In vitro mRNA delivery method using lipid nanoparticles

Abstract

To provide compositions and methods for introducing an mRNA into stem cells, such as HSPCs, and for delivering gene editing components to such cells in vitro.SOLUTION: Provided is a method of delivering an mRNA to a hematopoietic stem and / or progenitor cell (HSPC) or an HSPC population. The method comprises: a. pre-incubating a serum factor with an LNP composition comprising the mRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid; b. contacting the HSPC or the HSPC population with the pre-incubated LNP composition in vitro; and c. culturing the HSPC or the HSPC population in vitro, thereby delivering the mRNA to the HSPC or the HSPC population.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 566,232, filed September 29, 2017, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a method for in vitro delivery of mRNA using lipid nanoparticles. [Background technology]

[0003] There is interest in gene editing and gene therapy approaches in introducing genetic changes into stem cells, including hematopoietic stem cells (HSCs) and their progeny. Stem cells, such as HSCs, have proliferative potential that is lost in mature cells, making lineage-committed progenitor cells particularly useful for gene editing techniques. For example, the ability to modify HSCs and stem cells in vitro is important, and there is a need for methods to deliver biological factors to HSCs and other stem cells in culture. There is a particular need for delivery techniques in culture for human HSCs.

[0004] HSCs are essential for lifelong blood production. They can sustain long-term, functional hematopoiesis because they have the capacity to both differentiate and produce mature progeny of all myeloid and lymphoid blood lineages or to self-renew and replace cells that become progressively lineage-committed. HSCs can be used to restore blood and immune cells in transplant recipients, immunocompromised patients, or other patients. Specifically, autologous or allogeneic transplantation of HSCs can be used to reconstitute hematopoietic lineages and immune system defenses in the treatment of patients with inherited immunodeficiencies and autoimmune diseases, as well as various hematopoietic disorders. Summary of the Invention

[0005] Of particular interest are methods for delivering components of the CRISPR / Cas gene editing system to HSCs in culture. Provided herein are methods for delivering RNA containing CRISPR / Cas system components to hematopoietic cell cultures, such as HSCs. Such methods involve delivering active proteins to stem cells, such as HSCs, cultured in vitro, by contacting the cells with a lipid nanoparticle (LNP) composition that provides mRNA encoding such proteins. Additionally provided are methods for in vitro gene editing in stem cells, such as HSCs, and for generating engineered cells.

[0006] In some embodiments, methods for gene editing in HSCs in vitro and methods for generating engineered HSC cells are provided herein. In further embodiments, methods for delivering mRNA to hematopoietic stem and / or progenitor cells (HSPCs) or HSPC populations are provided herein. In some embodiments, the methods include preincubating serum factors with an LNP composition comprising mRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG-lipid. In some embodiments, the methods further include contacting the HSPCs or HSPC population with the preincubated LNP composition in vitro. In some embodiments, the methods further include culturing the HSPCs or HSPC population in vitro. In some embodiments, the methods result in mRNA delivery to the HSPCs or HSPC population.

[0007] In some embodiments, provided herein are methods for introducing Cas nuclease mRNA and gRNA into stem cells, e.g., HSPCs. In some embodiments, the methods include preincubating serum factors with an LNP composition comprising Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG-lipids. In some embodiments, the methods further include contacting the HSPCs in vitro with the preincubated LNP composition. In some embodiments, the methods further include culturing the HSPCs. In some embodiments, the methods result in the introduction of the Cas nuclease mRNA and gRNA into the HSPCs.

[0008] In some embodiments, provided herein are methods for generating genetically engineered stem cells, e.g., HSPCs, in vitro. In some embodiments, the methods include preincubating serum factors with an LNP composition comprising a Cas nuclease mRNA, a gRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG-lipid. In some embodiments, the methods further include contacting the HSPCs with the preincubated LNP composition in vitro. In some embodiments, the methods further include culturing the HSPCs in vitro. In some embodiments, the methods result in the generation of genetically engineered HSPCs.

[0009] In some embodiments, a method for delivering mRNA to HSPCs or HSPC populations is provided, the method comprising pre-incubating an LNP composition with serum factors, contacting the pre-incubated LNP composition with cells or populations in vitro, and culturing the cells or populations in vitro, thereby delivering mRNA to HSPCs. In some embodiments, the HSPCs are HSCs. In some embodiments, the method delivers mRNA, such as Cas nuclease mRNA, to HSPC populations (e.g., CD34+ cell populations). In certain embodiments, a guide RNA (gRNA), optionally combined with Cas nuclease mRNA, is delivered to the cells. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows the delivery of green fluorescent protein (GFP) mRNA in CD34+ bone marrow cells using LNPs. [Figure 2] We show that mRNA delivery in CD34+ bone marrow cells is dependent on preincubation with serum. [Figure 3] Figures 3A and 3B show B2M editing in CD34+ bone marrow cells following preincubation with serum; Figure 3A shows the percentage of B2M- (protein expression knocked down) cells, and Figure 3B graphs the editing rates achieved in the experiment. [Figure 4] Figures 4A and 4B show efficient delivery after preincubation with serum and ApoE3, with Figure 4A showing the percentage of B2M- cells and Figure 4B showing the editing rate achieved in the experiment. [Figure 5] 1 shows the effect of pre-incubating LNPs with various serum factor preparations on LNP delivery to CD34+ cells. [Figure 6A] Figure 1 shows the viability and compilation data of CD34+ cells exposed to LNP treatment at various intervals. Figure 2 shows the viability of CD34+ cells after 2, 6, and 24 hours of exposure to LNPs. [Figure 6B] Figure 1 shows viability and editing data for CD34+ cells exposed to LNP treatment at various intervals. Editing data are shown for each group at treatment times of 2 hours, 6 hours, and 24 hours. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure provides methods for using LNP compositions of RNA, such as RNA ("cargo") of CRISPR / Cas components, for in vitro delivery to CD34+ cells, e.g., HSC-containing cell populations. The methods may exhibit improved properties compared to conventional delivery techniques, e.g., the methods efficiently deliver RNA while reducing cell death caused by transfection.

[0012] In some embodiments, provided herein are methods for delivering mRNA to stem cells, e.g., HSPCs or HSPC populations. In some embodiments, the methods include preincubating serum factors with an LNP composition comprising mRNA, amine lipids, helper lipids, neutral lipids, and PEG-lipids. In some embodiments, the methods further include contacting the HSPCs or HSPC population with the preincubated LNP composition in vitro. In some embodiments, the methods further include culturing the HSPCs or HSPC population in vitro. In some embodiments, the methods result in mRNA delivery to the HSPCs or HSPC population. In some embodiments, the mRNA encodes a Cas nuclease.

[0013] In some embodiments, provided herein are methods for introducing Cas nuclease mRNA and gRNA into stem cells, e.g., HSPCs or HSPC populations. In some embodiments, the methods include preincubating serum factors with an LNP composition comprising Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG-lipids. In some embodiments, the methods further include contacting the HSPCs with the preincubated LNP composition in vitro. In some embodiments, the methods further include culturing the HSPCs. In some embodiments, the methods result in the introduction of the Cas nuclease mRNA and gRNA into the HSPCs.

[0014] In some embodiments, provided herein are methods for generating genetically engineered stem cells, e.g., HSPCs, in vitro. In some embodiments, the methods include preincubating serum factors with an LNP composition comprising a Cas nuclease mRNA, a gRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG-lipid. In some embodiments, the methods further include contacting the HSPCs with the preincubated LNP composition in vitro. In some embodiments, the methods further include culturing the HSPCs in vitro. In some embodiments, the methods result in the generation of genetically engineered HSPCs.

[0015] In some embodiments, the LNP composition further comprises a gRNA. In some embodiments, the mRNA encodes a Class 2 Cas nuclease. In certain embodiments, the cargo or RNA component comprises a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA. In certain embodiments, the cargo or RNA component comprises a CRISPR / Cas system gRNA or a nucleic acid encoding the gRNA. Methods of gene editing and producing engineered cells are also provided. In vitro methods

[0016] This method delivers RNA to CD34+ cells in vitro. "CD34+ cells" refers to cells that express the CD34 marker on their surface. CD34+ cells can be detected and enumerated, for example, using flow cytometry and a fluorescently labeled anti-human CD34 antibody.

[0017] In some embodiments, a method for delivering mRNA to stem cells, e.g., HSPCs or a population of HSPCs, is provided, the method comprising: (a) preincubating an LNP composition comprising mRNA, amine lipids, helper lipids, neutral lipids, and PEG-lipids with serum factors; (b) contacting the preincubated LNP composition with an HSPC or a population of HSPCs in vitro; and (c) culturing the HSPC or HSPC population in vitro, thereby delivering the mRNA to the HSPCs. In some embodiments, the mRNA encodes a Cas nuclease, such as a Class 2 Cas nuclease. In some aspects, the Class 2 Cas nuclease mRNA is Cas9 mRNA or Cpfl mRNA. In certain embodiments, the Class 2 Cas nuclease is S. pyogenes Cas9. In some embodiments, the LNP composition further comprises a gRNA. In a further embodiment, a method introduces Cas nuclease mRNA and gRNA into HSPCs, the method comprising: (a) pre-incubating an LNP composition comprising Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids with serum factors; (b) contacting the pre-incubated LNP composition with HSPCs in vitro; and (c) culturing the HSPCs, thereby introducing the Cas nuclease and gRNA into the HSPCs.

[0018] In various embodiments, the gRNA of the methods described herein can be a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

[0019] In some embodiments of the in vitro method, LNP transfection can reduce cell death of HSPCs or CD34+ cells compared to known techniques such as electroporation. In some embodiments, LNP transfection can cause less than 5%, less than 10%, less than 20%, less than 30%, or less than 40% cell death. In certain embodiments, cell survival after transfection is at least 60%, 70%, 80%, 90%, or 95%.

[0020] Stem cells are characterized by their ability to self-renew and differentiate into diverse cell types. Mammalian stem cells are broadly divided into two types: embryonic stem (ES) cells and adult stem cells. Adult stem cells or progenitor cells can replenish specialized cells. Most adult stem cells are lineage-restricted and can be referred to by their tissue of origin. ES cell lines are derived from the epiblast tissue of the inner cell mass of blastocyst- or early morula-stage embryos. ES cells are pluripotent and can give rise to cells derived from the three germ layers: ectoderm, endoderm, and mesoderm. Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state by forcing the expression of genes and factors important for maintaining the defining characteristics of embryonic stem cells. A "stem cell" can be, for example, an ESC, iPSC, progenitor cell, or HSPC.

[0021] The terms "hematopoietic stem and / or progenitor cells" and "HSPCs" are used interchangeably and refer to a population of cells that includes both HSCs and hematopoietic progenitor cells ("HPCs"). Such cells are characterized, for example, as CD34+. In an exemplary embodiment, HSPCs are isolated from bone marrow. In another exemplary embodiment, HSPCs are isolated from peripheral blood. In another exemplary embodiment, HSPCs are isolated from umbilical cord blood.

[0022] HSPCs may be derived from bone marrow, peripheral blood, or umbilical cord blood, and may be autologous (a patient's own stem cells) or allogeneic (stem cells obtained from a donor).

[0023] As used herein, the term "hematopoietic progenitor cell" or "HPC" refers to an undifferentiated hematopoietic cell that has limited self-renewal capacity and has multilineage (e.g., myeloid, lymphoid), single-lineage (e.g., myeloid or lymphoid) or cell-type restricted differentiation potential (e.g., erythroid progenitor cells) depending on where it is placed within the hematopoietic hierarchy (Doulatov et al., Cell Stem Cell 2012).

[0024] As used herein, the term "hematopoietic stem cells" or "HSCs" refers to immature blood cells that have the capacity to self-renew and differentiate into more mature blood cells, including granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), and monocytes (e.g., monocytes, macrophages). It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. CD34+ cells are believed to comprise a subpopulation of cells with the stem cell characteristics defined above. Transplantation of cell populations, such as HSPCs, containing pluripotent HSCs can be used to treat leukemia, lymphoma, and other disorders.

[0025] HSCs are pluripotent cells that can give rise to undifferentiated progenitor cells (e.g., multipotent progenitor cells) and / or committed progenitor cells to specific hematopoietic lineages (e.g., lymphoid progenitor cells). Committed stem cells can be stem cells of the T cell lineage, B cell lineage, dendritic cell lineage, Langerhans cell lineage, and / or lymphoid tissue-specific macrophage cell lineage. Furthermore, HSCs also refer to long-term HSCs (LT-HSCs) and short-term HSCs (ST-HSCs). ST-HSCs are more active and proliferative than LT-HSCs. However, while LT-HSCs self-renew indefinitely (i.e., survive throughout adulthood), ST-HSCs have limited self-renewal (i.e., survive only for a limited period of time). Any of these HSCs can be used in any of the methods described herein. Optionally, ST-HSCs are useful because they are highly proliferative, allowing the number of HSCs and their progeny to rapidly increase exponentially.

[0026] HSCs, HPCs, and HSPCs can optionally be obtained from blood products. Blood products include products obtained from the body or bodily organs containing cells of hematopoietic origin. Such sources include bone marrow, umbilical cord, peripheral blood (e.g., mobilized peripheral blood, e.g., mobilized using a mobilizing agent such as G-CSF or Plerixafor® (AMD3100)), liver, thymus, lymph, and spleen. All of the foregoing blood products (e.g., crude, unfractionated, or fractionated) can be enriched for cells with HSC characteristics using methods known to those skilled in the art. Similarly, the foregoing blood products can be enriched for characteristics of HPC and / or HSPC populations. In one embodiment, HSCs are characterized as CD34+ / CD38- / CD90+ / CD45RA-. In one embodiment, HSCs are characterized as CD34+ / CD90+ / CD49f+ cells. In further embodiments, HSCs are characterized as lineage- (negative) CD34+ / CD38- / CD90+ / CD45RA-. In embodiments, HSCs are characterized as lineage- (negative) CD34+ / CD90+ / CD49f+ cells, where "lineage" refers to markers that exclude terminally differentiated cells, e.g., T cells, B cells, etc. These can be removed by staining the cells with antibodies against surface markers expressed by committed hematopoietic cells. These include, but are not limited to, CD3 (T cells), CD19 (B cells), CD33 (myeloid), CD56 (NK cells), CD235a (erythroid cells), and CD71 (erythroid cells).

[0027] "Enriched" when used in reference to a cell population refers to a cell population that has been selected based on the presence of one or more markers, such as CD34+. A cell population, such as a stem cell or HSPC population, refers to eukaryotic mammalian, preferably human, cells that have been isolated from a biological source, such as a blood product or tissue, and that are derived from two or more cells.

[0028] The serum factors may be contacted with the LNP composition during pre-incubation before delivery to HSPC cells in vitro.

[0029] Some embodiments of the in vitro method include pre-incubating the serum factors with the LNP composition for about 30 seconds to overnight. In some embodiments, the pre-incubation step includes pre-incubating the serum factors with the LNP composition for about 1 minute to 1 hour. In some embodiments, such a step includes pre-incubating for about 1 to 30 minutes. In other embodiments, such a step includes pre-incubating for about 1 to 10 minutes. In yet other embodiments, such a step includes pre-incubating for about 5 minutes. In certain embodiments, the endpoints and values ​​of the above ranges can be ±0.5 minutes, 1 minute, 2 minutes, 3 minutes, or 4 minutes.

[0030] In certain embodiments, the pre-incubation step occurs at about 4° C. In certain embodiments, the pre-incubation step occurs at about 25° C. In certain embodiments, the pre-incubation step occurs at about 37° C. The pre-incubation step may include a buffer such as sodium bicarbonate or HEPES. In certain embodiments, the buffer may include HSPC medium. In further embodiments, the buffer may consist of HSPC medium.

[0031] Preincubation of the LNP composition with a serum factor may include preincubation with serum, a serum fraction, or an isolated serum factor. In some embodiments, the LNP composition is preincubated with serum. The serum may be mammalian, murine, primate, or human serum. In some embodiments, the LNP composition is preincubated with an isolated serum factor. In certain embodiments, the serum factor is ApoE. In certain embodiments, the serum factor is selected from ApoE2, ApoE3, and ApoE4. In further embodiments, the ApoE is a recombinant protein, such as a human recombinant protein. The ApoE may be human recombinant ApoE3. The ApoE may be human recombinant ApoE4.

[0032] In some embodiments, the method includes, after the pre-incubation step, contacting stem cells, e.g., HSPCs, or a stem cell population, e.g., an HSPC population, e.g., contacting the cells with a pre-incubated LNP composition. In some embodiments, the method includes, after the pre-incubation step, contacting a stem cell population, such as a population of ES or iPSCs, e.g., contacting the cells with a pre-incubated LNP composition. In some embodiments, the method includes contacting the cells with the pre-incubated LNP composition for about 1 minute to about 72 hours. In some embodiments, the method includes contacting the cells with the pre-incubated LNP composition for about 1 hour to about 24 hours. In some embodiments, the method includes contacting the cells with the pre-incubated LNP composition for about 4 hours to about 24 hours. In some embodiments, the method includes contacting the cells with the pre-incubated LNP composition for about 4 hours to about 12 hours. In some embodiments, the method includes contacting the cells with the pre-incubated LNP composition for about 2 hours to about 12 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 6 hours to about 8 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 6 hours to about 24 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 6 hours to about 24 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for about 4 hours to about 12 hours. In some embodiments, the method comprises contacting the cells with the pre-incubated LNP composition for at least about 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours. In some embodiments, the method comprises a washing step after the contacting step. The washing step may comprise medium.

[0033] In some embodiments, the method comprises a Cas nuclease mRNA. In some embodiments, the method comprises a Class 2 Cas nuclease mRNA. In some embodiments, the method comprises a gRNA nucleic acid, such as a gRNA. In certain embodiments, the method comprises at least two gRNA nucleic acids. In further embodiments, the method comprises three or more gRNA nucleic acids. In some embodiments, the mRNA, such as a Cas nuclease mRNA, and the gRNA are formulated into a single LNP composition. In some embodiments, the method comprises an mRNA, such as a Cas nuclease mRNA, and a gRNA nucleic acid co-encapsulated in an LNP composition. In further embodiments, the method comprises an mRNA and a gRNA nucleic acid encapsulated in separate LNPs. In certain embodiments, the mRNA is formulated into a first LNP composition and the gRNA nucleic acid is formulated into a second LNP composition. In some embodiments, the first and second LNP compositions are administered simultaneously. In other embodiments, the first and second LNP compositions are administered sequentially. In some embodiments of the in vitro method, the first and second LNP compositions are combined prior to the pre-incubation step. In some embodiments, the first and second LNP compositions are pre-incubated separately.

[0034] In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, may be administered to a cell or cell population, such as an HSPC or HSPC population, separately from the administration of a composition comprising a gRNA. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, and a gRNA may be administered to a cell, such as an HSPC or HSPC population, separately from the administration of a template nucleic acid. In one embodiment, an LNP composition comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, may be administered to an HSPC or HSPC population, followed by sequential administration of an LNP composition comprising a gRNA prior to administering the template to the cell or population. In embodiments in which an LNP composition comprising an mRNA encoding a Cas nuclease is administered before an LNP composition comprising a gRNA is administered, the administrations may be separated by about 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, or 72 hours, or about 1 day, 2 days, or 3 days.

[0035] In some embodiments of the in vitro methods described herein, stem cells, HSPCs, or HSPC populations may be cultured in vitro after transfection with LNPs.

[0036] In some embodiments, transfected stem cells, HSPCs, or HSPC populations are expanded in stem cell medium, such as HSPC medium. "Expansion" or "expanding," with respect to cells, refers to an increase in the number of a distinct cell type or cell types from an initial population of cells, which may or may not be identical. The initial cells used for expansion need not be the same as the cells from which the expansion occurred. Some embodiments of the in vitro method include culturing the HSPC or HSPC population in HSPC medium. Some embodiments further include expanding the HSPCs in HSPC medium containing a stem cell growth factor. See, for example, WO2010 / 059401 (e.g., the compound in Example 1), WO2013 / 110198, and WO2017115268, which are incorporated by reference herein for suitable compounds for stem cell expansion. A "stem cell growth factor" refers to a compound that causes cells, e.g., HSPCs, HSCs, and / or HPCs, to proliferate, e.g., increase in number, at a faster rate than the same cell type would in the absence of the substance. In one exemplary embodiment, the stem cell growth factor is an inhibitor of the aryl hydrocarbon receptor pathway.

[0037] In a further embodiment, the in vitro method further comprises changing the culture medium between the contacting step and the culturing step. In yet another embodiment, the culturing step comprises a cell culture medium comprising thrombopoietin (Tpo), Flt3-ligand (Flt-3L), and human stem cell factor (SCF). In embodiments, the cell culture medium further comprises human interleukin-6 (IL-6). In embodiments, the cell culture medium comprises thrombopoietin (Tpo), Flt3-ligand (Flt-3L), and human stem cell factor (SCF).

[0038] CRISPR / Cas cargo CRISPR / Cas cargo delivered via LNP formulations includes mRNA molecules encoding proteins of interest. For example, mRNAs for expressing proteins such as green fluorescent protein (GFP) and RNA-guided DNA-binding factors, or Cas nucleases, are included. LNP compositions are provided that include Cas nuclease mRNAs, such as class 2 Cas nuclease mRNAs, that enable intracellular expression of Cas9 proteins. Additionally, the cargo may contain one or more guide RNAs or nucleic acids encoding the guide RNAs. Template nucleic acids, for example, for repair or recombination, may also be included in the compositions, or the template nucleic acids may be used in the methods described herein.

[0039] "mRNA" refers to a non-DNA polynucleotide that contains an open reading frame that can be translated into a polypeptide (i.e., can be used as a substrate for translation by ribosomes and aminoacylated tRNAs). mRNA can include a phosphate-sugar backbone, such as ribose residues or analogs thereof, e.g., 2'-methoxyribose residues. In some embodiments, the sugars of the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof. Generally, mRNA does not contain substantial amounts of thymidine residues (e.g., 0 or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues, or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). mRNA can contain modified uridines at some or all of its uridine positions.

[0040] CRISPR / Cas nuclease system One component of the disclosed formulation is an mRNA encoding an RNA-guided DNA binding factor, such as a Cas nuclease.

[0041] As used herein, "RNA-guided DNA-binding factor" refers to a polypeptide or polypeptide complex having RNA and DNA-binding activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the sequence of the RNA. Exemplary RNA-guided DNA-binding factors include Cas cleavase / nickases and their inactive forms ("dCas DNA binders"). As used herein, "Cas nuclease" encompasses Cas cleavase, Cas nickase, and dCas DNA binders. Cas cleavase / nickases and dCas DNA binders include the Csm complex or Cmr complex of type III CRISPR systems, its subunits Cas10, Csm1, or Cmr2, the Cascade complex of type I CRISPR systems, its subunit Cas3, and class 2 Cas nucleases. As used herein, a "class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA-binding activity. Class 2 Cas nucleases include Class 2 Cas cleavase / nickases that additionally possess RNA-guided DNA cleavase or nickase activity (e.g., mutants H840A, D10A, or N863A), and Class 2 dCas DNA binders in which the cleavase / nickase activity has been inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 proteins (e.g., mutants N497A, R661A, Q695A, and Q926A), HypaCas9 proteins (e.g., mutants N692A, M694A, Q695A, and H698A), eSPCas9(1.0) proteins (e.g., mutants K810A, K1003A, and R1060A), and eSPCas9(1.1) proteins (e.g., mutants K848A, K1003A, and R1060A), and variants thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. The Cpf1 sequence of Zetsche is incorporated by reference in its entirety.See, e.g., Tables S1 and S3 of Zetsche. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015), Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0042] In some embodiments, the RNA-guided DNA-binding factor is a Class 2 Cas nuclease. In some embodiments, the RNA-guided DNA-binding factor has cleavage activity, which may also be referred to as double-stranded endonuclease activity. In some embodiments, the RNA-guided DNA-binding factor comprises a Cas nuclease, such as a Class 2 Cas nuclease (which may be, for example, a Type II, Type V, or Type VI Cas nuclease). Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins and variants thereof. Examples of Cas9 nucleases include those in Type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, for example, the list in the next paragraph), as well as modified forms (e.g., engineered or mutant) thereof. See, e.g., US2016 / 0312198 A1, US2016 / 0312199 A1. Other examples of Cas nucleases include the Csm complex or Cmr complex, or its subunits Cas10, Csm1, or Cmr2, of type III CRISPR systems, and the Cascade complex, or its subunit Cas3, of type I CRISPR systems. In some embodiments, the Cas nuclease can be of a type IIA, type IIB, or type IIC system. For a discussion of various CRISPR systems and Cas nucleases, see, e.g., Makarova et al., Nat. Rev. Microbiol. 9:467-477 (2011); Makarova et al., Nat. Rev. Microbiol, 13:722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0043] Non-limiting exemplary species from which Cas nucleases may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.、Crocosphaera watsonii、Cyanothece sp.、Microcystis aeruginosa、Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicellosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile. le, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromona s haloplanktis, Ktedonobacter racemifer, Methanohlobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina are mentioned.

[0044] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus sp.. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0045] Wild-type Cas9 has two nuclease domains, RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target DNA strand. In some embodiments, the Cas9 nuclease comprises two or more RuvC domains and / or two or more HNH domains. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 is capable of inducing a double-stranded break in the target DNA. In certain embodiments, the Cas nuclease may cleave dsDNA, cleave a single strand of dsDNA, or have no DNA cleavage or nickase activity. An exemplary Cas9 amino acid sequence is set forth as SEQ ID NO:3. An exemplary Cas9 mRNA ORF sequence, including the start and stop codons, is set forth as SEQ ID NO:4. An exemplary Cas9 mRNA coding sequence suitable for inclusion in a fusion protein is set forth as SEQ ID NO:10.

[0046] In some embodiments, chimeric Cas nucleases are used, in which one domain or region of such a protein is replaced with a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease, such as Fok1. In some embodiments, the Cas nuclease may be a modified nuclease.

[0047] In other embodiments, the Cas nuclease may be derived from a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a type I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be derived from a type III CRISPR / Cas system. In some embodiments, the Cas nuclease may have RNA cleavage activity.

[0048] In some embodiments, the RNA-guided DNA-binding factor has single-stranded nickase activity, i.e., it can cleave a single DNA strand, creating a single-stranded break, also known as a "nick." In some embodiments, the RNA-guided DNA-binding factor comprises a Cas nickase. A nickase is an enzyme that nicks dsDNA, i.e., cleaves one strand of a DNA double helix but not the other. In some embodiments, the Cas nickase is a variant of a Cas nuclease (e.g., a Cas nuclease described above) in which the endonucleolytic activity site has been inactivated, e.g., by one or more alterations (e.g., point mutations) in the catalytic domain. For a discussion of Cas nickases and exemplary catalytic domain modifications, see, e.g., U.S. Patent No. 8,889,356. In some embodiments, a Cas nickase, such as a Cas9 nickase, has an inactivated RuvC domain or HNH domain.

[0049] In some embodiments, the RNA-guided DNA-binding factor is modified to contain only one functional nuclease domain. For example, the factor protein may be modified to reduce its nucleic acid cleavage activity by mutating or completely or partially deleting one of the nuclease domains. In some embodiments, a nickase with a RuvC domain that has reduced activity is used. In some embodiments, a nickase with an inactive RuvC domain is used. In some embodiments, a nickase with an HNH domain that has reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0050] In some embodiments, conserved amino acids within the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2(CPF1_FRATN))).

[0051] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs complementary to the sense and antisense strands of a target sequence. In this embodiment, the guide RNA guides the nickase to the target sequence, where it introduces a DSB by nicking opposite strands of the target sequence (i.e., double nicking). In some embodiments, the use of double nicking can improve specificity and reduce off-target effects. In some embodiments, a nickase is used with two separate guide RNAs that target opposing DNA strands to create a double nick in the target DNA. In some embodiments, a nickase is used with two separate guide RNAs that are selected to be adjacent to each other to create a double nick in the target DNA.

[0052] In some embodiments, the RNA-guided DNA-binding factor lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding factor comprises a dCas DNA-binding polypeptide. The dCas polypeptide has DNA-binding activity but essentially no catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-binding factor or dCas DNA-binding polypeptide lacking cleavase and nickase activity is a variant of a Cas nuclease (e.g., a Cas nuclease described above), e.g., one or more alterations (e.g., point mutations) in its catalytic domain that inactivate its endonucleolytic activity. See, e.g., US2014 / 0186958A1, US2015 / 0166980A1.

[0053] In some embodiments, the RNA-guided DNA-binding factor comprises one or more heterologous functional domains (eg, is or comprises a fusion polypeptide).

[0054] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding factor into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding factor may be fused to one to ten NLS(s). In some embodiments, the RNA-guided DNA-binding factor may be fused to one to five NLS(s). In some embodiments, the RNA-guided DNA-binding factor may be fused to one NLS. When one NLS is used, the NLS may be linked at the N-terminus or C-terminus of the RNA-guided DNA-binding factor sequence. The NLS may also be inserted internally within the RNA-guided DNA-binding factor sequence. In other embodiments, the RNA-guided DNA-binding factor may be fused to two or more NLSs. In some embodiments, the RNA-guided DNA-binding factor may be fused to two, three, four, or five NLSs. In some embodiments, the RNA-guided DNA-binding factor may be fused to two NLSs. In certain circumstances, the two NLSs may be identical or different (e.g., two SV40 NLSs). In some embodiments, the RNA-guided DNA binding factor is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA binding factor may be fused to two NLSs, one linked at the N terminus and one linked at the C terminus. In some embodiments, the RNA-guided DNA binding factor may be fused to three NLSs. In some embodiments, the RNA-guided DNA binding factor may not be fused to an NLS. In some embodiments, the NLS may be a monopartite sequence, such as the SV40 NLS PKKKRKV or PKKKRRV. In some embodiments, the NLS may be a bipartite sequence, such as the nucleoplasmin NLS KRPAATKKAGQAKKKK. In certain embodiments, a single PKKKRKV NLS may be linked at the C terminus of the RNA-guided DNA binding factor. Optionally, one or more linkers are included at the fusion site.

[0055] In some embodiments, the heterologous functional domain may be capable of altering the intracellular half-life of the RNA-guided DNA-binding factor. In some embodiments, the heterologous functional domain may be capable of extending the half-life of the RNA-guided DNA-binding factor. In some embodiments, the half-life of the RNA-guided DNA-binding factor may be shortened. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding factor. In some embodiments, the heterologous functional domain may be capable of decreasing the stability of the RNA-guided DNA-binding factor. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding factor may be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP; also known as interferon-inducible gene 15 (ISG15)), ubiquitin-related modifier 1 (URM1), neural progenitor cell expressed and developmentally downregulated protein 8 (NEDD8; also called Rub1 in S. cerevisiae), human leukocyte antigen F-related (FAT10), autophagy 8 (ATG8) and autophagy 12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold modifier 1 (UFM1), and ubiquitin-like protein 5 (UBL5).

[0056] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagged GFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, m Examples of suitable fluorescent proteins include Kate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, JRed), and orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and / or an epitope tag.Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent protein.

[0057] In further embodiments, the heterologous functional domain may target the RNA-guided DNA-binding factor to a particular organelle, cell type, tissue, or organ, hi some embodiments, the heterologous functional domain may target the RNA-guided DNA-binding factor to mitochondria.

[0058] In further embodiments, the heterologous functional domain may be an effector domain. When targeting an RNA-guided DNA-binding factor to its target sequence, for example, when targeting a Cas nuclease to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be selected from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcription activation domain, or a transcription repressor domain. In some embodiments, the heterologous functional domain is a nuclease such as FokI nuclease. See, e.g., U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcription activator or a transcription repressor. See, for example, Qi et al., "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression," Cell 152:1173-83 (2013); Perez-Pinera et al., "RNA-guided gene activation by CRISPR-Cas9-based transcription factors," Nat. Methods 10:973-6 (2013); Mali et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nat. Biotechnol. 31:833-8 (2013); and Gilbert et al., "CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes," Cell 154:442-51 (2013). Thus, RNA-guided DNA-binding factors essentially become transcription factors that can be directed to bind to desired target sequences using guide RNAs.In certain embodiments, the DNA-modifying domain is a methylation domain, such as a demethylation domain or a methyltransferase domain. In certain embodiments, the effector domain is a DNA-modifying domain, such as a base-editing domain. In certain embodiments, the DNA-modifying domain is a nucleic acid-editing domain that introduces specific modifications into DNA, such as a deaminase domain. See, for example, WO2015 / 089406 and US2016 / 0304846. The nucleic acid-editing domains, deaminase domains, and Cas9 variants described in WO2015 / 089406 and US2016 / 0304846 are incorporated herein by reference.

[0059] Nucleases may contain at least one domain that interacts with a guide RNA ("gRNA"). Additionally, the nuclease may be directed to a target sequence by the gRNA. In Class 2 Cas nuclease systems, the gRNA interacts with the nuclease and the target sequence, thereby directing binding to the target sequence. In some embodiments, the gRNA provides specificity for targeted cleavage, while the nuclease may be versatile and may pair with different gRNAs to cleave different target sequences. Class 2 Cas nucleases may pair with gRNA scaffolds of the types, orthologs, and exemplary species listed above.

[0060] Guide RNA (gRNA) In some embodiments of the present disclosure, the cargo of the LNP formulation includes at least one gRNA. The gRNA may guide a Cas nuclease or a Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, the gRNA binds to a Class 2 Cas nuclease to provide specificity for cleavage by the Class 2 Cas nuclease. In some embodiments, the gRNA and Cas nuclease may form a ribonucleoprotein (RNP), such as a CRISPR / Cas complex, e.g., 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 may be paired with a specific gRNA. The gRNA scaffold structure paired with each Class 2 Cas nuclease varies depending on the individual CRISPR / Cas system.

[0061] "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to either crRNA (also known as CRISPR RNA) or the combination of crRNA and trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or as two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refer to each type. The trRNA may be either a naturally occurring sequence or a trRNA sequence that has modifications or variations compared to the naturally occurring sequence.

[0062] As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA-binding factor. A "guide sequence" may also be referred to as a "direction sequence" or a "spacer sequence." A guide sequence may be 20 base pairs in length, for example, in the case of Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. Shorter or longer sequences, e.g., 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, can be used as a guide. In some embodiments, the target sequence is, for example, within a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target region may be 100% complementary or identical. In other embodiments, the guide sequence and target region may contain at least one mismatch. For example, the guide sequence and target sequence may contain one, two, three, or four mismatches, in which case the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region may contain one to four mismatches, in which case the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain one, two, three, or four mismatches, in which case the guide sequence comprises 20 nucleotides.

[0063] Because the nucleic acid substrate for the Cas protein is a double-stranded nucleic acid, the target sequence for the Cas protein includes both the plus and minus strands of genomic DNA (i.e., the given sequence and the reverse complement of that sequence). Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that such a guide sequence can direct a guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, when a guide sequence binds to the reverse complement of a target sequence, such a guide sequence is identical to a particular nucleotide of the target sequence (e.g., a target sequence without a PAM) except that T in the guide sequence is replaced with U.

[0064] The length of the directional sequence can vary depending on the CRISPR / Cas system and components used. For example, different Class 2 Cas nucleases derived from different bacterial species have different optimal directional sequence lengths. Thus, the directional sequence can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the directional sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally occurring CRISPR / Cas system. In certain embodiments, the Cas nuclease and gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the directional sequence can comprise or consist of 18 to 24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides, hi some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0065] In some embodiments, the sgRNA is a "Cas9 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cas9 protein. In some embodiments, the sgRNA is a "Cpf1 sgRNA" capable of mediating RNA-guided DNA cleavage by the Cpf1 protein. In certain embodiments, the gRNA comprises a crRNA and a tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0066] Certain embodiments of the present invention also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. "Guide RNA nucleic acid," as used herein, refers to guide RNAs (e.g., sgRNAs or dgRNAs) and guide RNA expression cassettes, which are nucleic acids that encode one or more guide RNAs.

[0067] In some embodiments, the nucleic acid may be a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a directional sequence flanked by all or part of repeat sequences from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and tracrRNA may be encoded by two separate nucleic acids. In other embodiments, the crRNA and tracrRNA may be encoded by a single nucleic acid. In some embodiments, the crRNA and tracrRNA may be encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracrRNA may be encoded by the same strand of a single nucleic acid. In some embodiments, the gRNA nucleic acid encodes an sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

[0068] The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3'UTR, or a 5'UTR. In one example, the promoter is a tRNA promoter, e.g., a tRNA promoter. Lys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9; Scherer et al., Nucleic Acids Res. 2007 35:2620-2628. In certain embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters include the U6 promoter and the H1 promoter. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In certain embodiments, the gRNA nucleic acid includes a modified nucleoside or nucleotide. In some embodiments, the gRNA nucleic acid includes a 5'-end modification, such as a modified nucleoside or nucleotide that stabilizes the nucleic acid and prevents integration of the nucleic acid. In some embodiments, the gRNA nucleic acid includes double-stranded DNA with a 5'-end modification on each strand. In certain embodiments, the gRNA nucleic acid includes an inverted dideoxy-T or an inverted abasic nucleoside or nucleotide as a 5'-end modification. In some embodiments, the gRNA nucleic acid includes a label such as biotin, desthiobioten-TEG, digoxigenin, and a fluorescent marker, such as FAM, ROX, TAMRA, and AlexaFluor.

[0069] In certain embodiments, two or more gRNA nucleic acids, e.g., gRNAs, can be used with a CRISPR / Cas nuclease system. Each gRNA nucleic acid can contain a different targeting sequence so that two or more target sequences can be cleaved by the CRISPR / Cas system. In some embodiments, one or more gRNAs can have the same or different properties, such as activity or stability, within the CRISPR / Cas complex. When two or more gRNAs are used, each gRNA can be encoded on the same gRNA nucleic acid or on a different gRNA nucleic acid. The promoters used to drive expression of two or more gRNAs can be the same or different.

[0070] modified RNA In certain embodiments, the LNP composition comprises modified RNA.

[0071] Modified nucleosides or nucleotides can be present in RNA, such as gRNA or mRNA. For example, a gRNA or mRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" RNA, denoting the presence of one or more non-naturally occurring and / or naturally occurring components or structures used in place of, or in addition to, the standard residues A, G, C, and U. In some embodiments, the modified RNA is synthesized using non-standard nucleosides or nucleotides, referred to herein as "modified."

[0072] Modified nucleosides and nucleotides can include one or more of the following: (i) an alteration, e.g., substitution, of one or both of the non-linked oxygens of the phosphate group and / or one or more of the linked oxygens of the phosphate group in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) an alteration, e.g., substitution, of a component of the ribose sugar, such as the 2' hydroxyl of the ribose sugar (exemplary sugar modifications); (iii) a substantial replacement of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications); (iv) a modification or substitution of a naturally occurring nucleobase, including when a non-standard nucleobase is used (exemplary base modifications); (v) a substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., a modification that removes, modifies, or replaces the terminal phosphate group or attaches a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar modifications and / or backbone modifications); and (vii) a modification or substitution of the sugar (exemplary sugar modifications). Certain embodiments include 5'-end modifications to the mRNA, gRNA, or nucleic acid. Certain embodiments include 3'-end modifications to the mRNA, gRNA, or nucleic acid. The modified RNA may contain modifications at the 5' and 3' ends. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue.

[0073] As used herein, a first sequence is considered to "contain a sequence at least X% identical to" a second sequence if alignment of the two sequences shows that the first sequence matches X% or more of the positions across the entire second sequence. For example, the sequence AAGA contains a sequence that is 100% identical to the sequence AAG, because the alignment would show 100% identity, with matches at all three positions in the second sequence. As long as related nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for both thymidine, uridine, or modified uridine; as another example, cytosine and 5-methylcytosine both have guanosine or modified guanosine as their complements), differences between RNA and DNA (generally, uridine is replaced by thymidine, or vice versa) and the presence of nucleoside analogs, such as modified uridines, do not contribute to differences in identity or complementarity between polynucleotides. Thus, for example, the sequence 5'-AXG, where X is any modified uridine, such as pseudouridine, N1-methylpseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG, and both sequences are perfectly complementary to the same sequence (5'-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well known in the art. Those skilled in the art will understand which algorithm and parameter settings are appropriate for a given pair of sequences to be aligned, but for sequences that are generally similar in length and have an expected amino acid identity of greater than 50% or nucleotide identity of greater than 75%, it is generally appropriate to use the Needleman-Wunsch algorithm with the default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server.

[0074] mRNA In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF), such as an ORF encoding an RNA-guided DNA-binding factor, such as a Cas nuclease or Class 2 Cas nuclease described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA-binding factor, such as a Cas nuclease or Class 2 Cas nuclease, is provided, used, or administered. In some embodiments, the ORF encoding the RNA-guided DNA-binding factor is a "modified RNA-guided DNA-binding factor ORF" or simply a "modified ORF," which is used as shorthand to indicate that the ORF is modified in one or more of the following ways: (1) the modified ORF has a uridine content ranging from its minimum uridine content to 150% of such minimum uridine content; (2) the modified ORF has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of such minimum uridine dinucleotide content; or (3) the modified ORF has a uridine dinucleotide content ranging from the sequence (4) the modified ORF is composed of a set of codons in which at least 75% of the codons are minimal uridine codon(s) for a given amino acid, e.g., the codon(s) with the fewest uridines (the minimal uridine codons are typically zero or one, except for the codon for phenylalanine, which has two uridines); or (5) the modified ORF contains at least one modified uridine. In some embodiments, the modified ORF is modified in at least two, three, or four of the methods described above. In some embodiments, the modified ORF contains at least one modified uridine and is modified in at least one, two, three, or all of the methods described above.

[0075] As used herein, the term "modified uridine" refers to a nucleoside other than thymidine in which the hydrogen bond acceptor is the same as in uridine and which has one or more structural differences from uridine. In some embodiments, the modified uridine is a substituted uridine, i.e., a uridine in which one or more aprotic substituents (e.g., alkoxy, such as methoxy) replace a proton. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more aprotic substituents (e.g., alkyl, such as methyl) replace a proton. In some embodiments, the modified uridine is either a substituted uridine, a pseudouridine, or a substituted pseudouridine.

[0076] As used herein, a "uridine position" refers to a position in a polynucleotide that is occupied by a uridine or a modified uridine. Thus, for example, a polynucleotide with "100% modified uridines at uridine positions" contains a modified uridine at every position that would be a uridine in conventional RNA of the same sequence (wherein all bases are the standard bases A, U, C, or G). Unless otherwise specified, U in a polynucleotide sequence set forth in this disclosure or in a sequence table or sequence listing accompanying this disclosure can be a uridine or a modified uridine. [Table 1]

[0077] In any of the above embodiments, the modified ORF may be composed of a set of codons where at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons set forth in the table of minimal uridine codons. In any of the above embodiments, the modified ORF may comprise a sequence that is at least 90%, 95%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1, 4, 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.

[0078] In any of the above embodiments, the modified ORF may have a uridine content ranging from its minimum uridine content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of such minimum uridine content.

[0079] In any of the above embodiments, the modified ORF may have a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150%, 145%, 140%, 135%, 130%, 125%, 120%, 115%, 110%, 105%, 104%, 103%, 102%, or 101% of such minimum uridine dinucleotide content.

[0080] In any of the above-described embodiments, the modified ORF may include modified uridines at at least one, more than one, or all of the uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0081] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions of an mRNA according to the disclosure are modified uridines. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of an mRNA according to the disclosure are modified uridines, e.g., 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or combinations thereof. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are 5-methoxyuridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are pseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are N1-methylpseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are 5-iodouridine. In some embodiments, 10% to 25%, 15 to 25%, 25 to 35%, 35 to 45%, 45 to 55%, 55 to 65%, 65 to 75%, 75 to 85%, 85 to 95%, or 90 to 100% of the uridine positions in an mRNA according to the present disclosure are 5-methoxyuridine, and the remaining uridine positions are N1-methylpseudouridine.In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions in an mRNA according to the present disclosure are 5-iodouridine, and the remaining uridine positions are N1-methylpseudouridine.

[0082] In any of the above-described embodiments, the modified ORF may contain a low uridine dinucleotide (UU) content, such as the lowest possible content, e.g., an ORF that (a) uses minimal uridine codons (described above) at every position and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content can be expressed either as an absolute value, as the count of UU dinucleotides in the ORF, or as a ratio, as the percentage of positions occupied by uridine dinucleotides (e.g., in the case of AUUAU, two out of five positions are occupied by uridine dinucleotides, resulting in a uridine dinucleotide content of 40%). For purposes of assessing minimum uridine dinucleotide content, modified uridine residues are considered equivalent to uridine.

[0083] In some embodiments, the mRNA comprises at least one UTR derived from an expressed mammalian mRNA, such as a constitutively expressed mRNA. An mRNA is considered to be constitutively expressed in a mammal if it is continuously transcribed in at least one tissue of a healthy adult mammal. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, or both a 5' and a 3' UTR derived from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

[0084] In some embodiments, the mRNA comprises at least one UTR from 17-beta hydroxysteroid dehydrogenase 4 (HSD17B4 or HSD), such as a 5' UTR from an HSD. In some embodiments, the mRNA comprises at least one UTR from a globin mRNA, such as human alpha globin (HBA) mRNA, human beta globin (HBB) mRNA, or Xenopus laevis beta globin (XBG) mRNA. In some embodiments, the mRNA comprises a 5' UTR, a 3' UTR, or both 5' and 3' UTRs from a globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5' UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3' UTR derived from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises both 5' and 3' UTRs derived from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, XBG, heat shock protein 90 (Hsp90), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin, alpha-tubulin, tumor protein (p53), or epidermal growth factor receptor (EGFR).

[0085] In some embodiments, the mRNA comprises both the 5' and 3' UTRs derived from the same source, for example, a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.

[0086] In some embodiments, the mRNA does not include a 5' UTR, e.g., no additional nucleotides between the 5' cap and the start codon. In some embodiments, the mRNA includes a Kozak sequence (described below) between the 5' cap and the start codon, but no additional 5' UTR. In some embodiments, the mRNA does not include a 3' UTR, e.g., no additional nucleotides between the stop codon and the polyA tail.

[0087] In some embodiments, the mRNA contains a Kozak sequence. Kozak sequences can affect translation initiation and the overall yield of polypeptides translated from the mRNA. Kozak sequences include a methionine codon that can function as an initiation codon. A minimal Kozak sequence is NNNRUGN, where at least one of the following is true: the first N is A or G, and the second N is G. In nucleotide sequences, R represents a purine (A or G). In some embodiments, the Kozak sequence is RNNRUGN, NNNRUGG, RNNRUGG, RNNAUGN, NNNAUGG, or RNNAUGG. In some embodiments, the Kozak sequence is rccRUGg, with zero mismatches or up to one or two mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is rccAUGg, with zero mismatches or up to one or two mismatches relative to the lowercase positions. In some embodiments, the Kozak sequence is gccRccAUGG and has zero mismatches or up to one, two, or three mismatches at lowercase positions. In some embodiments, the Kozak sequence is gccAccAUG and has zero mismatches or up to one, two, three, or four mismatches at lowercase positions. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG and has zero mismatches or up to one, two, three, or four mismatches at lowercase positions.

[0088] In some embodiments, the mRNA comprising an ORF encoding an RNA-guided DNA-binding factor comprises a sequence at least 90% identical to SEQ ID NO: 43, where, optionally, the ORF of SEQ ID NO: 43 (i.e., SEQ ID NO: 4) is replaced with an alternative ORF. In some embodiments, the mRNA comprises any of SEQ ID NOs: 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66.

[0089] In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO: 43 is 95%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO: 4 is 98%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO: 43 is 99%. In some embodiments, the degree of identity to the optionally substituted sequence of SEQ ID NO: 43 is 100%.

[0090] In some embodiments, the mRNAs disclosed herein include a 5' cap, such as Cap0, Cap1, or Cap2. The 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, e.g., with an ARCA, as discussed below) linked via a 5'-triphosphate to the 5' position of the first nucleotide in the 5'-to-3' strand of the mRNA, i.e., the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA include a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both include a 2'-methoxy. See, for example, Katibah et al. (2014) Proc Natl Acad SciUSA 111(33):12025-30 and Abbas et al. (2017) Proc Natl Acad SciUSA 114(11):E2106-E2115. Most endogenous mRNAs in higher eukaryotes, including mammalian mRNAs such as human mRNAs, contain Cap1 or Cap2. Cap0 and other cap structures distinct from Cap1 and Cap2 can be immunogenic in mammals, including humans, because they are recognized as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, which can increase cytokine levels, including type I interferons. Components of the innate immune system, such as IFIT-1 and IFIT-5, can also compete with eIF4E for binding to mRNAs with caps other than Cap1 or Cap2, potentially inhibiting mRNA translation.

[0091] A cap can be incorporated co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific catalog number AM8045) is a cap analog that contains 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of a guanine ribonucleotide and can be incorporated into transcripts during in vitro transcription initiation. ARCA results in a Cap0 cap, in which the 2' position of the first cap-proximal 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. [ka]

[0092] The Cap1 structure can be obtained by co-transcription using CleanCap™ AG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies catalog number N-7113) or CleanCap™ GG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies catalog number N-7133). 3'-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies as catalog numbers N-7413 and N-7433, respectively. The CleanCap™ AG structure is shown below. [ka]

[0093] Alternatively, RNA can be capped post-transcriptionally. For example, vaccinia capping enzyme is commercially available (New England Biolabs, catalog number M2080S), which possesses RNA triphosphatase and guanylyltransferase activities provided by its D1 subunit and a guanine methyltransferase activity provided by its D12 subunit. Thus, in the presence of S-adenosylmethionine and GTP, this enzyme can add 7-methylguanine to RNA to give Cap0. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem. 269, 24472-24479.

[0094] In some embodiments, the mRNA further comprises a polyadenylation (polyA) tail. In some embodiments, the polyA tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the polyA tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some examples, the polyA tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more positions within the polyA tail. The polyA tail may comprise at least eight consecutive adenine nucleotides, but also includes one or more non-adenine nucleotides. As used herein, "non-adenine nucleotide" refers to any nucleotide, natural or unnatural, that does not contain adenine. Guanine, thymine, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly A tail of an mRNA described herein may comprise consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding factor or sequence of interest. In some instances, the poly A tail of an mRNA comprises non-consecutive adenine nucleotides located 3' to the nucleotides encoding the RNA-guided DNA-binding factor or sequence of interest, where the adenine nucleotides are interrupted at regularly or irregularly spaced intervals by non-adenine nucleotides.

[0095] 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 an mRNA described herein may include consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA-binding factor or a sequence of interest. In some examples, the polyA tail of an mRNA includes non-consecutive adenine nucleotides located 3' to nucleotides encoding an RNA-guided DNA-binding factor or a sequence of interest, where the adenine nucleotides are interrupted at regularly or irregularly spaced intervals by the non-adenine nucleotides.

[0096] In some embodiments, mRNA is purified. In some embodiments, mRNA is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, etc., or an equivalent method, such as, for example, a method described herein). In some embodiments, mRNA is purified using a chromatographic method, such as a method using HPLC, or an equivalent method (e.g., a method described herein). In some embodiments, mRNA is purified using both a precipitation method (e.g., LiCl precipitation) and a method using HPLC.

[0097] In some embodiments, at least one gRNA is provided in combination with the mRNA disclosed herein.In some embodiments, the gRNA is provided as a molecule separate from the mRNA.In some embodiments, the gRNA is provided as a part of the mRNA disclosed herein, for example, as a part of UTR.

[0098] gRNA In certain aspects, the disclosure provides methods of delivering a genome editing system (e.g., a zinc finger nuclease system, a TALEN system, a meganuclease system, or a CRISPR / Cas system) to a cell (or population of cells), e.g., an HSPC (or HSPC population), e.g., a CD34+ cell (or CD34+ cell population), wherein the resulting cell (or its progeny) has increased expression of fetal hemoglobin (e.g., when the cell differentiates into an erythrocyte). Disclosed herein are guide sequences useful in achieving such an effect. In embodiments, the genome editing system comprises one or more vectors, e.g., mRNA, encoding components of the genome editing system. In other embodiments, the genome editing system comprises one or more polypeptides. In preferred aspects, the method comprises delivering a CRISPR / Cas system. In embodiments, the CRISPR / Cas system comprises a gRNA and a Cas nuclease, e.g., complexed in the form of a ribonucleoprotein complex (RNP). In other embodiments, the CRISPR / Cas system includes one or more vectors encoding a gRNA and / or a Cas nuclease. In other embodiments, the CRISPR / Cas system includes one or more vectors, e.g., mRNA, encoding a Cas nuclease (e.g., a Class 2 Cas nuclease) and one or more gRNAs. In aspects, the CRISPR / Cas system includes a gRNA described in WO2017 / 115268, the entire contents of which are incorporated herein by reference. In aspects, the CRISPR / Cas system includes a gRNA that includes a guide sequence that is complementary to a target sequence within the BCL11a gene or a regulatory element thereof. In other aspects, the CRISPR / Cas system includes a gRNA that includes a guide sequence that is complementary to a target sequence within intron 2 of the BCL11a gene (e.g., within the intron 2 region of the BCL11a gene at or near the GATA1 binding site).In aspects, the CRISPR / Cas system includes a gRNA that includes a guide sequence complementary to a target sequence within the intron 2 region of the BCL11a gene, from ch2:60494000 to ch2:60498000 (according to hg38), e.g., within the intron 2 region of the BCL11a gene, from ch2:60494250 to ch2:60496300 (according to hg38). In embodiments, the CRISPR / Cas system includes a gRNA that includes a guide sequence set forth in Table 2 of U.S. Provisional Application No. 62 / 566,232, filed September 29, 2017, which is incorporated herein by reference.

[0099] An exemplary guide sequence for the gRNA is complementary to a target sequence within intron 2 of the BCL11a gene. +58, +62, and +55 refer to DNAse hypersensitive sites in the erythroid-specific enhancer region as described in Bauer et al., Science 2013;342(6155):253-257.

[0100] In other aspects, the CRISPR / Cas system includes a gRNA comprising a guide sequence complementary to a target sequence within the globin locus on chromosome 11. In one aspect, the CRISPR / Cas system includes a gRNA comprising a guide sequence complementary to a sequence within the HPFH region. As used herein, the term "HPFH region" refers to a genomic site that, when modified (e.g., mutated or deleted), causes increased HbF production in adult red blood cells, including HPFH regions identified in the literature (see, e.g., Online Mendelian Inheritance in Man: http: / / www.omim.org / entry / 141749, incorporated herein by reference). In an exemplary embodiment, the HPFH region is within or encompasses the beta-globin gene cluster on chromosome 11p15. In an exemplary embodiment, the HPFH region is within or encompasses at least a portion of the delta-globin gene and its regulatory elements. In an exemplary embodiment, the HPFH region is the promoter region of HBG1. In an exemplary embodiment, the HPFH region is the promoter region of HBG2. In an exemplary embodiment, the HPFH region is the region described in Sankaran VG et al. NEJM (2011) 365:807-814, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the HPFH region is the breakpoint deletion French HPFH described in Sankaran VG et al. NEJM (2011) 365:807-814. In an exemplary embodiment, the HPFH region is the Algerian HPFH described in Sankaran VG et al. NEJM (2011) 365:807-814. In an exemplary embodiment, the HPFH region is the Sri Lankan HPFH described in Sankaran VG et al. NEJM (2011) 365:807-814. In one exemplary embodiment, the HPFH region is HPFH-3 as described in Sankaran VG et al. NEJM (2011) 365:807-814. In one exemplary embodiment, the HPFH region is HPFH-2 as described in Sankaran VG et al. NEJM (2011) 365:807-814.In one embodiment, the HPFH-1 region is HPFH-3 as described in Sankaran VG et al. NEJM (2011) 365:807-814. In an exemplary embodiment, the HPFH region is Sri Lankan (δβ) 0-thalassemia HPFH as described in Sankaran VG et al. NEJM (2011) 365:807-814. In an exemplary embodiment, the HPFH region is Sicilian (δβ) 0-thalassemia HPFH as described in Sankaran VG et al. NEJM (2011) 365:807-814. In an exemplary embodiment, the HPFH region is Macedonian (δβ) 0-thalassemia HPFH as described in Sankaran VG et al. NEJM (2011) 365:807-814. In one exemplary embodiment, the HPFH region is Kurdish β0-thalassemia HPFH, as described in Sankaran VG et al. NEJM (2011) 365:807-814. In one exemplary embodiment, the HPFH region is a region located at Chr11:5213874-5214400 (hg18). In one exemplary embodiment, the HPFH region is a region located at Chr11:5215943-5215046 (hg18). In one exemplary embodiment, the HPFH region is a region located at Chr11:5234390-5238486 (hg38). In an embodiment, the CRISPR / Cas system includes a gRNA comprising a guide sequence comprising a sequence described in WO2017 / 077394, the entire contents of which are incorporated herein by reference. In embodiments, the CRISPR / Cas system includes a gRNA comprising a guide sequence that comprises a sequence selected from the guide sequences set forth in WO2017 / 077394. In embodiments, the CRISPR / Cas system includes a gRNA comprising a guide sequence set forth in Table 3 of U.S. Provisional Application No. 62 / 566,232, filed September 29, 2017, which is incorporated herein by reference.

[0101] An exemplary guide sequence was oriented toward French HPFH (Sankaran VG et al. A functional element necessary for fetal hemoglobin silencing. NEJM (2011) 365:807-814).

[0102] In embodiments, the CRISPR / Cas system includes a gRNA that includes a guide sequence as set forth in Table 4 of U.S. Provisional Application No. 62 / 566,232, filed September 29, 2017, which is incorporated herein by reference.

[0103] Exemplary guide sequences may be oriented to the promoter regions of HBG1 and / or HBG2.

[0104] Chemically modified gRNA In some embodiments, gRNAs are chemically modified. A gRNA that includes one or more modified nucleosides or nucleotides is referred to as a "modified" gRNA or a "chemically modified" gRNA, denoting the presence of one or more non-naturally occurring and / or naturally occurring components or structures that are substituted for or in addition to the standard residues A, G, C, and U. In some embodiments, modified gRNAs are synthesized using non-standard nucleosides or nucleotides, and are referred to herein as "modified." Modified nucleosides and nucleotides can include one or more of the following: (i) an alteration, e.g., substitution, of one or both of the non-linked oxygens of the phosphate group and / or the linked oxygen of the phosphate group in the phosphodiester backbone linkage (exemplary backbone modifications); (ii) an alteration, e.g., substitution, of a component of the ribose sugar, such as the 2' hydroxyl of the ribose sugar (exemplary sugar modifications); (iii) a substantial replacement of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications); (iv) a modification or substitution of a naturally occurring nucleobase, including when a non-standard nucleobase is used (exemplary base modifications); (v) a substitution or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) a modification of the 3' or 5' end of the oligonucleotide, e.g., a modification that removes, modifies, or replaces the terminal phosphate group or attaches a moiety, cap, or linker (such 3' or 5' cap modifications can include sugar modifications and / or backbone modifications); and (vii) a modification or substitution of the sugar (exemplary sugar modifications).

[0105] In some embodiments, the gRNA comprises modified uridines at some or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5-position, e.g., with a halogen or a C1-C6 alkoxy. In some embodiments, the modified uridine is a pseudouridine modified at the 1-position, e.g., with a C1-C6 alkyl. The modified uridine can be, for example, pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methylpseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0106] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the uridine positions of a gRNA according to the present disclosure are modified uridines. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of a gRNA according to the present disclosure are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or combinations thereof. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions of a gRNA according to the present disclosure are 5-methoxyuridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions of a gRNA according to the present disclosure are pseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions of a gRNA according to the present disclosure are N1-methylpseudouridine. In some embodiments, 10% to 25%, 15% to 25%, 25% to 35%, 35% to 45%, 45% to 55%, 55% to 65%, 65% to 75%, 75% to 85%, 85% to 95%, or 90% to 100% of the uridine positions of a gRNA according to the present disclosure are 5-iodouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions of a gRNA according to the present disclosure are 5-methoxyuridine, and the remaining uridine positions are N1-methylpseudouridine.In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions of a gRNA according to the present disclosure are 5-iodouridine, and the remaining uridine positions are N1-methylpseudouridine.

[0107] The above-listed chemical modifications can be combined to yield modified gRNAs containing nucleosides and nucleotides (collectively referred to as "residues") that may have two, three, four, or more modifications. For example, modified residues can have modified sugars and modified nucleobases. In some embodiments, every base of the gRNA is modified, e.g., all bases have modified phosphate groups, such as phosphorothioate groups. In certain embodiments, all or substantially all of the phosphate groups of the gRNA molecule are replaced with phosphorothioate groups. In some embodiments, the modified gRNA contains at least one modified residue at or near the 5' end of the RNA. In some embodiments, the modified gRNA contains at least one modified residue at or near the 3' end of the RNA.

[0108] In some embodiments, the gRNA comprises one, two, three, or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions of the modified gRNA are modified nucleosides or nucleotides.

[0109] Unmodified nucleic acids may be prone to degradation, for example, by nucleases found in cells or serum. For example, nucleases can hydrolyze phosphodiester bonds in nucleic acids. Thus, in one aspect, the gRNAs described herein contain one or more modified nucleosides or nucleotides to introduce stability against nucleases, for example, in cells or serum. In some embodiments, the modified gRNA molecules described herein may exhibit a reduced innate immune response when introduced into a cell population, either in vivo or ex vivo. The term "innate immune response" includes cellular responses to exogenous nucleic acids, such as single-stranded nucleic acids, resulting in the expression and release of cytokines, particularly interferon, and the induction of cell death.

[0110] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more oxygen atoms with different substituents.In addition, modified residues, for example, modified residues present in modified nucleic acids, can include substantial substitution of unmodified phosphate moieties with modified phosphate groups as described herein.In some embodiments, backbone modification of the phosphate backbone can include changes that result in either an uncharged linker or a charged linker with asymmetric charge distribution.

[0111] Examples of modified phosphate groups include phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. The phosphorus atom of an unmodified phosphate group is achiral. However, the phosphorus atom can be made chiral by replacing one of the non-bridging oxygens with one of the atoms or atomic groups listed above. The asymmetric phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacing the bridging oxygen (i.e., the oxygen connecting the phosphate group to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), or carbon (bridging methylene phosphonate). The replacement may occur at either or both of the linking oxygens.

[0112] In certain backbone modifications, the phosphate group can be replaced with a phosphorus-free connector. In some embodiments, the charged phosphate group can be replaced with a neutral moiety. Examples of moieties that can replace the phosphate group include, but are not limited to, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0113] In some embodiments, the invention includes sgRNAs comprising one or more modifications in one or more of the following regions: the 5'-terminal nucleotide, the lower stem region, the bulge region, the upper stem region, the nexus region, the hairpin 1 region, the hairpin 2 region, and the 3'-terminal nucleotide. In some embodiments, the modification comprises 2'-O-methyl (2'-O-Me) modified nucleotides. In some embodiments, the modification comprises 2'-fluoro (2'-F) modified nucleotides. In some embodiments, the modification comprises an internucleotide phosphorothioate (PS) linkage.

[0114] In some embodiments, the first 3 or 4 nucleotides at the 5' end and the last 3 or 4 nucleotides at the 3' end are modified. In some embodiments, the first 4 nucleotides at the 5' end and the last 4 nucleotides at the 3' end are linked with phosphorothioate (PS) linkages. In some embodiments, the modification comprises 2'-O-Me. In some embodiments, the modification comprises 2'-F.

[0115] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise 2'-O-Me modifications.

[0116] In some embodiments, the first four nucleotides at the 5' end and the last four nucleotides at the 3' end are linked by PS bonds, and the first three nucleotides at the 5' end and the last three nucleotides at the 3' end comprise 2'-F modifications.

[0117] In some embodiments, the sgRNA comprises the modification pattern of SEQ ID NO: 74 (mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmU*mU*mU*mU), where N is any natural or unnatural nucleotide. In some embodiments, the sgRNA comprises SEQ ID NO: 74. In certain embodiments, the sgRNA comprises 2'O-methyl modifications of the first three residues at its 5' end and has phosphorothioate linkages between residues 1-2, between residues 2-3, and between residues 3-4 of the RNA.

[0118] template nucleic acid The compositions and methods disclosed herein can include a template nucleic acid. The template can be used to alter or insert a nucleic acid sequence at or near a target site for a Cas nuclease. In some embodiments, the method includes introducing the template into a cell. In some embodiments, a single template can be provided. In other embodiments, two or more templates can be provided to allow editing to occur at two or more target sites. For example, different templates can be provided to edit a single gene in a cell or two different genes in a cell.

[0119] In some embodiments, the template may be used in homologous recombination. In some embodiments, homologous recombination may result in the incorporation of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template may be used in homologous recombination repair, in which DNA strand invasion occurs at the nucleic acid cleavage site. In some embodiments, homologous recombination repair may result in the template sequence being included in the edited target nucleic acid molecule. In yet another embodiment, the template may be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the cleavage site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes adjacent inverted terminal repeat (ITR) sequences.

[0120] In some embodiments, the template may include a first homology arm and a second homology arm (also referred to as a first nucleotide sequence and a second nucleotide sequence) that are complementary to sequences located upstream and downstream of the cleavage site, respectively. When the template contains two homology arms, each arm may be the same or different in length, and the sequence flanked by the homology arms may be substantially similar or identical to the target sequence flanked by the homology arms, or may be a completely unrelated sequence. In some embodiments, the degree of complementarity or percent identity between the first nucleotide sequence on the template and the sequence upstream of the cleavage site, and the degree of complementarity or percent identity between the second nucleotide sequence on the template and the sequence downstream of the cleavage site, may enable homologous recombination between the template and the target nucleic acid molecule, such as high-fidelity homologous recombination. In some embodiments, the degree of complementarity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be at least 98%, 99%, or 100%. In some embodiments, the degree of complementarity may be 100%. In some embodiments, the percent identity may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be about 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity may be at least 98%, 99%, or 100%. In some embodiments, the percent identity may be 100%.

[0121] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. Additionally or alternatively, the template sequence may correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or that is at a different location than its natural location in the genome of the cell. In some embodiments, the endogenous sequence may be a genomic sequence of the cell. In some embodiments, the endogenous sequence may be a chromosomal sequence or an extrachromosomal sequence. In some embodiments, the endogenous sequence may be a plasmid sequence of the cell. In some embodiments, the template sequence may be substantially identical to a portion of the endogenous sequence of the cell at or near the cleavage site, but contains at least one nucleotide change. In some embodiments, template editing of the cleaved target nucleic acid molecule may result in a mutation including an insertion, deletion, or substitution of one or more nucleotides of the target nucleic acid molecule. In some embodiments, the mutation may result in one or more amino acid changes in a protein expressed by a gene comprising the target sequence. In some embodiments, the mutation may result in one or more nucleotide changes in the RNA expressed by the target gene. In some embodiments, the mutation may change the expression level of the target gene. In some embodiments, the mutation may result in increased or decreased expression of the target gene. In some embodiments, the mutation may result in gene knockdown. In some embodiments, the mutation may result in gene knockout. In some embodiments, the mutation may result in restoration of gene function. In some embodiments, template editing of the cleaved target nucleic acid molecule may result in changes to exon sequences, intron sequences, regulatory sequences, transcriptional regulatory sequences, translational regulatory sequences, splicing sites, or non-coding sequences of the target nucleic acid molecule, such as DNA.

[0122] In other embodiments, the template sequence may comprise an exogenous sequence. In some embodiments, the exogenous sequence may comprise a protein- or RNA-encoding sequence operably linked to an exogenous promoter sequence, such that when the exogenous sequence is integrated into the target nucleic acid molecule, the cell can express the protein or RNA encoded by the integrated sequence. In other embodiments, when the exogenous sequence is integrated into the target nucleic acid molecule, expression of the integrated sequence may be controlled by the endogenous promoter sequence. In some embodiments, the exogenous sequence may provide a cDNA sequence encoding a protein or a portion of the protein. In yet other embodiments, the exogenous sequence may comprise or consist of an exon sequence, an intron sequence, a regulatory sequence, a transcriptional regulatory sequence, a translational regulatory sequence, a splice site, or a non-coding sequence. In some embodiments, integration of the exogenous sequence may result in restoration of gene function. In some embodiments, integration of the exogenous sequence may result in gene knock-in. In some embodiments, integration of the exogenous sequence may result in gene knock-out.

[0123] The template may be of any suitable length. In some embodiments, the template may comprise 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template may be a single-stranded nucleic acid. The template may be a double-stranded or partially double-stranded nucleic acid. In certain embodiments, the single-stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template may comprise a nucleotide sequence complementary to a portion of a target nucleic acid molecule comprising the target sequence (i.e., a "homology arm"). In some embodiments, the template may include homology arms that are complementary to sequences located upstream or downstream of the cleavage site on the target nucleic acid molecule.

[0124] In some embodiments, the template contains ssDNA or dsDNA flanked by inverted terminal repeat (ITR) sequences. In some embodiments, the template is provided as a vector, a plasmid, a minicircle, a nanocircle, or a PCR product.

[0125] Nucleic acid purification In some embodiments, nucleic acids are purified. In some embodiments, nucleic acids are purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, such as, for example, a method described herein). In some embodiments, nucleic acids are purified using a chromatographic method, such as a method using HPLC, or equivalent method (e.g., a method described herein). In some embodiments, nucleic acids are purified using both a precipitation method (e.g., LiCl precipitation) and a method using HPLC.

[0126] Target sequence In some embodiments, the CRISPR / Cas system of the present disclosure may be directed to and cleave a target sequence on a target nucleic acid molecule. For example, the target sequence may be recognized and cleaved by a Cas nuclease. In certain embodiments, the target sequence of the Cas nuclease is located near the specific PAM sequence of such nuclease. In some embodiments, a Class 2 Cas nuclease may be directed to a target sequence on a target nucleic acid molecule by a gRNA, where the gRNA hybridizes to the target sequence and a Class 2 Cas protein cleaves the target sequence. In some embodiments, a guide RNA hybridizes to a target sequence adjacent to or including the specific PAM of the Class 2 Cas nuclease, and the Class 2 Cas nuclease cleaves such target sequence. In some embodiments, the target sequence may be complementary to the targeting sequence of the guide RNA. In some embodiments, the degree of complementarity between the directional sequence of the guide RNA and the corresponding portion of the target sequence to which the guide RNA hybridizes may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the percent identity between the directional sequence of the guide RNA and the corresponding portion of the target sequence to which the guide RNA hybridizes may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the target homology region is flanked by a specific PAM sequence. In some embodiments, the target sequence may comprise a sequence that is 100% complementary to the directional sequence of the guide RNA. In other embodiments, the target sequence may contain at least one mismatch, deletion, or insertion compared to the target sequence of the guide RNA.

[0127] The length of the target sequence may vary depending on the nuclease system used. For example, the target sequence of a guide RNA for a CRISPR / Cas system may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides in length, with the target sequence being of a corresponding length and optionally flanked by a PAM sequence. In some embodiments, the target sequence may comprise 15-24 nucleotides in length. In some embodiments, the target sequence may comprise 17-21 nucleotides in length. In some embodiments, the target sequence may comprise 20 nucleotides in length. When using nickases, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave opposite strands of a DNA molecule. In some embodiments, the target sequence may comprise a pair of target sequences recognized by a pair of nickases that cleave the same strand of a DNA molecule, hi some embodiments, the target sequence may comprise a portion of a target sequence recognized by one or more Cas nucleases.

[0128] A target nucleic acid molecule can be any DNA or RNA molecule that is endogenous or exogenous to a cell. In some embodiments, the target nucleic acid molecule can be episomal DNA, a plasmid, genomic DNA, a viral genome, mitochondrial DNA, or chromosomal DNA from or within a cell. In some embodiments, the target sequence of the target nucleic acid molecule can be a genomic sequence from or within a cell, such as a human cell.

[0129] In further embodiments, the target sequence may be a viral sequence. In further embodiments, the target sequence may be a pathogen sequence. In yet another embodiment, the target sequence may be a synthetic sequence. In further embodiments, the target sequence may be a chromosomal sequence. In particular embodiments, the target sequence may comprise a translocation junction, e.g., a translocation associated with cancer. In some embodiments, the target sequence may be on a eukaryotic chromosome, such as a human chromosome. In particular embodiments, the target sequence is a liver-specific sequence, where the sequence is expressed in liver cells.

[0130] In some embodiments, the target sequence may be located within a coding sequence of a gene, within an intron sequence of a gene, within a regulatory sequence, within a transcriptional control sequence of a gene, within a translational control sequence of a gene, at a splice site, or within an intergenic non-coding sequence. In some embodiments, the gene may be a protein-coding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may comprise all or a portion of a disease-associated gene. In some embodiments, the target sequence may be located in a non-gene functional site of the genome, for example, a site that controls aspects of chromatin organization, such as a scaffolding site or locus control region.

[0131] In embodiments involving Class 2 Cas nucleases, such as Cas nucleases, the target sequence may be adjacent to a protospacer adjacent motif ("PAM"). In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides of the 3' end of the target sequence. The length and sequence of the PAM may vary depending on the Cas protein used. For example, the PAM may be selected from consensus sequences or specific PAM sequences for a particular Cas9 protein or Cas9 ortholog, including those disclosed in Figure 1 of Ran et al., Nature, 520:186-191 (2015) and Figure S5 of Zetsche 2015, the relevant disclosures of which are incorporated herein by reference. In some embodiments, the PAM may be 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Non-limiting exemplary PAM sequences include NGG, NGGNG, NG, NAAAAN, NNAAAAW, NNNNACA, GNNNCNNA, TTN, and NNNNGATT (where N is defined as any nucleotide and W is defined as either A or T). In some embodiments, the PAM sequence may be NGG. In some embodiments, the PAM sequence may be NGGNG. In some embodiments, the PAM sequence may be TTN. In some embodiments, the PAM sequence may be NNAAAAW.

[0132] Lipid preparations Disclosed herein are various embodiments of LNP formulations for biologically active substances, such as RNA, containing CRISPR / Cas cargo. Such LNP formulations include "amine lipids" or "biodegradable lipids," optionally along with one or more of helper lipids, neutral lipids, and stealth lipids, such as PEG-lipids. "Lipid nanoparticle" refers to a particle comprising multiple (i.e., two or more) lipid molecules physically associated with each other through intermolecular forces.

[0133] Amine lipids In certain embodiments, LNP compositions for delivering biologically active agents include "amine lipids," defined as lipid A or equivalents thereof, such as acetal analogs of lipid A.

[0134] In some embodiments, the amine lipid is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. Lipid A is [ka] It can be expressed as:

[0135] Lipid A may be synthesized according to WO2015 / 095340 (e.g., pages 84-86). In certain embodiments, the amine lipid is equivalent to lipid A.

[0136] In certain embodiments, the amine lipid is an analog of lipid A. In certain embodiments, the lipid A analog is an acetal analog of lipid A. In certain LNP compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In further embodiments, the acetal analog is a C5-C10 acetal analog. In further embodiments, the acetal analog is selected from C4, C5, C6, C7, C9, C10, C11, and C12 acetal analogs.

[0137] Amine lipids and other "biodegradable lipids" suitable for use in the LNPs described herein are biodegradable in vivo. Amine lipids have low toxicity (e.g., are tolerated in animal models at doses of 10 mg / kg or greater without adverse effects). In certain embodiments, LNPs comprising amine lipids include those in which at least 75% of the amine lipid is cleared from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days. In certain embodiments, LNPs comprising amine lipids include those in which at least 50% of the mRNA or gRNA is cleared from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days. In certain embodiments, LNPs comprising amine lipids include those in which at least 50% of the LNPs are cleared from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days, as measured, for example, by lipid (e.g., amine lipid), RNA (e.g., mRNA), or other components. In certain embodiments, the lipid, RNA, or nucleic acid components of the LNPs are measured both when lipid-encapsulated and when free.

[0138] Examples of biodegradable lipids include those described in WO / 2017 / 173054, WO2015 / 095340, and WO2014 / 136086.

[0139] Lipid clearance may be measured as described in the literature. See Maier, MA, et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21(8), 1570-78 ("Maier"). For example, in Maier, a 0.3 mg / kg LNP-siRNA system containing luciferase-directed siRNA was administered intravenously via a lateral tail vein to 6-8 week-old male C57Bl / 6 mice. Blood, liver, and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 hours after administration. Mice were perfused with saline, tissues were harvested, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. Furthermore, Maier describes a method for evaluating toxicity after administration of LNP-siRNA formulations. For example, luciferase-directed siRNA was administered to male Sprague-Dawley rats at 0 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, and 10 mg / kg (5 animals / group) via a single intravenous bolus injection in a dose volume of 5 mL / kg. After 24 hours, approximately 1 mL of blood was obtained from the jugular vein of conscious animals, and serum was isolated. Seventy-two hours after administration, all animals were euthanized for necropsy. Clinical signs, body weight, serum chemistry, organ weight, and histopathology were evaluated. Maier describes methods for evaluating siRNA-LNP formulations, and these methods may be applied to assess clearance, pharmacokinetics, and toxicity associated with the administration of the LNP compositions of the present disclosure.

[0140] Lipids can enhance clearance rates. In some embodiments, the clearance rate is a lipid clearance rate, e.g., the rate at which lipids are cleared from blood, serum, or plasma. In some embodiments, the clearance rate is an RNA clearance rate, e.g., the rate at which mRNA or gRNA is cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNPs are cleared from blood, serum, or plasma. In some embodiments, the clearance rate is the rate at which LNPs are cleared from tissues, such as liver tissue or spleen tissue. In certain embodiments, a high clearance rate results in a safety profile with substantially no adverse effects. Amine lipids and biodegradable lipids can reduce accumulation of LNPs in the circulation and in tissues. In some embodiments, reduced accumulation of LNPs in the circulation and in tissues results in a safety profile with substantially no adverse effects.

[0141] Lipid can be ionized according to the pH of the medium that contains it.For example, in a slightly acidic medium, lipid such as amine lipid can be protonated, and therefore can be positively charged.On the other hand, in a slightly basic medium, such as blood, whose pH is about 7.35, lipid such as amine lipid can not be protonated, and therefore can not be charged.

[0142] The ability of a lipid to carry a charge 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 bioavailable lipids of the present disclosure may independently have a pKa in the range of about 5.1 to about 7.4. 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. Lipids with a pKa in the range of about 5.1 to about 7.4 are effective in delivering cargo in vivo, e.g., to the liver. Furthermore, lipids with a pKa in the range of about 5.3 to about 6.4 have been found to be effective in delivering cargo in vivo, e.g., to tumors. See, e.g., WO2014 / 136086.

[0143] More lipids Suitable "neutral lipids" for use in the lipid compositions of the present disclosure include, for example, a wide variety of neutral lipids, uncharged lipids, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PM ... -stearoylphosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid may be distearoylphosphatidylcholine (DSPC).

[0144] "Helper lipids" include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid can be cholesterol. In one embodiment, the helper lipid can be cholesterol hemisuccinate.

[0145] A "stealth lipid" is a lipid that alters the length of time that nanoparticles can persist in vivo (e.g., in blood). Stealth lipids can aid in formulation processes, for example, by inhibiting particle aggregation and controlling particle size. Stealth lipids used herein can modulate the pharmacokinetic properties of LNPs. Stealth lipids suitable for use in the lipid compositions of the present disclosure include, but are not limited to, stealth lipids having a hydrophilic head group linked to the lipid moiety. Information regarding stealth lipids suitable for use in the lipid compositions of the present disclosure and the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Further suitable PEG-lipids are disclosed, for example, in WO 2006 / 007712.

[0146] In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from PEG-based polymers. The stealth lipid may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

[0147] In one embodiment, the stealth lipid comprises a polymer moiety selected from PEG-based polymers (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly[N-(2-hydroxypropyl)methacrylamide].

[0148] In one embodiment, the PEG-lipid comprises a PEG-based polymer moiety (sometimes referred to as poly(ethylene oxide)).

[0149] The PEG-lipid further comprises a lipid moiety. In some embodiments, the lipid moiety can be derived from a diacylglycerol or diacylglycamide, including those comprising, independently, a dialkylglycerol or dialkylglycamide group having an alkyl chain length of from about C4 to about C40 saturated or unsaturated carbon atoms, where such chain may comprise one or more functional groups, such as, for example, an amide or ester. In some embodiments, the alkyl chain length comprises from about C10 to C20. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups. The chain length can be symmetric or asymmetric.

[0150] Unless otherwise specified, the term "PEG," as used herein, refers to any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, PEG is substituted, for example, with one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. In one embodiment, the term includes PEG copolymers such as PEG-polyurethane or PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the PEG has a molecular weight of about 130 to about 50,000, in a subembodiment, about 150 to about 30,000, in a subembodiment, about 150 to about 20,000, in a subembodiment, about 150 to about 15,000, in a subembodiment, about 150 to about 10,000, in a subembodiment, about 150 to about 6,000, in a subembodiment, about 150 to about 5,000, in a subembodiment, about 150 to about 4,000, in a subembodiment, about 150 to about 3,000, in a subembodiment, about 300 to about 3,000, in a subembodiment, about 1,000 to about 3,000, and in a subembodiment, about 1,500 to about 2,500.

[0151] In certain embodiments, the PEG (e.g., attached to a lipid moiety or lipid, such as a stealth lipid) is "PEG-2K," also known as "PEG 2000," and has an average molecular weight of about 2,000 daltons. PEG-2K is herein represented by the following formula (I): [ka] where n is 45, meaning the number average degree of polymerization contains about 45 subunits. However, other PEG embodiments known in the art may be used, including, for example, embodiments containing a number average degree of polymerization of about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n may be from about 30 to about 60. In some embodiments, n may be from about 35 to about 55. In some embodiments, n may be from about 40 to about 50. In some embodiments, n may be from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

[0152] In any of the embodiments described herein, the PEG lipid may be PEG-dilaurylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (Cat. No. GM-020, manufactured by NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (Cat. No. DSPE-020CN, manufactured by NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristoylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycerol. glycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (catalog number 880150P, Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (catalog number 880120C, Avanti Polar The PEG lipid may be selected from PEG2k-DMG, PEG2k-DSG (manufactured by PEG2k-DSM, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, manufactured by NOF, Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA.In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027, which is disclosed in WO2016 / 010840 (paragraphs

[0240] to

[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.

[0153] LNP formulation LNPs may contain (i) a biodegradable lipid, (ii) an optional neutral lipid, (iii) a helper lipid, and (iv) a stealth lipid such as a PEG lipid. LNPs may contain a biodegradable lipid and one or more of a neutral lipid, a helper lipid, and a stealth lipid such as a PEG lipid.

[0154] LNPs may contain (i) amine lipids for encapsulation and endosomal escape, (ii) neutral lipids for stabilization, (iii) stabilizing and helper lipids, and (iv) stealth lipids such as PEG lipids. LNPs may contain amine lipids and one or more of neutral lipids, stabilizing and helper lipids, and stealth lipids such as PEG lipids.

[0155] In some embodiments, the LNP composition may comprise an RNA component including one or more of an RNA-guided DNA-binding factor, a Cas nuclease mRNA, a Class 2 Cas nuclease mRNA, a Cas9 mRNA, and a gRNA. In some embodiments, the LNP composition may comprise a Class 2 Cas nuclease and a gRNA as RNA components. In certain embodiments, the LNP composition may comprise an RNA component, an amine lipid, a helper lipid, a neutral lipid, and a stealth lipid. In certain LNP compositions, the helper lipid is cholesterol. In other compositions, the neutral lipid is DSPC. In further embodiments, the stealth lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the LNP composition comprises lipid A or a lipid A equivalent, a helper lipid, a neutral lipid, a stealth lipid, and a guide RNA. In certain compositions, the amine lipid is lipid A. In certain compositions, the amine lipid is lipid A or its acetal analog, the helper lipid is cholesterol, the neutral lipid is DSPC, and the stealth lipid is PEG2k-DMG.

[0156] In certain embodiments, the lipid composition is expressed according to the respective molar ratios of the component lipids in the formulation. Embodiments of the present disclosure provide lipid compositions expressed according to the respective molar ratios of the component lipids in the formulation. In one embodiment, the molar % of the amine lipids may be about 30 mol% to about 60 mol%. In one embodiment, the molar % of the amine lipids may be about 40 mol% to about 60 mol%. In one embodiment, the molar % of the amine lipids may be about 45 mol% to about 60 mol%. In one embodiment, the molar % of the amine lipids may be about 50 mol% to about 60 mol%. In one embodiment, the molar % of the amine lipids may be about 55 mol% to about 60 mol%. In one embodiment, the molar % of the amine lipids may be about 50 mol% to about 55 mol%. In one embodiment, the molar % of the amine lipids may be about 50 mol%. In one embodiment, the molar % of the amine lipids may be about 55 mol%. In some embodiments, the mole % of amine lipids in an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole %. In some embodiments, the mole % of amine lipids in an LNP batch will be ±4 mole %, ±3 mole %, ±2 mole %, ±1.5 mole %, ±1 mole %, ±0.5 mole %, or ±0.25 mole % of the target mole %. All mole % values ​​are given as a percentage of the lipid component of the LNP composition. In certain embodiments, the LNP lot-to-lot variation in the mole % of amine lipids will be less than 15%, less than 10%, or less than 5%.

[0157] In one embodiment, the mole % of neutral lipids may be about 5 mole % to about 15 mole %. In one embodiment, the mole % of neutral lipids may be about 7 mole % to about 12 mole %. In one embodiment, the mole % of neutral lipids may be about 9 mole %. In some embodiments, the mole % of neutral lipids for an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the mole % of the target neutral lipid. In certain embodiments, LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0158] In one embodiment, the mole % of helper lipid may be about 20 mole % to about 60 mole %. In one embodiment, the mole % of helper lipid may be about 25 mole % to about 55 mole %. In one embodiment, the mole % of helper lipid may be about 25 mole % to about 50 mole %. In one embodiment, the mole % of helper lipid may be about 25 mole % to about 40 mole %. In one embodiment, the mole % of helper lipid may be about 30 mole % to about 50 mole %. In one embodiment, the mole % of helper lipid may be adjusted based on the concentrations of amine lipids, neutral lipids, and PEG lipids to achieve a lipid component of 100 mole %. In some embodiments, the mole % of helper in an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the mole % of the target. In certain embodiments, LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0159] In one embodiment, the mole % of PEG lipids may be about 1 mole % to about 10 mole %. In one embodiment, the mole % of PEG lipids may be about 2 mole % to about 10 mole %. In one embodiment, the mole % of PEG lipids may be about 2 mole % to about 8 mole %. In one embodiment, the mole % of PEG lipids may be about 2 mole % to about 4 mole %. In one embodiment, the mole % of PEG lipids may be about 2.5 mole % to about 4 mole %. In one embodiment, the mole % of PEG lipids may be about 3 mole %. In one embodiment, the mole % of PEG lipids for an LNP batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mole % of PEG lipid. In certain embodiments, LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0160] In certain embodiments, the cargo includes an mRNA encoding an RNA-guided DNA-binding factor (e.g., a Cas nuclease, a class 2 Cas nuclease, or Cas9), and a gRNA or a nucleic acid encoding the gRNA, or a combination of an mRNA and a gRNA. In one embodiment, the LNP composition may include lipid A or an equivalent thereof. In some aspects, the amine lipid is lipid A. In some aspects, the amine lipid is a lipid A equivalent, e.g., an analog of lipid A. In certain aspects, the amine lipid is an acetal analog of lipid A. In various embodiments, the LNP composition includes an amine lipid, a neutral lipid, a helper lipid, and a PEG lipid. In certain embodiments, the helper lipid is cholesterol. In certain embodiments, the neutral lipid is DSPC. In specific embodiments, the PEG lipid is PEG2k-DMG. In some embodiments, the LNP composition may include lipid A, a helper lipid, a neutral lipid, and a PEG lipid. In some embodiments, the LNP composition includes an amine lipid, DSPC, cholesterol, and a PEG lipid. In some embodiments, the LNP composition comprises a PEG-lipid comprising DMG. In certain embodiments, the amine lipid is selected from lipid A and lipid A equivalents, such as acetal analogs of lipid A. In further embodiments, the LNP composition comprises lipid A, cholesterol, DSPC, and PEG2k-DMG.

[0161] Embodiments of the present disclosure also provide lipid compositions expressed according to the molar ratio of the positively charged amine groups (N) of the amine lipids to the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This can be mathematically represented by the formula N / P. In some embodiments, an LNP composition can include a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid, and a nucleic acid component, wherein the N / P ratio is about 3-10. In some embodiments, an LNP composition can include a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid, and an RNA component, wherein the N / P ratio is about 3-10. In one embodiment, the N / P ratio can be about 5-7. In one embodiment, the N / P ratio can be about 4.5-8. In one embodiment, the N / P ratio can be about 6. In one embodiment, the N / P ratio can be 6±1. In one embodiment, the N / P ratio can be about 6±0.5. In some embodiments, the N / P ratio will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target N / P ratio. In certain embodiments, the LNP lot-to-lot variation will be less than 15%, less than 10%, or less than 5%.

[0162] In some embodiments, the RNA component may comprise an mRNA, such as an mRNA encoding a Cas nuclease. In one embodiment, the RNA component may comprise a Cas9 mRNA. In some compositions comprising an mRNA encoding a Cas nuclease, the LNP further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the RNA component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the RNA component comprises a Class 2 Cas nuclease mRNA and a gRNA.

[0163] In certain embodiments, an LNP composition may comprise an 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 LNP compositions comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the neutral lipid is DSPC. In further embodiments comprising an mRNA encoding a Cas nuclease, such as a Class 2 Cas nuclease, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain compositions comprising an 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.

[0164] 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 LNP compositions including a gRNA, the helper lipid is cholesterol. In some compositions including a gRNA, the neutral lipid is DSPC. In further embodiments including 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.

[0165] In one embodiment, the LNP composition may comprise an sgRNA. In one embodiment, the LNP composition may comprise a Cas9 sgRNA. In one embodiment, the LNP composition may comprise a Cpf1 sgRNA. In some compositions comprising an sgRNA, the LNP comprises an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an sgRNA, the helper lipid is cholesterol. In other compositions comprising an sgRNA, the neutral lipid is DSPC. In further embodiments comprising an sgRNA, the PEG lipid is PEG2k-DMG or PEG2k-C11. In certain embodiments, the amine lipid is selected from lipid A and its equivalents, such as acetal analogs of lipid A.

[0166] In certain embodiments, the LNP composition comprises an mRNA encoding a Cas nuclease and a gRNA, which may be an sgRNA. In one embodiment, the LNP composition may comprise an amine lipid, an mRNA encoding a Cas nuclease, a gRNA, a helper lipid, a neutral lipid, and a PEG lipid. In certain compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the helper lipid is cholesterol. In some compositions comprising an mRNA encoding a Cas nuclease and a gRNA, the neutral lipid is DSPC. In further embodiments comprising an 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.

[0167] In certain embodiments, an LNP composition includes a Cas nuclease mRNA, such as a Class 2 Cas mRNA, and at least one gRNA. In certain embodiments, an LNP composition includes a gRNA to a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA, in a ratio of about 25:1 to about 1:25. In certain embodiments, an LNP formulation includes a gRNA to a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA, in a ratio of about 10:1 to about 1:10. In certain embodiments, an LNP formulation includes a gRNA to a Cas nuclease mRNA, such as a Class 2 Cas nuclease mRNA, in a ratio of about 8:1 to about 1:8. Ratios measured herein are by weight. In some embodiments, an LNP formulation includes a gRNA to a Cas nuclease mRNA, such as a Class 2 Cas mRNA, in a ratio of about 5:1 to about 1:5. In some embodiments, the ratio ranges are about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, or about 2:1 to 1:1. In some embodiments, the ratio of gRNA to mRNA is about 3:1 or about 2:1. In some embodiments, the ratio of gRNA to Cas nuclease mRNA, such as Class 2 Cas nuclease, is about 1:1. The ratio may be about 25:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, 1:10, or 1:25.

[0168] The LNP compositions disclosed herein can include a template nucleic acid. The template nucleic acid can be combined with an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease mRNA. In some embodiments, the template nucleic acid can be combined with a guide RNA. In some embodiments, the template nucleic acid can be combined with both an mRNA encoding a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid can be formulated separately from the mRNA encoding the Cas nuclease or the guide RNA. The template nucleic acid can be delivered with or separately from the LNP composition. In some embodiments, the template nucleic acid can be single-stranded or double-stranded depending on the desired repair mechanism. The template can have regions of homology to the target DNA or sequences flanking the target DNA.

[0169] In some embodiments, LNPs are formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution, such as 100% ethanol. Suitable solutions or solvents may include or contain water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, or isopropanol. A pharmacologically acceptable buffer may be used, for example, for in vivo administration of LNPs. In certain embodiments, a buffer is used to maintain the pH of a composition comprising LNPs at pH 6.5 or greater. In certain embodiments, a buffer is used to maintain the pH of a composition comprising LNPs at pH 7.0 or greater. In certain embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In further embodiments, the composition has a pH in the range of about 7.3 to about 7.7, or about 7.4 to about 7.6. In further embodiments, the composition has a pH of about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition may be measured with a micro pH probe. In certain embodiments, the composition includes a cryoprotectant. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as sucrose. In certain embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% cryoprotectant. In certain embodiments, the LNP composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% sucrose. In some embodiments, the LNP composition may include a buffer. In some embodiments, the buffer may include phosphate buffer (PBS), Tris buffer, citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer includes NaCl. In certain embodiments, NaCl is excluded. Exemplary amounts of NaCl can range from about 20 mM to about 45 mM. Exemplary amounts of NaCl can range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM. In some embodiments, the buffer is a Tris buffer.Exemplary amounts of Tris can range from about 20 mM to about 60 mM. Exemplary amounts of Tris can range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. In certain exemplary embodiments of the LNP composition, a Tris buffer containing 5% sucrose and 45 mM NaCl is included. In other exemplary embodiments, the composition contains about 5% w / v sucrose, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The amounts of salt, buffer, and cryoprotectant can be varied to maintain the overall osmolality of the formulation. For example, the final osmolality can be maintained below 450 mOsm / L. In further embodiments, the osmolality is between 350 mOsm / L and 250 mOsm / L. In certain embodiments, the final osmolality is 300 + / - 20 mOsm / L.

[0170] In some embodiments, microfluidic mixing, T-mixing, or cross-mixing is used. In certain aspects, flow rates, junction sizes, junction geometries, junction shapes, tubing diameters, solutions, and / or RNA and lipid concentrations may be varied. LNPs or LNP compositions may be concentrated or purified, for example, by dialysis, tangential flow filtration, or chromatography. LNPs may be stored, for example, as a suspension, emulsion, or lyophilized powder. In some embodiments, LNP compositions are stored at 2-8°C, and in certain aspects, LNP compositions are stored at room temperature. In further embodiments, LNP compositions are stored frozen, for example, at -20°C or -80°C. In other embodiments, LNP compositions are stored at temperatures ranging from about 0°C to about -80°C. Frozen LNP compositions may be thawed, for example, on ice, at 4°C, room temperature, or 25°C, prior to use. Frozen LNP compositions may be maintained at various temperatures, for example, on ice, at 4°C, room temperature, 25°C, or 37°C.

[0171] Methods for engineering stem cells, e.g., HSPCs; engineered stem cells, e.g., HSPCs The LNP compositions disclosed herein can be used in methods for engineering stem cells, such as HSPCs, for example, by in vitro CRISPR / Cas-based gene editing. In some embodiments, the engineered cell population is a CD34+ cell population. In some embodiments, methods for producing an in vitro engineered HSPC or CD34+ cell population are provided, including (a) preincubating an LNP composition for delivering Cas nuclease mRNA and gRNA with serum factors, (b) contacting the preincubated LNP composition with the HSPC or CD34+ cell population in vitro, and (c) culturing the HSPC or CD34+ cell population in vitro, thereby producing engineered HSPCs. In some embodiments, the methods involve contacting HSPCs or CD34+ cells with an LNP composition described herein according to the delivery methods described herein.

[0172] In some embodiments, engineered stem cells, such as HSPCs, are provided, e.g., engineered HSPCs or HSPC populations are provided. Such engineered cells are produced according to the methods described herein. In some embodiments, the engineered HSPCs are present in a tissue or organ, e.g., bone marrow, blood, or other tissue, within a subject, e.g., after transplantation of the engineered HSPCs.

[0173] In some of the methods and cells described herein, the cells comprise a modification, e.g., an insertion or deletion ("indel") or substitution, of a nucleotide in the target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides in the target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification comprises an indel that results in a frameshift mutation in the target sequence. In some embodiments, the modification comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a substitution of one or two nucleotides in the target sequence, hi some embodiments, the modification comprises one or more of an insertion, deletion, or substitution of a nucleotide resulting from incorporation of a template nucleic acid, e.g., any of the template nucleic acids described herein.

[0174] In some embodiments, cell populations comprising engineered cells are provided, e.g., cell populations comprising cells engineered according to the methods described herein. In some embodiments, the population comprises engineered cells cultured in vitro. In some embodiments, the population is present within a tissue or organ, such as the liver, within a subject. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more of the cells in the population are engineered. In certain embodiments, the methods disclosed herein result in an editing efficiency (or "editing rate") of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, as defined by the detection of indels. In other embodiments, the methods disclosed herein result in a DNA modification efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as defined by detection of sequence alterations, whether by insertion, deletion, substitution, or other means. In certain embodiments, the methods disclosed herein result in an editing or DNA modification efficiency level in a cell population of about 5% to about 100%, about 10% to about 50%, about 20 to about 100%, about 20 to about 80%, about 40 to about 100%, or about 40 to about 80%.

[0175] In some of the methods and cells described herein, the cells in the population comprise a modification, such as an indel or a substitution, in the target sequence. In some embodiments, the modification comprises an insertion of 1, 2, 3, 4, or 5 or more nucleotides in the target sequence. In some embodiments, the modification comprises an insertion of 1 or 2 nucleotides in the target sequence. In other embodiments, the modification comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in the target sequence. In some embodiments, the modification comprises a deletion of 1 or 2 nucleotides in the target sequence. In some embodiments, the modification results in a frameshift mutation in the target sequence. In some embodiments, the modification comprises an indel that results in a frameshift mutation in the target sequence. In some embodiments, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the engineered cells of the population contain frameshift mutations. In some embodiments, the modification comprises the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides of the target sequence. In some embodiments, the modification comprises the substitution of one or two nucleotides of the target sequence. In some embodiments, the modification comprises one or more of a nucleotide insertion, deletion, or substitution resulting from integration of a template nucleic acid, e.g., any of the template nucleic acids described herein.

[0176] Gene editing methods The methods disclosed herein may be used for in vitro gene editing of stem cells, HSPCs, or HSPC populations. In one embodiment, one or more LNP compositions described herein may be administered to stem cells, HSPCs, or HSPC populations. In one embodiment, one or more LNP compositions described herein may be contacted with stem cells, HSPCs, and HSCs or HPCs. In one embodiment, genetically engineered cells may be generated by contacting cells with an LNP composition according to the methods described herein. In some gene editing methods, the HSPCs or HSPC populations are maintained in culture. In some gene editing methods, the HSPCs or HSPC populations are transplanted into a patient. In some embodiments, the genetically engineered HSPCs reside within a tissue or organ, such as bone marrow, blood, or other tissue, within the patient, for example, after transplantation of the engineered HSPCs.

[0177] In some embodiments, the methods involve stem cells, HSPCs, or HSPC populations that are autologous with respect to the patient receiving the cells, hi some embodiments, the methods involve HSPCs or HSPC populations that are allogeneic with respect to the patient receiving the cells.

[0178] In various embodiments, the methods described herein achieve CRISPR-Cas gene editing in stem cells, HSPCs, or HSPC populations. In some embodiments, the methods further include detecting gene editing in the HSPC or HSPC population. In some embodiments, gene editing is measured as an editing rate. In some embodiments, gene editing is measured as a DNA modification rate. The methods may achieve at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% editing. The methods may achieve at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% DNA modification.

[0179] In one embodiment, an LNP composition comprising mRNA encoding a Class 2 Cas nuclease and gRNA can be administered to stem cells, HSPCs, or HSPC populations. In a further embodiment, a template nucleic acid is also introduced into cells. In a specific example, an LNP composition comprising a Class 2 Cas nuclease and sgRNA can be administered to cells.

[0180] In one embodiment, LNP compositions may be used to edit genes in stem cells, HSPCs, or HSPC populations to cause gene knockout. In one embodiment, LNP compositions may be used to edit genes in HSPCs or HSPC populations to cause gene knockdown, for example, in a cell population. Knockdown or knockout can be detected by measuring target protein levels. Knockdown or knockout can be detected by detecting target DNA. In another embodiment, LNP compositions may be used to edit genes in HSPCs or HSPC populations to cause gene correction. In a further embodiment, LNP compositions may be used to edit cells to cause gene insertion.

[0181] LNP compositions can be administered as a formulation with one or more pharmacologically acceptable additives. The term "additive" includes any component other than the compound(s) of the present disclosure, other lipid component(s), and biologically active substances. The additive may impart functional (e.g., drug release rate control) and / or non-functional (e.g., processing aid or diluent) characteristics to the formulation. The choice of additive will largely depend on factors such as the particular method of administration, the additive's effect on stem cell or HSPC culture and solubility and stability, and the nature of the dosage form.

[0182] When the formulation is aqueous, additives include sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably pH 3-9), although for some applications these may be more suitably formulated using sterile non-aqueous solutions or as a dry form for use in conjunction with a suitable vehicle such as sterile pyrogen-free water (WFI).

[0183] While the present invention will be described in conjunction with exemplary embodiments, it will be understood that they are not intended to limit the invention to the described embodiments. Rather, the present invention is intended to encompass all alternatives, modifications, and equivalents, including equivalents of specific features, which may be included within the scope of the present invention as defined by the appended claims.

[0184] Both the general and detailed descriptions set forth above, as well as the following examples, are illustrative and explanatory only and are not intended to be limiting of the teachings. The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any way. In the event that a document incorporated by reference conflicts with a term defined herein, the present specification controls. All ranges set forth in this application include endpoints unless otherwise specified.

[0185] It should be noted that, as used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a composition" includes a plurality of compositions, a reference to "a cell" includes a plurality of cells, and so on. The use of "or" is inclusive and means "and / or" unless specifically stated otherwise.

[0186] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximations, taking into account significant digits and error associated with the measurements. The terms "about" or "approximately" refer to an acceptable error for a particular value as determined by one of ordinary skill in the art, which may vary somewhat depending on how the value is measured or determined. The use of modifiers such as "about" before a range or a recited value modifies each endpoint of the range or each value in the recited value. For example, "about 50 to 55" includes "about 50 to about 55." Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not limiting.

[0187] Unless otherwise noted in the specification above, embodiments described herein as "comprising" various components are also intended to be "consisting of" or "consisting essentially of" the stated components; embodiments described herein as "consisting of" various components are also intended to be "comprising" or "consisting essentially of" the stated components; embodiments described herein as "about" various components are also intended to be "at" the stated components; and embodiments described herein as "consisting essentially of" various components are also intended to be "consisting of" or "comprising" the stated components (such interchangeability does not apply when using these terms in the claims). The present invention encompasses, for example, the following embodiments: [Embodiment 1] A method for delivering mRNA to a hematopoietic stem and / or progenitor cell (HSPC) or HSPC population, comprising: a. pre-incubating an LNP composition comprising the mRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG-lipid with serum factors; b. contacting the pre-incubated LNP composition with the HSPC or HSPC population in vitro; and c. culturing the HSPC or population of HSPCs in vitro; thereby delivering said mRNA to said HSPC or said population of HSPCs. [Embodiment 2] A method for delivering mRNA to HSPCs, comprising: a. pre-incubating an LNP composition comprising the mRNA and amine lipids with serum factors; b. contacting the pre-incubated LNP composition with the cells in vitro; and c. culturing the HSPCs in vitro; thereby delivering the mRNA to the HSPCs. [Embodiment 3] A method for delivering mRNA to a stem cell or stem cell population, comprising: a. pre-incubating the mRNA-containing LNP composition with serum factors; b. contacting the pre-incubated LNP composition with the stem cell population in vitro; and c. culturing the stem cell population in vitro; thereby delivering said mRNA to said stem cell population. [Embodiment 4] The method described in any one of embodiments 1 to 3, wherein the mRNA encodes a Cas nuclease. [Embodiment 5] A method for introducing Cas nuclease mRNA and gRNA into HSPCs, comprising: a. pre-incubating an LNP composition comprising the Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG-lipids with serum factors; b. contacting the pre-incubated LNP composition with the HSPCs in vitro; and c. Cultivating the HSPCs, The method thereby comprises introducing the Cas nuclease mRNA and gRNA into the HSPC. [Embodiment 6] A method for generating in vitro genetically engineered HSPCs, comprising: a. Pre-incubating an LNP composition comprising a Cas nuclease mRNA, a gRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid with a serum factor; b. contacting the pre-incubated LNP composition with the HSPCs in vitro; and c. culturing the HSPCs in vitro; thereby generating genetically engineered HSPCs. [Embodiment 7] A method for introducing Cas nuclease mRNA and gRNA into stem cells, comprising: a. pre-incubating an LNP composition comprising the Cas nuclease mRNA, gRNA, and amine lipids with a serum factor; b. contacting the pre-incubated LNP composition with the stem cells in vitro; and c. Culturing the stem cells; The method thereby comprises introducing the Cas nuclease mRNA and gRNA into the stem cell.

[0032] [Embodiment 8] A method for generating in vitro genetically engineered stem cells, such as HSPCs, comprising: a. Pre-incubating an LNP composition comprising a Cas nuclease mRNA, a gRNA, and a biodegradable lipid with a serum factor; b. contacting the pre-incubated LNP composition with the cells in vitro; and c. culturing the cells in vitro;

[0023] Embodiment 9: The method of embodiment 4, wherein the LNP composition further comprises a gRNA, thereby generating a genetically engineered stem cell, such as a HSPC. [Embodiment 10] The method described in any one of embodiments 4 to 9, wherein the Cas nuclease is a class 2 Cas nuclease. [Embodiment 11] The method of embodiment 10, wherein the Class 2 Cas nuclease is a Cas9 nuclease. [Embodiment 12] The method of embodiment 11, wherein the Cas9 nuclease is S. pyogenes Cas9. [Embodiment 13] The method described in embodiment 10, wherein the Class 2 Cas nuclease is a Cpf1 nuclease. [Embodiment 14] The method described in any one of embodiments 5 to 13, wherein the gRNA is a dual guide RNA (dgRNA). [Embodiment 15] The method described in any one of embodiments 5 to 13, wherein the gRNA is a single guide RNA (sgRNA). [Embodiment 16] The method of any preceding embodiment, further comprising a washing step after the contacting step. [Embodiment 17] The method of any preceding embodiment, wherein the contacting step takes from about 1 minute to about 72 hours. [Embodiment 18] The method of any preceding embodiment, wherein the contacting step takes from about 1 minute to about 24 hours. [Embodiment 19] The method of embodiment 17 or 18, wherein the contacting step is for about 2 hours to about 24 hours. [Embodiment 20] The method of any one of embodiments 17 to 19, wherein the contacting step is for about 4 hours to about 12 hours. [Embodiment 21] The method of any one of embodiments 17 to 20, wherein the contacting step is for about 6 hours to about 12 hours. [Embodiment 22] The method described in any of the preceding embodiments, wherein cell survival after transfection is at least 60%. [Embodiment 23] The method described in embodiment 22, wherein cell survival after transfection is at least 70%. [Embodiment 24] The method described in embodiment 22, wherein cell survival after transfection is at least 80%. [Embodiment 25] The method described in embodiment 22, wherein cell survival after transfection is at least 90%. [Embodiment 26] The method described in embodiment 22, wherein cell survival after transfection is at least 95%. [Embodiment 27] The method described in any of the preceding embodiments, further comprising pre-incubating the serum factors and the LNP composition for about 30 seconds to overnight. [Embodiment 28] The method described in embodiment 27, which comprises pre-incubating for about 1 minute to 1 hour. [Embodiment 29] The method described in embodiment 27, which comprises pre-incubating for about 1 to 30 minutes. [Embodiment 30] The method described in embodiment 27, which comprises pre-incubating for about 1 to 10 minutes. [Embodiment 31] The method of embodiment 27, comprising pre-incubating for about 5 minutes. [Embodiment 32] The method of embodiment 27 or embodiment 31, comprising pre-incubating for 5 minutes ± 2 minutes. [Embodiment 33] The method of any preceding embodiment, wherein the preincubation occurs at about 4°C. [Embodiment 34] The method of any preceding embodiment, wherein the preincubation occurs at about 25°C. [Embodiment 35] The method of any preceding embodiment, wherein the preincubation occurs at about 37°C. [Embodiment 36] The method of any preceding embodiment, wherein the pre-incubation step includes a buffer. [Embodiment 37] The method of embodiment 36, wherein the buffer comprises or consists of a medium for HSPCs. [Embodiment 38] The method of any of the preceding embodiments, wherein the LNP composition is preincubated with serum. [Embodiment 39] The method described in embodiment 38, wherein the serum is mammalian, murine, primate, or human serum. [Embodiment 40] The method described in any one of embodiments 1 to 37, wherein the LNP composition is pre-incubated with isolated serum factors. [Embodiment 41] The method described in embodiment 40, wherein the serum factor is ApoE. [Embodiment 42] The method described in embodiment 40, wherein the serum factor is selected from ApoE2, ApoE3, and ApoE4. [Embodiment 43] The method described in any one of embodiments 40 to 42, wherein the ApoE is a human recombinant protein. [Embodiment 44] The method of any of the preceding embodiments, wherein the culturing step comprises expanding the stem cells, HSPCs, or HSPC population in an HSPC culture buffer. [Embodiment 45] The method of any of the preceding embodiments, further comprising changing the medium between the contacting step and the culturing step. [Embodiment 46] The method described in any of the preceding embodiments, wherein the culturing step includes a stem cell growth factor. [Embodiment 47] The method described in any one of embodiments 1 to 2, 4 to 6, or 8 to 46, wherein the HSPC is a hematopoietic stem cell (HSC). [Embodiment 48] The method of any of the preceding embodiments, wherein the stem cells, HSPCs, or HSPC population are human cells or samples. [Embodiment 49] The method described in any one of embodiments 5 to 48, wherein the mRNA and the guide RNA nucleic acid are formulated into a single LNP composition. [Embodiment 50] A method described in any one of embodiments 5 to 48, wherein the mRNA and the gRNA are co-encapsulated in the LNP composition. [Embodiment 51] A method described in any one of embodiments 5 to 48, wherein the mRNA and the gRNA are encapsulated in separate LNPs. [Embodiment 52] The method described in any one of embodiments 5 to 48, wherein the mRNA is formulated in a first LNP composition and the guide RNA nucleic acid is formulated in a second LNP composition. [Embodiment 53] The method of embodiment 52, wherein the first and second LNP compositions are administered simultaneously. [Embodiment 54] The method of embodiment 52, wherein the first and second LNP compositions are administered sequentially. [Embodiment 55] The method of any of embodiments 52 to 54, wherein the first and second LNP compositions are combined prior to the pre-incubation step. [Embodiment 56] The method described in any one of embodiments 52 to 54, wherein the first and second LNP compositions are pre-incubated separately. [Embodiment 57] The method of any of the preceding embodiments, further comprising introducing a template nucleic acid into the cell. [Embodiment 58] The method of any of the preceding embodiments, wherein the LNP composition comprises an RNA component and a lipid component, wherein the lipid component comprises an amine lipid, a neutral lipid, a helper lipid, and a stealth lipid, and wherein the N / P ratio is about 1 to 10. [Embodiment 59] The method of embodiment 58, wherein the lipid component comprises lipid A or an acetal analog thereof. [Embodiment 60] The lipid component is about 40-60 mol % of an amine lipid; Approximately 5 to 15 mol % of neutral lipids; Approximately 1.5 to 10 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The method of embodiment 58, wherein the N / P ratio of the LNP composition is about 3-10. [Embodiment 61] The lipid component is about 50-60 mol % amine lipid; Approximately 8 to 10 mol% of neutral lipids; Approximately 2.5-4 mol% PEG lipid wherein the remainder of the lipid component is a helper lipid; The method of embodiment 58, wherein the N / P ratio of the LNP composition is about 3-8. [Embodiment 62] The lipid component is about 50-60 mol % amine lipid; about 5 to 15 mol % of DSPC; Approximately 2.5-4 mol% PEG lipid wherein the remainder of the lipid component is cholesterol; 57. The method of embodiment 56, wherein the N / P ratio of the LNP composition is about 3-8. [Embodiment 63] The lipid component is 48-53 mol% lipid A; about 8 to 10 mol % DSPC; 1.5-10 mol% PEG lipid wherein the remainder of the lipid component is cholesterol; 59. The method of embodiment 58, wherein the N / P ratio of the LNP composition is 3 to 8±0.2. [Embodiment 64] The method of any of the preceding embodiments, wherein the RNA is modified RNA. [Embodiment 65] The method described in embodiment 64, wherein the modified RNA is modified mRNA. [Embodiment 66] The method of any of the preceding embodiments, wherein the RNA comprises an open reading frame encoding an RNA-guided DNA binding factor, wherein the open reading frame has a uridine content ranging from its minimum uridine content to 150% of the minimum uridine content. [Embodiment 67] The composition of any of the preceding embodiments, wherein the RNA comprises an open reading frame encoding an RNA-guided DNA binding factor, wherein the open reading frame has a uridine dinucleotide content ranging from its minimum uridine dinucleotide content to 150% of the minimum uridine dinucleotide content. [Embodiment 68] The composition of any of the preceding embodiments, wherein the RNA comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 4, 10, 14, 15, 17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 50, 52, 54, 65, or 66, and wherein the mRNA comprises an open reading frame encoding an RNA-guided DNA binding factor. [Embodiment 69] The method described in any one of embodiments 5 to 68, wherein the gRNA is a modified gRNA. [Embodiment 70] The method described in embodiment 69, wherein the gRNA comprises a modification selected from 2'-O-methyl (2'-O-Me) modified nucleotides, internucleotide phosphorothioate (PS) linkages, and 2'-fluoro (2'-F) modified nucleotides. [Embodiment 71] The method described in embodiment 69 or 70, wherein the gRNA comprises a modification in one or more of the first five nucleotides at the 5' end. [Embodiment 72] The method described in any one of embodiments 69 to 71, wherein the gRNA comprises a modification in one or more of the last five nucleotides at the 3' end. [Embodiment 73] The method described in any one of embodiments 69 to 72, wherein the gRNA contains a PS bond between the first four nucleotides. [Embodiment 74] The method described in any one of embodiments 69 to 73, wherein the gRNA contains a PS bond between the last four nucleotides. [Embodiment 75] The method described in any one of embodiments 69 to 74, further comprising 2'-O-Me modified nucleotides in the first three nucleotides of the 5' end. [Embodiment 76] The method described in any one of embodiments 69 to 75, further comprising 2'-O-Me modified nucleotides in the last three nucleotides of the 3' end. [Embodiment 77] The method described in embodiments 1 to 2, 4 to 6, or 8 to 76, wherein the HSPC or HSPC population is CD34+. [Embodiment 78] The method described in embodiments 1 to 2, 4 to 6, or 8 to 77, wherein the HSPC or HSPC population is CD34+CD90+. [Embodiment 79] An engineered stem cell or stem cell population produced by the method of any of the preceding embodiments. [Embodiment 80] An engineered HSPC or HSPC population produced by the method of any of the preceding embodiments. [Embodiment 81] The HSPC or HSPC population of embodiment 78, wherein the engineered HSPC is present within a tissue or organ within a patient's body, e.g., bone marrow, blood, or other tissue, e.g., after transplantation of the engineered HSPC. [Embodiment 82] The method of any preceding embodiment, wherein the stem cells, HSPCs, or HSPC population are autologous with respect to the patient to whom the cells are to be administered. [Embodiment 83] The method of any preceding embodiment, wherein the stem cells, HSPCs, or HSPC population are allogeneic with respect to the patient to whom the cells are to be administered. [Embodiment 84] The method of any preceding embodiment, further comprising achieving CRISPR-Cas gene editing in the stem cell, HSPC, or HSPC population. [Embodiment 85] The method of any preceding embodiment, further comprising detecting gene editing in the stem cell, HSPC, or HSPC population. [Embodiment 86] The method of embodiment 84 or 85, wherein the gene editing is measured as an editing rate or DNA modification rate. [Embodiment 87] The method described in embodiment 86, wherein the editing rate is at least 40%. [Embodiment 88] The method described in embodiment 86, wherein the editing rate is at least 60%. [Embodiment 89] The method described in embodiment 86, wherein the editing rate is at least 70%. [Embodiment 90] The method described in embodiment 86, wherein the editing rate is at least 80%. [Embodiment 91] The method described in embodiment 86, wherein the editing rate is at least 90%. [Embodiment 92] The method described in embodiment 86, wherein the editing rate is at least 95%. [Embodiment 93] The method described in embodiment 86, wherein the DNA modification rate is at least 40%. [Embodiment 94] The method described in embodiment 86, wherein the DNA modification rate is at least 60%. [Embodiment 95] The method described in embodiment 86, wherein the DNA modification rate is at least 70%. [Embodiment 96] The method described in embodiment 86, wherein the DNA modification rate is at least 80%. [Embodiment 97] The method described in embodiment 86, wherein the DNA modification rate is at least 90%. [Embodiment 98] The method described in embodiment 86, wherein the DNA modification rate is at least 95%. [Embodiment 99] The method of any preceding embodiment, wherein the stem cells, HSPCs, or HSPC population are derived from a bone marrow sample. [Example]

[0188] Example 1 - Method cell culture Cryopreserved human CD34+ bone marrow cells were obtained from AllCells (catalog no. ABM017F) or StemCell Technologies (catalog no. 70008). After thawing and washing twice with 20 ml of StemSpan SFEM (Stem Cell technologies, catalog no. 09650), they were placed in StemSpan SFEM (StemCell Technologies, catalog no. 02922) containing thrombopoietin (TPO, 50 ng / ml; StemCell Technologies, catalog no. 02922), human Flt3 ligand (Flt3l, 50 ng / ml; StemCell Technologies, catalog no. 78137.2), human interleukin-6 (IL-6, 50 ng / ml; StemCell Technologies, catalog no. 78148.2), human stem cell factor (SCF, 50 ng / ml; StemCell Technologies, catalog no. 78155.2), and StemRegenin-1 (SR1, 0.75 μM). Cells were cultured for 48 hours in 100 μg / ml penicillin and 100 μg / ml streptomycin (P / S, 100 U / ml penicillin and 100 μg / ml streptomycin, Life Technologies, Cat. No. 15140122).

[0189] Lipid Nanoparticle ("LNP") Formulations Unless otherwise noted below, LNPs were formulated by dissolving lipid nanoparticle components in 100% ethanol at the following molar ratios: 45 mol% (12.7 mM) lipid amine (e.g., lipid A), 44 mol% (12.4 mM) helper lipid (e.g., cholesterol), 9 mol% (2.53 mM) neutral lipid (e.g., DSPC), and 2 mol% (0.563 mM) PEG lipid (e.g., PEG2k-DMG or PEG2k-C11). The N / P ratio (moles of lipid amine to moles of RNA) was 4.5. The LNP formulations were identified by the following identification numbers: LNP522, LNP525 (GFP mRNA), and LNP670, LNP926 (B2M single guide, Cas9 mRNA), and LNP899 (AAVS1 single guide, Cas9 mRNA). The RNA cargo was dissolved in 50 mM acetate buffer (pH 4.5) or 25 mM sodium citrate, 100 mM NaCl (pH 5.0) to a concentration of approximately 0.45 mg / mL of RNA cargo.

[0190] LNPs were generated by microfluidic mixing of lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument according to the manufacturer's operating instructions. Different flow rates were used during mixing, while maintaining a 2:1 aqueous-to-organic solvent ratio. After mixing, LNPs were collected and diluted (approximately 1:1) into phosphate-buffered saline (pH 7.4) (PBS) or 50 mM Tris (pH 7.5) (Tris) to reduce the ethanol content prior to further processing. Final buffer exchange was completed by dialysis overnight at 4°C under gentle agitation against PBS or Tris (100-fold excess of sample volume) using a 10 kDa Slide-a-Lyzer™ G2 Dialysis Cassette (ThermoFisher Scientific). Tris-treated formulations were diluted 1:1 into 100 mM Tris, 90 mM saline, 5% (w / v) sucrose, pH 7.5 (2x TSS). Alternatively, the LNP was collected after mixing, diluted in water, and held at room temperature for 1 hour before a second 1:1 dilution with water. Final buffer exchange into TSS was completed using a PD-10 desalting column (GE). If necessary, formulations from either process were concentrated by centrifugation in an Amicon 100 kDa centrifugal filter (Millipore). The resulting mixture was then filtered using a 0.2 μm sterile filter. The resulting filtrate was stored at 2–8°C if the final buffer was PBS or at -80°C if the final buffer was TSS.

[0191] In vitro transcription ("IVT") of nuclease mRNA and single guide RNA (sgRNA) Capped, polyadenylated Cas9 mRNA was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing a T7 promoter and a 100-residue poly(A / T) tract was linearized by incubation with XbaI at 37°C for 2 hours using the following conditions: 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1× reaction buffer. The reaction was heated at 65°C for 20 minutes to inactivate XbaI. The linearized plasmid was purified from the enzyme and buffer salts using a silica Maxi spin column (Epoch Life Sciences) and analyzed on an agarose gel to confirm linearization. IVT reactions to generate Cas9-modified mRNA were incubated for 4 hours at 37°C with the following conditions: 50 ng / μL linearized plasmid; 2 mM each of GTP, ATP, CTP, and N1-methylpseudo-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 using LiCl precipitation.

[0192] For all methods, transcript concentrations were determined by measuring absorbance at 260 nm (Nanodrop), and transcripts were analyzed by capillary electrophoresis with a Bioanlayzer (Agilent). sgRNA was chemically synthesized.

[0193] LNP transfection of human CD34+ bone marrow cells LNPs containing either GFP mRNA or Cas9 mRNA and a single guide targeting beta2-microglobulin (B2M) were added to 30,000 human CD34+ bone marrow cells at various concentrations ranging from 50.0 ng to 800.0 ng in a total volume of 100.0 μL. The sgRNA sequence targeting the B2M target sequence GGCCACGGAGCGAGACATCT (SEQ ID NO: 75) was mG*mG*mC*CACGGAGCGAGACAUCUGUUUUAGAmGmCmUmAmGmAmAmAmUmAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAmAmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 76). In this nucleic acid sequence, A, U, G, and C represent adenine, uracil, cytosine, and guanine, respectively, "m" indicates a 2'-O-methyl nucleotide, and "*" indicates a phosphorothioate bond.

[0194] For species-specific serum studies (Triple S studies), LNPs were incubated with 6.0% serum from M. musculus (Bioreclamation IVT, catalog no. MSESRM, lot no. MSE245821), M. fascicularis (Bioreclamation IVT, catalog no. CYNSRM, lot no. CYN197451), and H. sapiens (Sigma, pooled, H4522 ~ 20 ml, lot no. SLBR7629V; Bioreclamation IVT, catalog no. HMSRM, lot no. BRH1278638; Bioreclamation IVT, catalog no. HMSRM, lot no. BRH1227947) for 5 min at 37°C prior to cell transfection. Human recombinant apolipoprotein E3 (ApoE3, R&D Systems, Cat. No. 4144-AE) was used at a range of concentrations (01.0 μg / ml, 1.0 μg / ml, 10.0 μg / ml, and 50.0 μg / ml) in the recommended buffer under the same incubation conditions as above.

[0195] Flow cytometry reading of LNP-transfected human CD34+ bone marrow cells Cells were harvested for antibody staining 24 hours after LNP-GFP transfection or 5 days after LNP-B2M transfection. After washing with sample medium (PBS + 2% FBS + 2 mM EDTA), cells were blocked with Human TruStain FcX (Biolegend, Cat. No. 422302) for 5 minutes at room temperature (RT).

[0196] Cells were stained with the antibodies and labels shown in Table 2 below. [Table 2]

[0197] Cells were subjected to Beckman Coulter CytoflexS and analyzed using the FlowJo software package.

[0198] Cell viability was assessed by 7-AAD staining. Live cells were normalized based on 7-AAD intercalation in GC-rich DNA regions, followed by detection in a flow cytometry assay. Cell viability was calculated using the following formula: [(Sample 1 細胞イベント数 / Sample 1 ビーズイベント数 )*Sample 1 総添加ビーズ数 ] / average {[(control 1 細胞イベント数 / Control 1 ビーズイベント数 )*Control 1 総添加ビーズ数 ], [(Control 2 細胞イベント数 / Control 1 ビーズイベント数 )*Control 2 総添加ビーズ数 ], …, Control N}

[0199] In this formula, a "sample" is defined as any population of human CD34+ HSPCs that has been treated with LNPs, mRNA, gRNA, or any combination of the former for the duration of the experiment, and a "control" is defined as any population of human CD34+ HSPCs that has not been treated with LNPs, mRNA, gRNA, or any combination of the former for the duration of the experiment.

[0200] Next Generation Sequencing ("NGS") and Cleavage Efficiency Analysis To quantitatively determine the editing efficiency at the target locations in the genome, deep sequencing was used to identify the presence of insertions and deletions introduced by gene editing.

[0201] Cells were harvested 5 days post-transfection, and DNA was extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher Scientific, catalog no. K182002). Primers for the B2M target locus containing Illumina P5 and P7 adapter sequences were used to amplify the genomic site of interest in a standard PCR reaction.

[0202] PCR primers were designed around the B2M target site to amplify the genomic region of interest. Samples were subjected to sample preparation (Illumina MiSeq v2 Reagent Kit, 300 cycles, catalog number 15033624) and sequenced on an Illumina MiSeq instrument. Editing frequencies at the target loci of interest were analyzed using a custom pipeline. Briefly, additional PCR was performed according to the manufacturer's operating instructions (Illumina), and the necessary sequencing chemistry was added. Amplicons were sequenced on an Illumina MiSeq instrument. After removing low-quality scores, reads were aligned to a human reference genome (e.g., hg38). The resulting file containing the reads was mapped to the reference genome (BAM file), where reads that overlapped with the target region of interest were selected, and the ratio of wild-type reads to reads containing insertions, substitutions, or deletions was calculated.

[0203] The editing ratio (e.g., "editing efficiency" or "editing rate") is given as the total number of sequence reads containing an insertion or deletion relative to the total number of sequence reads, such as the wild type.

[0204] Pharmaceutical Analysis Methods LNP formulations are analyzed for mean particle size, polydispersity (pdi), total RNA content, and RNA encapsulation efficiency. Mean particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument. LNP samples are diluted 30X in PBS prior to DLS measurement. Z-mean diameter, an intensity-based measure of mean particle size, is reported along with number-mean diameter and pdi.

[0205] A fluorescence-based assay (Ribogreen®, ThermoFisher Scientific) was used to determine total RNA concentration and free RNA. Encapsulation efficiency was calculated as (total RNA - free RNA) / total RNA. LNP samples were appropriately diluted with 1x TE buffer containing 0.2% Triton-X 100 to determine total RNA, or with 1x TE buffer to determine free RNA. A standard curve was generated using the starting RNA solution used to make the formulation and then diluted in 1x TE buffer + / - 0.2% Triton-X 100. Diluted RiboGreen® dye (100x in 1x TE buffer according to the manufacturer's instructions) was then added to each standard and sample and incubated for 10 minutes at room temperature in the absence of light. Samples were read using a SpectraMax M5 plate reader (Molecular Devices) with excitation, autocutoff, and emission wavelengths set at 488 nm, 515 nm, and 525 nm, respectively. Total RNA and free RNA are determined using appropriate standard curves. The encapsulation rate is calculated as (total RNA - free RNA) / total RNA. A similar approach may be used to determine the encapsulation rate of DNA-based cargo components. Oligreen dye may be used for single-stranded DNA, and Picogreen dye may be used for double-stranded DNA.

[0206] Example 2 - Delivery of GFP to CD34+ bone marrow cells LNPs were formulated as described in Example 1 with GFP mRNA dissolved in PBS final buffer and added to 30,000 human CD34+ bone marrow cells in a total volume of 100.0 μl, yielding 0, 50.0 ng, 100.0 ng, and 200.0 ng of GFP mRNA in various reactions. Prior to administration to the cells, LNPs were preincubated with 6% (v / v) serum from M. musculus (Bioreclamation IVT, catalog number MSESRM, lot number MSE245821) at 37°C for 5 minutes. Cells were cultured as described in Example 1.

[0207] Twenty-four hours after LNP addition to human CD34+ cells, GFP+ cells were quantified by flow cytometry. The GFP+ cell population relative to the GFP- control (labeled "Control" in Figure 1) was determined using the FITC channel (excitation max 490, emission max 525, laser line 488). Figure 1 shows the percentage of GFP+ cells among total live cells in human CD34+ bone marrow cells 24 hours after LNP-mediated GFP mRNA delivery. LNP composition was as follows: Figure 1(A): 45% lipid A, 44% cholesterol, 9% DSPC, 2% PEG; Figure 1(B): 45% lipid A, 45% cholesterol, 9% DSPC, 1% PEG. Number of biological samples: n = 3.

[0208] The LNP composition exhibits dose-dependent mRNA delivery to CD34+ bone marrow cells in vitro.

[0209] Example 3 - Pre-incubation of LNPs enhances delivery The test was performed using human CD34 + We show that LNPs require incubation with 6% mouse serum (v / v) prior to transfection to efficiently deliver GFP mRNA to bone marrow cells. Cells were cultured and transfected with the LNP composition as described in Example 2 with the following modifications.

[0210] Figure 2A shows human CD34 cells supplemented with LNPs on day 0, immediately after thawing a vial of cryopreserved cells. + The percentage of GFP+ cells among all live cells in bone marrow samples is shown. LNPs with 50.0 ng, 100.0 ng, or 200.0 ng of GFP mRNA were added to cells with or without serum incubation prior to transfection.

[0211] Figure 2B shows the results of LNP-supplemented human CD34 cells on day 2 after thawing a vial of cryopreserved cells. + The percentage of GFP+ cells among all live cells in bone marrow samples is shown. LNPs with 50.0 ng, 100.0 ng, or 200.0 ng of GFP mRNA were added to cells with or without serum incubation before transfection. Number of biological samples: n=3. Example 4 - LNP-mediated delivery of Cas9 and guide RNA in CD34+ bone marrow cells Gene editing

[0212] LNPs were formulated using sgRNA (G529) and Cas9 mRNA as described in Example 1 in a 1:1 weight ratio in TSS final buffer as described in Example 1. The LNP composition was 45% lipid A, 44% cholesterol, 9% DSPC, and 2% PEG, with an N / P ratio of 4.5.

[0213] Human CD34 + Using the LNP delivery method to transfect bone marrow cells, Cas9 mRNA and B2M sgRNA were efficiently delivered into cells by preincubating M. musculus or M. fascicularis with increasing serum percentages (v / v). Active Cas9-sgRNA complexes were delivered by LNPs preincubated with various sera.

[0214] Figure 3A shows FACS analysis of transfected cells, showing the percentage of B2M-negative cells after addition of LNPs (400.0 ng of Cas9 mRNA and sgRNA (1:1 by weight)) incubated with mouse serum ("Mouse-S") at 6%, 30%, and 60% (v / v) or non-human primate serum ("Cyno-S") at 6%, 30%, and 60% (v / v). LNPs without serum preincubation ("LNP only") and untreated cells ("control") are used as indicators of inefficient delivery (measure of B2M expression knockdown). Preincubation with mouse or primate serum increases CD34 expression. + This facilitates efficient knockdown of B2M expression in myeloid cells.

[0215] Figure 3B shows genome-level editing frequencies, as determined by NGS, for human CD34+ bone marrow cells transfected with LNPs (400.0 ng of Cas9 mRNA and sgRNA (1:1 by weight)) incubated with mouse serum at 6%, 30%, and 60% (v / v) or non-human primate serum at 6%, 30%, and 60% (v / v). As shown in Figure 3(A), LNPs without serum preincubation and untreated cells do not demonstrate efficient delivery (measured as % editing). Insertions ("In", light gray) and deletions ("Del", black) are graphed on the Y-axis, and CD34 + The cells exhibited editing efficiencies of greater than about 60%, greater than about 70%, greater than about 80%, and greater than about 90%. The "LNP only" and "control" samples did not exhibit detectable levels of indels at the B2M locus. Number of biological samples: n=3.

[0216] Example 5 - Preincubation with isolated serum factor ApoE3 To investigate whether the serum pre-incubation step could be replaced with recombinant proteins, LNPs as described in Example 4 delivering Cas9 and B2M sgRNA were pre-incubated with human recombinant apolipoprotein E3 (ApoE3), mouse serum, or non-human primate serum during the LNP incubation step prior to cell transfection.

[0217] Figure 4A shows the percentage of B2M-negative cells after addition of LNPs (400 ng of Cas9 mRNA and sgRNA (1:1)) incubated with mouse serum ("mouse-S") at 6% (v / v), non-human primate serum ("cyno-S") at 6% (v / v), or ApoE3 at 0.1 μg / ml, 1.0 μg / ml, 10.0 μg / ml, and 50.0 μg / ml. Naive human CD34 + Bone marrow cells are used as a negative control ("Control"). ApoE3 has shown a dose-dependent increase in delivery to CD34+ cells, which can be used in the pre-incubation step.

[0218] Similarly, gene editing shows a dose-dependent response to ApoE3. Figure 4B shows the percentage editing of B2M targets determined by NGS for human CD34+ bone marrow cells transfected with 400.0 ng of LNPs incubated with 6% (v / v) mouse serum, 6% (v / v) non-human primate serum, or ApoE3 at 0.1 μg / ml, 1.0 μg / ml, 10.0 μg / ml, and 50.0 μg / ml. Untreated human CD34 + Bone marrow cells are used as a negative control, showing no detectable levels of indels at the B2M locus. Number of biological samples: n=3.

[0219] Example 6 - Preincubation with serum factors In this experiment, we tested pre-incubation of LNPs with a variety of different apolipoproteins and demonstrate in vitro LNP uptake with ApoE isoforms, measured as B2M knockdown levels and editing frequencies in HSPC populations. Prior to transfection, LNP (identification number LNP926) was incubated with 6% (v / v) M. fascicularis serum or various concentrations of the following apolipoproteins at 37°C for 5 min: recombinant human ApoA-I (Millipore Sigma, catalog number SRP4693), human plasma-derived ApoB (Millipore Sigma, catalog number A5353), human plasma-derived ApoC-I (Millipore Sigma, catalog number A7785), human recombinant ApoE2 (Millipore Sigma, catalog number SRP4760), human recombinant ApoE3 (Millipore Sigma, catalog number SRP4696), and human recombinant ApoE4 (Millipore Sigma, catalog number A3234). LNP was added to human CD34+ myeloid cells at a concentration of 200 ng of total RNA cargo (1:1 w / w ratio of Cas9 mRNA to single guide). On day 5 post-transfection, B2M expression at the protein level was determined by flow cytometry using the same antibodies as above. Data analysis was performed using the FlowJo software package. Data represent the mean + / - SD of replicate experiments from one biological sample (N=1).

[0220] Figure 5 shows B2M knockdown in CD34+ HSPC populations after transfection with LNPs preincubated with cynomolgus monkey (cyno) serum, ApoE2, ApoE3, and ApoE4. Untreated, no preincubation, and preincubation with ApoA-I, ApoB, and ApoC-I did not result in B2M knockdown. In this experiment, LNP preincubation with ApoE2 showed lower B2M knockdown compared to the other two isoforms of ApoE.

[0221] Example 7 - Time course of LNP exposure In this experiment, the duration of LNP exposure was examined for its effect on viability and editing rates. LNP899, delivering Cas9 mRNA targeting AAVS1 and G562, was preincubated with 6% (v / v) non-human primate serum for approximately 5 minutes at 37°C. LNPs were added to human CD34+ bone marrow cells at a concentration of 300 ng of total RNA cargo (1:1 w / w ratio of Cas9 mRNA to single guide). At 2, 6, or 24 hours post-transfection, cells were centrifuged and resuspended in fresh medium without LNP. Cell viability was assessed on days 3 and 8 using CountBright™ Absolute Counting Beads (Invitrogen, catalog C36950) on a CytoFLEXS flow cytometer (Beckman Coulter). Editing was measured by next-generation sequencing (NGS) as described in Example 1. Table 3 and Figure 6B show the editing frequency at day 8 post-transduction. Table 3 and Figure 6A show cell viability at days 3 and 8 after transduction. [Table 3] [Table 4] TIFF0007803917000009.tif243151TIFF0007803917000010.tif253160TIFF0007803917000011.tif60162

[0222] See the sequence listing below for the sequences themselves. The transcript sequences generally include GGG as the first three nucleotides for use with ARCA or AGG as the first three nucleotides for use with CleanCap™. Thus, the first three nucleotides can be modified for use with other capping methods, such as vaccinia capping enzyme. Promoters and polyA sequences are not included in the transcript sequences. Promoters such as the T7 promoter (SEQ ID NO: 31) and polyA sequences such as SEQ ID NO: 63 can be added to the disclosed transcript sequences at the 5' and 3' ends, respectively. While most nucleotide sequences are provided as DNA, they can be easily converted to RNA by changing T to U.

[0223] Sequence Listing The sequence listing below provides a list of the sequences disclosed herein, and it is understood that when a DNA sequence (containing T) is referenced in relation to RNA, T should be replaced with U (modified or unmodified as appropriate), and vice versa. [Table 5] TIFF0007803917000013.tif251164TIFF0007803917000014.tif253164TIFF0007803917000015.tif252163TIFF0007803917000016.tif255164TIFF0007803917000017.tif255166TIFF0007803917000018.tif253164TIFF0007803917000019.tif254162TIFF0007803917000020.tif254164TIFF0007803917000021.tif255166TIFF0007803917000022.tif253166TIFF0007803917000023.tif255165TIFF0007803917000024.tif255166TIFF0007803917000025.tif254166TIFF0007803917000026.tif254165TIFF0007803917000027.tif252163TIFF0007803917000028.tif253164TIFF0007803917000029.tif254164TIFF0007803917000030.tif254163TIFF0007803917000031.tif255162TIFF0007803917000032.tif253166TIFF0007803917000033.tif254163TIFF0007803917000034.tif255163TIFF0007803917000035.tif254163TIFF0007803917000036.tif253165TIFF0007803917000037.tif254165TIFF0007803917000038.tif253164TIFF0007803917000039.tif255164TIFF0007803917000040.tif254165TIFF0007803917000041.tif252164TIFF0007803917000042.tif253163TIFF0007803917000043.tif239154TIFF0007803917000044.tif255164TIFF0007803917000045.tif253164TIFF0007803917000046.tif255163TIFF0007803917000047.tif252164TIFF0007803917000048.tif255163TIFF0007803917000049.tif252165TIFF0007803917000050.tif239154TIFF0007803917000051.tif254163TIFF0007803917000052.tif254164TIFF0007803917000053.tif254165TIFF0007803917000054.tif252163TIFF0007803917000055.tif254165TIFF0007803917000056.tif253165TIFF0007803917000057.tif252164TIFF0007803917000058.tif253163TIFF0007803917000059.tif254165TIFF0007803917000060.tif254163TIFF0007803917000061.tif254163TIFF0007803917000062.tif253164TIFF0007803917000063.tif253164TIFF0007803917000064.tif253164TIFF0007803917000065.tif241154TIFF0007803917000066.tif255165TIFF0007803917000067.tif255165TIFF0007803917000068.tif253164TIFF0007803917000069.tif252164TIFF0007803917000070.tif240154TIFF0007803917000071.tif253164TIFF0007803917000072.tif252164TIFF0007803917000073.tif255163TIFF0007803917000074.tif253165TIFF0007803917000075.tif254164TIFF0007803917000076.tif254163TIFF0007803917000077.tif253163TIFF0007803917000078.tif253163TIFF0007803917000079.tif253164TIFF0007803917000080.tif252163TIFF0007803917000081.tif254164TIFF0007803917000082.tif254164TIFF0007803917000083.tif254163TIFF0007803917000084.tif254163TIFF0007803917000085.tif240154TIFF0007803917000086.tif240154TIFF0007803917000087.tif255165TIFF0007803917000088.tif255164TIFF0007803917000089.tif253162TIFF0007803917000090.tif253164TIFF0007803917000091.tif253163TIFF0007803917000092.tif255163TIFF0007803917000093.tif240154TIFF0007803917000094.tif238154TIFF0007803917000095.tif254164TIFF0007803917000096.tif253163TIFF0007803917000097.tif253164TIFF0007803917000098.tif253163TIFF0007803917000099.tif253163TIFF0007803917000100.tif252163TIFF0007803917000101.tif255163TIFF0007803917000102.tif254164TIFF0007803917000103.tif254164TIFF0007803917000104.tif255163TIFF0007803917000105.tif253164TIFF0007803917000106.tif255164TIFF0007803917000107.tif255164TIFF0007803917000108.tif252161TIFF0007803917000109.tif252163TIFF0007803917000110.tif252163TIFF0007803917000111.tif255165TIFF0007803917000112.tif252164TIFF0007803917000113.tif254163TIFF0007803917000114.tif255163TIFF0007803917000115.tif253164TIFF0007803917000116.tif253164TIFF0007803917000117.tif255164TIFF0007803917000118.tif253164TIFF0007803917000119.tif241154TIFF0007803917000120.tif241154TIFF0007803917000121.tif253164TIFF0007803917000122.tif255164TIFF0007803917000123.tif253162TIFF0007803917000124.tif253164TIFF0007803917000125.tif254162TIFF0007803917000126.tif253165TIFF0007803917000127.tif253162TIFF0007803917000128.tif254163TIFF0007803917000129.tif255164TIFF0007803917000130.tif254164TIFF0007803917000131.tif254164TIFF0007803917000132.tif252165TIFF0007803917000133.tif241154TIFF0007803917000134.tif254164TIFF0007803917000135.tif254163TIFF0007803917000136.tif254163TIFF0007803917000137.tif254164TIFF0007803917000138.tif241154TIFF0007803917000139.tif250165TIFF0007803917000140.tif249164TIFF0007803917000141.tif125164.

[0224] SEQUENCE LISTING <110> INTELLIA THERAPEUTICS, INC. <120> IN VITRO METHOD OF MRNA DELIVERY USING LIPID NANOPARTICLES <130> PA23-654 <150> US 62 / 566,232 <151> 2017-09-29 <160> 86 <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 ctggaaaa gcttcctggt cgaaagac aagaagcacg aagacaccc gatcttcgga 360 aacatcgtcg acgaagtcgc ataccacgaa aagtacccga caatctacca cctgagaaag 420 480 540 gtcgacaagc tgttcatcca gctggtccag acatacaacc agctgtttcga agaaaacccg 600 atcaacgcaa gcggagtcga cgcaaaggca atcctgagcg caagactgag caagagcaga 660 agatgaaa acctgatcgc acagctgccg ggagaaaga agaacggact gttcgggaaac 720 ctgatcgcac tgagcctggg actgacaccg aacttcaaga gcaacttcga cctggcagaa 780 gacgcaaagc tgcagctgag caaggacaca tacgacgacg acctggacaa cctgctggca 840 900 ctgctgagcg acatcctgag agtcaacaca gaatcacaa aggcaccgct gagcgcaagc 960 atgatcaaga gataggacga acaccaccag gacctgacac tgctgaaggc actggtcaga 1020 cagcagctgc cggaaaagta caaggaaatc ttcttcgacc agagcaagaa cggatacgca 1080 ggatacatcg acggaggagc aagccaggaa gaattctaca agttcatcaa gccgatcctg 1140 gaaaagatgg acggaacaga agaactgctg gtcaagctga acagagaaga cctgctgaga 1200 aagcagagaa cattcgacaa cggaagcatc ccgcaccaga tccacctggg agaactgcac 1260 gcaatcctga gaacagga agacttctac ccgttcctga agcaacaag agaaaagatc 1320 gaaaagatcc tgacattcag aatcccgtac tacgtcggac cgctggcaag aggaaacagc 1380 agattcgcat ggatgacaag aaagagcgaa gaacaatca caccgtggaa cttcgaagaa 1440 gtcgtcgaca agggagcaag cgcacagagc ttcatcgaaa gatgacaaa cttcgacaag 1500 aacctgccga acgaaaaggt cctgccgaag cacagcctgc tgtacgaata cttcacagtc 1560 tacaacgaac tgacaaggt caagtacgtc acagaaggaa tgagaagcc ggcattccctg 1620 agcggagaac agagaaggc aatcgtcgac ctgctgttca agacaacag aaggtcaca 1680 gtcaagcagc tgaggaag ctactcaag aagatcgaat gctcgacag cgtcgaaatc 1740 agcggagtcg agacagatt caacgcaagc ctgggaacat accacgacct gctgagatc 1800 atcaaggaca aggactcct ggacaacgaa gaaacgaag acatcctgga agacatcgtc 1860 ctgacactga cactgttcga agacagagaa atgatcgaag aaagactgaa gacatacgca 1920 cacctgttcg acgacaggt catgaagcag ctgagagaa gagatacac aggatggga 1980 agactgagca gaaagctgat spacegaatc agaactgaaa gaatcctg 2040 gacttcctga agagcgacgg attcgcaac agaacttca tgcagctgat ccacgacgac 2100 agcctgacat tcaggaag catchcagaag gcacaggtca gcggacaggg agacacctg 2160 cacgaacaca tcgcaacct ggcaggaagc ccggcaatca agaagggaat cctgcagaca 2220 gtcaggtcg tcgacgact ggtcaggtc atgggac acagccgga aaacatcgtc 2280 atcgaaatgg cagagaaaa cagahaaca cagaahaca cagahacaa 2340 atgagagaa tcgagaagg atcaaggaa ctgggaagcc agatcctgaa ggaacacccg 2400 gtcgaaaaca cacagctgca gaacgaaaag ctgtacctgt actacctgca gaacggaaga 2460 gatagtacg tcgaccagga actggacatc aacagactga gcgactacga cgtcgaccac 2520 atcgtcccgc agagcttcct gaggacgac agcatcgaca acaggtcct gaagaagc 2580 gagaaaa gaggaagg cgaacaacgtc ccgagcgaag aagtcgtcaa gaggagaag 2640 aactactgga gacagctgct gaacgcaaag ctgatcacac agaaagtt cgacaacctg 2700 acaaaggcag agaggagg actgagcgaa ctggacaagg caggattcat caagacag 2760 ctggtcgaaa caacagagat cacaaagcac gtcgcacaga tcctggacag cagaatgaac 2820 acaaagtacg acgaaaacga caagctgatc agaaagtca aggtcatcac actgaagc 2880 aagctggtca gcgacttcag aaagcttc cagttctaca aggtcagaga aatcaacaac 2940 3000 tacccgaagc tggaaagcga attcgtctac ggagactaca aggtctacga cgtcagaaag 3060 atgatcgcaa agagcgaaca ggaaatcgga aagcaacag caaagtactt cttctacagc 3120 aacatcatga acttcttcaa gacagaaatc acactggcaa acggagaaat cagaaagaga 3180 ccgctgatcg aaacaaacgg agaaacagga gaaatcgtct gggacaaggg aagagacttc 3240 gcaacagtca gaaggtcct gagcatgccg caggtcaa tcgtcagaa gagaagtc 3300 gaacaggag gattcaggca ggcagcatc ctgccgaga gaaacagcga caagctgatc 3360 gcaagaag aggactggga cccgaagaag tacggagt tcgacagccc ggagtcgca 3420 tacagcgtcc tggtcgtcgc aaaggtcgaa agggaaga gcaagaagct gaagagcgtc 3480 aaggaactgc tgggaatcac atcatggaa agaagcagct tcgaaagaa cccgatcgac 3540 ttcctggaag aaagggata aaggaagtc aagaaggacc tgatcatcaa gctgccgaag 3600 tacagcctgt tcgaactga aaacggaaga agagaatgc tggcaagcgc aggagactg 3660 cagaagggaa acgaactggc actgccgagc aagtacgtca acttcctgta cctggcaagc 3720 cactacgaaa agctgaaggg aagcccgaa cakacgaac agaagcagct gttcgtcgaa 3780 cagcacaagc actacctgga cgaaatcatc gaacagatca gcgaattcag cagagagtc 3840 atcctggcag acgcaaacct ggacaaggtc ctgagcgcat 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 ctgctgttg actccggcga aaccgcagaa 180 gcgacccggc tcaacgtac cgcgaggcga cgctacaccc ggcggaagaa tcgcatctgc 240 tatctgcaag agatctttc gaacgaatg gcaaggtcg acgacagctt cttccaccgc 300 ctggaagaat ctttcctggt ggaggac aagaagcatg aacggcatcc tatctttgga 360 aacatcgtcg acgaagtggc gtaccacgaa aagtacccga ccatctacca tctgcggaag 420 aagttggttg actcactga caggccgac ctcagattga tctactggc cctcgcccat 480 atgatcaaat tccgcggaca cttcctgatc gaggcgatc tgaccctga taactccgac 540 gtggataagc ttttcattca actggtgcag acctacaacc aactgttcga agaaaaccca 600 atcaatgcta gcggcgtcga tgccaaggcc atcctgtccg cccggctgtc gaagtcgcgg 660 cgcctcgaaa acctgatcgc acagctgccg ggagagaaaa agaacggact ttcggcaac 720 ttgatcgctc tctcactggg actcactccc aatttcaagt ccaattttga cctggccgag 780 gacgcgaagc tgcaactctc aaaggacacc tacgacgacg acttggacaa tttgctggca 840 caaattggcg atcagtacgc ggatctgttc cttgccgcta agaacctttc ggacgcaatc 900 ttgctgtccg atatcctgcg cgtgaacacc gaataacca aagcgccgct tagcgcctcg 960 atgattaagc ggtacgacga gcatcaccag gatctcacgc tgctcaaagc gctcgtgaga 1020 cagcaactgc ctgaaaagta caaggagatc ttcttcgacc agtccaagaa tgggtacgca 1080 gggtacatcg atggaggcgc tagccaggaa gagttctata agttcatcaa gccaatcctg 1140 gaaaagatgg acggaaccga agaactgctg gtcaagctga acagggaga tctgctccgg 1200 aaaagagaa cctttgacaa cggatccatt ccccaccaga tccatctggg tgagctgcac 1260 gccatcttgc ggcgccagga ggacttttac ccattcctca aggacaaccg ggaaaagatc 1320 gagaaaattc tgacgttccg catcccgtat tacgtgggcc cactggcgcg cggcaattcg 1380 cgcttcgcgt ggatgactag aaaatcagag gaaaccatca ctccttggaa ttcgaggaa 1440 gttgtggata agggagcttc ggcacaaagc ttcatcgaac gaatgaccaa cttcgacaag 1500 aatctcccaa acgagaaggt gcttcctaag cacagcctcc tttacgaata cttcactgtc 1560 tacaacgaac tgactaaagt gaaatacgtt actgaaggaa tgaggaagcc ggcctttctg 1620 tccggagaac agaagaaagc aattgtcgat ctgctgttca agaccaaccg caaggtgacc 1680 gtcaagcagc ttaaagagga ctacttcaag aagatcgagt gttcgactc agtggaaatc 1740 agcggggtgg aggacagatt caacgcttcg ctgggaacct atcatgatct cctgaagatc 1800 atcaaggaca aggacttcct tgacaacgag gagaacgagg acatcctgga agatatcgtc 1860 ctgaccttga cccttttcga ggatcgcgag atgatcgagg agaggcttaa gacctacgct 1920 catctcttcg acgataaggt catgaaacaa ctcaagcgcc gccggtacac tggttggggc 1980 cgcctctccc gcaagctgat caacggtatt cgcgataaac agagcggtaa aactatcctg 2040 gatttcctca aatcggatgg cttcgctaat cgtaacttca tgcaattgat ccacgacgac 2100 agcctgacct ttaaggagga catccaaaaa gcacaagtgt ccggacaggg agactcactc 2160 catgaacaca tcgcgaatct ggccggttcg ccggcgatta agaagggaat tctgcaaact 2220 gtgaaggtgg tcgacgagct ggtgaaggtc atgggacggc acaaaccgga gaatatcgtg 2280 attgaaatgg cccgagaaaa ccagactacc cagaagggcc agaaaaactc ccgcgaaagg 2340 atgaagcgga tcgaagaagg aatcaaggag ctgggcagcc agatcctgaa agagcacccg 2400 gtggaaaaca cgcagctgca gaacgagaag ctctacctgt actatttgca aaatggacgg 2460 gacatgtacg tggaccaaga gctggacatc aatcggttgt ctgattacga cgtggaccac 2520 atcgttccac agtcctttct gaaggatgac tcgatcgata acaaggtgtt gactcgcagc 2580 gacaagaaca gagggaagtc agataatgtg ccatcggagg aggtcgtgaa gaagatgaag 2640 aattactggc ggcagctcct gaatgcgaag ctgattaccc agagaaagtt tgacaatctc 2700 actaaagccg agcgcggcgg actctcagag ctggataagg ctggattcat caaacggcag 2760 ctggtcgaga ctcggcagat taccaagcac gtggcgcaga tcttggactc ccgcatgaac 2820 actaaatacg acgagaacga taagctcatc cgggaagtga aggtgattac cctgaaaagc 2880 aaacttgtgt cggactttcg gaaggacttt cagttttaca aagtgagaga aatcaacaac 2940 taccatcacg cgcatgacgc atacctcaac gctgtggtcg gtaccgccct gatcaaaaag 3000 taccctaaac ttgaatcgga gtttgtgtac ggagactaca aggtctacga cgtgaggaag 3060 atgatagcca agtccgaaca ggaaatcggg aaagcaactg cgaaatactt cttttactca 3120 aacatcatga actttttcaa gactgaaatt acgctggcca atggagaaat caggaagagg 3180 ccactgatcg aaactaacgg agaaacgggc gaaatcgtgt gggacaaggg cagggacttc 3240 3300 caaccggcg gattttcaaa ggaatcgatc ctcccaaaga gaatagcga caagctcatt 3360 gcacgcaaga aagactggga cccgaaag tacggaggat tcgattcgcc gactgtcgca 3420 tactccgtcc tcgtggtggc caaggtggag aaggaaga gcaaaaagct caaatccgtc 3480 aaagagctgc tggggattac catcatggaa cgatcctcgt tcgagaagaa cccgattgat 3540 ttcctcgagg cgaagggtta caaggaggtg aagaaggatc tgatcatcaa actccccaag 3600 tactcactgt tcgaactgga aaatggtcgg aagcgcatgc tggcttcggc cggagaactc 3660 caaaaaggaa atgagctggc cttgcctagc aagtacgtca acttctctcta 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> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <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 Leu To Asn Ala Lys With Thr Gln Arg Lys 885,890,895 Phe Asp Asp With Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu With Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Gly Gly Ser Pro Lys Lys Arg Lys Val 1370 1375 <210> 4 <211> 4140 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 4 auggacaaga agocagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 gcaacagac ugagagaac agcaucacaca gagaagaa cagaaucugc 240 uaccugcagg aaacuucag caacgaaug gcaaggucg acgacagcuu cuccacaga 300 neighborhood gcuuccuggu cgagagac aagaagcacg aagacaccc gaucuucgga 360 420 aagcuggucg acagcacaga caaggcagac cugacacuga ucuaccuggc acuggcacac 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agagaaggc aaucgucgac cugguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 agacugagca gaagcugau caacggaauc agacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 gcaagaaga aggacuggga cccgaagaag uacggaggau ucgacacccc gagucgca 3420 uacagcgucc uggucgucgc aaggucgaa agggaaa gcaagaagcu gagagcguc 3480 aaggaacugc ugggaucgac aaucauggaa aggaagcagcu ucgaaagaa cccgaucgac 3540 ooooooooooooooooooooooooooooooooooooooooooooooo back go back gcugccgag 3600 uacagccugu ucgaaaaaaaaaaaaaaggc agghaagcgc aggaaaagg 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagacaacagc acuaccugga cgaaaucauc gacagauca gcgaauucag cagagaguc 3840 auccuggcag acgshaaccu ggaaagguc cugagcgcau ahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagg 3900 ccgaucagag aacaggcaga aacaucauc 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 gaacgaaug gcaaggucg acgacagcuu cuccaccgc 300 neighbors cuckoooooooooogga ggaggaggac aaacuuugg 360 aacaucgucg acgaaguggc guaccacccgaa aaguacccga ccaucuacca ucugcggaag 420 aaguagguug acucacuga caggccgac cucagauuga ucuacuuggc ccucgcccau 480 augaucaaau uccgcggaca cuccugauc gaaggcgauc ugaacccuga uaacuccgac 540 guggauaagc uuuucauca acuggugcag accuaacc aacuguucga agaaaaccca 600 aucaugcua gcggcgucga ugccaaggcc accuguccg cccggcuguc gaagucgcgg 660 cgccucgaaa accugaucgc acagcugccg ggagagaaa agaacggacu uuucggcaac 720 uugaucgcuc ucucacuggg acucacuccc aauuucaagu ccaauuuuga ccuggccgag 780 gacgcgaagc ugcacucuc aaaggacacc uacgacgacg acuuggacha uuugcuggca 840 900 960 1020 cagcaacugc cugaaaagua caaggaac uucuucgacc aguccaagaa uggguacgca 1080 ggguacaucg auggaggcgc uagccaggaa gagucuauaa aguucaucaa gccaauccug 1140 gaaagaugg acggaaccga agaacugcug cucaagcuga acagggaga ucugcuccgg 1200 aaacagagaa ccuuugacaa cggauccauu cccaccaccaga uccaucuggg ugagcugcac 1260 gccaucuugc ggcgccagga ggacuuuac ccauuccuca aggacaaccg ggaaaagauc 1320 gagaaaauuc ugacguuccg caucccguau uacgugggcc cacuggcgcg cggcaauucg 1380 cgcuucgcgu ggaugacuag aaaaucagag gaacauuca cucuuggaa uuucgagaa 1440 guugugguaa agggagcuuc ggcacaaagc uucaucgaac gaaugaccaa cuucgacaag 1500 aaucucccaa acgagaaggu gcuuccuaag cacagccucc uuuacgaaua cuucacuguc 1560 uacaacgaac ugacuaaagu gaauacguu acugaaggaa uggaagcc ggccuuucug 1620 uccggagaac agagaaagc aauugucgau cugcuguuca agaccaaccg caaggugacc 1680 gucaagcagc uuaaagga cuacuucaag aagaucgagu guuucgacuc aguggaauc 1740 1800 aucaaggaca aggacuuccu ugacaacgag gagaacgagg acauccugga agauaucguc 1860 1920 caucucuucg acgauaaggu caugaacaa cucaagcgcc gccgguacac ugguugggggc 1980 cgccucccc gcaagcugau caacgguauu cgcgauaaac agagcgguaa aacuauccug 2040 gauuuccuca auucggaugg cuucgcuauu cguaacuuca ugcaauugau ccacgacgac 2100 agccugaccu uuaaggagga cauccaaaaa gcacaagugu ccggacaggg agcucacuc 2160 caugaacaca ucgcgaaucu ggccgguucg ccggcgauua agaagggaau ucugcaacu 2220 gugaaggugg ucgacgagcu ggugaagguc augggacggc aaaccgga gaauacgug 2280 auugaaaugg cccgagaaaaa ccagacuacc cagaagggcc agaaaaacuc ccgcgaaagg 2340 augaagcgga ucgagaagg augaaggag cuggcagcc agauccugaa agagcaccccg 2400 guggaaaaca cgcagcugca gaacgagaag cucuaccugu acuauuugca aauggacgg 2460 guauguacg uggaccaag gcuggauc auucgguug cugauuacga cguggaccac 2520 aucguuccac aguccuuucu gaaggagac ucgaucgaua aagguua gacucgcagc 2580 gaggaaaaaaaaaaaaaaaaaguaaaaaaaaaaaaaaaaaagugg ccaucggagg agggaaaaaaaaaaaagggaaaaagg 2640 aauuacuggc ggcagcuccu gaaugcgaag cugauuaccc agaaaguu ugacaaucuc 2700 acuaaagccg agcgcgggggg acucucagag cuggauaagg cuggauucau caaaccggcag 2760 cuggucgaga cucggcagau uaccaagcac guggcgcaga ucuuggacuc ccgcaugaac 2820 2880 2940 uaccaucacg cgcaugacgc auaccucaac gcuguggucg guaccgcccu gaucaaaaag 3000 uacccuaaac uugaaucgga guuuguguac ggagacuaca aggucuaca cgugagaag 3060 augauagcca aguccgaaca ggaaaucggg aaagcaacug cgaaauacuu cuuuuacuca 3120 aacaucauga acuuuuuucaa gacugaaauu acgcuggcca auggagaaau caggagaagg 3180 ccacugaucg aacuaacgg agaaacggc gaaucgugu gggacaaggg cagggacuuc 3240 caacuguuc caaagugcu cucuaugccg caaucaaua uugugaagaa aaccaagug 3300 caaccggcg gauuuucaaa ggaaucgauc cucccaaaga gaauagcga caagcuacauu 3360 ccacgcaaga aagacuggga cccgaaag uacggaggau ucgauucgcc gacugucgca 3420 uacuccgucc ucgugguggc caagguggag aagggaaaga gcaaaaagcu caaauccguc 3480 aaagagcugc ugggggauuac caucauggaa cgauccucgu ucgagaagaa cccgauugau 3540 uuccucgagg cgaaggguua caaggaggug aagaaggauc ugaucaucaa acuccccaag 3600 uacucacugu ucgaacugga aaauggucgg aagcgcaugc uggcuucggc cggagaacuc 3660 3720 3780 cagcacaagc auuaucugga ugaaaucauc gaacaaaucu ccgaguuuuc aaagcgcgug 3840 auccucgccg acgccaaccu cgacaaguc cugucggccu achauaagca uagagauaag 3900 ccgaucagag aacaggccga gacauuauuc cacuuguuca cccugacuaa ccugggagcc 3960 ccagccgccu ucaaguacuu cgauacuacu aucgaucgca aaagauacac guccaccaag 4020 gaaguucugg acgcgacccu gauccaccaa agcucacug gacucuacga acacgauc 4080 gaucugucgc agcugggggg cgaugggcggu gaucuccga aaagagag aaggguaa 4140 4143 <210> 6 <400> 6 000 <210> 7 <400> 7 000 <210> 8 <400> 8 000 <210> 9 <400> 9 000 <210> 10 <211> 4134 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 10 gapagaagu acagcaucgg acuggacauc ggaaaaca gcgucggaug ggcagucauc 60 acagacgaau acagguccc gaghagaag uucaggucc ugggaacac agacagac 120 shake shake agaaccugau cggashacug shake shake 180 accelerate accelerate accelerate accelerate accelerate 240 neighborhood ucuucagcaa cgaauggca aaggucgacg acagcuucuu ccacagacug 300 gagaaagcu squeeze agaaaaaaaaaaaaaaaaazzccgau cucggaac 360 aucgucgacg aagucgcaua ccacgaaag uacccgacaa ucuaccaccu gagaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gaggagcaag ccaggaga uacaucaagu ucaucaagcc gauccugga aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca auccugagaa gacaggaaga cuucuacccg auccugaagg acaacagaga aaagaucgaa aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggagaacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1740 ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaaucauc 1800 aagcaagg acuuccugga caacgaaa aacgaaca ucuggagaa caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaaaa gacugaagac aucgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaaaaggcaa gagaaaaaaaaaaaaaaaaaaaaaaaaaaaagg 2340 aagagaaucg aagaaggaau aacccgguc 2400 gaaaacacac agcugcaga cgaaaagcug uaccuguacu accugcaga cggagagac 2460 auguacgucg accaacuc agacugaccg accuacgacgu cgaccaac 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccag accuacaagg accucagaau storm 2940 caccacgcac acgacgcaua ccugaacgca gucgucggaa caccacugau caccaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ccgaagcugg cagaagaug 3060 aucgcaaag gcgaaga auucggaaag gcaacaaa aguacuucuu quacacaac 3120 aucaugaacu aucaucagac agaaucaca cuggcaacg gagaaucag aagagaccg 3180 cugauchgaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa mighte agaascoaa 3240 acagushagaa agguccugag caugccgcag agaguaucg agaguaggc agaguccag 3300 shakeshaggshaggshaggshaggshaggshaggshaggshaw gcugaucgca 3360 agaaagaagg acugggacccc gagaaguac ggaggauucg acagcccgac agacggquac 3420 agcguccugg ucgucgcaaa gggaaaag gggaagca agaagcuga gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaaagcaa aggguaacaa ggaagucaag aagaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag ccgggaagac aacgaacaga agcagcuguu cgucgaacag 3780 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaacccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaaccu gggagcaccg 3960 gcagcauuca aguacuucga cacaacaauc gacagaaaga gauacacaag cacaaaggaa 4020 4080 cugagccagc ugggaggaga cggaggagga agcccgaaga agaagagaaa gguc 4134 <210> 11 <400> 11 000 <210> 12 <400> 12 000 <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 Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 14 <211> 4107 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 14 auggacaaga agocagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 gcaacagac ugagagaac agcaucacaca gagaagaa cagaaucugc 240 uaccugcagg aaacuucag caacgaaug gcaaggucg acgacagcuu cuccacaga 300 neighborhood gcuuccuggu cgagagac aagaagcacg aagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccaccgaa aacaacccga caaucaacca ccugagaaag 420 aagcuggucg aaggshaga aaggshagac cugaaccuggc aaggcuggc 480 augaucaagu shake 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agagaaggc aaucgucgac cugguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 agacugagca gaagcugau caacggaauc agacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga ccaagaagcu gaagcguc 3480 aaagacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaaggguau caaggaaguc aagaaggacc ugaucaucaa cugccgaag 3600 uacagccugu ucgaaaaaaaaaaaaaaggc agghaagcgc aggaaaagg 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagacaacagc acuaccugga cgaaaucauc gacagauca gcgaauucag cagagaguc 3840 auccuggcag acgshaaccu ggaaagguc cugagcgcau ahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagg 3900 ccgaucagag aacaggcaga aacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaca aucgacagaa agagauacac aagcacaaag 4020 gaguccugg axaaaccu gauccaccag aacaxaac 4080 gaccugagcc agcugggagg agcuag 4107 <210> 15 <211> 4101 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 15 gapagaagu acagcaucgg acuggacauc ggaaaaca gcgucggaug ggcagucauc 60 acagacgaau acagguccc gaghagaag uucaaggucc ugggaacac agacagac 120 shake agaaccugau cggagshacug cuguaccug shake 180 accelerate accelerate accelerate accelerate accelerate 240 neighborhood ucuucagcaa cgaauggca aaggucgacg acagcuucuu ccacagacug 300 gagaaagcu aagaaaaaaaaaaaaazzaccgau cucggaac 360 aucgucgacg aagucgcaua ccacgaaag uacccgacaa ucuaccaccu gagaagaag 420 cuggucgaca gcacagacaa ggcagaccug agacugaucu accuggcacu ggcacacaug 480 aucaaguuca gaggacacuu ccugaucgaa ggagaccuga acccggacaa cagcgacguc 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagauggacg gaacagaaga acugcugguc aagcugaaca gagaagaccu gcugagaaag cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca auccugagaa gacaggaaga cuucuacccg auccugaagg acaacagaga aaagaucgaa aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc ggagucgaag acagauucaa cgcaagccug ggaacauacc acgaccugcu gaaucauc 1800 aagcaagg acuuccugga caacgaaa aacgaaca ucuggagaa caucguccug 1860 acacugacac uguucgaaga cagagaaaug aucgaaaa gacugaagac aucgcacac 1920 cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau aacccgguc 2400 gaaaacacac agcugcaga cgaaaagcug uaccuguacu accugcaga cggagagac 2460 auguacgucg accaacuc agacugaccg accuacgacgu cgaccaac 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccaag accuacaagg accucagaaau storm 2940 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ucuacgacgu cagaaagaug 3060 aucgcaaaga gcgaacagga aaucggaaag caacagcaa aguacuucuu cuacagcaac 3120 aucaugaacu ucuucaagac agaaaucaca cuggcaaacg gagaaucag aagagaccc 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acagucagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa cgugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 neighborhood agggaaaaaaaaaagaaaaaaaaaaaaacus gccgaaaaa 3600 agccuguucg aacuggaaaa cggaagaag agaugcugg caagcgcagg agacugcagg 3660 aagggaaacg aacuggcacu gccgagcaag uacgucacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaaggaag cccggaagc aacgaaaac agxaggcugu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg auucagca gagagucauc 3840 cuggcagacg aaccugga cuggcugg agcgcauaca acaaccacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaccu gggagcaccg 3960 gcagcauca aguacuucga cacaauc gagaaaga gauacacaag cacaaggaa 4020 guccuggacg hurricane ccaccagagc aucachaggac uguacgacg aagaaucgac 4080 cugagccagc uggaggaga c 4101 <210> 16 <211> 1368 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 16 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 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 Tyr Glu Tyr Phe Thr Val Tyr Asn Glu 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 Ile Glu Cys Phe Asp 565,570,575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580,585,590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Tyr Glu Thr Arg to Asp to Gln Ser to Gly Gly Asp 1355 1360 1365 <210> 17 <211> 4107 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 17 auggacaaga agocagcau cggacuggca aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 gcaacagac ugagagaac agcaucacaca gagaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccagaga 300 360 420 aagcuggucg acagcacaga caaggcagac cugacacuga ucuaccuggc acuggcacac 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agagaaggc aaucgucgac cugguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 agacugagca gaagcugau caacggaauc agacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaac agaacuuca agcagcugau ccacgacgac 2100 agccugacau ucaggaga cauccagaag gcacagguca gcggacaggg agcugccug 2160 cacgacaca ucgcaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 cuckaggucg ucgaacuc gguaacucg aaaacaucguc 2280 aucgaaaugg cagagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa has 2340 augaagaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaccg 2400 gucgaaaaca cacagcugca gaacgaaaag cuguaccugu acuaccugca gaacxaga 2460 qaugauguacg ucgaccagga acuggacuuc aaxagacuga gcgaquaacga cgucgaccac 2520 aucgucccgc shake shake shake shake shake shake shake 2580 gagaaa gagaagag cgaaaaaaack ccgaagaaaaaaaaaaaaaaackaaaaaagg 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga ccaagaagcu gaagcguc 3480 aaagacugc ugggaaucac aaucauggaa agaagcagcu ucgaaaagaa cccgaucgac 3540 uuccuggaag caaaggguau caaggaaguc aagaaggacc ugaucaucaa cugccgaag 3600 uacagccugu ucgaacugga aaacggaa aagagaaugc uggcaagcgc agggaaacug 3660 3720 3780 cagcaaagc auaccugga cgaaaucauc gaacagauca gcgaauucag caagagaguc 3840 auccuggcag acgcaaaccu ggacaagguc cugagcgcau acaacaagca cagagacaag 3900 ccgaucagag aacaggcaga aaacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaaca aucgacagaa agagauacac aagcacaaag 4020 gaaguccugg acgcaacacu gauccaccag agcaucacag gacuguacga aacaagaauc 4080 gaccugagcc agcugggagg agacuag 4107 <210> 18 <211> 4101 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 18 gacaagaagu acagcaucgg acuggcaauc ggaacaaaca gcgucggaug ggcagucauc 60 acagacgaau acaagguccc gagcaagaag uucaaggucc ugggaaacac agacagacac 120 agcaucaaga agaaccugau cggagcacug cuguucgaca gcggagaaac agcagaagca 180 acaagacuga agagaacagc aagagaaga uacacaagaa gaagaagacag aaucugcuac 240 cugcaggaaa ucuucagcaa cgaaauggca aaggucgacg acagcuucuu ccacagacug 300 gaaagcu ucuggucga agagacaag aagcacgaaa gacaccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaagaag 420 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagouggacg gaacagaaga aucugcugguc aagcugaaca gagaagaaccu cgugaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggagaa cuucuacccg uuccugaagg aacaagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga uucgcaugga ugacaagaa gagcgaaga acaaucacac cguggaacuu cgaagaaguc 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500. 1500 1560. cugccgaacg aaaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac aacgaacuga caaaggucaa guacgucaca caaaggauga gaaagccggc auuccugagc ggagaacaga agaaggcaau cgucgaccug cuguucaaga caaacagaaa ggucacaguc aagcagcuga aggaagacua cuucaagaag aucgaaugcu ucgacagcgu cgaaaucagc 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 aaggacaagg acuuccugga aacgaagaca accuggaga caucguccug acacugacac uguucgaaga cagagaaaug aucgaagaaa gacugaagac auacgcacac cuguucgacg acaaggucau gaagcagcug aagagaagaa gauacacagg auggggaaga 1980 cugagcagaa agcugaucaa cggaaucaga gacaagcaga gcggaaagac aauccuggac 2040 uuccugaaga gcgacggauu cgcaaacaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga ucugaagga acaccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcaga cggagagac 2460 2520 gucccgcaga gcuuccugaa ggacgacagc aucgacaca agguccugac aagaagcgac 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa-e has gauge 2640 uacuggagac accugac cgcaaagcug aucacacaga gaacuggac 2700 aggshock frequency frequency gaucauca frequency 2760 gucgaaacaa ghagaucac aaagcacguc gcacagaucc uggacagcag auugacaca 2820 aaaguacgacg aaacgacaa gcugaucaga gagucaagg ucaucacacu gagagacga 2880 cuggucagcg accucagaaa ggacuccaag accuacaagg accucagaaau storm 2940 caccacgcac acgacgcaua ccugaacgca gucgucgaa caccacugau caccaaguac 3000 ccgaagcugg aaagcgaauu cgucuacgga gacuacaagg ccgaagcugg cagaagaug 3060 aucgcaaag gcgaaga auucggaaag gcaacaaa aguacuucuu quacacaac 3120 aucaugaacu aucaucagac agaaucaca cuggcaacg gagaaucag aagagaccg 3180 cugauchgaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa mighte beachacoaaaaagghaa 3240 acagushagaa agguccugag caugccgcag agaguaucg agaguaggc agaguccag 3300 shakeshaggshaggshaggshaggshaggshaggshaggshaw gcugaucgca 3360 agaaagaagg acugggacccc gagaaguac ggaggauucg acagccgac agacggac 3420 agcguccugg ucgucgcaaa gggaaaag gggaagca agaagcuga gagcgucaag 3480 gaacugcugg gauccacaau cauggaaag agcagcuucg aaagaaccc gaucgacuuc 3540 neighborhood agggaaaaaaaaaagaaaaaaaaaaaaacus gccgaaaaa 3600 agccuguucg aacuggaaaa cggaagaag agaugcugg caagcgcagg agacugcagg 3660 aagggaaacg aacuggcacu gccgagcaag uacgucacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaaggaag cccggaagc aacgaaaac agxaggcugu cgucgaacag 3780 cacaagcacu accuggacga aaucaucgaa cagaucagcg auucagca gagagucauc 3840 cuggcagacg aaccugga cuggcugg agcgcauaca acaaccacag agacaagccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaccu gggagcaccg 3960 gcagcauca aguacuucga cacaauc gagaaaga gauacacaag cacaaggaa 4020 guccuggacg hurricane ccaccagagc aucachaggac uguacgacg aagaaucgac 4080 cugagccagc uggaggaga c 4101 <210> 19 <211> 1368 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide” <400> 19 Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Leu To Asn Ala Lys With Thr Gln Arg Lys 885,890,895 Phe Asp Asp With Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu With Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 20 <211> 4107 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic "polynucleotide" <400> 20 60 aucacagacg aauacaaggu cccgagcaag aaguucaagg uccugggaaa cacagacaga 120 180 ccaacaagac ugagagaac agcaagaaga aguaacacaa gaaaagaa cagaaucugc 240 uaccugcagg aaaucuucag caacgaaaug gcaaaggucg acgacagcuu cuuccagaga 300 360 420 aagcuggucg acagcacaga caaggcagac cugacacuga ucuaccuggc acuggcacac 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gaaucuaca aguucaucaa gccgauccug 1140 gaaagaugg acggaacaga agaacugcug cucaagcuga acagagaagaa ccugcugaga 1200 aagcagagaa cauucgacaa ccgaagcauc ccgcaccaga uccaccuggg agaacugcac 1260 ccauccuga gaacaagga agacuuac ccguuccuga agacaacag agaaaaagauc 1320 gaaagaucc ugacauucag aaucccguac uacgucggac cgcuggcaag aggaaacagc 1380 agauucgcau ggaugacaag aaagagcgaa gaacaauca caccguggaa cuucgaaa 1440 gucgucgaca agggagcaag cgcacagagc uucaucgaaa gaugacaaa cuucgacaag 1500 1560 uacaacgaac ugacaaaggu caaguacguc acagaaggaa ugagaaagcc ggcauuccug 1620 agcggagaac agaagaaggc aaucgucgac cugcuguuca agacaaacag aaaggucaca 1680 gucaagcagc ugaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaagauacac aggaugggga 1980 agacugagca gaaagcugau caacggaauc agagacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaggucgaa agggaaa gcaagaagcu gagagcguc 3480 aaggaacugc ugggaucgac aaucauggaa aggaagcagcu ucgaaagaa cccgaucgac 3540 ooooooooooooooooooooooooooooooooooooooooooooooo back go back gcugccgag 3600 uacagccugu ucgaaaaaaaaaaaaaaggc agghaagcgc aggaaaagg 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagacaacagc acuaccugga cgaaaucauc gacagauca gcgaauucag cagagaguc 3840 auccuggcag acgshaaccu ggaaagguc cugagcgcau ahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagg 3900 ccgaucagag aacaggcaga aacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaca aucgacagaa agagauacac aagcacaaag 4020 gaguccugg axaaaccu gauccaccag aacaxaac 4080 gaccugagcc agcugggagg agcuag 4107 <210> 21 <211> 4113 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 21 gapagaagu acagcaucgg acuggcauc ggaaaaca gcgucggaug ggcagucauc 60 acagacgaau acagguccc gaghagaag uucaggucc ugggaacac agacagac 120 shake shake agaaccugau cggashacug shake shake 180 accelerate accelerate accelerate accelerate accelerate 240 neighborhood ucuucagcaa cgaauggca aaggucgacg acagcuucuu ccacagacug 300 gaaagcu ucuggucga agagacaag aagcacgaaa gacaccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaagaag 420 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagouggacg gaacagaaga aucugcugguc aagcugaaca gagaagaaccu cgugaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggagaa cuucuacccg uuccugaagg aacaagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucaac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaacuu cgacaagaac 1500 cugccgaacg aaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga aaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 gggaacaga agaaggcaau cgucgaccug cugucagaaaaaa ggucacaguc 1680 aagshaaaugcua agghaaugcuaaaaaaaaggcua cgaaaaacagc 1740 ggagucgaag acagaucauc cgcaagccug ggacauacc acgaccugcu gagaucauc 1800 shakespeed shakespeed shakespeed shakespeed 1860 accugacac uguecgaga cagagaaug aucgaagaaa gacugagac auacgcacac 1920 cuguacgacg aaaggucau gaagcagcug agagagaa gauaccacagg augggaa 1980 2040 2040 2040 2040 - agcugauca cgaaaaaaaaaaaccugac uuccugaaga gcgacggauu cgchaaaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggucagcg gacagggaga cagccugcac 2160 gacacaucg caaaccuggc aggaagcccg caaucaaga agggaauccu gcagacaguc 2220 aaggucgucg acgaacuggu caaggucaug ggagacaca agccggaaaa caucgucauc 2280 gaauggcaa gagaaaacca gacaacacag aagggacaga agaacagcag agaaagaaug 2340 aagagaaucg aagaaggaau caaggaacug ggaagccaga ucugaagga acaccgguc 2400 gaaaacacac agcugcagaa cgaaaagcug uaccuguacu accugcaga cggagagac 2460 2520 gucccgcaga gcuuccuugaa ggacgacagc aucgacaaca agguccugac aagaagcgac 2580 aagaacagag gaagagcga caacguccg agcgaaag ucgucaagaa gaugaagaac 2640 2700 aaggcagagagaggagacu gagcgaacug gacaaggcag gauucaucaa gagacagcug 2760 gucgaaacaa gacagaucac aaagcacguc gcacagaucc uggacagcag aaugaacaca 2820 aaguacgacg aaaacgacaa gcaugaucaga 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 agaaucaca cuggcaaacg gagaaucag aaagagaccg 3180 cugaucgaaa caaacggaga aacaggagaa aucgucuggg acaagggaag agacuucgca 3240 acacagaa agguccugag caugccgcag gucaacaucg ucaagaagac agaaguccag 3300 acaggaggau ucagcaagga aagcauccug ccgaagagaa acagcgacaa cgugaucgca 3360 agaaagaagg acugggaccc gaagaaguac ggaggauucg acagcccgac agucgcauac 3420 agcguccugg ucgucgcaaa ggucgaaaag ggaaagagca agaagcugaa gagcgucaag 3480 gaacugcugg gaaucacaau cauggaaaga agcagcuucg aaaagaaccc gaucgacuuc 3540 cuggaaagcaa aggguaacaa ggaagucaag aagaccuga ucaucaagcu gccgaaguac 3600 agccuguucg aacuggaaaa cggagaaag agaaugcugg caagcgcagg agaacugcag 3660 aagggaaacg aacuggcacu gccgagcaag uacgucaacu uccuguaccu ggcaagccac 3720 uacgaaaagc ugaagggaag ccgggaagac aacgaacaga agcagcuguu cgucgaacag 3780 3840 cuggcagacg caaaccugga caagguccug agcgcauaca acaagcacag agacaacccg 3900 aucagagaac aggcagaaaa caucauccac cuguucacac ugacaaccu gggagcaccg 3960 gcagcauca aguacuucga cacaaac gagaaga gauacacaag cacaaggaa 4020 guccuggacg hurricane ccaccagagc aucachaggac uguacgacg aagaaucgac 4080 cugagccagc ugggaggaga cggaggagga agc 4113 <210> 22 <211> 1392 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide” <400> 22 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 Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915,920,925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930,935,940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965,970,975 Glu Ile Asn Asn Tyr His Ala His Asp Ala Tyr Leu Asn Ala Val 980,985,990 Val Gly Thr Ala Leu Ile Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 Gly Ser Gly Ser Pro Lys Lys Lys Arg Lys Val Asp Gly Ser Pro 1370 1375 1380 Lys Lys Lys Arg Lys Val Asp Ser Gly 1385 1390 <210> 23 <211> 4179 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 23 auggacaaga agocagcau cggacuggac aucggaacaa acagcgucgg augggcaguc 60 aucacagacg aauaacaggu cccgagcaag aagucaagg uccugggaaa cacagacaga 120 slow-wave slow-speed slow-speed slow-speed 180 gcaacagac ugagagaac agcaucacaca gagaagaa cagaaucugc 240 uaccugcagg aaacuucag caacgaaug gcaaggucg acgacagcuu cuccacaga 300 neighborhood gcuuccuggu cgagagac aagaagcacg aagacaccc gaucuucgga 360 aacaucgucg acgaagucgc auaccaccgaa aacaacccga caaucaacca ccugagaaag 420 aagcuggucg aaggshaga aaggshagac cugaaccuggc aaggcuggc 480 540 gucgacaagc uguucaucca gcugguccag acauacaacc agcuguucga agaaaacccg 600 aucaacgcaa gcggagucga cgcaaaggca auccugagcg caacacugag caagacuga 660 agacuggaa accugaucgc acagcugccg ggagaaaaaga agaacggacu guucggaaac 720 cugaucgcac ugagccugggg acugacaccg aacuucaaga caacauucga ccuggcagaa 780 gacgcaaagc ugcagcugag caaggacaca uacgacgacg accuggacaa ccugcuggca 840 900 cugcugagcg acauccugag agucaacaca gaaucacaa aggcaccgcu gagcgcaagc 960 1020 cagcagcugc cggaaaagua caaggaaauc uucuucgacc agagcaagaa cgguaacgca 1080 ggauacaucg acggaggagc aagccaggaa gauucuaca aguucauca gccgauccug 1140 gaaaagaugg shuffle agaacugcug shuffle shuffle ccugcuga 1200 aagcagagaa cauucgacaa cggaagcauc ccgcaccaga uccaccuggg agaacaccac 1260 gshake shake shake shake shake shake shake shake 1320 gaaaagaucc ugacaucag aaucccguac uacgucggac cgcuggcaag aggaacagc 1380 agauucgcau gagacaag aagagcgaa gaacauca caccgugga cucgagaa 1440 pushpuck agghag cg pushpuck uucaucaucaaa gaugahaa cuckhahag 1500 aaccugccga acgaaaggu ccugccgaag cacagccugc uguacgaaua cucacaguc 1560 uacaacgaac ugaaaaggu uacaacguc ugagaaagcc ggcauuccug 1620 agcgaaggc agagaaggc aaucgucgac cugcuguc agaaaagggaaaaggucaca 1680 gucaagcagc ugaaaggaaga cuacuucaag aagaucgaau gcuucgacag cgucgaaauc 1740 agcggagucg aagacagauu caacgcaagc cugggaacau accacgaccu gcugaagauc 1800 aucaaggaca aggacuuccu ggacaacgaa gaaaacgaag acauccugga agacaucguc 1860 cugacacuga cacuguucga agacagagaa augaucgaag aaagacugaa gacauacgca 1920 caccuguucg acgacaaggu caugaagcag cugaagagaa gaaguacac aggauggga 1980 agacugagca gaagcugau caacggaauc agacaagc agagcggaaa gacaauccug 2040 gacuuccuga agagcgacgg auucgcaaac agaaacuuca ugcagcugau ccacgacgac 2100 agccugacau ucaaggaaga cauccagaag gcacagguca gcggacaggg agacagccug 2160 cacgaacaca ucgcaaaccu ggcaggaagc ccggcaauca agaagggaau ccugcagaca 2220 gucaaggucg ucgacgaacu ggucaagguc augggaac acaagccgga aaacaucguc 2280 aucgaaugg caagaaaa ccagacaaca cagaagggac agaagaacag cagagaaaga 2340 augaagaa ucgaagaagg aaucaaggaa cugggaagcc agauccugaa ggaacacccg 2400 gucgaaaaca cacagcugca gacgaaaag cuguaccugu acuaccucca gacggaaga 2460 2520 2580 gacaagaaca gagaaagag cgacaacguc ccgagcgaag aagucgucaa gaaugaag 2640 aacuacugga gacagcugcu gacgcaaag cugaucacac agaaaguu cgacaaccug 2700 acaaaggcag agaggagg acugagcgaa cuggacaagg caggauucau caagacag 2760 cuggucgaaa caagacagau cacaaagcac gucgcacaga ucuggacag cagaaugaac 2820 acaaaguacg acgaaaacga caagcugauc agagauguca aggucaucac acugaagagc 2880 aagcugguca gcgacuucag aaaggacuuc cagucuaca aggucagaga aaucaacaac 2940 uaccaccacg cacacgacgc auaccugaac ggagucgucg gaagacacu gaucaagaag 3000 uacccgaagc uggaaagcga auucgucuac ggagacuaca aggucuacga cgucagaaag 3060 augaucgcaa agagcgaaca ggaaaucgga aagcaacag caaaguacuu cuucuacagc 3120 aacauacauga aucuuucaa gacagaaauc acacuggcaa acggagaaau cagaaagaga 3180 ccgcugaucg aaacaaacgg agaaacgga gaaucgucu gggacaaggg aagacacuuc 3240 ccaacaguca gaaagguccu gagcauggccg caggucaaca ucgucaagaa gacaagauguc 3300 cagacaggag gauucagcaa ggaaagcauc cugccgaaga gaacagcga caagcugauc 3360 ccaagaaaga aggacuggga cccgaaag uacggaggau ucgacagccc gacagucgca 3420 uacagcgucc uggucgucgc aaaggucgaa aagggaaaga ccaagaagcu gaagcguc 3480 aaggaacugc ugggaucgac aaucauggaa aggaagcagcu ucgaaagaa cccgaucgac 3540 ooooooooooooooooooooooooooooooooooooooooooooooo back go back gcugccgag 3600 uacagccugu ucgaaaaaaaaaaaaaaggc agghaagcgc aggaaaagg 3660 cagaagggaa acgaacuggc acugccgagc aaguacguca acuuccugua ccuggcaagc 3720 cacuacgaaa agcugaaggg aagccccgaaa agcuacgaaaagcccgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaccu says 3780 cagacaacagc acuaccugga cgaaaucauc gacagauca gcgaauucag cagagaguc 3840 auccuggcag acgshaaccu ggaaagguc cugagcgcau ahaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagg 3900 ccgaucagag aacaggcaga aacaucauc caccuguuca cacugacaaa ccugggagca 3960 ccggcagcau ucaaguacuu cgacacaca aucgacagaa agagauacac aagcacaaag 4020 gaguccugg axaaaccu gauccaccag aacaxaac 4080 gaccugagcc agcugggagg agacggaagc ggaagcccga agaagaag aaggcgac 4140 ggaagccccga agaagaag aaggcgac agcggauag 4179 <210> 24 <211> 4173 <212> RNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide” <400> 24 gapagaagu acagcaucgg acuggacauc ggaaaaca gcgucggaug ggcagucauc 60 acagacgaau acagguccc gaghagaag uucaggucc ugggaacac agacagac 120 shake shake agaaccugau cggashacug shake shake 180 accelerate accelerate accelerate accelerate accelerate 240 neighborhood ucuucagcaa cgaauggca aaggucgacg acagcuucuu ccacagacug 300 gaaagcu ucuggucga agagacaag aagcacgaaa gacaccgau cuucggaaac 360 aucgucgacg aagucgcaua ccacgaaaag uacccgacaa ucuaccaccu gagaagaag 420 480 540 gacaagcugu ucauccagcu gguccagaca uacaaccagc uguucgaaga aaacccgauc 600 aacgcaagcg gagucgacgc aaaggcaauc cugagcgcaa gacugagcaa gagcagaaga 660 cuggaaaacc ugaucgcaca cgucccggga gaaaagaaga acggacuguu cggaaaccug 720 aucgcacuga gccugggacu gacaccgaac uucaagagca acuucgaccu ggcagaagac 780 840 aucggagacc aguacgcaga ccuguuccug ccagcaaaga accugagcga cgcaauccug 900 cugagcgaca ucugagagu caacacagaa aucacaaagg caccgcugag cgcaagcaug 960 aucaagagau acgacgaaca ccaccaggac cugacacugc ugaaggcacu ggucagacag 1020 cagcugccgg aaaaguacaa ggaaaucuuc uucgaccaga gcaagaacgg auacgcagga 1080 uacaucgacg gagagcaag ccaggagaa uucuacaagu ucauacaagcc gauccuggaa 1140 aagouggacg gaacagaaga aucugcugguc aagcugaaca gagaagaaccu cgugaaag 1200 cagagaacau ucgacaacgg aagcaucccg caccagaucc accugggaga acugcacgca 1260 auccugagaa gacaggagaa cuucuacccg uuccugaagg aacaagaga aaagaucgaa 1320 aagauccuga cauucagaau cccguacuac gucggaccgc uggcaagagg aaacagcaga 1380 uucgcaugga ugacaagaaa gagcgaagaa acaaucaac cguggaacuu cgaagaaguc 1440 gucgacaagg gagcaagcgc acagagcuuc aucgaaagaa ugacaacuu cgacaagaac 1500 cugccgaacg aaagguccu gccgaagcac agccugcugu acgaauacuu cacagucuac 1560 aacgaacuga aaaggucaa guacgucaca gaaggaauga gaaagccggc auuccugagc 1620 gggaacaga agaaggcaau cgucgaccug cugucagaaaaaa ggucacaguc 1680 aagshaaaugcua agghaaugcuaaaaaaaaggcua cgaaaaacagc 1740 ggagucgaag acagaucauc cgcaagccug ggacauacc acgaccugcu gagaucauc 1800 shakespeed shakespeed shakespeed shakespeed 1860 accugacac uguecgaga cagagaaug aucgaagaaa gacugagac auacgcacac 1920 cuguacgacg aaaggucau gaagcagcug agagagaa gauaccacagg augggaa 1980 2040 2040 2040 2040 - agcugauca cgaaaaaaaaaaaccugac uuccugaaga gcgacggauu cgchaaaga aacuucaugc agcugaucca cgacgacagc 2100 cugacauuca aggagacau ccagaaggca caggu...

Claims

1. 1. A method for delivering mRNA to a hematopoietic stem and / or progenitor cell (HSPC) or HSPC population, comprising: a. pre-incubating an LNP composition comprising the mRNA, an amine lipid, a helper lipid, a neutral lipid, and a PEG lipid with ApoE; wherein the amine lipid is represented by the following formula (i): 【Chemistry 1】 (In the formula, R １ and R ２ are each independently a C4-C12 alkyl group), b. contacting the pre-incubated LNP composition with the HSPC or HSPC population in vitro; and c. Culturing the HSPC or the population of HSPCs in vitro; thereby comprising delivering said mRNA to said HSPC or said population of HSPCs.

2. 1. A method of delivering mRNA to HSPCs, comprising: a. pre-incubating an LNP composition comprising the mRNA and amine lipids with ApoE; wherein the amine lipid is represented by the following formula (i): 【Chemistry 2】 (In the formula, R １ and R ２ are each independently a C4-C12 alkyl group), b. contacting the pre-incubated LNP composition with the HSPCs in vitro; and c. culturing the HSPCs in vitro; thereby delivering the mRNA to the HSPCs.

3. 3. The method of claim 1 or 2, wherein the HSPC or HSPC population is CD34+ or CD34+CD90+.

4. The method of any one of claims 1 to 3, wherein the mRNA encodes a Cas nuclease.

5. 5. The method of claim 4, wherein the LNP composition further comprises a gRNA.

6. 1. A method of introducing Cas nuclease mRNA and gRNA into HSPCs, comprising: a. pre-incubating an LNP composition comprising the Cas nuclease mRNA, gRNA, amine lipids, helper lipids, neutral lipids, and PEG lipids with ApoE; wherein the amine lipid is represented by the following formula (i): 【Transformation 3】 (In the formula, R １ and R ２ are each independently a C4-C12 alkyl group), b. contacting the pre-incubated LNP composition with the HSPCs in vitro; and c. culturing the HSPC, The method thereby comprises introducing the Cas nuclease mRNA and gRNA into the HSPC.

7. 7. The method of claim 6, wherein the HSPC or HSPC population is CD34+ or CD34+CD90+.

8. The method of claim 6 or 7, wherein the mRNA and the gRNA are co-encapsulated in an LNP composition.

9. The method of claim 6 or 7, wherein the LNP composition comprises a first LNP and a second LNP, the mRNA is encapsulated in the first LNP, and the gRNA is encapsulated in the second LNP.

10. The method of any one of claims 6 to 9, wherein the gRNA is a dual guide RNA (dgRNA).

11. 10. The method of any one of claims 6 to 9, wherein the gRNA is a single guide RNA (sgRNA).

12. The method of any one of claims 6 to 11, wherein the gRNA is a modified gRNA.

13. 13. The method of claim 12, wherein the gRNA comprises modifications selected from 2'-O-methyl (2'-O-Me) modified nucleotides, phosphorothioate (PS) internucleotide linkages, and 2'-fluoro (2'-F) modified nucleotides.

14. the gRNA comprises a 5' end and a 3' end; (a) a modification in one or more of the first five nucleotides at the 5' end; (b) a modification in one or more of the last five nucleotides at the 3' end; (c) a PS bond between the first four nucleotides at the 5′ end, and (d) PS bond between the last four nucleotides at the 3' end 14. The method of claim 12 or 13, comprising at least one modification selected from:

15. the gRNA comprises a 5' end and a 3' end; (a) 2'-O-Me modified nucleotides in the first three nucleotides of the 5' end, and (b) 2'-O-Me modified nucleotides in the last three nucleotides of the 3' end The method according to any one of claims 12 to 14, comprising at least one modification selected from:

16. The method of any one of claims 4 to 15, wherein the Cas nuclease is a class 2 Cas nuclease.

17. 17. The method of claim 16, wherein the Class 2 Cas nuclease is a Cas9 nuclease or a Cpf1 nuclease.

18. 18. The method of claim 17, wherein the Class 2 Cas nuclease is a Cas9 nuclease, and the Cas9 nuclease is S. pyogenes Cas9.

19. The method of any one of claims 1 to 18, wherein cell survival after transfection is at least 60%.

20. 20. The method of any one of claims 1 to 19, comprising pre-incubating for 1 minute to 1 hour.

21. The method of any one of claims 1 to 20, wherein the LNP composition comprises a buffer.

22. The method of any one of claims 1 to 21, wherein the ApoE is selected from ApoE2, ApoE3, and ApoE4.

23. The method of any one of claims 1 to 22, wherein the ApoE is a human recombinant protein.

24. 24. The method of any one of claims 1 to 23, wherein the culturing step comprises contacting the HSPC or HSPC population with a stem cell growth factor.

25. The method of any one of claims 1 to 24, wherein the HSPC or the population of HSPCs is a human cell or sample.

26. 26. The method of any one of claims 1 to 25, further comprising introducing a template nucleic acid into the HSPC or the population of HSPCs.

27. 27. The method of any one of claims 1 to 26, wherein the N / P ratio is between 1 and 10, the N / P ratio being the ratio of positively charged amine groups (N) of the amine lipid to negatively charged phosphate groups (P) of the mRNA or mRNA and gRNA.

28. The amine lipid is 【Chemistry 4】 28. The method of claim 27, comprising administering to said patient a lipid A having the formula:

29. The method of claim 27 or 28, wherein the contents of amine lipids, neutral lipids, and PEG lipids relative to the total lipid content of the LNP composition are 40 to 60 mol%, 5 to 15 mol%, and 1.5 to 10 mol%, respectively, and the N / P ratio of the LNP composition is 3 to 10.

30. 30. The method of any one of claims 1 to 29, wherein the mRNA is modified mRNA.

31. 31. The method of any one of claims 1-30, wherein the mRNA comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 4, 10, 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.

32. 19. The method of any one of claims 4-18, wherein the gene editing is achieved and measured as an editing rate, and the editing rate is at least 40%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of total sequence reads.

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