins provided herein comprises a cytidine deaminase or adenosine deaminase and a Cas9 domain that are fused to each other via a linker. Various linker lengths and flexibilities between the cytidine or adenosine deaminase and the Cas9 domain can be utilized to achieve an optimal length for activity of the cytidine or adenosine deaminase nucleobase editor (e.g., (GGGS) n (SEQ ID NO: 246), (GGGGS) n (SEQ ID NO: 247) and very flexible linkers in the form of (G)n, to (EAAAK) n (SEQ ID NO: 248), (SGGS) n(SEQ ID NO: 355), SGSETPGTSESATPES (SEQ ID NO: 249) (see, for example, Guilinger JP, et al. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6):577-82, the entire contents of which are incorporated herein by reference), and in the form of a more rigid linker of (XP)n). In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the linker comprises a (GGS)n motif, where n is 1, 3, or 7. In some embodiments, the cytidine deaminase or adenosine deaminase and the Cas9 domain of any of the fusion proteins provided herein are fused via a linker comprising the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as an XTEN linker).

[0287] In some embodiments, the domain of the base editor has the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS (SEQ ID NO: 356), SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 357), or is fused via a linker comprising GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS (SEQ ID NO: 358).

[0288] In some embodiments, the domain of the base editor is fused via a linker that includes the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 249), which may also be referred to as an XTEN linker. In some embodiments, the linker includes the amino acid sequence SGGS (SEQ ID NO: 355). In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker includes the amino acid sequence SGGSSGGSSGSETPGTSESATPES (SEQ ID NO: 359). In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker includes the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS (SEQ ID NO: 360). In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker includes the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 361). In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker includes the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS (SEQ ID NO: 362).

[0289] In some embodiments, the linker comprises a plurality of proline residues and is 5-21, 5-14, 5-9, 5-7 amino acids in length, such as PAPAP (SEQ ID NO: 363), PAPAPA (SEQ ID NO: 364), PAPAPAP (SEQ ID NO: 365), PAPAPAPA (SEQ ID NO: 366), P(AP)4 (SEQ ID NO: 367), P(AP)7 (SEQ ID NO: 368), P(AP)10 (SEQ ID NO: 369) (see, e.g., Tan J, Zhang F, Karcher D, Bock R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat Commun. 2019 Jan 25; 10(1):439. The entire content thereof is incorporated herein by reference). Such proline-rich linkers are also referred to as "rigid" linkers.

[0290] Nucleic acid programmable DNA binding protein with guide RNA Compositions and methods for base editing within cells are provided herein. Compositions are further provided herein that include a guide polynucleotide sequence (e.g., a guide RNA sequence) or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more guide RNAs provided herein. In some embodiments, the compositions for base editing provided herein further include a polynucleotide encoding a base editor, e.g., a C base editor or an A base editor. For example, the composition for base editing can include an mRNA sequence encoding a combination of BE, BE4, ABE, and one or more of the guide RNAs provided. The composition for base editing can include a base editor polypeptide and a combination of one or more of any of the guide RNAs provided herein. Such compositions can be used to effect base editing in cells via different delivery approaches (e.g., electroporation, nucleofection, viral transduction, or transfection). In some embodiments, the composition includes an mRNA sequence encoding a base editor and a combination of one or more guide RNA sequences provided herein for electroporation.

[0291] Some aspects of the present disclosure provide a system comprising any of the fusion proteins or complexes provided herein and a guide RNA bound to a nucleic acid programmable DNA binding protein (napDNAbp) domain of the fusion protein or complex (e.g., Cas9 (e.g., dCas9, nuclease-active Cas9, or Cas9 nickase) or Cas12). These complexes are also referred to as ribonucleoprotein (RNP). In some embodiments, the guide nucleic acid (e.g., guide RNA) is 15 to 100 nucleotides in length and comprises a sequence of at least 10 consecutive nucleotides complementary to a target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is an RNA sequence. In some embodiments, the target sequence is a sequence of the genome of bacteria, yeast, fungi, insects, plants, or animals. In some embodiments, the target sequence is a sequence of the human genome. In some embodiments, the 3' end of the target sequence is directly adjacent to a canonical PAM sequence (NGG). In some embodiments, the 3' end of the target sequence is directly adjacent to a non-canonical PAM sequence (e.g., the sequences listed in Table 3 or 5'-NAA-3'). In some embodiments, the guide nucleic acid (e.g., guide RNA) is complementary to the sequence of a gene of interest (e.g., a gene associated with a disease or disorder).

[0292] Some aspects of the present disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of the present disclosure provide a method comprising contacting a DNA molecule with any of the fusion proteins or complexes provided herein and at least one guide RNA, the guide RNA being about 15 to 100 nucleotides in length and comprising a sequence of at least 10 consecutive nucleotides complementary to a target sequence.

[0293] The domains of the base editors disclosed herein can be arranged in any order.

[0294] The defined target region can be a deamination window. The deamination window can be a predetermined region where the base editor acts on and deaminates the target nucleotide. In some embodiments, the deamination window is within a 2, 3, 4, 5, 6, 7, 8, 9, or 10-base region. In some embodiments, the deamination window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 bases upstream of the PAM.

[0295] The base editors of the present disclosure can include any domain, feature, or amino acid sequence that facilitates the editing of a target polynucleotide sequence.

[0296] Methods of using a fusion protein or complex comprising a cytidine or adenosine deaminase and a Cas9 domain Some aspects of the present disclosure provide methods of using the fusion proteins or complexes provided herein. For example, some aspects of the present disclosure provide methods that include contacting a DNA molecule with any of the fusion proteins or complexes provided herein and contacting it with at least one guide RNA described herein.

[0297] In some embodiments, the fusion proteins or complexes of the present disclosure are used to edit a target gene of interest. In particular, the cytidine deaminase or adenosine deaminase nucleic acid base editors described herein can create multiple mutations within a target sequence. These mutations can affect the function of the target. For example, targeting a regulatory region using a cytidine deaminase or adenosine deaminase nucleic acid base editor changes the function of the regulatory region and reduces or eliminates the expression of downstream proteins.

[0298] Efficiency of base editors In some embodiments, the purpose of the methods provided by the present invention is to modify genes and / or gene products via gene editing. The nucleobase editing proteins provided herein can be used in vitro or in vivo for gene editing-based human therapeutics. It will be understood by those skilled in the art that the nucleobase editing proteins provided herein (e.g., fusion proteins or complexes comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nucleobase editing domain (e.g., an adenosine deaminase domain or a cytidine deaminase domain)) can be used to edit nucleotides from A to G or from C to T.

[0299] Advantageously, the base editor systems provided herein provide genome editing that does not generate double-stranded DNA breaks, does not require a donor DNA template, and does not induce excessive random insertions and deletions, since CRISPR can do so. In some embodiments, the present disclosure provides a base editor that efficiently generates "intended mutations", such as stop codons of nucleic acids (e.g., nucleic acids within the genome of a subject), without generating a significant number of unintended mutations (e.g., unintended point mutations).

[0300] The base editors of the present disclosure advantageously modify specific nucleotide bases encoding proteins without generating a significant proportion of indels (i.e., insertions or deletions). Such indels can result in frameshift mutations within the coding region of a gene.

[0301] In some embodiments, the base editors provided herein can generate a ratio of the intended mutation to indels greater than 1:1 (i.e., intended point mutation:unintended point mutation). In some embodiments, the base editors provided herein can generate a ratio of the intended mutation to indels of at least 1.5:1, at least 2:1, at least 2.5:1, at least 3:1, at least 3.5:1, at least 4:1, at least 4.5:1, at least 5:1, at least 5.5:1, at least 6:1, at least 6.5:1, at least 7:1, at least 7.5:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1, or more. The number of intended mutations and indels can be determined using any suitable method.

[0302] In some embodiments, the base editors provided herein can limit the formation of indels in a region of a nucleic acid. In some embodiments, this region is at the nucleotide targeted by the base editor or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor. In some embodiments, any of the base editors provided herein can limit the formation of indels in a region of a nucleic acid to less than 1%, less than 1.5%, less than 2%, less than 2.5%, less than 3%, less than 3.5%, less than 4%, less than 4.5%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, less than 10%, less than 12%, less than 15%, or less than 20%.

[0303] Base editing is often referred to as "modification" such as genetic modification, gene modification, and modification of nucleic acid sequences, and can be clearly understood based on the context in which the modification is base editing modification. Thus, base editing modification is, for example, as a result of deaminase activity discussed throughout this disclosure, a modification at the nucleotide base level, which in turn can lead to changes in the gene sequence and affect the gene product.

[0304] In some embodiments, a modification, e.g., a single base edit, results in a reduction of about or at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of gene target expression, or a reduction to undetectable levels.

[0305] The present disclosure provides variants of adenosine deaminase (e.g., ABE8 variants) having increased efficiency and specificity. In particular, the variants of adenosine deaminase described herein are more likely to edit a desired base within a polynucleotide and less likely to edit a base not intended to be modified (e.g., a "bystander").

[0306] In some embodiments, any of the base editor systems comprising one of the ABE8 base editor variants described herein has at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduced bystander editing or mutations compared to a base editor system comprising an ABE7 base editor, e.g., ABE7.10.

[0307] In some embodiments, any of the ABE8 base editor variants described herein have higher base editing efficiency compared to ABE7 base editors. In some embodiments, any of the ABE8 base editor variants described herein have at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 450%, or 500% higher base editing efficiency compared to an ABE7 base editor, such as ABE7.10.

[0308] The ABE8 base editor variants described herein can be delivered to a host cell via a plasmid, vector, LNP complex, or mRNA. In some embodiments, any of the ABE8 base editor variants described herein are delivered to a host cell as mRNA. In some embodiments, the methods described herein, such as base editing methods, have from minimal off-target effects to no off-target effects. In some embodiments, the methods described herein, such as base editing methods, have from minimal to chromosomal translocations.

[0309] In some embodiments, the base editing methods described herein result in a population of cells that are about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% normally edited.

[0310] In some embodiments, the percentage of viable cells in the cell population after base editing intervention is at least 60%, 70%, 80%, or more than 90% of the starting cell population at the time of the base editing event. In some embodiments, the percentage of viable cells in the edited cell population is about 70%. In some embodiments, the percentage of viable cells in the edited cell population is about 75%. In some embodiments, the percentage of viable cells in the edited cell population is about 80%. In some embodiments, the percentage of viable cells in the cell population described above is about 85%. In some embodiments, the percentage of viable cells in the cell population described above is about 90%, or about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the cells in the population at the time of the base editing event.

[0311] In an embodiment, the cell population is a population of cells that contacts the base editor, complex, or base editor system of the present disclosure. The number of intended mutations and indels can be determined using any suitable method, such as those described in International PCT Application Nos. PCT / US2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016), 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) and 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” Science Advances 3:eaao4774 (2017), the entire contents of which are incorporated herein by reference.

[0312] In some embodiments, to calculate the indel frequency, sequencing reads are scanned to exactly match two 10bp sequences flanking a window where an indel can occur. If an exact match is not found, the read is excluded from the analysis. If the length of this indel window exactly matches the reference sequence, the read is classified as indel-free. If the indel window is more than two bases longer or shorter than the reference sequence, the sequencing reads are classified as insertions or deletions, respectively. In some embodiments, the base editors provided herein may limit the formation of indels in a region of a nucleic acid. In some embodiments, this region is at the nucleotide targeted by the base editor or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of the nucleotide targeted by the base editor.

[0313] Multiplex editing In some embodiments, the base editor system provided by the present invention is capable of multiplex editing of multiple nucleic acid base pairs in one or more genes or polynucleotide sequences. In some embodiments, the multiple nucleic acid base pairs are located within the same gene or within one or more genes, and at least one gene is located within a different locus. In some embodiments, multiplex editing includes one or more guide polynucleotides. In some embodiments, multiplex editing includes one or more base editor systems. In some embodiments, multiplex editing includes one or more base editor systems having a single guide polynucleotide or multiple guide polynucleotides. In some embodiments, multiplex editing includes one or more guide polynucleotides having a single base editor system. It should be understood that the features of multiplex editing using any of the base editors described herein can be applied to any combination of methods using any of the base editors provided herein. It should be understood that multiplex editing using any of the base editors described herein can also include sequential editing of multiple nucleic acid base pairs.

[0314] In some embodiments, a base editor system capable of multiplex editing of multiple nucleic acid base pairs in one or more genes includes one of ABE7, ABE8, and / or ABE9 base editors.

[0315] Reduction of target gene expression in cells In some embodiments, cells having at least one modification in an endogenous gene or one or more of its regulatory elements (e.g., cells from the liver, eye, and / or central nervous system or components thereof) are provided herein. Also provided herein are methods, base editors, base editor systems, guide RNAs, and compositions for modifying cells. In some embodiments, the cells may include additional modifications in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more endogenous genes or their regulatory elements. In some embodiments, at least one modification is a single nucleic acid base modification. In some embodiments, at least one modification is generated by base editing. Base editing may be placed at any suitable position of the gene or in the regulatory element of the gene. Thus, it can be understood that a single base edit at the start codon can, for example, completely abolish gene expression. In some embodiments, base editing can be performed at a site within an exon. In some embodiments, base editing can be performed at sites in more than one exon. In some embodiments, base editing can be performed in any exon of multiple exons of a gene. In some embodiments, base editing may introduce a premature termination codon into the exon, resulting in a lack or truncation of the translation product (which may be misfolded and thereby eliminated by degradation) or production of an unstable mRNA (which is easily degraded). In some embodiments, the cells are hepatocytes and / or cells from the liver, eye, and / or central nervous system or components thereof.

[0316] In some embodiments, the gene is a C3 polynucleotide.

[0317] In some embodiments, editing of an endogenous gene reduces gene expression. In some embodiments, editing of an endogenous gene reduces gene expression by at least 50% compared to control cells without modification. In some embodiments, editing of an endogenous gene reduces gene expression by at least 60% compared to control cells without modification. In some embodiments, editing of an endogenous gene reduces gene expression by at least 70% compared to control cells without modification. In some embodiments, editing of an endogenous gene reduces gene expression by at least 80% compared to control cells without modification. In some embodiments, editing of an endogenous gene reduces gene expression by at least 90% compared to control cells without modification. In some embodiments, editing of an endogenous gene reduces gene expression by at least l00% compared to control cells without modification. In some embodiments, editing of an endogenous gene eliminates gene expression. <G

[0318] [[ID=S]]In some embodiments, base editing can be performed in an intron. For example, base editing may be performed in an intron. In some embodiments, base editing can be performed at a site within an intron. In some embodiments, base editing can be performed at sites in one or more introns. In some embodiments, base editing can be performed at any exon of multiple introns of a gene. In some embodiments, one or more base edits can be performed in an exon, an intron, or any combination of an exon and an intron.

[0319] In some embodiments, the modification or base editing can be within a promoter site. In some embodiments, base editing can be introduced into a selectable promoter site. In some embodiments, base editing can be within a 5' regulatory element such as an enhancer. In some embodiments, base editing can be introduced to disrupt the binding site of a nucleic acid binding protein. Exemplary nucleic acid binding proteins can be polymerase, nuclease, gyrase, topoisomerase, methylase or methyltransferase, transcription factor, enhancer, PABP, zinc finger protein, etc.

[0320] In some embodiments, base editing can be used for splice disruption to silence target protein expression. In some embodiments, base editing can generate splice acceptor-splice donor (SA-SD) sites. Targeted base editing that generates SA-SD, or targeted base editing at SA-SD sites, can result in reduced gene expression. In some embodiments, base editing (e.g., ABE, CBE, or CABE) is used to target dinucleotide motifs that constitute splice acceptor and splice donor sites (the first and last two nucleotides of each intron). In some embodiments, splice disruption is achieved with an adenosine base editor (ABE). In some embodiments, splice disruption is achieved with a cytidine base editor (CBE). In some embodiments, base editors (e.g., ABE, CBE, or CABE) are used to edit exons by creating a stop codon.

[0321] In some embodiments, the modification generates a premature stop codon in an endogenous gene. In some embodiments, the stop codon silences target protein expression. In some embodiments, the modification is a single base modification. In some embodiments, the modification is generated by base editing. Premature stop codons can be generated in exons, introns, or untranslated regions. In some embodiments, base editing can be used to introduce multiple stop codons into one or more alternative reading frames. In some embodiments, the stop codon is generated by an adenosine base editor (ABE). In some embodiments, the stop codon is generated by a cytidine base editor (CBE). In some embodiments, CBE generates any one of the following edits (shown in underlined font) to generate a stop codon:

[0322]

Chemical formula

[0323] In some embodiments, the modification / base editing can be introduced into the 3'-UTR, e.g., the polyadenylation (polyA) site. In some embodiments, the base editing can be performed on the 5'-UTR region.

[0324] Delivery system Nucleic acid-based delivery of base editor systems The nucleic acid molecule encoding the base editor system according to the present disclosure can be administered to a subject or delivered to cells in vitro or in vivo by methods known in the art or as described herein. For example, a base editor system comprising a deaminase (e.g., cytidine or adenine deaminase) can be delivered by a vector (e.g., a viral or non-viral vector) or by naked DNA, DNA complexes, lipid nanoparticles, or combinations of the foregoing compositions. The base editor system can be delivered to cells using any method available in the art, including but not limited to physical methods (e.g., electroporation, particle gun, calcium phosphate transfection), viral methods, non-viral methods (e.g., liposomes, cationic methods, lipid nanoparticles, polymeric nanoparticles), or biological non-viral methods (e.g., attenuated bacteria, engineered bacteriophage, mammalian virus-like particles, biological liposomes, erythrocyte ghosts, exosomes).

[0325] Organic or inorganic nanoparticles are useful in delivering base editor systems or components thereof. Nanoparticles are well known in the art, and any suitable nanoparticles can be used to deliver a base editor system or a component thereof, or a nucleic acid molecule encoding such a component. In one example, organic (e.g., lipid and / or polymer) nanoparticles are suitable for use as delivery vehicles in certain embodiments of the present disclosure. Non-limiting examples of lipid nanoparticles suitable for use in the methods of the present disclosure include those described in International Patent Application Publication Nos. WO2022140239, WO2022140252, WO2022140238, WO2022159421, WO2022159472, WO2022159475, WO2022159463, WO2021113365, and WO2021141969, the disclosures of each of which are hereby incorporated by reference in their entirety for all purposes.

[0326] Viral vector The base editors described herein can be delivered by viral vectors. In some embodiments, the base editors disclosed herein can be encoded on a nucleic acid contained in a viral vector. In some embodiments, one or more components of a base editor system can be encoded on one or more viral vectors.

[0327] Viral vectors can include lentiviral vectors (e.g., HIV and FIV-based vectors), adenoviral vectors (e.g., AD100), retroviral vectors (e.g., Moloney murine leukemia virus, MML-V), herpesvirus vectors (e.g., HSV-2), and adeno-associated virus (AAV) vectors, or other plasmid or viral vector types. For example, formulations and dosages from U.S. Patent No. 8,454,972 (formulations, dosages for adenovirus), U.S. Patent No. 8,404,658 (formulations, dosages for AAV), and U.S. Patent No. 5,846,946 (formulations, dosages for DNA plasmid), as well as formulations and dosages from clinical trials and publications regarding clinical trials involving lentivirus, AAV, and adenovirus are used. For example, in the case of AAV, the route of administration, formulation, and dosage can be similar to those in U.S. Patent No. 8,454,972 and clinical trials involving AAV. In the case of adenovirus, the route of administration, formulation, and dosage can be similar to those in U.S. Patent No. 8,404,658 and clinical trials involving adenovirus. In the case of plasmid delivery, the route of administration, formulation, and dosage can be similar to those in U.S. Patent No. 5,846,946 and clinical trials involving plasmid. Dosages can be based on an average 70 kg individual (e.g., adult human male) or can be estimated and adjusted for patients, subjects, mammals of different weights and species. The frequency of administration is within the purview of a physician or veterinarian (e.g., doctor, veterinarian) depending on usual factors including age, gender, general health status, other conditions of the patient or subject, and the specific condition or symptom being addressed. The viral vector can be injected into the tissue of interest. In the case of cell type-specific base editors, the expression of the base editor and optionally the guide nucleic acid can be driven by a cell type-specific promoter.

[0328] Viral vectors can be selected based on their use. For example, in the case of in vivo gene delivery, AAV may be more advantageous than other viral vectors. In some embodiments, AAV enables low toxicity, which may result from a purification method that does not require ultracentrifugation of cellular particles that can activate an immune response. In some embodiments, AAV is less likely to cause insertional mutagenesis because it does not integrate into the host genome. Adenoviruses are commonly used as vaccines because they induce a strong immunogenic response. The packaging capacity of viral vectors can limit the size of base editors that can be packaged into the vector. AAV has a packaging capacity of approximately 4.5Kb or 4.75Kb and contains two inverted terminal repeats (ITRs) of 145 bases each. This means that the disclosed base editors, as well as promoters and transcription terminators, can be adapted to a single viral vector. Constructs that exceed 4.5Kb or 4.75Kb can result in a significant reduction in virus production. For example, SpCas9 is quite large, with the gene itself exceeding 4.1Kb, making it difficult to package into AAV. Thus, embodiments of the present disclosure include the use of disclosed base editors that are shorter in length than conventional base editors. In some examples, the base editor is less than 4kb. The disclosed base editors can be less than 4.5kb, 4.4kb, 4.3kb, 4.2kb, 4.1kb, 4kb, 3.9kb, 3.8kb, 3.7kb, 3.6kb, 3.5kb, 3.4kb, 3.3kb, 3.2kb, 3.1kb, 3kb, 2.9kb, 2.8kb, 2.7kb, 2.6kb, 2.5kb, 2kb, or 1.5kb. In some embodiments, the length of the base editor of the present disclosure is 4.5kb or less.

[0329] The AAV can be AAV1, AAV2, AAV5, AAV6, or any combination thereof. The type of AAV can be selected with respect to the target cell. For example, AAV serotype 1, 2, 5 or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected to target the brain or neuronal cells, and AAV4 can be selected to target heart tissue. AAV8 is useful for delivery to the liver. A table of specific AAV serotypes for these cells can be found in Grimm, D. et al, J. Virol. 82:5887 - 5911 (2008).

[0330] In some embodiments, the lentiviral vector is used to transduce the target cell with a polynucleotide encoding a base editor or base editor system provided herein. Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both dividing and post - mitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), and it uses the envelope glycoproteins of other viruses to target a wide range of cell types.

[0331] In another embodiment, a minimal non - primate lentiviral vector based on equine infectious anemia virus (EIAV) is also contemplated. In another embodiment, RetinoStat® is a lentiviral gene therapy vector based on equine infectious anemia virus that expresses the angiogenesis - inhibiting proteins endostatin and angiostatin, which is contemplated to be delivered by subretinal injection. In another embodiment, the use of self - inactivating lentiviral vectors is contemplated.

[0332] Any RNA of the system, such as guide RNA or mRNA encoding a base editor, can be delivered in the form of RNA. The mRNA encoding the base editor can be generated using in vitro transcription. For example, nuclease mRNA can be synthesized using a PCR cassette containing the following elements: T7 promoter, optional kozak sequence (GCCACC), nuclease sequence, and a 3'UTR such as the 3'UTR derived from beta-globin-polyA tail. The cassette can be used for transcription by T7 polymerase. Guide polynucleotides (e.g., gRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence "GG", and the guide polynucleotide sequence.

[0333] Non-viral platform for gene transfer Non-viral platforms for introducing heterologous polynucleotides into target cells are known in the art.

[0334] For example, the present disclosure provides a method of inserting a heterologous polynucleotide into the genome of a cell using a Cas9 or Cas12 (e.g., Cas12b) ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP comprises a Cas9 or Cas12 nuclease domain and a guide RNA, the guide RNA specifically hybridizes to a target region of the cell's genome, and the Cas nuclease domain cleaves the target region to create an insertion site in the cell's genome. Then, the DNA template is used to introduce the heterologous polynucleotide. In embodiments, the DNA template is a double-stranded or single-stranded DNA template, the size of the DNA template is about 200 nucleotides or more than about 200 nucleotides, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequences adjacent to the insertion site. In some embodiments, the DNA template is a single-stranded circular DNA template. In embodiments, the molar ratio of RNP to DNA template in the complex is about 3:1 to about 100:1.

[0335] In some embodiments, the DNA template is a linear DNA template. In some examples, the DNA template is a single-stranded DNA template. In certain embodiments, the single-stranded DNA template is a pure single-stranded DNA template. In some embodiments, the single-stranded DNA template is a single-stranded oligodeoxynucleotide (ssODN).

[0336] In other embodiments, single-stranded DNA (ssDNA) can provide efficient HDR with minimal off-target integration. In one embodiment, an ssDNA phage is used to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors, when used with Cas9 or Cas12 (e.g., Cas12a, Cas12b), function as efficient HDR templates and have a higher integration frequency than linear ssDNA (lssDNA) donors.

[0337] intein An intein (intervening protein) is an auto-processing domain found in a variety of diverse organisms that perform a process known as protein splicing.

[0338] Non-limiting examples of inteins include any intein or intein pair known in the art, and the intein or intein pair includes synthetic inteins based on the dnaE intein, an intein pair of Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C), (e.g., as described in Stevens et al., J Am Chem Soc. 2016 Feb. 24;138(7):2162-5, which is incorporated herein by reference), and further includes DnaE. Non-limiting examples of intein pairs that can be used in accordance with the present disclosure include Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Patent No. 8,394,604, which is incorporated herein by reference). Exemplary nucleotide and amino acid sequences of inteins are provided in SEQ ID NOs: 370-377 and 389-424 in the Sequence Listing. Inteins suitable for use in embodiments of the present disclosure and methods of using them are described in U.S. Patent No. 10,526,401, International Patent Application Publication No. WO2013 / 045632, and U.S. Patent Application Publication No. US2020 / 0055900, the entire disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0339] Intein-N and Intein-C can be fused to the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9, respectively, for the binding of the N-terminal portion of split Cas9 and the C-terminal portion of split Cas9. For example, in some embodiments, Intein-N is fused to the C-terminus of the N-terminal portion of split Cas9, i.e., forming a structure of N-[N-terminal portion of split Cas9]-[Intein-N]-C. In some embodiments, Intein-C is fused to the N-terminus of the C-terminal portion of split Cas9, i.e., forming a structure of N-[Intein-C]-[C-terminal portion of split Cas9]-C. In embodiments, the base editor is encoded by two polynucleotides, one polynucleotide encodes a fragment of the base editor fused to Intein-N, and the other polynucleotide encodes a fragment of the base editor fused to Intein-C. Methods for designing and using inteins are known in the art and are described, for example, in WO2014004336, WO2017132580, WO2013045632A1, US20150344549, and US20180127780 (each of which is hereby incorporated by reference in its entirety).

[0340] In some embodiments, ABE is split into an N-terminal fragment and a C-terminal fragment at Ala, Ser, Thr, or Cys residues within selected regions of SpCas9. These regions correspond to loop regions identified by Cas9 crystal structure analysis.

[0341] The N-terminus of each fragment is fused to Intein-N, and the C-terminus of each fragment is fused to Intein-C at amino acid positions S303, T310, T313, S355, A456, S460, A463, T466, S469, T472, T474, C574, S577, A589, and S590, as referred to in SEQ ID NO: 197.

[0342] Pharmaceutical composition In one aspect, the present disclosure provides a pharmaceutical composition, which comprises any one of the cells, polynucleotides, vectors, base editors, base editor systems, guide polynucleotides, fusion proteins, complexes, or fusion protein-guide polynucleotide complexes described herein.

[0343] The pharmaceutical compositions of the present disclosure can be prepared according to known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed. 2005). Generally, the cells or a population thereof are mixed with a suitable carrier prior to administration or storage, and in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers generally include inert substances and assist in administering the pharmaceutical composition to a subject, assist in processing the pharmaceutical composition into a deliverable preparation, or assist in storing the pharmaceutical composition prior to administration. Pharmaceutically acceptable carriers can include agents that can modify the form, consistency, viscosity, pH, pharmacokinetics, solubility of the formulation for stabilization, optimization, or other modification. Such agents include buffers, wetting agents, emulsifying agents, diluents, encapsulating agents, and skin penetration enhancers. For example, carriers can include, but are not limited to, saline, buffered saline, dextrose, arginine, sucrose, water, glycerol, ethanol, sorbitol, dextran, sodium carboxymethyl cellulose, and combinations thereof.

[0344] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject. Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, intradermal, intracutaneous, intralesional, intra-articular, intraperitoneal, intravesical, transmucosal, gingival, intra-dental, intracochlear, intra-tympanic, intra-organ, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periorbital, intratumoral, intracerebral, and intraventricular administration.

[0345] In some embodiments, the pharmaceutical compositions described herein are administered locally to a disease site (e.g., the liver, the eye, or the central nervous system). In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, by catheter, by suppository, or by implant, where the implant is a porous, non-porous, or gel-like material that includes a membrane such as a sialastic membrane or fiber.

[0346] In some embodiments, any of the fusion proteins, gRNAs, and / or complexes described herein are provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes any of the fusion proteins or complexes provided herein. In some embodiments, the pharmaceutical composition includes a gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. In embodiments, the pharmaceutical composition includes lipid nanoparticles and a pharmaceutically acceptable excipient. In embodiments, the lipid nanoparticles contain a gRNA, a base editor, a complex, a base editing system or a component thereof, and / or one or more polynucleotides encoding the same. The pharmaceutical composition can optionally include one or more additional therapeutic active substances. The composition can be administered in an effective amount as described above. The effective amount depends on the mode of administration, the particular condition being treated, and the desired outcome. It can also depend on the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy (if any), and similar factors well known to physicians. It is an amount sufficient to achieve a medically desirable result in a therapeutic use.

[0347] In some embodiments, the compositions according to the present disclosure can be used for the treatment of any of a variety of diseases, disorders, and / or conditions.

[0348] Method of treatment Some aspects of the present disclosure provide a method of treating a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition described herein. In other embodiments, the methods of the present disclosure comprise expressing, or introducing into the cell, an intracellular base editor polypeptide and one or more guide RNAs capable of targeting a nucleic acid molecule encoding at least one polypeptide.

[0349] One of ordinary skill in the art will understand that multiple administrations of the pharmaceutical compositions contemplated in certain embodiments may be required to affect the desired therapy. For example, the composition may be administered to the subject one, two, three, four, five, six, seven, eight, nine, or ten times, or more, over a period of one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, ten years, or more.

[0350] Administration of the pharmaceutical compositions contemplated herein can be carried out using conventional techniques including, but not limited to, infusion, injection, or parenteral. In some embodiments, parenteral administration includes injection or infusion into the intravascular, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, and intrasternal.

[0351] In various embodiments, the methods of the present disclosure are associated with a reduction in complement activation in a subject. In some cases, the method is associated with a reduction in inflammation in the subject.

[0352] Combination therapy In various embodiments, the methods of the present disclosure comprise administering an inhibitor of complement component C3. In embodiments, the pharmaceutical composition of the present disclosure contains an inhibitor of complement component C3. In some embodiments, the complement inhibitor is compstatin or a compstatin analog or mimetic.

[0353] Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. U.S. Patent No. 6,319,897 describes a peptide having the sequence I[CVVQDWGHHRC]T (SEQ ID NO: 853) with a disulfide bond between two cysteines indicated in parentheses.

[0354] Morikis, et al., Protein Sci., 7(3):619-27, 1998) also describe compstatin. In some cases, compstatin is amidated at the C-terminus.Compstatin analogs, mimetics, derivatives thereof, and / or compositions containing them suitable for use in the methods and compositions of the present disclosure include those described in WO2021007111 (PCT / US2020 / 040741), WO2021011927 (PCT / US2020 / 042676), WO2004026328 (PCT / US2003 / 029653), Morikis, D., et al., Biochem Soc Trans.32 (Pt 1): 28-32,2004, Mallik, B., et al., J.Med.Chem., 274-286, 2005, Katragadda, M., et al. J.Med.Chem., 49:4616-4622, 2006, WO2007062249 (PCT / US2006 / 045539), WO2007044668 (PCT / US2006 / 039397), WO2009046198 (PCT / US2008 / 078593), WO2010127336 (PCT / US2010 / 033345), WO2012155107, WO2014078731, WO2019166411, WO2009121065, WO2021163654, WO2021142171, WO2017062879, WO2014152391, WO2014028861, WO2018187813, WO2019089653, WO2016049385, WO2018075373, WO2019118938, WO2022061304, WO2012006599, WO2011163394, WO2012178083, US9291622, US10407466, US8580735, and Hillmen, et al. “Pegcetacoplan versus Eculizumab in Paroxysmal Nocturnal Hemoglobinuria,” N Engl J Med. 2021 Mar 18; 384(11): 1028-1037, and the entire disclosures of all of them are incorporated herein by reference in their entirety for all purposes.In certain embodiments, the compstatin analog is pegcetacoplan ("APL-2") having the structure of the compound of FIG. 19 with PEG having an n of about 800 to about 1100 and / or an average molecular weight of about 40 kDa. Pegcetacoplan is poly(oxy-1,2-ethanediyl), α-hydroxy-ω-hydroxy-, N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-α-aspartyl-L-tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-N. 6 -carboxy-L-lysine amide cyclic(2-->12)-(disulfide) 15,15'-diester; or O,O'-bis[(S 2 ,S 12 -cyclo{N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-α-aspartyl-L-tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-L-lysine amide})-N 6.15 -carbonyl] polyethylene glycol (n = 800 - 1100), also referred to as such.

[0355] In some embodiments, the complement inhibitor is an antibody, such as an anti-C3 antibody, or a fragment thereof. In some embodiments, the antibody fragment can be used to inhibit C3 activation. The antibody fragment can be Fab', Fab'(2), Fv, or single-chain Fv. In some embodiments, the anti-C3 antibody is monoclonal. In some embodiments, the antibody is polyclonal. In some embodiments, the anti-C3 antibody is deimmunized. In some embodiments, the anti-C3 antibody is a fully human monoclonal antibody.

[0356] In some cases, the complement inhibitor is an inhibitory polynucleotide (e.g., siRNA) such as those described in WO2021163654, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

[0357] In some embodiments, the complement inhibitor is a polypeptide inhibitor and / or a nucleic acid aptamer (see, e.g., US Patent Application Publication No. 20030191084). Exemplary polypeptide inhibitors include enzymes that degrade C3 or C3b (see, e.g., US Patent No. 6,676,943).

[0358] Kit The present disclosure provides a kit for use in treating a subject to reduce complement activation. In some embodiments, the kit further comprises a base editor system or a polynucleotide encoding a base editor system, the base editor system comprising a nucleic acid programmable DNA binding protein (napDNAbp), a deaminase, and a guide RNA. In some embodiments, the napDNAbp is Cas9 or Cas12. In some embodiments, the polynucleotide encoding the base editor is an mRNA sequence. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase. In some embodiments, the kit comprises a guide RNA and / or a base editor system, and instructions for the use of the guide RNA and / or the base editor system.

[0359] The kit may further include a written instruction regarding the use of the base editor and / or base editor system described herein. In other embodiments, the instruction includes at least one of cautions, warnings, clinical trials, and / or references. The instruction may be printed directly on the container (if present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied within or with the container. In a further embodiment, the kit includes an instruction in the form of a label or a separate insert (accompanying document) for suitable operating parameters. In yet another embodiment, the kit includes one or more containers having appropriate positive and negative controls or control samples, which are used as standards (plural) for detection, calibration, or normalization. The kit may further include a second container containing a pharmaceutically acceptable buffer such as (sterile) phosphate buffered saline, Ringer's solution, or dextrose solution. This may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying documents including instructions for use.

[0360] In the practice of the embodiments of the present disclosure, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are used and are well within the scope of those skilled in the art. Such techniques are fully described in references such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989), “Oligonucleotide Synthesis” (Gait, 1984), “Animal Cell Culture” (Freshney, 1987), “Methods in Enzymology”, “Handbook of Experimental Immunology” (Weir, 1996), “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987), “Current Protocols in Molecular Biology” (Ausubel, 1987), “PCR: The Polymerase Chain Reaction”, (Mullis, 1994), “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure and can thus be considered in the making and practicing of the present disclosure. Particularly useful techniques for specific embodiments are discussed in the following sections.

[0361] The following examples are presented to provide a complete disclosure and description of how to make and use the assays, screening, and therapeutic methods of the present disclosure to those skilled in the art and are not intended to limit the scope of what the inventors regard as their invention.

Examples

[0362] Example 1: Complement Component 3 (C3) Knockout (KO) Guide Screening in Hek293T Cells Experiments were conducted to identify guide RNA sequences suitable for use in knockout expression of complement component 3 (C3) in cells. The 162 guides listed in Table 1A were designed to knockdown C3 protein expression (listed in Table 1A). These guides used ABE deaminase for disruption of splice sites or CBE deaminase for stop codon generation or disruption of splice sites. Twenty-six guides were screened with both ABE and CBE deaminases for disruption of splice sites. All 188 (i.e., 162 + 26) guide-editor combinations were screened for use in editing the C3 gene. Editor mRNA + sgRNA was transfected in triplicate in Hek293T cells with a total RNA dose of 800 ng (200 ng of guide + 600 ng of editor mRNA) for all samples. In addition to the guide-editor pair of interest (i.e., the base editor system), a positive control guide-editor pair, ABE8.8_sgRNA_088, which contains the spacer sequence CAGGAUCCGCACAGACUCCA (SEQ ID NO: 792) and is known to be effective in editing sites outside the C3 gene, was also tested. The untreated state was included as a negative control. Genomic DNA was recovered from the cells 3 days after transfection.

[0363] For the 188 guide-editor combinations screened, a wide range of editing rates were observed, and a subset of the guides showed favorable editing efficiency at the targeted sites (Figures 1 and 2). These included 29 guides screened with ABE8.8 deaminase that achieved an editing rate of 40% or more (Figure 1), and 26 guides screened with BE4 deaminase that achieved an editing rate of over 50% (Figure 2).

[0364] Example 2: Complement component 3 (C3) knockout (KO) guide screening and functional knockdown evaluation in hepatocytes extracted from humanized mouse liver (PXB cells) Twenty-two guide + editor combinations that achieved favorable editing in Hek293T cells were selected for screening in human primary hepatocytes to evaluate editing efficiency and the ability to functionally knockdown C3 protein expression. Editor mRNA + sgRNA was transfected into human hepatocytes extracted from humanized mouse livers (PXB cells, PhoenixBio) after 3 days of cell incubation. In addition to the 22 guide-editor pairs of interest, the positive control base editor system sgRNA_088_ABE8.8_SpCas9 was also transfected. The untreated state was included as a negative control. All conditions were performed in triplicate. To evaluate functional C3 knockdown, cell supernatants were collected and stored at -80°C. Such collections were performed 2 days before transfection (3-day incubation), as well as 4 days, 7 days, 10 days, 13 days, and 16 days after transfection. One day after transfection, additional medium exchanges were performed, but the supernatants were discarded. Genomic DNA was collected from the cells 16 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS). The human C3 ELISA assay was used to evaluate the C3 protein concentration in the cell supernatants before transfection, as well as 7 days, 13 days, and 16 days after transfection.

[0365] Before transfection, no significant difference in C3 protein concentration was observed among the samples (Figure 3). Seven days after transfection, a significant increase in C3 levels was observed for all conditions, but no significant difference was still observed among the samples (Figure 4). By 13 days after transfection, for gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9, a reduction of approximately 50% in C3 levels was observed compared to sgRNA_088_ABE8.8_SpCas9 that did not edit within the C3 gene (Figure 5). A similar trend was observed 16 days after transfection (Figure 6). C3 protein knockdown was positively correlated with the editing rate of the entire sample (Figures 5 and 6).

[0366] Example 3: Optimization of ABE deaminase Using ABE8.8 as in Examples 1 and 2, complement component 3 (C3) knockout (KO) guides in Hek293T cells and PXB cells were screened. Other eighth-generation ABE deaminase variants include ABE8.13, ABE8.17, and ABE8.20. Also, when the V82T mutation was incorporated into ABE8.20 (ABE9.51), potential editing benefits were also found. To optimize the editing performance for C3 knockout (KO) guides, CD KO guides showing potential C3 protein knockdown in PXB cells (i.e., gRNA676, gRNA696, gRNA701, gRNA662, gRNA661, gRNA695, and gRNA715) were screened in Hek293T cells using five eighth-generation ABE editor variants (i.e., ABE8.8, ABE8.13, ABE8.17, ABE8.20, and ABE8.20_V82T (ABE9.51)). A 400 ng dose of total RNA (100 ng of guide + 300 ng of editor mRNA) was transfected in triplicate for all samples. sgRNA_088_ABE8.8_SpCas9 was transfected as a positive control, and the untreated condition was also included as a negative control.

[0367] ABE8.8 performed similarly to or better than other ABE8 variants tested for gRNA676, gRNA696, gRNA701, gRNA662, gRNA661, and gRNA695 in terms of editing efficiency. For gRNA715, ABE8.13 achieved the highest editing efficiency (Figure 7).

[0368] Example 4: Optimization of spacer length for complement component 3 (C3) knockout (KO) guides To further optimize the editing performance for C3 KO guides, various spacer lengths (19, 20, 21, 22, and 23 bp) were screened for guides showing potential C3 protein knockdown (see Table 1C). Total RNA at a dose of 400 ng (100 ng of guide + 300 ng of editor mRNA) was transfected in triplicate for all samples. sgRNA_088_ABE8.8_SpCas9 was transfected as a positive control. The untreated condition was also included as a negative control. The standard 20-bp spacer performed similarly or better in terms of editing efficiency compared to other spacer lengths tested for gRNA676, gRNA696, gRNA701, gRNA662, gRNA661, and gRNA695. For gRNA715, the 21-bp protospacer achieved the highest editing efficiency (Figure 8).

[0369] Example 5: Evaluation of Repeated Editing and Functional Knockdown for Complement Component 3 (C3) Guides in PXB Cells Guides showing potential C3 knockdown: Transfection of PXB cells was repeated using gRNA676_ABE8.8_SpCas9, gRNA696_ABE8.8_SpCas9, gRNA701_ABE8.8_SpCas9, gRNA662_ABE8.8_SpCas9, gRNA661_ABE8.8_SpCas9, gRNA695_ABE8.8_SpCas9, and gRNA715_ABE8.13_SpCas9-VRQR (Figures 5 and 6). Editor mRNA + sgRNA (i.e., base editor system) was transfected in triplicate into PXB cells after 3 days of cell incubation. In addition to the seven guide-editor pairs of interest, two positive control guide-editor pairs were also transfected. These include sgRNA_088_ABE8.8_SpCas9, which results in high editing efficiency at a site outside the C3 gene, and gRNA1688_SpCas9, which has a spacer sequence of CAACAAGUUCGUGACCGUGC (SEQ ID NO: 793) and induces functional C3 knockdown. Doses of 800 ng (200 ng of guide + 600 ng of editor mRNA) and 1200 ng (300 ng of guide + 900 ng of editor mRNA) were transfected for most samples. Only the 800 ng dose was tested for gRNA1688_SpCas9, gRNA715_ABE8.13_SpCas9-VRQR, and gRNA695_ABE8.8_SpCas9. The untreated condition was also included as a negative control. All conditions were performed in triplicate. To evaluate functional C3 knockdown, cell supernatants were collected and stored at -80°C. Such collections were performed immediately before transfection (3-day incubation) and 4 days, 7 days, 10 days, and 13 days after transfection. One day after transfection, an additional medium change was performed, but the supernatant was discarded. Genomic DNA was harvested from the cells 13 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS). A human C3 ELISA assay was used to evaluate the C3 protein concentration in the cell supernatants before and 13 days after transfection.

[0370] Before transfection, no significant difference in C3 concentration was observed among the samples (Figure 9). By 13 days after transfection, more than 55% reduction in C3 levels was observed for gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 compared to sgRNA_088_ABE8.8_SpCas9 at both 800 ng and 1200 ng doses (Figure 10). The editing efficiency was high, more than 60% for most of the guides (Figure 10).

[0371] Example 6: Evaluation of complement component 3 (C3) knockout (KO) guide screening and functional knockdown in primary human hepatocyte (PHH) co - culture The editor mRNA + sgRNA (i.e., the base editor system) was transfected in triplicate in primary human hepatocyte (PHH) co-culture after 3 days of cell incubation. Guides (sgRNAs) that showed potential C3 knockdown in the first PXB cell experiment (see above) were used: gRNA676_ABE8.8_SpCas9, gRNA696_ABE8.8_SpCas9, gRNA701_ABE8.8_SpCas9, gRNA662_ABE8.8_SpCas9, gRNA661_ABE8.8_SpCas9, and gRNA715_ABE8.13_SpCas9-VRQR. In addition to the six guide-editor pairs (i.e., the base editor systems) of interest, two positive control guide-editor pairs were also transfected. These included sgRNA_088_ABE8.8_SpCas9, which results in high editing efficiency at a site outside the C3 gene, and gRNA1688_SpCas9, which contains the spacer sequence CAACAAGTTCGTGACCGTGC (SEQ ID NO: 793) and induces functional C3 knockdown using a nuclease-based strategy. For most samples, separate doses of 800 ng (200 ng of guide + 600 ng of editor mRNA) and 1200 ng (300 ng of guide + 900 ng of editor mRNA) were transfected, but for gRNA1688_SpCas9 and gRNA715_ABE8.13_SpCas9-VRQR, only the 800 ng dose was tested. The untreated condition was also included as a negative control. All conditions were performed in duplicate or triplicate. To evaluate functional C3 protein knockdown, cell supernatants were collected and stored at -80 °C. Such collections were performed immediately prior to transfection (3-day incubation), as well as 4, 7, 10, and 13 days after transfection. One day after transfection, an additional media change was performed, but the supernatant was discarded. Genomic DNA was harvested from the cells 13 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS). The human C3 ELISA assay was used to evaluate the C3 protein concentration in the cell supernatants before and 13 days after transfection.

[0372] Before transfection, no significant difference in C3 protein concentration was observed among the samples (Figure 11). By 13 days after transfection, more than 55% reduction in C3 protein levels was observed for gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 compared to sgRNA_088_ABE8.8_SpCas9 at both 800 ng and 1200 ng doses (Figure 12). For most guides, the editing efficiency was higher than 60% for most conditions (Figure 12).

[0373] Example 7: Evaluation of editing performance of complement component 3 (C3) knockout (KO) guides in primary cynomolgus monkey hepatocyte (PCH) monolayers For the guides that showed potential C3 knockdown in the first PXB cell experiment (see above), editor mRNA + sgRNA was transfected in triplicate into primary cynomolgus monkey (Macaca fascicularis) hepatocyte (PCH) monolayers: gRNA676_ABE8.8_SpCas9, gRNA696_ABE8.8_SpCas9, gRNA701_ABE8.8_SpCas9, gRNA662_ABE8.8_SpCas9, gRNA661_ABE8.8_SpCas9, and gRNA715_ABE8.13_SpCas9-VRQR. For gRNA676 and gRNA696, surrogate cynomolgus monkey guides, which were gRNA1793 and gRNA1798, respectively, were transfected (see Table 1B for surrogate spacer sequences). In addition to the six guide-editor pairs of interest (i.e., base editor systems), sgRNA_088_ABE8.8_SpCas9 was also transfected, and the untreated condition was included as a negative control. All conditions were performed in triplicate. Genomic DNA was harvested from the cells 3 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS).

[0374] The editing efficiency for all guides was over 35% (Figure 13). These editing rates were comparable to the positive control sgRNA_088_ABE8.8_SpCas9.

[0375] Example 8: Evaluation of editing and protein knockdown (KD) of complement component 3 (C3) guides in primary cynomolgus hepatocyte (PCH) co-cultures. The base editor systems gRNA1793_ABE8.8_SpCas9 and gRNA1798_ABE8.8_SpCas9, and their humanized versions (i.e., gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9), which showed high target base editing and functional C3 protein knockdown in PXB cells and PHH co-cultures, were transfected in triplicate in primary cynomolgus (Macaca fascicularis) hepatocyte (PCH) co-cultures. sgRNA_088_ABE8.8_SpCas9 was transfected as a positive control, and the untreated condition was included as a negative control. All conditions were performed in triplicate. To evaluate functional C3 knockdown, cell supernatants were collected and stored at -80 °C. Such collections were performed immediately before transfection (3-day incubation), and 4, 7, 10, and 13 days after transfection. One day after transfection, an additional medium exchange was performed, but the supernatant was discarded. Genomic DNA was harvested from the cells 13 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS). A modified C3 ELISA assay was used to evaluate the cynomolgus C3 protein concentration in the cell supernatants before transfection, and 7 and 13 days after transfection.

[0376] Before transfection, no significant difference in cynomolgus monkey C3 concentration was observed among samples (Figure 14). By 7 days after transfection, for gRNA1793_ABE8.8_SpCas9 and gRNA1798_ABE8.8_SpCas9, a reduction of approximately 70% in cynomolgus monkey C3 levels was observed compared to sgRNA_088_ABE8.8_SpCas9 that did not edit within the C3 gene (Figure 15). A similar trend was observed 13 days after transfection (Figure 16). The editing efficiencies for both gRNA1793_ABE8.8_SpCas9 and gRNA1798_ABE8.8_SpCas9 were high, approximately 70% (Figures 15 and 16). This was equivalent to the positive control sgRNA_088_ABE8.8_SpCas9 (Figures 15 and 16).

[0377] Example 9: Reduction of Complement Component 3 (C3) Protein and RNA in FRG Liver Humanized Mice after On-Target Editing Using a Base Editor System Experiments were conducted in FRG liver humanized mice using a base editor system to evaluate on-target editing and complement component 3 (C3) protein and RNA knockdown. The base editor system containing guide polynucleotide gRNA676 and base editor ABE8.8 containing the SpCas9 nucleic acid programmable DNA binding protein domain (i.e., gRNA676_ABE8.8_SpCas9) showed both high editing rates and high C3 protein knockdown in in vitro primary hepatocyte experiments, so the base editor system was further evaluated in mice in vivo. The base editor system was formulated with lipid nanoparticles (BL4 LNP) and administered to liver humanized Fah- / -Rag2- / -Il2rg- / - (FRG) mice with >70% human hepatocyte repopulation (Yecuris; donor: HHF04030) at doses of 2.0 and 0.3 mg / kg via retro-orbital (RO) tail vein injection. Tris-buffered saline (TBS) alone was also delivered to control animals. Serum samples were collected before dosing and 7 and 14 days after dosing. Liver fragments flash-frozen at the end of the study were recovered for next-generation sequencing (NGS), and liver fragments frozen in RNAlater were recovered to analyze C3 RNA levels. NGS was utilized to evaluate the editing level at the target locus, and the Mesoscale Discovery (MSD) assay was used to measure serum protein levels. RT-qPCR was used to measure C3 RNA levels in the liver.

[0378] Fourteen days after administration, in animals administered at 2.0 mg / kg, a reduction of approximately 90% in serum C3 protein levels was observed compared to pre-administration C3 protein levels (Figure 20). This reduction in C3 protein levels corresponded to approximately 90% A>G editing at the target site (splice site) in the harvested liver tissue and corresponded to a reduction of approximately 90% in liver C3 RNA levels compared to unedited control animals administered Tris-buffered saline (TBS) instead of the base editor system (Figures 20 and 21). Approximately 45-75% on-target editing was observed in animals administered at 0.3 mg / kg (Figure 20). The editing rate correlated well with the reduction in C3 protein and RNA levels in animals administered at 0.3 mg / kg, and animals showing higher editing also showed a greater reduction in C3 protein and RNA levels (Figures 20 and 21). The measured reduction in C3 RNA was confirmed using four different Taqman probes spanning the length of the C3 gene, including Hs01100912_m1, which binds upstream of the gRNA676 target splice site (exon 8 splice site donor) (Figure 21).

[0379] Example 10: Effect of Severe Chemical Modification on On-Target Editing and Complement Component 3 (C3) Protein Knockdown in FRG Liver Humanized Mice Using a Base Editor System Experiments were conducted to determine the effect of severe chemical modification of guide polynucleotides on on-target editing and reduction of C3 protein levels in FRG liver humanized mice using a base editing system. Since both base editor systems gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 showed high editing and C3 protein knockdown in in vitro primary hepatocyte experiments, and gRNA676_ABE8.8_SpCas9 showed higher efficacy (i.e., base editing level at a specific dose of the base editor system) in FRG liver humanized mice, experiments were conducted to determine the in vivo editing efficacy of the gRNA696_ABE8.8_SpCas9 base editor system and to determine the in vivo effect of severe chemical modification of the guide polynucleotide on the two base editor systems. To evaluate the in vivo performance for improvement of the efficacy of gRNA696_ABE8.8_SpCas9 and the base editor system related to various severe chemical modification schemes of the guide, regardless of the presence or absence of severe chemical modification of the guide polynucleotide (i.e., guide gRNA2202-gRNA2209), the base editor systems gRNA676_ABE8.8_SpCas9 and gRNA696_ABE8.8_SpCas9 were formulated with lipid nanoparticles (BL4 LNP) and administered to Fah- / -Rag2- / -Il2rg- / - (FRG) mice with liver humanization having more than 70% human hepatocyte repopulation (Yecuris; donor: HHF04030) at a dose of 0.3 mg / kg via intravenous retro-orbital plexus (RO) tail vein injection. sgRNA_088_ABE8.8_SpCas9 was formulated and administered as a control. Serum samples were collected before administration and 7 days and 14 days after administration. Flash-frozen liver fragments were collected at the end of the study for next-generation sequencing (NGS), and this was used to evaluate the editing level at the target locus. Serum C3 protein levels were measured using the human C3 Mesoscale Discovery (MSD) assay.

[0380] About variants of base editor systems containing guide polynucleotides with severe chemical modifications (gRNA2202 - gRNA2205) having gRNA676_ABE8.8_SpCas9 and gRNA2203_ABE8.8_SpCas9, which is associated with an increased editing rate compared to gRNA676_ABE8.8_SpCas9, about 65 - 80% A>G on - target editing was observed at the target splice site in the harvested liver tissue (Figure 22). This on - target editing corresponded to a reduction in serum C3 protein levels of about 55 - 65% 14 days after administration, compared to the pre - administration C3 protein levels, and gRNA2203_ABE8.8_SpCas9 was also associated with a greater reduction in C3 protein levels compared to those associated with gRNA676_ABE8.8_SpCas9 (Figure 22). The base editor system gRNA696_ABE8.8_SpCas9 or its variants (gRNA2206 - gRNA2209) containing guide polynucleotides with severe chemical modifications were associated with an on - target editing level of less than about 40% at the target splice site (Figure 22).

[0381] Example 11: Differences in base editing rates for human and cynomolgus monkey target sites in primary human hepatocytes (PHH) and primary cynomolgus monkey hepatocytes (PCH) To determine the difference in editing efficiency between a base editor system containing gRNA2202 targeting the human C3 gene and a base editor system containing gRNA2200 targeting the cynomolgus macaque C3 gene for base editing in primary human hepatocytes (PHH) and primary cynomolgus macaque hepatocytes (PCH), experiments were conducted. In previous experiments, gRNA2202_ABE8.8_SpCas9 resulted in approximately 70% editing and reduction of both C3 protein and RNA levels in FRG liver humanized mice administered at 0.3 mg / kg. The human sequence targeted by gRNA2202 for base editing is not conserved in cynomolgus macaques. In previous experiments, the base editor system gRNA2200_ABE8.8_SpCas9 containing gRNA2200 targeting the cynomolgus macaque C3 gene was associated with a base editing rate of approximately 20% at a dose of 1.5 mg / kg in cynomolgus macaques. To determine the potential difference in efficacy between cynomolgus macaque C3 gene targeting and human C3 gene targeting guides, primary cynomolgus macaque hepatocytes (PCH) were transfected with the base editor system gRNA2200_ABE8.8_SpCas9 and primary human hepatocytes (PHH) were transfected with the base editor system gRNA2202_ABE8.8_SpCas9. The base editor system sgRNA_088_ABE8.8_SpCas9 was used as a control for base editing in both cell types because the sequence targeted for editing by this base editor system is conserved between humans and cynomolgus macaques. Each base editor system was transfected into cells at a 3:1 editor-to-sgRNA ratio with doses of the base editor system (combined mass of mRNA encoding the guide polynucleotide and base editor) of 1000, 100, 50, 10, 5, 1, 0.5, 0.1, and 0.01 ng. All wells were normalized to a total mRNA dose of 1000 ng using Universal Human Reference RNA (Life Technologies, QS0639). All conditions were evaluated in duplicate. Genomic DNA was harvested from the cells 3 days after transfection and editing efficiency was evaluated by next-generation sequencing (NGS).The data was fitted to a variable slope (4-parameter) curve, and the EC50 was calculated for all base editor systems.

[0382] The on-target editing data was well-fitted to a variable gradient (4-parameter) curve, and the R 2 value was 0.99 or higher for all base editor systems evaluated (Figure 23). The comparison of EC50 revealed that the EC50 in PCH cells for gRNA2200_ABE8.8_SpCas9 was 13.63-fold higher than that observed for gRNA2202_ABE8.8_SpCas9 in PHH cells (65.44 ng vs. 4.80 ng) (Figure 23). The comparison of EC50 for the base editor system sgRNA_088_ABE8.8_SpCas9 revealed that the EC50 was also 9.71-fold higher in PCH cells compared to PHH cells (33.12 ng vs. 3.41 ng) despite the sequence targeted for base editing being conserved between PCH and PHH (Figure 23).

[0383] Example 12: Difference in base editing efficiency for human and cynomolgus monkey target sites in engineered Hek293T cell lines Using a similar base editing system, experiments were conducted to determine the cause of this difference in base editing efficiency considering the difference in base editing efficiency observed between PCH cells and PHH cells. For this purpose, engineered Hek293T cell lines containing the protospacer + PAM sequences for both the human sequence targeted by the gRNA2202 guide and the cynomolgus monkey sequence targeted by the gRNA2200 guide were generated, with the target sequences separated from each other by 50 bp. This cell line was separately transfected with the base editor systems gRNA2202_ABE8.8_SpCas9 and gRNA2200_ABE8.8_SpCas9 to evaluate the difference in base editing between the two base editor systems. Cells were transfected with the base editor systems in a 1:3 serial dilution series of a total base editing dose of 300 ng to 0.15 ng (editor to sgRNA ratio of 2:1), where the "total base editing dose" indicates the combined mass of the guide polynucleotide and the mRNA encoding the base editor. Non-translated ABE8.8 coding mRNA was used to normalize all wells to a total RNA dose of 300 - 400 ng. All conditions were evaluated in triplicate. Genomic DNA was harvested from the cells 3 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS). The data was fit to a variable slope (4-parameter) curve, and the EC50 was calculated for both base editor systems.

[0384] The on-target editing data fit well to a variable slope (4-parameter) curve, and the R 2 values were 0.98 - 0.99 for both base editor systems (Figure 24). Comparison of the EC50s revealed that the EC50 for gRNA2200_ABE8.8_SpCas9 was approximately 3.5-fold higher than that observed for gRNA2202_ABE8.8_SpCas9 (2.128 ng vs 0.6132 ng) (Figure 24).

[0385] Example 13: Optimization of ABE editors for gRNA676, gRNA696, gRNA661, and gRNA715 The base editors containing guide gRNAs 676, 696, 661, and 715 targeting the C3 gene for base editing showed high editing and C3 protein knockdown in PXB cells and PHH co-culture studies. Therefore, experiments were conducted to optimize the base editors used in the adenosine deaminase base editor (ABE) system containing these guides. For these guides, initial ABE optimization was performed as described in Example 3 above to optimize the editing performance. To further optimize the editing performance of the base editor system containing guide gRNA 676, 696, or 661, one of the 16 base editors containing the base editor system with the guide and the 8th and 9th generation TadA* deaminase domain variants and / or Cas9 variants was screened in HepG2 cells (Tables 8 - 10). To further optimize the editing performance of the base editor system containing guide gRNA 715, one of the 5 base editors containing the base editor system with the guide and the 8th and 9th generation TadA* deaminase domain variants was screened in HepG2 cells (Table 11). The base editor system sgRNA_088_ABE8.8_SpCas9 was used as a positive control for base editing, and the untreated state was also included as a negative control. The base editor systems were transfected in a 1:3 serial dilution series of a total base editing dose of 300 ng to 0.02 ng (editor to sgRNA ratio of 2:1), and the "total base editing dose" refers to the combined total mass of the guide polynucleotide and the mRNA encoding the base editor. All wells were normalized to a total RNA dose of 300 - 400 ng using mRNA encoding non-translated ABE8.8 or universal human reference RNA (Life Technologies, QS0639). All conditions were evaluated in triplicate. Genomic DNA was harvested from the cells 3 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS). The data was fitted to a variable slope (4-parameter) curve, and the EC50 was calculated for all guide + editor combinations.

[0386]

Table 8

[0387]

Table 9

[0388]

Table 10

[0389]

Table 11

[0390] Five editor variants (ABE8.13_SpCas9, ABE9.48_SpCas9, ABE9.50_SpCas9, ABE9.52_SpCas9) showed similar potency, where "potency" indicates the level of base editing achieved at a specific dose and the maximum editing relative to ABE8.8_SpCas9 when paired with gRNA676 (Table 8). Five editor variants (ABE8.13_SpCas9, ABE8.20_SpCas9, ABE9.51_SpCas9, ABE9.52_SpCas9, ABE8.13_SpCas9_A1283D_E1250K [KGPKPKKEESEK (SEQ ID NO: 940) linker]) showed similar or greater potency and maximum editing relative to ABE8.8_SpCas9 when paired with gRNA696 (Table 9). Seven editor variants (ABE8.13_SpCas9, ABE8.20_SpCas9, ABE9.47_SpCas9, ABE9.48_SpCas9, ABE9.51_SpCas9, ABE9.52_SpCas9, ABE8.13_SpCas9_A1283D_E1250K [KGPKPKKEESEK (SEQ ID NO: 940) linker]) showed similar or greater potency and maximum editing relative to ABE8.8_SpCas9 when paired with gRNA661 (Table 10). ABE9.51_SpCas9-VRQR showed higher potency relative to ABE8.13_SpCas9-VRQR when paired with gRNA715 (Table 11).

[0391] The editor variants identified above as showing good editing efficiency when combined with each guide were further screened in primary human hepatocyte (PHH) monolayers at sub-saturating doses.

[0392] Regarding the base editor system containing gRNA676, PHH monolayers were separately transfected with a total base editing dose of 2.5 ng and 10 ng. Conditions were normalized to a total RNA dose of 300 ng using Universal Human Reference RNA (Life Technologies, QS0639). Guide sgRNA_088 was used as a positive editing control, and the untransfected condition was used as a negative control. All conditions were evaluated in triplicate. Genomic DNA was harvested from all cells 3 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS).

[0393] Regarding the base editor system containing the gRNA696, gRNA661, and gRNA715 guides, PHH monolayers were separately transfected with a total base editing dose of 5 ng and 20 ng. Conditions were normalized to a total RNA dose of 300 ng using mRNA encoding non-translated ABE8.8. Guide sgRNA_088 was used as a positive editing control, and the untransfected condition was used as a negative control. All conditions were evaluated in triplicate. Genomic DNA was harvested from all cells 3 days after transfection, and the editing efficiency was evaluated by next-generation sequencing (NGS).

[0394] The base editor ABE9.48_SpCas9 was found to slightly improve the on-target editing efficiency compared to ABE8.8_SpCas9 when paired with gRNA676 at both total base editing doses of 2.5 ng and 10 ng (Figure 25). The base editor ABE8.13_SpCas9 was found to slightly improve the on-target editing efficiency compared to ABE8.8_SpCas9 when paired with gRNA696 at both base editing doses of 5 ng and 20 ng (Figures 26 and 27). The base editor ABE9.52_SpCas9 was found to significantly improve the on-target editing efficiency compared to ABE8.8_SpCas9 when paired with gRNA661 at both base editing doses of 5 ng and 20 ng (Figures 26 and 27). The base editor ABE9.51_SpCas9-VRQR did not improve the on-target editing efficiency compared to ABE8.13_SpCas9-VRQR wh...

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