Polynucleotides, compositions and methods for genome editing

Abstract

The present invention provides compositions and methods for gene editing. In some embodiments, a polynucleotide encoding Cas9 is provided that can provide one or more of improved editing efficiency, reduced immunogenicity, or other benefits.

Description

[0001] This application is a divisional application of Chinese application No. 2018800767113, whose application date is September 28, 2018 and whose invention name is “Polynucleotides, compositions and methods for genome editing”.

[0002] This application claims priority based on U.S. Provisional Application No. 62 / 556,144 filed on September 29, 2017, and the entire contents of which are incorporated by reference.

[0003] This application contains a sequence listing submitted electronically in ASCII format and its entire contents are incorporated herein by reference. This ASCII copy created on September 28, 2018 is named 2018-09-28_01155-0020-00PCT_ST25.txt and has a size of 963,200 bytes. Technical Field

[0004] The present invention relates to polynucleotides, compositions and methods for genome editing involving RNA-guided DNA binders, such as CRISPR-Cas systems and subunits thereof. Background Art

[0005] RNA-guided DNA binders, such as CRISPR-Cas systems, can be used to perform targeted genome editing, including in eukaryotic cells and in vivo. Such editing has been shown to be able to inactivate certain harmful alleles or correct certain harmful point mutations. The binder can be expressed in situ by providing the encoded mRNA. However, the editing efficiency provided by existing methods may be lower than the required editing efficiency or may have undesirable immunogenicity, for example, it may cause an increase in cytokine levels that is undesirable.

[0006] Therefore, there is a need for improved polynucleotides, compositions and methods for genome editing. The present invention aims to provide compositions and methods for genome editing, which provide one or more benefits, such as improved editing efficiency or at least one of reduced immunogenicity (e.g., reduced elevated amounts of cytokines after administration); or at least provide the public with a useful choice. In some embodiments, a polynucleotide encoding an RNA-guided DNA binder is provided, wherein one or more of its codon usage, non-coding sequences (e.g., UTRs), heterologous domains (e.g., NLSs), and / or nucleotide content are different from existing polynucleotides in a manner disclosed herein. Such features have been found to provide benefits such as those described above. In some embodiments, the improved editing efficiency occurs in or is specific to an organ or cell type of a mammal, such as the liver or hepatocytes. DETAILED DESCRIPTION OF THE INVENTION

[0008] Embodiment 1 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine content of the open reading frame is within the range of its minimum uridine content to 150% of the minimum uridine content.

[0009] Embodiment 2 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine dinucleotide content of the open reading frame is within the range of its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.

[0010] Embodiment 3 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the adenine content of the open reading frame is within the range of its minimum adenine content to 150% of the minimum adenine content.

[0011] Embodiment 4 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the adenine dinucleotide content of the open reading frame is within the range of its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content.

[0012] Embodiment 5 is an mRNA comprising a sequence having at least 90% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175, wherein the mRNA comprises an open reading frame encoding an RNA-guided DNA binder.

[0013] Embodiment 6 is an mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the open reading frame has at least 90% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175 over at least the first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides thereof.

[0014] Embodiment 7 is an mRNA as in any of the foregoing technical solutions, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are (i) codons listed in Table 1, Table 2 or Table 3 or (ii) a group of codons listed in Table 4.

[0015] Embodiment 8 is an mRNA encoding an RNA-guided DNA binder, which comprises an open reading frame encoding an RNA-guided DNA binder, wherein the open reading frame consists of a set of codons, wherein at least 75% of the codons are codons listed in Table 1, Table 2, Table 3 or (ii) a set of codons listed in Table 4.

[0016] Embodiment 9 is the mRNA of technical scheme 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the low U1 group in Table 4.

[0017] Embodiment 10 is the mRNA as described in technical solution 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the low A group in Table 4.

[0018] Embodiment 11 is the mRNA as described in technical solution 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the low A / U group in Table 4.

[0019] Embodiment 12 is the mRNA of technical scheme 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the long half-life group in Table 4.

[0020] Embodiment 13 is the mRNA of any one of technical schemes 7 to 12, wherein at least 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are (i) codons listed in Table 1, Table 2 or Table 3 or (ii) a group of codons listed in Table 4.

[0021] Embodiment 14 is the mRNA of any one of technical schemes 1 to 5 or 7 to 13, wherein the open reading frame has at least 90% identity with any one of SEQID NO:1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175 in at least the first 30, 50, 70, 100, 150, 200, 250 or 300 nucleotides thereof.

[0022] Embodiment 15 is an mRNA as any one of the aforementioned technical solutions, wherein the open reading frame has at least 90% identity with any one of SEQ ID NO:1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175 in at least the first 10%, 12%, 15%, 20%, 25%, 30% or 35% of its sequence.

[0023] Embodiment 16 is the mRNA of any one of technical schemes 1 to 4 or 6 to 15, wherein the mRNA comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0024] Embodiment 17 is the mRNA of any one of the foregoing technical schemes, wherein the uridine dinucleotide content of the open reading frame is in the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum uridine dinucleotide content to the minimum uridine dinucleotide content.

[0025] Embodiment 18 is the mRNA of any one of the foregoing technical schemes, wherein the uridine content of the open reading frame is in the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum uridine content to the minimum uridine content.

[0026] Embodiment 19 is the mRNA of any one of the foregoing technical schemes, wherein the adenine content of the open reading frame is in the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum adenine content to the minimum adenine content.

[0027] Embodiment 20 is the mRNA of any one of the foregoing technical schemes, wherein the adenine dinucleotide content of the open reading frame is in the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum adenine dinucleotide content to the minimum adenine dinucleotide content.

[0028] Embodiment 21 is an mRNA as described in any of the foregoing technical solutions, which comprises a 5'UTR that is at least 90% identical to any of SEQ ID NOs: 32, 34, 36, 38, 41 or 75-77.

[0029] Embodiment 22 is an mRNA as described in any of the foregoing technical solutions, which comprises a 3'UTR that is at least 90% identical to any of SEQ ID NO:33, 35, 37, 39 or 40.

[0030] Embodiment 23 is the mRNA of technical solution 21 or 22, wherein the mRNA comprises a 5'UTR and a 3'UTR from the same source.

[0031] Embodiment 24 is the mRNA of any one of the aforementioned technical solutions, which comprises a 5' cap structure selected from Cap0, Cap1 and Cap2.

[0032] Embodiment 25 is the mRNA of any one of the preceding technical solutions, wherein the open reading frame has codons that increase the translation of the mRNA in mammals.

[0033] Embodiment 26 is the mRNA of technical scheme 25, wherein the open reading frame has codons that improve the translation of mRNA in specific organs of mammals.

[0034] Example 27 is the mRNA of technical solution 26, wherein the organ is the liver.

[0035] Embodiment 28 is the mRNA of any one of technical schemes 25 to 27, wherein the mammal is human.

[0036] Embodiment 29 is the mRNA of any one of technical solutions 25 to 28, wherein the codon increases the translation of the mRNA in mammals relative to the translation of the mRNA comprising an ORF having a sequence consisting of SEQ ID NO: 5.

[0037] Embodiment 30 is an mRNA as described in any of the foregoing technical solutions, wherein when the mRNA is administered to a mammal in the form of a pharmaceutical composition, the mammal exhibits a cytokine response that is at least 5 times lower than that of a mammal administered with an mRNA comprising an ORF encoding a Cas9 nuclease having a minimum uridine content greater than 150%.

[0038] Embodiment 31 is the mRNA of technical scheme 30, wherein the mRNA containing the ORF encoding the Cas9 nuclease with a minimum uridine content greater than 150% has a sequence consisting of SEQ ID NO:5.

[0039] Embodiment 32 is the mRNA of any one of the foregoing technical solutions, wherein the RNA-guided DNA binder has double-stranded nuclease activity.

[0040] Embodiment 33 is the mRNA of technical scheme 32, wherein the RNA-guided DNA binder comprises Cas cleavage enzyme.

[0041] Embodiment 34 is the mRNA of any one of the foregoing technical schemes, wherein the RNA-guided DNA binder has nickase activity.

[0042] Embodiment 35 is the mRNA of technical scheme 34, wherein the RNA-guided DNA binder comprises Cas nickase.

[0043] Embodiment 36 is the mRNA of any one of technical schemes 1 to 31, wherein the RNA-guided DNA binder comprises a dCas DNA binding domain.

[0044] Embodiment 37 is the mRNA of any one of technical schemes 33 or 35 to 36, wherein the Cas lyase, Cas nickase or dCas DNA binding domain is a Cas9 lyase, Cas9 nickase, or dCas9 DNA binding domain.

[0045] Embodiment 38 is the mRNA of any one of the foregoing technical schemes, wherein the encoded RNA-guided DNA binder comprises a nuclear localization signal (NLS).

[0046] Embodiment 39 is the mRNA of technical scheme 38, wherein NLS is connected to the C-terminus of RNA-guided DNA binder.

[0047] Embodiment 40 is the mRNA of technical scheme 38, wherein NLS is connected to the N-terminus of RNA-guided DNA binder.

[0048] Embodiment 41 is the mRNA of any one of technical schemes 38 to 40, wherein the NLS comprises a sequence having at least 80%, 85%, 90% or 95% identity with any one of SEQ ID NOs: 78-91.

[0049] Embodiment 42 is the mRNA of any one of technical schemes 38 to 40, wherein the NLS comprises the sequence of any one of SEQ ID NO: 78-91.

[0050] Embodiment 43 is the mRNA of any one of technical schemes 38 to 42, wherein the NLS is encoded by a sequence having at least 80%, 85%, 90%, 95%, 98% or 100% identity with the sequence of any one of SEQ ID NOs: 92-104.

[0051] Embodiment 44 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO:4, 7 or 9.

[0052] Embodiment 45 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO:4, 7 or 9.

[0053] Embodiment 46 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 98% identical to SEQ ID NO:4, 7 or 9.

[0054] Embodiment 47 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is 100% identical to SEQ ID NO:4, 7 or 9.

[0055] Embodiment 48 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO:111, 114 or 117.

[0056] Embodiment 49 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO:111, 114 or 117.

[0057] Embodiment 50 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 98% identical to SEQ ID NO:111, 114 or 117.

[0058] Embodiment 51 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is 100% identical to SEQ ID NO:112, 122 or 125.

[0059] Embodiment 52 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO:112, 122 or 125.

[0060] Embodiment 53 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO:112, 122 or 125.

[0061] Embodiment 54 is an mRNA as any one of technical solutions 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0062] Embodiment 55 is an mRNA as any one of technical solutions 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0063] Embodiment 56 is an mRNA as any one of technical solutions 37 to 43, wherein the mRNA comprises a sequence that is at least 98% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0064] Embodiment 57 is the mRNA of any one of technical schemes 37 to 43, wherein the mRNA comprises a sequence that is 100% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0065] Embodiment 58 is the mRNA of any one of technical schemes 37 to 57, wherein the mRNA encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 8 or 186-196.

[0066] Embodiment 59 is an mRNA as described in any of the foregoing technical solutions, wherein the RNA-guided DNA binder further comprises a heterologous functional domain.

[0067] Example 60 is the mRNA of technical solution 59, wherein the heterologous functional domain is FokI nuclease.

[0068] Example 61 is the mRNA of technical solution 59, wherein the heterologous functional domain is a transcriptional regulatory domain.

[0069] Embodiment 62 is an mRNA as described in any of the foregoing technical solutions, wherein when an effective amount of the mRNA and a guide RNA targeting the TTR gene of the mammal are administered to a mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, an insertion / deletion mark (indel) is formed at the TTR locus in at least 50% of the genomic DNA obtained from the liver cells of the mammal.

[0070] Embodiment 63 is the mRNA of any one of the foregoing technical solutions, wherein when an effective amount of the mRNA and a guide RNA targeting the TTR gene of the mammal are administered to a mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, the concentration of TTR in the serum of the mammal is reduced by at least 50%.

[0071] Embodiment 64 is the mRNA of any one of the preceding technical solutions, wherein at least 10% of the uridines are replaced by modified uridines.

[0072] Embodiment 65 is the mRNA of technical scheme 64, wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine or 5-iodouridine.

[0073] Example 66 is the mRNA of technical solution 64, wherein the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.

[0074] Example 67 is the mRNA of technical solution 64, wherein the modified uridine is N1-methyl-pseudouridine.

[0075] Example 68 is the mRNA of technical solution 64, wherein the modified uridine is 5-methoxyuridine.

[0076] Embodiment 69 is the mRNA of any one of technical solutions 64 to 68, wherein 15% to 45% of the uridine is replaced by modified uridine.

[0077] Embodiment 70 is the mRNA of any one of technical solutions 64 to 68, wherein at least 20% or at least 30% of the uridine is substituted by modified uridine.

[0078] Embodiment 71 is the mRNA of technical scheme 70, wherein at least 80% or at least 90% of the uridine is replaced by modified uridine.

[0079] Example 72 is the mRNA of technical solution 70, wherein 100% of the uridine is replaced by modified uridine.

[0080] Embodiment 73 is an mRNA as described in any one of technical solutions 64 to 72, wherein when an effective amount of the mRNA and a guide RNA targeting the TTR gene of the mammal are administered to a mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, an insertion / deletion mark (indel) is formed at the TTR locus in at least 70% or at least 90% of the genomic DNA obtained from the liver cells of the mammal.

[0081] Embodiment 74 is the mRNA of any one of technical schemes 64 to 73, wherein when the mRNA and a guide RNA targeting the TTR gene of the mammal are administered to a mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, the concentration of TTR in the serum of the mammal is reduced by at least 70% or at least 90%.

[0082] Embodiment 75 is the mRNA of technical scheme 62, 63, 71 or 72, wherein the animal is a mouse and the guide RNA has a sequence consisting of SEQ ID NO:42.

[0083] Embodiment 76 is the mRNA of technical scheme 62, 63, 71 or 72, wherein the animal is a rat and the guide RNA has a sequence consisting of SEQ ID NO:69.

[0084] Embodiment 77 is an mRNA as in any of the foregoing technical solutions, wherein the mRNA comprises a sequence that is at least 90% identical to any of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.

[0085] Embodiment 78 is an mRNA as in any of the foregoing technical solutions, wherein the mRNA comprises a sequence that is at least 95% identical to any of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.

[0086] Embodiment 79 is an mRNA as in any of the foregoing technical solutions, wherein the mRNA comprises a sequence that is at least 98% identical to any of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.

[0087] Embodiment 80 is an mRNA as in any of the foregoing technical solutions, wherein the mRNA comprises a sequence that is at least 99% identical to any of SEQ ID NOs: 43, 44, 51, 53, 55-61 or 176-185.

[0088] Embodiment 81 is the mRNA of any one of the foregoing technical schemes, wherein the mRNA comprises a sequence that is 100% identical to any one of SEQ ID NO:43, 44, 51, 53, 55-61 or 176-185.

[0089] Embodiment 82 is an expression construct comprising a promoter operably linked to a sequence encoding an mRNA as described in any of the foregoing technical solutions.

[0090] Example 83 is a plasmid comprising an expression construct as in technical solution 82.

[0091] Embodiment 84 is a host cell comprising an expression construct as in technical solution 82 or a plasmid as in technical solution 83.

[0092] Embodiment 85 is a method for preparing mRNA, which comprises contacting an expression construct as in technical solution 82 or a plasmid as in technical solution 83 with a ribonucleic acid polymerase under conditions that allow transcription of the mRNA.

[0093] Embodiment 86 is a method as in technical solution 85, wherein the contacting step is performed in vitro.

[0094] Embodiment 87 is a composition comprising the mRNA of any one of technical solutions 1 to 81 and at least one guide RNA.

[0095] Embodiment 88 is a lipid nanoparticle comprising the mRNA of any one of technical schemes 1 to 81.

[0096] Embodiment 89 is a pharmaceutical composition comprising the mRNA of any one of technical solutions 1 to 81 and a pharmaceutically acceptable carrier.

[0097] Embodiment 90 is a lipid nanoparticle as in technical solution 88 or a pharmaceutical composition as in technical solution 89, which further comprises at least one guide RNA.

[0098] Embodiment 91 is a composition or lipid nanoparticle as any one of technical schemes 87 to 90, wherein at least one guide RNA targets TTR.

[0099] Embodiment 92 is a method for genome editing or modifying a target gene, which comprises contacting a cell with an mRNA, expression construct, composition or lipid nanoparticle as described in any one of technical solutions 1 to 83 or 87 to 91.

[0100] Embodiment 93 is the use of the mRNA, expression construct, composition or lipid nanoparticle of any one of technical schemes 1 to 83 or 87 to 91 for genome editing or modifying the target gene.

[0101] Embodiment 94 is the use of the mRNA, expression construct, composition or lipid nanoparticle of any one of technical schemes 1 to 83 or 87 to 91 for the manufacture of an agent for genome editing or modifying a target gene.

[0102] Embodiment 95 is a method or use as any one of technical schemes 92 to 94, wherein genome editing or modification of the target gene is performed in liver cells.

[0103] Embodiment 96 is the method or use of technical scheme 95, wherein the liver cells are hepatocytes.

[0104] Embodiment 97 is a method or use as any one of technical solutions 92 to 96, wherein genome editing or modification of the target gene is performed in vivo.

[0105] Embodiment 98 is a method or use as any one of technical solutions 92 to 97, wherein genome editing or modification of the target gene is performed in isolated or cultured cells.

[0106] Brief description of the revealed sequences

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[0116] For the sequences themselves, see the sequence listing below. Transcript sequences typically include GGG as the first three nucleotides for use with ARCA, or AGG as the first three nucleotides for use with CleanCap. TM The first three nucleotides are used together. Therefore, the first three nucleotides can be modified for use with other capping methods, such as vaccinia capping enzymes. The promoter and poly-A sequence are not included in the transcript sequence. A promoter, such as the T7 promoter (SEQ ID NO: 31) and a poly-A sequence, such as SEQ ID NO: 62 or 63, can be attached to the disclosed transcript sequence at the 5' and 3' ends, respectively. Most nucleotide sequences are provided in DNA form, but can be easily converted to RNA by changing Ts to Us. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figures 1A to 1D Shown are the levels of IFNα, IL-6, TNFα, and MCP-1 after administration of PBS or lipid nanoparticle (LNP) formulations LNP417 to LNP421 at 0.5 or 1 mg / kg (mpk).

[0119] FIG. 2A to FIG. 2BShown are serum TTR levels and percent liver editing following administration of PBS or LNP formulations LNP417 to LNP421 at 0.5 or 1 mpk.

[0120] Figure 3 In vitro transcription (IVT) yields of transcription from Cas9 DNA constructs are shown. Transcription was performed with unmodified uridine-5'-triphosphate (UTP) either alone (on the horizontal axis, 0), with N1-methyl-pseudoUTP mixed with the indicated ratios of 5-methoxyUTP (on the horizontal axis, 20 to 80), or with 100% 5-methoxyUTP (100). For each set of three bar graphs, the left bar graph uses N1-methyl-pseudoUTP and / or 5-methoxyUTP and SEQ ID NO: 2; the middle bar graph uses unmodified UTP and / or 5-methoxyUTP and SEQ ID NO: 2; and the right bar graph uses unmodified UTP and / or 5-methoxyUTP and SEQ ID NO: 1.

[0121] Figure 4 The purity of mRNA is shown as a result of in vitro transcription (IVT) of Cas9 (SEQ ID NO: 2) and optimized Cas9 (SEQ ID NO: 1) DNA constructs. The Cas9 sequence of SEQ ID NO: 2 was transcribed with unmodified uridine-5'-triphosphate (UTP) (squares) or with N1-methyl-pseudo-UTP (black circles) alone (0) or mixed with 5-methoxy UTP at the indicated ratios (20 to 80) or mixed with 100% 5-methoxy UTP (100). The Cas9 sequence of SEQ ID NO: 1 (light circles) was transcribed with unmodified UTP (0) or mixed with 5-methoxy UTP at the indicated ratios (20 to 80) or mixed with 100% 5-methoxy UTP (100). Each coding sequence includes a nuclear localization signal.

[0122] Figure 5 A to Figure 5 D shows the results of the anti-dsRNA antibody dot analysis. The results are generated with double-stranded RNA control (A), Cas9 transcribed in the presence of UTP and / or 5-methoxy UTP (B), Cas9 mRNA sequence comprising SEQ ID NO: 4 transcribed in the presence of UTP and / or 5-methoxy UTP (C), and Cas9 transcribed in the presence of N1-methyl-pseudo UTP and / or 5-methoxy UTP (D). Figures (B) to (D) were performed with 0% to 100% 5-methoxy UTP and 100% to 0% UTP or N1-methyl UTP.

[0123] Figure 6 A and Figure 6 B shows the in vitro editing efficiency of mRNA in neuroblastoma 2A cells (Neuro 2A cell) treated with Cas9 mRNA, which is presented as editing percentage (A) or editing EC50 (B). The effect of increasing the concentration of 5-methoxy-UTP in Cas9 mRNA was assessed. The Cas9 sequence of SEQ ID NO: 2 was transcribed with N1-methyl-pseudo-UTP (left group in A; dark circles in B) or with unmodified uridine-5'-triphosphate (UTP) (middle group in A; squares in B) alone (0) or mixed with 5-methoxy UTP in a specified ratio (20 to 80) or mixed with 100% 5-methoxy UTP (100). Transcription from the Cas9 sequence of SEQ ID NO: 1 (right panel in A; light circles in B) was performed with unmodified UTP (0) or unmodified UTP mixed with the indicated ratios of 5-methoxy UTP (20 to 80) or with 100% 5-methoxy UTP (100). Each coding sequence included a nuclear localization signal.

[0124] Figure 7 A to Figure 7 D shows serum cytokine levels 4 hours after administration of LNP formulations LNP720 to LNP724. Figure 7 The asterisk in A indicates that at least one independent measurement was below the detection limit.

[0125] Figure 8 A and Figure 8 B shows serum TTR levels (A) and TTR editing percentage in the liver (B) 7 days after dosing with LNP formulations LNP720 to LNP724. Figure 8 The asterisk in A indicates that at least one independent measurement was below the detection limit.

[0126] Fig. 9 Shown are the percent TTR editing in the spleen 7 days after dosing with LNP formulations LNP720 to LNP724 at 1 mpk.

[0127] Fig.10 Shown are the percent TTR editing in primary mouse hepatocytes (PMH) using LNP formulations LNP720 to LNP724 and LNP685.

[0128] Fig.11A and Fig. 11B Serum TTR levels following administration of a formulation comprising Cas9 mRNA in which the ORF has the sequence of SEQ ID NO: 5 or 4 are shown. TTR data are presented as serum levels (A) or as a percentage relative to TTR levels in TSS-treated animals (B).

[0129] Fig.12 Shown is the percentage of TTR editing in the liver following dosing at 5 mpk or 2 mpk of formulations comprising Cas9 mRNA in which the ORF has the sequence of SEQ ID NO: 5 or 4.

[0130] FIG. 13A to FIG. 13E Shown are serum TTR levels and percent TTR editing in the liver following administration of the indicated LNP formulations.

[0131] Fig.14 Shown are the percentages of TTR editing in primary mouse hepatocytes (PMH) treated with 0.3, 1, 3, or 10 ng of LNP815-821, 823, or 824.

[0132] FIG. 15A to FIG. 15B Shown are serum TTR levels following dosing of LNP formulations containing Cas9 mRNA at the indicated guide:Cas9 ratios and amounts, in which the ORF has the sequence of SEQ ID NO: 5 or 4.

[0133] FIG. 16A to FIG. 16B Shown are the percentages of TTR editing in the liver following dosing of LNP formulations containing Cas9 mRNA at the indicated guide:Cas9 ratios and amounts, in which the ORF had the sequence of SEQ ID NO: 5 or 4.

[0134] FIG. 17A to FIG. 17B Shown are the percentages of TTR editing in the spleen following dosing of LNP formulations containing Cas9 mRNA at the indicated guide:Cas9 ratios and amounts, in which the ORF had the sequence of SEQ ID NO: 5 or 4.

[0135] Fig.18 Shown are Western blots for Cas9 expression in the liver following dosing of LNP formulations containing Cas9 mRNA in which the ORF has the sequence of SEQ ID NO: 5 or 4 at the indicated guide:Cas9 ratios.

[0136] FIG. 19A to FIG. 19B Shown are serum TTR levels following dosing of the indicated LNP formulations in the indicated amounts.

[0137] Fig. 20 Shown are the percent TTR editing in the liver following dosing of the indicated LNP formulations at the indicated amounts.

[0138] FIG. 21A to FIG. 21CShown are liver editing levels (A) and serum TTR (B in μg / ml; C as a percentage of TSS control) after dosing of the indicated LNP formulations at the indicated amounts.

[0139] Figures 22A to 22D Shown are serum TTR and editing results after dosing of LNP formulations at the indicated ratios and amounts.

[0140] Fig.23 Shown is Cas9 protein expression in Hep2G cells after treatment with Cas9 mRNA in which the ORF has the sequence of the designated SEQ ID NO.

[0141] Fig.24 Shown are the editing percentages in Hep2G cells after treatment with Cas9 mRNA at the indicated concentrations, in which the ORF has the sequence of the indicated SEQ ID NO.

[0142] Fig.25 Shown is Cas9 expression in the liver following administration of LNP formulations with Cas9 mRNA in which the ORF has the sequence of the designated SEQ ID NO.

[0143] Fig.26 Shown are the results of in vivo editing at the TTR locus following administration of LNP formulations with Cas9 mRNA in which the ORF has the sequence of the indicated SEQ ID NO.

[0144] FIG. 27A to FIG. 27B Shown are serum TTR (A) and serum TTR (TSS%) (B) after administration of LNP formulations with Cas9 mRNA in which the ORF has the sequence of the indicated SEQ ID NO.

[0145] Fig.28 In vivo liver editing is shown following dosing of the indicated amounts of LNP formulations with Cas9 mRNA in which the ORF has the sequence of the indicated SEQ ID NO.

[0146] FIG. 29A to FIG. 29B Shown are serum TTR levels (A) and serum TTR (TSS%) (B) after dosing of the indicated amounts of LNP formulations with Cas9 mRNA in which the ORF has the sequence of the indicated SEQ ID NO.

[0147] FIG. 30A to FIG. 30BShown are serum TTR levels (A) and % editing in the liver (B) after administration of LNP formulations with Cas9 mRNA in which transcripts have the sequence of the indicated SEQ ID NO.

[0148] Fig.31 Shown are the percentages of TTR editing in the liver following administration of LNPs formulated with mRNAs having the indicated cap structures and transcript sequences at the indicated doses.

[0149] Fig.32 Shown are serum TTR levels following administration of LNPs formulated with mRNAs having the indicated cap structures and transcript sequences at the indicated doses.

[0150] Fig.33 Shown are the percentages of TTR editing in the liver following administration of LNPs formulated with mRNA encoding Cas9, in which the ORF has the sequence of the designated SEQ ID NO, including the NLS as indicated.

[0151] FIG. 34A to FIG. 34B Shown are serum TTR levels (A) and serum TTR (TSS%) (B) after administration of LNPs formulated with mRNA encoding Cas9, in which the ORF has the sequence of the designated SEQ ID NO, including the NLS as indicated.

[0152] Fig.35 The correlation between NLS activity and editing efficiency after administration of LNPs formulated with mRNA encoding Cas9 and including NLS sequences of different types and activity levels is shown.

[0153] Fig.36 Shown are the expression levels of Cas9 protein in HepG2 cells from mRNA transcripts with the indicated sequences and 5'UTR as indicated. Implementation

[0155] Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Although the present invention will be described in conjunction with the illustrated embodiments, it should be understood that it is not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents, which may be included in the present invention as defined by the appended claims.

[0156] Before describing the present teachings in detail, it should be understood that the invention is not limited to specific compositions or method steps and as such may vary. It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a conjugate" includes a plurality of conjugates and reference to "a cell" includes a plurality of cells and the like.

[0157] Numerical ranges include the numbers defining the range. Measurements and measurable values ​​should be understood to be approximate, taking into account significant figures and errors associated with the measurements. In addition, the use of "comprise / comprises / comprising", "contain / contains / containing" and "include / includes / including" is not intended to be limiting. It should be understood that the foregoing general description and detailed description are exemplary and explanatory only and do not limit the teachings.

[0158] The term "about" or "approximately" means an acceptable error for a particular value as determined by one of ordinary skill in the art, to some extent depending on how the value is measured or determined, or the degree of variation does not materially affect the properties of the subject matter (e.g., within 10%, 5%, 2% or 1%). Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the attached claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0159] Unless specifically stated in the above description, embodiments described in this specification as “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the components; embodiments described in this specification as “consisting of” various components are also contemplated as “comprising” the components or “consisting essentially of” the components; and embodiments described in this specification as “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the components (this interchangeability does not apply to the use of such terms in the claims).

[0160] The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. In the event that any document incorporated by reference conflicts with the contents of this specification (including but not limited to definitions), the contents of this specification shall prevail. Although the present teachings are described in conjunction with a number of embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, as will be appreciated by those skilled in the art, the present teachings encompass various alternatives, modifications, and equivalents.

[0161] A. Definition

[0162] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings:

[0163] As used herein, the term "or a combination thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular case, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or clauses are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will appreciate that, unless otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0164] As used herein, the term "kit" refers to a packaged set of related components, such as one or more polynucleotides or compositions and one or more related materials, such as a delivery device (e.g., a syringe), solvents, solutions, buffers, instructions, or desiccant.

[0165] Unless otherwise specified herein, "or" is used in an inclusive sense, ie, equivalent to "and / or".

[0166] "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs linked together along the backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers of analogs thereof. The nucleic acid "backbone" may be composed of a plurality of bonds, including one or more of sugar-phosphodiester bonds, peptide-nucleic acid bonds ("peptide nucleic acid" or PNA; PCT No. WO 95 / 32305), phosphorothioate bonds, methylphosphonate bonds, or combinations thereof. The sugar portion of the nucleic acid may be ribose, deoxyribose, or similar compounds having substitutions, such as 2' methoxy or 2' halide substitutions. The nitrogen-containing base may be a conventional base (A, G, C, T, U); an analog thereof (e.g., a modified uridine, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine or others); inosine; a derivative of a purine or pyrimidine (e.g., N 4 -methyl deoxyguanosine, deaza or aza purine, deaza or aza pyrimidine, a pyrimidine base having a substituent at the 5 or 6 position (e.g., 5-methylcytosine), a purine base having a substituent at the 2, 6 or 8 position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidine; U.S. Pat. No. 5,378,825 and PCT No. WO 93 / 13121). For a general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992. Nucleic acids may include one or more "abasic" residues, in which the backbone does not include a nitrogenous base for a polymer position (U.S. Pat. No. 5,585,481). Nucleic acids may include only conventional RNA or DNA sugars, bases, and linkages, or may include both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), an analog containing one or more LNA nucleotide monomers having a bicyclic furanose unit locked in RNA that mimics a sugar conformation, which enhances hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42): 13233-41). RNA and DNA have different sugar moieties and can be differentiated by the presence of uracil or its analog in RNA and thymine or its analog in DNA.

[0167] "Modified uridine" is used herein to refer to nucleosides other than thymidine that have the same hydrogen bond acceptors as uridine and that have one or more structural differences from uridine. In some embodiments, the modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substitutions (e.g., alkoxy groups, such as methoxy groups) replace a proton. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substitutions (e.g., alkyl groups, such as methyl groups) replace a proton. In some embodiments, the modified uridine is any of a substituted uridine, a pseudouridine, or a substituted pseudouridine.

[0168] As used herein, "uridine position" refers to a position in a polynucleotide that is occupied by uridine or a modified uridine. Thus, for example, a polynucleotide in which "100% of the uridine positions are modified uridines" contains a modified uridine at every position that would be a uridine in a conventional RNA of the same sequence (in which all bases are standard A, U, C, or G bases). Unless otherwise indicated, U in a polynucleotide sequence in the present invention or in a sequence table / sequence listing accompanying the present invention may be uridine or a modified uridine.

[0169] As used herein, a first sequence is considered to "comprise a sequence having at least X% identity to a second sequence" if an alignment of the first sequence with a second sequence shows that X% or more of the positions in the entire second sequence match the first sequence. For example, the sequence AAGA comprises a sequence having 100% identity to the sequence AAG, since matches occur at all three positions in the second sequence, resulting in 100% identity. Differences between RNA and DNA (generally, uridine is replaced by thymidine or vice versa) and the presence of nucleoside analogs (such as modified uridine) do not result in differences in identity or complementarity between polynucleotides, as long as the relevant nucleotides (such as thymidine, uridine or modified uridine) have the same complementary sequence (for example, adenosine for thymidine, uridine or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine as a complementary sequence). Thus, for example, the sequence 5'-AXG (wherein X is any modified uridine, such as pseudouridine, N1-methylpseudouridine or 5-methoxyuridine) is considered to have 100% identity to AUG, since both are fully complementary to the same sequence (5'-CAU). Examples of alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. One skilled in the art will appreciate which algorithm and parameter settings to choose for a given pair of sequences to be aligned; for sequences of generally similar length and expected amino acid identity of >50% or nucleotide identity of >75%, the Needleman-Wunsch algorithm with default settings provided by the EBI at www.ebi.ac.uk web server is generally appropriate.

[0170] "mRNA" is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and an aminoacylated tRNA). The mRNA may comprise a phosphate-sugar backbone comprising a ribose residue or an analog thereof, such as a 2'-methoxyribose residue. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of a ribose residue, a 2'-methoxyribose residue, or a combination thereof. Generally speaking, the mRNA does not contain a large number of thymidine residues (e.g., 0 residues or less than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). The mRNA may contain modified uridines at some or all of its uridine positions.

[0171] As used herein, "RNA-guided DNA binders" means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binders include Cas lyases / nickases and inactive forms thereof ("dCas DNA binders"). As used herein, "Cas nucleases" are also referred to as "Cas proteins", which encompass Cas lyases, Cas nickases, and dCas DNA binders. Cas lyases / nickases and dCas DNA binders include Csm or Cmr complexes of type III CRISPR systems, Cas10, Csm1, or Cmr2 subunits thereof, Cascade complexes of type I CRISPR systems, Cas3 subunits thereof, and class 2 Cas nucleases. As used herein, "class 2 Cas nucleases" are single-chain polypeptides having DNA binding activity guided by RNA, such as Cas9 nucleases or Cpf1 nucleases. Class 2 Cas nucleases include class 2 Cas lyases and class 2 Cas nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA lyase or nickase activity, and class 2 dCas DNA binders, in which the lyase / nickase activity is not activated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (1.1) (e.g., K848A, K1003A, R1060A variants) proteins and variants thereof. The Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated by reference in its entirety. See, e.g., Zetsche, Table S1 and Table S3. "Cas9" encompasses Spy Cas9, variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60: 385-397 (2015).

[0172] As used herein, the "minimum uridine content" of a given open reading frame (ORF) is the uridine content of an ORF that (a) uses a minimum uridine codon at each position and (b) encodes the same amino acid sequence as the given ORF. The minimum uridine codon for a given amino acid is the codon with the fewest uridines (usually 0 or 1, except for the codon for phenylalanine, in which the minimum uridine codon has 2 uridines). For the purpose of assessing the minimum uridine content, modified uridine residues are considered equivalent to uridine.

[0173] As used herein, the "minimum uridine dinucleotide content" of a given open reading frame (ORF) is the lowest possible uridine dinucleotide (UU) content of an ORF that (a) uses the minimum uridine codon (as discussed above) at each position and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed in an absolute sense as a count of UU dinucleotides in an ORF or based on a ratio, expressed as the percentage of positions occupied by uridine of uridine dinucleotides (e.g., a uridine dinucleotide content of AUUAU is 40% because uridine of uridine dinucleotides occupies 2 of the 5 positions). For the purpose of assessing the minimum uridine dinucleotide content, modified uridine residues are considered equivalent to uridine.

[0174] As used herein, the "minimum adenine content" of a given open reading frame (ORF) is the adenine content of an ORF that (a) uses the minimum adenine codon at each position and (b) encodes the same amino acid sequence as the given ORF. The minimum adenine codon for a given amino acid is the codon with the fewest adenines (usually 0 or 1, except for the codons for lysine and asparagine, where the minimum adenine codon has 2 adenines). For the purpose of assessing the minimum adenine content, modified adenine residues are considered equivalent to adenine.

[0175] As used herein, the "minimum adenine dinucleotide content" of a given open reading frame (ORF) is the lowest possible adenine dinucleotide (AA) content of an ORF that (a) uses the minimum adenine codon at each position (as discussed above) and (b) encodes the same amino acid sequence as the given ORF. The adenine dinucleotide (AA) content can be expressed in an absolute sense as a count of AA dinucleotides in the ORF or based on a ratio, expressed as the percentage of positions occupied by adenine of adenine dinucleotides (e.g., the adenine dinucleotide content of UAAUA is 40% because adenine of adenine dinucleotides occupies 2 of the 5 positions). For the purpose of assessing the minimum adenine dinucleotide content, modified adenine residues are considered equivalent to adenine.

[0176] "Guide RNA", "gRNA" and "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). The crRNA and trRNA can be associated in the form of a single RNA molecule (single guide RNA, sgRNA) or two independent RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA can be a naturally occurring sequence or a trRNA sequence that has modifications or changes compared to a naturally occurring sequence.

[0177] As used herein, "guide sequence" refers to a sequence in a guide RNA that is complementary to a target sequence and is guided to a target sequence by a DNA binder guided by RNA for binding or modification (e.g., cleavage). "Guide sequence" may also be referred to as a "target sequence" or "spacer sequence". The length of the guide sequence may be 20 base pairs, such as in Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / straight homologs. Shorter or longer sequences may also be used as guides, such as 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. In some embodiments, the target sequence is in, for example, a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or consistency between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1 to 4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, wherein the guide sequence comprises 20 nucleotides.

[0178] The target sequence for the Cas protein includes both the positive and negative strands of the genomic DNA (i.e., a given sequence and the reverse complement of the sequence), because the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Therefore, in the case of discussing that the guide sequence is "complementary to the target sequence", it should be understood that the guide sequence can guide the guide RNA to bind to the reverse complement of the target sequence. Therefore, in some embodiments, when the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence that does not include a PAM), except that U replaces T in the guide sequence.

[0179] As used herein, an "indel marker (indel)" refers to an insertion / deletion mutation consisting of multiple nucleotides that are inserted or deleted at a double strand break (DSB) site in a nucleic acid.

[0180] As used herein, "gene knockdown" refers to a decrease in the expression of a particular gene product (e.g., protein, mRNA, or both). Gene knockdown of a protein can be measured by detecting the protein secreted by a tissue or cell population (e.g., in serum or cell culture medium) or by detecting the total cellular amount of the protein from the relevant tissue or cell population. Methods for measuring gene knockdown of mRNA are known and include sequencing mRNA isolated from the relevant tissue or cell population. In some embodiments, "gene knockdown" may refer to some loss in expression of a particular gene product, such as a decrease in the amount of transcribed mRNA or a decrease in the amount of protein expressed or secreted by a cell population (including in vivo cell populations, such as those present in tissues).

[0181] As used herein, "gene knockout" refers to the loss of expression of a particular protein in a cell. Gene knockout can be measured by detecting the amount of protein secreted by a tissue or cell population (e.g., in serum or cell culture medium) or by detecting the total cellular amount of the protein in a tissue or cell population. In some embodiments, the methods of the invention "knock out" a target protein in one or more cells (e.g., in a cell population, including an in vivo cell population, such as those present in a tissue). In some embodiments, gene knockout does not result in the formation of a mutant target protein, such as by insertion or deletion of a marker (indel), but rather in the complete loss of expression of the target protein in the cell.

[0182] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a guide RNA and an RNA-guided DNA binder, such as a Cas lyase, a nickase, or a dCas DNA binder (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binder, such as Cas9, to a target sequence, and the guide RNA hybridizes to the target sequence and the binder binds to the target sequence; where the binder is a lyase or nickase, cleavage or nicking is performed after binding.

[0183] As used herein, "target sequence" refers to a nucleic acid sequence in a target gene that is complementary to the guide sequence of a gRNA. The interaction of the target sequence with the guide sequence guides the RNA-guided DNA binding agent to bind, and potentially breaks or cleaves in the target sequence (depending on the activity of the agent).

[0184] As used herein, "treatment" refers to any administration or administration of a therapeutic agent to a disease or disorder in a subject, and includes inhibiting the disease, arresting its progression, alleviating one or more symptoms of the disease, curing the disease, or preventing the recurrence of one or more symptoms of the disease.

[0185] B. Exemplary Polynucleotides and Compositions

[0186] 1. mRNA and ORF with low uridine content

[0187] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being within the range of its minimum uridine content to about 150% of its minimum uridine content. In some embodiments, the uridine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being equal to its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 150% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 145% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 140% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 135% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 130% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 125% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 120% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 115% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 110% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 105% of its minimum uridine content.In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 104% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 103% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 102% of its minimum uridine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine content of the ORF being less than or equal to about 101% of its minimum uridine content.

[0188] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being within the range of its minimum uridine dinucleotide content to 200% of its minimum uridine dinucleotide content. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 195%, 190%, 185%, 180%, 175%, 170%, 165%, 160%, 155%, 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being equal to its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 200% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 195% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 190% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 185% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 180% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) having a uridine dinucleotide content of less than or equal to about 175% of its minimum uridine dinucleotide content is provided. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) having a uridine dinucleotide content of less than or equal to about 170% of its minimum uridine dinucleotide content is provided. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) having a uridine dinucleotide content of less than or equal to about 165% of its minimum uridine dinucleotide content is provided.In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 160% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 155% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being equal to its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 150% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 145% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 140% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 135% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 130% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 125% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 120% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) having a uridine dinucleotide content of less than or equal to about 115% of its minimum uridine dinucleotide content is provided. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) having a uridine dinucleotide content of less than or equal to about 110% of its minimum uridine dinucleotide content is provided.In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 105% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 104% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 103% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 102% of its minimum uridine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the uridine dinucleotide content of the ORF being less than or equal to about 101% of its minimum uridine dinucleotide content.

[0189] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the uridine dinucleotide content of the ORF is within the range of its minimum uridine dinucleotide content to 90% or less than 90% of the maximum uridine dinucleotide content of a reference sequence encoding the same protein as the relevant mRNA. In some embodiments, the uridine dinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum uridine dinucleotide content of the reference sequence encoding the same protein as the relevant mRNA.

[0190] In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, wherein the uridine trinucleotide content of the ORF ranges from 0 uridine trinucleotides to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 uridine trinucleotides (wherein a longer string of uridines is counted as the number of unique triuridine segments therein, e.g., a uridine tetranucleotide contains two uridine trinucleotides, a uridine pentanucleotide contains three uridine trinucleotides, etc.). In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the uridine trinucleotide content of the ORF ranges from 0% uridine trinucleotides to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5% or 2% uridine trinucleotides, wherein the percentage content of uridine trinucleotides is calculated as the percentage of positions in the sequence occupied by uridines that form part of a uridine trinucleotide (or a longer string of uridines) such that the sequences UUUAAA and UUUUAAAA will each have a uridine trinucleotide content of 50%. For example, in some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 2%. For example, in some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 1.5%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 1%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.9%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.8%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.7%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.6%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.5%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.4%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.3%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.2%. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to 0.1%. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the ORF being free of uridine trinucleotides.

[0191] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the uridine trinucleotide content of the ORF is within the range of its minimum uridine trinucleotide content to 90% or less than 90% of the maximum uridine trinucleotide content of a reference sequence encoding the same protein as the relevant mRNA. In some embodiments, the uridine trinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum uridine trinucleotide content of the reference sequence encoding the same protein as the relevant mRNA.

[0192] In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the ORF having very few nucleotide homopolymers, such as strings of repeated identical nucleotides. For example, in some embodiments, when a minimal uridine codon is selected from the codons listed in Table 1, the mRNA is constructed by selecting a minimal uridine codon that reduces the number and length of nucleotide homopolymers (e.g., for alanine, GCA is selected instead of GCC; or for glycine, GGA is selected instead of GGG; or for lysine, AAG is selected instead of AAA).

[0193] The uridine content or uridine dinucleotide content or uridine trinucleotide content of a given ORF can be reduced, for example, by using minimal uridine codons in a sufficient portion of the ORF. For example, the amino acid sequence of the RNA-guided DNA binder can be translated back into the ORF sequence by converting the amino acids into codons, wherein some or all of the ORFs use the exemplary minimal uridine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons in the ORF are the codons listed in Table 1.

[0194] Table 1. Exemplary minimal uridine codons

[0195] Amino Acids Minimal uridine codon A Alanine GCA or GCC or GCG G Glycine GGA or GGC or GGG V Valine GUC or GUA or GUG D Aspartic acid GAC E Glutamate GAA or GAG I Isoleucine AUC or AUA T Threonine ACA or ACC or ACG N Asparagine AAC K Lysine AAG or AAA S Serine AGC R Arginine AGA or AGG L Leucine CUG or CUA or CUC P Proline CCG or CCA or CCC H Histidine CAC Q Glutamine CAG or CAA F Phenylalanine UUC Y Tyrosine UAC C Cysteine UGC W Tryptophan UGG M Methionine AUG

[0196] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) consisting of a set of codons in which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are listed in Table 1 is provided.

[0197] 2. mRNA and ORF with low adenine content

[0198] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine content of the ORF being within the range of its minimum adenine content to about 150% of its minimum adenine content. In some embodiments, the adenine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine content of the ORF being equal to its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine content of the ORF being less than or equal to about 150% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 145% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 140% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 135% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 130% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 125% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) having an adenine content of less than or equal to about 120% of its minimum adenine content is provided. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) having an adenine content of less than or equal to about 115% of its minimum adenine content is provided. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) having an adenine content of less than or equal to about 110% of its minimum adenine content is provided.In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 105% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 104% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 103% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 102% of its minimum adenine content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine content of the ORF is less than or equal to about 101% of its minimum adenine content.

[0199] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being within the range of its minimum adenine dinucleotide content to 200% of its minimum adenine dinucleotide content. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 195%, 190%, 185%, 180%, 175%, 170%, 165%, 160%, 155%, 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being equal to the minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 200% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 195% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 190% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 185% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 180% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 175% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 170% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 165% of its minimum adenine dinucleotide content.In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 160% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 155% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being equal to the minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 150% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 145% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 140% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 135% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 130% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 125% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 120% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 115% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 110% of its minimum adenine dinucleotide content.In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 105% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 104% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 103% of its minimum adenine dinucleotide content. In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the adenine dinucleotide content of the ORF being less than or equal to about 102% of its minimum adenine dinucleotide content. In some embodiments, provided is an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) having an adenine dinucleotide content less than or equal to about 101% of its minimum adenine dinucleotide content.

[0200] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine dinucleotide content of the ORF is within the range of its minimum adenine dinucleotide content to an adenine dinucleotide content that is 90% or less than 90% of the maximum adenine dinucleotide content of a reference sequence encoding the same protein as the relevant mRNA. In some embodiments, the adenine dinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum adenine dinucleotide content of the reference sequence encoding the same protein as the relevant mRNA.

[0201] In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, wherein the adenine trinucleotide content of the ORF ranges from 0 adenine trinucleotides to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 adenine trinucleotides (wherein a longer run of adenines is counted as the number of unique three adenine segments therein, e.g., an adenine tetranucleotide contains two adenine trinucleotides, an adenine pentanucleotide contains three adenine trinucleotides, etc.). In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine trinucleotide content of the ORF is in the range of 0% adenine trinucleotide to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5% or 2% adenine trinucleotide, wherein the percentage content of adenine trinucleotide is calculated as the percentage of positions in the sequence occupied by adenines that form part of an adenine trinucleotide (or a longer string of adenines) such that the sequences UUUAAA and UUUUAAAA will each have an adenine trinucleotide content of 50%. For example, in some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 2%. For example, in some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 1.5%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 1%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.9%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.8%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.7%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.6%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.5%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.4%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.3%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.2%. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to 0.1%. In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the ORF being free of adenine trinucleotides.

[0202] In some embodiments, an mRNA encoding an RNA-guided DNA-binding agent comprising an open reading frame (ORF) is provided, the ORF having very few nucleotide homopolymers, such as strings of repeated identical nucleotides. For example, in some embodiments, when a minimal adenine codon is selected from the codons listed in Table 1, the mRNA is constructed by selecting a minimal adenine codon that reduces the number and length of nucleotide homopolymers (e.g., for alanine, GCA is selected instead of GCC; or for glycine, GGA is selected instead of GGG; or for lysine, AAG is selected instead of AAA).

[0203] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, wherein the adenine trinucleotide content of the ORF is within the range of its minimum adenine trinucleotide content to an adenine trinucleotide content that is 90% or less than 90% of the maximum adenine trinucleotide content of a reference sequence encoding the same protein as the relevant mRNA. In some embodiments, the adenine trinucleotide content of the ORF is less than or equal to about 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the maximum adenine trinucleotide content of the reference sequence encoding the same protein as the relevant mRNA.

[0204] The adenine content or adenine dinucleotide content or adenine trinucleotide content of a given ORF can be reduced, for example, by using minimal adenine codons in a sufficient portion of the ORF. For example, the amino acid sequence of the RNA-guided DNA binder can be translated back into the ORF sequence by converting the amino acids into codons, wherein some or all of the ORFs use the exemplary minimal adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons in the ORF are codons listed in Table 2.

[0205] Table 2. Exemplary minimal adenine codons

[0206]

[0207]

[0208] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) consisting of a set of codons in which at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are listed in Table 2 is provided.

[0209] 3. mRNA and ORF with low adenine and low uridine content

[0210] To the extent feasible, any of the features described above with respect to low adenine content may be combined with any of the features described above with respect to low uridine content. For example, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) can be provided, wherein the uridine content of the ORF is within the range of its minimum uridine content to about 150% of its minimum uridine content (e.g., the uridine content of the ORF is less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum uridine content), and the adenine content of the ORF is within the range of its minimum adenine content to about 150% of its minimum adenine content (e.g., less than or equal to about 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of its minimum adenine content). The same is true for uridine and adenine dinucleotides. Similarly, the content of uridine nucleotides and adenine dinucleotides in ORF can be as described above. Similarly, the content of uridine dinucleotides and adenine nucleotides in ORF can be as described above.

[0211] The uridine and adenine nucleotide and / or dinucleotide content of a given ORF can be reduced, for example, by using minimal uridine and adenine codons in a sufficient portion of the ORF. For example, the amino acid sequence of the RNA-guided DNA binder can be translated back into the ORF sequence by converting the amino acids into codons, wherein some or all of the ORF uses the exemplary minimal uridine and adenine codons shown below. In some embodiments, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons in the ORF are codons listed in Table 3.

[0212] Table 3. Exemplary minimal uridine and adenine codons

[0213]

[0214]

[0215] In some embodiments, an mRNA encoding an RNA-guided DNA binder comprising an open reading frame (ORF) is provided, the ORF consisting of a set of codons wherein at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are codons listed in Table 3. As can be seen in Table 3, each of the three listed serine codons contains one A or one U. In some embodiments, for serine, uridine minimization is performed by prioritizing using AGC codons. In some embodiments, for serine, adenine minimization is performed by prioritizing using UCC and / or UCG codons.

[0216] 4. Codons that improve translation and / or correspond to highly expressed tRNAs; Exemplary codon sets

[0217] In some embodiments, the mRNA comprises an ORF with a codon that increases translation in a mammal, such as a human. In other embodiments, the mRNA comprises an ORF with a codon that increases translation in an organ, such as a liver, of a mammal, such as a human. In other embodiments, the mRNA comprises an ORF with a codon that increases translation in a cell type, such as a hepatocyte, of a mammal, such as a human. The increase in translation in a mammal; in a cell type, an organ of a mammal (human); in a human organ, etc., can be measured relative to the degree of translation of the wild-type sequence of the ORF or relative to the following ORF: the ORF has a codon distribution that matches the codon distribution of the organism from which the ORF is derived or the organism that contains the most similar ORF at a certain amino acid content, such as Streptococcus pyogenes, Staphylococcus aureus (S. aureus), or another prokaryotic organism (when the case may be a Cas nuclease derived from a prokaryotic organism, such as a Cas nuclease from another prokaryotic organism described below). Alternatively, in some embodiments, the increase in translation of a Cas9 sequence in a mammal; a cell type, an organ of a mammal (human); a human organ, etc. is measured relative to the translation of an ORF having SEQ ID NO: 5, while being identical in all other respects (including any applicable point mutations, heterologous domains, and the like). Codons suitable for increasing expression in humans, including human liver and human hepatocytes, can be codons corresponding to tRNAs that are highly expressed in human liver / hepatocytes, as discussed in Dittmar KA, PLos Genetics 2(12):e221(2006). In some embodiments, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the codons in the ORF are codons corresponding to highly expressed tRNAs (e.g., the most highly expressed tRNA for each amino acid) in mammals, such as humans. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the codons in the ORF are codons corresponding to highly expressed tRNAs (e.g., the highest expressed tRNA for each amino acid) in mammalian organs, such as human organs. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the codons in the ORF are codons corresponding to highly expressed tRNAs (e.g., the highest expressed tRNA for each amino acid) in mammalian liver, such as human liver.In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons corresponding to highly expressed tRNAs (e.g., the most highly expressed tRNA for each amino acid) in mammalian hepatocytes, such as human hepatocytes.

[0218] Alternatively, codons corresponding to highly expressed tRNAs in an organism such as humans can generally be used.

[0219] Any of the foregoing codon selection methods can be combined with the minimal uridine and / or adenine codons indicated above, for example, by starting with a codon from Table 1, Table 2, or Table 3, and then, where more than one choice is available, using codons corresponding to more highly expressed tRNAs in a general organism (e.g., humans), or in a relevant organ or cell type, such as the liver or hepatocytes (e.g., human liver or human hepatocytes).

[0220] In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons from the codon groups shown in Table 4 (e.g., low U1, low A, or low A / U codon groups). Codon usage in the low U1, low G, low C, low A, and low A / U groups minimizes the number of nucleotides specified, while also using codons corresponding to highly expressed tRNAs where more than one option is available. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons from the low U1 codon group shown in Table 4. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons from the low A codon group shown in Table 4. In some embodiments, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the ORF are codons from the low A / U codon group shown in Table 4.

[0221] Table 4. Exemplary codon groups.

[0222]

[0223] 5. Encoded RNA-guided DNA binders

[0224] In some embodiments, the RNA-guided DNA binder is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA binder has a lyase activity, which may also be referred to as a double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA binder comprises a Cas nuclease, such as a Class 2 Cas nuclease (which may be, for example, a Type II, Type V, or Type VI Cas nuclease). Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and modified forms thereof. Examples of Cas9 nucleases include those of the Type II CRISPR system of Streptococcus pyogenes, Staphylococcus aureus, and other prokaryotes (see, for example, the list in the next paragraph) and their modified (e.g., engineered or mutant) forms. See, for example, US2016 / 0312198A1; US2016 / 0312199 A1. Other examples of Cas nucleases include Csm or Cmr complexes of type III CRISPR systems or Cas10, Csm1 or Cmr2 subunits thereof; and Cascade complexes of type I CRISPR systems or Cas3 subunits thereof. In some embodiments, the Cas nuclease may be from a type IIA, type IIB or type IIC system. For a discussion of different CRISPR systems and Cas nucleases, see, e.g., Makarova et al. NAT. REV. MICROBIOL. 9: 467-477 (2011); Makarova et al., NAT. REV. MICROBIOL, 13: 722-36 (2015); Shmakov et al., MOLECULAR CELL, 60: 385-397 (2015).

[0225] Non-limiting exemplary species from which Cas nucleases can be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridans viridochromogenes), Streptosporangiumroseum, Alicyclobacillus acidocaldarius, Bacilluspseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii), Cyanothecesp.), Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacilluscaldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp.), Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatori asp.), Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006 and Acaryochloris marina.

[0226] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidococcus genus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae ND2006. In other embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0227] Wild-type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves non-target DNA strands, and the HNH domain cleaves target DNA strands. In some embodiments, the Cas9 nuclease comprises more than one RuvC domain and / or more than one HNH domain. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing double-strand breaks in target DNA. In certain embodiments, the Cas nuclease can cleave dsDNA, it can cleave one strand of dsDNA, or it may not have DNA cleavage enzyme or nickase activity. An exemplary Cas9 amino acid sequence is provided in the form of SEQ ID NO: 3. An exemplary Cas9 mRNA ORF sequence including start and stop codons is provided in the form of SEQ ID NO: 4. An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is provided in the form of SEQ ID NO: 10.

[0228] In some embodiments, chimeric Cas nucleases are used in which one domain or region of the protein is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

[0229] In other embodiments, the Cas nuclease may be from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of a Cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0230] In some embodiments, the RNA-guided DNA binder has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a "nick". In some embodiments, the RNA-guided DNA binder comprises a Cas nickase. A nickase is an enzyme that causes a nick in dsDNA, i.e., cuts one strand but does not cut the other strand of the DNA double helix. In some embodiments, the Cas nickase is a form of a Cas nuclease in which the endonuclease active site is inactivated, such as by one or more changes (e.g., point mutations) in the catalytic domain (e.g., the Cas nucleases discussed above). See, e.g., U.S. Pat. No. 8,889,356 for a discussion of Cas nickases and exemplary catalytic domain changes. In some embodiments, a Cas nickase, such as a Cas9 nickase, has an inactive RuvC or HNH domain. An exemplary Cas9 nickase amino acid sequence is provided in the form of SEQ ID NO: 6. An exemplary Cas9 nickase mRNA ORF sequence including start and stop codons is provided in the form of SEQ ID NO: 7. An exemplary Cas9 nickase mRNA coding sequence suitable for inclusion in a fusion protein is provided as SEQ ID NO:11.

[0231] In some embodiments, the RNA-guided DNA binder is modified to contain only one functional nuclease domain. For example, the binder protein may be modified so that one of the nuclease domains is mutated or completely or partially missing to reduce its nucleic acid cleavage activity. In some embodiments, a nickase with a RuvC domain with reduced activity is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with a HNH domain with reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0232] In some embodiments, the conservative amino acids in the Cas protein nuclease domain are replaced to reduce or change the nuclease activity. In some embodiments, the Cas nuclease may include an amino acid substitution in a RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in RuvC or RuvC-like nuclease domains include D10A (based on Streptococcus pyogenes Cas9 protein). See, for example, Zetsche et al. (2015) Cell Oct 22: 163 (3): 759-771. In some embodiments, the Cas nuclease may include an amino acid substitution in a HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in a HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on Streptococcus pyogenes Cas9 protein). See, for example, Zetsche et al. (2015). Other exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2 (CPF1_FRATN)).

[0233] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs complementary to the sense strand and antisense strand of the target sequence, respectively. In this embodiment, the guide RNA guides the nickase to the target sequence and introduces DSB (i.e., double nicking) by creating a nick on the relative strand of the target sequence. In some embodiments, using double nicking can improve specificity and reduce off-target effects. In some embodiments, nickase is used together with two independent guide RNAs of the relative strands of the target DNA to produce double nicks in the target DNA. In some embodiments, nickase is used together with two independent guide RNAs selected to be very close to produce double nicks in the target DNA.

[0234] In some embodiments, the RNA-guided DNA binder lacks lyase and nickase activity. In some embodiments, the RNA-guided DNA binder comprises a dCas DNA-binding polypeptide. The dCas polypeptide has DNA binding activity, but substantially lacks catalytic (lyase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA binder or dCas DNA-binding polypeptide lacking lyase and nickase activity is in the form of a Cas nuclease in which the endonuclease active site is inactivated, for example, by one or more changes (e.g., point mutations) in the catalytic domain (e.g., the Cas nuclease discussed above). See, e.g., US2014 / 0186958 A1; US2015 / 0166980 A1. An exemplary dCas9 amino acid sequence is provided in the form of SEQ ID NO: 8. An exemplary dCas9mRNA ORF sequence including start and stop codons is provided in the form of SEQ ID NO: 9. An exemplary dCas9mRNA coding sequence suitable for inclusion in a fusion protein is provided in the form of SEQ ID NO: 12.

[0235] 6. Heterologous functional domain; nuclear localization signal

[0236] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (eg, is or comprises a fusion polypeptide).

[0237] In some embodiments, the heterologous functional domain can facilitate the delivery of the RNA-guided DNA binder to the nucleus. For example, the heterologous functional domain can be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA binder can be fused to 1 to 10 NLSs. In some embodiments, the RNA-guided DNA binder can be fused to 1 to 5 NLSs. In some embodiments, the RNA-guided DNA binder can be fused to one NLS. In the case of using one NLS, the NLS can be connected to the N-terminus or C-terminus of the RNA-guided DNA binder sequence. In some embodiments, the RNA-guided DNA binder can be fused to at least one NLS at the C-terminus. The NLS can also be inserted into the RNA-guided DNA binder sequence. In other embodiments, the RNA-guided DNA binder can be fused to more than one NLS. In some embodiments, the RNA-guided DNA binder can be fused to 2, 3, 4 or 5 NLSs. In some embodiments, the RNA-guided DNA binder can be fused to two NLSs. In some cases, the two NLSs can be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binder is fused to two SV40 NLS sequences connected at the carboxyl termini. In some embodiments, the RNA-guided DNA binder may be fused to two NLSs, one NLS connected at the N-terminus and one connected at the C-terminus. In some embodiments, the RNA-guided DNA binder may be fused to three NLSs. In some embodiments, the RNA-guided DNA binder may not be fused to an NLS. In some embodiments, the NLS may be a monopartite sequence, such as the SV40 NLS, PKKKRKV (SEQ ID NO: 78) or PKKKRRV (SEQ ID NO: 90). In some embodiments, the NLS may be a doublet sequence, such as the NLS of the nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 91). In some embodiments, the NLS sequence may comprise LAAKRSRTT (SEQ ID NO:79), QAAKRSRTT (SEQ ID NO:80), PAPAKRERTT (SEQ ID NO:81), QAAKRPRTT (SEQ ID NO:82), RAAKRPRTT (SEQ ID NO:83), AAAKRSWSMAA (SEQ ID NO:84), AAAKRVWSMAF (SEQ ID NO:85), AAAKRSWSMAF (SEQ ID NO:86), AAAKRKYFAA (SEQ ID NO:87), RAAKRKAFAA (SEQ ID NO:88), or RAAKRKYFAV (SEQ ID NO:89).In a specific embodiment, a single PKKKRKV (SEQ ID NO: 78) NLS may be attached to the C-terminus of the RNA-guided DNA binder. One or more linkers are optionally included at the fusion site. In some embodiments, one or more NLSs according to any of the preceding embodiments are present in the RNA-guided DNA binder in combination with one or more additional heterologous functional domains, such as any of the heterologous functional domains described below.

[0238] In some embodiments, the heterologous functional domain may be able to adjust the intracellular half-life of the RNA-guided DNA binder. In some embodiments, the half-life of the RNA-guided DNA binder may be improved. In some embodiments, the half-life of the RNA-guided DNA binder may be reduced. In some embodiments, the heterologous functional domain may be able to increase the stability of the RNA-guided DNA binder. In some embodiments, the heterologous functional domain may be able to reduce the stability of the RNA-guided DNA binder. In some embodiments, the heterologous functional domain may serve as a signal peptide for protein degradation. In some embodiments, protein degradation may be mediated by proteolytic enzymes, such as proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA binder may be modified by adding ubiquitin or polyubiquitin chains. In some embodiments, ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also known as Rub1 in S. cerevisiae), human leukocyte antigen F-related (FAT10), autophagy-8 (ATG8) and autophagy-12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin folding modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0239] In some embodiments, the heterologous functional domain may be a tag domain. Non-limiting examples of tag domains include fluorescent proteins, purification tags, antigenic determinant tags, and reporter gene sequences. In some embodiments, the tag domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), enhanced blue fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midorishi enhanced blue (Midor The marker domain may be a purification tag and / or an antigenic determinant tag.Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly (NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6×His, 8×His, biotin carboxyl carrier protein (BCCP), poly His and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase or fluorescent protein.

[0240] In other embodiments, the heterologous functional domain can target the RNA-guided DNA-binding agent to a specific organelle, cell type, tissue or organ. In some embodiments, the heterologous functional domain can target the RNA-guided DNA-binding agent to mitochondria.

[0241] In other embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA binder is guided to its target sequence, for example, when the Cas nuclease is guided to the target sequence by gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be selected from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, for example, U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or an inhibitor. See, e.g., Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression", Cell 152: 1173-83 (2013); Perez-Pinera et al., "RNA-guided gene activation by CRISPR-Cas9-based transcription factors", Nat. Methods 10: 973-6 (2013); Mali et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering", Nat. Biotechnol. 31: 833-8 (2013); Gilbert et al., "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes", Cell 154: 442-51 (2013). Thus, RNA-guided DNA binders essentially become transcription factors that can be guided using guide RNAs to bind to desired target sequences. In certain embodiments, the DNA modification domain is a methylation domain, such as a demethylation or methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain, such as a base editing domain. In a particular embodiment, the DNA modification domain is a nucleic acid editing domain that introduces specific modifications into the DNA, such as a deaminase domain. See, e.g., WO 2015 / 089406; US 2016 / 0304846. The nucleic acid editing domains, deaminase domains, and Cas9 variants described in WO 2015 / 089406 and US2016 / 0304846 are incorporated herein by reference.

[0242] 7.UTR; Kozak sequence

[0243] In some embodiments, the mRNA comprises at least one UTR from hydroxysteroid 17-β dehydrogenase 4 (HSD17B4 or HSD), such as a 5'UTR from HSD. In some embodiments, the mRNA comprises at least one UTR from a hemoglobin mRNA, such as human α hemoglobin (HBA) mRNA, human β hemoglobin (HBB) mRNA, or clawed frog β hemoglobin (XBG) mRNA. In some embodiments, the mRNA comprises a 5'UTR, a 3'UTR, or a 5' and 3'UTR from a hemoglobin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5'UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3'UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises 5' and 3' UTRs from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, α-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).

[0244] In some embodiments, the mRNA comprises 5' and 3' UTRs from the same source (eg, a constitutively expressed mRNA such as actin, albumin, or hemoglobin (HBA, HBB, or XBG)).

[0245] In some embodiments, the mRNA disclosed herein comprises a 5'UTR having at least 90% identity to any one of SEQ ID NOs: 32, 34, 36, 38, or 41. In some embodiments, the mRNA disclosed herein comprises a 3'UTR having at least 90% identity to any one of SEQ ID NOs: 33, 35, 37, 39, or 40. In some embodiments, any of the foregoing degrees of identity is at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the mRNA disclosed herein comprises a 5'UTR having a sequence of any one of SEQ ID NOs: 32, 34, 36, 38, or 41. In some embodiments, the mRNA disclosed herein comprises a 3'UTR having a sequence of any one of SEQ ID NOs: 33, 35, 37, 39, or 40.

[0246] In some embodiments, the mRNA does not include a 5'UTR, e.g., there are no additional nucleotides between the 5' cap structure and the start codon. In some embodiments, the mRNA includes a Kozak sequence (described below) between the 5' cap structure and the start codon, but does not have any additional 5'UTR. In some embodiments, the mRNA does not include a 3'UTR, e.g., there are no additional nucleotides between the stop codon and the poly-A tail.

[0247] In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect the initiation of translation and the total yield of polypeptides translated by the mRNA. The Kozak sequence includes a methionine codon that can be used as a start codon. The minimum Kozak sequence is NNNRUGN, wherein at least one of the following is true: the first N is A or G and the second N is G. In the nucleotide sequence, R means purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg with zero mismatches or a position in lowercase letters with at most one or two mismatches. In some embodiments, the Kozak sequence is rccAUGg with zero mismatches or a position in lowercase letters with at most one or two mismatches. In some embodiments, the Kozak sequence is gccRccAUGG with zero mismatches or at most one, two, or three mismatches at positions in lower case (nucleotides 4 to 13 of SEQ ID NO: 105). In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or at most one, two, three, or four mismatches at positions in lower case. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG with zero mismatches or at most one, two, three, or four mismatches at positions in lower case (SEQ ID NO: 105).

[0248] 8. Exemplary sequences

[0249] In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binder, wherein the ORF comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175. In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binder, wherein the RNA-guided DNA binder comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 3, 6, 8, 13, 16, 19, 22, 25, 28, 68, or 186-196:, wherein the uridine content of the ORF ranges from its minimum uridine content to 150% of its minimum uridine content, and / or its uridine dinucleotide content ranges from its minimum uridine dinucleotide content to 150% of its minimum uridine dinucleotide content. In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA-binding agent, wherein the RNA-guided DNA-binding agent comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 3, 6, 8, 13, 16, 19, 22, 25, 28, 68, or 186-196:, wherein the adenine content of the ORF is within the range of its minimum adenine content to 150% of the minimum adenine content, and / or its adenine dinucleotide content is within the range of its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content. In some such embodiments, both the adenine content and the uridine nucleotide content are less than or equal to 150% of their respective minimum values. In some embodiments, both the adenine content and the uridine dinucleotide content are less than or equal to 150% of their respective minimum values. In some embodiments, the mRNA comprises a sequence at least 90% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 67, wherein the sequence comprises an ORF encoding an RNA-guided DNA binder. In some embodiments, the mRNA comprises a sequence at least 90% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 67, wherein the sequence comprises an ORF encoding an RNA-guided DNA binder, wherein the first three nucleotides of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 67 are omitted. In some embodiments, any of the foregoing degrees of identity is at least 95%, at least 98%, at least 99%, or 100%.

[0250] In some embodiments, the mRNA comprises an ORF encoding an RNA-guided DNA binder, wherein the ORF is at least 90% identical to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175 over at least the first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides thereof. The first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides are measured from the first nucleotide of the start codon (typically ATG) such that A is the first nucleotide, T is the second nucleotide, etc. In some embodiments, the open reading frame has at least 90% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175 over at least the first 10%, 12%, 15%, 20%, 25%, 30%, or 35% of its sequence. The length of the sequence of an ORF is the number of nucleotides starting from the start codon and ending at the stop codon, and the first 10%, 12%, 15%, 20%, 25%, 30%, or 35% of its sequence corresponds to the number of nucleotides starting from the first nucleotide of the start codon and constituting the specified percentage of the length of the total sequence.

[0251] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:43, optionally wherein the ORF of SEQ ID NO:43 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0252] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:44, optionally wherein the ORF of SEQ ID NO:44 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0253] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:56, optionally wherein the ORF of SEQ ID NO:56 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0254] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:57, optionally wherein the ORF of SEQ ID NO:57 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0255] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:58, optionally wherein the ORF of SEQ ID NO:58 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0256] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:59, optionally wherein the ORF of SEQ ID NO:59 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0257] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:60, optionally wherein the ORF of SEQ ID NO:60 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0258] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO:61, optionally wherein the ORF of SEQ ID NO:61 (i.e., SEQ ID NO:4) is replaced by an ORF replaced by any one of SEQ ID NOs:7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0259] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 176, optionally wherein the ORF of SEQ ID NO: 176 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0260] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 177, optionally wherein the ORF of SEQ ID NO: 177 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0261] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 178, optionally wherein the ORF of SEQ ID NO: 178 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0262] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 179, optionally wherein the ORF of SEQ ID NO: 179 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0263] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 180, optionally wherein the ORF of SEQ ID NO: 180 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0264] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 181, optionally wherein the ORF of SEQ ID NO: 181 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0265] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 182, optionally wherein the ORF of SEQ ID NO: 182 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0266] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 183, optionally wherein the ORF of SEQ ID NO: 183 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175:.

[0267] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 184, optionally wherein the ORF of SEQ ID NO: 184 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0268] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binder comprises a sequence having at least 90% identity to SEQ ID NO: 185, optionally wherein the ORF of SEQ ID NO: 185 is replaced by an ORF replaced by any one of SEQ ID NOs: 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0269] In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43, 44, 56-61 or 176-185 is at least 95%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43, 44, 56-61 or 176-185 is at least 98%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43, 44, 56-61 or 176-185 is at least 99%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO 43, 44, 56-61 or 176-185 is 100%.

[0270] 9. Poly-A tail

[0271] In some embodiments, the mRNA further comprises a poly-A tail. In some cases, the poly-A tail is interrupted by one or more non-adenine nucleotide "anchors" at one or more positions in the poly-A tail. The poly-A tail may comprise at least 8 consecutive adenine nucleotides and also comprise one or more non-adenine nucleotides. As used herein, "non-adenine nucleotides" refers to any natural or non-natural nucleotide that does not comprise adenine. Guanine, thymine and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly-A tail on the mRNA described herein may comprise consecutive adenine nucleotides located 3' to the nucleotide encoding the RNA-guided DNA-binding agent or a related sequence. In some cases, the poly-A tail on the mRNA comprises non-continuous adenine nucleotides located 3' to the nucleotide encoding the RNA-guided DNA-binding agent or a related sequence, wherein the non-adenine nucleotides interrupt the adenine nucleotides at regular or irregular intervals.

[0272] In some embodiments, the poly-A tail is encoded in the plasmid used to transcribe the mRNA in vitro and becomes part of the transcript. The poly-A sequence encoded in the plasmid, i.e., the number of consecutive adenine nucleotides in the poly-A sequence, may not be exact, for example, 100 poly-A sequences in the plasmid may not produce exactly 100 poly-A sequences in the transcribed mRNA. In some embodiments, the poly-A tail is not encoded in the plasmid and is added by PCR tailing or enzymatic tailing, for example, using E. coli poly (A) polymerase.

[0273] In some embodiments, one or more non-adenine nucleotides are positioned to interrupt consecutive adenine nucleotides so that the poly (A) binding protein can bind to a stretch of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8, 9, 10, 11 or 12 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8 to 50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 8 to 100 consecutive adenine nucleotides. In some embodiments, the non-adenine nucleotide is after one, two, three, four, five, six or seven adenine nucleotides and is followed by at least 8 consecutive adenine nucleotides.

[0274] The poly-A tail of the present invention may comprise a sequence of consecutive adenine nucleotides, followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.

[0275] In some embodiments, the poly-A tail comprises or contains a non-adenine nucleotide or a continuous segment of 2 to 10 non-adenine nucleotides. In some embodiments, the non-adenine nucleotide is located after at least 8, 9, 10, 11 or 12 continuous adenine nucleotides. In some cases, one or more non-adenine nucleotides are located after at least 8 to 50 continuous adenine nucleotides. In some embodiments, one or more non-adenine nucleotides are located after at least 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 continuous adenine nucleotides.

[0276] In some embodiments, the non-adenine nucleotide is guanine, cytosine or thymine. In some cases, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some cases where there is more than one non-adenine nucleotide, the non-adenine nucleotide can be selected from: a) guanine and thymine nucleotides; b) guanine and cytosine nucleotides; c) thymine and cytosine nucleotides; or d) guanine, thymine and cytosine nucleotides. An exemplary poly-A tail comprising non-adenine nucleotides is provided in the form of SEQ ID NO: 62.

[0277] 10. Modified Nucleotides

[0278] In some embodiments, the mRNA comprises modified uridines at some or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5 position, for example, with a halogen or C1-C3 alkoxy group. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, for example, with a C1-C3 alkyl group. The modified uridine may be, for example, a pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is a 5-methoxyuridine. In some embodiments, the modified uridine is a 5-iodouridine. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is an N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl-pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0279] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the uridine positions in the mRNA according to the present invention are modified uridines. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the present invention are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methyl pseudouridine, pseudouridine or a combination thereof. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the invention are 5-methoxyuridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the invention are pseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the present invention are N1-methyl pseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the present invention are 5-iodouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the present invention are 5-methoxyuridine, and the rest are N1-methyl pseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95% or 90% to 100% of the uridine positions in the mRNA according to the present invention are 5-iodouridine, and the rest are N1-methyl pseudouridine.

[0280] 11.5' cap structure

[0281] In some embodiments, the mRNA disclosed herein comprises a 5' cap structure, such as Cap0, Cap1, or Cap2. The 5' cap structure is typically a 7-methylguanine ribonucleotide (which may be further modified, as discussed below, for example, with respect to ARCA) attached to the first nucleotide of the 5' to 3' strand of the mRNA via a 5'-triphosphate. In Cap0, the ribose of the first and second cap proximal nucleotides of the mRNA both comprise a 2'-hydroxyl group. In Cap1, the ribose of the first and second transcribed nucleotides of the mRNA comprise a 2'-methoxy group and a 2'-hydroxyl group, respectively. In Cap2, the ribose of the first and second cap proximal nucleotides of the mRNA both comprise a 2'-methoxy group. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs (such as human mRNAs), contain Cap1 or Cap2. Cap0 and other cap structures different from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, which may lead to elevated levels of cytokines, including type I interferons. Components of the innate immune system, such as IFIT-1 and IFIT-5, may also compete with eIF4E for binding to mRNAs with cap structures other than Cap1 or Cap2, which may inhibit translation of the mRNA.

[0282] Cap structures can be included in a co-transcriptional manner. For example, ARCA (anti-reverse cap analog; ThermoFisher Scientific catalog number AM8045) is a cap analog comprising 7-methylguanine 3'-methoxy-5'-triphosphate attached to the 5' position of a guanine ribonucleotide, which can be incorporated into transcripts in vitro at the beginning. ARCA produces a Cap0 cap structure in which the 2' position of the first cap proximal nucleotide is a hydroxyl group. See, for example, Stepinski et al. (2001) "Synthesis and properties of mRNAs containing the novel'anti-reverse'cap analogs 7-methyl(3'-O-methyl)GpppG and7-methyl(3'deoxy)GpppG", RNA 7:1486-1495. The ARCA structure is shown below.

[0283]

[0284] CleanCap TM AG (m7G (5') ppp (5') (2'OMeA) pG; TriLink Biotechnologies Cat. No. N-7113) or CleanCap TM GG (m7G (5') ppp (5') (2'OMeG) pG; TriLink Biotechnologies Cat. No. N-7133) can be used to co-transcriptionally deliver the Cap1 construct. TM AG and CleanCap TM The 3'-O-methylated forms of GG are also available from TriLink Biotechnologies as catalog numbers N-7413 and N-7433, respectively. TM The AG structure is shown below. TM Constructs are sometimes referred to herein using the last three digits of the catalog number listed above (e.g., for TriLink Biotechnologies catalog number N-7113, use "CleanCap TM 113”).

[0285]

[0286] Alternatively, the cap structure can be added to the RNA in a post-transcriptional manner. For example, the vaccinia capping enzyme is commercially available (New England Biolabs catalog number M2080S) and has RNA triphosphatase and guanyl acyltransferase activities provided by its D1 subunit and guanine methyltransferase activity provided by its D12 subunit. Therefore, in the presence of S-adenosylmethionine and GTP, 7-methylguanine can be added to RNA to produce CapO. See, for example, Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479. For further discussion of cap structures and methods of adding cap structures, see, for example, WO 2017 / 053297 and Ishikawa et al., Nucl. Acids. Symp. Ser. (2009) No. 53, 129-130.

[0287] 12. Guide RNA

[0288] In some embodiments, at least one guide RNA is provided in combination with an mRNA disclosed herein. In some embodiments, the guide RNA is provided as a molecule separate from the mRNA. In some embodiments, the guide RNA is provided as a portion of an mRNA disclosed herein, such as a portion of a UTR. In some embodiments, at least one guide RNA targets a TTR.

[0289] In some embodiments, the guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in SEQ ID NO: 74, wherein N is any natural or non-natural nucleotide, and wherein all N' comprise a guide sequence. For example, SEQ ID NO: 74 is encompassed herein, wherein N' is replaced by any one of the guide sequences disclosed herein. Although N' is replaced by the nucleotide of the guide, the modification is still as shown in SEQ ID NO: 74. That is, although the nucleotide of the guide replaces "N'", the first three nucleotides are still modified by 2'OMe, and there is a phosphorothioate bond between the first nucleotide and the second nucleotide, the second nucleotide and the third nucleotide, and the third nucleotide and the fourth nucleotide.

[0290] 13. Lipids; Formulations; Delivery

[0291] In some embodiments, the mRNA described herein, alone or with one or more guide RNAs, is formulated in or administered via lipid nanoparticles; see, for example, PCT / US2017 / 024973, filed on March 30, 2017, claiming priority to USSN 62 / 315,602, filed on March 30, 2016, and entitled "LIPID NANOPARTICLE FORMULATIONS FOR CRISPR / CAS COMPONENTS", the contents of which are incorporated herein by reference in their entirety. The RNA described herein can be administered using any lipid nanoparticle (LNP) known to those skilled in the art that is capable of delivering nucleotides to an individual, in some embodiments, the RNA is accompanied by one or more guide RNAs. In some embodiments, the mRNA described herein, alone or with one or more guide RNAs, is formulated in or administered via liposomes, nanoparticles, extracellular bodies, or microvesicles. Emulsions, micelles and suspensions may be suitable compositions for topical and / or topical delivery.

[0292] Disclosed herein are LNP formulations of RNA, including multiple embodiments of CRISPR / Cas cargo. Such LNP formulations may include (i) CCD lipids, such as amine lipids, (ii) neutral lipids, (iii) helper lipids, and (iv) stealth lipids, such as PEG lipids. Some embodiments of LNP formulations include "amine lipids" as well as helper lipids, neutral lipids, and stealth lipids, such as PEG lipids. "Lipid nanoparticle" means a particle comprising a plurality (i.e., more than one) lipid molecules physically associated with each other by intermolecular forces.

[0293] CCD lipids

[0294] The lipid composition for delivering CRISPR / Cas mRNA and guide RNA components to liver cells comprises CCD lipids.

[0295] In some embodiments, the CCD lipid is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecadienoate, also known as (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecadienoate.

[0296] Lipid A can be depicted as:

[0297]

[0298] Lipid A can be synthesized according to WO2015 / 095340 (eg pages 84-86).

[0299] In some embodiments, the CCD lipid is lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), also known as ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Lipid B can be depicted as:

[0300]

[0301] Lipid B can be synthesized according to WO2014 / 136086 (eg pages 107-109).

[0302] In some embodiments, the CCD lipid is lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadecadienoate).

[0303] Lipid C can be depicted as:

[0304]

[0305] In some embodiments, the CCD lipid is lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecanoate.

[0306] Lipid D can be depicted as:

[0307]

[0308] Lipid C and lipid D can be synthesized according to WO2015 / 095340.

[0309] The CCD lipid may also be an equivalent of lipid A, lipid B, lipid C, or lipid D. In certain embodiments, the CCD lipid is an equivalent of lipid A, an equivalent of lipid B, an equivalent of lipid C, or an equivalent of lipid D.

[0310] Amine lipids

[0311] In some embodiments, LNP compositions for delivery of biologically active agents comprise an "amine lipid," which is defined as lipid A or its equivalent, including acetal analogs of lipid A.

[0312] In some embodiments, the amine lipid is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecadienoate, also known as (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadecadienoate.

[0313] Lipid A can be depicted as:

[0314]

[0315] Lipid A can be synthesized according to WO2015 / 095340 (eg, pages 84-86). In certain embodiments, the amine lipid is an equivalent of lipid A.

[0316] In certain embodiments, the amine lipid is an analog of lipid A. In certain embodiments, the lipid A analog is an acetal analog of lipid A. In certain LNP compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In other embodiments, the acetal analog is a C5-C10 acetal analog. In other embodiments, the acetal analog is selected from C4, C5, C6, C7, C9, C10, C11 and C12 acetal analogs.

[0317] Amine lipids suitable for use in LNPs described herein are biodegradable in vivo. Amine lipids have low toxicity (e.g., tolerated in animal models in an amount greater than or equal to 10 mg / kg without adverse effects). In certain embodiments, LNPs comprising amine lipids include LNPs wherein at least 75% of amine lipids are removed from plasma within 8, 10, 12, 24, or 48 hours or within 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising amine lipids include LNPs wherein at least 50% of mRNA or gRNA are removed from plasma within 8, 10, 12, 24, or 48 hours or within 3, 4, 5, 6, 7, or 10 days. In certain embodiments, LNPs comprising amine lipids include LNPs wherein at least 50% of LNPs are removed from plasma within 8, 10, 12, 24, or 48 hours or within 3, 4, 5, 6, 7, or 10 days, for example, by measuring lipids (e.g., amine lipids), RNA (e.g., mRNA), or other components. In certain embodiments, the lipid encapsulated component versus the free lipid component, RNA component, or nucleic acid component of the LNP is measured.

[0318] Lipid clearance can be measured as described in the literature. See Maier, MA et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21 (8), 1570-78 ("Maier"). For example, in Maier, 0.3 mg / kg was injected intravenously via the lateral tail vein to six to eight week old male C57BL / 6 mice with an LNP-siRNA system containing siRNA targeting luciferase. Blood, liver and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96 and 168 hours after administration. Before collecting tissues, mice were perfused with saline and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. In addition, Maier describes a procedure for assessing the toxicity after administering LNP-siRNA formulations. For example, siRNA targeting luciferase was administered to male Sprague-Dawley rats at 0, 1, 3, 5 and 10 mg / kg (5 animals / group) via a single intravenous rapid injection at a dose volume of 5 mL / kg. After 24 hours, about 1 mL of blood was obtained from the jugular vein of awake animals and serum was separated. 72 hours after administration, all animals were euthanized for autopsy. Clinical symptoms, body weight, serum chemistry, organ weights and histopathology were evaluated. Although Maier describes methods for evaluating siRNA-LNP formulations, these methods can also be applied to evaluate the clearance, pharmacokinetics and toxicity of the administration of the LNP compositions of the present invention.

[0319] Amine lipids cause increased clearance rates. In some embodiments, the clearance rate is a lipid clearance rate, such as the rate at which amine lipids are cleared from blood, serum or plasma. In some embodiments, the clearance rate is an RNA clearance rate, such as the rate at which mRNA or gRNA is cleared from blood, serum or plasma. In some embodiments, the clearance rate is the rate at which LNPs are cleared from blood, serum or plasma. In some embodiments, the clearance rate is the rate at which LNPs are cleared from tissues, such as liver tissue or spleen tissue. In certain embodiments, a high rate of clearance rate can produce a safe distribution without significant adverse effects. Amine lipids reduce LNP accumulation in the circulation and in tissues. In some embodiments, reduced LNP accumulation in the circulation and in tissues can produce a safe distribution without significant adverse effects.

[0320] The amine lipids of the invention may be ionized depending on the pH of the medium in which they are located. For example, in a weakly acidic medium, the amine lipids may be protonated and thus carry a positive charge. Conversely, in a weakly alkaline medium, such as blood, where the pH is about 7.35, the amine lipids may not be protonated and thus carry no charge. In some embodiments, the amine lipids of the invention may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the invention may be protonated at a pH of at least about 10.

[0321] The ability of an amine lipid to carry a charge is related to its intrinsic pKa. For example, the amine lipids of the present invention may each independently have a pKa in the range of about 5.8 to about 6.2. For example, the amine lipids of the present invention may each independently have a pKa in the range of about 5.8 to about 6.5. When it has been found that cationic lipids with a pKa in the range of about 5.1 to about 7.4 are effective for delivering a load in vivo to, for example, the liver, this may be advantageous. In addition, it has been found that cationic lipids with a pKa in the range of about 5.3 to about 6.4 are effective for delivering in vivo to, for example, a tumor. See, for example, WO2014 / 136086.

[0322] Other lipids

[0323] "Neutral lipids" suitable for use in the lipid compositions of the present invention include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present invention include, but are not limited to, 5-heptadecaylbenzene-1,3-diol (resorcinol), disalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphorylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoylphosphatidylcholine (PE), ...phosphatidylcholine (PMPC), 1-palmitoylphosphatidylcholine (PMPC), 1-palmitoylphosphatidylcholine (PMPC), 1-palmitoylphosphatidylcholine (PMPC), 1-palmito In one embodiment, the neutral phospholipids may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0324] "Helper lipids" include steroids, sterols and alkylresorcinols. Helper lipids suitable for use in the present invention include, but are not limited to, cholesterol, 5-heptadecaylresorcinol and cholesterol hemisuccinate. In one embodiment, the helper lipid may be cholesterol. In one embodiment, the helper lipid may be cholesterol hemisuccinate.

[0325] "Stealth lipids" are lipids that change the length of time that nanoparticles can exist in vivo (e.g., in the blood). Stealth lipids can assist the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein can adjust the pharmacokinetic properties of LNPs. Stealth lipids suitable for use in the present invention include, but are not limited to, stealth lipids having a hydrophilic head group connected to a lipid portion. Stealth lipids suitable for use in lipid compositions of the present invention and information on the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Additional suitable PEG lipids are disclosed in, for example, WO 2006 / 007712.

[0326] In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from a PEG-based polymer. The stealth lipid may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

[0327] In one embodiment, the stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly[N-(2-hydroxypropyl)methacrylamide].

[0328] In one embodiment, the PEG lipid comprises a PEG (sometimes referred to as poly(ethylene oxide)) based polymer portion.

[0329] PEG lipids further comprise a lipid moiety. In some embodiments, the lipid moiety may be derived from diacylglycerol or diacylglycerol amide, including those comprising dialkyl glyceryl or dialkyl glyceryl amide, the group having an alkyl chain length independently comprising about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups, such as amide or ester groups. In some embodiments, the alkyl chain length comprises about C10 to C20. Dialkyl glycerol or dialkyl glyceryl amide may further comprise one or more substituted alkyl groups. The chain length may be symmetrical or asymmetrical.

[0330] Unless otherwise indicated, as used herein, the term "PEG" means any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG is an optionally substituted straight or branched chain polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups. In one embodiment, the term includes PEG copolymers, such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the molecular weight of PEG is from about 130 to about 50,000, in one subembodiment, from about 150 to about 30,000, in one subembodiment, from about 150 to about 20,000, in one subembodiment, from about 150 to about 15,000, in one subembodiment, from about 150 to about 10,000, in one subembodiment, from about 150 to about 6,000, in one subembodiment, from about 150 to about 5,000, in one subembodiment, from about 150 to about 4,000, in one subembodiment, from about 150 to about 3,000, in one subembodiment, from about 300 to about 3,000, in one subembodiment, from about 1,000 to about 3,000 and in one subembodiment, from about 1,500 to about 2,500.

[0331] In certain embodiments, PEG (e.g., in combination with a lipid moiety or lipid, such as a stealth lipid), is "PEG-2K," also known as "PEG2000," which has an average molecular weight of about 2,000 Daltons. PEG-2K is represented herein by the following formula (I), wherein n is 45, meaning that the number-averaged degree of polymerization comprises about 45 subunits. However, other PEG embodiments known in the art may also be used, including, for example, those in which the number-averaged degree of polymerization comprises about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n may be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

[0332] In any of the embodiments described herein, the PEG lipid can be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (Catalog No. GM-020, from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (Catalog No. DSPE-020CN, NOF, Tokyo, Japan), PEG-dilauroylglyceramide, PEG-dimyristoylglyceramide, PEG-dipalmitoylglyceramide, and PEG-distearoylglyceramide, PEG-cholesterol (1-[8' -(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctane]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (Cat. No. 880150P, Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (Cat. No. 880120C, Avanti Polar Lipids, Alabaster, Alabama, USA) Lipids), 1,2-distearoyl-sn-glycerol, methoxy polyethylene glycol (PEG2k-DSG; GS-020, NOF, Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, which is disclosed in WO2016 / 010840 (paragraphs

[00240] to

[00244] ). In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.

[0333] LNPs may contain (i) amine lipids for encapsulation and for endosomal escape, (ii) neutral lipids for stabilization, (iii) helper lipids also for stabilization, and (iv) stealth lipids, such as PEG lipids.

[0334] In some embodiments, the LNP composition may include an RNA component including one or more of the following: an RNA-guided DNA binder, a Cas nuclease mRNA, a class 2 Cas nuclease mRNA, a Cas9 mRNA, and a gRNA. In some embodiments, the LNP composition may include class 2 Cas nucleases and gRNA as RNA components. In certain embodiments, the LNP composition may include an RNA component, an amine lipid, an auxiliary lipid, a neutral lipid, and a stealth lipid. In certain LNP compositions, the auxiliary lipid is cholesterol. In other compositions, the neutral lipid is DSPC. In other embodiments, the stealth lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the LNP composition comprises lipid A or an equivalent of lipid A; an auxiliary lipid; a neutral lipid; a stealth lipid; and a guide RNA. In certain compositions, the amine lipid is lipid A. In certain compositions, the amine lipid is lipid A or an acetal analog thereof; the auxiliary lipid is cholesterol; the neutral lipid is DSPC; and the stealth lipid is PEG2k-DMG.

[0335] In certain embodiments, lipid composition is described according to the corresponding mol ratio of lipid component in formulation.Embodiments of the present invention provide lipid composition described according to the corresponding mol ratio of lipid component in formulation.In one embodiment, the mol% of amine lipid can be about 30mol% to about 60mol%.In one embodiment, the mol% of amine lipid can be about 40mol% to about 60mol%.In one embodiment, the mol% of amine lipid can be about 45mol% to about 60mol%.In one embodiment, the mol% of amine lipid can be about 50mol% to about 60mol%.In one embodiment, the mol% of amine lipid can be about 55mol% to about 60mol%.In one embodiment, the mol% of amine lipid can be about 50mol% to about 55mol%.In one embodiment, the mol% of amine lipid can be about 50mol%.In one embodiment, the mol% of amine lipid can be about 50mol%.In one embodiment, the mol% of amine lipid can be about 55mol%.In some embodiments, the mol% of LNP batch material amine lipid mol% will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of target mol%. In some embodiments, the amine lipid mol% of the LNP batch will be ±4mol%, ±3mol%, ±2mol%, ±1.5mol%, ±1mol%, ±0.5mol% or ±0.25mol% of the target mol%. All mol% numbers are given by a certain fraction of the lipid component of the LNP composition. In certain embodiments, the LNP batch-to-batch variability of the amine lipid mol% will be less than 15%, less than 10% or less than 5%.

[0336] In one embodiment, the mol% of neutral lipids may be about 5mol% to about 15mol%. In one embodiment, the mol% of neutral lipids may be about 7mol% to about 12mol%. In one embodiment, the mol% of neutral lipids may be about 9mol%. In some embodiments, the neutral lipid mol% of LNP batches will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target neutral lipid mol%. In certain embodiments, the LNP batch-to-batch variability will be less than 15%, less than 10% or less than 5%.

[0337] In one embodiment, the mol% of the auxiliary lipid may be about 20mol% to about 60mol%. In one embodiment, the mol% of the auxiliary lipid may be about 25mol% to about 55mol%. In one embodiment, the mol% of the auxiliary lipid may be about 25mol% to about 50mol%. In one embodiment, the mol% of the auxiliary lipid may be about 25mol% to about 40mol%. In one embodiment, the mol% of the auxiliary lipid may be about 30mol% to about 50mol%. In one embodiment, the mol% of the auxiliary lipid may be about 30mol% to about 40mol%. In one embodiment, the mol% of the auxiliary lipid is adjusted based on the concentration of amine lipids, neutral lipids and PEG lipids to make the lipid component reach 100mol%. In some embodiments, the mol% of the auxiliary lipid of the LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol%. In certain embodiments, the LNP batch-to-batch variation rate will be less than 15%, less than 10% or less than 5%.

[0338] In one embodiment, the mol% of PEG lipids may be from about 1mol% to about 10mol%. In one embodiment, the mol% of PEG lipids may be from about 2mol% to about 10mol%. In one embodiment, the mol% of PEG lipids may be from about 2mol% to about 8mol%. In one embodiment, the mol% of PEG lipids may be from about 2mol% to about 4mol%. In one embodiment, the mol% of PEG lipids may be from about 2.5mol% to about 4mol%. In one embodiment, the mol% of PEG lipids may be about 3mol%. In one embodiment, the mol% of PEG lipids may be about 2.5mol%. In some embodiments, the mol% of PEG lipids of LNP batches will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target PEG lipid mol%. In certain embodiments, the LNP batch-to-batch variation rate will be less than 15%, less than 10% or less than 5%.

[0339] In certain embodiments, the load includes mRNA encoding a DNA binder guided by RNA (e.g., Cas nuclease, 2 types of Cas nuclease or Cas9), and gRNA or nucleic acid encoding gRNA, or a combination of mRNA and gRNA. In one embodiment, the LNP composition may include lipid A or its equivalent. In some aspects, the amine lipid is lipid A. In some aspects, the amine lipid is a lipid A equivalent, such as an analog of lipid A. In some aspects, the amine lipid is an acetal analog of lipid A. In various embodiments, the LNP composition includes amine lipids, neutral lipids, auxiliary lipids and PEG lipids. In certain embodiments, the auxiliary lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In a particular embodiment, the PEG lipid is PEG2k-DMG. In some embodiments, the LNP composition may include lipid A, auxiliary lipids, neutral lipids and PEG lipids. In some embodiments, the LNP composition includes amine lipids, DSPC, cholesterol and PEG lipids. In some embodiments, the LNP composition includes PEG lipids, and the lipid includes DMG. In certain embodiments, the amine lipid is selected from lipid A, and an equivalent of lipid A, including an acetal analog of lipid A. In other embodiments, the LNP composition comprises lipid A, cholesterol, DSPC, and PEG2k-DMG.

[0340] Embodiments of the present invention also provide lipid compositions described according to the molar ratio between the positively charged amine groups (N) and the negatively charged phosphate groups (P) of the amine lipids of the nucleic acid to be encapsulated. This can be mathematically represented by the equation N / P. In some embodiments, the LNP composition may include a lipid component comprising an amine lipid, an auxiliary lipid, a neutral lipid, and a stealth lipid; and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP composition may include a lipid component comprising an amine lipid, an auxiliary lipid, a neutral lipid, and a stealth lipid; and an RNA component, wherein the N / P ratio is about 3 to 10. In one embodiment, the N / P ratio may be about 5 to 7. In one embodiment, the N / P ratio may be about 4.5 to 8. In one embodiment, the N / P ratio may be about 6. In one embodiment, the N / P ratio may be 6±1. In one embodiment, the N / P ratio may be 6±0.5. In some embodiments, the N / P ratio will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N / P ratio. In certain embodiments, the LNP batch-to-batch variability will be less than 15%, less than 10%, or less than 5%.

[0341] In some embodiments, the RNA component may include mRNA, such as mRNA disclosed herein, e.g., mRNA encoding a Cas nuclease. In one embodiment, the RNA component may include Cas9 mRNA. In some compositions comprising mRNA encoding a Cas nuclease, the LNP further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the RNA component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the RNA component comprises 2 types of Cas nuclease mRNAs and gRNAs.

[0342] In certain embodiments, the LNP composition may include mRNA encoding Cas nucleases (such as Class 2 Cas nucleases), amine lipids, auxiliary lipids, neutral lipids, and PEG lipids. In some LNP compositions comprising mRNA encoding Cas nucleases (such as Class 2 Cas nucleases), the auxiliary lipid is cholesterol. In other compositions comprising mRNA encoding Cas nucleases (such as Class 2 Cas nucleases), the neutral lipid is DSPC. In other embodiments comprising mRNA encoding Cas nucleases (such as Class 2 Cas nucleases), the PEG lipid is PEG2k-DMG or PEG2k-C11. In a specific composition comprising mRNA encoding Cas nucleases (such as Class 2 Cas nucleases), the amine lipid is selected from lipid A and its equivalents, such as acetal analogs of lipid A.

[0343] In some embodiments, the LNP composition may include gRNA. In certain embodiments, the LNP composition may include amine lipids, gRNA, helper lipids, neutral lipids and PEG lipids. In some LNP compositions comprising gRNA, the helper lipid is cholesterol. In some compositions comprising gRNA, the neutral lipid is DSPC. In other embodiments comprising gRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalent, such as acetal analogs of lipid A.

[0344] In one embodiment, the LNP composition may include sgRNA. In one embodiment, the LNP composition may include Cas9 sgRNA. In one embodiment, the LNP composition may include Cpf1 sgRNA. In some compositions comprising sgRNA, the LNP includes amine lipids, auxiliary lipids, neutral lipids and PEG lipids. In some compositions comprising sgRNA, the auxiliary lipid is cholesterol. In other compositions comprising sgRNA, the neutral lipid is DSPC. In other embodiments comprising sgRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalents, such as acetal analogs of lipid A.

[0345] In certain embodiments, the LNP composition comprises mRNA disclosed herein, such as mRNA encoding Cas nuclease, and gRNA, which may be sgRNA. In one embodiment, the LNP composition may comprise amine lipids, mRNA encoding Cas nuclease, gRNA, auxiliary lipids, neutral lipids and PEG lipids. In some compositions comprising mRNA and gRNA encoding Cas nuclease, the auxiliary lipid is cholesterol. In some compositions comprising mRNA and gRNA encoding Cas nuclease, the neutral lipid is DSPC. In other embodiments comprising mRNA and gRNA encoding Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalents, such as acetal analogs of lipid A.

[0346] In certain embodiments, the LNP composition includes Cas nuclease mRNA, such as 2 types of Cas mRNA, and at least one gRNA. In certain embodiments, the LNP composition includes a ratio of about 25: 1 to about 1: 25 gRNA and Cas nuclease mRNA, such as 2 types of Cas nuclease mRNA. In certain embodiments, the LNP formulation includes a ratio of about 10: 1 to about 1: 10 gRNA and Cas nuclease mRNA, such as 2 types of Cas nuclease mRNA. In certain embodiments, the LNP formulation includes a ratio of about 8: 1 to about 1: 8 gRNA and Cas nuclease mRNA, such as 2 types of Cas nuclease mRNA. As measured herein, the ratio is by weight. In some embodiments, the LNP formulation includes a ratio of about 5: 1 to about 1: 5 gRNA and Cas nuclease mRNA, such as 2 types of Cas mRNA. In some embodiments, the ratio ranges from about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments, the ratio of gRNA to mRNA is about 3:1 or about 2:1. In some embodiments, the ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease, is about 1:1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10, or 1:25.

[0347] The LNP compositions disclosed herein may include template nucleic acids. The template nucleic acids may be co-deployed with mRNA encoding Cas nucleases, such as class 2 Cas nuclease mRNAs. In some embodiments, the template nucleic acids may be co-deployed with guide RNAs. In some embodiments, the template nucleic acids may be co-deployed with both mRNA encoding Cas nucleases and guide RNAs. In some embodiments, the template nucleic acids may be separately deployed with mRNA encoding Cas nucleases and guide RNAs. The template nucleic acids may be delivered together with the LNP compositions or separately therefrom. In some embodiments, the template nucleic acids may be single-stranded or double-stranded, depending on the desired repair mechanism. The template may have a region homologous to the target DNA or to a sequence adjacent to the target DNA.

[0348] Any of the LNPs and LNP formulations described herein are suitable for delivering mRNA encoding RNA-guided DNA binders, such as Cas nucleases, alone or together with one or more guide RNAs. In some embodiments, a LNP composition comprising: an RNA component and a lipid component, wherein the lipid component comprises an amine lipid, a neutral lipid, a helper lipid, and a stealth lipid; and wherein the N / P ratio is about 1 to 10 is contemplated.

[0349] In some cases, the lipid component comprises lipid A or an acetal analog thereof, cholesterol, DSPC, and PEG-DMG; and wherein the N / P ratio is about 1 to 10. In some embodiments, the lipid component comprises: about 40 to 60 mol% amine lipids; about 5 to 15 mol% neutral lipids; and about 1.5% to 10 mol% PEG lipids, wherein the remainder of the lipid component is a helper lipid, and wherein the N / P ratio of the LNP composition is about 3 to 10. In some embodiments, the lipid component comprises: about 50 to 60 mol% amine lipids; about 8 to 10 mol% neutral lipids; and about 2.5% to 4 mol% PEG lipids, wherein the remainder of the lipid component is a helper lipid, and wherein the N / P ratio of the LNP composition is about 3 to 8. In some cases, the lipid component comprises: about 50 to 60 mol% amine lipids; about 5 to 15 mol% DSPC; and about 2.5% to 4 mol% PEG lipids, wherein the remainder of the lipid component is cholesterol, and wherein the N / P ratio of the LNP composition is about 3 to 8. In some cases, the lipid component comprises: 48 to 53 mol% lipid A; about 8 to 10 mol% DSPC; and about 1.5% to 10 mol% PEG lipid, wherein the remainder of the lipid component is cholesterol, and wherein the N / P ratio of the LNP composition is 3 to 8±0.2.

[0350] In some embodiments, LNP is formed by mixing RNA aqueous solution with organic solvent-based lipid solution, such as 100% ethanol. Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. Pharmaceutically acceptable buffers can be used for in vivo administration of LNPs, for example. In certain embodiments, the buffer is used to maintain the pH of the composition comprising LNP at or above pH 6.5. In certain embodiments, the buffer is used to maintain the pH of the composition comprising LNP at or above pH 7.0. In certain embodiments, the pH of the composition is in the range of about 7.2 to about 7.7. In other embodiments, the pH of the composition is in the range of about 7.3 to about 7.7 or in the range of about 7.4 to about 7.6. In other embodiments, the pH of the composition is about 7.2, 7.3, 7.4, 7.5, 7.6 or 7.7. The pH of the composition can be measured with a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectants, such as sucrose. In certain embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% cryoprotectants. In certain embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may include a phosphate buffer (PBS), a Tris buffer, a citrate buffer and a mixture thereof. In certain exemplary embodiments, the buffer includes NaCl. In certain embodiments, NaCl is omitted. An exemplary amount of NaCl may be in the range of about 20mM to about 45mM. An exemplary amount of NaCl may be in the range of about 40mM to about 50mM. In some embodiments, the amount of NaCl is about 45mM. In some embodiments, the buffer is a Tris buffer. An exemplary amount of Tris may be in the range of about 20mM to about 60mM. An exemplary amount of Tris may be in the range of about 40mM to about 60mM. In some embodiments, the amount of Tris is about 50mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of LNP compositions contain a Tris buffer of 5% sucrose and 45mM NaCl. In other exemplary embodiments, the composition contains sucrose in an amount of about 5% w / v, about 45mM NaCl, and about 50mM Tris at pH 7.5. The amount of salt, buffer, and cryoprotectant may vary to maintain the weight molar osmotic concentration of the overall formulation. For example, the final weight molar osmotic concentration may be maintained below 450mOsm / L.In other embodiments, the osmolarity is between 350 and 250 mOsm / L. Certain embodiments have a final osmolarity of 300 + / - 20 mOsm / L.

[0351] In some embodiments, microfluidic mixing, T-mixing or staggered mixing is used. In some aspects, flow rate, joint size, joint geometry, joint shape, caliber, solution and / or RNA and lipid concentrations may vary. LNP or LNP composition can be concentrated or purified, for example, via dialysis, tangential flow filtration or chromatography. LNP can be stored in the form of, for example, a suspension, an emulsion or a lyophilized powder. In some embodiments, the LNP composition is stored at 2 to 8°C, and in some aspects, the LNP composition is stored at room temperature. In other embodiments, frozen storage, for example, storage of the LNP composition at -20°C or -80°C. In other embodiments, the LNP composition is stored at a temperature ranging from about 0°C to about -80°C. The frozen LNP composition can be melted, for example, on ice, at 4°C, at room temperature or at 25°C before use. The frozen LNP composition can be maintained at different temperatures, for example, on ice, at 4°C, at room temperature, at 25°C or at 37°C.

[0352] In some embodiments, the LNP composition has an encapsulation rate greater than about 80%. In some embodiments, the particle size of the LNP composition is less than about 120 nm. In some embodiments, the pdi of the LNP composition is less than about 0.2. In some embodiments, at least two of these characteristics are present. In some embodiments, each of these three characteristics is present. Analytical methods for determining these parameters are discussed in the General Reagents and Methods section below.

[0353] In some embodiments, LNPs associated with mRNA disclosed herein are used to prepare a medicament.

[0354] Electroporation is a well-known means of delivering a payload, and any electroporation method can be used to deliver any of the gRNAs disclosed herein. In some embodiments, electroporation can be used to deliver an mRNA and one or more guide RNAs disclosed herein.

[0355] In some embodiments, a method for delivering the mRNA disclosed herein to an ex vivo cell is provided, wherein the mRNA is associated with or not associated with LNP. In some embodiments, the mRNA / LNP or mRNA is also associated with one or more guide RNAs.

[0356] In some embodiments, when the mRNA disclosed herein is administered to a mammal in the form of a pharmaceutical composition, the mammal exhibits a cytokine response that is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times lower than that of a mammal administered an mRNA encoding a Cas9 nuclease having a minimum uridine content greater than 150%. The cytokine response can be determined as described in the examples. The difference between the cytokine responses can be measured in the form of an average change in a group of cytokines, such as at least one, two, three, or four of the following cytokines: IFNα, IL-6, TNFα, and MCP-1. In some embodiments, when an mRNA disclosed herein is administered to a mammal in the form of a pharmaceutical composition, the mammal exhibits a cytokine response that is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times lower than that of a mammal administered an mRNA having an ORF encoding a Cas9 nuclease, wherein the sequence of the ORF consists of SEQ ID NO: 5. In some embodiments, the uridine in the ORF having a sequence consisting of SEQ ID NO: 5 is unmodified. It is generally understood that the characteristics of the comparative composition other than the mRNA should remain constant, including the dosage, and the dosage should be in an appropriate range, such as 0.1 to 5 mpk or other ranges described herein (e.g., as discussed in the determination of mRNA efficacy section).

[0357] In some embodiments, the nucleotide sequence encoding the guide RNA may be located on the same vector, transcript, or mRNA comprising the nucleotide sequence encoding the RNA-guided DNA binder. In some embodiments, the expression of the guide RNA and the expression of the RNA-guided DNA binder may be driven by their own corresponding promoters. In some embodiments, the expression of the guide RNA may be driven by the same promoter that drives the expression of the RNA-guided DNA binder. In some embodiments, the guide RNA and the ORF encoding the RNA-guided DNA binder may be contained in a single transcript. For example, the guide RNA may be in the untranslated region (UTR) of the RNA-guided DNA binder transcript. In some embodiments, the guide RNA may be in the 5'UTR of the RNA-guided DNA binder transcript. In some embodiments, the guide RNA may be in the 3'UTR of the RNA-guided DNA binder transcript. In some embodiments, the intracellular half-life of the RNA-guided DNA binder transcript may be reduced by including the guide RNA in the 3'UTR of the transcript and thereby shortening the length of its 3'UTR. In other embodiments, the guide RNA may be in an intron of the RNA-guided DNA binder transcript. In some embodiments, a suitable splice site can be added at the intron where the guide RNA is located so that the guide RNA is properly spliced ​​out from the transcript. In some embodiments, the expression of the RNA-guided DNA binder and the guide RNA in close proximity on the same vector can promote more efficient formation of the ribonucleoprotein complex of the RNA-guided DNA binder and the guide RNA.

[0358] In some embodiments, a pharmaceutical formulation comprising the mRNA of the present invention is provided. In some embodiments, a pharmaceutical formulation comprising at least one lipid, such as a LNP comprising the mRNA of the present invention is provided. Any LNP suitable for delivering RNA, such as the LNP described above, can be used; other exemplary LNPs are described in PCT / US2017 / 024973 filed on March 30, 2017. The pharmaceutical formulation may further include a pharmaceutically acceptable carrier, such as water or a buffer. The pharmaceutical formulation may further include one or more pharmaceutically acceptable excipients, such as stabilizers, preservatives, bulking agents, or the like. The pharmaceutical formulation may further include one or more pharmaceutically acceptable salts, such as sodium chloride. In some embodiments, the pharmaceutical formulation is formulated for intravenous administration. In some embodiments, the pharmaceutical formulation is formulated for delivery to the liver circulation.

[0359] C. Determination of mRNA efficacy

[0360] In some embodiments, the efficacy of the mRNA is determined when expressed with other components of the RNP, e.g., at least one gRNA, such as a gRNA targeting TTR.

[0361] DNA binders guided by RNA with lyase activity can cause double-strand breaks in DNA. Non-homologous end joining (NHEJ) is a method by which double-strand breaks (DSBs) in DNA are repaired via reconnection of the broken ends, which may produce errors in the form of insertion / deletion (indel) mutations. Before reconnecting the ends, the DNA ends of DSBs often undergo enzyme treatment, which causes the addition or removal of nucleotides at one or two strands. Such additions or removals before reconnection cause insertion or deletion (indel) mutations at the NHEJ repair site in the DNA sequence. A variety of mutations due to indels change the reading frame or introduce stop codons prematurely, and thus produce non-functional proteins.

[0362] In some embodiments, the efficacy of the mRNA encoding the nuclease is determined based on an in vitro model. In some embodiments, the in vitro model is HEK293 cells. In some embodiments, the in vitro model is HUH7 human hepatoma cells. In some embodiments, the in vitro model is primary hepatocytes, such as primary human or mouse hepatocytes.

[0363] In some embodiments, the efficacy of the RNA is measured by the percent editing of TTR. An exemplary procedure for determining the percent editing is given in the following examples. In some embodiments, the percent editing of TTR is compared to the percent editing obtained when the mRNA comprises an ORF of SEQ ID NO: 5 with unmodified uridine and all others are identical.

[0364] In some embodiments, the efficacy of the mRNA is determined using serum TTR concentration in mice after administration of LNPs comprising mRNA and a gRNA targeting TTR, e.g., SEQ ID NO: 42. In some embodiments, the efficacy of the mRNA is determined using serum TTR concentration in rats after administration of LNPs comprising mRNA and a gRNA targeting TTR, e.g., SEQ ID NO: 69. The serum TTR concentration can be expressed in absolute terms or as a % gene reduction relative to a sham-treated control. In some embodiments, the efficacy of the mRNA is determined using the percentage of editing in the liver in mice after administration of LNPs comprising mRNA and a gRNA targeting TTR, e.g., SEQ ID NO: 42. In some embodiments, the effective amount is capable of achieving at least 50% editing or 50% gene reduction of serum TTR. Exemplary effective amounts range from 0.1 to 10 mg / kg (mpk), e.g., 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk, or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, or 10 mpk.

[0365] In some embodiments, detection of gene editing events, such as the formation of insertion / deletion ("indel") mutations and homology-directed repair (HDR) events in target DNA utilizes linear amplification (hereinafter referred to as "LAM-PCR" or "linear amplification (LA)" method) accompanied by tagged primers and separation of tagged amplification products.

[0366] In some embodiments, the method comprises isolating cellular DNA from a cell that has been induced to have a double-strand break (DSB) and, optionally, has an HDR template to repair the DSB; performing at least one round of linear amplification of the DNA with a tagged primer; isolating the linear amplification product comprising the tag, thereby discarding any amplification product amplified with a non-tagged primer; further amplifying the separated product as appropriate; and analyzing the linear amplification product or the further amplified product to determine whether there is an editing event in the target DNA, such as a double-strand break, an insertion, a deletion, or an HDR template sequence. In some cases, the editing event can be quantified. As used herein (including in the context of HDR and non-HDR editing events, such as insertion-deletion markers (indels)), quantification and the like include detecting the number and / or type of editing events in a population.

[0367] In some embodiments, only one round of linear amplification is performed.

[0368] In some cases, the tagged primers comprise a molecular barcode. In some embodiments, the tagged primers comprise a molecular barcode and only one round of linear amplification is performed.

[0369] In some embodiments, the analyzing step comprises sequencing the linear amplification product or the further amplified product. Sequencing may comprise any method known to those skilled in the art, including next generations sequencing and cloning the linear amplification product or the further amplified product in a plasmid and sequencing the plasmid or a portion of the plasmid. In other aspects, the analyzing step comprises performing digital PCR (dPCR) or droplet digital PCR (ddPCR) on the linear amplification product or the further amplified product. In other cases, the analyzing step comprises contacting the linear amplification product or the further amplified product with a nucleic acid probe designed to identify DNA comprising an HDR template sequence, and detecting the probe that has been bound to the linear amplification product or the further amplified product. In some embodiments, the method further comprises determining the position of the HDR template in the target DNA.

[0370] In certain embodiments, the method further comprises determining the sequence of an insertion site in the target DNA, wherein the insertion site is the position at which the HDR template is incorporated into the target DNA, and wherein the insertion site may include a portion of the target DNA sequence and a portion of the HDR template sequence.

[0371] In some embodiments, linear amplification of target DNA using tagged primers is performed for 1 to 50 rounds, 1 to 60 rounds, 1 to 70 rounds, 1 to 80 rounds, 1 to 90 rounds, or 1 to 100 rounds.

[0372] In some embodiments, linear amplification of target DNA using tagged primers comprises a denaturation step to separate the DNA duplex, an annealing step to allow primers to bind, and an extension step. In some embodiments, the linear amplification is isothermal (no temperature change is required). In some embodiments, the isothermal linear amplification is a loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification, or a nickase amplification reaction.

[0373] In some embodiments, the tagged primer is ligated to a target DNA that is at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, at least 300, at least 1,000, at least 5,000, or at least 10,000 nucleotides away from the location of a desired editing event, e.g., an insertion, deletion, or template insertion site.

[0374] In some embodiments, the tagged primer comprises a molecular barcode. In some embodiments, the molecular barcode comprises a sequence that is not complementary to the target DNA. In some embodiments, the molecular barcode comprises 6, 8, 10, or 12 nucleotides.

[0375] In some embodiments, the label on the primer is biotin, streptavidin, digoxigenin, a DNA sequence, or fluorescent isothiocyanate (FITC).

[0376] In some embodiments, the linear amplification product is separated using a capture reagent that is specific to the label on the primer. In some embodiments, the capture reagent is on a bead, a solid support, a matrix, or a column. In some embodiments, the separation step comprises contacting the linear amplification product with a capture reagent that is specific to the label on the primer. In some embodiments, the capture reagent is biotin, streptavidin, digoxigenin, a DNA sequence, or fluorescent isothiocyanate (FITC).

[0377] In some embodiments, the label is biotin and the capture reagent is streptavidin. In some embodiments, the label is streptavidin and the capture reagent is biotin. In some embodiments, the label is at the 5' end of the primer, at the 3' end of the primer, or inside the primer. In some embodiments, the label and / or capture reagent are removed after the separation step. In some embodiments, the label and / or capture reagent are not removed, and further amplification and analysis steps are performed in the presence of the label and / or capture reagent.

[0378] In some embodiments, the further amplification is non-linear. In some embodiments, the further amplification is digital PCR, qPCR or RT-PCR. In some embodiments, the sequencing is next generation sequencing (NGS).

[0379] In some embodiments, the target DNA is genomic or mitochondrial. In some embodiments, the target DNA is genomic DNA of prokaryotic or eukaryotic cells. In some embodiments, the target DNA is mammalian DNA. The target DNA may be from a non-dividing cell or a dividing cell. In some embodiments, the target DNA may be from a primary cell. In some embodiments, the target DNA is from a replicating cell.

[0380] In some cases, the cellular DNA is sheared prior to linear amplification. In some embodiments, the average size of the sheared DNA is between 0.5 kb and 20 kb. In some cases, the cellular DNA is sheared to 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, 10.0, 10.25, 10.5, 10.75, In some cases, the cellular DNA is sheared to an average size of about 1.5 kb.

[0381] D. Exemplary Uses, Methods, and Treatments

[0382] In some embodiments, mRNA, LNP or pharmaceutical composition is used for genome editing, such as editing target genes. In some embodiments, mRNA, LNP or pharmaceutical composition is used for modifying target genes, such as changing their sequence or epigenetic status. In some embodiments, mRNA, LNP or pharmaceutical composition is used for inducing double strand breaks (DSB) in target genes. In some embodiments, mRNA, LNP or pharmaceutical composition is used for inducing insertion and deletion markers (indel) in target genes. In some embodiments, the use of mRNA, LNP or pharmaceutical composition disclosed herein is provided, which is used to prepare a medicament for genome editing, such as editing a target gene. In some embodiments, the use of mRNA, LNP or pharmaceutical composition disclosed herein is provided, which is used to prepare a medicament for modifying a target gene, such as changing its sequence or epigenetic status. In some embodiments, the use of mRNA, LNP or pharmaceutical composition disclosed herein is provided, which is used to prepare a medicament for inducing double strand breaks (DSB) in a target gene. In some embodiments, the use of mRNA, LNP or pharmaceutical compositions disclosed herein is provided for the preparation of a medicament for inducing insertion and deletion markers (indel) in a target gene. In some embodiments, the target gene is in an individual, such as a mammal, such as a human. In some embodiments, the target gene is in an organ, such as a liver, such as a mammalian liver, such as a human liver. In some embodiments, the target gene is in a liver cell, such as a mammalian liver cell, such as a human liver cell. In some embodiments, the target gene is in a liver cell, such as a mammalian liver cell, such as a human liver cell. In some embodiments, the liver cell or liver cell is in situ. In some embodiments, the liver cell or liver cell is isolated in, for example, a culture, such as a primary culture. A method corresponding to the use disclosed herein is also provided, comprising administering to an individual an mRNA, LNP or pharmaceutical composition disclosed herein or contacting a cell, such as a cell described above, with an mRNA, LNP or pharmaceutical composition disclosed herein.

[0383] In some embodiments, the mRNA, LNP or pharmaceutical composition is for use in therapy or treatment of a disease, such as amyloidosis associated with TTR (ATTR). In some embodiments, the use of the mRNA disclosed herein (e.g., in the form of a composition provided herein) is provided for the preparation of a medicament, such as for treating an individual with amyloidosis associated with TTR (ATTR).

[0384] In some embodiments, the mRNA, LNP, or pharmaceutical composition is administered intravenously for any of the uses discussed above for an organism, an organ, or a cell in situ. In some embodiments, the mRNA, LNP, or pharmaceutical composition is administered at a dose of 0.01 to 10 mg / kg (mpk), e.g., 0.01 to 0.1 mpk, 0.1 to 0.3 mpk, 0.3 to 0.5 mpk, 0.5 to 1 mpk, 1 to 2 mpk, 2 to 3 mpk, 3 to 5 mpk, 5 to 10 mpk, or 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5, or 10 mpk.

[0385] In any of the foregoing embodiments involving a subject, the subject can be a mammal. In any of the foregoing embodiments involving a subject, the subject can be a human. In any of the foregoing embodiments involving a subject, the subject can be a cow, pig, monkey, sheep, dog, cat, fish, or poultry.

[0386] In some embodiments, the mRNA, LNP, or pharmaceutical composition disclosed herein is administered intravenously or used for intravenous administration. In some embodiments, the guide RNA, composition, and formulation are administered to or used for administration in the hepatic circulation.

[0387] In some embodiments, a single administration of an mRNA, LNP, or pharmaceutical composition disclosed herein is sufficient to genetically attenuate the expression of a target gene product. In some embodiments, a single administration of an mRNA, LNP, or pharmaceutical composition disclosed herein is sufficient to genetically knock out the expression of a target gene product. In other embodiments, more than one administration of an mRNA, LNP, or pharmaceutical composition disclosed herein may be beneficial for maximizing editing, modification, indel formation, DSB formation, or the like via a cumulative effect.

[0388] In some embodiments, the therapeutic efficacy of the mRNA, LNP, or pharmaceutical composition disclosed herein is seen 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years after delivery.

[0389] In some embodiments, treatment slows or halts disease progression.

[0390] In some embodiments, treatment results in improvement, stabilization, or slowing of changes in organ function or symptoms of a disease of an organ, such as the liver.

[0391] In some embodiments, the efficacy of the treatment is measured by increasing the survival time of the subject. E. Exemplary DNA molecules, vectors, expression constructs, host cells, and manufacturing methods

[0392] In certain embodiments, the present invention provides a DNA molecule comprising a sequence encoding any one of the mRNAs encoding the RNA-guided DNA binders described herein. In some embodiments, in addition to the RNA-guided DNA binder sequence, the DNA molecule further comprises a nucleic acid that does not encode the RNA-guided DNA binder. The nucleic acid that does not encode the RNA-guided DNA binder includes, but is not limited to, a promoter, an enhancer, a regulatory sequence, and a nucleic acid encoding a guide RNA.

[0393] In some embodiments, the DNA molecule further comprises a nucleotide sequence encoding crRNA, trRNA or crRNA and trRNA. In some embodiments, the nucleotide sequence encoding crRNA, trRNA or crRNA and trRNA comprises or consists of: a guide sequence flanked by all or part of a repetitive sequence from a naturally occurring CRISPR / Cas system. The nucleic acid comprising or consisting of crRNA, trRNA or crRNA and trRNA may further constitute a vector sequence, wherein the vector sequence comprises or consists of: a nucleic acid that will not be found naturally with crRNA, trRNA or crRNA and trRNA. In some embodiments, crRNA and trRNA are encoded by non-continuous nucleic acids in a vector. In other embodiments, crRNA and trRNA may be encoded by continuous nucleic acids. In some embodiments, crRNA and trRNA are encoded by relative strands of a single nucleic acid. In other embodiments, crRNA and trRNA are encoded by the same strands of a single nucleic acid.

[0394] In some embodiments, the DNA molecule further comprises a promoter operably linked to a sequence encoding any one of the mRNAs encoding the RNA-guided DNA binders described herein. In some embodiments, the DNA molecule is an expression construct suitable for expression in mammalian cells, such as human cells or mouse cells, such as human hepatocytes or rodent (e.g., mouse) hepatocytes. In some embodiments, the DNA molecule is an expression construct suitable for expression in a mammalian organ, such as a human liver or a rodent (e.g., mouse) liver cell. In some embodiments, the DNA molecule is a plasmid or an episome. In some embodiments, the DNA molecule is contained in a host cell, such as a bacterium or a cultured eukaryotic cell. Exemplary bacteria include Proteobacteria, such as Escherichia coli. Exemplary cultured eukaryotic cells include primary hepatocytes, including hepatocytes of rodent (e.g., mouse) or human origin; hepatocyte cell lines, including hepatocytes of rodent (e.g., mouse) or human origin; human cell lines; rodent (e.g., mouse) cell lines; CHO cells; microbial fungi, such as fission or budding yeast, such as yeast, such as Saccharomyces cerevisiae; and insect cells.

[0395] In some embodiments, a method for manufacturing mRNA disclosed herein is provided. In some embodiments, such methods include contacting a DNA molecule described herein with an RNA polymerase under conditions that allow transcription. In some embodiments, in vitro, for example, contact is performed in a cell-free system. In some embodiments, the RNA polymerase is a phage-derived RNA polymerase, such as a T7 RNA polymerase. In some embodiments, an NTP comprising at least one modified nucleotide as discussed above is provided. In some embodiments, the NTP comprises at least one modified nucleotide as discussed above and does not comprise UTP.

[0396] In some embodiments, the mRNA disclosed herein alone or together with one or more guide RNAs may be included in or delivered by the vector system of one or more vectors. In some embodiments, one or more or all of the vectors in the vector may be DNA vectors. In some embodiments, one or more or all of the vectors in the vector may be RNA vectors. In some embodiments, one or more or all of the vectors in the vector may be circular. In some embodiments, one or more or all of the vectors in the vector may be linear. In some embodiments, one or more or all of the vectors in the vector may be encapsulated in lipid nanoparticles, liposomes, non-lipid nanoparticles or viral capsids. Non-limiting exemplary vectors include plasmids, phagemids, cosmids, artificial chromosomes, minichromosomes, transposons, viral vectors and expression vectors.

[0397] Non-limiting exemplary viral vectors include adeno-associated virus (AAV) vectors, lentiviral vectors, adenoviral vectors, helper-dependent adenoviral vectors (HDAd), herpes simplex virus (HSV-1) vectors, bacteriophage T4, baculoviral vectors, and retroviral vectors. In some embodiments, the viral vector may be an AAV vector. In other embodiments, the viral vector may be a lentiviral vector. In some embodiments, the lentivirus may be non-integrative. In some embodiments, the viral vector may be an adenoviral vector. In some embodiments, the adenovirus may be a high cloning capacity or "gutless" adenovirus, in which all viral coding regions except the 5' and 3' inverted terminal repeats (ITRs) and the packaging signal ('I') are deleted from the virus to increase its packaging capacity. In yet other embodiments, the viral vector may be an HSV-1 vector. In some embodiments, HSV-1-like vectors are helper-dependent, and in other embodiments, they are non-helper-dependent. For example, an amplicon vector that retains only the packaging sequence requires a helper virus with structural components for packaging, while a 30kb HSV-1 vector that removes non-essential viral functions does not require a helper virus. In other embodiments, the viral vector may be bacteriophage T4. In some embodiments, when the viral head is emptied, bacteriophage T4 may be able to package any linear or circular DNA or RNA molecule. In other embodiments, the viral vector may be a baculovirus vector. In yet other embodiments, the viral vector may be a retroviral vector. In embodiments using AAV or lentiviral vectors with smaller cloning capacity, it may be necessary to use more than one vector to deliver all components of the vector system as disclosed herein. For example, one AAV vector may contain a sequence encoding a Cas protein, and a second AAV vector may contain one or more guide sequences.

[0398] In some embodiments, the vector may be able to drive the expression of one or more coding sequences in the cell, such as the coding sequence of the mRNA disclosed herein. In some embodiments, the cell may be a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as a yeast, plant, insect or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters for driving expression in different types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or more effective expression. In yet other embodiments, the promoter may be truncated but still retain its function. For example, the promoter may have a normal size or a reduced size suitable for appropriately packaging the vector in a virus.

[0399] In some embodiments, the vector system may comprise one copy of a nucleotide sequence encoding an RNA-guided DNA binder. In other embodiments, the vector system may comprise more than one copy of a nucleotide sequence encoding an RNA-guided DNA binder. In some embodiments, the nucleotide sequence encoding an RNA-guided DNA binder may be operably linked to at least one transcriptional or translational control sequence. In some embodiments, the nucleotide sequence encoding a nuclease may be operably linked to at least one promoter.

[0400] In some embodiments, the promoter may be constitutive, inducible or tissue-specific. In some embodiments, the promoter may be a constitutive promoter. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late promoter (MLP), Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-α (EF1a) promoter, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, a combination of any one of the functional fragments or the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EF1a promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include inducible promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics or alcohol. In some embodiments, the inducible promoter may be one with a low basal (non-induced) expression level, such as Promoter (Clontech).

[0401] In some embodiments, the promoter may be a tissue-specific promoter, such as a promoter specific for expression in the liver.

[0402] The vector may further comprise a nucleotide sequence encoding at least one guide RNA. In some embodiments, the vector comprises a copy of one guide RNA. In other embodiments, the vector comprises more than one copy of a guide RNA. In embodiments with more than one guide RNA, the guide RNAs may be different so that they target different target sequences, or may be the same so that they target the same target sequence. In some embodiments in which the vector comprises more than one guide RNA, each guide RNA may have other different properties, such as activity or stability in a ribonucleoprotein complex with an RNA-guided DNA binder. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or translational control sequence, such as a promoter, 3'UTR or 5'UTR. In one embodiment, the promoter may be a tRNA promoter, such as a tRNA Lys3 , or tRNA chimera. See Mefferd et al., RNA.201521:1683-9; Scherer et al., Nucleic Acids Res.2007 35:2620-2628. In some embodiments, the promoter can be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include U6 and H1 promoters. In some embodiments, the nucleotide sequence encoding the guide RNA can be operably connected to a mouse or human U6 promoter. In other embodiments, the nucleotide sequence encoding the guide RNA can be operably connected to a mouse or human H1 promoter. In embodiments with more than one guide RNA, the promoters used to drive expression may be the same or different. In some embodiments, the nucleotides encoding the crRNA of the guide RNA and the nucleotides encoding the trRNA of the guide RNA may be provided on the same vector. In some embodiments, the nucleotides encoding the crRNA and the nucleotides encoding the trRNA may be driven by the same promoter. In some embodiments, crRNA and trRNA may be transcribed into a single transcript. For example, crRNA and trRNA may be processed by a single transcript to form a double-molecule guide RNA. Alternatively, crRNA and trRNA can be transcribed as single-molecule guide RNA. In other embodiments, crRNA and trRNA can be driven by their corresponding promoters on the same vector. In yet other embodiments, crRNA and trRNA can be encoded by different vectors.

[0403] In some embodiments, the composition comprises a vector system, wherein the system comprises more than one vector. In some embodiments, the vector system may comprise a single vector. In other embodiments, the vector system may comprise two vectors. In other embodiments, the vector system may comprise three vectors. When different guide RNAs are used for multiplexing or when multiple copies of guide RNAs are used, the vector system may comprise more than three vectors.

[0404] In some embodiments, the vector system may include an inducible promoter to initiate expression only after it is delivered to the target cell. Non-limiting exemplary inducible promoters include inducible promoters that can be induced by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohols. In some embodiments, the inducible promoter may be an inducible promoter with a low basal (non-induced) expression level, such as Promoter (Clontech).

[0405] In other embodiments, the vector system may contain a tissue-specific promoter to initiate expression only after it is delivered to a specific tissue.

[0406] 1. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine content of the open reading frame is within the range of its minimum uridine content to 150% of the minimum uridine content.

[0407] 2. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine dinucleotide content of the open reading frame is within the range of its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.

[0408] 3. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the adenine content of the open reading frame is within the range of its minimum adenine content to 150% of the minimum adenine content.

[0409] 4. An mRNA comprising an open reading frame encoding an RNA-guided DNA-binding agent, wherein the adenine dinucleotide content of the open reading frame is within the range of its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content.

[0410] 5. An mRNA comprising a sequence that is at least 90% identical to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107 to 175, wherein the mRNA comprises an open reading frame encoding an RNA-guided DNA binder.

[0411] 6. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the open reading frame has at least 90% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107 to 175 over at least the first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides thereof.

[0412] 7. The mRNA of any one of the preceding embodiments, wherein the open reading frame consists of a set of codons, wherein at least 75% of the codons are (i) codons listed in Table 1, Table 2 or Table 3 or (ii) codons listed in a set of Table 4.

[0413] 8. An mRNA encoding an RNA-guided DNA binder, the RNA-guided DNA binder comprising an open reading frame encoding the RNA-guided DNA binder, wherein the open reading frame consists of a set of codons, wherein at least 75% of the codons are codons listed in Table 1, Table 2, Table 3 or (ii) a set of codons listed in Table 4.

[0414] 9. The mRNA of embodiment 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the low U1 group in Table 4.

[0415] 10. The mRNA of embodiment 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the low A group in Table 4.

[0416] 11. The mRNA of embodiment 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the low A / U group in Table 4.

[0417] 12. The mRNA of embodiment 7 or 8, wherein the open reading frame consists of a group of codons, wherein at least 75% of the codons are codons of the long half-life group in Table 4.

[0418] 13. The mRNA of any one of embodiments 7 to 12, wherein at least 80%, 85%, 90%, 95%, 98%, 99% or 100% of the codons are (i) codons listed in Table 1, Table 2 or Table 3 or (ii) a group of codons listed in Table 4.

[0419] 14. The mRNA of any one of embodiments 1 to 5 or 7 to 13, wherein the open reading frame is at least 90% identical to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107 to 175 over at least the first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides thereof.

[0420] 15. The mRNA of any one of the preceding embodiments, wherein the open reading frame is at least 90% identical to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107 to 175 over at least the first 10%, 12%, 15%, 20%, 25%, 30%, or 35% of its sequence.

[0421] 16. The mRNA of any one of embodiments 1 to 4 or 6 to 15, wherein the mRNA comprises a sequence that is at least 90% identical to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, SEQ ID NO: 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66, or 107 to 175.

[0422] 17. The mRNA of any one of the preceding embodiments, wherein the uridine dinucleotide content of the open reading frame is within the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum uridine dinucleotide content to the minimum uridine dinucleotide content.

[0423] 18. The mRNA of any of the preceding embodiments, wherein the uridine content of the open reading frame is within the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum uridine content to the minimum uridine content.

[0424] 19. The mRNA of any of the preceding embodiments, wherein the adenine content of the open reading frame is within the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum adenine content to the minimum adenine content.

[0425] 20. The mRNA of any of the preceding embodiments, wherein the adenine dinucleotide content of the open reading frame is within the range of 101%, 102%, 103%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145% or 150% of its minimum adenine dinucleotide content to the minimum adenine dinucleotide content.

[0426] 21. The mRNA of any of the preceding embodiments, comprising a 5'UTR that is at least 90% identical to any one of SEQ ID NOs: 32, 34, 36, 38, 41 or 75 to 77.

[0427] 22. The mRNA of any one of the preceding embodiments, comprising a 3'UTR that is at least 90% identical to any one of SEQ ID NOs: 33, 35, 37, 39 or 40.

[0428] 23. The mRNA of embodiment 21 or 22, wherein the mRNA comprises a 5'UTR and a 3'UTR from the same source.

[0429] 24. The mRNA of any one of the preceding embodiments, comprising a 5' cap structure selected from Cap0, Cap1 and Cap2.

[0430] 25. The mRNA of any one of the preceding embodiments, wherein the open reading frame has codons that increase translation of the mRNA in mammals.

[0431] 26. The mRNA of embodiment 25, wherein the open reading frame has codons that increase translation of the mRNA in specific organs of a mammal.

[0432] 27. The mRNA of embodiment 26, wherein the organ is the liver.

[0433] 28. The mRNA of any one of embodiments 25 to 27, wherein the mammal is a human.

[0434] 29. The mRNA of any one of embodiments 25 to 28, wherein the codon increases translation of the mRNA in the mammal relative to translation of an mRNA comprising an ORF having a sequence consisting of SEQ ID NO: 5.

[0435] 30. The mRNA of any of the preceding embodiments, wherein when the mRNA is administered to a mammal in the form of a pharmaceutical composition, the mammal exhibits a cytokine response that is at least 5-fold lower than that of a mammal administered with an mRNA comprising an ORF encoding a Cas9 nuclease having greater than 150% of the minimum uridine content.

[0436] 31. The mRNA of embodiment 30, wherein the mRNA comprises an ORF encoding a Cas9 nuclease having a minimum uridine content of more than 150%, and the mRNA has a sequence consisting of SEQ ID NO: 5.

[0437] 32. The mRNA of any one of the preceding embodiments, wherein the RNA-guided DNA binder has double-stranded endonuclease activity.

[0438] 33. The mRNA of embodiment 32, wherein the RNA-guided DNA binder comprises a Cas lyase.

[0439] 34. The mRNA of any one of the preceding embodiments, wherein the RNA-guided DNA binder has nickase activity.

[0440] 35. The mRNA of embodiment 34, wherein the RNA-guided DNA binder comprises a Cas nickase.

[0441] 36. The mRNA of any one of embodiments 1 to 31, wherein the RNA-guided DNA binder comprises a dCas DNA binding domain.

[0442] 37. The mRNA of any one of embodiments 33 or 35 to 36, wherein the Cas lyase, Cas nickase or dCas DNA binding domain is a Cas9 lyase, Cas9 nickase, or dCas9 DNA binding domain.

[0443] 38. The mRNA of any of the preceding embodiments, wherein the encoded RNA-guided DNA binder comprises a nuclear localization signal (NLS).

[0444] 39. The mRNA of embodiment 38, wherein the NLS is linked to the C-terminus of the RNA-guided DNA binder.

[0445] 40. The mRNA of embodiment 38, wherein the NLS is linked to the N-terminus of the RNA-guided DNA binder.

[0446] 41. The mRNA of any one of embodiments 38 to 40, wherein the NLS comprises a sequence that is at least 80%, 85%, 90% or 95% identical to any one of SEQ ID NOs: 78-91.

[0447] 42. The mRNA of any one of embodiments 38 to 40, wherein the NLS comprises the sequence of any one of SEQ ID NOs: 78-91.

[0448] 43. The mRNA of any one of embodiments 38 to 42, wherein the NLS is encoded by a sequence that is at least 80%, 85%, 90%, 95%, 98% or 100% identical to the sequence of any one of SEQ ID NOs: 92-104.

[0449] 44. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 4, 7, or 9.

[0450] 45. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO: 4, 7, or 9.

[0451] 46. ​​The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 98% identical to SEQ ID NO: 4, 7, or 9.

[0452] 47. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is 100% identical to SEQ ID NO: 4, 7, or 9.

[0453] 48. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 111, 114, or 117.

[0454] 49. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO: 111, 114, or 117.

[0455] 50. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 98% identical to SEQ ID NO: 111, 114, or 117.

[0456] 51. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is 100% identical to SEQ ID NO: 112, 122, or 125.

[0457] 52. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 112, 122, or 125.

[0458] 53. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO: 112, 122 or 125.

[0459] 54. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 90% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0460] 55. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 95% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0461] 56. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is at least 98% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0462] 57. The mRNA of any one of embodiments 37 to 43, wherein the mRNA comprises a sequence that is 100% identical to SEQ ID NO: 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107-175.

[0463] 58. The mRNA of any one of embodiments 37 to 57, wherein the mRNA encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, 6, 8, or 186-196.

[0464] 59. The mRNA of any one of the preceding embodiments, wherein the RNA-guided DNA binder further comprises a heterologous functional domain.

[0465] 60. The mRNA of embodiment 59, wherein the heterologous functional domain is FokI nuclease.

[0466] 61. The mRNA of embodiment 59, wherein the heterologous functional domain is a transcriptional regulatory domain.

[0467] 62. The mRNA of any one of the preceding embodiments, wherein when an effective amount of the mRNA and a guide RNA targeting the TTR gene of the mammal are administered together to a mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, an insertion-deletion mark (indel) is formed at the TTR locus in at least 50% of the genomic DNA obtained from the liver cells of the mammal.

[0468] 63. The mRNA of any one of the preceding embodiments, wherein when an effective amount of the mRNA and a guide RNA targeting the TTR gene of the mammal are administered together to the mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, the concentration of TTR in the serum of the mammal is reduced by at least 50%.

[0469] 64. The mRNA of any of the preceding embodiments, wherein at least 10% of the uridines are substituted with modified uridines.

[0470] 65. The mRNA of embodiment 64, wherein the modified uridine is one or more of N1-methyl-pseudouridine, pseudouridine, 5-methoxyuridine, or 5-iodouridine.

[0471] 66. The mRNA of embodiment 64, wherein the modified uridine is one or both of N1-methyl-pseudouridine or 5-methoxyuridine.

[0472] 67. The mRNA of embodiment 64, wherein the modified uridine is N1-methyl-pseudouridine.

[0473] 68. The mRNA of embodiment 64, wherein the modified uridine is 5-methoxyuridine.

[0474] 69. The mRNA of any one of embodiments 64 to 68, wherein 15% to 45% of the uridines are substituted with modified uridines.

[0475] 70. The mRNA of any one of embodiments 64 to 68, wherein at least 20% or at least 30% of the uridines are substituted with modified uridines.

[0476] 71. The mRNA of embodiment 70, wherein at least 80% or at least 90% of the uridines are substituted with modified uridines.

[0477] 72. The mRNA of embodiment 70, wherein 100% of the uridines are substituted with modified uridines.

[0478] 73. The mRNA of any one of embodiments 64 to 72, wherein when an effective amount of the mRNA and a guide RNA targeting a TTR gene of the mammal are administered together to a mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, an insertion-deletion mark (indel) is formed at the TTR locus in at least 70% or at least 90% of genomic DNA obtained from liver cells of the mammal.

[0479] 74. The mRNA of any one of embodiments 64 to 73, wherein when the mRNA and a guide RNA targeting a TTR gene of the mammal are administered together to the mammal in the form of a pharmaceutical composition comprising lipid nanoparticles, the TTR concentration in the serum of the mammal is reduced by at least 70% or at least 90%.

[0480] 75. The mRNA of embodiments 62, 63, 71 or 72, wherein the animal is a mouse and the guide RNA has a sequence consisting of SEQ ID NO: 42.

[0481] 76. The mRNA of embodiments 62, 63, 71 or 72, wherein the animal is a rat and the guide RNA has a sequence consisting of SEQ ID NO: 69.

[0482] 77. The mRNA of any of the preceding embodiments, wherein the mRNA comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 176-185.

[0483] 78. The mRNA of any one of the preceding embodiments, wherein the mRNA comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 176-185.

[0484] 79. The mRNA of any of the preceding embodiments, wherein the mRNA comprises a sequence that is at least 98% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 176-185.

[0485] 80. The mRNA of any one of the preceding embodiments, wherein the mRNA comprises a sequence that is at least 99% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 176-185.

[0486] 81. The mRNA of any one of the preceding embodiments, wherein the mRNA comprises a sequence that is 100% identical to any one of SEQ ID NOs: 43, 44, 51, 53, 55-61, or 176-185.

[0487] 82. An expression construct comprising a promoter operably linked to a sequence encoding the mRNA of any of the preceding embodiments.

[0488] 83. A plasmid comprising the expression construct of embodiment 82.

[0489] 84. A host cell comprising the expression construct of embodiment 82 or the plasmid of embodiment 83.

[0490] 85. A method of preparing mRNA, the method comprising contacting the expression construct of embodiment 82 or the plasmid of embodiment 83 with an RNA polymerase under conditions that allow transcription of the mRNA.

[0491] 86. The method of embodiment 85, wherein the contacting step is performed in vitro.

[0492] 87. A composition comprising the mRNA of any one of embodiments 1 to 81 and at least one guide RNA.

[0493] 88. A lipid nanoparticle comprising the mRNA of any one of embodiments 1 to 81.

[0494] 89. A pharmaceutical composition comprising the mRNA of any one of embodiments 1 to 81 and a pharmaceutically acceptable carrier.

[0495] 90. The lipid nanoparticle of embodiment 88 or the pharmaceutical composition of embodiment 89, further comprising at least one guide RNA.

[0496] 91. The composition or lipid nanoparticle of any one of embodiments 87 to 90, wherein at least one guide RNA targets TTR.

[0497] 92. A method for genome editing or modifying a target gene, comprising contacting a cell with an mRNA, expression construct, composition, or lipid nanoparticle as described in any one of embodiments 1 to 83 or 87 to 91.

[0498] 93. Use of an mRNA, expression construct, composition or lipid nanoparticle as described in any one of embodiments 1 to 83 or 87 to 91 for genome editing or modifying a target gene.

[0499] 94. Use of an mRNA, expression construct, composition or lipid nanoparticle as described in any one of embodiments 1 to 83 or 87 to 91 for the manufacture of a medicament for genome editing or modifying a target gene.

[0500] 95. The method or use of any one of embodiments 92 to 94, wherein the genome editing or modification of the target gene is performed in liver cells.

[0501] 96. The method or use of embodiment 95, wherein the liver cell is a hepatocyte.

[0502] 97. The method or use of any one of embodiments 92 to 96, wherein genome editing or modification of the target gene is performed in vivo.

[0503] 98. The method or use of any one of embodiments 92 to 97, wherein genome editing or modification of the target gene is performed in isolated or cultured cells.

[0504] Examples

[0505] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the invention in any way.

[0506] General Reagents and Methods. Unless otherwise indicated, mRNA was synthesized by in vitro transcription (IVT) using a linearized plasmid DNA template and T7 ribonucleic acid polymerase. Transcription was generally performed by a construct comprising a T7 promoter; a transcript sequence disclosed herein, such as SEQ ID NO:43 (which comprises SEQ ID NO:1 and encodes the RNA ORF of SEQ ID NO:4) or SEQ ID NO:48 (which comprises SEQ ID NO:2 and encodes the RNA ORF of SEQ ID NO:5) and a poly-A tail (SEQ ID NO:63) encoded in the plasmid. Experiments in which multiple UTRs were tested used similar constructs except that transcript sequences such as SEQ ID NO:58 and SEQ ID NO:59 were used. Plasmid DNA containing a T7 promoter and a 100 nt poly(A / T) region was linearized by incubation with XbaI for 2 hours at 37°C using the following conditions: 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1× reaction buffer. XbaI was inactivated by heating the reaction at 65° C. for 20 min. The linearized plasmid was purified from enzyme and buffer salts using a silica max spin column (Epoch Life Sciences) and analyzed by agarose gel to confirm linearization. IVT reactions for the production of Cas9-modified mRNA were incubated for 4 hours at 37°C under the following conditions: 50 ng / μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and UTP (or, if indicated, a modified triphosphate nucleotide such as N1-methyl pseudoUTP (Trilink) in place of CTP or UTP); 10 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL murine RNase inhibitor (NEB); 0.004 U / μL inorganic E. coli pyrophosphatase (NEB); and 1× reaction buffer. After the 4-hour incubation, TURBO deoxyribonuclease (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reaction was incubated for an additional 30 minutes to remove the DNA template. Cas9 was purified from enzymes and nucleotides using the MegaClear Transcription Cleanup Kit according to the manufacturer's protocol (ThermoFisher). mRNA. The mRNA was purified via a precipitation protocol (which in some cases was followed by HPLC-based purification). In brief, after DNase digestion, the mRNA was precipitated by adding 0.21× volume of 7.5M LiCl solution and mixing, and the precipitated mRNA was assembled by centrifugation. After removing the supernatant, the mRNA was rehydrated. The mRNA was precipitated again using ammonium acetate and ethanol. 5M ammonium acetate was added to the mRNA solution together with 2× volume of 100% EtOH to a final concentration of 2M.The solution was mixed and incubated at -20°C for 15 minutes. The precipitated mRNA was again assembled by centrifugation, the supernatant was removed and the mRNA was rehydrated. The mRNA was precipitated using sodium acetate and ethanol as a final step. 1 / 10 volume of 3M sodium acetate (pH 5.5) was added to the solution together with 2× volume of 100% EtOH. The solution was mixed and incubated at -20°C for 15 minutes. The precipitated mRNA was again assembled by centrifugation, the supernatant was removed, the aggregated particles were washed with 70% cold ethanol and air-dried. The mRNA was rehydrated. For HPLC-purified mRNA, after LiCl precipitation and rehydration, the mRNA was purified by RP-IP HPLC (see, e.g., Kariko et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). The fractions selected for collection were combined and desalted by sodium acetate / ethanol precipitation as described above.

[0507] For all methods, transcript concentrations were determined by measuring absorbance at 260 nm (Nanodrop) and transcripts were analyzed by capillary electrophoresis using a Bioanlayzer (Agilent).

[0508] Unless otherwise indicated, in vivo editing experiments were performed with CD-1 female mice and Sprague-Dawley rats from Charles River Laboratories. Unless otherwise indicated, analysis of serum TTR levels in mice was performed as follows. Blood was collected and serum was separated as indicated.

[0509] In the case of the applicable example, the cytokine induction in the treated mice is also measured. For this analysis, about 50 to 100 μL of blood is collected by tail vein incision for serum cytokine measurement. The blood is allowed to coagulate for about 2 hours at room temperature, and then centrifuged at 1000 × g for 10 minutes, and then serum is collected. The Luminex-based magnetic bead multitasking analysis (Affymetrix ProcartaPlus, catalog number Exp040-00000-801) for measuring IL-6, TNF-α, IFN-α and MCP-1 is used to collect the cytokine analysis in the sample. The kit reagents and standards are prepared as directed in the manufacturer's scheme. The mouse serum is diluted 4 times using the sample diluent provided, and 50 μL is added to the hole containing the magnetic beads coated with 50 μL dilution antibodies. The plate is incubated for 2 hours at room temperature and then washed. Diluted biotin antibody (50 μL) is added to beads and incubated for 1 hour at room temperature. The beads were washed again, after which 50 μL of diluted streptavidin-PE was added to each well, followed by incubation for 30 minutes. The beads were washed again and then suspended in 100 μL of wash buffer and read on a Bio-Plex 200 instrument (Bio-Rad). The data were analyzed using the Bioplex Manager version 6.1 analysis software package by using a five-parameter logistic curve fit to the cytokine concentrations calculated from the standard curve.

[0510] Unless otherwise specified, unmodified ATP, GTP, CTP and UTP were used. Unless otherwise specified, all mRNAs encode a nuclear localization signal.

[0511] By using the Precision Nanosystems NanoAssemblr TM Benchtop instrument, according to the manufacturer's protocol, microflow mixing or cross-flow mixing of lipid and RNA solutions to form LNPs, as described below. Unless otherwise specified, LNPs contained 45% lipid A, 9% DSPC, 44% cholesterol and 2% PEG2k-DMG and an N:P ratio of 4.5.

[0512] LNP Preparation - NanoAssemblr

[0513] Typically, lipid nanoparticle components are dissolved in 100% ethanol at different lipid component molar ratios. RNA load is dissolved in 25mM citrate, 100mM NaCl (pH 5.0) to produce an RNA load concentration of approximately 0.45mg / mL. LNPs are formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 4.5 or about 6, and a ratio of mRNA to gRNA of 1:1 by weight.

[0514] By using the Precision Nanosystems NanoAssemblr TM Benchtop instrument, according to the manufacturer's protocol, lipid and RNA solution microfluidization is mixed to form LNP. Use differential flow rate to maintain a 2: 1 ratio of aqueous phase to organic solvent during mixing. After mixing, LNP is collected, diluted in water (approximately 1: 1v / v), kept at room temperature for 1 hour, and further diluted with water (approximately 1: 1v / v), and then the final buffer exchange is performed. The final buffer exchange is completed with PD-10 desalting column (GE) to 50mM Tris, 45mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS). If necessary, the formulation is concentrated by centrifugation with Amicon100kDa centrifugal filter (Millipore). The resulting mixture is then filtered using a 0.2μm sterile filter. Before further use, the final LNP is stored at -80°C.

[0515] LNP blending - cross flow

[0516] For LNPs prepared using cross-flow technology, LNPs are formed by impinging jet mixing of an ethanol solution of lipids with two volumes of RNA solution and one volume of water. The ethanol solution of lipids is mixed with two volumes of RNA solution via a mixing cross tube. The fourth water flow is mixed with the outlet flow of the cross tube via a T-shaped tube along the line (see WO2016010840 Fig. 2.). The LNPs are kept at room temperature for 1 hour and further diluted with water (approximately 1: 1v / v). The diluted LNPs are concentrated on a flat filter cartridge (Sartorius, 100kD MWCO) using tangential flow filtration, and then by diafiltration, the buffer is exchanged with 50mMTris, 45mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS). Alternatively, the final buffer exchange is completed with a PD-10 desalting column (GE) to TSS. If necessary, the formulation is concentrated by centrifugation with an Amicon 100kDa centrifugal filter (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNPs were stored at 4°C or -80°C prior to further use.

[0517] Formulation Analysis

[0518] Dynamic light scattering ("DLS") is used to characterize the polydispersity index ("pdi") and size of the LNPs of the present invention. DLS measures the scattering of light produced by placing a sample under a light source. As determined from the DLS measurement, the PDI represents the distribution of particle sizes in a population (around the average particle size), where the PDI of a completely uniform population is zero. The average particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer instrument. Prior to measurement by DLS, the LNP sample is diluted 30× in PBS. The Z-average diameter, which is an intensity-based measure of the average particle size, is reported together with the number average diameter and pdi. The Malvern Zetasizer instrument is also used to measure the zeta potential of the LNPs. Prior to measurement, the sample is diluted 1:17 (50 μL in 800 μL) in 0.1× PBS, pH 7.4.

[0519] Using fluorescence-based assays ( Total RNA concentration and free RNA were determined using a PCR amplification kit (ThermoFisher Scientific). Encapsulation efficiency was calculated as (total RNA - free RNA) / total RNA. LNP samples were appropriately diluted with 1× TE buffer containing 0.2% Triton-X 100 for total RNA or 1× TE buffer for free RNA. A standard curve was prepared using the starting RNA solution used to make the formulation and diluted in 1× TE buffer + / - 0.2% Triton-X 100. The diluted Dye (according to the manufacturer's instructions) was added to each of the standards and samples and allowed to incubate at room temperature for approximately 10 minutes in the absence of light. Samples were read using a SpectraMax M5 microplate reader (Molecular Devices) with excitation, automatic cutoff, and emission wavelengths set to 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA were determined according to appropriate standard curves.

[0520] The encapsulation efficiency is calculated as (total RNA - free RNA) / total RNA. The same procedure can be used to determine the encapsulation efficiency of DNA-based cargo components. For single-stranded DNA, Oligreen dye can be used, and for double-stranded DNA, Picogreen dye can be used.

[0521] Typically, when LNPs are prepared, the encapsulation efficiency is >80%, the particle size is <120 nm and the pdi is <0.2.

[0522] LNP delivery in vivo

[0523] Unless otherwise noted, CD-1 female mice in the range of 6 to 10 weeks of age were used in each study. Animals were weighed and grouped according to body weight to prepare dosing solutions based on group average body weight. LNPs were dosed via the lateral tail vein in a volume of 0.2 mL (approximately 10 mL per kg of body weight) per animal. Animal side effects were observed approximately 6 hours after administration. Body weight was measured twenty-four hours after dosing, and animals were euthanized at various time points by bleeding via cardiac puncture under isoflurane anesthesia. Blood was collected in serum separation tubes or in tubes containing buffered sodium citrate for plasma as described herein. For studies involving in vivo editing, liver tissue was collected from the middle lobe or three independent leaves (e.g., right middle, left middle, and left lateral leaves) from each animal for DNA extraction and analysis.

[0524] The mouse groups were assessed for liver editing by next generation sequencing (NGS) and serum TTR levels (data not shown).

[0525] Transthyretin (TTR) ELISA analysis

[0526] Blood was collected and serum was separated as indicated. Total mouse TTR serum levels were determined using a mouse prealbumin (transthyretin) ELISA kit (Aviva Systems Biology, catalog number OKIA00111). Rat TTR serum levels were measured using a rat-specific ELISA kit (Aviva Systems Biology catalog number OKIA00159) according to the manufacturer's protocol. Briefly, serum was serially diluted with the kit sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to the ELISA plate and then analyzed as indicated.

[0527] NGS Sequencing

[0528] Briefly, to quantitatively determine editing efficiency at target locations in the genome, genomic DNA was isolated and deep sequencing was used to identify the presence of insertions and deletions caused by gene editing.

[0529] PCR primers are designed around the target site (e.g., TTR), and the relevant genomic region is amplified. Primer sequences are provided below. Additional PCR is performed according to the manufacturer's protocol (Illumina) to add the required chemistry for sequencing. The amplicons are sequenced on an Illumina MiSeq instrument. After eliminating reads with low quality scores, the reads are aligned to a human reference genome (e.g., hg38). The resulting archive containing the reads is mapped to a reference genome (BAM archive), wherein reads overlapping the relevant target region are selected and the number of wild-type reads is calculated relative to the number of reads containing insertions, substitutions, or deletions.

[0530] The editing percentage (eg, "editing efficiency" or "editing %") is defined as the total number of sequence reads with an insertion or deletion compared to the total number of sequence reads that include the wild type.

[0531] 1. In vivo characterization of Cas9 mRNA with modified nucleotides

[0532] mRNAs containing the ORF as set forth in SEQ ID NO: 5 were prepared with different modified nucleotide contents as shown in Table 5 below. The mRNAs were combined with a guide RNA (G282; SEQ ID NO: 42) targeting the thyroxine transporter gene (TTR) and incorporated into LNPs. Unmodified cytidine was used in all LNPs except LNP420.

[0533] Table 5. LNP417 to LNP421 used for in vivo studies

[0534]

[0535] LNP417 to LNP421 were administered to mice at a dose of 0.5 mg / kg (mpk) or 1 mpk. Cytokine (IFNα, IL-6, TNFα and MCP-1) induction was measured 4 hours post-dose (hpd). The results are shown in Figures 1A to 1D middle.

[0536] At necropsy 7 days after dosing, serum and liver were collected for serum TTR measurement and analysis of editing efficacy, respectively. The results are shown in FIG. 2A to FIG. 2B middle.

[0537] It was observed that the use of pseudouridine and 5-methyl CTP almost completely abolished cytokine induction. The use of N1-methylpseudouridine at 60% (LNP421) or 100% (LNP417) also caused less cytokine induction than unmodified Cas9 mRNA, and the degree of reduction at 60% N1-methylpseudouridine was similar to that at 100%.

[0538] All modified Cas9 constructs had similar effects in reducing serum TTR and were more effective than unmodified constructs, likely due to increased stability. Constructs using pseudouridine and N1-methylpseudouridine were equally effective based on liver editing data. Constructs with pseudouridine and 5-methylcytidine were significantly less effective than constructs with pseudouridine alone. Constructs with 60% N1-methylpseudouridine were likely slightly less effective than constructs with 100% N1-methylpseudouridine.

[0539] 2. Development and in vitro characterization of modified mRNA encoding Cas9

[0540] The Cas9 sequence (SEQ ID NO: 1) is designed to improve liver expression and minimize uridine. Codons are selected based on the least possible uridine content and the maximum expression of the corresponding tRNA in the liver. For liver tRNA expression, see Dittmar KA, PLos Genetics 2 (12): e221 (2006). Reducing the uridine content of Cas9 mRNA is intended to reduce the innate immune response to mRNA and / or provide other benefits. Table 6 shows the best liver codons based on tRNA content and the codons with the least possible number of uridines. The situation where the minimum uridine codon is different from the best liver codon is in bold italic form. The table also shows the number of each amino acid in the amino acid sequence of Streptococcus pyogenes Cas9 (SEQ ID NO: 3).

[0541] Table 6: Codon optimization parameters

[0542] Amino Acids Optimal liver codons Minimal uridine codon Cas9 frequency A Alanine GCA GCA 73 G Glycine GGA GGA 73 V Valine GTC GTC 74 D Aspartic acid GAT GAC 100 E Glutamate GAA GAA 111 I Isoleucine ATC ATC 93 T Threonine ACA ACA 66 N Asparagine AAC AAC 70 K Lysine AAG AAG 155 S Serine TCG AGC 79 R Arginine AGA AGA 79 L Leucine CTG CTG 148 P Proline CCG CCG 36 H Histidine CAC CAC 32 Q Glutamine CAG CAG 52 F Phenylalanine TTC TTC 64 Y Tyrosine TAC TAC 55 C Cysteine TGC TGC 2 W Tryptophan TGG TGG 7 M Methionine ATG ATG 22

[0543] In the case of aspartate and serine, the liver codons corresponding to the most highly expressed tRNAs contain thymidine, which will be transcribed as uridine in the corresponding mRNA. Minimal uridine codons were selected for aspartate and serine (GAC and AGC, respectively). The Cas9 ORF sequence is 4140 nt long, contains 528 Us (12.8% uridine content), and any junctions with 3 or more consecutive uridines are avoided in the ORF. There are 63 instances of UU dinucleotides in the sequence (126 / 4140 = 3% uridine dinucleotide content). SEQ ID NO: 2 provides an alternative Cas9 sequence containing 19.6% uridine as an RNA ORF.

[0544] SEQ ID NO: 3 provides the amino acid sequence of Cas9 encoded by both SEQ ID NO: 1 and SEQ ID NO: 2 as a novel design of the Cas9 ORF without changing the encoded amino acid sequence. SEQ ID NO: 4 is the RNA form of the ORF of SEQ ID NO: 1. SEQ ID NO: 5 is the RNA form of the ORF of SEQ ID NO: 2.

[0545] The effect of modified nucleotides was also evaluated. Modified UTPs used for transcription of Cas9 transcription included N1-methyl-pseudo-UTP and 5-methoxy-UTP.

[0546] The structure of N1-methyl-pseudoUTP is:

[0547]

[0548] The structure of 5-methoxy-UTP is:

[0549]

[0550] In vitro transcription (IVT) yields were determined for mRNAs comprising the ORFs of SEQ ID NO:4 and SEQ ID NO:5. Both encode nuclear localization signals (NLS). In the presence of either unmodified UTP or N1-methyl-pseudoUTP, the sequence comprising SEQ ID NO:5 was transcribed. In the presence of unmodified UTP, the sequence comprising SEQ ID NO:4 was transcribed. IVT was also performed in increasing percentages of 5-methoxy-UTP, such as Figure 3 , which shows the yield of each of these constructs as determined spectrophotometrically.

[0551] These results show that when the 5-methoxyuridine content of the mRNA is increased, the yield is slightly reduced, but the mRNA yield is acceptable under all conditions. Therefore, Cas9 mRNA with acceptable yield can be generated for two Cas9 sequences through the conditions tested.

[0552] The purity of in vitro transcribed mRNA was calculated using area under the curve (AUC) analysis from mRNA capillary electrophoresis (CE) traces obtained using an Agilent Bioanalyzer 2100. Figure 4 ). The purity of SEQ ID NO:5Cas9 mRNA generated using unmodified UTP generally improves with increasing 5-methoxy-UTP substitutions, while the same construct made with N1-methyl-pseudoUTP is less affected by increasing 5-methoxy-UTP substitutions.

[0553] SEQ ID NO: 4 Cas9 made with unmodified UTP appears to be relatively unaffected by 5-methoxy-UTP substitution, with slightly improved purity at 5-methoxy-UTP substitution degrees between 0% and 20%.

[0554] The immunogenicity of different mRNAs was assessed by blot analysis using anti-dsRNA antibodies as a measure of double-stranded (ds) mRNA characteristics, which is an indicator of potential immunogenicity ( Figure 5 A to Figure 5 D). Figure 5 B and Figure 5 D uses a Cas9 mRNA sequence comprising SEQ ID NO: 5 and Figure 5 C used a Cas9 mRNA sequence comprising SEQ ID NO: 4. For constructs generated using unmodified UTP ( Figure 5 B to Figure 5C), with increasing 5-methoxy-UTP content, the double-stranded form usually decreases significantly. mRNA generated using N1-methyl-pseudo-UTP ( Figure 5 D) shows that the binding to the anti-dsRNA antibody decreases, but the binding to the antibody also decreases with the increase of 5-methoxy-UTP content.

[0555] Editing efficiency was then assessed in vitro by transfecting mRNA into Neuro 2A cells together with a guide sequence targeting transthyretin (TTR) (G209; SEQ ID NO: 64) and measuring the editing percentage.

[0556] like Figure 6 As shown in A, Cas9 mRNA transcribed from a construct comprising SEQ ID NO: 2 with N1-methyl-pseudo-UTP and 2 nuclear localization sequences and an HA tag (group indicated by the leftmost bracket), Cas9 mRNA transcribed from a construct comprising SEQ ID NO: 2 with UTP and 2 nuclear localization sequences and an HA tag (group indicated by the middle bracket), and Cas9 mRNA transcribed from a construct comprising SEQ ID NO: 1 with UTP (group indicated by the rightmost bracket) were evaluated. For each group, different concentrations of mRNA ranging from 0.1 ng to 100 ng were assessed for transcription with increasing amounts of 5-methoxy-UTP from 0% to 100% as indicated on the X-axis. Untreated cells showed no measurable editing. Figure 6 B shows editing efficiency data expressed as EC50 values ​​(ng).

[0557] Increased 5-methoxy-UTP content during transcription appeared to have an adverse effect on editing efficiency under both SEQ ID NO:5 conditions, with transcripts also containing N1-methyl-pseudo-UTP being more robust than transcripts containing UTP (e.g., at 60% and 80% 5-methoxy-UTP). In contrast, the editing efficiency of the Cas9 mRNA sequence comprising SEQ ID NO:4 showed little (if any) effect on increased 5-methoxy-UTP content. Thus, according to this system, the Cas9 mRNA sequence comprising SEQ ID NO:4 mRNA can provide similar editing efficiency to that of the form containing unmodified uridine with up to 100% 5-methoxy-uridine.

[0558] 3. In vivo Characterization of Cas9-encoding mRNA

[0559] The in vivo efficacy of the Cas9 mRNA sequence comprising SEQ ID NO: 4 on the Cas9 mRNA sequence comprising SEQ ID NO: 5 and the effect of transcription of the Cas9 mRNA sequence comprising SEQ ID NO: 4 in the presence of unmodified UTP, N1-methyl-pseudo-UTP, 40% 5-methoxy-UTP + 60% unmodified UTP or 100% 5-methoxy-UTP were evaluated. Table 7 provides information on these in vivo study groups. Each mRNA was administered in a lipid nanoparticle (LNP) formulation.

[0560] Table 7. LNP720 to LNP724 used for in vivo studies

[0561]

[0562] The in vivo study design is as follows. CD-1 female mice were from Charles River (n=5 per group). Animals were dosed intravenously (iv) at 1 mg per kilogram (mpk) or 0.5 mpk together with a single guide RNA (SEQ ID NO: 42) for thyroxine transporter (TTR). Blood was drawn from animals receiving the 1 mpk dose at 4 hours post-dose (hpd) for cytokine analysis of MCP-1, IL-6, IFN-α, and TNF-α. Animals were assessed for overall health at 24 hpd. Necropsy was performed 7 days after dosing, with blood collected for serum TTR analysis and liver collected for next generation sequencing (NGS) editing analysis.

[0563] Serum from animals dosed at 1 mpk was collected at 4 hpd and serum was prepared and run according to the manufacturer's instructions. Mouse 4-plex assay (Thermo Fisher). The results for serum levels of MCP-1, IL-6, IFN-α, and TNF-α are shown in Figure 7 A to Figure 7 D. These results indicate that the Cas9 mRNA sequence (LNP721, LNP723 or LNP724) comprising SEQ ID NO: 4 prepared with modified UTP showed relatively low levels of cytokine production.

[0564] The TTR level in serum was also assessed 7 days after administration. Figure 8 A and shown in Table 8. TSS (ie, 5% sucrose, 45 mM NaCl, 50 mM Tris (pH 7.5)) samples indicate TTR levels without LNP treatment. All LNP formulations are described in Table 7.

[0565] Table 8: Results of serum TTR levels after administration of LNP720 to LNP724

[0566]

[0567]

[0568] Table 9 and Figure 8 B provides results regarding the editing percentage of TTR in the liver as measured by next generation sequencing (NGS).

[0569] Table 9: Results showing the percent editing of TTR in the liver after dosing with LNP720 to LNP724

[0570]

[0571] All LNPs containing Cas9 showed reduced serum TTR levels and editing above baseline compared to TSS control samples. When comparing standard Cas9 mRNA (SEQ ID NO:5, LNP720) and Cas9 mRNA sequences containing SEQ ID NO:4 mRNA (SEQ ID NO:4, LNP721), both transcribed with N1-methyl-pseudoUTP, the Cas9 mRNA sequence containing SEQ ID NO:4 showed improved activity (lower TTR and higher editing%). For the Cas9 mRNA sequence containing SEQ ID NO:4, activity was highest with N1-methyl-pseudoUTP, and transcription with 40% 5-methoxy-UTP + 60% unmodified UTP (LNP723) produced higher activity than 100% 5-methoxy-UTP (LNP724).

[0572] As a measure of off-target effects, editing was also measured in the spleen of animals dosed with 1 mpk of the LNP formulation described above, as Figure 7 and as shown in Table 10. For all LNP formulations, >20-fold higher editing was seen in the liver, regardless of whether it was Cas9 or optimized Cas9 ( Figure 6 A).

[0573] Table 10: Results on the editing percentage of TTR in spleen after dosing 1 mpk of LNPs containing sgRNA and different Cas9

[0574]

[0575]

[0576] 4. Characterization of the efficacy of mRNA encoding Cas9 in primary mouse hepatocytes

[0577] The efficacy of different LNPs was evaluated in vitro in primary mouse hepatocytes (PMH).

[0578] At 100 ng, all LNPs described in Table 5 supported editing of TTR, as Fig.10 As expected, untreated cells showed no measurable editing of TTR.

[0579] Table 11 shows the Fig.10 EC50 values ​​of each LNP calculated based on the data presented in .

[0580] Table 11: Estimated EC50 values ​​for gene editing of TTR in PMH (ng)

[0581]

[0582] 5. In vivo Characterization of Cas9 mRNA-containing LNPs in Rats

[0583] The in vivo efficacy of a Cas9 mRNA sequence comprising SEQ ID NO: 4 versus a Cas9 mRNA sequence comprising SEQ ID NO: 5 was evaluated in rats. Table 12 provides information on these in vivo study groups. The standard Cas9 mRNA refers to SEQ ID NO: 5, while the mRNA lacking U (U-dep) refers to SEQ ID NO: 4. Each mRNA was administered in a lipid nanoparticle (LNP) formulation.

[0584] Details of LNP716 (standard Cas9) and LNP738 (lacking U) LNP formulations are shown in Table 12.

[0585] Table 12: LNP formulation characterization

[0586]

[0587] PDI = Polydispersity Index

[0588] N:P = N:P ratio, as described above

[0589] Serum TTR was measured as previously described.

[0590] In rats, Cas9 mRNA having the ORF of SEQ ID NO: 5 was co-administered with Cas9 mRNA having the ORF of SEQ ID NO: 4 at a dose of 2 mpk and 5 mpk ( FIG. 11A to FIG. 11B ) for comparison, such as Fig.11Aand as shown in Table 13. These data indicate that at both 2 mpk and 5 mpk, the Cas9 ORF of SEQ ID NO: 4 caused a more significant reduction in serum TTR compared to the Cas9 ORF of SEQ ID NO: 5. Fig. 11B These results are shown as percentages relative to the values ​​of TSS-treated controls in Table 13. A 5 mpk dose of U-dep Cas9 LNPs caused a greater than 90% reduction in serum TTR levels.

[0591] Table 13: Serum TTR levels after dosing with LNP716 and LNP738 Cas9 formulations

[0592]

[0593] KD% = % attenuation of gene compared to mean serum concentration of TSS samples

[0594] Fig.10 Table 14 shows liver editing of TTR after dosing with LNP716 (standard) and LNP738 (U-dep) formulations at 2 mpk and 5 mpk. Although TSS showed negligible editing, both LNP716 and LNP738 formulations caused liver editing of TTR. Among the comparative formulations, LNP738 formulations containing U-depletion caused twice as much editing as LNP716 formulations containing standard Cas9.

[0595] Table 14: Liver editing of TTR following dosing with U-deficient and standard Cas9 formulations

[0596]

[0597] These data indicate that Cas9 mRNA lacking U significantly improves the extent of TTR editing in the liver.

[0598] 6. Characterization of mRNAs with different UTRs

[0599] mRNA encoding Cas9 with UTR and + / - hemagglutinin (HA) tag as indicated in Table 15 was formulated into LNPs with guide RNA targeting TTR (G282; SEQ ID NO: 42). TMLNPs containing 45% lipid A, 9% DSPC, 44% cholesterol and 2% PEG2k-DMG were prepared and purified using an Amicon PD10 filter and used at a concentration of 0.5 mg / ml (LNP concentration). CD-1 female mice (n=5 per group) were dosed intravenously at 0.5 or 1.0 mpk. Seven days after dosing, the animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured.

[0600] Table 15. Description of LNP662 to LNP669 mRNA and results of serum TTR and liver editing analysis

[0601]

[0602]

[0603] Unless otherwise specified, the UTR in the mRNA is HSD / Alb. HBA: human alpha hemoglobin; HBB: human beta hemoglobin (HBB); XBG: Xenopus beta hemoglobin (XBG). Unless otherwise specified, the mRNA contains 100% N1-methylpseudouridine instead of uridine.

[0604] FIG. 13A to FIG. 13E Serum TTR (in Fig.13A The values ​​are shown as μg / ml and in Fig. 13B All LNP662 to LNP669 liver editing ( Fig. 13C ); Liver editing of LNP663 to LNP666 in which only the UTR was changed ( Fig.13D ); and liver editing of LNP662 and LNP667 to LNP669 in which only the mRNA sequence and UTP modification were changed ( Fig.13E ).

[0605] The UTRs for human albumin, human α-globulin, human β-globulin, and Xenopus β-globulin were roughly equally effective; the value for human α-globulin was slightly lower but it was not clear whether the difference was significant.

[0606] The ORF of SEQ ID NO: 4 containing less uridine increases the amount of editing in the liver. Cas9 mRNA made with N1-methylpseudouridine is more efficient than Cas9 mRNA made with unmodified uridine.

[0607] 7. In vitro and in vivo editing using different guide:Cas9 ratios

[0608] mRNA comprising an ORF according to SEQ ID NO: 4 or SEQ ID NO: 5 and a guide RNA targeting TTR were formulated into LNPs at different guide:Cas9 mRNA weight ratios as shown in Table 16. Cas9 mRNA was made by IVT synthesis as indicated above with N1-methylpseudouridine triphosphate instead of uridine triphosphate, HSD 5'UTR, human albumin 3'UTR, and a poly-A tail.

[0609] Table 16. LNP815 to LNP824 used for in vitro and in vivo studies

[0610] LNP Cas9 SEQ ID NO RNA ratio (guide:Cas9) LNP815 5 2:1 LNP816 5 1:1 LNP817 5 1:2 LNP818 5 1:4 LNP819 5 1:8 LNP820 4 2:1 LNP821 4 1:1 LNP822 4 1:2 LNP823 4 1:4 LNP824 4 1:8

[0611] Primary mouse hepatocytes (PMH) were plated in medium supplemented with 3% cynomolgus monkey serum for 24 hours and then treated with 0.3, 1, 3 or 10 ng of LNPs as shown in Table 16. Cells were lysed after 48 hours and the % editing was determined by NGS. The results are shown in Fig.14 And Table 17.

[0612] Table 17. In vitro editing in PMH

[0613]

[0614] For in vivo characterization, LNPs were administered to mice at 0.2, 0.5, or 1 mpk (n=5 per group). Eight days after dosing, animals were sacrificed, blood and liver and spleen were collected, and serum TTR, liver editing, and spleen editing were measured. Serum TTR results are shown in FIG. 15A to FIG. 15B and Table 18. The liver editing results are shown in FIG. 16A to FIG. 16B and Table 19. The spleen editing results are shown in FIG. 17A to FIG. 17B and in Table 20. Negative control mice were dosed with vehicle (Transformation and Storage Solution; "TSS"). Independent controls were run for experiments using LNP815 to LNP819 and experiments using LNP820 to LNP824.

[0615] Table 18. Serum TTR levels after dosing with LNP815 to LNP824

[0616]

[0617] The KD% provides the % attenuation of the gene in TTR content relative to the TSS control.

[0618] Table 19. Liver editing after dosing with LNP815 to LNP824

[0619]

[0620] LNP820-LNP824 generally produced liver editing results greater than or approximately equal to their LNP815-LNP819 counterparts at the same ratios. LNP820-LNP824 showed constant potency over the ratio range tested at 0.5 and 1 mpk and at ratios of 2:1 to 1:4 at 0.2 mpk.

[0621] Table 20. Spleen edited after dosing with LNP815 to LNP824

[0622]

[0623] Additional groups of mice (n=2) were dosed with each formulation at 3 mpk and sacrificed at 6 hpd for determination of protein expression in the liver. Western blots of liver proteins from mice treated with 3 mpk 1:1 and 1:4 ratio formulations (LNP816, LNP818, LNP821, and LNP823) are shown in Fig.18 The primary Ab for Western blotting was Immunoprecise TM Rabbit anti-Cas9, and the secondary Ab was Dylight at 1:12,500 TM Goat anti-rabbit. Cas9 protein expression was significantly higher in LNPs using mRNA with the ORF of SEQ ID NO:4.

[0624] 8. Characterization of the Effects of Modified Nucleotides

[0625] mRNA encoding Cas9 and containing modified nucleotides as indicated in Table 21 was formulated into LNPs with a guide RNA targeting TTR (G282; SEQ ID NO: 42). LNP1034 contains Cas9 mRNA commercially available from Trilink Biotechnologies, LLC and includes CleanCap TM (Cap1 structure in which the first nucleotide after the 7-methylguanine cap structure is 2'-O-methylated). LNP1027 to LNP1033 contain mRNAs comprising the ORF according to SEQ ID NO: 4 and ARCA (anti-reverse cap analog) Cap0. Nano Assemblr TMLNPs were formulated containing 45% lipid A, 9% DSPC, 44% cholesterol and 2% PEG2k-DMG, purified using an Amicon PD10 filter, and suspended in TSS buffer. The N:P (nitrogen to phosphate) ratio in the LNPs was 4.5 and the RNA concentration of the formulation was 0.4 mg / ml. CD-1 female mice (n=5 per group) were dosed intravenously at 0.1 or 0.3 mpk. Seven days after dosing, the animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured.

[0626] Table 21. LNP1027 to LNP1034 used for in vivo studies

[0627]

[0628]

[0629] For LNPs where modified uridine and / or cytidine nucleotides are listed at 25% or 50%, the remainder of the uridine and / or cytidine, respectively, are unmodified.

[0630] Serum TTR results are shown in FIG. 19A to FIG. 19B (Serum TTR results are expressed as μg / mL and % of TSS control, respectively); Fig. 20 (Liver edit); and Table 22.

[0631] Table 22. Serum TTR and liver editing results of LNP1027 to LNP1034

[0632]

[0633] The N1-methylpseudouridine-containing mRNA of LNP1027 had slightly higher editing efficiency than the pseudouridine-containing mRNA of LNP1032. The mRNA containing both pseudouridine and 5-methylcytosine nucleosides (LNP1033) had significantly reduced efficacy. The mRNA containing 25% 5-iodouridine showed equivalent editing efficiency to the mRNA containing N1-methylpseudouridine. At 50% 5-iodouridine, the efficacy was reduced. The 5-methoxyuridine mRNA from Trilink showed lower activity.

[0634] 9. Characterization of the Effects of mRNAs with Different UTRs in Rats

[0635] This study evaluated the in vivo efficacy of ARCA-capped Cas9 mRNA with HBB (human β-hemoglobin) 5' and 3' UTRs; XBG (Xenopus β-hemoglobin) 5' and 3' UTRs; or with human HSD17B4 (HSD) 5' UTR and albumin (ALB) 3' UTR in rats.

[0636] Formulations containing guide RNA targeting rat TTR gene (G534; SEQ ID NO: 72) and Cas9 mRNA at a 1:1 molar ratio in LNPs were prepared using the cross-flow method described above and plated on the VivaFlow TM 50 membranes were filtered. The LNPs contained cationic lipids (lipid A), cholesterol, DSPC and PEG2k-DMG in a molar ratio of 45:9:43:3 and had an N:P ratio of 6.0. The formulations were dosed at 1 mpk and 0.3 mpk. All rats were Sprague-Dawley females from Charles River, n=5 per group. At necropsy (7 days after dosing), serum was collected for TTR analysis and liver was collected for editing analysis. In LNP1058, the mRNA contained the HBB UTR. In LNP1059, the mRNA contained the XBG UTR. In LNP1060, the mRNA contained the HSD and ALB 5' and 3' UTRs, respectively. In all cases, the mRNA coding sequence is according to SEQ ID NO:4.

[0637] Liver editing and serum TTR results are shown in FIG. 21A to FIG. 21C And Table 23.

[0638] Table 23. Liver editing and serum TTR results in rats using LNP1058 to LNP1060.

[0639]

[0640] The results showed that all tested mRNAs in LNP1058 to LNP1060 were able to support editing. The highest degree of editing and the most significant reduction in serum TTR were seen with the mRNA containing the XBG UTR in LNP1059.

[0641] 10. RNA loading: co-deployment of mRNA and gRNA

[0642] This study evaluated the in vivo efficacy of different gRNA to mRNA ratios in mice. CleanCap was prepared by IVT synthesis as indicated in Example 1 using N1-methylpseudouridine triphosphate instead of uridine triphosphate. TM The capped Cas9 mRNA has the ORF of SEQ ID NO: 4, HSD 5'UTR, human albumin 3'UTR, Kozak sequence and poly-A tail.

[0643] LNP formulations were prepared from the mRNA and sg282 (SEQ ID NO: 42; G282) as described in Example 2 with lipid A, cholesterol, DSPC, and PEG2k-DMG at a 55:33:9:3 molar ratio and at an N:P ratio of 6. The gRNA:Cas9 mRNA weight ratios of the formulations are shown in Table 24.

[0644] Table 24. Characterization of LNP1110 to LNP1116.

[0645]

[0646] For in vivo characterization, the above LNPs were administered to mice at 0.1 mg total RNA (mg guide RNA + mg mRNA) per kg (n=5 per group). Animals were sacrificed 7-9 days after dosing, blood and liver were collected, and serum TTR and liver editing were measured as described above. Serum TTR and liver editing results are shown in Fig.22A and Fig. 22B Negative control mice were dosed with TSS vehicle.

[0647] In addition, the above LNPs were administered to mice at a constant mRNA dose of 0.05 mg mRNA per kilogram, while the gRNA dose was changed from 0.06 mg / kg to 0.4 mg / kg (n=5 per group). 7 to 9 days after dosing, the animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured. Serum TTR and liver editing results are shown in Fig. 22C and Fig.22D Negative control mice were dosed with TSS vehicle.

[0648] 11. Characterization of the codon scheme

[0649] Cas9 sequences using different codon schemes were designed to be tested for improved protein expression. Each sequence was designed to encode the Cas9 amino acids of SEQ ID No:3 using a unique set of codons. In each open reading frame sequence, a single codon was used to encode each amino acid. The sequence was changed based on the frequency of occurrence of codons in the entire protein-coding gene in Homo sapiens based on NCBI-GenBank Flat File Release 160.0 (Nakamura et al. (2000) Nucl. Acids Res. 28, 292; Benson et al. (2006) Nucleic Acids Res. 34 (database period), D16-20) and the abundance of specific nucleotides in codons. Based on the codon scheme shown in Table 4, seven different Cas9 open reading frames (SEQ ID NO: 52, SEQ ID NO: 54 and SEQ ID NO: 108 to SEQ ID NO: 112) encoding the Cas9 protein of SEQ ID NO: 3 were constructed. These were incorporated into a construct that also contained an HSD 5'UTR (SEQ ID NO: 41), an albumin 3'UTR, a T7 promoter, and a poly A tail. An exemplary sequence containing an albumin 3'UTR and a poly A tail is SEQ ID NO: 53, wherein the 3'UTR and poly A tail follow the HSD 5'UTR and the ORF of SEQ ID NO: 52. A similarly constructed construct encoding the Cas9 protein of SEQ ID NO: 3 (SEQ ID NO: 107, using the long half-life codon set of Table 4) using a codon scheme based on optimal codons for improved mRNA half-life as described by Presnyak and colleagues (2015) was also included in these evaluations.

[0650] Messenger RNA for each construct was generated by IVT. 800 ng of each Cas9 mRNA was used with Lipofectamine TM MessengerMAX TMTransfection agent (ThermoFisher) transfected HepG2 cells. Six hours after transfection, cells were lysed by freeze-thaw and cleared by centrifugation. Cas9 protein content was determined by ELISA analysis. In brief, total protein concentration was determined by bicinchoninic acid (protein) assay. MSD GOLD 96-well streptavidin SECTOR plates (Meso Scale Diagnostics, catalog number L15SA-1) were prepared according to the manufacturer's protocol using Cas9 mouse antibodies (Origene, catalog number CF811179) as capture antibodies and Cas9 (7A9-3A3) mouse mAb (Cell Signaling Technology, catalog number 14697) as detection antibodies. In 1×Halt 500 μL of EDTA-free medium, 10% PBS was added to the plate. The plate was then plated with 1% PBS and 1% PBS. TM Cas9 protein at 0, 0.12, 0.49, 1.95, 7.81, 31.25, 125 and 500 ng / mL in diluent 39 of protease inhibitor cocktail (ThermoFisher, catalog number 78437) was used as calibration standard. ELISA plates were read using a Meso Quickplex SQ120 instrument (Meso Scale Discovery) and data were analyzed with the Discovery Workbench 4.0 software package (Meso Scale Discovery).

[0651] Editing efficiency was assessed in vitro by transfecting mRNA and a guide targeting thyroxine transporter (TTR) (G502; SEQ ID NO: 70) into HepG2 cells and measuring the percentage of editing. Cas9 mRNAs comprising the SEQ ID Nos indicated in Table 25 were assessed at mRNA concentrations ranging from 3 ng to 100 ng. Untreated cells showed no measurable editing. Figure 23 to Figure 24 And Table 25 shows the effects of different codon groups on Cas9 protein expression and in vitro editing.

[0652] Table 25. In vitro editing and expression of ORFs with different codon sets.

[0653]

[0654] To determine the in vivo effectiveness of the codon scheme, Cas9 protein expression was measured when the mRNA encoding Cas9 was expressed in vivo using the codon scheme described in Table 4. Messenger RNAs as indicated in Table 26 were formulated into LNPs with guide RNAs targeting TTR (G282; SEQ ID NO: 42). LNPs were assembled using a cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had an N:P ratio of 6.0. LNPs were purified using an Amicon PD-10 filter (GE Healthcare) and used at a concentration of 0.32 mg / ml (LNP concentration). CD-1 female mice (n=5 per group) were dosed intravenously at 1 mpk. At 3 hours after dosing, the animals were sacrificed, the livers were collected, and Cas9 expression in the liver was measured. Cas9 protein expression in the liver was measured using the Meso Scale Discovery ELISA assay described above. Approximately 40 mg of liver tissue was homogenized by bead mill in RIPA buffer (Boston Bioproducts BP-115) with 1× Complete protease inhibitor tablet (Roche, cat. no. 11836170001). Fig.25 Table 26 shows the results of Cas9 expression in the liver. The mRNA of low A and low A / U codon schemes (ORFs of SEQ ID NO: 111 and SEQ ID NO: 112) showed the highest Cas9 expression of the ORFs tested. The Cas9 protein expression of the negative control and the ORF of SEQ ID NO: 54 was less than the lower limit of quantification (LLOQ).

[0655] Table 26

[0656] ORF Average Cas9 (nanograms per gram of liver) Standard Deviation TSS <LLOQ 0.0 SEQ ID No:4 1644 1172 SEQ ID NO:52 1562 951 SEQ ID NO:54 <LLOQ 0.0 SEQ ID NO:111 2630 730 SEQ ID NO:112 2134 362

[0657] To determine the in vivo effectiveness of the codon scheme, genome editing was measured in vivo from mRNA encoding Cas9 using different codon schemes. Messenger RNAs as indicated in Table 27 were formulated into LNPs with guide RNAs targeting TTR (G282; SEQ ID NO: 42). LNPs were assembled using a cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had an N:P ratio of 6.0. LNPs were purified using an Amicon PD-10 filter (GE Healthcare) and used at a concentration of 0.05 mg / ml (LNP concentration). CD-1 female mice (n=5 per group, except for the group treated with SEQ ID NO: 52, n=4) were dosed intravenously at 0.1 mpk. Six days after dosing, the animals were sacrificed, blood and livers were collected, and serum TTR and liver editing were measured. Table 27 and Fig.26 The results of in vivo editing are shown in Table 27 and FIG. 27A to FIG. 27B Display serum TTR content.

[0658] Table 27

[0659]

[0660]

[0661] In order to determine the efficacy of the codon scheme at different mRNA concentrations, an in vivo dose-response experiment was performed. The messenger RNA as indicated in Table 28 was formulated into LNPs with a guide RNA targeting TTR (G282; SEQ ID NO: 42). The LNPs were assembled using a cross-flow method and contained 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG. The LNPs were purified using an Amicon PD-10 filter (GE Healthcare) and used at a concentration of 0.7 mg / ml (LNP concentration). CD-1 female mice (n=5 per group) were dosed intravenously at 0.03, 0.1, or 0.3 mpk. Seven days after dosing, the animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured. Table 28 and Fig.28 The results of in vivo editing are shown in Table 28 and FIG. 29A to FIG. 29B Display serum TTR content.

[0662] Table 28

[0663]

[0664] To determine the effectiveness of codon schemes with different UTRs, genome editing was measured in vivo after administration of mRNA encoding Cas9. Messenger RNAs as indicated in Table 29 were formulated into LNPs with guide RNAs targeting TTR (G282; SEQ ID NO: 42). LNPs were assembled using a cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had an N:P ratio of 6.0. LNPs were purified using an Amicon PD-10 filter (GE Healthcare) and used at a concentration of 0.05 mg / ml (LNP concentration). CD-1 female mice (n=5 per group; n=4 for SEQ ID NO: 43 editing) were dosed intravenously at 0.1 mpk. Six days after dosing, the animals were sacrificed, blood and livers were collected, and serum TTR and liver editing were measured. Table 29 and FIG. 30A to FIG. 30B In vivo editing (B) and serum TTR results (A) are shown.

[0665] Table 29

[0666] mRNA constructs edit% Standard Deviation Serum TTR (μg / ml) Standard Deviation TSS 0 0 1274 214 SEQ ID No:43 28 4 630 152 SEQ ID No:176 35 8 482 138 SEQ ID No:177 37 9 316 143 SEQ ID No:178 42 6 524 192

[0667] 12. Characterization of the Effect of Capping Structure

[0668] mRNA encoding Cas9 and containing a cap structure, UTR, and poly A tail as indicated in Table 30 was formulated into LNPs with a guide RNA targeting TTR (G282; SEQ ID NO: 42). LNPs were assembled using a cross-flow procedure, containing 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and having an N:P ratio of 6.0. LNPs were purified using an Amicon PD-10 filter (GE Healthcare) and used at a concentration of 0.06 mg / ml (LNP concentration). CD-1 female mice (n=5 per group) were dosed intravenously at 0.1 or 0.3 mpk. Seven days after dosing, the animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured.

[0669] Fig.31 Table 30 shows that at 0.1 mpk, mRNA with Cap 1 had an average editing that was about 10% higher than mRNA with Cap 0. At 0.3 mpk, mRNA with XBG UTR had a slightly higher average editing than mRNA with HSD UTR, except for enzyme cap 0. Serum TTR results are shown in Fig.32 (Serum TTR results are expressed as μg / mL and % of TSS control, respectively); Fig.31(Liver edit); and Table 30.

[0670] Table 30. Serum TTR and liver editing results for in vivo capping studies

[0671]

[0672]

[0673] 13. Characterization of Nuclear Localization Signals

[0674] Cas9 sequences using several nuclear localization signals (NLS) were designed and tested to determine efficacy. Eleven atypical NLSs of varying strength were selected from the NLSs identified by Kosugi et al. (2009) Journal of Biological Chemistry, 284(1), 478-485, as shown in Table 31. These amino acid sequences were added to the carboxyl terminus of the Cas9 amino acid sequence (SEQ ID NO: 13). The control sequence encodes SEQ ID No. 4.

[0675] Table 31

[0676]

[0677]

[0678] mRNA encoding Cas9 with NLS as indicated in Table 31 was formulated into LNPs with a guide RNA targeting TTR (G282; SEQ ID NO: 42). LNPs were assembled using a cross-flow procedure and contained 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG in a molar ratio of 50:38:9:3, respectively, and had an N:P ratio of 6.0. LNPs were purified using an Amicon PD-10 filter (GE Healthcare) and used at a concentration of 0.07 mg / ml (LNP concentration). CD-1 female mice (n=5 per group) were dosed intravenously at 0.1 mpk. Seven days after dosing, the animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured. The results are shown in Tables 32 and Fig.33 For SEQ ID NOs corresponding to the NLS listed in Table 32, see Table 31.

[0679] Table 32 - Liver editing with different nuclear localization signals

[0680]

[0681]

[0682] NLS5 showed a statistically significant increase compared to the SV40 NLS (one-way ANOVA, p=0.006). NLS4 and NLS8 each showed a possible trend toward increased editing compared to the SV40 NLS, but the differences were not statistically significant in this experiment. FIG. 34A to FIG. 34B Serum TTR levels after administration of nuclear localization signal variants are shown. Kosugi et al. (2009) (see above) rated the activity of NLS for the degree of nuclear localization ("NLS intensity" in Table 32), where 10 is completely in the nucleus and 1 is diffuse throughout the cell. The NLS activity rated in this literature is positively correlated with editing efficiency, such as Fig.35 as shown in .

[0683] 14. In vitro characterization of the effects of UTR

[0684] Table 33 and Fig.36 Cas9 expression from transcripts with different 5'UTRs is shown. All constructs used a 3' human albumin UTR. Messenger RNA for each construct was generated by IVT. Messenger RNA for SEQ ID No: 179 was generated using a linearized plasmid, and all others were generated using PCR products as templates. Lipofectamine was used with 100 ng of each Cas9 mRNA and a guide targeting transthyretin (TTR) (G502; SEQ ID NO: 70) at a final concentration of 25 nM. TM MessengerMAX TM HepG2 cells were transfected with 1% paraformaldehyde (1% paraformaldehyde) and 1% paraformaldehyde (1% paraformaldehyde) using a transfection agent (ThermoFisher). Cells were lysed using the HiBiT lysis assay (Promega). Nano-Glo HiBiT extracellular detection system (Promega, catalog number N2420) was used to measure the Cas9 protein content. Fig.36 Cas9 expression from transcripts with different 5'UTRs is shown.

[0685] Table 33: Cas9 expression

[0686]

[0687]

[0688] 15. LNP Delivery to Non-Human Primates

[0689] Three studies were conducted with LNP formulations prepared using the X-flow / TFF method as described above. Specific molar amounts and loadings are provided in Tables 34 to 36. Each formulation containing Cas9 mRNA and guide RNA (gRNA) has a mRNA:gRNA ratio of 1:1 by weight. The dosage of LNP (in mg / kg, total RNA content), route of administration, and whether the animal received pretreatment with dexamethasone are indicated in the table. For animals pretreated with dexamethasone (Dex), Dex was administered at 2 mg / kg by IV rapid injection 1 hour before administration of LNP or vehicle.

[0690] For blood chemistry analysis, animals were bled at the time indicated in the table for each factor measured. Cytokine induction was measured in NHP before and after treatment. A minimum of 0.5 mL of whole blood was collected in a 4 ml serum separation tube from the peripheral vein of a restrained awake animal. The blood was clotted for a minimum of 30 minutes at room temperature, then centrifuged at 2000 × g for 15 minutes. Serum was aliquoted in 2 120 μL polypropylene microtubes, and each was stored at -60°C to -86°C before analysis. The non-human primate U-Plex cytokine custom kit from Meso Scale Discovery (MSD) was used to analyze. The following parameters were included in the analysis: INF-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, MCP-1 and TNF-α, with a focus on IL-6 and MCP-1. Kit reagents and standards were prepared as directed in the manufacturer's protocol. NHP serum was used in pure form. The plates were run on an MSD Sector Imager 6000 with analysis performed using MSD Discovery work bench software version 4012.

[0691] Complement content was measured by enzyme immunoassay in animals before and after treatment. Whole blood of 0.5 mL volume was collected in 0.5 mL k2EDTA tubes from a peripheral vein of restrained conscious animals. The blood was centrifuged at 2000 × g for 15 minutes. The plasma was aliquoted in 2 120 μL polypropylene microtubes and stored at -60°C to -86°C before analysis. The Quidel MicroVue Complement Plus EIA kit (C3a-Cat No. A031) or (Bb-Cat No. A027) was used for analysis. The kit reagents and standards were prepared as directed in the manufacturer's protocol. The plate was operated on an MSDSector Imager 6000 at an optical density of 450 nm. The results were analyzed using a 4-parameter curve fit.

[0692] Data for cytokine induction and complement activation are provided in the table below. "BLQ" means less than the limit of quantitation.

[0693] Table 34, Study 1

[0694]

[0695] Table 35, Study 2

[0696]

[0697]

[0698]

[0699] Table 36, Study 3

[0700]

[0701]

[0702] Table 37. IL-6 Measurements According to Study 1

[0703] Treatment Group Before blood collection 6 hours 24 hours (1) TSS (medium) 5.71±2.70 29.1±20.37 7.05±3.49 (2)LNP699G502 9.73±8.34 1296.41±664.71 5.43±7.68 (3)LNP688G506 16.83±4.08 1749.47±1727.22 38.57±39.39 (4)LNP689G509 18.11±11.51 1353.49±766.66 32.42±18.40 (5)LNP690G510 13.95±1.85 11838±17161.74 90.07±96.02

[0704] Table 38. MCP-1 Measurements According to Study 1

[0705] Treatment Group Before blood collection 6 hours 24 hours (1) TSS (medium) 810.49±178.27 1351.16±397.31 745.25±56.49 (2)LNP699G502 842.31±350.65 19298.49±11981.14 2092.89±171.21 (3)LNP688G506 1190.79±383.64 13500.17±12691.60 1414.71±422.43 (4)LNP689G509 838.63±284.42 14427.7±8715.48 1590±813.23 (5)LNP690G510 785.32±108.97 52557.24±48034.68 6319.77±983.37

[0706] Table 39. Complement C3a Measurements According to Study 1

[0707] Treatment Group Before blood collection 6 hours Day 7 (1) TSS (medium) 23.9±11.95 25.51±14.79 30.67±18.36 (2)LNP699G502 32.36±11.29 94.33±58.45 38.50±12.69 (3)LNP688G506 22.30±1.73 127.00±22.34 37.80±6.86 (4)LNP689G509 35.83±21.94 174.00±44.51 50.83±21.92 (5)LNP690G510 36.30±8.21 163.00±40.60 42.50±12.44

[0708] Table 40. Complement bb measurements according to Study 1

[0709] Treatment Group 04-bb Before blood collection 6 hours Day 7 (1) TSS (medium) Comparison 1.53±0.19 3.37±2.13 1.43±0.71 (2)LNP699G502 G502 1.45±0.39 9.01±5.28 1.57±0.54 (3)LNP688G506 G506 1.45±0.78 11.78±2.33 1.78±0.84 (4)LNP689G509 G509 1.95±0.99 15.73±2.23 2.83±0.88 (5)LNP690G510 G510 2.12±0.44 13.57±1.23 2.21±0.72

[0710] Table 41. IL-6 measurements according to Study 2

[0711] Treatment Group Before blood collection 90 minutes 6 hours 24 hours Day 7 (1) TSS (medium) 1.77 11.46 4.2 2.76 3.01 (2) TSS (medium) 5.23 18.11 20.36 13.2 6.36 (3)LNP898G502 2.02 1305.75 1138.22 383.32 16.02 (4)LNP898G502 2.34 37.19 91.59 14.11 3.07 (5)LNP897G502 2.1 55.79 6.89 2.26 2.01 (6)LNP897G502 6.8 10.1 44.72 5.4 2.01 (7)LNP897G502 1.97 44.87 32.61 2.97 1.11 (8)LNP897G502 3.14 37.68 73.41 8.58 2.22 (9) LNP916GFP 1.6 BLQ 95.32 27.58 BLQ (10)LNP916GFP 2.43 BLQ 883.01 66.71 BLQ

[0712] Table 42. MCP-1 Measurements According to Study 2

[0713]

[0714]

[0715] Table 43. Complement C3a Measurements According to Study 2

[0716] Treatment Group Before blood collection 90 minutes 6 hours 24 hours Day 7 (1) TSS (medium) 0.087 0.096 0.048 0.033 0.038 (2) TSS (medium) 0.369 0.311 0.146 0.1 0.106 (3)LNP898G502 0.087 0.953 0.647 0.277 0.065 (4)LNP898G502 0.099 0.262 0.123 0.049 0.044 (5)LNP897G502 0.067 0.479 0.209 0.036 0.036 (6)LNP897G502 0.141 0.433 0.34 0.11 0.074 (7)LNP897G502 0.1 0.345 0.396 0.096 0.127 (8)LNP897G502 0.261 0.458 0.409 0.244 0.313 (9) LNP916GFP 0.149 BLQ 0.714 0.382 BLQ (10)LNP916GFP 0.117 BLQ 0.752 0.723 BLQ

[0717] Table 44. Complement bb measurements according to Study 2

[0718] Treatment Group Before blood collection 90 minutes 6 hours 24 hours Day 7 (1) TSS (medium) 0.087 0.096 0.048 0.033 0.038 (2) TSS (medium) 0.369 0.311 0.146 0.1 0.106 (3)LNP898G502 0.087 0.953 0.647 0.277 0.065 (4)LNP898G502 0.099 0.262 0.123 0.049 0.044 (5)LNP897G502 0.067 0.479 0.209 0.036 0.036 (6)LNP897G502 0.141 0.433 0.34 0.11 0.074 (7)LNP897G502 0.1 0.345 0.396 0.096 0.127 (8)LNP897G502 0.261 0.458 0.409 0.244 0.313 (9) LNP916GFP 0.149 BLQ 0.714 0.382 BLQ (10)LNP916GFP 0.117 BLQ 0.752 0.723 BLQ

[0719] Table 45. IL-6 measurements according to Study 3

[0720]

[0721]

[0722] Table 46. MCP-1 Measurements According to Study 2

[0723]

[0724] Table 47. Complement C3a Measurements According to Study 3

[0725]

[0726]

[0727] Table 48. Complement bb measurements according to Study 3

[0728] Treatment Group Before blood collection 90 minutes 6 hours 24 hours Day 7 (1)TSS 1.46±0.70 2.18±0.78 1.96±0.64 0.945±0.15 1.34±0.50 (2)LNP1021G502 1.77±0.60 6.51±3.66 11.00±4.85 3.59±2.25 2.07±0.93 (3)LNP1021G502 1.24 2.90 11.50 2.97 1.24 (4)LNP1022G502 1.52±0.34 5.67±2.28 10.2±3.36 3.66±1.68 1.84±0.24 (5)LNP1023G502 1.65±0.94 4.4±1 7.68±4.67 2.64±1.18 2.08±1.32 (6)LNP1024G509 1.61±0.13 4.52±1.81 4.50±3.22 1.63±0.84 1.63±0.32 <h2 style=";text-align:left;direction:ltr">(7)LNP1024G509 0.96 2.99 2.64 1.13 1.07 <h2 style=";text-align:left;direction:ltr"> (8)LNP1025G509 1.37±0.17 4.9±4.51 3.79±3.84 1.66±1.43 1.35±0.44 <h2 style=";text-align:left;direction:ltr"> (9)LNP1021G502 1.41 5.67 11.50 4.64 1.38 <h2 style=";text-align:left;direction:ltr"> (10)LNP1022G502 1.28 5.22 14.10 5.64 1.87

[0729] 16. Comparison of Cas9 expression of different mRNAs in mouse liver

[0730] Cas9 expression was measured in vivo after administration of different mRNAs encoding Cas9. Messenger RNAs as indicated in Table 49 were formulated into LNPs with mouse sgRNA targeting the mouse TTR gene (sgRNA:mRNA weight ratio of 1:2). LNPs with 50% lipid A, 9% DSPC, 38% cholesterol, and 3% PEG2k-DMG and an N:P ratio of 6.0 were assembled using a cross-flow procedure. LNPs were purified using Sartocon Slice 200 (Sartorius) and used at a concentration of 1.53 mg / ml (RNA concentration). LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content, and encapsulation efficiency of RNA as described above (data not shown).

[0731] CD-1 female mice (n=5 per group) were dosed intravenously at 0.3 mpk. At 1 hour, 3 hours and 6 hours after dosing, the animals were sacrificed, liver tissues were collected, and Cas9 protein levels were measured by MSD ELISA as described in Example 11. Table 49 shows Cas9 protein levels. At each time point, more Cas9 protein was detected in animals treated with SEQ ID NO: 177 than in animals treated with SEQ ID NO: 43.

[0732] Table 49

[0733]

[0734] 17. Comparison of dose responses of different mRNAs

[0735] Comparison of in vivo dose response curves of different mRNAs encoding Cas9. LNP formulations were prepared with mRNAs of SEQ ID No. 43 and SEQ ID No. 177 and sg502 (SEQ ID NO: 70; G502), formulated as described in Example 16. The lipid nanoparticle components were dissolved in 100% ethanol, wherein the lipid component molar ratio was 50 / 9 / 38 / 3 (LP01 / DSPC / cholesterol / PEG-DMG). LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6 and a gRNA to mRNA ratio of 1:2 by weight. LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content, and encapsulation efficiency of RNA as described above (data not shown).

[0736] For in vivo characterization, CD-1 female mice (n=5 per group) were dosed intravenously at 0.03, 0.1, or 0.3 mg total RNA (mg guide RNA + mg mRNA) per kg (n=5 per group). Seven days after dosing, animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured as described in Example 1. Negative control animals were dosed with TSS vehicle. Editing data are provided in Table 50 below. For SEQ ID NO:43, an average of 8 in vivo experiments with 5 animals each is provided. For SEQ ID NO:177, an average of in vivo experiments with 5 animals at each dose is provided. At each dose, the % editing in animals treated with SEQ ID NO:177 was higher than that in animals treated with SEQ ID NO:43.

[0737] Table 50

[0738]

[0739] Sequence Listing

[0740] The following sequence listing provides a list of the sequences disclosed herein. It should be understood that if a DNA sequence (comprising Ts) is referred to with respect to RNA, the Ts should be replaced by Us (which may be modified or unmodified as appropriate), and vice versa.

[0741]

[0742]

[0743]

[0744]

[0745]

[0746]

[0747]

[0748]

[0749]

[0750]

[0751]

[0752]

[0753]

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776]

[0777]

[0778]

[0779]

[0780]

[0781]

[0782]

[0783]

[0784]

[0785]

[0786]

[0787]

[0788]

[0789]

[0790]

[0791]

[0792]

[0793]

[0794]

[0795]

[0796]

[0797]

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827]

[0828]

[0829]

[0830]

[0831]

[0832]

[0833]

[0834]

[0835]

[0836]

[0837]

[0838]

[0839]

[0840]

[0841]

[0842]

[0843]

[0844]

[0845]

[0846]

[0847]

[0848]

[0849]

[0850]

[0851]

[0852]

[0853]

[0854]

[0855]

[0856]

[0857]

[0858]

[0859]

[0860]

[0861]

[0862]

[0863]

[0864]

[0865]

[0866]

[0867]

[0868]

[0869]

[0870]

[0871]

[0872]

[0873]

[0874]

[0875]

[0876]

[0877]

[0878]

[0879]

[0880]

[0881]

[0882]

[0883]

[0884]

[0885]

[0886]

[0887]

[0888]

[0889] * = PS bond; 'm' = 2'-O-Me nucleotide

Claims

1. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine content of the open reading frame is within the range of its minimum uridine content to 150% of the minimum uridine content.

2. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the uridine dinucleotide content of the open reading frame is within the range of its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content.

3. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the adenine content of the open reading frame is within the range of its minimum adenine content to 150% of the minimum adenine content.

4. An mRNA comprising an open reading frame encoding an RNA-guided DNA-binding agent, wherein the adenine dinucleotide content of the open reading frame is within the range of its minimum adenine dinucleotide content to 150% of the minimum adenine dinucleotide content.

5. An mRNA comprising a sequence that is at least 90% identical to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107 to 175, wherein the mRNA comprises an open reading frame encoding an RNA-guided DNA binder.

6. An mRNA comprising an open reading frame encoding an RNA-guided DNA binder, wherein the open reading frame has at least 90% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, 66, or 107 to 175 over at least the first 30, 50, 70, 100, 150, 200, 250, or 300 nucleotides thereof.

7. An mRNA encoding an RNA-guided DNA binder, the RNA-guided DNA binder comprising an open reading frame encoding the RNA-guided DNA binder, wherein the open reading frame consists of a set of codons, wherein at least 75% of the codons are codons listed in Table 1, Table 2, Table 3 or (ii) a set of codons listed in Table 4.

8. An expression construct comprising a promoter operably linked to a sequence encoding the mRNA as claimed in any one of the preceding claims.

9. A plasmid comprising the expression construct of claim 8.

10. A host cell comprising the expression construct of claim 8 or the plasmid of claim 9.

11. A method for preparing mRNA, the method comprising contacting the expression construct of claim 8 or the plasmid of claim 9 with an RNA polymerase under conditions that allow transcription of the mRNA.

12. A composition comprising the mRNA of any one of claims 1 to 7 and at least one guide RNA.

13. A lipid nanoparticle comprising the mRNA according to any one of claims 1 to 7. 14 . A pharmaceutical composition comprising the mRNA according to claim 1 and a pharmaceutically acceptable carrier.

15. A method for genome editing or modifying a target gene, comprising contacting a cell with the mRNA, expression construct, composition or lipid nanoparticle of any one of claims 1 to 7 or 8 to 13.

16. Use of an mRNA, expression construct, composition or lipid nanoparticle as described in any one of claims 1 to 7 or 8 to 13 for genome editing or modifying a target gene.

17. Use of an mRNA, expression construct, composition or lipid nanoparticle as described in any one of claims 1 to 7 or 8 to 13 for the manufacture of a medicament for genome editing or modifying a target gene.

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