formulation

Abstract

To provide compositions using lipid nanoparticles, having properties useful for delivery of CRISPR / Cas gene editing components.SOLUTION: A lipid nanoparticle (LNP) composition comprises: an RNA component; about 50-60 mol% of amine lipid; about 8-10 mol% of neutral lipid; and about 2.5-4 mol% of PEG lipid, with the remainder of the lipid component being helper lipid, and the N / P ratio of the LNP composition being about 6.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 566,240, filed September 29, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Provided herein are lipid nanoparticle ("LNP") compositions with improved properties for delivering biologically active agents, particularly RNA, mRNA, and guide RNA. The LNP compositions facilitate the delivery of RNA agents across cell membranes, and in certain embodiments, such compositions introduce gene-editing components and compositions into living cells. [Background technology]

[0003] Biologically active agents that are particularly difficult to deliver into cells include proteins, nucleic acid-based drugs, and their derivatives. Of particular interest are compositions for delivering promising gene editing technologies into cells, such as CRISPR / Cas9 system components.

[0004] Numerous components and systems currently exist for editing cellular genes in vivo, offering enormous potential for the treatment of disease. The CRISPR / Cas gene editing system is active within cells as a ribonucleoprotein complex. RNA-dependent nucleases bind to and induce breaks in DNA sequences within the cell. This site-specific nuclease activity facilitates gene editing through the cell's own natural processes. For example, cells respond to double-stranded DNA breaks (DSBs) using an error-prone repair process known as non-homologous end joining (NHEJ). During NHEJ, cells may add or remove nucleotides at the DNA end, resulting in an altered sequence from the broken sequence. In other situations, cells repair DSBs through mechanisms known as homology-directed repair (HDR) or homologous recombination (HR), which can use endogenous or exogenous templates to guide the repair of the break. Some of these editing techniques exploit the cellular machinery that repairs single-strand breaks (SSBs) or DSBs.

[0005] There is a need for compositions for delivering CRISPR / Cas protein and nucleic acid components to cells, such as patient cells. Of particular interest are compositions for delivering mRNA encoding CRISPR protein components and compositions for delivering CRISPR guide RNA. Of particular interest are compositions that can stabilize and deliver RNA components and have properties useful for in vitro and in vivo delivery. Summary of the Invention

[0006] Provided herein are compositions that utilize lipid nanoparticles that have useful properties, particularly properties that are useful for the delivery of CRISPR / Cas gene editing components.

[0007] In certain embodiments, an LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises (1) about 50-60 mol% amine lipid, (2) about 8-10 mol% neutral lipid, and (3) about 2.5-4 mol% PEG lipid, with the remainder of the lipid component being helper lipid, and the N / P ratio of the LNP composition is about 6. In further embodiments, the LNP composition comprises (1) an RNA component, (2) about 50-60 mol% amine lipid, (3) about 27-39.5 mol% helper lipid, (4) about 8-10 mol% neutral lipid, and (5) about 2.5-4 mol% PEG lipid, wherein the N / P ratio of the LNP composition is about 5-7.

[0008] In another embodiment, the LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises (1) about 50-60 mol% amine lipid, (2) about 5-15 mol% neutral lipid, and (3) about 2.5-4 mol% PEG lipid, with the remainder of the lipid component being helper lipid, and the N / P ratio of the LNP composition is about 3 to 10. In a further embodiment, the LNP composition comprises a lipid component comprising (1) about 40-60 mol% amine lipid, (2) about 5-15 mol% neutral lipid, and (3) about 2.5-4 mol% PEG lipid, with the remainder of the lipid component being helper lipid, and the N / P ratio of the LNP composition is about 6. In another embodiment, the LNP composition comprises a lipid component comprising: (1) about 50-60 mol% amine lipid; (2) about 5-15 mol% neutral lipid; and (3) about 1.5-10 mol% PEG lipid, wherein the remainder of the lipid component is a helper lipid, and the N / P ratio of the LNP composition is about 6.

[0009] In some embodiments, an LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises (1) about 40-60 mol% amine lipids, (2) about 0-5 mol% neutral lipids, e.g., phospholipids, and (3) about 1.5-10 mol% PEG lipids, with the remainder of the lipid component being helper lipids, and the N / P ratio of the LNP composition is about 3-10. In some embodiments, an LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises (1) about 40-60 mol% amine lipids, (2) less than about 1 mol% neutral lipids, e.g., phospholipids, and (3) about 1.5-10 mol% PEG lipids, with the remainder of the lipid component being helper lipids, and the N / P ratio of the LNP composition is about 3-10. In certain embodiments, the LNP composition is essentially free of neutral lipids. In some embodiments, an LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises (1) about 40-60 mol % amine lipids and (2) about 1.5-10 mol % PEG lipids, with the remainder of the lipid component being helper lipids, the N / P ratio of the LNP composition being about 3-10, and the LNP composition is free of neutral lipids, e.g., phospholipids. In certain embodiments, the LNP composition is essentially free of, or free of, neutral phospholipids. In certain embodiments, the LNP composition is essentially free of, or free of, neutral lipids, e.g., phospholipids.

[0010] In certain embodiments, the RNA component comprises an mRNA, such as an RNA-guided DNA-binding factor (e.g., a Cas nuclease or a class 2 Cas nuclease). In certain embodiments, the RNA component comprises a gRNA. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the rate of TTR gene editing achieved in mouse liver after delivery of the CRISPR / Cas gene editing components, Cas9 mRNA and gRNA, in an LNP composition, following a single dose of 1 mpk (Figure 1A) or 0.5 mpk (Figure 1B). [Figure 2] 1 shows particle distribution data for LNP compositions containing Cas9 mRNA and gRNA. [Figure 3] The physicochemical properties of the LNP compositions are shown, comparing the logarithmic molar mass difference of the compositions (Figure 3A) and the average molecular weight measurements of the compositions (Figure 3B). [Figure 4] Polydispersity calculations analyzing the LNP compositions of Figure 3 are shown in Figure 4A and Burchard-Stockmeyer analysis in Figure 4B. [Figure 5]

[0049] Figure 5 describes the results of experiments evaluating the effect of LNP compositions with increasing PEG lipid concentrations on serum TTR knockdown, liver gene editing, and cytokine MCP-1 levels in rats after a single dose. Figure 5A graphically depicts serum TTR levels, Figure 5B graphically depicts editing rates in liver samples, and Figure 5C depicts MCP-1 levels in pg / mL. [Figure 6-1] We demonstrate that gene editing efficacy is maintained across a variety of PEG-lipids in LNP compositions, as measured by serum TTR levels (Figures 6A and 6B) and editing rates (Figure 6C). [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7-1] In mice, lipid A analogs are shown to effectively deliver gene-editing cargo in LNP compositions as measured by % editing in the liver after a single dose. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. [Figure 7-4] This is a continuation of Figure 7-3. [Figure 8-1] 1 shows dose-response curves of editing rates for various LNP compositions in primary cynomolgus monkey hepatocytes. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9-1] Figures 9A and 9B show serum TTR and editing rates after a single dose in mice with varying gRNA to mRNA ratios, and Figures 9C and 9D show serum TTR and editing rates in the liver after a single dose in mice with varying gRNA but constant amounts of Cas9 mRNA. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 10] Figures 10A and 10B show the results of serum TTR and liver editing after administration of LNP compositions with and without neutral lipids. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present disclosure provides embodiments of lipid nanoparticle (LNP) compositions of RNA, such as CRISPR / Cas component RNA ("cargo"), for delivery to cells and methods of their use. LNP compositions may exhibit improved properties compared to conventional delivery techniques. LNP compositions may contain an RNA component and a lipid component, as defined herein. In certain embodiments, the RNA component includes a Cas nuclease, such as a Class 2 Cas nuclease. In certain embodiments, the cargo or RNA component includes an mRNA encoding the Class 2 Cas nuclease and a guide RNA or a nucleic acid encoding the guide RNA. Methods of gene editing and generating engineered cells are also provided.

[0013] CRISPR / Cas cargo The CRISPR / Cas cargo delivered via the LNP formulation may include an mRNA molecule encoding a protein of interest. For example, it may include mRNA for expressing a protein such as green fluorescent protein (GFP) and an RNA-guided DNA-binding factor, or a Cas nuclease. LNP compositions are provided that include a Cas nuclease mRNA, such as a class 2 Cas nuclease mRNA, that enables intracellular expression of the Cas9 protein. Additionally, the cargo may contain one or more guide RNAs or nucleic acids encoding the guide RNAs. Template nucleic acids, for example, for repair or recombination, may also be included in the composition, or the template nucleic acids may be used in the methods described herein.

[0014] "mRNA" refers to a polynucleotide containing an open reading frame that can be translated into a polypeptide (i.e., can be used as a substrate for translation by ribosomes and aminoacylated tRNAs). mRNA can include a phosphate-sugar backbone, such as ribose residues or analogs thereof, e.g., 2'-methoxyribose residues. In some embodiments, the sugars of the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. Generally, mRNA does not contain substantial amounts of thymidine residues (e.g., 0 or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues, or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). mRNA can contain modified uridines at some or all of its uridine positions.

[0015] CRISPR / Cas nuclease system One component of the disclosed formulation is an mRNA encoding an RNA-guided DNA-binding factor, such as a Cas nuclease.

[0016] As used herein, "RNA-guided DNA-binding factor" refers to a polypeptide or polypeptide complex having RNA and DNA-binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA-binding factors include Cas cleavase / nickases and their inactive forms ("dCas DNA binders"). As used herein, "Cas nuclease" encompasses Cas cleavase, Cas nickase, and dCas DNA binders. Cas cleavase / nickases and dCas DNA binders include the Csm complex or Cmr complex of type III CRISPR systems, its subunits Cas10, Csm1, or Cmr2, the Cascade complex of type I CRISPR systems, its subunit Cas3, and class 2 Cas nucleases. As used herein, a "class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA-binding activity. Class 2 Cas nucleases include Class 2 Cas cleavase / nickases that additionally possess RNA-guided DNA cleavase or nickase activity (e.g., mutants H840A, D10A, or N863A), and Class 2 dCas DNA binders in which the cleavase / nickase activity has been inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 proteins (e.g., mutants N497A, R661A, Q695A, and Q926A), HypaCas9 proteins (e.g., mutants N692A, M694A, Q695A, and H698A), eSPCas9(1.0) proteins (e.g., mutants K810A, K1003A, and R1060A), and eSPCas9(1.1) proteins (e.g., mutants K848A, K1003A, and R1060A), 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. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety.See, e.g., Tables S1 and S3 of Zetsche. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015), Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0017] In some embodiments, the RNA-guided DNA-binding factor is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA-binding factor has cleavage activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA-binding factor 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 variants thereof. Examples of Cas9 nucleases include those in Type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, for example, the list in the next paragraph), as well as modified forms (e.g., engineered or mutant) thereof. See, e.g., US2016 / 0312198 A1, US2016 / 0312199 A1. Other examples of Cas nucleases include the Csm complex or Cmr complex, or its subunits Cas10, Csm1, or Cmr2, of type III CRISPR systems, and the Cascade complex, or its subunit Cas3, of type I CRISPR systems. In some embodiments, the Cas nuclease can be of a type IIA, type IIB, or type IIC system. For a discussion of various 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).

[0018] Non-limiting exemplary species from which Cas nucleases may 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 viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.、Crocosphaera watsonii、Cyanothece sp.、Microcystis aeruginosa、Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohlobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp.、Lachnospiraceae bacterium ND2006、、、Acaryochloris marina is included。.

[0019] 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 Acidaminococcus sp.. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, 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.

[0020] Wild-type Cas9 has two nuclease domains, RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target DNA strand. In some embodiments, the Cas9 nuclease comprises two or more RuvC domains and / or two or more HNH domains. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing a double-stranded break in the target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, cleave a single strand of dsDNA, or have no DNA cleavage or nickase activity. An exemplary Cas9 amino acid sequence is set forth as SEQ ID NO:3. An exemplary Cas9 mRNA ORF sequence, including the start and stop codons, is set forth as SEQ ID NO:4. An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is set forth as SEQ ID NO:10.

[0021] In some embodiments, chimeric Cas nucleases are used, in which one domain or region of such a protein is replaced with a portion of a different protein. In some embodiments, a 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.

[0022] In other embodiments, the Cas nuclease may be derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the 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 derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0023] In some embodiments, the RNA-guided DNA-binding factor has single-stranded nickase activity, i.e., it can cleave a single DNA strand, creating a single-stranded break, also known as a "nick." In some embodiments, the RNA-guided DNA-binding factor comprises a Cas nickase. A nickase is an enzyme that nicks dsDNA, i.e., cleaves one strand of a DNA double helix but not the other. In some embodiments, the Cas nickase is a variant of a Cas nuclease (e.g., a Cas nuclease described above), in which the endonucleolytic activity site has been inactivated, e.g., by one or more alterations (e.g., point mutations) in the catalytic domain. For a discussion of Cas nickases and exemplary catalytic domain modifications, see, e.g., U.S. Patent No. 8,889,356. In some embodiments, a Cas nickase, such as a Cas9 nickase, has an inactivated RuvC domain or HNH domain. The amino acid sequence of an exemplary Cas9 nickase is set forth as SEQ ID NO: 6. The ORF sequence of an exemplary Cas9 nickase mRNA, including start and stop codons, is set forth as SEQ ID NO: 7. The coding sequence of an exemplary Cas9 nickase mRNA suitable for inclusion in a fusion protein is set forth as SEQ ID NO: 11.

[0024] In some embodiments, the RNA-guided DNA-binding factor is modified to contain only one functional nuclease domain. For example, the factor protein may be modified to reduce its nucleic acid cleavage activity by mutating or completely or partially deleting one of the nuclease domains. In some embodiments, a nickase with a RuvC domain that has reduced activity is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with an HNH domain that has reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0025] In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2(CPF1_FRATN))).

[0026] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs complementary to the sense and antisense strands of a target sequence. In this embodiment, the guide RNA guides the nickase to the target sequence, where it introduces a DSB by nicking opposite strands of the target sequence (i.e., double nicking). In some embodiments, the use of double nicking can improve specificity and reduce off-target effects. In some embodiments, a nickase is used with two separate guide RNAs that target opposing DNA strands to create a double nick in the target DNA. In some embodiments, a nickase is used with two separate guide RNAs that are selected to be adjacent to each other to create a double nick in the target DNA.

[0027] In some embodiments, the RNA-guided DNA-binding factor lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding factor comprises a dCas DNA-binding polypeptide. The dCas polypeptide has DNA-binding activity but essentially no catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding factor or dCas DNA-binding polypeptide lacking cleavase and nickase activity is a variant of a Cas nuclease (e.g., a Cas nuclease described above), e.g., in which one or more alterations (e.g., point mutations) in its catalytic domain have inactivated its endonucleolytic activity. See, e.g., US2014 / 0186958 A1 and US2015 / 0166980 A1. An exemplary dCas9 amino acid sequence is set forth as SEQ ID NO:8. An exemplary Cas9 mRNA ORF sequence, including a start codon and a stop codon, is set forth as SEQ ID NO:9. An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is set forth as SEQ ID NO:12.

[0028] In some embodiments, the RNA-guided DNA-binding factor comprises one or more heterologous functional domains (eg, is or comprises a fusion polypeptide).

[0029] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding factor into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding factor may be fused to one to ten NLS(s). In some embodiments, the RNA-guided DNA-binding factor may be fused to one to five NLS(s). In some embodiments, the RNA-guided DNA-binding factor may be fused to one NLS. When one NLS is used, the NLS may be linked at the N-terminus or C-terminus of the RNA-guided DNA-binding factor sequence. The NLS may also be inserted internally within the RNA-guided DNA-binding factor sequence. In other embodiments, the RNA-guided DNA-binding factor may be fused to two or more NLSs. In some embodiments, the RNA-guided DNA-binding factor may be fused to two, three, four, or five NLSs. In some embodiments, the RNA-guided DNA-binding factor may be fused to two NLSs. In certain circumstances, the two NLSs may be identical or different (e.g., two SV40 NLSs). In some embodiments, the RNA-guided DNA binding factor is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA binding factor may be fused to two NLSs, one linked at the N terminus and one linked at the C terminus. In some embodiments, the RNA-guided DNA binding factor may be fused to three NLSs. In some embodiments, the RNA-guided DNA binding factor may not be fused to an NLS. In some embodiments, the NLS may be a monopartite sequence, such as the SV40 NLS PKKKRKV or PKKKRRV. In some embodiments, the NLS may be a bipartite sequence, such as the nucleoplasmin NLS KRPAATKKAGQAKKKK. In certain embodiments, a single PKKKRKV NLS may be linked at the C terminus of the RNA-guided DNA binding factor. Optionally, one or more linkers are included at the fusion site.

[0030] In some embodiments, the heterologous functional domain may be capable of altering the intracellular half-life of the RNA-guided DNA-binding factor. In some embodiments, the heterologous functional domain may be capable of extending the half-life of the RNA-guided DNA-binding factor. In some embodiments, the half-life of the RNA-guided DNA-binding factor may be shortened. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding factor. In some embodiments, the heterologous functional domain may be capable of decreasing the stability of the RNA-guided DNA-binding factor. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes such as, for example, 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-binding factor may be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the 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-inducible gene 15 (ISG15)), ubiquitin-related modifier 1 (URM1), neural progenitor cell expressed and developmentally downregulated protein 8 (NEDD8; also called 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 fold modifier 1 (UFM1), and ubiquitin-like protein 5 (UBL5).

[0031] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagged GFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, monomeric Azami Green, 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), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, m Examples of suitable fluorescent proteins include Kate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, JRed), and orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and / or an epitope 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, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, 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), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent protein.

[0032] In further embodiments, the heterologous functional domain may target the RNA-guided DNA-binding factor to a particular organelle, cell type, tissue, or organ, hi some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding factor to mitochondria.

[0033] In further embodiments, the heterologous functional domain may be an effector domain. When targeting an RNA-guided DNA-binding factor to its target sequence, for example, when targeting a Cas nuclease to a target sequence by a 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 transcription activation domain, or a transcription repressor domain. In some embodiments, the heterologous functional domain is a nuclease such as FokI nuclease. See, e.g., U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcription activator or a transcription repressor. See, for example, 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); and Gilbert et al., "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes," Cell 154:442-51 (2013). Thus, RNA-guided DNA-binding factors essentially become transcription factors that can be directed to bind to desired target sequences using guide RNAs.In certain embodiments, the DNA-modifying domain is a methylation domain, such as a demethylation domain or a methyltransferase domain. In certain embodiments, the effector domain is a DNA-modifying domain, such as a base-editing domain. In certain embodiments, the DNA-modifying domain is a nucleic acid-editing domain that introduces specific modifications into DNA, such as a deaminase domain. See, for example, WO2015 / 089406 and US2016 / 0304846. The nucleic acid-editing domains, deaminase domains, and Cas9 variants described in WO2015 / 089406 and US2016 / 0304846 are incorporated herein by reference.

[0034] Nucleases may contain at least one domain that interacts with a guide RNA ("gRNA"). Additionally, the nuclease may be directed to a target sequence by the gRNA. In Class 2 Cas nuclease systems, the gRNA interacts with the nuclease and the target sequence, thereby directing binding to the target sequence. In some embodiments, the gRNA provides specificity for targeted cleavage, while the nuclease may be versatile and may pair with different gRNAs to cleave different target sequences. Class 2 Cas nucleases may pair with gRNA scaffolds of the types, orthologs, and exemplary species listed above.

[0035] Guide RNA (gRNA) In some embodiments of the present disclosure, the cargo of the LNP formulation includes at least one gRNA. The gRNA may guide a Cas nuclease or a Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a Class 2 Cas nuclease to provide specificity for cleavage by the Class 2 Cas nuclease. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex, that can be delivered by the LNP composition. In some embodiments, the CRISPR / Cas complex may be a Type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a Type V CRISPR / Cas complex, such as a Cpf1 / guide RNA complex. The Cas nuclease may be paired with a specific gRNA. The gRNA scaffold structure paired with each Class 2 Cas nuclease varies depending on the individual CRISPR / Cas system.

[0036] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or the combination of crRNA and trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refer to each type. The trRNA may be either a naturally occurring sequence or a trRNA sequence that has modifications or variations compared to the naturally occurring sequence.

[0037] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding factor. A "guide sequence" may also be referred to as a "direction sequence" or a "spacer sequence." A guide sequence may be 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences, e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, can be used as a guide. In some embodiments, the target sequence is, for example, within a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity 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 target region may be 100% complementary or identical. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain one, two, three, or four mismatches, in which case 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 target region may contain one to four mismatches, in which case 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 one, two, three, or four mismatches, in which case the guide sequence comprises 20 nucleotides.

[0038] Because the nucleic acid substrate for the Cas protein is a double-stranded nucleic acid, the target sequence for the Cas protein includes both the plus and minus strands of genomic DNA (i.e., the given sequence and the reverse complement of that sequence). Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that such a guide sequence can direct a guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, when a guide sequence binds to the reverse complement of a target sequence, such a guide sequence is identical to a particular nucleotide of the target sequence (e.g., a target sequence without a PAM) except that T in the guide sequence is replaced with U.

[0039] The length of the directional sequence can vary depending on the CRISPR / Cas system and components used. For example, different Class 2 Cas nucleases derived from different bacterial species have different optimal directional sequence lengths. Thus, the directional sequence can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the directional sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally occurring CRISPR / Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the directional sequence can comprise or consist of 18 to 24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides, hi some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0040] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cpf1 protein. In certain embodiments, the gRNA comprises a crRNA and a tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0041] Certain embodiments of the invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. A "guide RNA nucleic acid" is a nucleic acid that encodes one or more guide RNAs, which is used herein to refer to guide RNAs (e.g., sgRNAs or dgRNAs) and guide RNA expression cassettes.

[0042] In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a directional sequence flanked by all or part of repeat sequences from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and tracrRNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and tracrRNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and tracrRNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracrRNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

[0043] The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3'UTR, or a 5'UTR. In one example, the promoter is a tRNA promoter, e.g., a tRNA promoter. Lys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35:2620-2628. In certain embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include the U6 promoter and the H1 promoter. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In certain embodiments, the gRNA nucleic acid includes a modified nucleoside or nucleotide. In some embodiments, the gRNA nucleic acid includes a 5'-end modification, such as a modified nucleoside or nucleotide that stabilizes the nucleic acid and prevents integration of the nucleic acid. In some embodiments, the gRNA nucleic acid includes double-stranded DNA with a 5'-end modification on each strand. In certain embodiments, the gRNA nucleic acid includes an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as a 5'-end modification. In some embodiments, the gRNA nucleic acid includes a label such as biotin, desthiobioten-TEG, digoxigenin, and a fluorescent marker, such as FAM, ROX, TAMRA, and AlexaFluor.

[0044] In certain embodiments, two or more gRNA nucleic acids, e.g., gRNAs, can be used with a CRISPR / Cas nuclease system. Each gRNA nucleic acid can contain a different targeting sequence so that two or more target sequences can be cleaved by the CRISPR / Cas system. In some embodiments, one or more gRNAs can have the same or different properties, such as activity or stability, within the CRISPR / Cas complex. When two or more gRNAs are used, each gRNA can be encoded on the same gRNA nucleic acid or on a different gRNA nucleic acid. The promoters used to drive expression of two or more gRNAs can be the same or different.

[0045] modified RNA In certain embodiments, the LNP composition comprises modified RNA.

[0046] Modified nucleosides or nucleotides can be present in RNA, such as gRNA or mRNA. For example, a gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" RNA, denoting the presence of one or more non-naturally occurring and / or naturally occurring components or structures used in place of, or in addition to, the standard residues A, G, C, and U. In some embodiments, the modified RNA is synthesized using non-standard nucleosides or nucleotides, referred to herein as "modified."

[0047] Modified nucleosides and nucleotides can include one or more of the following: (i) an alteration, e.g., substitution, of one or both of the non-linked oxygens of the phosphate group and / or one or more of the linked oxygens of the phosphate group in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) an alteration, e.g., substitution, of a component of the ribose sugar, such as the hydroxyl at the 2' position of the ribose sugar (exemplary sugar modifications); (iii) a substantial replacement of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications); (iv) a modification or substitution of a naturally occurring nucleobase, such as with a non-standard nucleobase (exemplary base modifications); (v) a substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., a modification to remove, modify, or replace the terminal phosphate group or to attach a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar modifications and / or backbone modifications); and (vii) a modification or substitution of the sugar (exemplary sugar modifications). Certain embodiments include 5'-end modifications to the mRNA, gRNA, or nucleic acid. Certain embodiments include 3'-end modifications to the mRNA, gRNA, or nucleic acid. The modified RNA may contain modifications at the 5' and 3' ends. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue.

[0048] As used herein, a first sequence is considered to "contain a sequence at least X% identical to" a second sequence if alignment of the two sequences shows that the first sequence matches X% or more of the positions across the entire second sequence. For example, the sequence AAGA contains a sequence that is 100% identical to the sequence AAG, because the alignment would show 100% identity, with matches at all three positions in the second sequence. As long as related nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for both thymidine, uridine, or modified uridine; as another example, cytosine and 5-methylcytosine both have guanosine or modified guanosine as their complements), differences between RNA and DNA (generally, uridine is replaced by thymidine, or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity between polynucleotides. Thus, for example, the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG, and both sequences are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand which algorithm and parameter settings are appropriate for a given pair of sequences to be aligned, but for sequences that are generally similar in length and have an expected amino acid identity of greater than 50% or nucleotide identity of greater than 75%, it is generally appropriate to use the Needleman-Wunsch algorithm with the default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server.

[0049] mRNA In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA-binding factor, such as a Cas nuclease or a Class 2 Cas nuclease, as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding factor, such as a Cas nuclease or a Class 2 Cas nuclease, is provided, used, or administered. In some embodiments, the ORF encoding the RNA-guided DNA-binding factor is a "modified RNA-guided DNA-binding factor ORF" or simply a "modified ORF," which is used as shorthand to indicate that the ORF is modified in one or more of the following ways: (1) the modified ORF has a uridine content ranging from its minimum uridine content to 150% of such minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of such minimum uridine dinucleotide content; or (3) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of such minimum uridine dinucleotide content. , 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66; (4) the modified ORF consists of a set of codons in which at least 75% of the codons are minimal uridine codon(s) for a given amino acid, e.g., the codon(s) with the fewest uridines (the minimal uridine codons are typically zero or one, except for the codon for phenylalanine, which has two uridines); or (5) the modified ORF contains at least one modified uridine. In some embodiments, the modified ORF is modified in at least two, three, or four of the methods described above. In some embodiments, the modified ORF contains at least one modified uridine and is modified in at least one, two, three, or all of the methods described above.

[0050] As used herein, the term "modified uridine" refers to a nucleoside other than thymidine in which the hydrogen bond acceptor is the same as in uridine and which has 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 aprotic substituents (e.g., alkoxy, such as methoxy) 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 aprotic substituents (e.g., alkyl, such as methyl) replace a proton. In some embodiments, the modified uridine is either a substituted uridine, a pseudouridine, or a substituted pseudouridine.

[0051] As used herein, a "uridine position" refers to a position in a polynucleotide that is occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide with "100% modified uridines at uridine positions" contains a modified uridine at every position that would be a uridine in conventional RNA of the same sequence (wherein all bases are the standard bases A, U, C, or G). Unless otherwise specified, U in a polynucleotide sequence set forth in this disclosure or in a sequence table or sequence listing accompanying this disclosure can be a uridine or a modified uridine. [Table 1]

[0052] In any of the above embodiments, the modified ORF may be composed of a set of codons where at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons set forth in the table of minimal uridine codons. In any of the above embodiments, the modified ORF may comprise a sequence that is at least 90%, 95%, 98%, 99%, or 100% 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, or 66.

[0053] In any of the above embodiments, the modified ORF may comprise a sequence that is at least 90%, 95%, 98%, 99%, or 100% 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, or 66.

[0054] In any of the above embodiments, the modified ORF may have a uridine content ranging from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of such minimum uridine content.

[0055] In any of the above embodiments, the modified ORF may have a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of such minimum uridine dinucleotide content.

[0056] In any of the above-described embodiments, the modified ORF may include modified uridines at at least one, more than one, or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is 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-methylpseudouridine 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.

[0057] 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 of an mRNA according to the disclosure are modified uridines. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of an mRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or combinations 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 an mRNA according to the present disclosure 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 an mRNA according to the present disclosure 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 an mRNA according to the present disclosure are N1-methylpseudouridine. 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 an mRNA according to the present disclosure 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 an mRNA according to the present disclosure are 5-methoxyuridine, and the remaining uridine positions are N1-methylpseudouridine.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 an mRNA according to the present disclosure are 5-iodouridine, and the remaining uridine positions are N1-methylpseudouridine.

[0058] In any of the above-described embodiments, the modified ORF may contain a low uridine dinucleotide (UU) content, such as the lowest possible content, e.g., an ORF that (a) uses minimal uridine codons (described above) at every position and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed either as an absolute value, as the count of UU dinucleotides in the ORF, or as a ratio, as the percentage of positions occupied by uridine dinucleotides (e.g., in the case of AUUAU, two out of five positions are occupied by uridine dinucleotides, resulting in a uridine dinucleotide content of 40%). For purposes of assessing minimum uridine dinucleotide content, modified uridine residues are considered equivalent to uridine.

[0059] In some embodiments, the mRNA comprises at least one UTR derived from an expressed mammalian mRNA, such as a constitutively expressed mRNA. An mRNA is considered to be constitutively expressed in a mammal if it is continuously transcribed in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, or both a 5' and a 3' UTR derived from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

[0060] In some embodiments, the mRNA comprises at least one UTR from 17-beta hydroxysteroid dehydrogenase 4 (HSD17B4 or HSD), such as a 5' UTR from an HSD. In some embodiments, the mRNA comprises at least one UTR from a globin mRNA, such as human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, or both 5' and 3' UTRs from a globin 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 derived from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises both 5' and 3' UTRs derived from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).

[0061] In some embodiments, the mRNA comprises both the 5' and 3' UTRs from the same source, such as a constitutively expressed mRNA such as actin, albumin, or globin, e.g., HBA, HBB, or XBG.

[0062] In some embodiments, the mRNA does not include a 5' UTR, e.g., no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA includes a Kozak sequence (described below) between the 5' cap and the start codon, but no additional 5' UTR. In some embodiments, the mRNA does not include a 3' UTR, e.g., no additional nucleotides between the stop codon and the polyA tail.

[0063] In some embodiments, the mRNA contains a Kozak sequence. Kozak sequences can affect translation initiation and the overall yield of polypeptides translated from the mRNA. Kozak sequences include a methionine codon that can function as an initiation codon. A minimal Kozak sequence is NNNRUGN, where at least one of the following is true: the first N is A or G, and the second N is G. In nucleotide sequences, R represents a 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 up to one or two mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is rccAUGg, with zero mismatches or up to one or two mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is gccRccAUGG and has zero mismatches or up to one, two, or three mismatches at lowercase positions. In some embodiments, the Kozak sequence is gccAccAUG and has zero mismatches or up to one, two, three, or four mismatches at lowercase positions. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG and has zero mismatches or up to one, two, three, or four mismatches at lowercase positions.

[0064] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 43, wherein, optionally, the ORF of SEQ ID NO: 43 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0065] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 44, wherein, optionally, the ORF of SEQ ID NO: 44 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0066] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 56, wherein, optionally, the ORF of SEQ ID NO: 56 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0067] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 57, wherein, optionally, the ORF of SEQ ID NO: 57 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0068] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 58, wherein, optionally, the ORF of SEQ ID NO: 58 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0069] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 59, wherein, optionally, the ORF of SEQ ID NO: 59 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0070] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 60, wherein, optionally, the ORF of SEQ ID NO: 60 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0071] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence that is at least 90% identical to SEQ ID NO: 61, wherein, optionally, the ORF of SEQ ID NO: 61 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF of 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, or 66.

[0072] In some embodiments, the mRNA comprises an alternative ORF of 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, or 66.

[0073] In some embodiments, the degree of identity to the sequence of SEQ ID NO: 43, 44, or 56-61, optionally substituted, is 95%. In some embodiments, the degree of identity to the sequence of SEQ ID NO: 43, 44, or 56-61, optionally substituted, is 98%. In some embodiments, the degree of identity to the sequence of SEQ ID NO: 43, 44, or 56-61, optionally substituted, is 99%. In some embodiments, the degree of identity to the sequence of SEQ ID NO: 43, 44, or 56-61, optionally substituted, is 100%.

[0074] In some embodiments, the mRNAs disclosed herein include a 5' cap, such as Cap0, Cap1, or Cap2. The 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, e.g., with an ARCA, as discussed below) linked via a 5'-triphosphate to the 5' position of the first nucleotide in the 5'-to-3' strand of the mRNA, i.e., the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA include a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-methoxy. See, for example, Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30 and Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous mRNAs in higher eukaryotes, including mammalian mRNAs such as human mRNAs, contain Cap1 or Cap2. Cap0 and other cap structures distinct from Cap1 and Cap2 can be immunogenic in mammals, including humans, because they are recognized as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, which can increase cytokine levels, including type I interferons. Components of the innate immune system, such as IFIT-1 and IFIT-5, can also compete with eIF4E for binding to mRNAs with caps other than Cap1 or Cap2, potentially inhibiting mRNA translation.

[0075] A cap can be incorporated co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific catalog number AM8045) is a cap analog that contains 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of a guanine ribonucleotide and can be incorporated into transcripts during in vitro transcription initiation. In ARCA, the 2' position of the first cap-proximal nucleotide is hydroxyl, resulting in a Cap0 cap. 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 and 7-methyl(3'deoxy)GpppG," RNA 7:1486-1495. The structure of ARCA is shown below. [ka]

[0076] The Cap1 structure can be obtained by co-transcription using CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies catalog number N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies catalog number N-7133). 3'-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as catalog numbers N-7413 and N-7433, respectively. The CleanCap™ AG structure is shown below. [ka]

[0077] Alternatively, RNA can be capped post-transcriptionally. For example, vaccinia capping enzyme is commercially available (New England Biolabs, catalog number M2080S), which possesses RNA triphosphatase and guanylyltransferase activities provided by its D1 subunit and a guanine methyltransferase activity provided by its D12 subunit. Thus, in the presence of S-adenosylmethionine and GTP, this enzyme can add 7-methylguanine to RNA to give Cap0. See, e.g., 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.

[0078] In some embodiments, the mRNA further comprises a polyadenylation (polyA) tail. In some embodiments, the polyA tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the polyA tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some examples, the polyA tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more positions within the polyA tail. The polyA tail may comprise at least eight consecutive adenine nucleotides, but also includes one or more non-adenine nucleotides. As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly A tail of an mRNA described herein may comprise consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding factor or sequence of interest. In some instances, the poly A tail of an mRNA comprises non-consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding factor or sequence of interest, where the adenine nucleotides are interrupted at regularly or irregularly spaced intervals by non-adenine nucleotides.

[0079] In some embodiments, the mRNA further comprises a polyadenylation (polyA) tail. In some embodiments, the polyA tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the polyA tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some examples, the polyA tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more positions within the polyA tail. The polyA tail may comprise at least eight consecutive adenine nucleotides, but also includes one or more non-adenine nucleotides. As used herein, "non-adenine nucleotide" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly A tail of an mRNA described herein may comprise consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding factor or sequence of interest. In some instances, the poly A tail of an mRNA comprises non-consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding factor or sequence of interest, where the adenine nucleotides are interrupted at regularly or irregularly spaced intervals by non-adenine nucleotides.

[0080] In some embodiments, one or more non-adenine nucleotides are inserted to interrupt the stretch of consecutive adenine nucleotides to allow binding of poly(A) binding proteins to the stretch. In some embodiments, at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides are followed by one or more non-adenine nucleotide(s). In some embodiments, at least 8-50 consecutive adenine nucleotides are followed by one or more non-adenine nucleotides. In some embodiments, at least 8-100 consecutive adenine nucleotides are followed by one or more non-adenine nucleotides. In some embodiments, the non-adenine nucleotide is inserted after 1, 2, 3, 4, 5, 6, or 7 adenine nucleotides, followed by at least 8 consecutive adenine nucleotides.

[0081] A poly-A tail may comprise a sequence of consecutive adenine nucleotides, followed by one or more non-adenine nucleotides, optionally followed by a further adenine nucleotide.

[0082] In some embodiments, the poly-A tail comprises or contains a single non-adenine nucleotide or a long stretch of 2-10 consecutive non-adenine nucleotides. In some embodiments, at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides are followed by a non-adenine nucleotide(s). In some examples, at least 8-50 consecutive adenine nucleotides are followed by one or more non-adenine nucleotides. In some embodiments, 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 consecutive adenine nucleotides are followed by one or more non-adenine nucleotides.

[0083] In some embodiments, the non-adenine nucleotides are guanine, cytosine, or thymine. In some examples, the non-adenine nucleotides are guanine nucleotides. In some embodiments, the non-adenine nucleotides are cytosine nucleotides. In some embodiments, the non-adenine nucleotides are thymine nucleotides. In some examples where two or more non-adenine nucleotides are present, the non-adenine nucleotides may be selected from a) guanine nucleotides and thymine nucleotides, b) guanine nucleotides and cytosine nucleotides, c) thymine nucleotides and cytosine nucleotides, or d) guanine nucleotides, thymine nucleotides, and cytosine nucleotides. An exemplary poly-A tail comprising non-adenine nucleotides is set forth as SEQ ID NO: 62.

[0084] In some embodiments, mRNA is purified. In some embodiments, mRNA is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, etc., or an equivalent method, such as, for example, a method described herein). In some embodiments, mRNA is purified using a chromatographic method, such as a method using HPLC, or an equivalent method (e.g., a method described herein). In some embodiments, mRNA is purified using both a precipitation method (e.g., LiCl precipitation) and a method using HPLC.

[0085] In some embodiments, at least one gRNA is provided in combination with the mRNA disclosed herein.In some embodiments, the gRNA is provided as a molecule separate from the mRNA.In some embodiments, the gRNA is provided as a part of the mRNA disclosed herein, for example, as a part of UTR.

[0086] Chemically modified gRNA In some embodiments, gRNAs are chemically modified. A gRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or a "chemically modified" gRNA, denoting the presence of one or more non-naturally occurring and / or naturally occurring components or structures that are substituted for or in addition to the standard residues A, G, C, and U. In some embodiments, modified gRNAs are synthesized using non-standard nucleosides or nucleotides, and are referred to herein as "modified." Modified nucleosides and nucleotides can include one or more of the following: (i) an alteration, e.g., substitution, of one or both of the non-linked oxygens of the phosphate group and / or one or more of the linked oxygens of the phosphate group in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) an alteration, e.g., substitution, of a component of the ribose sugar, such as the hydroxyl at the 2' position of the ribose sugar (exemplary sugar modifications); (iii) a substantial replacement of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications); (iv) a modification or substitution of a naturally occurring nucleobase, such as with a non-standard nucleobase (exemplary base modifications); (v) a substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., a modification to remove, modify, or replace the terminal phosphate group or to attach a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar modifications and / or backbone modifications); and (vii) a modification or substitution of the sugar (exemplary sugar modifications).

[0087] In some embodiments, the gRNA comprises modified uridines at some or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, e.g., with a halogen or a C1-C6 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, e.g., with a C1-C6 alkyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is 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-methylpseudouridine 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.

[0088] 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 of a gRNA according to the present disclosure are modified uridines. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of a gRNA according to the present disclosure are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or combinations 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 of a gRNA according to the present disclosure 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 of a gRNA according to the present disclosure 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 of a gRNA according to the present disclosure are N1-methylpseudouridine. 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 of a gRNA according to the present disclosure are 5-iodouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions of a gRNA according to the present disclosure are 5-methoxyuridine, and the remaining uridine positions are N1-methylpseudouridine.In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions of a gRNA according to the present disclosure are 5-iodouridine, and the remaining uridine positions are N1-methylpseudouridine.

[0089] The above-listed chemical modifications can be combined to yield modified gRNAs containing nucleosides and nucleotides (collectively referred to as "residues") that may have two, three, four, or more modifications. For example, modified residues can have modified sugars and modified nucleobases. In some embodiments, every base of the gRNA is modified, e.g., all bases have modified phosphate groups, such as phosphorothioate groups. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA contains at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA contains at least one modified residue at or near the 3' end of the RNA.

[0090] In some embodiments, the gRNA comprises one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions of the modified gRNA are modified nucleosides or nucleotides.

[0091] Unmodified nucleic acids may be prone to degradation, for example, by nucleases found in cells or serum. For example, nucleases can hydrolyze phosphodiester bonds in nucleic acids. Thus, in one aspect, the gRNAs described herein contain one or more modified nucleosides or nucleotides to introduce stability against nucleases, for example, in cells or serum. In some embodiments, the modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a cell population, either in vivo or ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, such as single-stranded nucleic acids, resulting in the expression and release of cytokines, particularly interferon, and the induction of cell death.

[0092] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more oxygen atoms with different substituents.In addition, modified residues, for example, modified residues present in modified nucleic acids, can include substantial substitution of unmodified phosphate moieties with modified phosphate groups as described herein.In some embodiments, backbone modification of the phosphate backbone can include changes that result in either an uncharged linker or a charged linker with asymmetric charge distribution.

[0093] Examples of modified phosphate groups include phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. The phosphorus atom of an unmodified phosphate group is achiral. However, the phosphorus atom can be made chiral by replacing one of the non-bridging oxygens with one of the atoms or atomic groups listed above. The asymmetric phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacing the bridging oxygen (i.e., the oxygen connecting the phosphate group to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), or carbon (bridging methylene phosphonate). The replacement may occur at either or both of the linking oxygens.

[0094] In certain backbone modifications, the phosphate group can be replaced with a phosphorus-free connector. In some embodiments, the charged phosphate group can be replaced with a neutral moiety. Examples of moieties that can replace the phosphate group include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0095] template nucleic acid The compositions and methods disclosed herein can include a template nucleic acid. The template can be used to alter or insert a nucleic acid sequence at or near a target site for a Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template can be provided. In other embodiments, two or more templates can be provided to allow editing to occur at two or more target sites. For example, different templates can be provided to edit a single gene in a cell or two different genes in a cell.

[0096] In some embodiments, the template may be used in homologous recombination. In some embodiments, homologous recombination may result in the incorporation of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template may be used in homologous recombination repair, in which DNA strand invasion occurs at the nucleic acid cleavage site. In some embodiments, homologous recombination repair may result in the template sequence being included in the edited target nucleic acid molecule. In yet another embodiment, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes adjacent inverted terminal repeat (ITR) sequences.

[0097] In some embodiments, the template may include a first homology arm and a second homology arm (also referred to as a first nucleotide sequence and a second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. When the template contains two homology arms, each arm may be the same or different in length, and the sequence flanked by the homology arms may be substantially similar or identical to the target sequence flanked by the homology arms, or may be a completely unrelated sequence. In some embodiments, the degree of complementarity or percent identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and the degree of complementarity or percent identity between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, may enable homologous recombination between the template and the target nucleic acid molecule, such as high-fidelity homologous recombination. In some embodiments, the degree of complementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%.

[0098] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. Additionally or alternatively, the template sequence may correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or that is at a different location than its natural location in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal sequence or an extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence of the cell at or near the cleavage site, but contains at least one nucleotide change. In some embodiments, template editing of the cleaved target nucleic acid molecule may result in a mutation including an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the mutation may result in one or more amino acid changes in a protein expressed by a gene comprising the target sequence. In some embodiments, the mutation may result in one or more nucleotide changes in the RNA expressed by the target gene. In some embodiments, the mutation may change the expression level of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. In some embodiments, the mutation may result in gene knockdown. In some embodiments, the mutation may result in gene knockout. In some embodiments, the mutation may result in restoration of gene function. In some embodiments, template editing of the cleaved target nucleic acid molecule may result in changes to exon sequences, intron sequences, regulatory sequences, transcriptional regulatory sequences, translational regulatory sequences, splice sites, or non-coding sequences of the target nucleic acid molecule, such as DNA.

[0099] In other embodiments, the template sequence may comprise an exogenous sequence. In some embodiments, the exogenous sequence may comprise a protein- or RNA-encoding sequence operably linked to an exogenous promoter sequence, such that when the exogenous sequence is integrated into the target nucleic acid molecule, the cell can express the protein or RNA encoded by the integrated sequence. In other embodiments, when the exogenous sequence is integrated into the target nucleic acid molecule, expression of the integrated sequence may be controlled by the endogenous promoter sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence encoding a protein or a portion of the protein. In yet other embodiments, the exogenous sequence may comprise or consist of an exon sequence, an intron sequence, a regulatory sequence, a transcriptional regulatory sequence, a translational regulatory sequence, a splice site, or a non-coding sequence. In some embodiments, integration of the exogenous sequence may result in restoration of gene function. In some embodiments, integration of the exogenous sequence may result in gene knock-in. In some embodiments, integration of the exogenous sequence may result in gene knock-out.

[0100] The template may be of any suitable length. In some embodiments, the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template may be a single-stranded nucleic acid. The template may be a double-stranded or partially double-stranded nucleic acid. In certain embodiments, the single-stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template may comprise a nucleotide sequence complementary to a portion of the target nucleic acid molecule that comprises the target sequence (i.e., a "homology arm"). In some embodiments, the template may comprise a homology arm complementary to a sequence located upstream or downstream of the cleavage site on the target nucleic acid molecule.

[0101] In some embodiments, the template contains ssDNA or dsDNA flanked by inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0102] Nucleic acid purification In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, such as, for example, a method described herein). In some embodiments, nucleic acids are purified using a chromatographic method, such as a method using HPLC, or equivalent method (e.g., a method described herein). In some embodiments, nucleic acids are purified using both a precipitation method (e.g., LiCl precipitation) and a method using HPLC.

[0103] Target sequence In some embodiments, the CRISPR / Cas system of the present disclosure may be directed to and cleave a target sequence on a target nucleic acid molecule. For example, the target sequence may be recognized and cleaved by a Cas nuclease. In certain embodiments, the target sequence of the Cas nuclease is located near the specific PAM sequence of such nuclease. In some embodiments, a Class 2 Cas nuclease may be directed to a target sequence on a target nucleic acid molecule by a gRNA, where the gRNA hybridizes to the target sequence and a Class 2 Cas protein cleaves the target sequence. In some embodiments, a guide RNA hybridizes to a target sequence adjacent to or including the specific PAM of the Class 2 Cas nuclease, and the Class 2 Cas nuclease cleaves such target sequence. In some embodiments, the target sequence may be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity between the directional sequence of the guide RNA and the corresponding portion of the target sequence to which the guide RNA hybridizes may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity between the directional sequence of the guide RNA and the corresponding portion of the target sequence to which the guide RNA hybridizes may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the target homology region is flanked by a specific PAM sequence. In some embodiments, the target sequence may comprise a sequence that is 100% complementary to the directional sequence of the guide RNA. In other embodiments, the target sequence may contain at least one mismatch, deletion, or insertion compared to the target sequence of the guide RNA.

[0104] The length of the target sequence may vary depending on the nuclease system used. For example, the target sequence of a guide RNA for a CRISPR / Cas system may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length, with the target sequence being of a corresponding length and optionally flanked by a PAM sequence. In some embodiments, the target sequence may comprise 15-24 nucleotides in length. In some embodiments, the target sequence may comprise 17-21 nucleotides in length. In some embodiments, the target sequence may comprise 20 nucleotides in length. When using nickases, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave opposite strands of a DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strand of a DNA molecule, hi some embodiments, the target sequence may comprise a portion of a target sequence recognized by one or more Cas nucleases.

[0105] A target nucleic acid molecule can be any DNA or RNA molecule that is endogenous or exogenous to a cell. In some embodiments, the target nucleic acid molecule can be episomal DNA, a plasmid, genomic DNA, a viral genome, mitochondrial DNA, or chromosomal DNA from or within a cell. In some embodiments, the target sequence of the target nucleic acid molecule can be a genomic sequence from or within a cell, such as a human cell.

[0106] In further embodiments, the target sequence may be a viral sequence. In further embodiments, the target sequence may be a pathogen sequence. In yet another embodiment, the target sequence may be a synthetic sequence. In further embodiments, the target sequence may be a chromosomal sequence. In particular embodiments, the target sequence may comprise a translocation junction, e.g., a translocation associated with cancer. In some embodiments, the target sequence may be on a eukaryotic chromosome, such as a human chromosome. In particular embodiments, the target sequence is a liver-specific sequence, where the sequence is expressed in liver cells.

[0107] In some embodiments, the target sequence may be located within a coding sequence of a gene, within an intron sequence of a gene, within a regulatory sequence, within a transcriptional control sequence of a gene, within a translational control sequence of a gene, at a splice site, or within an intergenic non-coding sequence. In some embodiments, the gene may be a protein-coding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may comprise all or a portion of a disease-associated gene. In some embodiments, the target sequence may be located in a non-gene functional site of the genome, for example, a site that controls aspects of chromatin organization, such as a scaffolding site or locus control region.

[0108] In embodiments involving Cas nucleases, such as Class 2 Cas nucleases, the target sequence may be adjacent to a protospacer adjacent motif ("PAM"). In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides of the 3' end of the target sequence. The length and sequence of the PAM may vary depending on the Cas protein used. For example, the PAM may be selected from consensus sequences or specific PAM sequences for a particular Cas9 protein or Cas9 ortholog, including those disclosed in Figure 1 of Ran et al., Nature, 520:186-191 (2015) and Figure S5 of Zetsche 2015, the relevant disclosures of which are incorporated herein by reference. In some embodiments, the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (where N is defined as any nucleotide and W is defined as either A or T). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be TTN. In some embodiments, the PAM sequence may be NNAAAAW.

[0109] Lipid preparations Disclosed herein are various embodiments of LNP formulations of RNA, such as CRISPR / Cas cargo. Such LNP formulations include an "amine lipid" along with a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, such LNP formulations include an "amine lipid" along with a helper lipid and a PEG lipid. In some embodiments, the LNP formulation includes less than 1 percent neutral phospholipids. In some embodiments, the LNP formulation includes less than 0.5 percent neutral phospholipids. "Lipid nanoparticle" refers to a particle comprising multiple (i.e., two or more) lipid molecules physically associated with each other by intermolecular forces.

[0110] Amine lipids LNP compositions for delivering biologically active agents include "amine lipids," defined as lipid A or equivalents thereof, such as acetal analogs of lipid A.

[0111] In some embodiments, the amine lipid is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Lipid A is [ka] It can be expressed as:

[0112] Lipid A may be synthesized according to WO2015 / 095340 (e.g., pages 84-86). In certain embodiments, the amine lipid is equivalent to lipid A.

[0113] 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 further embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is selected from C4, C5, C6, C7, C9, C10, C11, and C12 acetal analogs.

[0114] Amine lipids suitable for use in the LNPs described herein are biodegradable in vivo and suitable for delivering biologically active substances, such as RNA, to cells. Amine lipids have low toxicity (e.g., RNA cargo amounts of 10 mg / kg or greater are tolerated in animal models without adverse effects). In certain embodiments, LNPs containing amine lipids include those in which at least 75% of the amine lipids are cleared from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days. In certain embodiments, LNPs containing amine lipids include those in which at least 50% of the mRNA or gRNA is cleared from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days. In certain embodiments, LNPs comprising amine lipids include those in which at least 50% of the LNPs are cleared from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days, as measured by, for example, lipid (e.g., amine lipid), RNA (e.g., mRNA), or another component. In certain embodiments, the lipid, RNA, or nucleic acid component of the LNP is measured both when encapsulated in lipids and when free.

[0115] Lipid clearance may 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, a 0.3 mg / kg LNP-siRNA system containing luciferase-directed siRNA was administered intravenously via a lateral tail vein to 6-8 week-old male C57Bl / 6 mice. 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. Mice were perfused with saline, tissues were harvested, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Furthermore, Maier describes a method for evaluating toxicity after administration of LNP-siRNA formulations. For example, luciferase-directed siRNA was administered to male Sprague-Dawley rats at 0 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, and 10 mg / kg (5 animals / group) via a single intravenous bolus injection in a dose volume of 5 mL / kg. After 24 hours, approximately 1 mL of blood was obtained from the jugular vein of conscious animals, and serum was isolated. Seventy-two hours after administration, all animals were euthanized for necropsy. Clinical signs, body weight, serum chemistry, organ weight, and histopathology were evaluated. Maier describes methods for evaluating siRNA-LNP formulations, and these methods may be applied to assess clearance, pharmacokinetics, and toxicity associated with the administration of the LNP compositions of the present disclosure.

[0116] Amine lipids can cause an increased clearance rate. In some embodiments, the clearance rate is a lipid clearance rate, for example, the rate at which lipids disappear from blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, for example, the rate at which mRNA or gRNA disappears from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNPs disappear from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNPs disappear from tissues, such as liver tissue or spleen tissue. In certain embodiments, a high clearance rate results in a safety profile with substantially no adverse effects. Amine lipids can reduce the accumulation of LNPs in the circulation and in tissues. In some embodiments, reduced accumulation of LNPs in the circulation and in tissues results in a safety profile with substantially no adverse effects.

[0117] The amine lipids of the present disclosure may be ionized (e.g., may form salts) depending on the pH of the medium in which they are contained. For example, in a slightly acidic medium, the amine lipids may be protonated and therefore positively charged. Conversely, in a slightly basic medium, such as blood, with a pH of about 7.35, the amine lipids may not be protonated and therefore may not be charged. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 9. In some embodiments, the amine lipids of the present disclosure may be protonated at a pH of at least about 10.

[0118] The pH at which an amine lipid is predominantly protonated is related to its intrinsic pKa. In some embodiments, each amine lipid of the present disclosure may independently have a pKa in the range of about 5.1 to about 7.4. In some embodiments, each amine lipid of the present disclosure may independently have a pKa in the range of about 5.5 to about 6.6. In some embodiments, each amine lipid of the present disclosure may independently have a pKa in the range of about 5.6 to about 6.4. In some embodiments, each amine lipid of the present disclosure may independently have a pKa in the range of about 5.8 to about 6.2. For example, each amine lipid of the present disclosure may independently have a pKa in the range of about 5.8 to about 6.5. The pKa of the amine lipid may be an important consideration in formulating LNPs, as cationic lipids with a pKa in the range of about 5.1 to about 7.4 have been found to be effective in delivering cargo in vivo, e.g., to the liver. Furthermore, cationic lipids with a pKa in the range of about 5.3 to about 6.4 have been found to be effective in delivering them in vivo, for example, to tumors, etc. See, for example, WO2014 / 136086.

[0119] More lipids Suitable "neutral lipids" for use in the lipid compositions of the present disclosure include, for example, a wide variety of neutral lipids, uncharged lipids, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PM ... -stearoylphosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).In another embodiment, the neutral phospholipid may be dipalmitoylphosphatidylcholine (DPPC).

[0120] "Helper lipids" include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid can be cholesterol. In one embodiment, the helper lipid can be cholesterol hemisuccinate.

[0121] PEG-lipids are stealth lipids that alter the length of time nanoparticles can exist in vivo (e.g., in the blood). PEG-lipids can aid in the formulation process, for example, by inhibiting particle aggregation and controlling particle size. PEG-lipids used herein can adjust the pharmacokinetic properties of LNPs. Typically, PEG-lipids include a lipid portion and a PEG-based polymer portion.

[0122] In some embodiments, the lipid moiety can be derived from a diacylglycerol or diacylglycamide, including those that independently comprise a dialkylglycerol or dialkylglycamide group having an alkyl chain length of from about C4 to about C40 saturated or unsaturated carbon atoms, where such chains may include one or more functional groups, such as, for example, amides or esters. In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglycamide group can further include one or more substituted alkyl groups. The chain length can be symmetric or asymmetric.

[0123] Unless otherwise specified, the term "PEG," as used herein, refers to any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, the PEG moiety is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. Alternatively, the PEG moiety may be substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the PEG moiety includes a PEG copolymer such as a PEG-polyurethane or a PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); alternatively, the PEG moiety does not include a PEG copolymer, e.g., a PEG monopolymer. In one embodiment, the PEG has a molecular weight of about 130 to about 50,000, and in a subembodiment, about 150 to about 30,000, and in a subembodiment, about 150 to about 20,000, and in a subembodiment, about 150 to about 15,000, and in a subembodiment, about 150 to about 10,000, and in a subembodiment, about 150 to about 6,000, and in a subembodiment, about 150 to about 5,000, and in a subembodiment, about 150 to about 4,000, and in a subembodiment, about 150 to about 3,000, and in a subembodiment, about 300 to about 3,000, and in a subembodiment, about 1,000 to about 3,000, and in a subembodiment, about 1,500 to about 2,500.

[0124] In certain embodiments, the PEG (e.g., attached to a lipid moiety or lipid, such as a stealth lipid) is "PEG-2K," also known as "PEG 2000," and has an average molecular weight of about 2,000 daltons. PEG-2K is herein represented by the following formula (I): [ka] where n is 45, meaning the number average degree of polymerization contains about 45 subunits. However, other PEG embodiments known in the art may be used, including, for example, embodiments containing a number average degree of polymerization of about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n may be from about 30 to about 60. In some embodiments, n may be from about 35 to about 55. In some embodiments, n may be from about 40 to about 50. In some embodiments, n may be from 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.

[0125] In any of the embodiments described herein, the PEG lipid may be PEG-dilaurylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (Cat. No. GM-020, manufactured by NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (Cat. No. DSPE-020CN, manufactured by NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristoylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycerol. glycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (catalog number 880150P, Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (catalog number 880120C, Avanti Polar The PEG lipid may be selected from PEG2k-DMG, PEG2k-DSG (manufactured by PEG2k-DSM, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, manufactured by NOF, Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-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 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 paragraphs

[0240] to

[0244] of WO2016 / 010840. 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.

[0126] LNP formulation Embodiments of the present disclosure provide lipid compositions expressed according to the respective molar ratios of component lipids in the formulation. In one embodiment, the molar % of amine lipids may be about 30 mol% to about 60 mol%. In one embodiment, the molar % of amine lipids may be about 40 mol% to about 60 mol%. In one embodiment, the molar % of amine lipids may be about 45 mol% to about 60 mol%. In one embodiment, the molar % of amine lipids may be about 50 mol% to about 60 mol%. In one embodiment, the molar % of amine lipids may be about 55 mol% to about 60 mol%. In one embodiment, the molar % of amine lipids may be about 50 mol% to about 55 mol%. In one embodiment, the molar % of amine lipids may be about 50 mol%. In one embodiment, the molar % of amine lipids in an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar %. In some embodiments, the mole % of amine lipid in an LNP batch is within ±4 mole %, ±3 mole %, ±2 mole %, ±1.5 mole %, ±1 mole %, ±0.5 mole %, or ±0.25 mole % of the target mole %. All mole % values ​​are given as a percentage of the lipid component of the LNP composition. In certain embodiments, the mole % of amine lipid varies less than 15%, less than 10%, or less than 5% between LNP lots.

[0127] In one embodiment, the molar percentage of the neutral lipid, such as a neutral phospholipid, may be about 5 mol% to about 15 mol%. In one embodiment, the molar percentage of the neutral lipid, such as a neutral phospholipid, may be about 7 mol% to about 12 mol%. In one embodiment, the molar percentage of the neutral lipid, such as a neutral phospholipid, may be about 0 mol% to about 5 mol%. In one embodiment, the molar percentage of the neutral lipid, such as a neutral phospholipid, may be about 0 mol% to about 10 mol%. In one embodiment, the molar percentage of the neutral lipid, such as a neutral phospholipid, may be about 5 mol% to about 10 mol%. In one embodiment, the molar percentage of the neutral lipid, such as a neutral phospholipid, may be about 8 mol% to about 10 mol%.

[0128] In one embodiment, the mol% of neutral lipids, such as neutral phospholipids, can be about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. In one embodiment, the mol% of neutral lipids, such as neutral phospholipids, can be about 9 mol%.

[0129] In one embodiment, the molar percentage of neutral lipids, such as neutral phospholipids, can be about 1 mol% to about 5 mol%. In one embodiment, the molar percentage of neutral lipids can be about 0.1 mol% to about 1 mol%. In one embodiment, the molar percentage of neutral lipids, such as neutral phospholipids, can be about 0.1 mol%, about 0.2 mol%, about 0.5 mol%, 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, or about 5 mol%.

[0130] In one embodiment, the mole percent of neutral lipids, such as neutral phospholipids, may be less than about 1 mole percent. In one embodiment, the mole percent of neutral lipids, such as neutral phospholipids, may be less than about 0.5 mole percent. In one embodiment, the mole percent of neutral lipids, such as neutral phospholipids, may be about 0 mole percent, about 0.1 mole percent, about 0.2 mole percent, about 0.3 mole percent, about 0.4 mole percent, about 0.5 mole percent, about 0.6 mole percent, about 0.7 mole percent, about 0.8 mole percent, about 0.9 mole percent, or about 1 mole percent. In some embodiments, the formulations disclosed herein are free of neutral lipids (i.e., 0 mole percent neutral lipids). In some embodiments, the formulations disclosed herein are essentially free of neutral lipids (i.e., about 0 mole percent neutral lipids). In some embodiments, the formulations disclosed herein are free of neutral phospholipids (i.e., 0 mole percent neutral phospholipids). In some embodiments, the formulations disclosed herein are essentially free of neutral phospholipids (i.e., about 0 mol % neutral phospholipids).

[0131] In some embodiments, the mole % neutral lipid of an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the mole % target neutral lipid. In certain embodiments, the LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0132] In one embodiment, the mole percent of the helper lipid may be about 20 mole percent to about 60 mole percent. In one embodiment, the mole percent of the helper lipid may be about 25 mole percent to about 55 mole percent. In one embodiment, the mole percent of the helper lipid may be about 25 mole percent to about 50 mole percent. In one embodiment, the mole percent of the helper lipid may be about 25 mole percent to about 40 mole percent. In one embodiment, the mole percent of the helper lipid may be about 30 mole percent to about 50 mole percent. In one embodiment, the mole percent of the helper lipid may be about 30 mole percent to about 40 mole percent. In one embodiment, the mole percent of the helper lipid is adjusted based on the concentrations of the amine lipid, neutral lipid, and PEG lipid so that the lipid component is 100 mole percent. In one embodiment, the mole percent of the helper lipid is adjusted based on the concentrations of the amine lipid and PEG lipid so that the lipid component is 100 mole percent. In one embodiment, the mole percent of the helper lipid is adjusted based on the concentrations of the amine lipid and PEG lipid so that the lipid component is at least 99 mole percent. In some embodiments, the mole % of helper for an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the mole % of target. In certain embodiments, LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0133] In one embodiment, the mole % of PEG lipids may be about 1 mole % to about 10 mole %. In one embodiment, the mole % of PEG lipids may be about 2 mole % to about 10 mole %. In one embodiment, the mole % of PEG lipids may be about 2 mole % to about 8 mole %. In one embodiment, the mole % of PEG lipids may be about 2 mole % to about 4 mole %. In one embodiment, the mole % of PEG lipids may be about 2.5 mole % to about 4 mole %. In one embodiment, the mole % of PEG lipids may be about 3 mole %. In one embodiment, the mole % of PEG lipids for an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole % of PEG lipid. In certain embodiments, LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0134] In certain embodiments, the cargo includes an mRNA encoding an RNA-guided DNA-binding factor (e.g., a Cas nuclease, a class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding the gRNA, or a combination of an mRNA and a gRNA. In one embodiment, the LNP composition may include lipid A or an equivalent thereof. In some aspects, the amine lipid is lipid A. In some aspects, the amine lipid is a lipid A equivalent, e.g., an analog of lipid A. In certain aspects, the amine lipid is an acetal analog of lipid A. In various embodiments, the LNP composition includes an amine lipid, a neutral lipid, a helper lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, the PEG lipid is PEG2k-DMG. In some embodiments, the LNP composition may include lipid A, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, the LNP composition includes an amine lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG-lipid comprising DMG. In certain embodiments, the amine lipid is selected from lipid A and lipid A equivalents, such as acetal analogs of lipid A. In further embodiments, the LNP composition comprises lipid A, cholesterol, DSPC, and PEG2k-DMG.

[0135] In various embodiments, the LNP composition comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In various embodiments, the LNP composition comprises an amine lipid, a helper lipid, a neutral phospholipid, and a PEG lipid. In various embodiments, the LNP composition comprises a lipid component consisting of an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In various embodiments, the LNP composition comprises an amine lipid, a helper lipid, and a PEG lipid. In certain embodiments, the LNP composition does not comprise a neutral lipid, such as a neutral phospholipid. In various embodiments, the LNP composition comprises a lipid component consisting of an amine lipid, a helper lipid, and a PEG lipid. In certain embodiments, the neutral lipid is selected from one or more of DSPC, DPPC, DAPC, DMPC, DOPC, DOPE, and DSPE. In certain embodiments, the neutral lipid is DSPC. In certain embodiments, the neutral lipid is DPPC. In certain embodiments, the neutral lipid is DAPC. In certain embodiments, the neutral lipid is DMPC. In certain embodiments, the neutral lipid is DOPC. In certain embodiments, the neutral lipid is DOPE. In certain embodiments, the neutral lipid is DSPE. In certain embodiments, the helper lipid is cholesterol. In a specific embodiment, the PEG lipid is PEG2k-DMG. In some embodiments, the LNP composition may comprise lipid A, a helper lipid, and a PEG lipid. In some embodiments, the LNP composition may comprise a lipid component consisting of lipid A, a helper lipid, and a PEG lipid. In some embodiments, the LNP composition comprises an amine lipid, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a lipid component consisting of an amine lipid, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from lipid A and lipid A equivalents, such as acetal analogs of lipid A. In certain embodiments, the amine lipid is a C5-C12 or C4-C12 acetal analog of lipid A. In further embodiments, the LNP composition comprises lipid A, cholesterol, and PEG2k-DMG.

[0136] Embodiments of the present disclosure also provide lipid compositions expressed according to the molar ratio of the positively charged amine group (N) of the amine lipid to the negatively charged phosphate group (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the formula N / P. In some embodiments, an LNP composition can include a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid, and a nucleic acid component, wherein the N / P ratio is about 3-10. In some embodiments, an LNP composition can include a lipid component comprising an amine lipid, a helper lipid, and a PEG lipid, and a nucleic acid component, wherein the N / P ratio is about 3-10. In some embodiments, an LNP composition can include a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid, and an RNA component, wherein the N / P ratio is about 3-10. In some embodiments, an LNP composition can include a lipid component comprising an amine lipid, a helper lipid, and a PEG lipid, and an RNA component, wherein the N / P ratio is about 3-10. In one embodiment, the N / P ratio can be about 5-7. In one embodiment, the N / P ratio may be about 3-7. In one embodiment, the N / P ratio may be about 4.5-8. 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, LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0137] In some embodiments, the nucleic acid component, e.g., the RNA component, may comprise an mRNA, such as an mRNA encoding a Cas nuclease. The RNA component includes RNA, optionally with additional nucleic acids and / or proteins, such as RNP cargo. In one embodiment, the RNA comprises a Cas9 mRNA. In some compositions including an 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 a Class 2 Cas nuclease mRNA and a gRNA.

[0138] In certain embodiments, LNP compositions may comprise mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain embodiments, LNP compositions may comprise mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, an amine lipid, a helper lipid, and a PEG lipid. In certain LNP compositions comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the neutral lipid is DSPC. In further embodiments comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain compositions comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the amine lipid is selected from lipid A and its equivalents, such as acetal analogs of lipid A.

[0139] In some embodiments, the LNP composition may comprise a gRNA. In certain embodiments, the LNP composition may comprise an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain embodiments, the LNP composition may comprise an amine lipid, a gRNA, a helper lipid, and a PEG lipid. In certain LNP compositions comprising a gRNA, the helper lipid is cholesterol. In some compositions comprising a gRNA, the neutral lipid is DSPC. In further embodiments comprising a gRNA, 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.

[0140] In one embodiment, the LNP composition may comprise an sgRNA. In one embodiment, the LNP composition may comprise a Cas9 sgRNA. In one embodiment, the LNP composition may comprise a Cpf1 sgRNA. In some compositions comprising an sgRNA, the LNP comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In some compositions comprising an sgRNA, the LNP comprises an amine lipid, a helper lipid, and a PEG lipid. In certain compositions comprising an sgRNA, the helper lipid is cholesterol. In other compositions comprising an sgRNA, the neutral lipid is DSPC. In further embodiments comprising an 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.

[0141] In certain embodiments, an LNP composition comprises an mRNA encoding a Cas nuclease and a gRNA, which may be an sgRNA. In one embodiment, an LNP composition may comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In one embodiment, an LNP composition may comprise a lipid component consisting of an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid, and a nucleic acid component consisting of an mRNA encoding a Cas nuclease and a gRNA. In one embodiment, an LNP composition may comprise a lipid component consisting of an amine lipid, a helper lipid, and a PEG lipid, and a nucleic acid component consisting of an mRNA encoding a Cas nuclease and a gRNA. In certain compositions comprising an mRNA and a gRNA encoding a Cas nuclease, the helper lipid is cholesterol. In some compositions comprising an mRNA and a gRNA encoding a Cas nuclease, the neutral lipid is DSPC. Certain compositions comprising an mRNA and a gRNA encoding a Cas nuclease comprise less than about 1 mol% neutral lipid, e.g., a neutral phospholipid. Certain compositions comprising mRNA and gRNA encoding a Cas nuclease contain less than about 0.5 mol% neutral lipids, e.g., neutral phospholipids. In certain compositions, the LNPs are free of neutral lipids, e.g., neutral phospholipids. In further embodiments comprising mRNA and gRNA encoding a 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.

[0142] In certain embodiments, an LNP composition includes a Cas nuclease mRNA, such as a Class 2 Cas mRNA, and at least one gRNA. In certain embodiments, an LNP composition includes a gRNA and a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA, in a ratio of about 25:1 to about 1:25. In certain embodiments, an LNP formulation includes a gRNA and a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA, in a ratio of about 10:1 to about 1:10. In certain embodiments, an LNP formulation includes a gRNA and a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA, in a ratio of about 8:1 to about 1:8. Ratios measured herein are by weight. In some embodiments, an LNP formulation includes a gRNA and a Cas nuclease mRNA, such as a Class 2 Cas mRNA, in a ratio of about 5:1 to about 1:5. In some embodiments, the ratio ranges are 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, or 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.

[0143] The LNP compositions disclosed herein can include a template nucleic acid. The template nucleic acid can be combined with an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid can be combined with a guide RNA. In some embodiments, the template nucleic acid can be combined with both an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid can be formulated separately from the mRNA encoding the Cas nuclease or the guide RNA. The template nucleic acid can be delivered with or separately from the LNP composition. In some embodiments, the template nucleic acid can be single-stranded or double-stranded depending on the desired repair mechanism. The template can have regions of homology to the target DNA or sequences flanking the target DNA.

[0144] In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, such as 100% ethanol. Suitable solutions or solvents may include or contain water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, or isopropanol. A pharmacologically acceptable buffer may be used, for example, for in vivo administration of LNPs. In certain embodiments, a buffer is used to maintain the pH of a composition comprising LNPs at pH 6.5 or greater. In certain embodiments, a buffer is used to maintain the pH of a composition comprising LNPs at pH 7.0 or greater. In certain embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In further embodiments, the composition has a pH in the range of about 7.3 to about 7.7, or about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition may be measured with a micro pH probe. In certain embodiments, the composition includes a cryoprotectant. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as sucrose. In certain embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9, or 10% cryoprotectant. 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 phosphate buffer (PBS), Tris buffer, citrate buffer, or a mixture thereof. In certain exemplary embodiments, the buffer includes NaCl. In certain embodiments, NaCl is excluded. Exemplary amounts of NaCl can range from about 20 mM to about 45 mM. Exemplary amounts of NaCl can range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer.Exemplary amounts of Tris can range from about 20 mM to about 60 mM. Exemplary amounts of Tris can range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. In certain exemplary embodiments of the LNP composition, a Tris buffer containing 5% sucrose and 45 mM NaCl is included. In other exemplary embodiments, the composition contains about 5% w / v sucrose, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amounts of salt, buffer, and cryoprotectant can be varied to maintain the overall osmolality of the formulation. For example, the final osmolality can be maintained below 450 mOsm / L. In further embodiments, the osmolality is between 350 mOsm / L and 250 mOsm / L. In certain embodiments, the final osmolality is 300 + / - 20 mOsm / L.

[0145] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, the flow rate, junction size, junction location, junction shape, tubing diameter, solution, and / or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, for example, by dialysis, tangential flow filtration, or chromatography. LNPs may be stored, for example, as a suspension, emulsion, or lyophilized powder. In some embodiments, LNP compositions are stored at 2-8°C, and in certain aspects, LNP compositions are stored at room temperature. In further embodiments, LNP compositions are stored frozen, for example, at -20°C or -80°C. In other embodiments, LNP compositions are stored at a temperature ranging from about 0°C to about -80°C. Frozen LNP compositions may be thawed, for example, on ice, at room temperature, or at 25°C, prior to use.

[0146] LNPs may be, for example, microparticles (unilamellar and multilamellar vesicles, e.g., "liposomes," which in some embodiments are substantially spherical, comprise a lipid bilayer of a lamellar phase, and in certain embodiments may contain an aqueous core, e.g., a substantial portion of RNA molecules), the dispersed phase of an emulsion, a micelle, or the internal phase of a suspension.

[0147] Furthermore, the LNP compositions are biodegradable and do not accumulate in vivo to cytotoxic concentrations at therapeutically effective doses. In some embodiments, the LNP compositions do not elicit a natural immune response that results in substantial adverse effects at therapeutic doses. In some embodiments, the LNP compositions provided herein do not cause toxicity at therapeutic doses.

[0148] In some embodiments, pdi may range from about 0.005 to about 0.75. In some embodiments, pdi may range from about 0.01 to about 0.5. In some embodiments, pdi may range from about zero to about 0.4. In some embodiments, pdi may range from about zero to about 0.35. In some embodiments, pdi may range from about zero to about 0.35. In some embodiments, pdi may range from about zero to about 0.3. In some embodiments, pdi may range from about zero to about 0.25. In some embodiments, pdi may be less than about 0.08, less than 0.1, less than 0.15, less than 0.2, or less than 0.4.

[0149] The LNPs disclosed herein have a size (e.g., Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs are about 10 to about 200 nm in size. In further embodiments, the LNPs are about 20 to about 150 nm in size. In some embodiments, the LNPs are about 50 to about 150 nm in size. In some embodiments, the LNPs are about 50 to about 100 nm in size. In some embodiments, the LNPs are about 50 to about 120 nm in size. In some embodiments, the LNPs are about 60 to about 100 nm in size. In some embodiments, the LNPs are about 75 to about 150 nm in size. In some embodiments, the LNPs are about 75 to about 120 nm in size. In some embodiments, the LNPs are about 75 to about 100 nm in size. Unless otherwise specified, all sizes referred to herein are the average size (diameter) of fully formed particles as measured by dynamic light scattering on a Malvern Zetasizer. Dilute nanoparticle samples in phosphate-buffered saline (PBS) to achieve a count rate of approximately 200–400 kcps. Data are presented as a weighted average of intensity measurements (Z-mean diameter).

[0150] In some embodiments, LNPs are formed with an average encapsulation efficiency in the range of about 50% to about 100%. In some embodiments, LNPs are formed with an average encapsulation efficiency in the range of about 50% to about 70%. In some embodiments, LNPs are formed with an average encapsulation efficiency in the range of about 70% to about 90%. In some embodiments, LNPs are formed with an average encapsulation efficiency in the range of about 90% to about 100%. In some embodiments, LNPs are formed with an average encapsulation efficiency in the range of about 75% to about 95%.

[0151] In some embodiments, LNPs are formed with an average molecular weight ranging from about 1.00E+05 g / mol to about 1.00E+10 g / mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 5.00E+05 g / mol to about 7.00E+07 g / mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 1.00E+06 g / mol to about 1.00E+10 g / mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 1.00E+07 g / mol to about 1.00E+09 g / mol. In some embodiments, LNPs are formed with an average molecular weight ranging from about 5.00E+06 g / mol to about 5.00E+09 g / mol.

[0152] In some embodiments, the polydispersity (Mw / Mn; the ratio of weight-average molar mass (Mw) to number-average molar mass (Mn)) may range from about 1.000 to about 2.000. In some embodiments, Mw / Mn may range from about 1.00 to about 1.500. In some embodiments, Mw / Mn may range from about 1.020 to about 1.400. In some embodiments, Mw / Mn may range from about 1.010 to about 1.100. In some embodiments, Mw / Mn may range from about 1.100 to about 1.350.

[0153] Methods for engineering cells; engineered cells The LNP compositions disclosed herein may be used in methods for engineering cells by gene editing, both in vivo and in vitro, hi some embodiments, the methods involve contacting cells with an LNP composition described herein.

[0154] In some embodiments, the method involves contacting a cell of a subject, such as a mammal, such as a human. In some embodiments, the cell is in an organ, e.g., a liver, e.g., a mammalian liver, e.g., a human liver. In some embodiments, the cell is a liver cell, e.g., a mammalian liver cell, e.g., a human liver cell. In some embodiments, the cell is a hepatocyte, e.g., a mammalian hepatocyte, e.g., a human hepatocyte. In some embodiments, the liver cell is a stem cell. In some embodiments, the human liver cell can be a liver sinusoidal endothelial cell (LSEC). In some embodiments, the human liver cell can be a Kupffer cell. In some embodiments, the human liver cell can be a hepatic stellate cell. In some embodiments, the human liver cell can be a tumor cell. In some embodiments, the human liver cell can be a liver stem cell. In further embodiments, the cell comprises an ApoE-binding receptor. In some embodiments, the liver cell, e.g., a hepatocyte, is in situ. In some embodiments, the liver cell, e.g., a hepatocyte, has been isolated, e.g., in culture, e.g., a primary culture. Also provided are methods corresponding to the uses disclosed herein, which include administering an LNP composition disclosed herein to a subject or contacting such cells with an LNP composition disclosed herein.

[0155] In some embodiments, engineered cells are provided, e.g., engineered cells derived from any one of the cell types in the preceding paragraphs. Such engineered cells are produced according to the methods described herein. In some embodiments, the engineered cells are located within a tissue or organ, such as the liver, within a subject.

[0156] In some of the methods and cells described herein, the cells comprise a modification, e.g., an insertion or deletion ("indel") or substitution, of a nucleotide in the target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides in the target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises an indel that results in a frameshift mutation in the target sequence. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a substitution of one or two nucleotides in the target sequence, hi some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of a nucleotide resulting from incorporation of a template nucleic acid, e.g., any of the template nucleic acids described herein.

[0157] In some embodiments, cell populations comprising engineered cells are provided, e.g., cell populations comprising cells engineered according to the methods described herein. In some embodiments, the population comprises engineered cells cultured in vitro. In some embodiments, the population is within a tissue or organ within a subject, such as the liver. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the cells in the population are engineered. In certain embodiments, the methods disclosed herein result in an editing efficiency (or "editing rate") of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, as defined by the detection of indels. In other embodiments, the methods disclosed herein result in a DNA modification efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as defined by detection of sequence alterations, whether by insertion, deletion, substitution, or other means. In certain embodiments, the methods disclosed herein result in an editing or DNA modification efficiency level in a cell population of about 5% to about 100%, about 10% to about 50%, about 20 to about 100%, about 20 to about 80%, about 40 to about 100%, or about 40 to about 80%.

[0158] In some of the methods and cells described herein, the cells in the population comprise a modification, such as an indel or a substitution, in the target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides in the target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification results in a frameshift mutation in the target sequence. In some embodiments, the modification comprises an indel that results in a frameshift mutation in the target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells of the population contain frameshift mutations. In some embodiments, the modification comprises the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides of the target sequence. In some embodiments, the modification comprises the substitution of one or two nucleotides of the target sequence. In some embodiments, the modification comprises one or more of a nucleotide insertion, deletion, or substitution resulting from integration of a template nucleic acid, e.g., any of the template nucleic acids described herein.

[0159] Gene editing methods The LNP compositions disclosed herein may be used to perform gene editing in vivo and in vitro. In one embodiment, one or more LNP compositions described herein may be administered to a subject in need thereof. In one embodiment, one or more LNP compositions described herein may be contacted with cells. In one embodiment, a therapeutically effective amount of a composition described herein may be contacted with cells of a subject in need thereof. In one embodiment, genetically engineered cells may be produced by contacting cells with an LNP composition described herein. In various embodiments, the method includes introducing a template nucleic acid into a cell or a subject as described above.

[0160] In some embodiments, the method involves administering an LNP composition to cells associated with liver damage. In some embodiments, the method involves treating liver damage. In certain embodiments, the method involves contacting hepatocytes with the LNP composition. In certain embodiments, the method involves contacting hepatocytes with the LNP composition. In some embodiments, the method involves contacting ApoE-binding cells with the LNP composition.

[0161] In one embodiment, an LNP composition comprising an mRNA encoding a Class 2 Cas nuclease and a gRNA may be administered to a cell, such as an ApoE-binding cell. In a further embodiment, a template nucleic acid is also introduced into the cell. In a specific example, an LNP composition comprising a Class 2 Cas nuclease and an sgRNA may be administered to a cell, such as an ApoE-binding cell. In one embodiment, an LNP composition comprising an mRNA encoding a Class 2 Cas nuclease, a gRNA, and a template may be administered to a cell. In a specific example, an LNP composition comprising a Cas nuclease and an sgRNA may be administered to a liver cell. In some cases, the liver cell is within a subject.

[0162] In certain embodiments, a subject may receive a single dose of the LNP composition. In other instances, a subject may receive multiple doses of the LNP composition. In some embodiments, the LNP composition is administered two to five times. When two or more doses are administered, the doses may be administered about 1, 2, 3, 4, 5, 6, 7, 14, 21, or 28 days apart, about 2, 3, 4, 5, or 6 months apart, or about 1, 2, 3, 4, or 5 years apart. In certain embodiments, upon re-administration of the LNP composition, editing improves.

[0163] In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, may be administered to a cell separately from the administration of a composition comprising a gRNA. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, and a gRNA may be administered to a cell separately from the administration of a template nucleic acid to a cell. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, may be administered to a cell, followed by sequential administration of an LNP composition comprising a gRNA, and then administration of the template to the cell. In embodiments in which an LNP composition comprising an mRNA encoding a Cas nuclease is administered followed by administration of an LNP composition comprising a gRNA, the administrations may be separated by approximately 4 hours, 6 hours, 8 hours, 12 hours, or 24 hours, or 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[0164] In one embodiment, LNP compositions can be used to edit genes to cause gene knockout.In one embodiment, LNP compositions can be used to edit genes to cause gene knockdown in a cell population.In another embodiment, LNP compositions can be used to edit genes to cause gene correction.In a further embodiment, LNP compositions can be used to edit cells to cause gene insertion.

[0165] In one embodiment, administration of an LNP composition can result in gene editing that produces a durable response. For example, administration can result in a response duration of one day, one month, one year, or longer. As used herein, "duration of response" means that after a cell is edited using an LNP composition disclosed herein, the resulting modification is still present for a period of time after administration of the LNP composition. The modification can be detected by measuring target protein levels. The modification can be detected by detecting target DNA. In some embodiments, the duration of the response can be at least one week. In other embodiments, the duration of the response can be at least two weeks. In one embodiment, the duration of the response can be at least one month. In some embodiments, the duration of the response can be at least two months. In one embodiment, the duration of the response can be at least four months. In one embodiment, the duration of the response can be at least six months. In certain embodiments, the duration of the response can be about 26 weeks. In some embodiments, the duration of the response can be at least one year. In some embodiments, the duration of the response can be at least five years. In some embodiments, the duration of the response can be at least 10 years. In some embodiments, a sustained response is detectable after at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, or 24 months, either by measuring target protein levels or detecting target DNA. In some embodiments, a sustained response is detectable after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 years, either by measuring target protein levels or detecting target DNA.

[0166] LNP compositions can be administered parenterally. They may be administered directly into the bloodstream, tissue, muscle, or internal organs. Administration may be systemic, for example, by injection or infusion. Administration may be local. Suitable administration means include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, subretinal, intravitreal, intracameral, intramuscular, intrasynovial, intradermal, and subcutaneous. Suitable administration devices include needle (including microneedle) injectors, needleless, osmotic pumps, and infusion techniques.

[0167] LNP compositions are typically, but not necessarily, administered as a formulation containing one or more pharmacologically acceptable excipients. The term "excipient" includes any component other than the compound(s) of the present disclosure, other lipid component(s), and biologically active agents. The excipient may impart either functional (e.g., drug release rate control) and / or non-functional (e.g., processing aid or diluent) characteristics to the formulation. The choice of excipient will vary widely depending on factors such as the particular method of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0168] Parenteral formulations are typically aqueous or oily solutions or suspensions. When the formulation is aqueous, additives include sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably pH 3-9), although for some applications they may be more suitably formulated using sterile non-aqueous solutions or as a dry form for use in combination with a suitable vehicle such as sterile pyrogen-free water (WFI).

[0169] While the present invention will be described in conjunction with exemplary embodiments, it will be understood that they are not intended to limit the invention to the described embodiments. Rather, the present invention is intended to encompass all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the scope of the present invention as defined by the appended claims.

[0170] Both the general and detailed descriptions set forth above, as well as the following examples, are illustrative and explanatory only and are not intended to be limiting of the teachings. 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 a document incorporated by reference conflicts with a term defined herein, the present specification controls. All ranges set forth in this application include endpoints unless otherwise specified.

[0171] It should be noted that, as used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a composition" includes a plurality of compositions, a reference to "a cell" includes a plurality of cells, and so on. The use of "or" is inclusive and means "and / or" unless specifically stated otherwise.

[0172] Numerical ranges are inclusive of the numbers defining the range. It is understood that measurements and measurable values ​​are approximations and should take into account significant figures and error associated with measurement. The terms "about" or "approximately" refer to an acceptable error for a particular value as determined by one of ordinary skill in the art, which may vary somewhat depending on how the value is measured or determined. The use of modifiers such as "about" before a range or a recited value modifies each endpoint of the range or each value in the recited value. "About" also includes the value or endpoint. For example, "about 50 to 55" includes "about 50 to about 55." Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "include," and "including" is not limiting.

[0173] Unless otherwise indicated in the specification above, embodiments described herein as "comprising" various components are also intended to be "consisting of" or "consisting essentially of" the stated components; embodiments described herein as "consisting of" various components are also intended to be "consisting of" or "consisting essentially of" the stated components; embodiments described herein as "about" various components are also intended to be "at" the stated components; and embodiments described herein as "consisting essentially of" various components are also intended to be "consisting of" or "comprising" the stated components (such interchangeability is understood to be reflected in the use of these terms in the claims). does not apply in this case). The present invention encompasses, for example, the following embodiments: [Embodiment 1] An RNA component, and a lipid component, wherein the lipid component comprises: about 50-60 mol % of an amine lipid; Approximately 8 to 10 mol% of neutral lipids; Approximately 2.5-4 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The lipid nanoparticle ("LNP") composition, wherein the N / P ratio of the LNP composition is about 6. [Embodiment 2] An RNA component, about 50-60 mol % of an amine lipid; About 27 to 39.5 mol% of a helper lipid; Approximately 8 to 10 mol% of neutral lipids; Approximately 2.5-4 mol% PEG lipid wherein the N / P ratio of the LNP composition is about 5-7. [Embodiment 3] The LNP composition of embodiment 2, wherein the N / P ratio is about 6. [Embodiment 4] An RNA component, and a lipid component, wherein the lipid component comprises: about 50-60 mol % of an amine lipid; Approximately 5 to 15 mol % of neutral lipids; Approximately 2.5-4 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The LNP composition, wherein the N / P ratio of the LNP composition is about 3 to 10. [Embodiment 5] An RNA component, and a lipid component, wherein the lipid component comprises: about 40-60 mol % of an amine lipid; Approximately 5 to 15 mol % of neutral lipids; Approximately 2.5-4 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The LNP composition, wherein the N / P ratio of the LNP composition is about 6. [Embodiment 6] An RNA component, and a lipid component, wherein the lipid component comprises: about 50-60 mol % of an amine lipid; Approximately 5 to 15 mol % of neutral lipids; Approximately 1.5 to 10 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The LNP composition, wherein the N / P ratio of the LNP composition is about 6. [Embodiment 7] An RNA component, and a lipid component, wherein the lipid component comprises: about 40-60 mol % of an amine lipid; Approximately 0 to 10 mol% of a neutral lipid; Approximately 1.5 to 10 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The LNP composition, wherein the N / P ratio of the LNP composition is about 3 to 10. [Embodiment 8] An RNA component, and a lipid component, wherein the lipid component comprises: about 40-60 mol % of an amine lipid; less than about 1 mol % neutral lipids; Approximately 1.5 to 10 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The LNP composition, wherein the N / P ratio of the LNP composition is about 3 to 10. [Embodiment 9] An RNA component, and a lipid component, wherein the lipid component comprises: about 40-60 mol % of an amine lipid; Approximately 1.5 to 10 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The N / P ratio of the LNP composition is about 3-10, and The LNP composition, wherein the LNP composition is essentially free of or free of neutral phospholipids. [Embodiment 10] An RNA component, and a lipid component, wherein the lipid component comprises: about 50-60 mol % of an amine lipid; Approximately 8 to 10 mol% of neutral lipids; Approximately 2.5-4 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The LNP composition, wherein the N / P ratio of the LNP composition is about 3 to 7. [Embodiment 11] The composition of any preceding embodiment, wherein the RNA component comprises mRNA. [Embodiment 12] The composition of any preceding embodiment, wherein the RNA component comprises an RNA-guided DNA-binding factor, such as a Cas nuclease mRNA. [Embodiment 13] The composition of any preceding embodiment, wherein the RNA component comprises a Class 2 Cas nuclease mRNA. [Embodiment 14] The composition of any preceding embodiment, wherein the RNA component comprises a Cas9 nuclease mRNA. [Embodiment 15] The composition described in any one of embodiments 11 to 14, wherein the mRNA is a modified mRNA. [Embodiment 16] The composition of any preceding embodiment, wherein the RNA component comprises RNA comprising an open reading frame encoding an RNA-guided DNA-binding factor, wherein the open reading frame has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content. [Embodiment 17] The composition of any preceding embodiment, wherein the RNA component comprises an mRNA comprising an open reading frame encoding an RNA-guided DNA binding factor, wherein the open reading frame has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content. [Embodiment 18] The composition of any preceding embodiment, wherein the RNA component comprises an mRNA comprising a sequence 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, or 66, wherein the mRNA comprises an open reading frame encoding an RNA-guided DNA-binding factor. [Embodiment 19] The composition of any of the preceding embodiments, wherein the RNA component comprises a gRNA nucleic acid. [Embodiment 20] The composition described in embodiment 19, wherein the gRNA nucleic acid is a gRNA. [Embodiment 21] The composition of any preceding embodiment, wherein the RNA component comprises a Class 2 Cas nuclease mRNA and a gRNA. [Embodiment 22] The composition of any of embodiments 19 to 21, wherein the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA). [Embodiment 23] A composition described in any of embodiments 19 to 21, wherein the gRNA nucleic acid is or encodes an sgRNA. [Embodiment 24] The composition described in any one of embodiments 19 to 23, wherein the gRNA is modified. [Embodiment 25] The composition of embodiment 24, wherein the gRNA comprises a modification selected from 2'-O-methyl (2'-O-Me) modified nucleotides, internucleotide phosphorothioate (PS) linkages, and 2'-fluoro (2'-F) modified nucleotides. [Embodiment 26] The composition described in any of embodiments 24 to 25, wherein the gRNA comprises a modification in one or more of the first five nucleotides at the 5' end. [Embodiment 27] The composition described in any of embodiments 24 to 26, wherein the gRNA comprises a modification in one or more of the last five nucleotides at the 3' end. [Embodiment 28] A composition described in any one of embodiments 24 to 27, wherein the gRNA contains a PS bond between the first four nucleotides. [Embodiment 29] A composition described in any of embodiments 24 to 28, wherein the gRNA contains a PS bond between the last four nucleotides. [Embodiment 30] The composition according to any one of embodiments 24 to 29, further comprising 2'-O-Me modified nucleotides in the first three nucleotides of the 5' end. [Embodiment 31] A composition described in any one of embodiments 24 to 30, further comprising 2'-O-Me modified nucleotides in the last three nucleotides of the 3' end. [Embodiment 32] A composition described in any of embodiments 19 to 31, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio ranging from about 10:1 to about 1:10 by weight. [Embodiment 33] A composition described in any of embodiments 19 to 31, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio ranging from about 5:1 to about 1:5 by weight. [Embodiment 34] A composition described in any of embodiments 19 to 33, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio ranging from about 3:1 to about 1:1 by weight. [Embodiment 35] A composition described in any of embodiments 19 to 34, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio ranging from about 2:1 to about 1:1 by weight. [Embodiment 36] A composition described in any of embodiments 19 to 35, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio of approximately 2:1 by weight. [Embodiment 37] A composition described in any of embodiments 19 to 35, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio of approximately 1:1 by weight. [Embodiment 38] The composition of any preceding embodiment, further comprising at least one template. [Embodiment 39] The composition of any preceding embodiment, wherein the mole percent of the PEG lipid is about 3. [Embodiment 40] The composition of any preceding embodiment, wherein the mole percent of the amine lipid is about 50. [Embodiment 41] The composition of any preceding embodiment, wherein the mole percent of the amine lipid is about 55. [Embodiment 42] The composition of any preceding embodiment, wherein the mole percent of the amine lipid is ±3 mole percent. [Embodiment 43] The composition of any preceding embodiment, wherein the mole percent of the amine lipid is ±2 mole percent. [Embodiment 44] The composition of any preceding embodiment, wherein the amine lipid molar percentage is 47-53 mol%. [Embodiment 45] The composition of any preceding embodiment, wherein the amine lipid molar percentage is 48-53 mol%. [Embodiment 46] The composition of any preceding embodiment, wherein the amine lipid molar percentage is 53-57 mol%. [Embodiment 47] The composition of any preceding embodiment, wherein the N / P ratio is 6 ± 1. [Embodiment 48] The composition of any preceding embodiment, wherein the N / P ratio is 6 ± 0.5. [Embodiment 49] The composition of any preceding embodiment, wherein the amine lipid is lipid A. [Embodiment 50] The composition of any preceding embodiment, wherein the amine lipid is a lipid A analog. [Embodiment 51] The composition of embodiment 50, wherein the analog is an acetal analog. [Embodiment 52] The composition of embodiment 51, wherein the acetal analog is a C4-C12 acetal analog. [Embodiment 53] The composition of embodiment 50, wherein the acetal analog is a C5-C12 acetal analog. [Embodiment 54] The composition of embodiment 50, wherein the acetal analog is a C5-C10 acetal analog. [Embodiment 55] The composition of embodiment 50, wherein the acetal analog is selected from a C4 analog, a C5 analog, a C6 analog, a C7 analog, a C9 analog, a C10 analog, a C11 analog, and a C12 analog. [Embodiment 56] The composition of any preceding embodiment, wherein the helper lipid is cholesterol. [Embodiment 57] The composition of any preceding embodiment, wherein the neutral lipid is DSPC. [Embodiment 58] The composition of any preceding embodiment, wherein the neutral lipid is DPPC. [Embodiment 59] The composition of any preceding embodiment, wherein the PEG lipid comprises dimyristoylglycerol (DMG). [Embodiment 60] The composition of any preceding embodiment, wherein the PEG lipid comprises PEG-2k. [Embodiment 61] The composition of any preceding embodiment, wherein the PEG lipid is PEG-DMG. [Embodiment 62] The composition described in embodiment 61, wherein the PEG-DMG is PEG2k-DMG. [Embodiment 63] The composition of embodiment 9, wherein the LNP composition is essentially free of neutral lipids. [Embodiment 64] The composition described in embodiment 63, wherein the neutral lipid is a phospholipid. [Embodiment 65] A gene editing method comprising contacting a cell with an LNP composition described in any one of embodiments 12 to 64. [Embodiment 66] A gene editing method comprising delivering a Class 2 Cas nuclease mRNA and a guide RNA nucleic acid to a cell, wherein the Class 2 Cas mRNA and the guide RNA nucleic acid are formulated as at least one LNP composition described in any of embodiments 13 to 64. [Embodiment 67] A method for producing genetically engineered cells, comprising contacting cells with at least one LNP composition described in any one of embodiments 12 to 64. [Embodiment 68] The method of any one of embodiments 65 to 67, wherein the LNP composition is administered at least twice. [Embodiment 69] The method described in embodiment 68, wherein the LNP composition is administered 2 to 5 times. [Embodiment 70] The method of embodiment 68 or 69, wherein editing improves upon re-administration. [Embodiment 71] The method described in any one of embodiments 65 to 70, further comprising introducing at least one template nucleic acid into the cell. [Embodiment 72] The method described in any one of embodiments 65 to 71, wherein the mRNA is formulated in a first LNP composition and the guide RNA nucleic acid is formulated in a second LNP composition. [Embodiment 73] The method of embodiment 72, wherein the first and second LNP compositions are administered simultaneously. [Embodiment 74] The method of embodiment 72, wherein the first and second LNP compositions are administered sequentially. [Embodiment 75] The method described in any one of embodiments 65 to 73, wherein the mRNA and the guide RNA nucleic acid are formulated into a single LNP composition. [Example]

[0174] Example 1 - LNP Composition for In Vivo Editing in Mice Small-scale preparations of various LNP compositions were prepared and characterized. For liver editing rate assays in mice, Cas9 mRNA and chemically modified sgRNA directed to the mouse TTR sequence were formulated into LNPs with varying mole % PEG, mole % lipid A, and N:P ratios as listed in Table 2 below. [Table 2]

[0175] In Figure 1, LNP formulations are identified on the X-axis based on their lipid A mole % and N:P ratio, represented as "% CL;N:P." As indicated in the legend to Figure 1, PEG-2k-DMG at concentrations of 2 mol%, 2.5 mol%, 3 mol%, 4 mol%, or 5 mol% was formulated with: (1) 45 mol% lipid A; N:P ratio of 4.5 ("45;4.5"), (2) 45 mol% lipid A; N:P ratio of 6 ("45;6"), (3) 50 mol% lipid A; N:P ratio of 4.5 ("50;4.5"), (4) 50 mol% lipid A; N:P ratio of 6 ("50;6"), (5) 55 mol% lipid A; N:P ratio of 4.5 ("55;4.5"), and (6) 55 mol% lipid A; N:P ratio of 6 ("55;6"). The mole % of DSPC was kept constant at 9 mole %, and cholesterol (mole %) was added to make the remaining lipid component of each formulation 100 mole %. Each of the 30 formulations was formulated as described below and administered as a single dose at a total RNA dose of 1 mg per kg or 0.5 mg per kg (Figures 1A and 1B, respectively).

[0176] LNP Formulation - NanoAssemblr Lipid nanoparticle components were prepared by dissolving the lipid components in 100% ethanol at the molar ratios listed above. The RNA cargo was dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg / mL. LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of approximately 4.5 or 6, and a 1:1 mRNA to gRNA ratio by weight.

[0177] LNPs were generated by microfluidic mixing of lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument according to the manufacturer's operating instructions. Different flow rates were used during mixing, while maintaining a 2:1 aqueous-to-organic solvent ratio. After mixing, the LNPs were collected, diluted in water (approximately 1:1 v / v), and held at room temperature for 1 hour. They were then further diluted in water (approximately 1:1 v / v) before a final buffer exchange. A final buffer exchange into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) was completed using a PD-10 desalting column (GE). If necessary, the formulation was concentrated by centrifugation through an Amicon 100 kDa centrifugal filter (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNPs were stored at -80°C until further use.

[0178] Pharmaceutical Analysis Methods Dynamic light scattering ("DLS") is used to characterize the polydispersity index ("pdi") and size of the LNPs of the present disclosure. DLS measures the scattering of light that results when a sample is exposed to a light source. The PDI determined from a DLS measurement represents the particle size (approximate mean particle size) distribution in a population; a completely homogeneous population would have a PDI of zero.

[0179] Electrophoretic light scattering is used to characterize the surface charge of LNPs at a specific pH. Surface charge, or zeta potential, is a measure of the magnitude of electrostatic repulsion / attraction between particles in an LNP suspension.

[0180] Asymmetric flow field-flow fractionation-multiangle light scattering (AF4-MALS) is used to separate particles in formulations by hydrodynamic radius, followed by measurement of the molecular weight, hydrodynamic radius, and root-mean-square radius of the fractionated particles. This allows for the assessment of molecular weight and size distributions as well as secondary features, such as Burchard-Stockmeyer plots (the ratio of root-mean-square ("rms") radius to hydrodynamic radius over time, which indicates the particle's core density) and rms conformation plots (the logarithm of rms radius versus the logarithm of molecular weight, where the slope of the resulting linear fit indicates the degree of compactness and elongation).

[0181] Nanoparticle Tracking Analysis (NTA, Malvern Nanosight) can be used to determine the particle size distribution and particle concentration of a formulation. An LNP sample is appropriately diluted and injected onto a microscope slide. A camera records the scattered light as the particles are slowly poured through the field of view. Once a movie is acquired, Nanoparticle Tracking Analysis processes the movie by tracking the pixels and calculating the diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. The instrument also provides the particle concentration by counting the number of individual particles counted during the analysis.

[0182] Cryo-electron microscopy ("cryo-EM") can be used to determine the particle size, morphology, and structural characteristics of LNPs.

[0183] The lipid composition of LNPs can be determined by liquid chromatography followed by charged aerosol detection (LC-CAD), which provides a comparison of the actual lipid content versus the theoretical lipid content.

[0184] LNP formulations are analyzed for mean particle size, polydispersity index (pdi), total RNA content, RNA encapsulation efficiency, and zeta potential. LNP formulations may be further characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. Mean particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. Prior to DLS measurement, LNP samples were diluted 30X in PBS. The Z-average diameter, an intensity-based measure of mean particle size, was reported along with the number-average diameter and pdi. The Malvern Zetasizer instrument is also used to measure the zeta potential of LNPs. Prior to measurement, samples were diluted 1:17 (50 μL to 800 μL) in 0.1X PBS (pH 7.4).

[0185] A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) was used to determine total RNA concentration and free RNA. Encapsulation efficiency was calculated as (total RNA - free RNA) / total RNA. LNP samples were appropriately diluted with 1x TE buffer containing 0.2% Triton-X 100 to determine total RNA, or with 1x TE buffer to determine free RNA. A standard curve was generated using the starting RNA solution used to make the formulation and then diluted in 1x TE buffer + / - 0.2% Triton-X 100. Diluted RiboGreen® dye (according to the manufacturer's instructions) was then added to each standard and sample, and the mixture was incubated for approximately 10 minutes at room temperature in the absence of light. Samples were read using a SpectraMax M5 plate reader (Molecular Devices) with excitation, autocutoff, and emission wavelengths set at 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA were determined using the appropriate standard curve.

[0186] The encapsulation rate is calculated as (total RNA - free RNA) / total RNA. Similar methods can be used to determine the encapsulation rate of DNA-based or nucleic acid-containing cargo components. Oligreen dye can be used for single-stranded DNA, and Picogreen dye can be used for double-stranded DNA.

[0187] AF4-MALS was used to investigate molecular weight and size distribution, as well as their calculated second-order statistics. LNPs were appropriately diluted and injected into the AF4 separation channel using an HPLC autosampler, where they were collected, and then eluted with an exponential gradient in a crossflow across the channel. All fluids were driven by an HPLC pump and a Wyatt Eclipse instrument. Particles eluted from the AF4 channel flow and passed through a UV detector, a multi-angle light scattering detector, a quasi-elastic light scattering detector, and a differential refractive index detector. The raw data were processed using the Debeye model to determine molecular weight and rms radius from the detector signals.

[0188] The lipid components in LNPs were quantitatively analyzed by HPLC coupled to a charged aerosol detector (CAD). Chromatographic separation of the four lipid components was achieved by reversed-phase HPLC. CAD is a mass-based detector that detects nonvolatile compounds without omission, and the signal remains constant regardless of the analyte structure.

[0189] Cas9 mRNA and gRNA cargo Cas9 mRNA cargo was prepared by in vitro transcription. Capped, polyadenylated Cas9 mRNA containing a 1X NLS (SEQ ID NO: 48) was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter and a 100-nt poly(A / T) tract was linearized by incubating XbaI with the following conditions at 37°C for 2 hours: 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1X reaction buffer. XbaI was inactivated by heating the reaction at 65°C for 20 minutes. The linearized plasmid was purified from the enzyme and buffer salts using a silica maxi spin column (Epoch Life Sciences) and analyzed on an agarose gel to confirm linearization. The IVT reaction to generate Cas9-modified mRNA was incubated for 4 hours at 37°C with the following conditions: 50 ng / μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methyl pseudo-UTP (Trilink); 10 mM ARCA (Trilink); 5 U / μL T7 RNA polymerase (NEB); 1 U / μL mouse RNAse inhibitor (NEB); 0.004 U / μL E. coli inorganic pyrophosphatase (NEB); and 1x reaction buffer. After 4 hours of incubation, TURBO DNase (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 mRNA was purified with enzymes and nucleotides using the MegaClear Transcription Clean-up Kit according to the manufacturer's instructions (ThermoFisher). Alternatively, Cas9 mRNA was purified by LiCl precipitation.

[0190] The sgRNA in this example was chemically synthesized and supplied by a commercial supplier. The sg282 sequence is set forth below, with 2'-O-methyl modifications and phosphorothioate linkages indicated as follows (m=2'-OMe; *=phosphorothioate): mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU. (SEQ ID NO: 42).

[0191] LNP The final LNPs were characterized to determine the encapsulation rate, polydispersity index, and mean particle size according to the analytical methods described above.

[0192] Mice were administered LNP (single dose at 1 mg / kg or 0.5 mg / kg), and genomic DNA was isolated and subjected to NGS analysis as described below.

[0193] In vivo LNP delivery Each study used female CD-1 mice aged 6 to 10 weeks. Animals were weighed and divided into groups according to their body weight, and the dosing solution was prepared based on the average weight of the group. LNP was administered via the lateral tail vein at a volume of 0.2 mL per animal (approximately 10 mL per kilogram of body weight). Approximately 6 hours after administration, animals were observed for adverse effects. 24 hours after administration, animals were weighed, and at various time points, animals were euthanized by exsanguination via cardiac puncture under isoflurane anesthesia. Blood was collected into serum separator tubes or into tubes containing buffered sodium citrate for plasma as described herein. For in vivo editing studies, liver tissue was collected from the median lobe or three independent lobes (e.g., right median lobe, left median lobe, and left lateral lobe) of each animal for DNA extraction and analysis.

[0194] A cohort of mice was measured for editing in the liver by next-generation sequencing (NGS) and serum TTR levels (data not shown).

[0195] Transthyretin (TTR) ELISA analysis Blood was collected and serum isolated as indicated. Mouse serum total TTR levels were determined using a mouse prealbumin (transthyretin) ELISA kit (Aviva Systems Biology, catalog number OKIA00111). Rat serum TTR levels were measured using a rat-specific ELISA kit (Aviva Systems Biology, catalog number OKIA00159) according to the manufacturer's instructions. Briefly, serum was serially diluted with the kit's sample diluent to a final dilution of 1:10,000. The diluted samples were then added to an ELISA plate and the assay was performed according to the manufacturer's instructions.

[0196] NGS sequencing Briefly, to quantitatively determine the editing efficiency at the target locations in the genome, genomic DNA was isolated and deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing.

[0197] PCR primers were designed around the target site (e.g., TTR) to amplify the genomic region of interest. Primer sequences are provided below. Further PCR was performed according to the manufacturer's operating instructions (Illumina), adding the necessary sequencing chemistry. Amplicons were sequenced on an Illumina MiSeq instrument. After removing low quality scores, reads were aligned to a human reference genome (e.g., hg38). The resulting file containing the reads was mapped to the reference genome (BAM file), where reads that overlapped with the target region of interest were selected, and the ratio of wild-type reads to reads containing insertions, substitutions, or deletions was calculated.

[0198] The editing ratio (e.g., "editing efficiency" or "editing rate") is defined as the total number of sequence reads containing insertions or deletions relative to the total number of sequence reads, such as the wild type.

[0199] Figure 1 shows the percentage of editing in mouse livers as measured by next-generation sequencing (NGS). As shown in Figure 1A, when 1 mg of RNA was administered per kg, in vivo editing rates ranged from approximately 20% to over 60% in the liver. In Figure 1B, when 0.5 mg per kg was administered, approximately 10% to 60% editing in the liver was observed. In this in vivo study in mice, all compositions effectively delivered Cas9 mRNA and gRNA to hepatocytes, and each LNP composition demonstrated active CRISPR / Cas nuclease activity at the target site as measured by next-generation sequencing (NGS). LNPs containing 5% PEG lipids had lower encapsulation rates (data not shown) and were somewhat less potent.

[0200] Example 2 - LNP Composition Analysis Method Characterization of LNPs shows improved physicochemical parameters in LNPs formulated with increased amounts of lipid A and PEG lipids. Compositions containing 2 mol% or 3 mol% PEG lipid (PEG2k-DMG) are listed in Table 3 below. [Table 3]

[0201] LNP Formulation - Crossflow Lipid-containing ethanol was impinging-jet mixed with two volumes of RNA solution and one volume of water to form LNPs. Lipid-containing ethanol was mixed with two volumes of RNA solution through a mixing intersection. A fourth stream of water was mixed with the outlet stream of the intersection through an in-line T-junction (see Figure 2 in WO2016010840). The LNPs were maintained at room temperature for 1 hour and then further diluted with water (approximately 1:1 v / v). The diluted LNPs were concentrated using tangential flow filtration with a flat-sheet cartridge (Sartorius, 100 kD MWCO), and then buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) by hemodiafiltration. Alternatively, the final buffer exchange to TSS was completed with a PD-10 desalting column (GE). If necessary, the formulation was concentrated by centrifugation using an Amicon 100 kDa centrifugal filter (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNP was stored at 4°C or -80°C until further use.

[0202] Cas9 mRNA and sgRNA were prepared as described in Example 1, except that the capped and polyadenylated Cas9 U-deleted (Cas9 Udep) mRNA contained SEQ ID NO: 43. Sg282 is described in Example 1, and the sequence of sg534 ("G534") is set forth below: mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 72)

[0203] The LNP formulations were analyzed for mean particle size, polydispersity index (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1.

[0204] Analysis of mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation rate are shown in Table 4. In addition to the theoretical lipid concentrations of the LNP compositions, lipid analysis revealed the actual lipid mole % levels as shown in Table 5 below. [Table 4] [Table 5] TIFF0007645929000010.tif99155

[0205] To further analyze their physicochemical properties, LNP897, LNP898, LNP966, and LNP969 were subjected to asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS) analysis. The AF4-MALS instrument measures particle size and molecular weight distribution, providing information about particle conformation and density.

[0206] LNPs are injected into the AF4 separation channel using an HPLC autosampler where they are collected and then eluted with an exponential gradient in crossflow across the channel. All fluids are driven by HPLC pumps and a Wyatt Eclipse instrument. Particles elute from the AF4 channel flow and pass through a UV detector, a Wyatt Heleos II multi-angle light scattering detector, a quasi-elastic light scattering detector, and a Wyatt Optilab T-rEX differential refractive index detector. Raw data are processed in Wyatt Astra 7 software using the Debeye model to determine molecular weight and rms radius from the detector signals.

[0207] The logarithmic molar mass difference plot for LNPs is shown in Figure 2A. Briefly, the X-axis shows molar mass (g / mol) and the Y-axis shows differential number fraction. The logarithmic molar mass difference plot shows the distribution of different molecular weights measured for a particular formulation. This provides data on the molecular weight in the formulation as well as the overall molecular weight distribution, giving a better picture of particle heterogeneity than the average molecular weight.

[0208] The heterogeneity of various LNP formulations is determined by measuring different molar mass moments and calculating the ratio of weight-average molar mass (Mw) to number-average molar mass (Mn) to obtain the polydispersity Mw / Mn. The polydispersity graphs for these various formulations are shown in Figure 2B.

[0209] As shown in Figure 2A, the data indicate that the particle distribution becomes tighter at N / P 6.0 with 3 mol% PEG and 50 and 55 mol% lipid A. This is reflected in the tighter polydispersity shown in Figure 2B.

[0210] Example 3 - AF4 MALS Data - Further Formulations Characterization of LNPs shows improved physicochemical parameters in LNPs formulated with increased amounts of lipid A. Compositions containing either 45 mol%, 50 mol%, or 55 mol% lipid A with two different gRNAs are listed in Table 6 below. [Table 6]

[0211] LNPs were formed as described in Example 2.

[0212] Cas9 mRNA and sgRNA were prepared as described above.

[0213] The LNP compositions were characterized to determine the encapsulation efficiency, polydispersity index, and mean particle size as described in Example 1.

[0214] The analyses of mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation rate are shown in Table 7. In addition to the theoretical lipid concentrations of the LNP compositions, lipid analysis revealed the actual lipid mole % levels as shown in Table 8 below. [Table 7] TIFF0007645929000013.tif46159 [Table 8] TIFF0007645929000015.tif36156

[0215] To further analyze their physicochemical properties, LNP1021, LNP1022, LNP1023, LNP1024, and LNP1025 were subjected to asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS) analysis. The AF4-MALS instrument measures particle size and molecular weight distribution, providing information about particle conformation and density.

[0216] The LNPs were subjected to AF4-MALS as described in Example 1.

[0217] The logarithmic molar mass difference plot for LNPs is shown in Figure 3A. Briefly, the X-axis shows molar mass (g / mol) and the Y-axis shows differential number fraction. The logarithmic molar mass difference plot shows the distribution of different molecular weights calculated for a particular formulation. This provides data on the molecular weight in the formulation as well as the overall distribution of molecular weights, giving a better picture of particle heterogeneity than the average molecular weight.

[0218] Figure 3B plots the average molecular weight. The average molecular weight is an average across the distribution, but does not provide information about the shape of that distribution. LNP1022 and LNP1025 have the same average molecular weight, but the distribution is slightly broader for LNP1022.

[0219] The heterogeneity of various LNP formulations is calculated by examining different molar mass moments and calculating the ratio of weight-average molar mass (Mw) to number-average molar mass (Mn) to obtain the polydispersity of Mw / Mn. The polydispersity graphs for these various formulations are shown in Figure 4A.

[0220] Additionally, the Burchard-Stockmeyer plots for the LNP formulations are shown in Figure 4B. The Burchard-Stockmeyer plot shows the ratio of the rms radius to the hydrodynamic radius for the entire formulation elution from the AF4 channel, providing information about the internal density of the lipid nanoparticles. Figure 4B shows that LNP1021, LNP1022, and LNP1023 have distinct profiles in this measurement.

[0221] Example 4 - PEG lipid augmentation maintains efficacy and reduces cytokine response In another study, PEG-DMG lipids were compared in LNP formulations containing either 2 mol% or 3 mol% PEG-lipid. Compositions containing either 2 mol% or 3 mol% PEG-DMG are listed in Table 9 below. [Table 9] TIFF0007645929000017.tif51145

[0222] LNPs were formed as described in Example 2.

[0223] Cas9 mRNA and sgRNA were prepared as described in Example 1, and the sequence of sg390 (“G390”) is set forth below: mG*mC*mC*GAGUCUGGAGAGCUGCAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 69).

[0224] The LNP formulations were analyzed for mean particle size, polydispersity index (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1.

[0225] The analyses of mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation rate are shown in Table 10. In addition to the theoretical lipid concentrations of the LNP compositions, lipid analysis revealed the actual lipid mole % levels as shown in Table 11 below. [Table 10] [Table 11] TIFF0007645929000020.tif112160

[0226] Rat serum cytokines were assessed using a Luminex magnetic bead multiplex assay (Milliplex MAP magnetic bead assay, Millipore Sigma, catalog number RECYTMAG-65K) to analyze MCP-1, IL-6, TNF-alpha, and IFN-gamma. Assay beads were read on a BioRad BioPlex-200, and cytokine concentrations were calculated from the standard curve using a four-parameter logistic fit in BioPlex Manager software version 6.1. Data are graphed in Figure 5. See Figure 5A (serum TTR), Figure 5B (liver ED), and Figure 5C (cytokine pMCP1).

[0227] Rat serum TTR levels were measured using a rat-specific ELISA kit (Aviva Systems Biology, catalog number OKIA00159) according to the manufacturer's instructions. Briefly, serum was serially diluted with the kit's sample diluent to a final dilution of 1:10,000. The diluted samples were then added to an ELISA plate and the assay was performed according to the manufacturer's instructions.

[0228] Genomic DNA was isolated from approximately 10 mg of liver tissue and analyzed using NGS as described above. The PCR primer sequences for amplification are listed below.

[0229] Figures 5A and 5B show that the 2 mol% and 3 mol% PEG formulations achieved sufficient serum TTR knockdown and liver editing, while Figure 5C shows that the 3 mol% PEG formulation resulted in a reduced MCP-1 response.

[0230] Example 5 - LNP delivery to non-human primates Three studies were performed using LNP formulations prepared as described in Example 1. Specific molar amounts and cargos are listed in Tables 12-26. Each formulation contained Cas9 mRNA and guide RNA (gRNA) at a 1:1 mRNA:gRNA ratio by weight. The LNP dose (unit: mg / kg, total RNA content), route of administration, and whether animals were pretreated with dexamethasone are listed in the tables. For animals pretreated with dexamethasone (Dex), Dex was administered via IV bolus injection at 2 mg / kg 1 hour before LNP or vehicle administration.

[0231] For blood chemistry analysis, blood was collected from animals at the times indicated in the table below for each factor measured. Cytokine induction was measured in NHPs before and after treatment. A minimum of 0.5 mL of whole blood was collected from a peripheral vein of restrained, conscious animals into a 4 mL serum separator tube. Blood was allowed to clot for a minimum of 30 minutes at room temperature and then centrifuged at 2000 x g for 15 minutes. Serum was aliquoted into two 120 μL polypropylene microtubes and stored at -60 to -86°C until analysis. Analysis was performed using a non-human primate U-Plex Cytokine custom kit from Meso Scale Discovery (MSD). The following parameters were included in the analysis, with an emphasis on IL-6 and MCP-1: IFN-γ, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, MCP-1, and TNF-α. Kit reagents and standard solutions were prepared as directed in the manufacturer's operating procedures. NHP serum was used as received. Plates were read on an MSD Sector Imager 6000 and analysis was performed with MSD Discovery Workbench software version 4012.

[0232] Complement levels were measured by enzyme immunoassay in animals before and after treatment. Whole blood (0.5 mL) was collected from a peripheral vein of restrained, conscious animals into tubes containing 0.5 mL of k₂EDTA. Blood was centrifuged at 2000 x g for 15 minutes. Plasma was aliquoted into two 120 μL polypropylene microtubes and stored at -60 to -86°C until analysis. Analysis was performed using the Quidel MicroVue Complement Plus EIA Kit (C3a - Catalog No. A031) or (Bb - Catalog No. A027). Kit reagents and standards were prepared as directed in the manufacturer's operating instructions. Plates were read on an MSD Sector Imager 6000 at an optical density of 450 nm. Results were analyzed using a four-parameter curve fit.

[0233] Data for cytokine induction and complement activation are listed in the table below. "BLQ" means below limit of quantitation.

Table 12

Table 13

Table 14

Table 15

Table 17

Table 18

Table 19

[0234] Example 6 - PEG lipid screening Another study compared alternative PEG-lipids in LNP formulations containing 2 mol% or 3 mol% PEG-lipid.

[0235] Three types of PEG lipids were used in the study, and lipid 1 (DMG-PEG2k; Nof) is represented as follows: [ka]

[0236] Lipid 2, synthesized as described in Heyes, et al., J. Controlled Release, 107 (2005), pp. 278-279 (see "Synthesis of PEG2000-C-DMA"), can be represented as follows: [ka] Lipid 3 disclosed in WO2016 / 010840 (compound S027, see paragraphs

[0240] to

[0244] ) and WO2011 / 076807 can be represented as follows: [ka]

[0237] Lipid A was formulated using 2 mol% and 3 mol% of each PEG-lipid. Lipid nanoparticle components were prepared by dissolving the lipid components in 100% ethanol at the molar ratios listed above. Briefly, the RNA cargo was prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, resulting in an RNA cargo concentration of approximately 0.45 mg / mL. LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of approximately 4.5 and a 1:1 mRNA to gRNA ratio by weight. [Table 27] TIFF0007645929000052.tif93170

[0238] Cas9 mRNA, sg282, and LNP were prepared as described in Example 1.

[0239] LNP compositions containing lipid 1, lipid 2, or lipid 3 were administered to female CD-1 mice at 1 mg / kg body weight and 0.5 mg / kg body weight and evaluated as described in Example 1. Cohorts of mice were assayed for liver editing by next-generation sequencing (NGS) and serum TTR levels according to the methods of Example 1.

[0240] Figures 6A and 6B compare serum TTR levels between PEG-lipid formulations. Figure 6A shows serum TTR in μg / mL, and Figure 6B shows the data as percent knockdown (TSS%). Figure 6C shows the editing rate achieved in the liver. The data show that LNP compositions containing each of the tested PEG-lipids showed efficacy at 2 mol% and 3 mol%, with lipid 1 consistently outperforming lipid 2 and lipid 3.

[0241] Example 7 - Lipid A Analogues A number of structural analogs of lipid A were synthesized and tested in the LNP compositions described herein.

[0242] Synthesis: Lipid A is prepared by reacting 4,4-bis(octyloxy)butanoic acid ("Intermediate 13b" in Example 13 of WO 2015 / 095340) with (9Z,12Z)-3-hydroxy-2-(hydroxymethyl)propyloctadeca-9,12-dienoate ("Intermediate 13c"), followed by reaction of the product of Intermediate 13b and Intermediate 13c with 3-diethylamino-1-propanol to add the head group (see pages 84-86 of WO 2015 / 095340).

[0243] Intermediate 13b (4,4-bis(octyloxy)butanoic acid) of WO2015 / 095340 was synthesized from 4,4-bis(octyloxy)butanenitrile as follows.

[0244] Intermediate 13a: 4,4-bis(octyloxy)butanenitrile [ka]

[0245] To a mixture of 4,4-diethoxybutanenitrile (9.4 g, 60 mmol) and octan-1-ol (23.1 g, 178 mmol) was added pyridinium p-toluenesulfonate (748 mg, 3.0 mmol) at room temperature. The mixture was heated to 105 °C and stirred for 18 h in an open, open-air reactor without a reflux condenser. The reaction mixture was then cooled to room temperature and purified on silica gel (ethyl acetate in hexanes, 0-5% gradient) to give 10.1 g (31.0 mmol) of intermediate 13a as a clear oil. 1 H NMR (400 MHz, CDCl3) δ 4.55 (t, J = 5.3 Hz, 1H), 3.60 (dt, J = 9.2, 6.6 Hz, 2H), 3.43 (dt, J = 9.2, 6.6 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 1.94 (td, J = 7.4, 5.3 Hz, 2H), 1.63 - 1.50 (m, 4H), 1.38 - 1.19 (m, 20H), 0.93 - 0.82 (m, 6H) ppm.

[0246] Next, to a solution of intermediate 13a (8.42 g, 31 mmol) in ethanol (30 mL) was added 31 mL of aqueous potassium hydroxide (2.5 M, 30.9 mL, 77.3 mmol) at room temperature. The reaction vessel was fitted with a reflux condenser, and the mixture was heated to 110 °C and stirred for 24 hours. The mixture was then cooled to room temperature, acidified to pH 5 with aqueous hydrochloric acid (1 N), and extracted three times into hexane. The combined organic extracts were washed with water (twice) and brine, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure to give 8.15 g (23.6 mmol) of intermediate 13b as a clear oil, which was used without further purification. 1 H NMR (400 MHz, CDCl3) δ 4.50 (t, J = 5.5 Hz, 1H), 3.57 (dt, J = 9.4, 6.7 Hz, 2H), 3.41 (dt, J = 9.3, 6.7 Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H), 1.92 (td, J = 7.4, 5.3 Hz, 2H), 1.56 (m, 4H), 1.37 - 1.21 (m, 20H), 0.92 - 0.83 (m, 6H) ppm (structure below).

[0247] Intermediate 13b [ka]

[0248] Using the above method, C(C5, 6, 7, 9, and 10)-acetal acidic intermediates (referred to as intermediates B3-F3 and depicted below) were prepared using the appropriate alkan-1-ol reagent.

[0249] Intermediate B3, 4,4-bis(pentyloxy)butanoic acid [ka]

[0250] 1H NMR (400 MHz, CDCl3) δ 4.52 (t, J = 5.5 Hz, 1H), 3.58 (dt, J = 9.3, 6.6 Hz, 2H), 3.41 (dt, J = 9.3, 6.7 Hz, 2H), 2.45 (t, J = 7.4 Hz, 2H), 1.94 (m, 2H), 1.57 (m, 4H), 1.32 (m, J = 3.7 Hz, 8H), 0.95 - 0.83 (m, 6H) ppm.

[0251] Intermediate C3: 4,4-Hydroic acid

change

[0252] 1 H NMR (400 MHz, CDCl3) δ 4.44 (t, J = 5.6 Hz, 1H), 3.49 (dt, J = 9.3, 6.9 Hz, 2H), 3.39 (dt, J = 9.3, 6.8 Hz, 2H), 2.12 (t, J = 7.6 Hz, 2H), 1.79 (q, J = 7.0 Hz, 2H), 1.54 (m, 4H), 1.29 (m, 12H), 0.94 - 0.82 (m, 6H) ppm.

[0253] Intermediate D3: 4,4-ビス(ヘプチルオキシ)ブタン acid

change

[0254] 1H NMR (400 MHz, CDCl3) δ 8.85 (br s, 1H), 4.46 (t, J = 5.6 Hz, 1H), 3.52 (dt, J = 9.4, 6.8 Hz, 2H), 3.39 (dt, J = 9.3, 6.8 Hz, 2H), 2.26 (t, J = 7.6 Hz, 2H), 1.85 (q, J = 7.0 Hz, 2H), 1.53 (m, 4H), 1.29 (m, 16H), 0.94 - 0.80 (m, 6H) ppm.

[0255] Intermediate E3: 4,4-ビス(ノニルオキシ)ブタン acid

change

[0256] 1 H NMR (400 MHz, CDCl3) δ 5.32 (br s, 1H), 4.44 (t, J = 5.6 Hz, 1H), 3.49 (dt, J = 9.3, 6.9 Hz, 2H), 3.38 (dt, J = 9.4, 6.9 Hz, 2H), 2.10 (t, J = 7.6 Hz, 2H), 1.78 (q, J = 7.0 Hz, 2H), 1.53 (m, 4H), 1.27 (m, 24H), 0.88 (t, J = 6.6 Hz, 6H) ppm.

[0257] Intermediate F3: 4,4-ビス(デシルオキシ) acid:

change

[0258] 1H NMR (400 MHz, CDCl3) δ 4.48 (t, J = 5.5 Hz, 1H), 3.55 (m, 2H), 3.42 (m, 2H), 2.29 (dd, J = 10.8, 7.5 Hz, 2H), 1.90 - 1.82 (m, 2H), 1.55 (m, 4H), 1.27 (m, 28H), 0.88 (t, J = 6.7 Hz, 6H) ppm.

[0259] The acetal analogs of lipid A (C(8)) were synthesized by reacting the C(C5, 6, 7, 9, or 10)-acetal acidic intermediates (B3-F3) with intermediate 13c, followed by reaction of the product of that step with 3-diethylamino-1-propanol (see WO2015 / 095340, pages 84-86). Each analog was synthesized and 1 Characterization was carried out by 1 H NMR (data not shown).

[0260] The C7, C9, and C10 analogs were formulated at an N:P ratio of 4.5 with 45 mol% lipid A analog, 2 mol% DMG-PEG2k, 9 mol% DSPC, and 44 mol% cholesterol. Each analog was also formulated at an N:P ratio of 6 with 55 mol% lipid A analog, 2.5 mol% DMG-PEG2k, 9 mol% DSPC, and 38.5 mol% cholesterol. The lipid nanoparticle components were prepared by dissolving the lipid components in 100% ethanol at the above molar ratios. The RNA cargo was prepared in 25 mM citrate, 100 mM NaCl, pH 5.0, to a concentration of approximately 0.45 mg / mL.

[0261] The RNA cargo included Cas9 mRNA containing SEQ ID NO: 43 and sg282, prepared as described above. LNPs were formed as described in Example 1.

[0262] An expanded panel of acetal analogs was tested alongside the previous panel, including LNP compositions containing the C(5) and C(6) lipid A analogs. The two new analogs were formulated as described above with 55 mol% lipid A analog, 2.5 mol% DMG-PEG2k, 9 mol% DPSC, and 33.5 mol% cholesterol at an N / P ratio of 6. Analysis showed that the LNP sizes were all below 120 nm, PDIs were less than 0.2, and the percent RNA encapsulated was greater than 80%. The analytical results of the formulations are listed below in Table 28. [Table 28]

[0263] Analogs were evaluated for pKa using 6-(p-toluidino)-6-naphthalenesulfonic acid ("TNS") dissolved in water. For this assay, 0.1 M phosphate buffers were prepared at different pH values ​​ranging from 4.5 to 10.5. Each analog was individually prepared in 100% ethanol. The lipid and TNS were then added to the individual pH buffers, transferred to a plate, and analyzed on a SpectraMax microplate reader at wavelengths of 321-488 nm. The values ​​were plotted to generate pKa. The logarithm of IC 50 is used as the pKa.

[0264] Female CD-1 mice were administered 0.3 mg / kg (Figures 7A-7E) or 0.1 mg / kg (Figures 7F-7G) as described in Example 1. Briefly, female CD-1 mice (n=5 / group) obtained from Charles River Laboratories were administered various doses of the LNP composition. At necropsy (7 days post-administration), serum was collected for TTR analysis, and livers were harvested for editing analysis. Serum TTR and editing rate assays were performed as described in Example 1. Serum TTR levels and liver editing in Figures 7A-7E show that all analogs performed comparable to lipid A at 0.3 milligrams per kilogram of body weight. Figures 7F-7G show that lipid A was the most potent, but all newly synthesized analogs demonstrated adequate TTR knockdown and liver editing.

[0265] Example 8 - Dose-Response Curve - Primary Cyno Hepatocytes Primary hepatocytes from the liver. Primary cynomolgus monkey liver hepatocytes (PCH) (Gibco) were thawed and resuspended in supplemented Hepatocyte Thawing Medium (Gibco, Cat. CM7000) followed by centrifugation at 80g for 4 minutes. The supernatant was discarded, and the pelleted cells were resuspended in supplemented Hepatocyte Seeding Medium Pack (Invitrogen, Cat. A1217601 and CM3000). Cells were counted and seeded at a density of 50,000 cells / well onto Bio-coat collagen I-coated 96-well plates (ThermoFisher, Cat. 877272). The seeded cells were allowed to settle and adhere for 24 hours in a tissue culture incubator (37°C, 5% CO2 atmosphere) before administration of LNPs. After incubation, cells were checked for monolayer formation and the medium was replaced with Hepatocyte Medium and Serum-Free Supplement Pack (Invitrogen, Cat. A1217601 and CM4000).

[0266] The LNP formulations for this study (LNP1021, LNP1022, LNP1023, LNP1024, LNP1025, and LNP897) were prepared as described above.

[0267] Lipid nanoparticle formulations containing modified sgRNA were tested at various doses in primary cynomolgus monkey hepatocytes to generate dose-response curves. After seeding and culturing for 24 hours, LNPs were incubated in hepatocyte maintenance medium containing 6% cynomolgus monkey serum for 5 minutes at 37°C. After incubation, LNPs were added to primary cynomolgus monkey hepatocytes starting with 100 ng of mRNA in an 8.2-fold dose-response curve. Cells were lysed 72 hours after treatment and subjected to NGS analysis as described in Example 1. Data on the editing rates determined for various LNP compositions are graphed in Figure 8A. The editing rates (%) for Cas9 mRNA (SEQ ID NO: 48) and U-deleted Cas9 mRNA (SEQ ID NO: 43) are listed in Figure 8B. LNP compositions are listed in Table 2 (LNP897) and Table 5 (LNP1021, LNP1022, LNP1023, LNP1024, and LNP1025).

[0268] The results represent a quantitative assessment of comparative potency evaluation, demonstrating that both mRNA and LNP composition influence potency.

[0269] Example 9 - RNA cargo: mRNA and gRNA combination This study evaluated the in vivo efficacy in mice of different gRNA to mRNA ratios. CleanCap™ capped Cas9 mRNA with ORF SEQ ID NO:4, HSD 5'UTR, human albumin 3'UTR, Kozak sequence, and poly-A tail was generated by IVT synthesis using N1-methylpseudouridine triphosphate instead of uridine triphosphate as shown in Example 1.

[0270] LNP formulations were prepared as described in Example 2 from the indicated mRNA and sg282 (SEQ ID NO:42; G282) in a molar ratio of 50:38:9:3 for lipid A, cholesterol, DSPC, and PEG2k-DMG, with an N:P ratio of 6. The gRNA:Cas9 mRNA weight ratios of the formulations were as shown in Table 29. [Table 29]

[0271] For in vivo characterization, the LNPs were administered to mice (n=5 / group) at 0.1 mg of total RNA (guide RNA (mg) + mRNA (mg)) per kg. Seven to nine days after administration, animals were sacrificed, blood and liver were collected, and serum TTR and liver editing were measured as described in Example 1. The results for serum TTR and liver editing are shown in Figures 9A and 9B. Negative control mice received the TSS vehicle.

[0272] Furthermore, the LNPs were administered to mice at a fixed mRNA dose of 0.05 mg per kg (n = 5 per group) and varying doses of gRNA ranging from 0.06 mg per kg to 0.4 mg per kg. Seven to nine days after administration, the animals were sacrificed, and blood and liver samples were collected to measure serum TTR and liver editing. The results are shown in Figures 9C and 9D. Negative control mice were administered the TSS vehicle.

[0273] Example 10 - Neutral lipids To evaluate the in vivo efficacy of LNPs, LNP formulations were prepared as described in Example 2 using the mRNA from Example 2 and sg534 (SEQ ID NO: 72; G534). The lipid nanoparticle components were dissolved in 100% ethanol at the following lipid component molar ratios: Briefly, the RNA cargo was prepared in a buffer of 25 mM citrate and 100 mM NaCl (pH 5.0) to a concentration of approximately 0.45 mg / mL. LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of approximately 6 and an mRNA to gRNA ratio of 1:2 by weight.

[0274] The LNP formulations were analyzed for mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency as described in Example 1. The analyses of mean particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency are shown in Table 30. The lipid molar ratio is listed as amine lipid (lipid A) / neutral lipid / helper lipid (cholesterol) / PEG lipid (PEG2k-DMG). The neutral lipid was DSP, DPPC, or absent, as indicated. [Table 30] TIFF0007645929000063.tif184166

[0275] For in vivo characterization, female Sprague Dawley rats were intravenously administered the above LNPs at 0.3 mg of total RNA (guide RNA and mRNA) per kg of body weight. There were five rats per group. Seven days after administration, the 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 administered the TSS vehicle. The results of serum TTR and liver editing are shown in Figures 10A and 10B and Table 30 (above). [Table 31] TIFF0007645929000065.tif254163TIFF0007645929000066.tif250164TIFF0007645929000067.tif255165TIFF0007645929000068.tif83167

[0276] See the sequence listing below for the sequences themselves. The transcript sequence generally includes GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCap™. Thus, the first three nucleotides can be modified for use with other capping methods, such as vaccinia capping enzyme. Promoters and polyA sequences are not included in the transcript sequence. Promoters such as the T7 promoter (SEQ ID NO: 31) and polyA sequences such as SEQ ID NO: 62 or 63 can be added to the disclosed transcript sequences at the 5' and 3' ends, respectively. While most nucleotide sequences are provided as DNA, they can be easily converted to RNA by changing T to U.

[0277] Sequence Listing The sequence listing below provides a list of the sequences disclosed herein, and it is understood that when a DNA sequence (containing T) is referenced in relation to RNA, T should be replaced with U (modified or unmodified as appropriate), and vice versa. [Table 32] TIFF0007645929000070.tif255165TIFF0007645929000071.tif255165TIFF0007645929000072.tif253163TIFF0007645929000073.tif254167TIFF0007645929000074.tif252165TIFF0007645929000075.tif255165TIFF0007645929000076.tif251166TIFF0007645929000077.tif255166TIFF0007645929000078.tif252170TIFF0007645929000079.tif255166TIFF0007645929000080.tif255167TIFF0007645929000081.tif253164TIFF0007645929000082.tif254164TIFF0007645929000083.tif253166TIFF0007645929000084.tif253166TIFF0007645929000085.tif252166TIFF0007645929000086.tif252168TIFF0007645929000087.tif255166TIFF0007645929000088.tif254164TIFF0007645929000089.tif254164TIFF0007645929000090.tif255165TIFF0007645929000091.tif254166TIFF0007645929000092.tif253167TIFF0007645929000093.tif255164TIFF0007645929000094.tif255167TIFF0007645929000095.tif255165TIFF0007645929000096.tif253164TIFF0007645929000097.tif251165TIFF0007645929000098.tif253165TIFF0007645929000099.tif255164TIFF0007645929000100.tif254166TIFF0007645929000101.tif254166TIFF0007645929000102.tif254168TIFF0007645929000103.tif253167TIFF0007645929000104.tif251166TIFF0007645929000105.tif254166TIFF0007645929000106.tif254167TIFF0007645929000107.tif251166TIFF0007645929000108.tif253164TIFF0007645929000109.tif253166TIFF0007645929000110.tif254165TIFF0007645929000111.tif253168TIFF0007645929000112.tif253166TIFF0007645929000113.tif250166TIFF0007645929000114.tif252167TIFF0007645929000115.tif251166TIFF0007645929000116.tif253167TIFF0007645929000117.tif253167TIFF0007645929000118.tif251165TIFF0007645929000119.tif254166TIFF0007645929000120.tif253166TIFF0007645929000121.tif253164TIFF0007645929000122.tif254166TIFF0007645929000123.tif253164TIFF0007645929000124.tif253164TIFF0007645929000125.tif253166TIFF0007645929000126.tif255165TIFF0007645929000127.tif253166TIFF0007645929000128.tif254167TIFF0007645929000129.tif254165TIFF0007645929000130.tif253165TIFF0007645929000131.tif255166TIFF0007645929000132.tif255166TIFF0007645929000133.tif253166TIFF0007645929000134.tif253163TIFF0007645929000135.tif252166TIFF0007645929000136.tif253165TIFF0007645929000137.tif253165TIFF0007645929000138.tif251166TIFF0007645929000139.tif254166TIFF0007645929000140.tif254166TIFF0007645929000141.tif255166TIFF0007645929000142.tif253164TIFF0007645929000143.tif254166TIFF0007645929000144.tif253163TIFF0007645929000145.tif254165TIFF0007645929000146.tif253165TIFF0007645929000147.tif254166TIFF0007645929000148.tif254164TIFF0007645929000149.tif253165TIFF0007645929000150.tif253167TIFF0007645929000151.tif253165TIFF0007645929000152.tif255167TIFF0007645929000153.tif252163TIFF0007645929000154.tif253164TIFF0007645929000155.tif254166TIFF0007645929000156.tif253165TIFF0007645929000157.tif254164TIFF0007645929000158.tif253167TIFF0007645929000159.tif253164TIFF0007645929000160.tif254165TIFF0007645929000161.tif255164TIFF0007645929000162.tif254166TIFF0007645929000163.tif254166TIFF0007645929000164.tif253166TIFF0007645929000165.tif253168TIFF0007645929000166.tif253166TIFF0007645929000167.tif254167TIFF0007645929000168.tif254166TIFF0007645929000169.tif254166TIFF0007645929000170.tif253165TIFF0007645929000171.tif254166TIFF0007645929000172.tif253166TIFF0007645929000173.tif253165TIFF0007645929000174.tif254166TIFF0007645929000175.tif253167TIFF0007645929000176.tif253165TIFF0007645929000177.tif253166TIFF0007645929000178.tif253167TIFF0007645929000179.tif252166TIFF0007645929000180.tif253168TIFF0007645929000181.tif253165TIFF0007645929000182.tif253166TIFF0007645929000183.tif255168TIFF0007645929000184.tif254166TIFF0007645929000185.tif254166TIFF0007645929000186.tif253165TIFF0007645929000187.tif253163TIFF0007645929000188.tif252166TIFF0007645929000189.tif252165TIFF0007645929000190.tif254164TIFF0007645929000191.tif253164TIFF0007645929000192.tif254166TIFF0007645929000193.tif253166TIFF0007645929000194.tif254165TIFF0007645929000195.tif254166TIFF0007645929000196.tif253164TIFF0007645929000197.tif253168TIFF0007645929000198.tif253165TIFF0007645929000199.tif254166TIFF0007645929000200.tif253164TIFF0007645929000201.tif254163TIFF0007645929000202.tif254166TIFF0007645929000203.tif255165TIFF0007645929000204.tif254164TIFF0007645929000205.tif253164TIFF0007645929000206.tif254165TIFF0007645929000207.tif251163TIFF0007645929000208.tif254163TIFF0007645929000209.tif253166TIFF0007645929000210.tif254164TIFF0007645929000211.tif252166TIFF0007645929000212.tif254166TIFF0007645929000213.tif253164TIFF0007645929000214.tif253164TIFF0007645929000215.tif253165TIFF0007645929000216.tif253165TIFF0007645929000217.tif253165TIFF0007645929000218.tif255164TIFF0007645929000219.tif254166TIFF0007645929000220.tif253166TIFF0007645929000221.tif254164TIFF0007645929000222.tif254167TIFF0007645929000223.tif253167TIFF0007645929000224.tif253165TIFF0007645929000225.tif253165TIFF0007645929000226.tif253165TIFF0007645929000227.tif254163TIFF0007645929000228.tif254165TIFF0007645929000229.tif253167TIFF0007645929000230.tif253166TIFF0007645929000231.tif254165TIFF0007645929000232.tif254165TIFF0007645929000233.tif251165TIFF0007645929000234.tif253167TIFF0007645929000235.tif254166TIFF0007645929000236.tif254165TIFF0007645929000237.tif253168TIFF0007645929000238.tif25216 6TIFF0007645929000239.tif253163TIFF0007645929000240.tif253163TIFF0007645929000241.tif254167TIFF0007645929000242.tif255166TIFF0007 645929000243.tif255164TIFF0007645929000244.tif253164TIFF0007645929000245.tif253165TIFF0007645929000246.tif254166TIFF0007645929000247.tif253164TIFF0007645929000248.tif255165TIFF0007645929000249.tif254163TIFF0007645929000250.tif134165* = PS bond; "m" = 2'-O-Me nucleotide. Mouse G000282 NGS primer sequence Forward primer: CACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTTGTTCCAGAGTCTATCACCG Reverse primer: GGAGTTCAGACGTGTGCTCTTCCGATCTACACGAATAAGAGCAAATGGGAAC Rat G000390 NGS primer sequence Forward primer: CACTCTTTCCCTACACGACGCTCTTCCGATCTTGCATTTCATGAGACCGAAAACA Reverse primer: GGAGTTCAGACGTGTGCTCTTCCGATCTGCTACAGTAGAGCTGTACATAAAACTT GFP sequence:

[0278] Sequence Listing SEQUENCE LISTING <110> INTELLIA THERAPEUTICS, INC. <120> FORMULATIONS <130> PA23-236 <150> US 62 / 566,240 <151> 2017-09-29 <160> 84 <170> PatentIn version 3.5 <210> 1 <211> 4140 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 1 atggacaaga agtacagcat cggactggac atcggaacaa acagcgtcgg atgggcagtc 60 atcacagacg aatacaaggt cccgagcaag aagttcaagg tcctgggaaa cacagacaga 120 cacagcatca agaagaacct gatcggagca ctgctgttcg acagcggaga aacagcagaa 180 ccaacaagac tgaagagaac agcaagaga agatacaacaa gaaaagaa cagaatctgc 240 300 ctggaaaa gcttcctggt cgaaagac aagaagcacg aagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 480 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgtttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agatgaaa acctgatcgc acagctgccg ggagaaaga agaacggact gttcgggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 900 ctgctgagcg acatcctgag agtcaacaca gaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gataggacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaacagga agacttctac ccgttcctga agcaacaag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaggt caagtacgtc acagaaggaa tgagaagcc ggcattccctg 1620 agcggagaac agagaaggc aatcgtcgac ctgctgttca agacaacag aaggtcaca 1680 gtcaagcagc tgaggaag ctactcaag aagatcgaat gctcgacag cgtcgaaatc 1740 agcggagtcg agacagatt caacgcaagc ctgggaacat accacgacct gctgagatc 1800 atcaaggaca aggactcct ggacaacgaa gaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaggt catgaagcag ctgagagaa gagatacac aggatggga 1980 agactgagca gaaagctgat spacegaatc agaactgaaa gaatcctg 2040 gacttcctga agagcgacgg attcgcaac agaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaggaag catchcagaag gcacaggtca gcggacaggg agacacctg 2160 cacgaacaca tcgcaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaggtcg tcgacgact ggtcaggtc atgggac acagccgga aaacatcgtc 2280 atcgaaatgg cagagaaaa cagahaaca cagaahaca cagahacaa 2340 atgagagaa tcgagaagg atcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca gaacggaaga 2460 gatagtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaggacgac agcatcgaca acaggtcct gaagaagc 2580 gacaagaaca gagaagag cgacaacgtc ccgagcgaag aagtcgtcaa gaagtgaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agaaagtt cgacaacctg 2700 acaaaggcag agaggagg actgagcgaa ctggacaagg caggattcat caagacag 2760 ctggtcgaaa caacagagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agaaagtca aggtcatcac actgaagc 2880 aagctggtca gcgacttcag aaagcttc cagttctaca aggtcagaga aatcaacaac 2940 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aagcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaaaacgg agaacagga gaatcgtct gggacaaggg agagacttc 3240 gcaacagtca gaaggtcct gagcatgccg caggtcaa tcgtcagaa gagaagtc 3300 gaacaggag gattcaggca ggcagcatc ctgccgaga gaaacagcga caagctgatc 3360 gcaagaag aggactggga cccgaagaag tacggagt tcgacagccc ggagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa agggaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac atcatggaa agaagcagct tcgaaagaa cccgatcgac 3540 ttcctggaag aaagggata aaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactga aaacggaaga agagaatgc tggcaagcgc aggagactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccgaa cakacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag cagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacagca cagagacaag 3900 ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa cctgggagca 3960 ccggcagcat tcaagtactt cgacacaca atcgacagaa agagatac aagcacaaag 4020 gaagtcctgg acgcacact gatccaccag agcacacag gactgtacga aacaagaatc 4080 gacctgagcc agctgggagg agacggagga ggaagcccga agagagag aaggtctag 4140 <210> 2 <211> 4143 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 2 atggataaga agtactcaat cgggctggat atcggaacta attccgtggg tggggcagtg 60 atcacggatg atacaagt gccgtccaag aagttcagg tcctggggaa caccgataga 120 cacagcatca agaaaaatct catcggagcc ctgctgttg actccggcga aaccgcagaa 180 gcgacccggc tcaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag agatctttc gaacgaatg gcaaggtcg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtcg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcactga caggccgac ctcagattga tctactggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaggcgatc tgaccctga taactccgac 540 gtggataagc ttttcattca actggtgcag acctacaacc aactgttcga agaaaaccca 600 atcaatgcta gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc gaagtcgcgg 660 cgcctcgaaa acctgatcgc acagctgccg ggagagaaaa agaacggact tttcggcaac 720 ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga cctggccgag 780 gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa tttgctggca 840 caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc ggacgcaatc 900 ttgctgtccg atatcctgcg cgtgaacacc gaataacca aagcgccgct tagcgcctcg 960 atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc gctcgtgaga 1020 cagcaactgc ctgaaaagta caaggagatc ttcttcgacc agtccaagaa tgggtacgca 1080 gggtacatcg atggaggcgc tagccaggaa gagttctata agttcatcaa gccaatcctg 1140 gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggaga tctgctccgg 1200 aaaagagaa cctttgacaa cggatccatt ccccaccaga tccatctggg tgagctgcac 1260 gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg ggaaaagatc 1320 gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg cggcaattcg 1380 cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa ttcgaggaa 1440 gttgtggata agggagcttc ggcacaaagc ttcatcgaac gaatgaccaa cttcgacaag 1500 aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata cttcactgtc 1560 tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc ggcctttctg 1620 tccggagaac agaagaaagc aattgtcgat ctgctgttca agaccaaccg caaggtgacc 1680 gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gttcgactc agtggaaatc 1740 agcggggtgg aggacagatt caacgcttcg ctgggaacct atcatgatct cctgaagatc 1800 atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga agatatcgtc 1860 ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa gacctacgct 1920 catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac tggttggggc 1980 cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa aactatcctg 2040 gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcaattgat ccacgacgac 2100 agcctgacct ttaaggagga catccaaaaa gcacaagtgt ccggacaggg agactcactc 2160 catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat tctgcaaact 2220 gtgaaggtgg tcgacgagct ggtgaaggtc atgggacggc acaaaccgga gaatatcgtg 2280 attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaaaaactc ccgcgaaagg 2340 atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa agagcacccg 2400 gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca aaatggacgg 2460 gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga cgtggaccac 2520 atcgttccac agtcctttct gaaggatgac tcgatcgata acaaggtgtt gactcgcagc 2580 gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa gaagatgaag 2640 aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt tgacaatctc 2700 actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat caaacggcag 2760 ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcttggactc ccgcatgaac 2820 actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac cctgaaaagc 2880 aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga aatcaacaac 2940 taccatcacg cgcatgacgc atacctcaac gctgtggtcg gtaccgccct gatcaaaaag 3000 taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga cgtgaggaag 3060 atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt cttttactca 3120 aacatcatga actttttcaa gactgaaatt acgctggcca atggagaaat caggaagagg 3180 ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg cagggacttc 3240 3300 caaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aaggaaga gcaaaaagct caaatccgtc 3480 aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa cccgattgat 3540 ttcctcgagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa actccccaag 3600 tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc cggagaactc 3660 caaaaaggaa atgagctggc cttgcctagc aagtacgtca acttcctcta tcttgcttcg 3720 cactacgaaa aactcaaagg gtcaccggaa gataacgaac agaagcagct tttcgtggag 3780 cagcacaagc attatctgga tgaaatcatc gaacaaatct ccgagttttc aaagcgcgtg 3840 atcctcgccg acgccaacct cgacaaagtc ctgtcggcct acaataagca tagagataag 3900 ccgatcagag aacaggccga gaacattatc cacttgttca ccctgactaa cctgggagcc 3960 ccagccgcct tcaagtactt cgatactact atcgatcgca aaagatacac gtccaccaag 4020 gaagttctgg acgcgaccct gatccaccaa agcatcactg gactctacga aactaggatc 4080 gatctgtcgc agctgggtgg cgatggcggt ggatctccga aaaagaagag aaaggtgtaa 4140 tga 4143 <210> 3 <211> 1379 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: Cas9 sequence" <400> 3 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Tyr Glu Thr Arg to Asp to Gln Ser to Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Arg Lys Val 1370 1375 <210> 4 <211> 4140 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 4 auggacaaga agocagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 gcaacagac ugagagaac agcaucacaca gagaagaa cagaaucugc 240 uaccugcagg aaacuucag caacgaaug gcaaggucg acgacagcuu cuccacaga 300 360 420 aagcuggucg acagcacaga caaggcagac cugacacuga ucuaccuggc acuggcacac 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agagaaggc aaucgucgac cugguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 agacugagca gaagcugau caacggaauc agacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga ccaagaagcu gaagcguc 3480 aaagacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaaggguau caaggaaguc aagaaggacc ugaucaucaa cugccgaag 3600 uacagccugu ucgaacugga aaacggaa aagagaaugc uggcaagcgc agggaaacug 3660 3720 3780 cagcaaagc auaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaca aucgacagaa agagauacac aagcacaaag 4020 gaguccugg axaaaccu gauccaccag axaaauc 4080 gaccugagcc agcugggagg aggagggagg ggaagcccga agagagag aaggucuag 4140 <210> 5 <211> 4143 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 5 auggauaaga aguacucaau cgggcuggau aucggaacua auuccguggg ugggcagug 60 aucacggaug auacaagu gccguccaag aucacggaag uccuggga caccgauaga 120 cacagcauca agaaaaaucu caucggagcc cugcuguuug acuccggcga aaccgcagaa 180 gcgacccggc ucaacguac cgcgaggcga cgcuacaccc ggcggaagaa ucgcaucugc 240 uaucugcaag agaucuuuuc gaacgaaug gcaaggucg acgacagcuu cuccaccgc 300 neighbors cuckoooooooooogga ggaggaggac aaacuuugg 360 aacaucgucg acgaaguggc guaccacccgaa aaguacccga ccaucuacca ucugcggaag 420 aaguuggug acucacuga caggccgac cucagauuga ucuacuuggc ccucgcccau 480 augaucaaau uccgcggaca cuccugauc gaaggcgauc ugaacccuga uaacuccgac 540 guggauaagc uuuucauca acuggugcag accuaacc aacuguucga agaaaaccca 600 aucaugcua gcggcgucga ugccaaggcc accuguccg cccggcuguc gaagucgcgg 660 cgccucgaaa accugaucgc acagcugccg ggagagaaa agaacggacu uuucggcaac 720 uugaucgcuc ucucacuggg acucacuccc aauuucaagu ccaauuuuga ccuggccgag 780 gacgcgaagc ugcaacucuc aaaggacacc uacgacgacg auuggcaa uuugcuggca 840 caaauggcg aucaguacgc ggaucuguc cuugccgcua agaaccuuuc ggacgcaauc 900 960 1020 cagcaacugc cugaaaagua caaggaac uucuucgacc aguccaagaa uggguacgca 1080 ggguacaucg auggaggcgc uagccaggaa gagucuauaa aguucaucaa gccaauccug 1140 gaaagaugg acggaaccga agaacugcug cucaagcuga acagggaga ucugcuccgg 1200 aaacagagaa ccuuugacaa cggauccauu cccaccaccaga uccaucuggg ugagcugcac 1260 gccaucuugc ggcgccagga ggacuuuuac ccauuccuca aggacaaccg ggaaaagauc 1320 gagaaaauuc ugacguuccg caucccguau uacgugggcc cacuggcgcg cggcaauucg 1380 cgcuucgcgu ggaugacuag aaaaucagag gaacauuca cucuuggaa uuucgagaa 1440 guugugguaa agggagcuuc ggcacaaagc uucaucgaac gaaugaccaa cuucgacaag 1500 aaucucccaa acgagaaggu gcuuccuaag cacagccucc uuuacgaaua cuucacuguc 1560 uacaacgaac ugacuaaagu gaauacguu acugaaggaa uggaagcc ggccuuucug 1620 uccggagaac agagaaagc aauugucgau cugcuguuca agaccaaccg caaggugacc 1680 gucaagcagc uuaaagga cuacuucaag aagaucgagu guuucgacuc aguggaauc 1740 agcgggggggg aggagagauu caacgcuucg cugggaaccu aucaugaucau ccugaagauc 1800 aucaaggaca aggacuuccu ugacaacgag gagaacgagg acauccugga agauaucguc 1860 1920 caucucuucg acgauaaggu caugaacaa cucaagcgcc gccgguacac ugguugggggc 1980 cgccucuccc gcaagcugau caacgguauu cgcgauaaac agagcgguaa aacuauccug 2040 gauuuccuca aaucggaugg cuucgcuaau cguaacuuca ugcaauugau ccacgacgac 2100 agccugaccu uuaaggagga cauccaaaaa gcacaagugu ccggacaggg agacucacuc 2160 caugaacaca ucgcgaaucu ggccgguucg ccggcgauua agaagggaau ucugcaaacu 2220 gugaaggugg ucgacgagcu ggugaagguc augggacggc acaaaccgga gaauaucgug 2280 auugaaaugg cccgagaaaa ccagacuacc cagaagggcc agaaaaacuc ccgcgaaagg 2340 augaagcgga ucgaagaagg aaucaaggag cugggcagcc agauccugaa agagcacccg 2400 guggaaaaca cgcagcugca gaacgagaag cucuaccugu acuauuugca aaauggacgg 2460 gacauguacg uggaccaaga gcuggacauc aaucgguugu cugauuacga cguggaccac 2520 aucguuccac aguccuuucu gaaggaugac ucgaucgaua acaagguguu gacucgcagc 2580 2640 aauuacuggc ggcagcuccu gaaugcgaag cugauuaccc agaaaguu ugacaaucuc 2700 acuaaagccg agcgcgggggg acucucagag cuggauaagg cuggauucau caaaccggcag 2760 cuggucgaga cucggcagau uaccaagcac guggcgcaga ucuuggacuc ccgcaugaac 2820 2880 2940 uaccaucacg cgcaugacgc auaccucaac gcuguggucg guaccgcccu gaucaaaaag 3000 uacccuaaac uugaaucgga guuuguguac ggagacuaca aggucuaca cgugagaag 3060 augauagcca aguccgaaca ggaaaucggg aaagcaacug cgaaauacuu cuuuuacuca 3120 aacaucauga acuuuuuucaa gacugaaauu acgcuggcca auggagaaau caggagaagg 3180 ccacugaucg aacuaacgg agaaacggc gaaucgugu gggacaaggg cagggacuuc 3240 caacuguuc caaagugcu cucuaugccg caaucaaua uugugaagaa aaccaagug 3300 caaccggcg gauuuucaaa ggaaucgauc cucccaaaga gaauagcga caagcuacauu 3360 ccacgcaaga aagacuggga cccgaaag uacggaggau ucgauucgcc gacugucgca 3420 uacuccgucc ucgugguggc caagguggag aagggaaaga gcaaaaagcu caaauccguc 3480 aaagagcugc ugggggauuac caucauggaa cgauccucgu ucgagaagaa cccgauugau 3540 uuccucgagg cgaaggguua caaggaggug aagaaggauc ugaucaucaa acuccccaag 3600 uacucacugu ucgaacugga aaauggucgg aagcgcaugc uggcuucggc cggagaacuc 3660 3720 3780 cagcacaagc auuaucugga ugaaucauc gaacaaucu ccgaguuuuc aaagcgcgug 3840 auccucgccg acgccaaccu cgacaaguc cugucggccu achauaagca uagagauaag 3900 ccgaucagag aacaggccga gacauuauuc cacuuguuca cccugacuaa ccugggagcc 3960 ccagccgccu ucaaguacuu cgauacuacu aucgaucgca aaagauacac guccaccaag 4020 gaaguucugg acgcgacccu gauccaccaa agcucacug gacucuacga acacgauc 4080 gaucugucgc agcugggggg cgaugggcggu gaucuccga aaagagag aaggguaa 4140 4143 <210> 6 <211> 1379 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide” <400> 6 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asp With Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu With Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> 7 <211> 4140 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic "polynucleotide" <400> 7 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 180 ccaacaagac ugagagaac agcaagaaga aguaacacaa gaaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccagaga 300 360 420 aagcuggucg acagcacaga caaggcagac cugacacuga ucuaccuggc acuggcacac 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaggucgaa agggaaa gcaagaagcu gagagcguc 3480 aaggaacugc ugggaucgac aaucauggaa aggaagcagcu ucgaaagaa cccgaucgac 3540 ooooooooooooooooooooooooooooooooooooooooooooooo back go back gcugccgag 3600 uacagccugu ucgaaaaaaaaaaaaaaggc agghaagcgc aggaaaagg 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagacaacagc acuaccugga cgaaaucauc gacagauca gcgaauucag cagagaguc 3840 auccuggcag acgshaaccu ggaaagguc cugagcgcau ahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagg 3900 ccgaucagag aacaggcaga aacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 4140 <210> 8 <211> 1379 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 8 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu With Arg Lys Arg Pro Leu Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Lys Arg Lys Val 1370 1375 <210> 9 <211> 4140 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 9 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 gcaacagac ugagagaac agcaucacaca gagaagaa cagaaucugc 240 uaccugcagg aaacuucag caacgaaug gcaaggucg acgacagcuu cuccacaga 300 neighborhood gcuuccuggu cgagagac aagaagcacg aagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccaccgaa aacaacccga caaucaacca ccugagaaag 420 aagcuggucg aaggshaga aaggshagac cugaaccuggc aaggcuggc 480 augaucaagu shake 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggaaggcaaaccugagcg 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agagaaggc aaucgucgac cugguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 agacugagca gaagcugau caacggaauc agacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga ccaagaagcu gaagcguc 3480 aaagacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaaggguau caaggaaguc aagaaggacc ugaucaucaa cugccgaag 3600 uacagccugu ucgaacugga aaacggaa aagagaaugc uggcaagcgc agggaaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagacaacagc acuaccugga cgaaaucauc gacagauca gcgaauucag cagagaguc 3840 auccuggcag acgshaaccu ggaaagguc cugagcgcau ahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagg 3900 ccgaucagag aacaggcaga aacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaca aucgacagaa agagauacac aagcacaaag 4020 gaguccugg axaaaccu gauccaccag axaaauc 4080 gaccugagcc agcugggagg aggagggagg ggaagcccga agagagag aaggucuag 4140 <210> 10 <211> 4134 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic "polynucleotide" <400> 10 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagagaaga uacacaagaa gaagaagacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaaagcu ucuggucga agagacaag aagcacgaaa gacaccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaagaag 420 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagouggacg gaacagaaga aucugcugguc aagcugaaca gagaagaaccu cgugaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca auccugagaa gacaggaaga cuucuacccg auccugaagg acaacagaga aaagaucgaa aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 aagcaagg acuuccugga caacgaaa aacgaaca ucuggagaa caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaaaa gacugaagac aucgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga ucugaagga acaccgguc 2400 gaaaacacac agcugcaga cgaaaagcug uaccuguacu accugcaga cggagagac 2460 auguacgucg accaacuc agacugaccg accuacgacgu cgaccaac 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccag accuacaagg accucagaau storm 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa caccacugau caccaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag caacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaucag aagagaccc 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa cgugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaaagcaa aggguaacaa ggaagucaag aagaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaag agaugcugg caagcgcagg agacugcagg 3660 aagggaaacg aacuggcacu gccgagcaag uacgucacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaaggaag cccggaagc aacgaaaac agxaggcugu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg auucagca gagagucauc 3840 cuggcagacg aaccugga cuggcugg agcgcauaca acaaccacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaccu gggagcaccg 3960 gcagcauca aguacuucga cacaauc gagaaaga gauacacaag cacaaggaa 4020 guccuggacg hurricane ccaccagagc aucachaggac uguacgacg aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaga agagagaaa gguc 4134 <210> 11 <211> 4134 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 11 gapagaagu acagcaucgg acuggcauc ggaaaaca gcgucggaug ggcagucauc 60 acagacgaau acagguccc gaghagaag uucaaggucc ugggaacac agacagac 120 shake agaaccugau cggagshacug cuguaccug shake 180 accelerate accelerate accelerate accelerate accelerate 240 neighborhood ucuucagcaa cgaauggca aaggucgacg acagcuucuu ccacagacug 300 gagaaagcu aagaaaaaaaaaaaaazzaccgau cucggaac 360 aucgucgacg aagucgcaua ccacgaaag uacccgacaa ucuaccaccu gagaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca auccugagaa gacaggaaga cuucuacccg auccugaagg acaacagaga aaagaucgaa aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaaucauc 1800 aagcaagg acuuccugga caacgaaa aacgaaca ucuggagaa caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaaaa gacugaagac aucgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau aacccgguc ggaagccaagg aacccgguc 2400 gaaaacacac agcugcaga cgaaaagcug uaccuguacu accugcaga cggagagac 2460 auguacgucg accaacuc agacugaccg accuacgacgu cgaccaac 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccag accuacaagg accucagaau storm 2940 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag caacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaucag aagagaccc 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa cgugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 neighborhood agggaaaaaaaagaaaaaaaaaaaaaacus gccgaaaaa 3600 agccuguucg aacuggaaaa cggaagaag agaugcugg caagcgcagg agacugcagg 3660 aagggaaacg aacuggcacu gccgagcaag uacgucacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaaggaag cccggaagc aacgaaaac agxaggcugu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg auucagca gagagucauc 3840 cuggcagacg aaccugga cuggcugg agcgcauaca acaaccacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaccu gggagcaccg 3960 gcagcauca aguacuucga cacaauc gagaaaga gauacacaag cacaaggaa 4020 guccuggacg hurricane ccaccagagc aucachaggac uguacgacg aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaga agagagaaa gguc 4134 <210> 12 <211> 4134 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 12 gapagaagu acagcaucgg acuggcauc ggaaaaca gcgucggaug ggcagucauc 60 acagacgaau acagguccc gaghagaag uucaaggucc ugggaacac agacagac 120 shake agaaccugau cggagshacug cuguaccug shake 180 accelerate accelerate accelerate accelerate accelerate 240 neighborhood ucuucagcaa cgaauggca aaggucgacg acagcuucuu ccacagacug 300 gagaaagcu aagaaaaaaaaaaaaazzaccgau cucggaac 360 aucgucgacg aagucgcaua ccacgaaag uacccgacaa ucuaccaccu gagaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaga uacaucaagu ucaucaagcc gauccugga aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca auccugagaa gacaggaaga cuucuacccg auccugaagg acaacagaga aaagaucgaa aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggagaacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaaucauc 1800 aagcaagg acuuccugga caacgaaa aacgaaca ucuggagaa caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaaaa gacugaagac aucgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaaaaggcaa gagaaaaaaaaaaaaaaaaaaaaaaaaaaaagg 2340 aagagaaucg aagaaggaau aacccgguc ggaagccaagg aacccgguc 2400 gaaaacacac agcugcaga cgaaaagcug uaccuguacu accugcaga cggagagac 2460 auguacgucg accaacu ggaacacac agacugagcg acacgacgu cgacgcauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccag accuacaagg accucagaau storm 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa caccacugau caccaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ccgaagcugg cagaagaug 3060 aucgcaaag gcgaaga auucggaaag gcaacaaa aguacuucuu quacacaac 3120 aucaugaacu aucaucagac agaaucaca cuggcaacg gagaaucag aagagaccg 3180 cugauchgaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa mighte beachacoaaaaagghaa 3240 acagushagaa agguccugag caugccgcag agaguaucg agaguaggc agaguccag 3300 shakeshaggshaggshaggshaggshaggshaggshaggshaw gcugaucgca 3360 agaaagaagg acugggacccc gagaaguac ggaggauucg acagccgac agacggac 3420 agcguccugg ucgucgcaaa gggaaaag gggaagca agaagcuga gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaaagcaa aggguaacaa ggaagucaag aagaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag ccgggaagac aacgaacaga agcagcuguu cgucgaacag 3780 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaacccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> 13 <211> 1368 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 13 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu With Arg Lys Arg Pro Leu Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Tyr Glu Thr Arg to Asp to Gln Ser to Gly Gly Asp 1355 1360 1365 <210> 14 <211> 4107 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 14 auggacaaga agocagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 ccaacaagac ugagagaac agcaagaaga aguaacacaa gaaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccagaga 300 360 420 aagcuggucg acagcacaga caaggcagac cugacacuga ucuaccuggc acuggcacac 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agagaaggc aaucgucgac cugguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 freak haggcugau storm freak freak freak freak storm 2040 gacuuccuga agagcgacgg auucgcaac agaacuuca agcagcugau ccacgacgac 2100 agccugacau ucaggaga cauccagaag gcacagguca gcggacaggg agcugccug 2160 cacgacaca ucgcaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 cuckaggucg ucgaacuc gguaacucg aaaacaucguc 2280 aucgaaaugg cagagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa has 2340 augaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacxaga 2460 qaugauguacg ucgaccagga acuggacuuc aaxagacuga gcgaquaacga cgucgaccac 2520 aucgucccgc shake shake shake shake shake shake shake 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaaaacgg agaacagga gaaacgucu gggacaaggg agagacuuc 3240 gcaacaguca gaaagguccu gagaaugccg caggucaca gaaagguc 3300 frequency frequency frequency frequency frequency 3360 gcaagaaga aggacuggga cccgaagaag uacggaggau ucgacacccc gagucgca 3420 uacagcgucc uggucgucgc aaggucgaa agggaaa gcaagaagcu gagagcguc 3480 aaggaacugc ugggaucgac aaucauggaa aggaagcagcu ucgaaagaa cccgaucgac 3540 ooooooooooooooooooooooooooooooooooooooooooooooo back go back gcugccgag 3600 uacagccugu ucgaaaaaaaaaaaaaaggc agghaagcgc aggaaaagg 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagcaaagc auaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agaguacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> 15 <211> 4101 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic "polynucleotide" <400> 15 gacaagaagu acagcaucgg acuggacauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagagaaga uacacaagaa gaagaagacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaaagcu ucuggucga agagacaag aagcacgaaa gacaccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaagaag 420 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagouggacg gaacagaaga aucugcugguc aagcugaaca gagaagaaccu cgugaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg auccugaagg acaacagaga aaagaucgaa aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 aaggacaagg acuuccugga aacgaagaca accuggaga caucguccug acacugacac uguucgaaga cagagaaaug aucgaaaa gacugaagac aucgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga ucugaagga acaccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcaga cggagagac 2460 auguacgucg accaacuc agacugaccg accuacgacgu cgaccaac 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccag accuacaagg accucagaau storm 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa caccacugau caccaaguac ...

Claims

1. 1. A lipid nanoparticle ("LNP") composition comprising: an RNA component comprising (i) an mRNA encoding an RNA-guided DNA-binding factor, and (ii) a gRNA nucleic acid; Lipid components and Including, Where: The lipid component is 40-60 mol % amine lipid; 0 to 10 mol % of a neutral lipid; 1.5 to 10 mol% PEG lipid; Including, wherein the remainder of the lipid component is a helper lipid; The N / P ratio of the LNP composition is 4.5 to 8; The amine lipid is lipid A or an acetal analog of lipid A, where lipid A has the following structural formula: 【Chemistry 1】 Represented by The composition.

2. The composition of claim 1 , wherein the RNA-guided DNA-binding factor is a Cas nuclease.

3. The composition of claim 1 or 2, wherein the RNA-guided DNA binding factor is a class 2 Cas nuclease.

4. The composition described in claim 3, wherein the class 2 Cas nuclease is a Cas9 nuclease.

5. 5. The composition of any one of claims 1-4, wherein the mRNA comprises a sequence that 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, or 66, wherein the mRNA comprises an open reading frame encoding the RNA-guided DNA binding factor.

6. The composition of any one of claims 1 to 5, wherein the gRNA nucleic acid is a gRNA.

7. The composition of claim 6, wherein the gRNA is modified.

8. The composition of claim 7, wherein the gRNA comprises modifications selected from 2'-O-methyl (2'-O-Me) modified nucleotides, internucleotide phosphorothioate (PS) linkages, and 2'-fluoro (2'-F) modified nucleotides.

9. The composition of any one of claims 1 to 8, wherein the RNA-guided DNA-binding factor is a class 2 Cas nuclease, and the gRNA and class 2 Cas nuclease mRNA are present in a ratio ranging from 10:1 to 1:10 by weight.

10. The composition described in claim 9, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio ranging from 5:1 to 1:5, 3:1 to 1:1, or 2:1 to 1:1 by weight.

11. The composition described in claim 9 or 10, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio of 2:1, 1:1 or 1:2 by weight.

12. The composition of any one of claims 1 to 11, further comprising at least one template nucleic acid.

13. The composition of any one of claims 1 to 12, wherein the N / P ratio of the LNP composition is 6±1.

14. The composition of claim 13, wherein the N / P ratio is 6±0.

5.

15. The composition of any one of claims 1 to 14, wherein the mol% of the PEG lipid is between 2 mol% and 10 mol% of the lipid component, or between 2 mol% and 8 mol% of the lipid component, or between 2 mol% and 4 mol% of the lipid component.

16. The composition of claim 15, wherein the mol% of the PEG lipid is 2.5 mol% of the lipid component.

17. The composition of any one of claims 1 to 14, wherein the mole percent of the PEG lipid is 1.5 mole percent of the lipid component.

18. 18. The composition of any one of claims 1 to 17, wherein the mol% of the amine lipid is from 45 mol% to 60 mol%, or from 50 mol% to 55 mol%, or from 47 to 53 mol%, or from 48 to 53 mol% of the lipid component.

19. The composition of claim 18, wherein the mol% of the amine lipid is 50 mol%, 45 mol%, 47 mol%, 48 mol%, 53 mol%, or 55 mol% of the lipid component.

20. 20. The composition of claim 1, wherein the mol% of the neutral lipid is from 5 mol% to 10 mol% or from 0 mol% to 5 mol% of the lipid component.

21. The composition of claim 20, wherein the mol% of the neutral lipid is 9 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, or 10 mol% of the lipid component.

22. 22. The composition according to any one of claims 1 to 21, wherein the mol % of the helper lipid is between 30 mol % and 50 mol %, or between 30 mol % and 40 mol % of the lipid component.

23. The composition of any one of claims 1 to 22, wherein the amine lipid is lipid A.

24. 23. The composition of any one of claims 1 to 22, wherein the amine lipid is a C4-C12 acetal analog of lipid A.

25. The composition according to any one of claims 1 to 24, wherein the helper lipid is a steroid, a sterol or an alkylresorcinol.

26. The composition of claim 25, wherein the helper lipid is cholesterol.

27. The composition of any one of claims 1 to 26, wherein the neutral lipid is a neutral phospholipid.

28. The composition described in claim 27, wherein the neutral lipid is DSPC or DPPC.

29. 29. The composition of any one of claims 1 to 28, wherein the PEG lipid comprises dimyristoylglycerol (DMG), PEG-2k, or PEG-DMG.

30. The composition of claim 29, wherein the PEG lipid is PEG-DMG.

31. The composition of claim 30, wherein the PEG lipid is PEG2k-DMG.

32. 32. Use of a composition according to any one of claims 1 to 31 in (a) gene editing in vitro or (b) generating genetically engineered cells in vitro.

33. A composition according to any one of claims 1 to 31 for use in therapy.

34. The treatment comprising: (a) Gene editing in a subject; or (b) Creation of genetically engineered cells in a subject.

34. The composition of claim 33, comprising:

35. 35. The use of claim 32 or the composition of claim 34, wherein producing the genetically engineered cell further comprises introducing at least one template nucleic acid into the cell.

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