formulations

Abstract

To provide compositions using a lipid nanoparticle ("LNP") with properties useful for delivery of CRISPR / Cas gene editing components.SOLUTION: Provided is a lipid nanoparticle composition comprising an RNA component and a lipid component, the lipid component including about 50 to 60 mol% amine lipids, about 8 to 10 mol% neutral lipids, and about 2.5 to 4 mol% PEG lipids, the remainder of the lipid component being helper lipids, and an N / P ratio of the LNP composition being about 6.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 566,240, filed Sep. 29, 2017, the contents of which are hereby incorporated by reference in their 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 are used to introduce components and compositions for gene editing into living cells.

Background Art

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

[0004] Currently, there are numerous components and systems for in vivo editing of cellular genes, offering great potential for the treatment of diseases. The CRISPR / Cas gene editing system is active as a ribonucleoprotein complex within cells. The RNA-dependent nuclease binds to DNA sequences within the cell and induces cleavage. This site-specific nuclease activity promotes gene editing through the cell's own natural processes. For example, in response to double-strand DNA breaks (DSBs), cells use an error-prone repair process known as non-homologous end joining ("NHEJ"). During NHEJ, the cell adds or removes nucleotides at the DNA ends, which may result in a sequence that has changed from the cleaved sequence. In other situations, the cell repairs DSBs by mechanisms such as homologous recombination repair ("HDR") or homologous recombination ("HR"), where an endogenous or exogenous template can be used to direct the repair of the cleavage. In some of these editing techniques, cellular mechanisms that repair single-strand breaks (SSBs) or DSBs are utilized.

[0005] Compositions are needed for delivering CRISPR / Cas protein and nucleic acid components to cells, such as the cells of a patient. In particular, compositions for delivering mRNA encoding the CRISPR protein component, and compositions for delivering CRISPR guide RNA are of particular interest. Compositions having properties useful for delivery in vitro and in vivo, which can stabilize and deliver the RNA components, are also of particular interest. SUMMARY OF THE INVENTION

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

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

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

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

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

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] The present disclosure provides embodiments of lipid nanoparticle (LNP) compositions of RNAs, such as CRISPR / Cas component RNAs (''cargo'') for delivery to cells, and methods of using them. The LNP compositions may exhibit improved properties compared to conventional delivery techniques. The 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 a class 2 Cas nuclease, and a guide RNA or a nucleic acid encoding a guide RNA. Also provided are gene editing methods and methods of making engineered cells.

[0013] CRISPR / Cas Cargo The CRISPR / Cas cargo delivered via the LNP formulation may include an mRNA molecule encoding a target protein. For example, it includes an mRNA for expressing a protein such as green fluorescent protein (GFP) and an RNA-induced DNA binding factor, or a Cas nuclease. Provided are LNP compositions containing a Cas nuclease mRNA that enables intracellular expression of the Cas9 protein, such as a class 2 Cas nuclease mRNA. Further, the cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs. For example, a template nucleic acid for repair or recombination may also be included in the composition, or the template nucleic acid 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 ribose residue or an analog thereof, such as a 2'-methoxyribose residue, etc., and a phosphate-sugar backbone. In some embodiments, the sugar of the mRNA phosphate-sugar backbone consists essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. Generally, mRNA does not contain a substantial amount of thymidine residues (e.g., the thymidine residue is 0 or less than 30, 20, 10, 5, 4, 3, or 2, or the thymidine content is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). mRNA can contain modified uridine at some or all of its uridine positions.

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

[0016] As used herein, "RNA-guided DNA binding factor" means a polypeptide or complex of polypeptides having RNA and DNA binding activities, 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 base / nickase and its inactive form ("dCas DNA binder"). As used herein, "Cas nuclease" includes Cas base, Cas nickase, and dCas DNA binder. Cas base / nickase and dCas DNA binder include the Csm complex or Cmr complex of the type III CRISPR system, Cas10, Csm1, or Cmr2 of its subunits, the Cascade complex of the type I CRISPR system, Cas3 of its subunits, and class 2 Cas nuclease. As used herein, "class 2 Cas nuclease" is a single-stranded polypeptide having RNA-guided DNA binding activity. Class 2 Cas nucleases further include class 2 Cas base / nickase (e.g., mutants such as H840A, D10A, or N863A) having RNA-guided DNA cleavage activity or nickase activity, and class 2 dCas DNA binder inactivated for cleavage / nickase activity. Examples of class 2 Cas nucleases include proteins such as Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., mutants such as N497A, R661A, Q695A, Q926A), HypaCas9 protein (e.g., mutants such as N692A, M694A, Q695A, H698A), eSPCas9(1.0) protein (e.g., mutants such as K810A, K1003A, R1060A), and eSPCas9(1.1) protein (e.g., mutants such as K848A, K1003A, R1060A), and variants thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains an RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated herein by reference in its entirety.For example, refer to Table S1 and Table S3 of Zetsche. For example, refer to 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, V, or VI Cas nuclease). Examples of class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and variants thereof. Examples of Cas9 nucleases include those of the 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 thereof (e.g., engineered or mutants). For example, refer to U.S. 2016 / 0312198 A1, U.S. 2016 / 0312199 A1. Other examples of Cas nucleases include Cas10, Csm1, or Cmr2 of the Csm complex or Cmr complex of the type III CRISPR system or subunits thereof, and the Cascade complex of the type I CRISPR system, or Cas3 of subunits thereof. In some embodiments, the Cas nuclease may be of a type IIA, IIB, or IIC system. For discussion of various CRISPR systems and Cas nucleases, see, for example, 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 the Cas nuclease can be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces 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, Methanohalobium 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, and Acaryochloris marina are 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 derived 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 can induce double-strand breaks in target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, cleave one strand of dsDNA, or have neither cleavage activity nor nickase activity on DNA. An exemplary Cas9 amino acid sequence is set forth as SEQ ID NO: 3. An exemplary Cas9 mRNA ORF sequence, including the start codon and stop codon, 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, a chimeric Cas nuclease is used, in which case one domain or region of such a protein is replaced with a portion of a different protein. In some embodiments, the Cas nuclease domain may be replaced with a domain derived 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 the type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be the 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-strand nickase activity, i.e., it can cleave a single DNA strand to create a single-strand 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 the DNA double helix but not the other. In some embodiments, the Cas nickase is a variant of a Cas nuclease (e.g., the Cas nucleases described above), e.g., the endonuclease cleavage active site is inactivated by one or more changes (e.g., point mutations) within the catalytic domain. For 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 Cas9 nickase, has an inactivated RuvC domain or HNH domain. An exemplary amino acid sequence of a Cas9 nickase is set forth as SEQ ID NO: 6. The ORF sequence of an exemplary Cas9 nickase mRNA including the start codon and stop codon 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 protein of the factor may be modified to mutate or completely or partially delete one of the nuclease domains to reduce its nucleic acid cleavage activity. In some embodiments, a nickase having a reduced-activity RuvC domain is used. In some embodiments, a nickase having an inactivated RuvC domain is used. In some embodiments, a nickase having a reduced-activity HNH domain is used. In some embodiments, a nickase having an inactivated 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 include 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 include 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, the mRNA encoding the nickase is provided in combination with a pair of guide RNAs that are complementary to each of the sense and antisense strands of the target sequence. In this embodiment, the guide RNAs direct the nickase to the target sequence and introduce a DSB by creating a nick in the opposite strand 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, the 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, the nickase is used with two separate guide RNAs that are selected to be proximal to create a double nick in the target DNA.

[0027] In some embodiments, the RNA-guided DNA binding factor has no cleavage activity and no 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 (cleavage / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA binding factor or the dCas DNA binding polypeptide having no cleavage activity and no nickase activity is a variant of a Cas nuclease (e.g., the Cas nuclease described above), e.g., the endonuclease cleavage active site thereof is inactivated by one or more changes (e.g., point mutations) within its catalytic domain. See, e.g., U.S. 2014 / 0186958 A1, U.S. 2015 / 0166980 A1. An exemplary dCas9 amino acid sequence is set forth as SEQ ID NO: 8. An exemplary Cas9 mRNA ORF sequence including the start codon and the 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 (e.g., is or comprises a fusion polypeptide).

[0029] In some embodiments, the heterologous functional domain may facilitate the transport of the RNA-guided DNA binding factor into the nucleus of the 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 with 1 to 10 NLSs (plural possible). In some embodiments, the RNA-guided DNA binding factor may be fused with 1 to 5 NLSs (plural possible). In some embodiments, the RNA-guided DNA binding factor may be fused with one NLS. When using one NLS, such NLS may be ligated at the N-terminus or C-terminus of the RNA-guided DNA binding factor sequence. Such one NLS may also be inserted inside the RNA-guided DNA binding factor sequence. In other embodiments, the RNA-guided DNA binding factor may be fused with two or more NLSs. In some embodiments, the RNA-guided DNA binding factor may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-guided DNA binding factor may be fused with two NLSs. In certain situations, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-guided DNA binding factor is ligated and fused at the carboxy terminus with two SV40 NLS sequences. In some embodiments, the RNA-guided DNA binding factor may be fused with two NLSs, wherein one is ligated at the N-terminus and one is ligated at the C-terminus. In some embodiments, the RNA-guided DNA binding factor may be fused with three NLSs. In some embodiments, the RNA-guided DNA binding factor may not need to be fused with an NLS. In some embodiments, the NLS may be a monopartite sequence such as PKKKRKV or PKKKRRV of, for example, the SV40 NLS. In some embodiments, the NLS may be a bipartite sequence such as KRPAATKKAGQAKKKK of the NLS of nucleoplasmin. In certain embodiments, a single PKKKRKV NLS may be ligated 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 modifying the intracellular half-life of the RNA-guided DNA binding factor. In some embodiments, it 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 proteolytic signal peptide. In some embodiments, proteolysis may be mediated by a proteolytic enzyme such as, for example, a proteasome, a lysosomal protease, or a calpain protease. 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 a polyubiquitin chain. 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 precursor cell expressed, developmentally down-regulated 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 protein (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent protein (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent protein (e.g., mKate, mKate2, 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 a fluorescent protein.

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

[0033] In further embodiments, the heterologous functional domain may be an effector domain. When orienting an RNA-guided DNA binding factor to its target sequence, for example, when orienting 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 transcriptional activation domain, or a transcriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease such as FokI nuclease. See, for example, U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or a transcriptional 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), Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51 (2013). Thus, the RNA-guided DNA binding factor essentially becomes a transcription factor that can be oriented to bind to a desired target sequence using a guide RNA.In certain embodiments, the DNA modification domain is a methylation domain such as a demethylation domain or a methyltransferase domain. In certain embodiments, the effector domain is a DNA modification domain such as a base editing domain. In certain embodiments, the DNA modification domain is a nucleic acid editing domain that introduces a specific modification into DNA, such as a deaminase domain. See, e.g., WO2015 / 089406, U.S.2016 / 0304846. The nucleic acid editing domains, deaminase domains, and Cas9 variants described in WO2015 / 089406 and U.S.2016 / 0304846 are incorporated herein by reference.

[0034] The nuclease may comprise at least one domain that interacts with a guide RNA (“gRNA”). Further, the nuclease may be oriented to a target sequence by the gRNA. In class 2 Cas nuclease systems, the gRNA interacts with the nuclease and the target sequence, thereby leading to binding to the target sequence. In some embodiments, the gRNA provides specificity for the targeted cleavage, while the nuclease may be promiscuous and pair with different gRNAs to cleave different target sequences. Class 2 Cas nucleases may pair with the gRNA scaffold structures 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 direct 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 the Cas nuclease may form a ribonucleoprotein (RNP) that can be delivered by the LNP composition, such as a CRISPR / Cas complex such as a CRISPR / Cas9 complex. 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 and a specific gRNA may be paired. The gRNA scaffold structure that pairs 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 and refer to either a crRNA (also known as CRISPR RNA) or a combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and the 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" refers to each type. The trRNA may be a naturally occurring sequence or a trRNA sequence having modifications or variations compared to a naturally occurring sequence.

[0037] As used herein, a "guide sequence" is 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 "directing sequence" or "spacer sequence". For example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs, the guide sequence can be 20 base pairs in length. Shorter or longer sequences, such as sequences 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, can be used as guides. 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 the guide sequence and its corresponding target sequence can be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, in which case the full length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region may contain 1 to 4 mismatches, in which case the guide sequence contains at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, in which case the guide sequence contains 20 nucleotides.

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

[0039] The length of the targeting sequence may 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 targeting sequence lengths. Therefore, the targeting sequence may include 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 targeting sequence length may be 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 the gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may include or consist of 18 to 24 nucleotides. In some embodiments, the targeting sequence may include or consist of 19 to 21 nucleotides. In some embodiments, the targeting sequence may include or consist of 20 nucleotides.

[0040] In some embodiments, the sgRNA is a "Cas9 sgRNA" that can mediate RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" that can mediate 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 encoding the gRNAs described herein, such as expression cassettes. "Guide RNA nucleic acid" is a nucleic acid encoding one or more guide RNAs, used herein to refer to guide RNAs (e.g., sgRNA or dgRNA) 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 targeting sequence flanked by all or a portion of a repeat sequence derived 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, 3’UTR, or 5’UTR, for example. In one example, the promoter is a tRNA promoter, such as tRNA Lys3or may be 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 also 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 contains modified nucleosides or nucleotides. In some embodiments, the gRNA nucleic acid contains 5' end modifications, such as modified nucleosides or nucleotides that stabilize the nucleic acid and prevent integration of the nucleic acid. In some embodiments, the gRNA nucleic acid contains double-stranded DNA with 5' end modifications on each strand. In certain embodiments, the gRNA nucleic acid contains inverted dideoxy-T or inverted abasic nucleosides or nucleotides as 5' end modifications. In some embodiments, the gRNA nucleic acid contains labels such as biotin, desthiobiotin-TEG, digoxigenin, and fluorescent markers such as FAM, ROX, TAMRA, and Alexa Fluor.

[0044] In certain embodiments, two or more gRNA nucleic acids, such as gRNAs, can be used with the CRISPR / Cas nuclease system. Each gRNA nucleic acid may contain a different targeting sequence such that two or more target sequences are cleaved by the CRISPR / Cas system. In some embodiments, one or more gRNAs may have the same or different properties, such as activity or stability, within the CRISPR / Cas complex. When using two or more gRNAs, each gRNA can be encoded either on the same gRNA nucleic acid or on different gRNA nucleic acids. The promoters used to induce the expression of two or more gRNAs may 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, for example, gRNA or mRNA. For example, a gRNA or mRNA comprising one or more modified nucleosides or nucleotides is referred to as “modified” RNA and represents the use of non-naturally occurring components or configurations and / or the presence of one or more naturally occurring components or configurations 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 “modifications”.

[0047] Modified nucleosides and modified nucleotides include (i) one or both of the unlinked oxygens of the phosphate group and / or one or more changes in the linked oxygens of the phosphate group in the phosphodiester backbone linkage, e.g., substitution (exemplary backbone modifications), (ii) changes in the components of the ribose sugar, e.g., changes such as the hydroxyl at the 2'-position of the ribose sugar, e.g., substitution (exemplary sugar modifications), (iii) substantial substitution of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications), (iv) modification or substitution of the naturally occurring nucleobases, e.g., using non-standard nucleobases (exemplary base modifications), (v) substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications), (vi) modification of the 3'- or 5'-terminus of the oligonucleotide, e.g., removing, modifying or substituting the terminal phosphate group, or modifying by attaching a moiety, cap or linker (such 3'- or 5'-cap modifications may include sugar modifications and / or backbone modifications), and (vii) one or more of sugar modifications or substitutions (exemplary sugar modifications). Certain embodiments include 5'-terminal modifications to mRNA, gRNA, or nucleic acids. Certain embodiments include 3'-terminal modifications to mRNA, gRNA, or nucleic acids. Modified RNA may contain 5'- and 3'-terminal modifications. Modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA contains at least one modified residue. In certain embodiments, the mRNA contains at least one modified residue.

[0048] As used herein, when an alignment of a first array and a second array shows that the first array matches at least X% of the positions of the entire second array, the first array is considered to "include an array having at least X% identity to the second array". For example, the array AAGA includes an array having 100% identity to the array AAG, because this is considered to show an identity of 100% with matches seen for all three positions of the second array upon alignment. As long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., for any of thymidine, uridine, or modified uridine, it is adenosine, and in another example, both cytosine and 5-methylcytosine have guanosine or modified guanosine as their complement), the differences between RNA and DNA (generally, the exchange of uridine for thymidine, or vice versa) and the presence of nucleoside analogs such as modified uridine 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, etc., is considered 100% identical to AUG, and both sequences are completely 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 understand which algorithm and parameter settings are appropriate for a given pair of sequences to be aligned, but generally for sequences of similar length and with expected amino acid identity over 50% or nucleotide identity over 75%, it is generally appropriate to use the Needleman-Wunsch algorithm with the default settings of the Needleman-Wunsch algorithm interface provided by EBI at the www.ebi.ac.uk web server.

[0049] mRNA In some embodiments, the compositions or formulations disclosed herein comprise mRNA comprising an open reading frame (ORF) encoding a Cas nuclease as described herein, or an RNA-guided DNA binding factor such as a Class 2 Cas nuclease. In some embodiments, an mRNA comprising an ORF encoding a Cas nuclease or an RNA-guided DNA binding factor such as 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 a shorthand to indicate that the ORF is modified in one or more of the following ways: (1) the modified ORF has a uridine content in the range from its minimum uridine content to 150% of such minimum uridine content; (2) the modified ORF has a uridine dinucleotide content in the range from its minimum uridine dinucleotide content to 150% of such minimum uridine dinucleotide content; (3) the modified ORF has at least 90% identity to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66; (4) at least 75% of the codons of the modified ORF are the minimum uridine codon(s) for a given amino acid, e.g., the codon(s) with the least uridine (the minimum uridine codon is usually 0 or 1, except for the codon of phenylalanine having 2 uridines), consisting of a set of codons, 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 above-described ways. 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 ways of (1)-(4) above.

[0050] As used herein, "modified uridine" is used to refer to nucleosides other than thymidine, where the hydrogen bond acceptor is the same as that of uridine and there is one or more structural differences from uridine. In some embodiments, the modified uridine is a substituted uridine, i.e., a uridine in which one or more non-proton substituents (e.g., alkoxy such as methoxy) replace the proton. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl such as methyl) replace the proton. In some embodiments, the modified uridine is either a substituted uridine, pseudouridine, or a substituted pseudouridine.

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

[0052] In any of the above embodiments, the modified ORF may be composed of a set of codons in which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are the codons listed in the table of minimal uridine codons. In any of the above embodiments, the modified ORF may include a sequence having at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.

[0053] In any of the above embodiments, the modified ORF may include a sequence having at least 90%, 95%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.

[0054] In any of the above embodiments, the modified ORF may have a uridine content in the range 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 in the range 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 embodiments, the modified ORF may contain modified uridines at at least one, a plurality, or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, for example, with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, for example, 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 in the mRNA 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 in the mRNA according to the present disclosure are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or a combination thereof. 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 in the mRNA according to the present disclosure are 5-methoxyuridine. 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 in the mRNA according to the present disclosure are pseudouridine. 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 in the mRNA according to the present disclosure 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 in the mRNA 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 in the 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 of the mRNA according to the present disclosure are 5-iodouridine, and the remaining uridine positions are N1-methylpseudouridine.

[0058] In any of the above embodiments, the modified ORF may include a low uridine nucleotide content, such as a uridine dinucleotide (UU) content as low as possible. For example, (a) using the minimum uridine codon (described above) at any position, and (b) an ORF encoding the same amino acid sequence as a given ORF. The uridine dinucleotide (UU) content can be expressed as an absolute value as the count of UU dinucleotides within the ORF, or as a ratio as the proportion of positions occupied by uridine of the uridine dinucleotide (for example, in the case of AUUAU, since 2 out of 5 positions are occupied by uridine of the uridine dinucleotide, the uridine dinucleotide content is 40%). To evaluate the minimum uridine dinucleotide content, modified uridine residues are considered equivalent to uridine.

[0059] In some embodiments, the mRNA includes 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 includes a 5'UTR, a 3'UTR, or both 5' and 3' UTRs 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 derived from 17-beta-hydroxysteroid dehydrogenase 4 (HSD17B4 or HSD), such as a 5’UTR derived from HSD. In some embodiments, the mRNA comprises at least one UTR derived from 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 derived from globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5’UTR derived 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 5’ and 3’ UTRs derived from the same source, such as both 5’ and 3’ UTRs derived from constitutively expressed mRNAs such as actin, albumin, or globin, such as HBA, HBB, or XBG.

[0062] In some embodiments, the mRNA does not contain a 5’ UTR. For example, there are no additional nucleotides between the 5’ cap and the start codon. In some embodiments, the mRNA contains a Kozak sequence (described below) between the 5’ cap and the start codon, but there is no additional 5’ UTR. In some embodiments, the mRNA does not contain a 3’ UTR. For example, there are no additional nucleotides between the stop codon and the polyA tail.

[0063] In some embodiments, the mRNA contains a Kozak sequence. The Kozak sequence can affect translation initiation and the overall yield of the polypeptide translated from the mRNA. The Kozak sequence contains a methionine codon that can function as a start codon. The minimal Kozak sequence is NNNRUGN, and at least one of the following is true, i.e., the first N is A or G, and the second N is G. In a nucleotide sequence, R means 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 at most one or two mismatches relative to the positions of the lowercase letters. In some embodiments, the Kozak sequence is rccAUGg, with zero mismatches or at most one or two mismatches relative to the positions of the lowercase letters. In some embodiments, the Kozak sequence is gccRccAUGG, with zero mismatches or at most one, two, or three mismatches relative to the positions of the lowercase letters. In some embodiments, the Kozak sequence is gccAccAUG, with zero mismatches or at most one, two, three, or four mismatches relative to the positions of the lowercase letters. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG, with zero mismatches or at most one, two, three, or four mismatches relative to the positions of the lowercase letters.

[0064] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA binding factor comprises a sequence having at least 90% identity to SEQ ID NO: 43, wherein optionally, the ORF of SEQ ID NO: 43 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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 having at least 90% identity to SEQ ID NO: 44, wherein optionally, the ORF of SEQ ID NO: 44 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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 having at least 90% identity to SEQ ID NO: 56, wherein optionally, the ORF of SEQ ID NO: 56 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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 having at least 90% identity to SEQ ID NO: 57, wherein optionally, the ORF of SEQ ID NO: 57 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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-induced DNA binding factor comprises a sequence having at least 90% identity to SEQ ID NO: 58, wherein optionally, the ORF of SEQ ID NO: 58 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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-induced DNA binding factor comprises a sequence having at least 90% identity to SEQ ID NO: 59, wherein optionally, the ORF of SEQ ID NO: 59 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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-induced DNA binding factor comprises a sequence having at least 90% identity to SEQ ID NO: 60, wherein optionally, the ORF of SEQ ID NO: 60 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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-induced DNA binding factor comprises a sequence having at least 90% identity to SEQ ID NO: 61, wherein optionally, the ORF of SEQ ID NO: 61 (i.e., SEQ ID NO: 4) is replaced with any one of the alternative ORFs of SEQ ID NO: 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 NO: 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 sequences of SEQ ID NO: 43, 44, or 56 - 61, optionally substituted, is 95%. In some embodiments, the degree of identity to the sequences of SEQ ID NO: 43, 44, or 56 - 61, optionally substituted, is 98%. In some embodiments, the degree of identity to the sequences of SEQ ID NO: 43, 44, or 56 - 61, optionally substituted, is 99%. In some embodiments, the degree of identity to the sequences of SEQ ID NO: 43, 44, or 56 - 61, optionally substituted, is 100%.

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

[0075] The cap can be included by co-transcription. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific catalog number AM8045) is a cap analog that contains 7-methylguanosine 3'-methoxy-5'-triphosphate linked to the 5'-position of guanine ribonucleotide and can be incorporated into the transcription product during in vitro transcription initiation. In ARCA, it becomes a Cap0 cap where the 2'-position of the first cap-neighboring nucleotide is hydroxyl. 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. [Chemical formula]

[0076] When 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), a Cap1 structure can be obtained by co-transcription. The 3’-O-methylated forms 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. [Chemical formula]

[0077] Alternatively, a cap can be added to the RNA after transcription. For example, the vaccinia capping enzyme is commercially available (New England Biolabs catalog number M2080S), which has RNA triphosphatase activity and guanylyltransferase activity provided by its D1 subunit, as well as guanine methyltransferase provided by its D12 subunit. Thus, this enzyme can add 7-methylguanine to the RNA to give Cap0 in the presence of S-adenosylmethionine and GTP. See, for example, Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027, Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479.

[0078] In some embodiments, the mRNA further comprises a polyadenylated (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" by one or more non-adenine nucleotide "anchors" at one or more positions within the polyA tail. The polyA tail may comprise at least 8 consecutive adenine nucleotides, but also comprises 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 polyA tail of the mRNA described herein may comprise consecutive adenine nucleotides located 3' to the RNA-induced DNA-binding factor or the nucleotide encoding the target sequence. In some examples, the polyA tail of the mRNA comprises non-consecutive adenine nucleotides located 3' to the RNA-induced DNA-binding factor or the nucleotide encoding the target sequence, where the adenine nucleotides are interrupted at regular or irregular intervals by non-adenine nucleotides.

[0079] In some embodiments, the mRNA further comprises a polyadenylated (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" by one or more non-adenine nucleotide "anchors" at one or more positions within the polyA tail. The polyA tail may comprise at least 8 consecutive adenine nucleotides, but also comprises 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 polyA tail of the mRNA described herein may comprise consecutive adenine nucleotides located 3' to the RNA-induced DNA-binding factor or the nucleotide encoding the target sequence. In some examples, the polyA tail of the mRNA comprises non-consecutive adenine nucleotides located 3' to the RNA-induced DNA-binding factor or the nucleotide encoding the target sequence, where the adenine nucleotides are interrupted at regular or irregular intervals by non-adenine nucleotides.

[0080] In some embodiments, one or more non-adenine nucleotides are arranged to interrupt consecutive adenine nucleotides so that poly(A)-binding proteins can bind to a long extended region of consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) are positioned after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) are positioned after at least 8 to 50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) are positioned after at least 8 to 100 consecutive adenine nucleotides. In some embodiments, a non-adenine nucleotide is after 1, 2, 3, 4, 5, 6, or 7 adenine nucleotides, followed by at least 8 consecutive adenine nucleotides.

[0081] The poly-A tail may include one sequence of consecutive adenine nucleotides, followed by one or more non-adenine nucleotides, optionally followed by additional adenine nucleotides.

[0082] In some embodiments, the poly-A tail includes or contains one non-adenine nucleotide or a long region where 2 to 10 non-adenine nucleotides are consecutive. In some embodiments, one or more non-adenine nucleotide(s) are positioned after at least 8, 9, 10, 11, or 12 consecutive adenine nucleotides. In some examples, one or more non-adenine nucleotide(s) are positioned after at least 8 to 50 consecutive adenine nucleotides. In some embodiments, one or more non-adenine nucleotide(s) are positioned after at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive adenine nucleotides.

[0083] In some embodiments, the non-adenine nucleotide is guanine, cytosine, or thymine. In some examples, the non-adenine nucleotide is a guanine nucleotide. In some embodiments, the non-adenine nucleotide is a cytosine nucleotide. In some embodiments, the non-adenine nucleotide is a thymine nucleotide. In some 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 polyA tail containing non-adenine nucleotides is described as SEQ ID NO: 62.

[0084] In some embodiments, the mRNA is purified. In some embodiments, precipitation methods (e.g., LiCl precipitation, alcohol precipitation, etc., or equivalent methods, e.g., the methods described herein) are used to purify the mRNA. In some embodiments, methods using chromatography, such as methods using HPLC or equivalent methods (e.g., the methods described herein), are used to purify the mRNA. In some embodiments, both a precipitation method (e.g., LiCl precipitation) and a method using HPLC are used to purify the mRNA.

[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 separate molecule from the mRNA. In some embodiments, the gRNA is provided as part of the mRNA disclosed herein, e.g., as part of the UTR.

[0086] Chemically modified gRNA In some embodiments, the gRNA is chemically modified. A gRNA containing one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or a "chemically modified" gRNA, and represents that non-naturally occurring components or configurations are used in place of or in addition to the standard residues A, G, C, and U, and / or that one or more naturally occurring components or configurations are present. In some embodiments, the modified gRNA is synthesized using non-standard nucleosides or nucleotides, which are referred to herein as "modifications". Modified nucleosides and modified nucleotides include (i) one or both of the non-bridging oxygens of the phosphate group in the phosphodiester backbone bond and / or one or more changes in the bridging oxygens of the phosphate group, such as substitution (exemplary backbone modifications), (ii) changes in the components of the ribose sugar, such as the hydroxyl at the 2'-position of the ribose sugar, such as substitution (exemplary sugar modifications), (iii) substantial substitution of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications), (iv) modification or substitution of naturally occurring nucleobases, such as using non-standard nucleobases (exemplary base modifications), (v) substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications), (vi) modification of the 3'- or 5'-end of the oligonucleotide, such as removing, modifying or substituting the terminal phosphate group, or attaching a moiety, cap or linker (such 3'- or 5'-cap modifications may include sugar modifications and / or backbone modifications), and (vii) one or more of sugar modifications or substitutions (exemplary sugar modifications).

[0087] In some embodiments, the gRNA comprises a modified uridine at some or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, for example, with a halogen or a C1-C6 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, for example, 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 the 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 the gRNA according to the present disclosure are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or a combination thereof. 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 the gRNA according to the present disclosure are 5-methoxyuridine. 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 the gRNA according to the present disclosure are pseudouridine. 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 the gRNA according to the present disclosure 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 the 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 the gRNA 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 of the gRNA according to the present disclosure are 5-iodouridine, and the remaining uridine positions are N1-methylpseudouridine.

[0089] By combining the chemical modifications as described above, modified gRNAs can be obtained that contain nucleosides and nucleotides (collectively referred to as "residues") having two, three, four, or more modifications. For example, the modified residues can have modified sugars and modified nucleobases. In some embodiments, all bases of the gRNA are modified, for example, all bases have a modified phosphate group such as a phosphorothioate group. 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 contains 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 can tend to be degraded, for example, by nucleases found intracellularly or in serum. For example, nucleases can hydrolyze the phosphodiester bonds of nucleic acids. Thus, in one aspect, the gRNAs described herein can contain one or more modified nucleosides or nucleotides to introduce, for example, stability against nucleases intracellularly or in serum. In some embodiments, the modified gRNA molecules described herein can exhibit a low innate immune response when introduced into a cell population, either in vivo or ex vivo. The term "innate immune response" includes cellular responses to foreign nucleic acids such as single-stranded nucleic acids, and includes the expression and release of cytokines, particularly the release of interferons, as well as 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 of the oxygens with different substituents. Further, modified residues, for example, those present in a modified nucleic acid, can include substantial replacement of the unmodified phosphate moiety with the modified phosphate groups 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 an asymmetric charge distribution.

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

[0094] In certain backbone modifications, the phosphate group can be replaced with a phosphorus-free linker. In some embodiments, the charged phosphate group can be replaced with a neutral moiety. Examples of moieties that can replace the phosphate group include, without limitation, for example, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylene methylimino, methylene hydrazo, methylene dimethylhydrazo, and methylene oxymethylimino.

[0095] template nucleic acid The compositions and methods disclosed herein may include a template nucleic acid. The template may be used to alter or insert a nucleic acid sequence at or near the target site of a Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template may be provided. In other embodiments, two or more templates may be provided such that editing occurs at two or more target sites. For example, different templates may be provided to edit a single gene of a cell or two different genes of 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 where DNA strand invasion occurs at the cleavage site of the nucleic acid. In some embodiments, homologous recombination repair may result in the template sequence being included within 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 terminal inverted 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, respectively, of each cleavage site. When the template contains two homology arms, each arm may be of the same or different lengths, 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 an entirely 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 array may correspond to, include, or consist of the endogenous sequence of the target cell. Additionally, or alternatively, the template array may correspond to, include, 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 a sequence whose native location in the genome of the cell is at a different location. In some embodiments, the endogenous sequence may be the 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 array may be substantially identical to a portion of the endogenous sequence of the cell at or near the cleavage site, but includes at least one nucleotide change. In some embodiments, editing the cleaved target nucleic acid molecule using the template can result in a mutation that includes the insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the mutation can result in a change in one or more amino acids in the protein expressed by the gene containing the target sequence. In some embodiments, the mutation can result in a change in one or more nucleotides in the RNA expressed by the target gene. In some embodiments, the mutation can change the expression level of the target gene. In some embodiments, the mutation can result in an increase or decrease in the expression of the target gene. In some embodiments, the mutation can result in gene knockdown. In some embodiments, the mutation can result in gene knockout. In some embodiments, the mutation can result in the restoration of gene function. In some embodiments, editing the cleaved target nucleic acid molecule using the template can result in a change in the exon sequence, intron sequence, regulatory sequence, transcriptional regulatory sequence, translational regulatory sequence, splicing site, or non-coding sequence of the target nucleic acid molecule, such as DNA.

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

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

[0101] In some embodiments, the template contains ssDNA or dsDNA in which terminal inverted repeat (ITR) sequences are contained adjacent to each other. In some embodiments, the template is provided as a vector, plasmid, minicircle, nanocircle, or PCR product.

[0102] Purification of Nucleic Acids In some embodiments, nucleic acids are purified. In some embodiments, precipitation methods (e.g., LiCl precipitation, alcohol precipitation, or equivalent methods, such as the methods described herein) are used to purify nucleic acids. In some embodiments, chromatography methods or equivalent methods (e.g., the methods described herein), such as methods using HPLC, are used to purify nucleic acids. In some embodiments, both a precipitation method (e.g., LiCl precipitation) and a method using HPLC are used to purify nucleic acids.

[0103] Target sequence In some embodiments, the CRISPR / Cas system of the present disclosure may be oriented 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 in the vicinity of the specific PAM sequence of such nuclease. In some embodiments, a class 2 Cas nuclease is oriented to the target sequence of the target nucleic acid molecule by a gRNA, where the gRNA hybridizes to the target sequence and the class 2 Cas protein may cleave the target sequence. In some embodiments, the 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 targeting 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 targeting 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 adjacent to the specific PAM sequence. In some embodiments, the target sequence may include a sequence that is 100% complementary to the targeting sequence of the guide RNA. In other embodiments, the target sequence may include at least one mismatch, deletion, or insertion compared to the targeting sequence of the guide RNA.

[0104] The length of the target sequence may vary depending on the nuclease system used. For example, the targeting sequence of a guide RNA for the CRISPR / Cas system may include a nucleotide length of 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, the target sequence is of the corresponding length, and optionally adjacent to the PAM sequence. In some embodiments, the target sequence may include a length of 15 to 24 nucleotides. In some embodiments, the target sequence may include a length of 17 to 21 nucleotides. In some embodiments, the target sequence may include a length of 20 nucleotides. When using a nickase, the target sequence may include 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 include a pair of target sequences recognized by a pair of nickases that cleave the same strand of a DNA molecule. In some embodiments, the target sequence may include a portion of a target sequence recognized by one or more Cas nucleases.

[0105] The target nucleic acid molecule may be any DNA molecule or RNA molecule that is endogenous or exogenous to the cell. In some embodiments, the target nucleic acid molecule may be cell-derived or intracellular episomal DNA, plasmid, genomic DNA, viral genome, mitochondrial DNA, or chromosomal DNA. In some embodiments, the target sequence of the target nucleic acid molecule may be a genomic sequence derived 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 certain embodiments, the target sequence may include a translocation junction, e.g., a translocation associated with cancer. In some embodiments, the target sequence may be on the chromosome of a eukaryote such as a human chromosome. In certain embodiments, the target sequence is a liver-specific sequence, in which case the sequence is expressed in hepatocytes.

[0107] In some embodiments, the target sequence may be located within the coding sequence of a gene, within the intron sequence of a gene, within a regulatory sequence, within the transcriptional regulatory sequence of a gene, within the translational regulatory sequence of a gene, at a splicing site, or within the non-coding sequence between genes. In some embodiments, the gene may be a gene encoding a protein. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may include all or part of a disease-related gene. In some embodiments, the target sequence may be located at a non-gene functional site of the genome, for example, a site that controls the flank of chromatin organization such as a scaffold site or a locus control region.

[0108] In embodiments involving a Cas nuclease, such as a Class 2 Cas nuclease, the target sequence may be adjacent to a protospacer adjacent motif (PAM). In some embodiments, the PAM may be adjacent to the 3’ end of the target sequence or within 1, 2, 3, or 4 nucleotides thereof. The length and sequence of the PAM may vary depending on the Cas protein used. For example, the PAM may be selected from a consensus sequence for a particular Cas9 protein or Cas9 ortholog or a particular PAM sequence, including those disclosed in FIG. 1 of Ran et al., Nature, 520:186-191 (2015) and FIG. S5 of Zetsche 2015, each of which is 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, NNNAAAW, 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 NNNAAAW.

[0109] Lipid formulation This specification discloses various embodiments of LNP formulations of RNA, such as CRISPR / Cas cargo. Such LNP formulations include "amine lipids" together with helper lipids, neutral lipids, and PEG lipids. In some embodiments, such LNP formulations include "amine lipids" together with helper lipids and PEG lipids. In some embodiments, the LNP formulation includes less than 1 percent neutral phospholipid. In some embodiments, the LNP formulation includes less than 0.5 percent neutral phospholipid. "Lipid nanoparticles" mean particles comprising a plurality (i.e., two or more) of lipid molecules physically associated with each other by intermolecular forces.

[0110] Amine lipid An LNP composition for delivering a biologically active agent includes an "amine lipid" defined as lipid A or its equivalent, such as an acetal analog 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 and is also referred to 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

Chemical formula

[0112] Lipid A may be synthesized according to WO2015 / 095340 (e.g., pages 84-86). In certain embodiments, the amine lipid is an equivalent of 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 the acetal analogs of C4, C5, C6, C7, C9, C10, C11, and C12.

[0114] The amine lipids suitable for use in the LNPs described herein are biodegradable in vivo and are suitable for delivering biologically active substances such as RNA to cells. The amine lipids are of low toxicity (e.g., tolerated in animal models with an RNA cargo amount of 10 mg / kg or more and having no adverse effects). In certain embodiments, the LNPs containing the amine lipid include those in which at least 75% of the amine lipid disappears from the plasma within 8 hours, within 10 hours, within 12 hours, within 24 hours, or within 48 hours, or within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, or within 10 days. In certain embodiments, the LNPs containing the amine lipid include those in which at least 50% of the mRNA or gRNA disappears from the plasma within 8 hours, within 10 hours, within 12 hours, within 24 hours, or within 48 hours, or within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, or within 10 days. In certain embodiments, the LNPs containing the amine lipid include those in which, for example, at least 50% of the LNP (measuring lipids (e.g., amine lipids), RNA (e.g., mRNA), or another component) disappears from the plasma within 8 hours, within 10 hours, within 12 hours, within 24 hours, or within 48 hours, or within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, or within 10 days. In certain embodiments, the lipid, RNA, or nucleic acid component of the LNP is measured in both the encapsulated and free forms with respect to the lipid.

[0115] Lipid clearance may be measured as described in the literature. See Maier, M.A., 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, an LNP-siRNA system containing luciferase-directed siRNA was administered to 6- to 8-week-old male C57Bl / 6 mice at 0.3 mg / kg via intravenous bolus injection through the lateral tail vein. Samples of blood, liver, and spleen were collected 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 96 hours, and 168 hours after administration. The mice were perfused with saline and then the tissues were collected, and the blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Further, Maier describes methods for toxicity assessment after administration of the LNP-siRNA formulation. 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) at a dose volume of 5 mL / kg by single intravenous bolus injection. After 24 hours, approximately 1 mL of blood was obtained from the jugular vein of the awake 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 the evaluation of clearance, pharmacokinetics, and toxicity regarding administration of the LNP compositions of the present disclosure.

[0116] Amine lipids can cause an increase in clearance rate. In some embodiments, the clearance rate is the lipid clearance rate, e.g., the rate at which lipids disappear from blood, serum, or plasma. In some embodiments, the clearance rate is the RNA clearance rate, e.g., 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 circulation and in tissues. In some embodiments, the reduction in the accumulation of LNPs in circulation and in tissues results in a safety profile with substantially no adverse effects.

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

[0118] The pH at which the amine lipid is predominantly protonated is related to its intrinsic pKa. In some embodiments, each of the amine lipids of the present disclosure may independently have a pKa in the range of about 5.1 to about 7.4. In some embodiments, each of the amine lipids of the present disclosure may independently have a pKa in the range of about 5.5 to about 6.6. In some embodiments, each of the amine lipids of the present disclosure may independently have a pKa in the range of about 5.6 to about 6.4. In some embodiments, each of the amine lipids of the present disclosure may independently have a pKa in the range of about 5.8 to about 6.2. For example, each of the amine lipids of the present disclosure may independently have a pKa in the range of about 5.8 to about 6.5. Since 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, for example, to the liver, etc., the pKa of the amine lipid can be an important consideration in the formulation of LNPs. 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 in vivo, for example, to tumors. See, for example, WO2014 / 136086.

[0119] Additional lipid Examples of "neutral lipids" suitable 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), phosphatidylcholine (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 (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diarachidonoyl-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, but are not limited thereto. 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 lipid may be dipalmitoyl phosphatidylcholine (DPPC).

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

[0121] The PEG lipid is a stealth lipid that changes the length of time the nanoparticles can exist in vivo (e.g., in the blood). The PEG lipid can assist in the formulation process, for example, by suppressing particle aggregation and controlling the particle size. The pharmacokinetic properties of the LNP can be regulated by the PEG lipid used herein. Typically, the PEG lipid includes a lipid moiety and a polymer moiety based on PEG.

[0122] In some embodiments, the lipid moiety can be obtained from a dialkylglycerol or a dialkylglycamide, which independently includes a dialkylglycerol group or a dialkylglycamide group containing saturated or unsaturated carbon atoms with an alkyl chain length of about C4 to about C40, where such chains may include one or more functional groups such as, for example, an amide or an ester. In some embodiments, the alkyl chain length includes from about C10 to C20. The dialkylglycerol group or the dialkylglycamide group can further include one or more substituted alkyl groups. The chain length may be symmetric or asymmetric.

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

[0124] In certain embodiments, the PEG (e.g., attached to a lipid moiety such as a stealth lipid or a lipid) is "PEG-2K", also referred to as "PEG 2000", and has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (I)

Chemical formula

[0125] In any of the embodiments described herein, the PEG lipid may be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog number GM-020, manufactured by NOF (Tokyo, Japan)), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog number DSPE-020CN, manufactured by NOF (Tokyo, Japan)), PEG-dilaurylglycamide, PEG-dimyristoylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8’-(cholesta-5-en-3[beta]-oxy)carboxamido-3’,6’-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (catalog number 880150P, manufactured by Avanti Polar Lipids (Alabaster, Alabama, USA)), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (catalog number 880120C, manufactured by Avanti Polar Lipids (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 disclosed in WO2016 / 010840 from paragraph

[0240] to paragraph

[0244] . 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 a lipid composition represented according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the mole % of the amine lipid may be from about 30 mole % to about 60 mole %. In one embodiment, the mole % of the amine lipid may be from about 40 mole % to about 60 mole %. In one embodiment, the mole % of the amine lipid may be from about 45 mole % to about 60 mole %. In one embodiment, the mole % of the amine lipid may be from about 50 mole % to about 60 mole %. In one embodiment, the mole % of the amine lipid may be from about 55 mole % to about 60 mole %. In one embodiment, the mole % of the amine lipid may be from about 50 mole % to about 55 mole %. In one embodiment, the mole % of the amine lipid may be about 50 mole %. In one embodiment, the mole % of the amine lipid may be about 55 mole %. In some embodiments, the mole % of the amine lipid in the LNP batch is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole %. In some embodiments, the mole % of the amine lipid in the LNP batch is ±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 relative to the lipid components of the LNP composition. In certain embodiments, the variation in the mole % of the amine lipid between LNP lots is less than 15%, less than 10% or less than 5%.

[0127] In one embodiment, the mole percentage of neutral lipids, such as neutral phospholipids, etc., may be from about 5 mole % to about 15 mole %. In one embodiment, the mole percentage of neutral lipids, such as neutral phospholipids, etc., may be from about 7 mole % to about 12 mole %. In one embodiment, the mole percentage of neutral lipids, such as neutral phospholipids, etc., may be from about 0 mole % to about 5 mole %. In one embodiment, the mole percentage of neutral lipids, such as neutral phospholipids, etc., may be from about 0 mole % to about 10 mole %. In one embodiment, the mole percentage of neutral lipids, such as neutral phospholipids, etc., may be from about 5 mole % to about 10 mole %. In one embodiment, the mole percentage of neutral lipids, such as neutral phospholipids, etc., may be from about 8 mole % to about 10 mole %.

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

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

[0130] In one embodiment, the mole % of neutral lipids, such as neutral phospholipids, etc., may be less than about 1 mole %. In one embodiment, the mole % of neutral lipids, such as neutral phospholipids, etc., may be less than about 0.5 mole %. In one embodiment, the mole % of neutral lipids, such as neutral phospholipids, etc., may be about 0 mole %, about 0.1 mole %, about 0.2 mole %, about 0.3 mole %, about 0.4 mole %, about 0.5 mole %, about 0.6 mole %, about 0.7 mole %, about 0.8 mole %, about 0.9 mole %, or about 1 mole %. In some embodiments, the formulations disclosed herein do not contain neutral lipids (i.e., the neutral lipids are 0 mole %). In some embodiments, the formulations disclosed herein essentially do not contain neutral lipids (i.e., the neutral lipids are about 0 mole %). In some embodiments, the formulations disclosed herein do not contain neutral phospholipids (i.e., the neutral phospholipids are 0 mole %). In some embodiments, the formulations disclosed herein essentially do not contain neutral phospholipids (i.e., the neutral phospholipids are about 0 mole %).

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

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

[0133] In one embodiment, the molar percentage of the PEG lipid may be from about 1 mol% to about 10 mol%. In one embodiment, the molar percentage of the PEG lipid may be from about 2 mol% to about 10 mol%. In one embodiment, the molar percentage of the PEG lipid may be from about 2 mol% to about 8 mol%. In one embodiment, the molar percentage of the PEG lipid may be from about 2 mol% to about 4 mol%. In one embodiment, the molar percentage of the PEG lipid may be from about 2.5 mol% to about 4 mol%. In one embodiment, the molar percentage of the PEG lipid may be about 3 mol%. In one embodiment, the molar percentage of the PEG lipid may be about 2.5 mol%. In some embodiments, the molar percentage of the PEG lipid in the LNP batch is within ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target molar percentage of the PEG lipid. In certain embodiments, the LNP lot-to-lot variation is less than 15%, less than 10%, or less than 5%.

[0134] In certain embodiments, the cargo includes 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 a gRNA, or a combination of mRNA and 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 a specific embodiment, 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 includes a PEG lipid comprising DMG. In certain embodiments, the amine lipid is selected from lipid A equivalents such as lipid A and acetal analogs of lipid A. In a further embodiment, the LNP composition includes 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 contain 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 equivalents such as lipid A and acetal analogs of lipid A. In certain embodiments, the amine lipid is an acetal analog of lipid A having C5-C12 or C4-C12. In a further embodiment, the LNP composition comprises lipid A, cholesterol, and PEG2k-DMG.

[0136] Embodiments of the present disclosure also provide lipid compositions represented according to the molar ratio of the positively charged amine group (N) of the aminolipid 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, the LNP composition may comprise a lipid component including an aminolipid, a helper lipid, a neutral lipid, and a PEG lipid, and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP composition may comprise a lipid component including an aminolipid, a helper lipid, and a PEG lipid, and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP composition may comprise a lipid component including an aminolipid, a helper lipid, a neutral lipid, and a helper lipid, and an RNA component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP composition may comprise a lipid component including an aminolipid, a helper lipid, and a PEG lipid, and an RNA component, wherein the N / P ratio is about 3 to 10. In one embodiment, the N / P ratio may be about 5 to 7. In one embodiment, the N / P ratio may be about 3 to 7. In one embodiment, the N / P ratio may be about 4.5 to 8. In one embodiment, the N / P ratio may be about 6. In one embodiment, the N / P ratio may be 6 ± 1. In one embodiment, the N / P ratio may be 6 ± 0.5. In some embodiments, the N / P ratio is ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, or ± 2.5% of the target N / P ratio. In certain embodiments, the LNP lot-to-lot variation is less than 15%, less than 10% or less than 5%.

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

[0138] In certain embodiments, the LNP composition may include 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, the LNP composition may include mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, an amine lipid, and a PEG lipid. In certain LNP compositions including mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions including mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the neutral lipid is DSPC. In a further embodiment including mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain compositions including 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 include a gRNA. In certain embodiments, the LNP composition may include an amine lipid, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain embodiments, the LNP composition may include an amine lipid, a gRNA, a helper lipid, and a PEG lipid. In certain LNP compositions containing a gRNA, the helper lipid is cholesterol. In some compositions containing a gRNA, the neutral lipid is DSPC. In further embodiments containing 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, etc.

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

[0141] In certain embodiments, the LNP composition comprises mRNA encoding a Cas nuclease and a gRNA, which may be an sgRNA. In one embodiment, the LNP composition may comprise an amine lipid, mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In one embodiment, the 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 mRNA encoding a Cas nuclease and a gRNA. In one embodiment, the 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 mRNA encoding a Cas nuclease and a gRNA. In certain compositions comprising mRNA encoding a Cas nuclease and a gRNA, the helper lipid is cholesterol. In some compositions comprising mRNA encoding a Cas nuclease and a gRNA, the neutral lipid is DSPC. Certain compositions comprising mRNA encoding a Cas nuclease and a gRNA comprise less than about 1 mol% neutral lipid, such as neutral phospholipid. Certain compositions comprising mRNA encoding a Cas nuclease and a gRNA comprise less than about 0.5 mol% neutral lipid, such as neutral phospholipid. In certain compositions, the LNP does not contain a neutral lipid, such as neutral phospholipid. In a further embodiment comprising mRNA encoding a Cas nuclease and 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, etc.

[0142] In certain embodiments, the LNP composition comprises a Cas nuclease mRNA, such as a Class 2 Cas mRNA, and at least one gRNA. In certain embodiments, the LNP composition comprises the gRNA and the Cas nuclease mRNA, such as a Class 2 Cas mRNA, in a ratio of from about 25:1 to about 1:25. In certain embodiments, the LNP formulation comprises the gRNA and the Cas nuclease mRNA, such as a Class 2 Cas mRNA, in a ratio of from about 10:1 to about 1:10. In certain embodiments, the LNP formulation comprises the gRNA and the Cas nuclease mRNA, such as a Class 2 Cas mRNA, in a ratio of from about 8:1 to about 1:8. The ratios measured herein are on a weight basis. In some embodiments, the LNP formulation comprises the gRNA and the Cas nuclease mRNA, such as a Class 2 Cas mRNA, in a ratio of from about 5:1 to about 1:5. In some embodiments, the ratio ranges are from about 3:1 to 1:3, from about 2:1 to 1:2, from about 5:1 to 1:2, from about 5:1 to 1:1, from about 3:1 to 1:2, from about 3:1 to 1:1, about 3:1, from 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 the Cas nuclease mRNA, such as a Class 2 Cas nuclease, is about 1:1. The ratio can 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 may contain a template nucleic acid. The template nucleic acid may be formulated together with an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid may be formulated together with a guide RNA. In some embodiments, the template nucleic acid may be formulated together with both an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid may be formulated separately from the mRNA encoding a Cas nuclease or the guide RNA. The template nucleic acid may be delivered together with the LNP composition or separately from the LNP composition. In some embodiments, the template nucleic acid may be single-stranded or double-stranded depending on the desired repair mechanism. The template may have a homology region to the target DNA or a sequence adjacent to 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, isopropanol. A pharmaceutically 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 the composition containing the LNPs at pH 6.5 or higher. In certain embodiments, a buffer is used to maintain the pH of the composition containing the LNPs at pH 7.0 or higher. 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 contains a cryoprotectant. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may contain up to 10% of a cryoprotectant, such as sucrose. In certain embodiments, the LNP composition may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9, or 10% of a cryoprotectant. In certain embodiments, the LNP composition may contain about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9, or 10% of sucrose. In some embodiments, the LNP composition may contain 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 contains NaCl. In certain embodiments, NaCl is excluded. Exemplary amounts of NaCl may range from about 20 mM to about 45 mM. Exemplary amounts of NaCl may 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 Tris buffer.Exemplary amounts of Tris may range from about 20 mM to about 60 mM. Exemplary amounts of Tris may 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 contains NaCl and Tris. In certain exemplary embodiments of the LNP composition, a Tris buffer in which 5% sucrose and 45 mM NaCl are dissolved is included. In other exemplary embodiments, the composition contains sucrose in an amount of about 5 w / v%, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amounts of salt, buffer, and cryoprotectant may be varied such that the osmotic pressure of the overall formulation is maintained. For example, the final osmotic pressure may be maintained below 450 mOsm / L. In further embodiments, the osmotic pressure is between 350 mOsm / L and 250 mOsm / L. In certain embodiments, the final osmotic pressure 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, relative position of the junctions, shape of the junctions, tube diameter, solution, and / or RNA concentration and lipid concentration may be varied. The LNP or LNP composition may be concentrated or purified, for example, by dialysis, tangential flow filtration, or chromatography. The LNP may be stored, for example, as a suspension, emulsion, or lyophilized powder. In some embodiments, the LNP composition is stored at 2 - 8°C, and in certain aspects, the LNP composition is stored at room temperature. In further embodiments, the LNP composition is stored frozen, for example, at -20°C or -80°C. In other embodiments, the LNP composition is stored at a temperature in the range of about 0°C to about -80°C. The frozen LNP composition may be thawed, for example, on ice, at room temperature, or at 25°C, prior to use.

[0146] The LNP may be, for example, microparticles (including unilamellar vesicles and multilamellar vesicles, such as lamellar phase lipid bilayers which are substantially spherical in some embodiments and are referred to as "liposomes", and which may further include, in certain embodiments, an aqueous core, for example, a substantial portion of RNA molecules), the dispersed phase of an emulsion, micelles, or the internal phase of a suspension, and the like.

[0147] Furthermore, the LNP composition is biodegradable and does not accumulate in vivo to cytotoxic concentrations at therapeutically effective amounts. In some embodiments, the LNP composition does not elicit an innate immune response that results in substantial adverse effects at therapeutic doses. In some embodiments, the LNP compositions provided herein are non-toxic at therapeutic doses.

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

[0149] The LNPs disclosed in this specification have a size (e.g., Z-average diameter) of about 1 to about 250 nm. In some embodiments, the LNPs have a size of about 10 to about 200 nm. In further embodiments, the LNPs have a size of about 20 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 150 nm. In some embodiments, the LNPs have a size of about 50 to about 100 nm. In some embodiments, the LNPs have a size of about 50 to about 120 nm. In some embodiments, the LNPs have a size of about 60 to about 100 nm. In some embodiments, the LNPs have a size of about 75 to about 150 nm. In some embodiments, the LNPs have a size of about 75 to about 120 nm. In some embodiments, the LNPs have a size of about 75 to about 100 nm. Unless otherwise specified, all sizes mentioned in this specification are the average size (diameter) of fully formed particles measured by dynamic light scattering using a Malvern Zetasizer. Dilute the nanoparticle sample with phosphate-buffered saline (PBS) so that the counting rate is about 200 - 400 kcps. The data is presented as the weighted average of the intensity measurements (Z-average diameter).

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

[0151] In some embodiments, the LNP is formed with an average molecular weight in the range of about 1.00E+05 g / mol to about 1.00E+10 g / mol. In some embodiments, the LNP is formed with an average molecular weight in the range of about 5.00E+05 g / mol to about 7.00E+07 g / mol. In some embodiments, the LNP is formed with an average molecular weight in the range of about 1.00E+06 g / mol to about 1.00E+10 g / mol. In some embodiments, the LNP is formed with an average molecular weight in the range of about 1.00E+07 g / mol to about 1.00E+09 g / mol. In some embodiments, the LNP is formed with an average molecular weight in the range of 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 of 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. In some embodiments, the method involves contacting the cells with an LNP composition described herein.

[0154] In some embodiments, the method involves contacting cells of a subject such as a mammal, such as a human. In some embodiments, the cells are in an organ, such as the liver, such as the liver of a mammal, such as the human liver. In some embodiments, the cells are liver cells, such as liver cells of a mammal, such as human liver cells. In some embodiments, the cells are hepatocytes, such as hepatocytes of a mammal, such as human hepatocytes. In some embodiments, the liver cells are stem cells. In some embodiments, the human liver cells may be liver sinusoidal endothelial cells (LSEC) of the liver. In some embodiments, the human liver cells may be Kupffer cells. In some embodiments, the human liver cells may be hepatic stellate cells. In some embodiments, the human liver cells may be tumor cells. In some embodiments, the human liver cells may be liver stem cells. In further embodiments, the cells comprise an ApoE binding receptor. In some embodiments, liver cells such as hepatocytes are in situ. In some embodiments, liver cells such as hepatocytes are isolated, such as in a culture such as a primary culture. Methods corresponding to the uses disclosed herein are also provided, which include administering the LNP compositions disclosed herein to a subject or contacting cells as described above with the LNP compositions disclosed herein.

[0155] In some embodiments, engineered cells are provided, such as 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 inside a tissue or organ in a subject, such as the liver.

[0156] In some of the methods and cells described herein, the cell comprises a modification of the nucleotides of the target sequence, such as an insertion or deletion ("indel") or substitution. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides of the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides of 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 of the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides of 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 of the target sequence. In some embodiments, the modification comprises a substitution of 1 or 2 nucleotides of the target sequence. In some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid, such as any of the template nucleic acids described herein.

[0157] In some embodiments, a cell population comprising engineered cells is provided, such as a cell population 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 within 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%, which is 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%, which is defined by the detection of sequence changes, whether by insertion, deletion, substitution, or other means. In certain embodiments, the methods disclosed herein result in an editing efficiency level or DNA modification efficiency level in the cell population of from about 5% to about 100%, from about 10% to about 50%, from about 20 to about 100%, from about 20 to about 80%, from about 40 to about 100%, or from about 40 to about 80%.

[0158] In some of the methods and cells described herein, the cells within the population contain modifications in the target sequence, such as indels or substitutions. In some embodiments, the modification includes the insertion of 1, 2, 3, 4, or 5 or more nucleotides of the target sequence. In some embodiments, the modification includes the insertion of 1 or 2 nucleotides of the target sequence. In other embodiments, the modification includes the deletion 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 includes the deletion of 1 or 2 nucleotides of the target sequence. In some embodiments, the modification results in a frameshift mutation in the target sequence. In some embodiments, the modification includes 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 a frameshift mutation. In some embodiments, the modification includes 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 includes the substitution of 1 or 2 nucleotides of the target sequence. In some embodiments, the modification includes one or more of an insertion, deletion, or substitution of a nucleotide resulting from the incorporation of a template nucleic acid, such as any of the template nucleic acids described herein.

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

[0160] In some embodiments, the method involves administration of an LNP composition to cells associated with liver disorders. In some embodiments, the method involves treatment of liver disorders. In certain embodiments, the method involves contact of hepatocytes with an LNP composition. In certain embodiments, the method involves contact of hepatocytes with an LNP composition. In some embodiments, the method involves contact of ApoE-binding cells with an LNP composition.

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

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

[0163] In one embodiment, an LNP composition comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, may be administered to cells separately from the administration of a composition comprising gRNA. In one embodiment, an LNP composition comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, and gRNA may be administered to cells separately from the administration of template nucleic acid to the cells. In one embodiment, an LNP composition comprising mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, may be administered to cells, and then an LNP composition comprising gRNA may be sequentially administered, followed by administration of the template to the cells. In embodiments where an LNP composition comprising mRNA encoding a Cas nuclease is administered followed by an LNP composition comprising gRNA, the time between administrations may be about 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, an LNP composition may be used to edit a gene that causes gene knockout. In one embodiment, an LNP composition may be used to edit a gene that causes gene knockdown in a cell population. In another embodiment, an LNP composition may be used to edit a gene that causes gene correction. In a further embodiment, an LNP composition may be used to edit a cell that causes gene insertion.

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

[0166] The LNP composition is suitable for parenteral administration. The LNP composition may be directly administered into the bloodstream, tissue, muscle, or viscera. Administration may be systemic, for example, by injection or infusion. Administration may be local. Suitable means of administration include intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, subretinal, intravitreal, intracameral, intramuscular, intrasynovial, intradermal, and subcutaneous. Suitable administration devices include needle (including microneedle) injectors, needle-free, osmotic pumps, and infusion techniques.

[0167] Although not necessarily, the LNP composition is generally administered as a formulation containing one or more pharmaceutically acceptable additives. The term "additive" includes any component other than the compound(s) of the present disclosure, other lipid component(s), and biologically active agents. The additives may impart either functional (e.g., drug release rate control) characteristics and / or non-functional (e.g., processing aid or diluent) characteristics to the formulation. The choice of additives varies greatly depending on factors such as the individual method of administration, the effect of the additives 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, the additives are sugars (including, but not limited to, glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably with a pH of 3 to 9). For some applications, these may be more preferably formulated in a dry form using a sterile non-aqueous solution or in combination with a suitable vehicle such as sterile pyrogen-free water (WFI).

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

[0170] Both the foregoing general description and the detailed description, as well as the following examples, are illustrative and explanatory only and do not limit the teachings. The section headings used herein are for the purpose of organization only and are not to be construed as limiting the desired subject matter in any way. To the extent that documents incorporated by reference conflict with the terms defined herein, the present specification shall control. All ranges recited in this application include their endpoints, unless otherwise specified.

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

[0172] Numeric ranges include the numbers defining the range. Measured and measurable values are approximate and should be understood to take into account significant figures and errors associated with the measurement. The terms “about” or “approximately” mean an acceptable error for a particular value as determined by one of ordinary skill in the art and vary somewhat depending on the method of measurement or determination of the value. The use of a modifier such as “about” before a range or an enumerated value modifies each endpoint of the range or each value in the enumeration. “About” includes the value or endpoint. For example, “about 50-55” includes “about 50-about 55.” Also, the use of “comprise,” “comprises,” “comprising,” “contain,” “contains,” “containing,” “include,” “includes,” and “including” is not limiting.

[0173] Unless otherwise specified in the above specification, in the specification, embodiments with descriptions of various components as "comprising" are also intended to "consist of" or "consist essentially of" the described components, and in the specification, embodiments with descriptions of various components as "consisting of" are also intended to "comprise" or "consist essentially of" the described components, and in the specification, embodiments with descriptions of various components as "about" are also intended to be "at" the described components, and in the specification, embodiments with descriptions of various components as "consisting essentially of" are also intended to "consist of" or "comprise" the described components (such interchangeability does not apply when using these terms in claims). (It is not applied in practice). The present invention includes, for example, the following embodiments: [Embodiment 1] An RNA component and, a lipid component, wherein the lipid component comprises about 50 to 60 mol% of amine lipid, about 8 to 10 mol% of neutral lipid, about 2.5 to 4 mol% of PEG lipid and, wherein the balance 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 and, about 50 to 60 mol% of amine lipid, about 27 to 39.5 mol% of helper lipid, about 8 to 10 mol% of neutral lipid, about 2.5 to 4 mol% of PEG lipid and, wherein the N / P ratio of the LNP composition is about 5 to 7, the LNP composition. [Embodiment 3] The LNP composition according to 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 to 60 mol% of an amine lipid, about 5 to 15 mol% of a neutral lipid, about 2.5 to 4 mol% of a PEG lipid and, wherein the remainder of the lipid component is a helper lipid, and 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 to 60 mol% of an amine lipid, about 5 to 15 mol% of a neutral lipid, about 2.5 to 4 mol% of a PEG lipid and, wherein the remainder of the lipid component is a helper lipid, and 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 to 60 mol% of an amine lipid, about 5 to 15 mol% of a neutral lipid, about 1.5 to 10 mol% of a PEG lipid and, wherein the remainder of the lipid component is a helper lipid, and 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 to 60 mol% of an amine lipid, about 0 to 10 mol% of a neutral lipid, about 1.5 to 10 mol% of a PEG lipid and, wherein the remainder of the lipid component is a helper lipid, and the N / P ratio of the LNP composition is about 3 to 10. [Embodiment 8] An RNA component and comprising a lipid component, wherein the lipid component is about 40 to 60 mol% of amine lipid, and less than about 1 mol% of neutral lipid, and about 1.5 to 10 mol% of PEG lipid and wherein the balance of the lipid component is helper lipid, and the LNP composition has an N / P ratio of about 3 to 10. [Embodiment 9] An RNA component and a lipid component, wherein the lipid component is about 40 to 60 mol% of amine lipid, and about 1.5 to 10 mol% of PEG lipid and wherein the balance of the lipid component is helper lipid, the LNP composition has an N / P ratio of about 3 to 10, and the LNP composition essentially does not contain or does not contain neutral phospholipids. [Embodiment 10] An RNA component and a lipid component, wherein the lipid component is about 50 to 60 mol% of amine lipid, and about 8 to 10 mol% of neutral lipid, and about 2.5 to 4 mol% of PEG lipid and wherein the balance of the lipid component is helper lipid, and the LNP composition has an N / P ratio of about 3 to 7. [Embodiment 11] The composition according to any one of the preceding embodiments, wherein the RNA component comprises mRNA. [Embodiment 12] The composition according to any one of the preceding embodiments, wherein the RNA component comprises an RNA-guided DNA binding factor, such as Cas nuclease mRNA, etc. [Embodiment 13] The composition according to any one of the preceding embodiments, wherein the RNA component comprises class 2 Cas nuclease mRNA. [Embodiment 14] The composition according to any one of the preceding embodiments, wherein the RNA component comprises Cas9 nuclease mRNA. [Embodiment 15] The composition according to any one of Embodiments 11 to 14, wherein the mRNA is a modified mRNA. [Embodiment 16] The composition according to any of the preceding embodiments, wherein the RNA component comprises an RNA comprising an open reading frame encoding an RNA-induced 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 according to any of the preceding embodiments, wherein the RNA component comprises an mRNA comprising an open reading frame encoding an RNA-induced 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 according to any of the preceding embodiments, wherein the RNA component comprises an mRNA comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 1, 4, 7, 9, 10, 11, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66, wherein the mRNA comprises an open reading frame encoding an RNA-induced DNA-binding factor. [Embodiment 19] The composition according to any of the preceding embodiments, wherein the RNA component comprises a gRNA nucleic acid. [Embodiment 20] The composition according to Embodiment 19, wherein the gRNA nucleic acid is a gRNA. [Embodiment 21] The composition according to any of the preceding embodiments, wherein the RNA component comprises a class 2 Cas nuclease mRNA and a gRNA. [Embodiment 22] The composition according to any of Embodiments 19 to 21, wherein the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA). [Embodiment 23] The composition according to any of Embodiments 19 to 21, wherein the gRNA nucleic acid is or encodes a sgRNA. [Embodiment 24] The composition according to any of Embodiments 19 to 23, wherein the gRNA is modified. [Embodiment 25] The gRNA is the composition according to Embodiment 24, comprising a modification selected from 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) bonds between nucleotides, and 2'-fluoro (2'-F) modified nucleotides. [Embodiment 26] The gRNA is the composition according to any one of Embodiments 24 to 25, comprising a modification in one or more of the first 5 nucleotides at the 5' end. [Embodiment 27] The gRNA is the composition according to any one of Embodiments 24 to 26, comprising a modification in one or more of the last 5 nucleotides at the 3' end. [Embodiment 28] The gRNA is the composition according to any one of Embodiments 24 to 27, comprising a PS bond between the first 4 nucleotides. [Embodiment 29] The gRNA is the composition according to any one of Embodiments 24 to 28, comprising a PS bond between the last 4 nucleotides. [Embodiment 30] The composition according to any one of Embodiments 24 to 29, further comprising 2'-O-Me modified nucleotides at the first 3 nucleotides at the 5' end. [Embodiment 31] The composition according to any one of Embodiments 24 to 30, further comprising 2'-O-Me modified nucleotides at the last 3 nucleotides at the 3' end. [Embodiment 32] The composition according to any one of Embodiments 19 to 31, wherein the gRNA and the class 2 Cas nuclease mRNA are present in a ratio in the range of about 10:1 to about 1:10 by weight. [Embodiment 33] The composition according to any one of Embodiments 19 to 31, wherein the gRNA and the class 2 Cas nuclease mRNA are present in a ratio in the range of about 5:1 to about 1:5 by weight. [Embodiment 34] The composition according to any one of Embodiments 19 to 33, wherein the gRNA and the class 2 Cas nuclease mRNA are present in a ratio in the range of about 3:1 to about 1:1 by weight. [Embodiment 35] The composition according to any one of Embodiments 19 to 34, wherein the gRNA and the class 2 Cas nuclease mRNA are present in a ratio in the range of about 2:1 to about 1:1 by weight. [Embodiment 36] The composition according to any one of Embodiments 19 to 35, wherein the gRNA and the Class 2 Cas nuclease mRNA are present in a ratio of about 2:1 by weight. [Embodiment 37] The composition according to any one of Embodiments 19 to 35, wherein the gRNA and the Class 2 Cas nuclease mRNA are present in a ratio of about 1:1 by weight. [Embodiment 38] The composition according to any one of the preceding embodiments, further comprising at least one template. [Embodiment 39] The composition according to any one of the preceding embodiments, wherein the mol% of the PEG lipid is about 3. [Embodiment 40] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is about 50. [Embodiment 41] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is about 55. [Embodiment 42] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is ±3 mol%. [Embodiment 43] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is ±2 mol%. [Embodiment 44] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is 47 to 53 mol%. [Embodiment 45] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is 48 to 53 mol%. [Embodiment 46] The composition according to any one of the preceding embodiments, wherein the mol% of the amine lipid is 53 to 57 mol%. [Embodiment 47] The composition according to any one of the preceding embodiments, wherein the N / P ratio is 6 ± 1. [Embodiment 48] The composition according to any one of the preceding embodiments, wherein the N / P ratio is 6 ± 0.5. [Embodiment 49] The composition according to any one of the preceding embodiments, wherein the amine lipid is lipid A. [Embodiment 50] The composition according to any one of the preceding embodiments, wherein the amine lipid is an analog of lipid A. [Embodiment 51] The composition according to Embodiment 50, wherein the analog is an acetal analog. [Embodiment 52] The composition according to Embodiment 51, wherein the acetal analog is a C4-C12 acetal analog. [Embodiment 53] The composition according to Embodiment 50, wherein the acetal analog is a C5-C12 acetal analog. [Embodiment 54] The composition according to Embodiment 50, wherein the acetal analog is a C5-C10 acetal analog. [Embodiment 55] The composition according to 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 according to any of the preceding embodiments, wherein the helper lipid is cholesterol. [Embodiment 57] The composition according to any of the preceding embodiments, wherein the neutral lipid is DSPC. [Embodiment 58] The composition according to any of the preceding embodiments, wherein the neutral lipid is DPPC. [Embodiment 59] The composition according to any of the preceding embodiments, wherein the PEG lipid contains dimyristoyl glycerol (DMG). [Embodiment 60] The composition according to any of the preceding embodiments, wherein the PEG lipid contains PEG-2k. [Embodiment 61] The composition according to any of the preceding embodiments, wherein the PEG lipid is PEG-DMG. [Embodiment 62] The composition according to Embodiment 61, wherein the PEG-DMG is PEG2k-DMG. [Embodiment 63] The composition according to Embodiment 9, wherein the LNP composition essentially does not contain neutral lipid. [Embodiment 64] The composition according to Embodiment 63, wherein the neutral lipid is a phospholipid. [Embodiment 65] A gene editing method comprising contacting a cell with the LNP composition according to any 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 according to any one of Embodiments 13 to 64, said method. [Embodiment 67] A method for producing a genetically engineered cell, comprising contacting a cell with at least one LNP composition according to any one of Embodiments 12 to 64. [Embodiment 68] The method according to any one of Embodiments 65 to 67, wherein the LNP composition is administered at least twice. [Embodiment 69] The method according to Embodiment 68, wherein the LNP composition is administered 2 to 5 times. [Embodiment 70] The method according to Embodiment 68 or 69, wherein editing is improved upon re-administration. [Embodiment 71] The method according to any one of Embodiments 65 to 70, further comprising introducing at least one template nucleic acid into the cell. [Embodiment 72] The method according to 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 according to Embodiment 72, wherein the first and second LNP compositions are administered simultaneously. [Embodiment 74] The method according to Embodiment 72, wherein the first and second LNP compositions are administered sequentially. [Embodiment 75] The method according to any one of Embodiments 65 to 73, wherein the mRNA and the guide RNA nucleic acid are formulated in 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 their properties were examined. In assays for the editing ratio in the liver in mice, Cas9 mRNA and chemically modified mouse TTR sequence-directed sgRNA were formulated in LNP, with the mol% of PEG, the mol% of lipid A, and the N:P ratio varied as described in Table 2 below.

Table 2

[0175] In Figure 1, the LNP formulations are identified on the x-axis based on their lipid A mol% and N:P ratio, represented as “%CL;N:P”. As shown in the legend of 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 4.5 (“45;4.5”), (2) 45 mol% lipid A; N:P ratio 6 (“45;6”), (3) 50 mol% lipid A; N:P ratio 4.5 (“50;4.5”), (4) 50 mol% lipid A; N:P ratio 6 (“50;6”), (5) 55 mol% lipid A; N:P ratio 4.5 (“55;4.5”), and (6) 55 mol% lipid A; N:P ratio 6 (“55;6”). The mol% of DSPC was kept constant at 9 mol%, and cholesterol (mol%) was added so that the remainder of the lipid component of each formulation was 100 mol%. Each of the 30 formulations was formulated as described below and administered as a single dose at a total RNA dose of 1 mg / kg or 0.5 mg / kg (Figure 1A and Figure 1B, respectively).

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

[0177] The Precision Nanosystems NanoAssemblr (trademark) Benchtop Instrument was used according to the manufacturer's operating procedures to generate LNPs by microfluidic mixing of lipids and RNA solution. Different flow rates were used while maintaining the ratio of aqueous solvent to organic solvent at 2:1 during mixing. After mixing, the LNPs were recovered, diluted in water (approx. 1:1 v / v), held at room temperature for 1 hour, further diluted in water (approx. 1:1 v / v), and then the final buffer exchange was performed. The final buffer exchange to 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 using an Amicon 100 kDa centrifugal filter (Millipore). Subsequently, the resulting mixture was filtered using a 0.2 μm sterile filter. The final LNPs were stored at -80 °C until further use.

[0178] Formulation analysis method For the characterization of the polydispersity index ("pdi") and size of the LNPs of the present disclosure, dynamic light scattering method ("DLS") is used. DLS measures the scattering of light obtained by irradiating a sample with a light source. The PDI determined from DLS measurements represents the particle size (approximate average particle size) distribution in the population, and in the case of a completely homogeneous population, the PDI is zero.

[0179] Electrophoretic light scattering method is used for the characterization of the surface charge of LNPs at a specific pH. The surface charge, or zeta potential, is a measure of the magnitude of the electrostatic repulsive / attractive forces between particles in an LNP suspension.

[0180] Using asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS), particles in the formulation are separated by hydrodynamic radius, and then the molecular weight, hydrodynamic radius, and root mean square radius of the fractionated particles are measured. This enables the evaluation of the molecular weight and particle size distribution as well as secondary characteristics such as the Burchard-Stockmeyer plot (the ratio of the root mean square ("rms") radius to the hydrodynamic radius over time, which suggests the core density of the particle) and the rms conformation plot (the ratio of the logarithm of the rms radius to the logarithm of the molecular weight, where the slope of the resulting linear fit gives 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 the formulation. The LNP sample is appropriately diluted and injected onto a microscope slide. As the particles slowly flow through the field of view, the camera records the scattered light. Once a movie is acquired, nanoparticle tracking analysis processes the movie by tracking pixels and calculating the diffusion coefficient. This diffusion coefficient can be translated into the hydrodynamic radius of the particle. The device also gives the particle concentration by counting the number of individual particles counted during the analysis.

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

[0183] The lipid composition analysis of LNP can be determined by liquid chromatography followed by charged particle detection (LC-CAD). This analysis provides a comparison of the actual lipid content with the theoretical lipid content.

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

[0185] A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) is used for the determination of total RNA concentration and free RNA. The encapsulation efficiency is calculated as (total RNA - free RNA) / total RNA. The LNP sample is appropriately diluted using 1× TE buffer containing 0.2% Triton-X 100 to determine total RNA or 1× TE buffer to determine free RNA. A standard curve is created using the starting RNA solution that was used to make the formulation and then diluted in 1× TE buffer + / - 0.2% Triton-X 100. Subsequently, the diluted RiboGreen® dye (according to the manufacturer's instructions) is added to each of the standards and samples and incubated at room temperature for approximately 10 minutes in the dark. The samples are read using a SpectraMax M5 plate reader (Molecular Devices) with the excitation, autocaloff, and emission wavelengths set at 488 nm, 515 nm, and 525 nm, respectively. The total RNA and free RNA are determined using an appropriate standard curve.

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

[0187] Use AF4-MALS to examine the molecular weight, particle size distribution, and secondary statistics based on their calculated values. Appropriately dilute the LNPs and inject them into the AF4 separation channel using the HPLC autosampler in which they are collected, and then elute them with an exponential gradient in the cross-flow across the channel. All fluids are driven by an HPLC pump and a Wyatt Eclipse device. The particles elute from the channel flow of AF4 and pass through a UV detector, a multi-angle light scattering detector, a quasi-elastic light scattering detector, and a differential refractive index detector. Use the Debeye model to process the raw data and determine the molecular weight and rms radius from the detector signals.

[0188] Quantitatively analyze the lipid components in the LNP by HPLC connected to a charged aerosol detector (CAD). Separation of the four lipid components by chromatography was achieved by reverse-phase HPLC. CAD is a mass-based detector that detects non-volatile compounds without leakage, and the signal is constant regardless of the structure of the analyte.

[0189] Cargo of Cas9 mRNA and gRNA Cas9 mRNA cargo was prepared by in vitro transcription. Capped and polyadenylated Cas9 mRNA containing 1X NLS (SEQ ID NO: 48) was generated by in vitro transcription using linearized plasmid DNA template and T7 RNA polymerase. XbaI was used at 37 °C for 2 hours under the conditions of the following 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1× reaction buffer to linearize the plasmid DNA containing the T7 promoter and 100 nt poly(A / T) region. The reaction was heated at 65 °C for 20 minutes to inactivate XbaI. The linearized plasmid was purified from enzymes and buffer salts using a silica maxi spin column (Epoch Life Sciences), analyzed by agarose gel, and linearization was confirmed. The IVT reaction for generating Cas9 modified mRNA was incubated at 37 °C for 4 hours under 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 1× 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 from enzymes and nucleotides using the MegaClear Transcription Clean-up kit according to the manufacturer's (ThermoFisher) protocol. Alternatively, Cas9 mRNA was purified by LiCl precipitation method.

[0190] The sgRNA in this example was chemically synthesized and supplied by a commercial vendor. The sg282 sequence is described below, and the 2'-O-methyl modification and phosphorothioate bond are represented as follows (m = 2'-OMe; *= phosphorothioate): mU*mU*mA*CAGCCACGUCUACAGCAGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU. (SEQ ID NO: 42).

[0191] LNP The final LNP was characterized, and the encapsulation efficiency, polydispersity index, and average particle size were determined according to the above analysis methods.

[0192] LNP was administered to mice (single dose at 1 mg / kg or 0.5 mg / kg), and genomic DNA was isolated and subjected to the following NGS analysis.

[0193] In vivo LNP delivery In each experiment, female CD-1 mice aged 6 to 10 weeks were used. The body weights of the animals were measured, and they were divided into groups according to their body weights, and the dosing solutions were prepared based on the average body weight of the groups. 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, the animals were observed for adverse effects. The body weights were measured 24 hours after administration, and at various time points, the animals were euthanized by exsanguination via cardiac puncture under isoflurane anesthesia. Blood was collected into tubes containing serum separator or sodium citrate buffer tubes for plasma as described herein. For the tests to perform in vivo editing, liver tissue was collected from the middle lobe or three independent lobes (e.g., right middle lobe, left middle lobe, and left lobe) of each animal and subjected to DNA extraction and analysis.

[0194] Mouse cohorts were measured for liver editing by next-generation sequencing (NGS) and serum TTR levels (data not shown).

[0195] Transthyretin (TTR) ELISA analysis Blood was collected and serum was isolated as shown. Mouse prealbumin (transthyretin) ELISA kit (Aviva Systems Biology, catalog OKIA00111) was used to determine the total TTR levels in mouse serum. A rat-specific ELISA kit (Aviva Systems Biology, catalog number OKIA00159) was used according to the manufacturer's protocol to measure rat serum TTR levels. Briefly, the serum was serially diluted with the kit's sample diluent to a final dilution of 10,000-fold. Then, this diluted sample was added to an ELISA plate and the assay was performed according to the instructions.

[0196] NGS sequencing Briefly, to quantitatively determine the editing efficiency at the target locus in the genome, genomic DNA was isolated and deep sequencing was utilized 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. The primer sequences are provided below. Further PCR was performed according to the manufacturer's protocol (Illumina), and the chemistry required for sequencing was added. Sequencing of the amplicons was performed on an Illumina MiSeq instrument. After removing those with low quality scores, the reads were aligned against the human reference genome (e.g., hg38). The resulting file containing the reads was mapped against the reference genome (BAM file), where reads overlapping the target region of interest were selected, and the ratio of the number of wild-type reads to the number of 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 wild-type.

[0199] Figure 1 shows the editing ratio in mouse liver measured by NGS. As shown in Figure 1A, when 1 mg of RNA per kg was administered, the in vivo editing ratio was more than about 20% - 60% in the liver. In Figure 1B where 0.5 mg per kg was administered, editing in about 10% - 60% of the liver was observed. In this in vivo test in mice, Cas9 mRNA and gRNA were effectively delivered to hepatocytes in all compositions, and active CRISPR / Cas nuclease activity at the target site, measured by NGS of each LNP composition, was observed. The LNP containing 5% PEG lipid had a low encapsulation rate (not shown in the data figure) and somewhat lower efficacy.

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

[0201] LNP Formulation - Cross - Flow Lipid-containing ethanol was mixed with 2 volumes of RNA solution and 1 volume of water by impinging jet mixing to form LNP. The lipid-containing ethanol was mixed with 2 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 of WO2016010840). The LNP was maintained at room temperature for 1 hour and then further diluted with water (about 1:1 v / v). The diluted LNP was concentrated using tangential flow filtration with a flat sheet cartridge (Sartorius, 100 kD MWCO), and then the buffer was exchanged to 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-depleted (Cas9 Udep) mRNA contains SEQ ID NO: 43. Sg282 was described in Example 1, and the sequence of sg534 ("G534") is described below: mA*mC*mG*CAAAUAUCAGUCCAGCGGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 72)

[0203] The LNP formulation was analyzed for average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1.

[0204] Analysis of the average particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency is shown in Table 4. In addition to the theoretical lipid concentration of the LNP composition, lipid analysis showed the actual molar % levels of the lipids as shown in Table 5 below. [Table 4] [Table 5] TIFF2025084955000010.tif99155

[0205] To further analyze the 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 the particle size and molecular weight distribution and provides information about the particle conformation and density.

[0206] The LNPs were injected into the AF4 separation channel using the HPLC autosampler in which they were collected and then eluted with an exponential gradient in the cross-flow across the channel. All fluids were driven by HPLC pumps and a Wyatt Eclipse instrument. The particles eluted from the channel flow of AF4 and passed 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 were processed with Wyatt Astra 7 software using the Debye model to determine the molecular weight and rms radius from the detector signals.

[0207] The logarithmic molar mass difference plot for the LNP is shown as Figure 2A. Briefly, the X-axis represents the molar mass (g / mol), and the Y-axis represents the differential number fraction. The logarithmic molar mass difference plot shows the distribution of different molecular weights measured for a specific formulation. This provides data on the molecular weight and the overall distribution of molecular weights in the formulation, and a better picture of the 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 the weight-average molar mass (Mw) to the number-average molar mass (Mn) to obtain the polydispersity of 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 is dense at N / P 6.0 when PEG is 3 mol% and lipid A is 50 mol% and 55 mol%. This is reflected in the dense polydispersity as shown in Figure 2B.

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

Table 6

[0211] The LNP was formed as described in Example 2.

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

[0213] The LNP composition was characterized, and the encapsulation efficiency, polydispersity index, and average particle size were determined as described in Example 1.

[0214] Analysis of the average particle size, polydispersity index (PDI), total RNA content, and RNA encapsulation efficiency is shown in Table 7. In addition to the theoretical lipid concentration of the LNP composition, lipid analysis revealed the actual molar % levels of the lipids as shown in Table 8 below. [Table 7] TIFF2025084955000013.tif46159 [Table 8] TIFF2025084955000015.tif36156

[0215] To further analyze the 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 apparatus measures the particle size and molecular weight distribution and provides information about the particle conformation and density.

[0216] AF4-MALS was performed on the LNP as described in Example 1.

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

[0218] The average molecular weight was plotted in Figure 3B. The average molecular weight is the average of the entire distribution, but no information about the shape of the distribution is obtained. 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 the weight-average molar mass (Mw) to the number-average molar mass (Mn) to obtain the polydispersity of Mw / Mn. The polydispersity graphs of these various formulations are shown in Figure 4A.

[0220] Furthermore, the Burchard-Stockmeyer plot of the LNP formulation is shown as Figure 4B. In the Burchard-Stockmeyer plot, the ratio of the rms radius to the hydrodynamic radius in the entire eluate of the formulation from the AF4 channel is shown. Thereby, information regarding the internal density of the lipid nanoparticles can be obtained. Figure 4B shows that in this measurement, LNP1021, LNP1022, and LNP1023 have different profiles.

[0221] In Example 4 - with increased PEG lipid, efficacy is maintained and cytokine response decreases In another test, LNP formulations containing 2 mol% or 3 mol% of PEG lipid were compared with PEG DMG lipid. Compositions containing either 2 mol% or 3 mol% of PEG DMG are listed in Table 9 below. [Table 9] TIFF2025084955000017.tif51145

[0222] LNPs were formed by the method described in Example 2.

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

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

[0225] Analysis of average particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency is shown in Table 10. In addition to the theoretical lipid concentration of the LNP composition, lipid analysis showed the actual lipid mole % levels as shown in Table 11 below.

Table 10

Table 11

[0226] Rat serum cytokines were analyzed and evaluated for MCP-1, IL-6, TNF-alpha, and IFN-gamma using a Luminex magnetic bead multiplex assay (Milliplex MAP magnetic bead assay, manufactured by Millipore Sigma, catalog number RECYTMAG-65K). The assay beads were read on a BioRad BioPlex-200, and cytokine concentrations were calculated from the standard curve using a 4-parameter logistic fit with BioPlex Manager software version 6.1. The data are graphed in Figure 5. See Figure 5A (serum TTR), Figure 5B (editing in the liver), and Figure 5C (cytokine pMCP1).

[0227] A rat-specific ELISA kit (Aviva Systems Biology, catalog number OKIA00159) was used according to the manufacturer's protocol to measure rat serum TTR levels. Briefly, serum was serially diluted with the kit's sample diluent to a final dilution of 10,000-fold. This diluted sample was then added to an ELISA plate, and the assay was performed according to the 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 serum TTR knockdown and editing in the liver were sufficient in each formulation with 2 mol% and 3 mol% PEG. Figure 5C shows that the MCP-1 response was decreased when the formulation with 3 mol% PEG was used.

[0230] Example 5 - LNP Delivery to Non-Human Primates Three tests were conducted using the LNP formulation prepared as described in Example 1. The specific molar amounts and cargos are listed in Tables 12 to 26. Each formulation containing Cas9 mRNA and guide RNA (gRNA) had an mRNA:gRNA ratio of 1:1 by weight. The LNP dosage (unit: mg / kg, total RNA content), administration route, and the presence or absence of pretreatment with dexamethasone in the animals are shown in the table. For animals pretreated with dexamethasone (Dex), Dex was administered by IV bolus injection at 2 mg / kg 1 hour before the administration of LNP or vehicle.

[0231] For blood chemical analysis, blood was collected from the animals at the times shown in the following table 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 the peripheral vein of the restrained awake animal into a tube containing 4 mL of serum separator. The blood was allowed to clot at room temperature for at least 30 minutes and then centrifuged at 2000 x g for 15 minutes. The serum was dispensed 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 manufactured by Meso Scale Discovery (MSD). Focusing on IL-6 and MCP-1, the following parameters, namely INF-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, MCP-1 and TNF-α, were included in the analysis. The kit reagents and standards were prepared as instructed in the manufacturer's operating procedure. NHP serum was used as is. The plate was read using an MSD Sector Imager 6000 and the analysis was performed using 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 the peripheral vein of the restrained awake animal into a tube containing 0.5 mL of 2 EDTA. The blood was centrifuged at 2000 x g for 15 minutes. The plasma was dispensed into two 120 μL polypropylene microtubes and stored at -60 to -86 °C until analysis. Analysis was performed using a Quidel MicroVue Complement Plus EIA kit (C3a - catalog number A031) or (Bb - catalog number A027). The kit reagents and standards were prepared as instructed in the manufacturer's operating procedure. The optical density was set to 450 nm and the plate was read using an MSD Sector Imager 6000. The results were analyzed using 4-parameter curve fitting.

[0233] Data on cytokine induction and complement activation are presented in the following table. "BLQ" means below the limit of quantification.

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

[0234] Example 6 - PEG Lipid Screening In another test, alternative PEG lipids were compared in LNP formulations containing 2 mol% or 3 mol% of PEG lipid.

[0235] Three PEG lipids were used in the test, and lipid 1 (DMG - PEG2k; Nof) is represented as follows.

Chem.

[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,

Chem.

[0240] to

[0244] ) and WO2011 / 076807, can be represented as follows.

Chem.

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

Table 27

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

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

[0240] In Figures 6A and 6B, serum TTR levels are compared between PEG lipid formulations. Figure 6A shows serum TTR in μg / mL units, and Figure 6B shows data as the percentage of knockdown (TSS%). Figure 6C shows the editing rate achieved in the liver. The data indicate that LNP compositions containing each of the test PEG lipids show efficacy at 2 mol% and 3 mol%, and lipid 1 consistently performs better than lipid 2 and lipid 3.

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

[0242] Synthesis: Lipid A is made by reacting 4,4-bis(octyloxy)butanoic acid (“Intermediate 13b” in Example 13 of WO2015 / 095340) with (9Z,12Z)-3-hydroxy-2-(hydroxymethyl)propyl octadeca-9,12-dienoate (“Intermediate 13c”), and then reacting the product of Intermediate 13b and Intermediate 13c with 3-diethylamino-1-propanol to add a head group. (See pages 84-86 of WO2015 / 095340).

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

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

[0245] Pyridinium p-toluenesulfonate (748 mg, 3.0 mmol) was added to a mixture of 4,4-diethoxybutanenitrile (9.4 g, 60 mmol) and octan-1-ol (23.1 g, 178 mmol) at room temperature. The mixture was warmed to 105 °C, the reaction vessel was opened, and the mixture was stirred for 18 hours without attaching a reflux condenser while being exposed to air. Then, the reaction mixture was cooled to room temperature and purified with silica gel (gradient of hexane containing ethyl acetate 0-5%) to obtain 10.1 g (31.0 mmol) of Intermediate 13a as a clear oil. 1 H NMR (400 MHz, CDCl 3) 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, 31 mL of aqueous potassium hydroxide (2.5 M, 30.9 mL, 77.3 mmol) was added to an ethanol solution (30 mL) of intermediate 13a (8.42 g, 31 mmol) at room temperature. When a reflux condenser was attached to the reaction vessel, the mixture was heated to 110 °C and stirred for 24 hours. Thereafter, the mixture was cooled to room temperature, acidified with aqueous hydrochloric acid (1 N) to pH 5, and extracted three times into hexane. The combined organic extracts were washed with water (twice) and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain 8.15 g (23.6 mmol) of intermediate 13b as a clear oil, which was used without further purification. 1 H NMR (400 MHz, CDCl 3 ) 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 (the following structure).

[0247] Intermediate 13b

Chemical Structure

[0248] Using the above method, C (carbon number 5, 6, 7, 9, and 10)-acetal acidic intermediates (referred to as intermediates B3 to F3 and shown below) were prepared using appropriate alkane-1-ol reagents.

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

Chemical formula

[0250] 1 H 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-bis(hexyloxy)butanoic acid

Chemical formula

[0252] 1 H NMR (400 MHz, CDCl 3 ) 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-bis(heptyloxy)butanoic acid

Chemical formula

[0254] 1 H NMR (400 MHz, CDCl 3 ) 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-Bis(nonyloxy)butanoic acid

Chem.

[0256] 1 H NMR (400 MHz, CDCl 3 ) 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-Bis(decyloxy)butanoic acid

Chem.

[0258] 1 H NMR (400 MHz, CDCl 3) 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 analog of lipid A (C(8)) was synthesized by reacting a C(5, 6, 7, 9, or 10)-acetal acidic intermediate (B3 - F3) with intermediate 13c and then reacting the product of that step with 3-diethylamino-1-propanol. (See pages 84 - 86 of WO2015 / 095340). Each analog was synthesized and 1 characterized by HNMR (data not shown).

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

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

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

Table 28

[0263] 6-(p-Toluidino)-6-naphthalenesulfonic acid ("TNS") dissolved in water was used to evaluate the analogs for pKa. In this assay, 0.1 M phosphate buffer was prepared at different pH values in the range of 4.5 - 10.5. Each analog was individually prepared in 100% ethanol. Lipids and TNS were then added to the individual pH buffers and transferred to plates and analyzed at wavelengths of 321 - 488 nm using a SpectraMax microplate reader. Values were plotted to generate the pKa. The logarithm of the IC 50 was used as the pKa.

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

[0265] Example 8 - Dose - response curve - Primary Cyno hepatocytes Primary hepatocytes of the liver. Primary cynomolgus monkey liver hepatocytes (PCH) (Gibco) were thawed and resuspended in hepatocyte thawing medium containing additives (Gibco, catalog CM7000), and then centrifuged at 80 g for 4 minutes. The supernatant was discarded, and the pelleted cells were resuspended in hepatocyte seeding medium pack containing additives (Invitrogen, catalog A1217601 and CM3000). The cells were counted and seeded at a density of 50,000 cells / well in a Bio - coat collagen I - coated 96 - well plate (ThermoFisher, catalog 877272). The seeded cells were allowed to settle and placed in a tissue culture incubator (37 °C, 5% CO 2 atmosphere) for 24 hours to adhere, and then the LNP was administered. After incubation, the cells were checked for monolayer formation and the medium was replaced with hepatocyte medium and a serum - free additive pack (Invitrogen, catalog 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 cynomolgus monkey primary hepatocytes, and a dose-response curve was generated. After seeding and culturing for 24 hours, the LNP was incubated with hepatocyte maintenance medium containing 6% cynomolgus monkey serum at 37 °C for 5 minutes. After incubation, the LNP was added to cynomolgus monkey primary hepatocytes starting with 100 ng of mRNA with an 8.2-fold dose-response curve. Cells were lysed 72 hours after treatment, and NGS analysis was performed as described in Example 1. Data determining the editing rate for various LNP compositions are graphed in Figure 8A. The editing rates (%) with Cas9 mRNA (SEQ ID NO: 48) and U-removed Cas9 mRNA (SEQ ID NO: 43) are described in Figure 8B. The LNP compositions are described in Table 2 (LNP897) and Table 5 (LNP1021, LNP1022, LNP1023, LNP1024, and LNP1025).

[0268] The results show a quantitative assessment of comparative efficacy, indicating that both the mRNA and the LNP composition affect efficacy.

[0269] Example 9 - RNA Cargo: mRNA and gRNA Cocktails In this study, the in vivo efficacy in mice was evaluated when the ratio of gRNA to mRNA was different. CleanCap™-capped Cas9 mRNA having the ORF of SEQ ID NO: 4, HSD 5'UTR, human albumin 3'UTR, Kozak sequence, and polyA tail was prepared by IVT synthesis using N1-methylpseudouridine triphosphate instead of uridine triphosphate as shown in Example 1.

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

Table 29

[0271] For in vivo characterization, the above LNPs were administered to mice at a total RNA (guide RNA (mg) + mRNA (mg)) of 0.1 mg per kg (n = 5 / group). On days 7 to 9 after administration, the animals were sacrificed and blood and liver were collected, and serum TTR and editing in the liver were measured as described in Example 1. The results of serum TTR and liver editing are shown in FIGS. 9A and 9B. The negative control mice were administered the TSS vehicle.

[0272] Furthermore, the above LNPs were administered to mice, and at that time, the mRNA was set at a fixed dose of 0.05 mg of mRNA per kg (n = 5 / group), and the dose of gRNA was changed from 0.06 mg per kg to 0.4 mg per kg. On days 7 to 9 after administration, the animals were sacrificed and blood and liver were collected, and serum TTR and editing in the liver were measured. The results of serum TTR and liver editing are shown in FIGS. 9C and 9D. The negative control mice were administered the TSS vehicle.

[0273] Example 10-Neutral Lipid To evaluate the in vivo efficacy of the LNP, an LNP formulation was prepared as described in Example 2 using the mRNA of 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, an RNA cargo was prepared in a buffer of 25 mM citrate and 100 mM NaCl (pH 5.0), and the concentration of the RNA cargo was adjusted to approximately 0.45 mg / mL. The LNP was formulated with a molar ratio of lipid amine to RNA phosphate (N:P) of approximately 6 and a ratio of mRNA to gRNA of 1:2 by weight.

[0274] The LNP formulations were analyzed for average particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency as described in Example 1. The analysis of average particle size, polydispersity (PDI), total RNA content, and RNA encapsulation efficiency is shown in Table 30. The molar ratio of lipids is described 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

[0275] For in vivo characterization, female Sprague Dawley rats were intravenously administered the above LNP with 0.3 mg total RNA (guide RNA and mRNA) per kg body weight. The rats were 5 per group. On day 7 after administration, the animals were sacrificed and blood and liver were collected, and serum TTR and editing in the liver were measured as described in Example 1. The TSS vehicle was administered to negative control animals. The results of serum TTR and editing in the liver are shown in Figures 10A and 10B, and Table 30 (above).

Table 31

[0276] For the array itself, refer to the following Sequence Listing. In general, the transcript array contains GGG as the first 3 nucleotides for use with ARCA, or AGG as the first 3 nucleotides for use with CleanCap™. Thus, the first 3 nucleotides can be modified for use with other capping methods, such as the vaccinia capping enzyme. The promoter and polyA sequences are not included in the transcript array. 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 array at the 5' and 3' ends, respectively. Most nucleotide sequences are provided as DNA, but they can be easily converted to RNA by changing T to U.

[0277] Sequence Listing The following Sequence Listing provides a list of the sequences disclosed herein. It is understood that when a DNA sequence (including T) is referred to in relation to RNA, T must be replaced by U (either modified or unmodified depending on the situation), and vice versa. [Table 32] TIFF2025084955000070.tif255165TIFF2025084955000071.tif255165TIFF2025084955000072.tif253163TIFF2025084955000073.tif254167TIFF2025084955000074.tif252165TIFF2025084955000075.tif255165TIFF2025084955000076.tif251166TIFF2025084955000077.tif255166TIFF2025084955000078.tif252170TIFF2025084955000079.tif255166TIFF2025084955000080.tif255167TIFF2025084955000081.tif253164TIFF2025084955000082.tif254164TIFF2025084955000083.tif253166TIFF2025084955000084.tif253166TIFF2025084955000085.tif252166TIFF2025084955000086.tif252168TIFF2025084955000087.tif255166TIFF2025084955000088.tif254164TIFF2025084955000089.tif254164TIFF2025084955000090.tif255165TIFF2025084955000091.tif254166TIFF2025084955000092.tif253167TIFF2025084955000093.tif255164TIFF2025084955000094.tif255167TIFF2025084955000095.tif255165TIFF2025084955000096.tif253164TIFF2025084955000097.tif251165TIFF2025084955000098.tif253165TIFF2025084955000099.tif255164TIFF2025084955000100.tif254166TIFF2025084955000101.tif254166TIFF2025084955000102.tif254168TIFF2025084955000103.tif253167TIFF2025084955000104.tif251166TIFF2025084955000105.tif254166TIFF2025084955000106.tif254167TIFF2025084955000107.tif251166TIFF2025084955000108.tif253164TIFF2025084955000109.tif253166TIFF2025084955000110.tif254165TIFF2025084955000111.tif253168TIFF2025084955000112.tif253166TIFF2025084955000113.tif250166TIFF2025084955000114.tif252167TIFF2025084955000115.tif251166TIFF2025084955000116.tif253167TIFF2025084955000117.tif253167TIFF2025084955000118.tif251165TIFF2025084955000119.tif254166TIFF2025084955000120.tif253166TIFF2025084955000121.tif253164TIFF2025084955000122.tif254166TIFF2025084955000123.tif253164TIFF2025084955000124.tif253164TIFF2025084955000125.tif253166TIFF2025084955000126.tif255165TIFF2025084955000127.tif253166TIFF2025084955000128.tif254167TIFF2025084955000129.tif254165TIFF2025084955000130.tif253165TIFF2025084955000131.tif255166TIFF2025084955000132.tif255166TIFF2025084955000133.tif253166TIFF2025084955000134.tif253163TIFF2025084955000135.tif252166TIFF2025084955000136.tif253165TIFF2025084955000137.tif253165TIFF2025084955000138.tif251166TIFF2025084955000139.tif254166TIFF2025084955000140.tif254166TIFF2025084955000141.tif255166TIFF2025084955000142.tif253164TIFF2025084955000143.tif254166TIFF2025084955000144.tif253163TIFF2025084955000145.tif254165TIFF2025084955000146.tif253165TIFF2025084955000147.tif254166TIFF2025084955000148.tif254164TIFF2025084955000149.tif253165TIFF2025084955000150.tif253167TIFF2025084955000151.tif253165TIFF2025084955000152.tif255167TIFF2025084955000153.tif252163TIFF2025084955000154.tif253164TIFF2025084955000155.tif254166TIFF2025084955000156.tif253165TIFF2025084955000157.tif254164TIFF2025084955000158.tif253167TIFF2025084955000159.tif253164TIFF2025084955000160.tif254165TIFF2025084955000161.tif255164TIFF2025084955000162.tif254166TIFF2025084955000163.tif254166TIFF2025084955000164.tif253166TIFF2025084955000165.tif253168TIFF2025084955000166.tif253166TIFF2025084955000167.tif254167TIFF2025084955000168.tif254166TIFF2025084955000169.tif254166TIFF2025084955000170.tif253165TIFF2025084955000171.tif254166TIFF2025084955000172.tif253166TIFF2025084955000173.tif253165TIFF2025084955000174.tif254166TIFF2025084955000175.tif253167TIFF2025084955000176.tif253165TIFF2025084955000177.tif253166TIFF2025084955000178.tif253167TIFF2025084955000179.tif252166TIFF2025084955000180.tif253168TIFF2025084955000181.tif253165TIFF2025084955000182.tif253166TIFF2025084955000183.tif255168TIFF2025084955000184.tif254166TIFF2025084955000185.tif254166TIFF2025084955000186.tif253165TIFF2025084955000187.tif253163TIFF2025084955000188.tif252166TIFF2025084955000189.tif252165TIFF2025084955000190.tif254164TIFF2025084955000191.tif253164TIFF2025084955000192.tif254166TIFF2025084955000193.tif253166TIFF2025084955000194.tif254165TIFF2025084955000195.tif254166TIFF2025084955000196.tif253164TIFF2025084955000197.tif253168TIFF2025084955000198.tif253165TIFF2025084955000199.tif254166TIFF2025084955000200.tif253164TIFF2025084955000201.tif254163TIFF2025084955000202.tif254166TIFF2025084955000203.tif255165TIFF2025084955000204.tif254164TIFF2025084955000205.tif253164TIFF2025084955000206.tif254165TIFF2025084955000207.tif251163TIFF2025084955000208.tif254163TIFF2025084955000209.tif253166TIFF2025084955000210.tif254164TIFF2025084955000211.tif252166TIFF2025084955000212.tif254166TIFF2025084955000213.tif253164TIFF2025084955000214.tif253164TIFF2025084955000215.tif253165TIFF2025084955000216.tif253165TIFF2025084955000217.tif253165TIFF2025084955000218.tif255164TIFF2025084955000219.tif254166TIFF2025084955000220.tif253166TIFF2025084955000221.tif254164TIFF2025084955000222.tif254167TIFF2025084955000223.tif253167TIFF2025084955000224.tif253165TIFF2025084955000225.tif253165TIFF2025084955000226.tif253165TIFF2025084955000227.tif254163TIFF2025084955000228.tif254165TIFF2025084955000229.tif253167TIFF2025084955000230.tif253166TIFF2025084955000231.tif254165TIFF2025084955000232.tif254165TIFF2025084955000233.tif251165TIFF2025084955000234.tif253167TIFF2025084955000235.tif254166TIFF2025084955000236.tif254165TIFF2025084955000237.tif253168TIFF2025084955000238.tif252166TIFF2025084955000239.tif253163TIFF2025084955000240.tif253163TIFF2025084955000241.tif254167TIFF2025084955000242.tif255166TIFF2025084955000243.tif255164TIFF2025084955000244.tif253164TIFF2025084955000245.tif253165TIFF2025084955000246.tif254166TIFF2025084955000247.tif253164TIFF2025084955000248.tif255165TIFF2025084955000249.tif254163TIFF2025084955000250.tif134165*=PS combination; "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> PA25-085 <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 gcaacaagac tgaagagaac agcaagaaga agatacacaa gaagaaagaa cagaatctgc 240 tacctgcagg aaatcttcag caacgaaatg gcaaaggtcg acgacagctt cttccacaga 300 ctggaagaaa gcttcctggt cgaagaagac aagaagcacg aaagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 aagctggtcg acagcacaga caaggcagac ctgagactga tctacctggc actggcacac 480 atgatcaagt tcagaggaca cttcctgatc gaaggagacc tgaacccgga caacagcgac 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agactggaaa acctgatcgc acagctgccg ggagaaaaga agaacggact gttcggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag cgacgcaatc 900 cagatcggag accagtacgc agacctgttc ctggcagcaa agaacctgag cgacgcaatc 900 ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct gagcgcaagc 960 ctgctgagcg acatcctgag agtcaacaca gaaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 atgatcaaga gatacgacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag agaaaagatc 1320 gcaatcctga gaagacagga agacttctac ccgttcctga aggacaacag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gaatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaaggt caagtacgtc acagaaggaa tgagaaagcc ggcattcctg 1620 agcggagaac agaagaaggc aatcgtcgac ctgctgttca agacaaacag aaaggtcaca 1680 gtcaagcagc tgaaggaaga ctacttcaag aagatcgaat gcttcgacag cgtcgaaatc 1740 agcggagtcg aagacagatt caacgcaagc ctgggaacat accacgacct gctgaagatc 1800 atcaaggaca aggacttcct ggacaacgaa gaaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaaggt catgaagcag ctgaagagaa gaagatacac aggatgggga 1980 agactgagca gaaagctgat caacggaatc agagacaagc agagcggaaa gacaatcctg 2040 gacttcctga agagcgacgg attcgcaaac agaaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaaggaaga catccagaag gcacaggtca gcggacaggg agacagcctg 2160 cacgaacaca tcgcaaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaaggtcg tcgacgaact ggtcaaggtc atgggaagac acaagccgga aaacatcgtc 2280 atcgaaatgg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 atgaagagaa tcgaagaagg aatcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca gaacggaaga 2460 gacatgtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaaggacgac agcatcgaca acaaggtcct gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacgtc ccgagcgaag aagtcgtcaa gaagatgaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agagaaagtt cgacaacctg 2700 acaaaggcag agagaggagg actgagcgaa ctggacaagg caggattcat caagagacag 2760 ctggtcgaaa caagacagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agagaagtca aggtcatcac actgaagagc 2880 aagctggtca gcgacttcag aaaggacttc cagttctaca aggtcagaga aatcaacaac 2940 taccaccacg cacacgacgc atacctgaac gcagtcgtcg gaacagcact gatcaagaag 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aaggcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg aagagacttc 3240 gcaacagtca gaaaggtcct gagcatgccg caggtcaaca tcgtcaagaa gacagaagtc 3300 cagacaggag gattcagcaa ggaaagcatc ctgccgaaga gaaacagcga caagctgatc 3360 gcaagaaaga aggactggga cccgaagaag tacggaggat tcgacagccc gacagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa aagggaaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac aatcatggaa agaagcagct tcgaaaagaa cccgatcgac 3540 ttcctggaag caaagggata caaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactgga aaacggaaga aagagaatgc tggcaagcgc aggagaactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccggaa gacaacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag caagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat acaacaagca cagagacaag 3900 ccgatcagag aacaggcaga aaacatcatc cacctgttca cactgacaaa cctgggagca 3960 ccggcagcat tcaagtactt cgacacaaca atcgacagaa agagatacac aagcacaaag 4020 gaagtcctgg acgcaacact gatccaccag agcatcacag gactgtacga aacaagaatc 4080 gacctgagcc agctgggagg agacggagga ggaagcccga agaagaagag aaaggtctag 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 ttgggcagtg 60 atcacggatg aatacaaagt gccgtccaag aagttcaagg tcctggggaa caccgataga 120 cacagcatca agaaaaatct catcggagcc ctgctgtttg actccggcga aaccgcagaa 180 gcgacccggc tcaaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag agatcttttc gaacgaaatg gcaaaggtcg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtcg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcaactga caaggccgac ctcagattga tctacttggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaaggcgatc tgaaccctga 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 gaaataacca 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 acagggagga tctgctccgg 1200 aaacagagaa 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 tttcgaggaa 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 gtttcgactc 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 gcaactgttc gcaaagtgct ctctatgccg caagtcaata ttgtgaagaa aaccgaagtg 3300 caaaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaagaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aagggaaaga 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 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> 4 <211> 4140 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 4 auggacaaga aguacagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 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 augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 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 uugggcagug 60 aucacggaug aauacaaagu gccguccaag aaguucaagg uccuggggaa caccgauaga 120 cacagcauca agaaaaaucu caucggagcc cugcuguuug acuccggcga aaccgcagaa 180 gcgacccggc ucaaacguac cgcgaggcga cgcuacaccc ggcggaagaa ucgcaucugc 240 uaucugcaag agaucuuuuc gaacgaaaug gcaaaggucg acgacagcuu cuuccaccgc 300 cuggaagaau cuuuccuggu ggaggaggac aagaagcaug aacggcaucc uaucuuugga 360 aacaucgucg acgaaguggc guaccacgaa aaguacccga ccaucuacca ucugcggaag 420 aaguugguug acucaacuga caaggccgac cucagauuga ucuacuuggc ccucgcccau 480 augaucaaau uccgcggaca cuuccugauc gaaggcgauc ugaacccuga uaacuccgac 540 guggauaagc uuuucauuca acuggugcag accuacaacc aacuguucga agaaaaccca 600 aucaaugcua gcggcgucga ugccaaggcc auccuguccg cccggcuguc gaagucgcgg 660 cgccucgaaa accugaucgc acagcugccg ggagagaaaa agaacggacu uuucggcaac 720 uugaucgcuc ucucacuggg acucacuccc aauuucaagu ccaauuuuga ccuggccgag 780 gacgcgaagc ugcaacucuc aaaggacacc uacgacgacg acuuggacaa uuugcuggca 840 caaauuggcg aucaguacgc ggaucuguuc cuugccgcua agaaccuuuc ggacgcaauc 900 uugcuguccg auauccugcg cgugaacacc gaaauaacca aagcgccgcu uagcgccucg 960 augauuaagc gguacgacga gcaucaccag gaucucacgc ugcucaaagc gcucgugaga 1020 cagcaacugc cugaaaagua caaggagauc uucuucgacc aguccaagaa uggguacgca 1080 ggguacaucg auggaggcgc uagccaggaa gaguucuaua aguucaucaa gccaauccug 1140 gaaaagaugg acggaaccga agaacugcug gucaagcuga acagggagga ucugcuccgg 1200 aaacagagaa ccuuugacaa cggauccauu ccccaccaga uccaucuggg ugagcugcac 1260 gccaucuugc ggcgccagga ggacuuuuac ccauuccuca aggacaaccg ggaaaagauc 1320 gagaaaauuc ugacguuccg caucccguau uacgugggcc cacuggcgcg cggcaauucg 1380 cgcuucgcgu ggaugacuag aaaaucagag gaaaccauca cuccuuggaa uuucgaggaa 1440 guuguggaua agggagcuuc ggcacaaagc uucaucgaac gaaugaccaa cuucgacaag 1500 aaucucccaa acgagaaggu gcuuccuaag cacagccucc uuuacgaaua cuucacuguc 1560 uacaacgaac ugacuaaagu gaaauacguu acugaaggaa ugaggaagcc ggccuuucug 1620 uccggagaac agaagaaagc aauugucgau cugcuguuca agaccaaccg caaggugacc 1680 gucaagcagc uuaaagagga cuacuucaag aagaucgagu guuucgacuc aguggaaauc 1740 agcggggugg aggacagauu caacgcuucg cugggaaccu aucaugaucu ccugaagauc 1800 aucaaggaca aggacuuccu ugacaacgag gagaacgagg acauccugga agauaucguc 1860 cugaccuuga cccuuuucga ggaucgcgag augaucgagg agaggcuuaa gaccuacgcu 1920 caucucuucg acgauaaggu caugaaacaa cucaagcgcc gccgguacac ugguuggggc 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 gacaagaaca gagggaaguc agauaaugug ccaucggagg aggucgugaa gaagaugaag 2640 aauuacuggc ggcagcuccu gaaugcgaag cugauuaccc agagaaaguu ugacaaucuc 2700 acuaaagccg agcgcggcgg acucucagag cuggauaagg cuggauucau caaacggcag 2760 cuggucgaga cucggcagau uaccaagcac guggcgcaga ucuuggacuc ccgcaugaac 2820 acuaaauacg acgagaacga uaagcucauc cgggaaguga aggugauuac ccugaaaagc 2880 aaacuugugu cggacuuucg gaaggacuuu caguuuuaca aagugagaga aaucaacaac 2940 uaccaucacg cgcaugacgc auaccucaac gcuguggucg guaccgcccu gaucaaaaag 3000 uacccuaaac uugaaucgga guuuguguac ggagacuaca aggucuacga cgugaggaag 3060 augauagcca aguccgaaca ggaaaucggg aaagcaacug cgaaauacuu cuuuuacuca 3120 aacaucauga acuuuuucaa gacugaaauu acgcuggcca auggagaaau caggaagagg 3180 ccacugaucg aaacuaacgg agaaacgggc gaaaucgugu gggacaaggg cagggacuuc 3240 gcaacuguuc gcaaagugcu cucuaugccg caagucaaua uugugaagaa aaccgaagug 3300 caaaccggcg gauuuucaaa ggaaucgauc cucccaaaga gaaauagcga caagcucauu 3360 gcacgcaaga aagacuggga cccgaagaag uacggaggau ucgauucgcc gacugucgca 3420 uacuccgucc ucgugguggc caagguggag aagggaaaga gcaaaaagcu caaauccguc 3480 aaagagcugc uggggauuac caucauggaa cgauccucgu ucgagaagaa cccgauugau 3540 uuccucgagg cgaaggguua caaggaggug aagaaggauc ugaucaucaa acuccccaag 3600 uacucacugu ucgaacugga aaauggucgg aagcgcaugc uggcuucggc cggagaacuc 3660 caaaaaggaa augagcuggc cuugccuagc aaguacguca acuuccucua ucuugcuucg 3720 cacuacgaaa aacucaaagg gucaccggaa gauaacgaac agaagcagcu uuucguggag 3780 cagcacaagc auuaucugga ugaaaucauc gaacaaaucu ccgaguuuuc aaagcgcgug 3840 auccucgccg acgccaaccu cgacaaaguc cugucggccu acaauaagca uagagauaag 3900 ccgaucagag aacaggccga gaacauuauc cacuuguuca cccugacuaa ccugggagcc 3960 ccagccgccu ucaaguacuu cgauacuacu aucgaucgca aaagauacac guccaccaag 4020 gaaguucugg acgcgacccu gauccaccaa agcaucacug gacucuacga aacuaggauc 4080 gaucugucgc agcugggugg cgauggcggu ggaucuccga aaaagaagag aaagguguaa 4140 uga 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 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 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 auggacaaga aguacagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 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 augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgaccac 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca 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 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 Methionine, Isoleucine, Lysine, Arginine, Tyrosine, Aspartic Acid, Glutamic Acid, Histidine, Histidine, Glutamine, Aspartic Acid, Leucine, Threonine, Leucine, Leucine, Lysine 325 330 335 Alanine, Leucine, Valine, Arginine, Glutamine, Glutamine, Leucine, Proline, Glutamic Acid, Lysine, Tyrosine, Lysine, Glutamic Acid, Isoleucine, Phenylalanine, Phenylalanine 340 345 350 Aspartic Acid, Glutamine, Serine, Lysine, Asparagine, Glycine, Tyrosine, Alanine, Glycine, Tyrosine, Isoleucine, Aspartic Acid, Glycine, Glycine, Alanine, Serine 355 360 365 Glutamine, Glutamic Acid, Glutamic Acid, Phenylalanine, Tyrosine, Lysine, Phenylalanine, Isoleucine, Lysine, Proline, Isoleucine, Leucine, Glutamic Acid, Lysine, Methionine, Aspartic Acid 370 375 380 Glycine, Threonine, Glutamic Acid, Glutamic Acid, Leucine, Leucine, Valine, Lysine, Leucine, Asparagine, Arginine, Glutamic Acid, Aspartic Acid, Leucine, Leucine, Arginine 385 390 395 400 Lysine, Glutamine, Arginine, Threonine, Phenylalanine, Aspartic Acid, Asparagine, Glycine, Serine, Isoleucine, Proline, Histidine, Glutamine, Isoleucine, Histidine, Leucine 405 410 415 Glycine, Glutamic Acid, Leucine, Histidine, Alanine, Isoleucine, Leucine, Arginine, Arginine, Glutamine, Glutamic Acid, Aspartic Acid, Phenylalanine, Tyrosine, Proline, Phenylalanine 420 425 430 Leucine, Lysine, Aspartic Acid, Asparagine, Arginine, Glutamic Acid, Lysine, Isoleucine, Glutamic Acid, Lysine, Isoleucine, Leucine, Threonine, Phenylalanine, Arginine, Isoleucine 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 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 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> 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 aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 cacagcauca agaagaaccu gaucggagca cugcuguucg acagcggaga aacagcagaa 180 gcaacaagac ugaagagaac agcaagaaga agauacacaa gaagaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccacaga 300 cuggaagaaa gcuuccuggu cgaagaagac aagaagcacg aaagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccacgaa aaguacccga caaucuacca ccugagaaag 420 aagcuggucg acagcacaga caaggcagac cugagacuga ucuaccuggc acuggcacac 480 augaucaagu ucagaggaca cuuccugauc gaaggagacc ugaacccgga caacagcgac 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caagacugag caagagcaga 660 agacuggaaa accugaucgc acagcugccg ggagaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccuggg acugacaccg aacuucaaga gcaacuucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 cagaucggag accaguacgc agaccuguuc cuggcagcaa agaaccugag cgacgcaauc 900 cugcugagcg acauccugag agucaacaca gaaaucacaa aggcaccgcu gagcgcaagc 960 augaucaaga gauacgacga acaccaccag gaccugacac ugcugaaggc acuggucaga 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cggauacgca 1080 ggauacaucg acggaggagc aagccaggaa gaauucuaca aguucaucaa gccgauccug 1140 gaaaagaugg acggaacaga agaacugcug gucaagcuga acagagaaga ccugcugaga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 gcaauccuga gaagacagga agacuucuac ccguuccuga aggacaacag agaaaagauc 1320 gaaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaaacaauca caccguggaa cuucgaagaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaaugacaaa cuucgacaag 1500 aaccugccga acgaaaaggu ccugccgaag cacagccugc uguacgaaua cuucacaguc 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 augggaagac acaagccgga aaacaucguc 2280 aucgaaaugg caagagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacggaaga 2460 gacauguacg ucgaccagga acuggacauc aacagacuga gcgacuacga cgucgacgca 2520 aucgucccgc agagcuuccu gaaggacgac agcaucgaca acaagguccu gacaagaagc 2580 gacaagaaca gaggaaagag cgacaacguc ccgagcgaag aagucgucaa gaagaugaag 2640 aacuacugga gacagcugcu gaacgcaaag cugaucacac agagaaaguu cgacaaccug 2700 acaaaggcag agagaggagg acugagcgaa cuggacaagg caggauucau caagagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga uccuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagaaguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc caguucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac gcagucgucg gaacagcacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aaggcaacag caaaguacuu cuucuacagc 3120 aacaucauga acuucuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacagga gaaaucgucu gggacaaggg aagagacuuc 3240 gcaacaguca gaaagguccu gagcaugccg caggucaaca ucgucaagaa gacagaaguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaaacagcga caagcugauc 3360 gcaagaaaga aggacuggga cccgaagaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga gcaagaagcu gaagagcguc 3480 aaggaacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaagggaua caaggaaguc aagaaggacc ugaucaucaa gcugccgaag 3600 uacagccugu ucgaacugga aaacggaaga aagagaaugc uggcaagcgc aggagaacug 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagcccggaa gacaacgaac agaagcagcu guucgucgaa 3780 cagcacaagc acuaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacggagga ggaagcccga agaagaagag aaaggucuag 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 aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 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 gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> 11 <211> 4134 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 11 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 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 gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgaccacauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> 12 <211> 4134 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 12 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagaagaaga uacacaagaa gaaagaacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaagaaagcu uccuggucga agaagacaag aagcacgaaa gacacccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaaagaag 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 gcugccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 gcaaagcugc agcugagcaa ggacacauac gacgacgacc uggacaaccu gcuggcacag 840 aucggagacc aguacgcaga ccuguuccug gcagcaaaga accugagcga cgcaauccug 900 cugagcgaca uccugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaagaa uucuacaagu ucaucaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggaaga cuucuacccg uuccugaagg acaacagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucacac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaaacuu cgacaagaac 1500 cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga caaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc 1680 aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaagaucauc 1800 aaggacaagg acuuccugga caacgaagaa aacgaagaca uccuggaaga caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggaagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gaacacaucg caaaccuggc aggaagcccg gcaaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggaagacaca agccggaaaa caucgucauc 2280 gaaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga uccugaagga acacccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcagaa cggaagagac 2460 auguacgucg accaggaacu ggacaucaac agacugagcg acuacgacgu cgacgcaauc 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaaagagcga caacgucccg agcgaagaag ucgucaagaa gaugaagaac 2640 uacuggagac agcugcugaa cgcaaagcug aucacacaga gaaaguucga caaccugaca 2700 aaggcagaga gaggaggacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcugaucaga gaagucaagg ucaucacacu gaagagcaag 2880 cuggucagcg acuucagaaa ggacuuccag uucuacaagg ucagagaaau caacaacuac 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa cagcacugau caagaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag gcaacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa gcugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaagcaa agggauacaa ggaagucaag aaggaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggaagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag cccggaagac aacgaacaga agcagcuguu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg aauucagcaa gagagucauc 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 guccuggacg caacacugau ccaccagagc aucacaggac uguacgaaac aagaaucgac 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 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 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 ...

Claims

1. An RNA component; and a lipid component, wherein the lipid component comprises: About 50-60 mole % amine lipid; About 8-10 mol % neutral lipids; About 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.

2. An RNA component; About 50-60 mole % amine lipid; About 27-39.5 mol % of a helper lipid; About 8-10 mol % neutral lipids; About 2.5-4 mol% PEG lipid; wherein the N / P ratio of the LNP composition is about 5-7.

3. 3. The LNP composition of claim 2, wherein the N / P ratio is about 6.

4. An RNA component; and a lipid component, wherein the lipid component comprises: About 50-60 mole % amine lipid; About 5-15 mol % neutral lipids; About 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.

5. An RNA component; and a lipid component, wherein the lipid component comprises: About 40-60 mole % amine lipid; About 5-15 mol % neutral lipids; About 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.

6. An RNA component; and a lipid component, wherein the lipid component comprises: About 50-60 mole % amine lipid; About 5-15 mol % neutral lipids; About 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.

7. An RNA component; and a lipid component, wherein the lipid component comprises: About 40-60 mole % amine lipid; About 0-10 mol % neutral lipids; About 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.

8. An RNA component; and a lipid component, wherein the lipid component comprises: About 40-60 mole % amine lipid; less than about 1 mol % neutral lipids; About 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.

9. An RNA component; and a lipid component, wherein the lipid component comprises: About 40-60 mole % amine lipid; About 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 to 10; and The LNP composition, wherein the LNP composition is essentially free of or free of neutral phospholipids.

10. An RNA component; and a lipid component, wherein the lipid component comprises: About 50-60 mole % amine lipid; About 8-10 mol % neutral lipids; About 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.

11. 2. A composition according to any preceding claim, wherein the RNA component comprises mRNA.

12. 2. A composition according to any preceding claim, wherein the RNA component comprises an RNA-guided DNA binding factor, such as a Cas nuclease mRNA.

13. 2. The composition of any preceding claim, wherein the RNA component comprises a class 2 Cas nuclease mRNA.

14. 2. The composition of any preceding claim, wherein the RNA component comprises a Cas9 nuclease mRNA.

15. The composition of any one of claims 11 to 14, wherein the mRNA is a modified mRNA.

16. The composition of any preceding claim, 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.

17. The composition of any preceding claim, 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.

18. 10. The composition of any preceding claim, 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.

19. 2. The composition of any preceding claim, wherein the RNA component comprises a gRNA nucleic acid.

20. 20. The composition of claim 19, wherein the gRNA nucleic acid is a gRNA.

21. 2. The composition of any preceding claim, wherein the RNA component comprises a class 2 Cas nuclease mRNA and a gRNA.

22. The composition of any of claims 19 to 21, wherein the gRNA nucleic acid is or encodes a dual guide RNA (dgRNA).

23. The composition of any of claims 19 to 21, wherein the gRNA nucleic acid is or encodes an sgRNA.

24. The composition of any one of claims 19 to 23, wherein the gRNA is modified.

25. 25. The composition of claim 24, wherein the gRNA comprises a modification selected from 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) internucleotide linkages, and 2'-fluoro (2'-F) modified nucleotides.

26. The composition of any of claims 24 to 25, wherein the gRNA comprises a modification in one or more of the first five nucleotides at the 5' end.

27. The composition of any one of claims 24 to 26, wherein the gRNA comprises a modification in one or more of the last five nucleotides at the 3' end.

28. The composition of any one of claims 24 to 27, wherein the gRNA comprises a PS bond within the first four nucleotides.

29. The composition of any one of claims 24 to 28, wherein the gRNA comprises a PS bond between the last four nucleotides.

30. The composition of any one of claims 24 to 29, further comprising 2'-O-Me modified nucleotides within the first three nucleotides of the 5' end.

31. The composition of any one of claims 24 to 30, further comprising 2'-O-Me modified nucleotides at the last three nucleotides of the 3' end.

32. The composition of any one of claims 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.

33. The composition of any one of claims 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.

34. The composition of any one of claims 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.

35. The composition of any one of claims 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.

36. The composition of any one of claims 19 to 35, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio of about 2:1 by weight.

37. The composition of any one of claims 19 to 35, wherein the gRNA and class 2 Cas nuclease mRNA are present in a ratio of about 1:1 by weight.

38. 20. A composition according to any preceding claim, further comprising at least one template.

39. 2. The composition of any preceding claim, wherein the mole percent of PEG lipid is about 3.

40. 2. The composition of any preceding claim, wherein the mole percent of the amine lipid is about 50.

41. 2. The composition of any preceding claim, wherein the mole percent of the amine lipid is about 55.

42. 2. The composition of any preceding claim, wherein the mole percentage of the amine lipid is ±3 mole percent.

43. 2. The composition of any preceding claim, wherein the mole percentage of the amine lipid is ±2 mole percent.

44. 10. The composition of any preceding claim, wherein the mole percentage of the amine lipid is 47-53 mole percent.

45. 10. The composition of any preceding claim, wherein the mole percentage of the amine lipid is 48-53 mole percent.

46. 10. A composition according to any preceding claim, wherein the mole percentage of the amine lipid is from 53 to 57 mole percent.

47. 10. A composition according to any preceding claim, wherein the N / P ratio is 6±1.

48. 10. A composition according to any preceding claim, wherein the N / P ratio is 6±0.

5.

49. 2. A composition according to any preceding claim, wherein the amine lipid is lipid A.

50. 2. A composition according to any preceding claim, wherein the amine lipid is an analogue of lipid A.

51. 51. The composition of claim 50, wherein the analog is an acetal analog.

52. 52. The composition of claim 51, wherein the acetal analog is a C4-C12 acetal analog.

53. 51. The composition of claim 50, wherein the acetal analog is a C5-C12 acetal analog.

54. 51. The composition of claim 50, wherein the acetal analog is a C5-C10 acetal analog.

55. 51. The composition of claim 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.

56. 2. A composition according to any preceding claim, wherein the helper lipid is cholesterol.

57. 2. The composition of any preceding claim, wherein the neutral lipid is DSPC.

58. 2. A composition according to any preceding claim, wherein the neutral lipid is DPPC.

59. 2. The composition of any preceding claim, wherein the PEG lipid comprises dimyristoylglycerol (DMG).

60. 2. A composition according to any preceding claim, wherein the PEG lipid comprises PEG-2k.

61. 2. A composition according to any preceding claim, wherein the PEG lipid is PEG-DMG.

62. 62. The composition of claim 61, wherein the PEG-DMG is PEG2k-DMG.

63. The composition of claim 9 , wherein the LNP composition is essentially free of neutral lipids.

64. 64. The composition of claim 63, wherein the neutral lipid is a phospholipid.

65. A gene editing method comprising contacting a cell with an LNP composition according to any one of claims 12 to 64.

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 according to any one of claims 13 to 64.

67. 65. A method of producing a genetically engineered cell, comprising contacting a cell with at least one LNP composition according to any one of claims 12 to 64.

68. The method of any one of claims 65 to 67, wherein the LNP composition is administered at least twice.

69. 69. The method of claim 68, wherein the LNP composition is administered 2 to 5 times.

70. 70. The method of claim 68 or 69, wherein editing is improved upon re-administration.

71. The method of any one of claims 65 to 70, further comprising introducing at least one template nucleic acid into the cell.

72. The method of any of claims 65-71, wherein the mRNA is formulated in a first LNP composition and the guide RNA nucleic acid is formulated in a second LNP composition.

73. 73. The method of claim 72, wherein the first and second LNP compositions are administered simultaneously.

74. 73. The method of claim 72, wherein the first and second LNP compositions are administered sequentially.

75. The method of any of claims 65 to 73, wherein the mRNA and the guide RNA nucleic acid are formulated into a single LNP composition.

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