CN117377464A - Compositions and methods for delivering nucleic acids - Google Patents
Compositions and methods for delivering nucleic acids Download PDFInfo
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- CN117377464A CN117377464A CN202280016755.3A CN202280016755A CN117377464A CN 117377464 A CN117377464 A CN 117377464A CN 202280016755 A CN202280016755 A CN 202280016755A CN 117377464 A CN117377464 A CN 117377464A
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Abstract
The present disclosure provides compositions and methods for genetically modifying cells, including but not limited to hepatocytes. The compositions and methods can include lipid nanoparticles, wherein the lipid nanoparticles include at least one bioreducable ionizable cationic lipid, at least one structural lipid, at least one phospholipid, and at least one pegylated lipid.
Description
Cross reference to related applications
The present application claims the benefit of U.S. provisional application No. 63/152,517 filed on day 23 of 2 months 2021, U.S. provisional application No. 63/156,649 filed on day 4 of 3 months 2021, U.S. provisional application No. 63/164,174 filed on day 22 of 3 months 2021, and U.S. provisional application No. 63/197,946 filed on day 7 of 6 months 2021. The contents of each of the above-mentioned patent applications are incorporated herein by reference in their entirety.
Sequence listing
The present application contains a sequence listing that has been submitted via EFS-Web in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 2022, 2/23, named "pots-066_001wo_seqlist.txt" and was about 320,338 bytes in size.
Technical Field
The present disclosure relates generally to novel lipid nanoparticles ("LNPs") comprising bioreducable ionizable cationic lipids, methods of making these LNPs, and uses of these LNPs for gene therapy and cell-based therapy applications.
Background
There is a long felt, but unmet need in the art for compositions and methods for delivering nucleic acids to cells, and for genetically modifying cells in vivo, ex vivo, and in vitro. Widely accepted techniques of gene delivery and modification, such as the use of viral vectors, including AAV, can cause acute toxicity and deleterious side effects in patients. The present disclosure provides improved compositions, methods, and kits for delivering nucleic acids to various types of cells, including hepatocytes, in vivo, ex vivo, and in vitro. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods of using the same. These lipid nanoparticle compositions and methods allow for the delivery of specific types of nucleic acids (e.g., RNA) to liver cells with high efficiency and low toxicity. Furthermore, the lipid nanoparticle compositions of the present disclosure exhibit improved storage stability, which is advantageous in clinical and commercial settings. Thus, the compositions and methods of the present disclosure can be used in a wide variety of fields, including gene therapy and the production of cell-based therapeutics.
Disclosure of Invention
In some aspects, novel lipid nanoparticles ("LNPs") are provided that include bioreducable ionizable cationic lipids. In one aspect, the bioreducable ionizable cationic lipid is a coatname SS-OP.
In one aspect, a pharmaceutical composition is provided comprising a composition of the present disclosure and at least one pharmaceutically acceptable excipient or diluent.
In one aspect, a method of delivering at least one nucleic acid to at least one cell is provided, the method comprising contacting the at least one cell with at least one composition of the present disclosure.
In one aspect, a method of genetically modifying at least one cell is provided, the method comprising contacting the at least one cell with at least one composition of the present disclosure.
In one aspect, there is provided a method of treating at least one disease or disorder in a subject in need thereof, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.
In one aspect, a method of delivering at least one nucleic acid to at least one cell is provided, the method comprising contacting the at least one cell with at least one composition of the present disclosure.
In one aspect, there is provided a cell modified according to the methods of the present disclosure.
Any of the above aspects may be combined with any other aspect.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification, the singular forms also include the plural unless the context clearly dictates otherwise; as an example, the terms "a/an" and "the" are to be understood as being singular or plural, and the term "or" is to be interpreted as inclusive. For example, "an element" means one or more elements. Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step or group of elements, integers or steps, but not the exclusion of any other element, integer or step or group of elements, integers or groups of steps. About may be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values provided herein are modified by the term "about.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description, and from the claims.
Drawings
The above described and additional features will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings.
Fig. 1 is a series of graphs showing human FVIII expression in adult mice administered with LNPs of the present disclosure.
Fig. 2 is a series of graphs showing human FIX expression in mice administered with LNP of the present disclosure.
FIG. 3 is a series of graphs showing the average diameter and polydispersity index (PDI) of various LNP compositions of the disclosure during a 7 day incubation at a temperature of 0.1mg/mL and 4 ℃.
Fig. 4 is a graph showing human FVIII expression in mice administered with LNP of the present disclosure.
Fig. 5 is a graph showing expression of human erythropoietin (hEPO) in a non-human primate (NHP) to which the LNP of the present disclosure is administered.
Fig. 6 is a series of graphs showing liver enzyme levels in NHPs to which LNPs of the present disclosure were administered.
Fig. 7 shows the survival of mice administered with human OTC (httc) transposon AAV viral vector particles and human OTC (httc) transposon AAV viral vector particles in combination with SPB LNPs of the present disclosure.
Fig. 8 is a series of graphs showing integrated Viral Copy Number (VCN) and httc mRNA levels in mice administered with human OTC (httc) transposon AAV viral vector particles in combination with the SPB LNP of the present disclosure.
Fig. 9 is a series of graphs showing survival and orotic acid levels of mice administered with human OTC (httc) transposon AAV viral vector particles in combination with SPB LNPs of the present disclosure.
Detailed Description
The present disclosure provides novel Lipid Nanoparticle (LNP) compositions comprising bioreducable ionizable cationic lipids, methods for preparing the compositions, and methods of using the compositions. In a non-limiting example, the compositions and methods of the present disclosure can be used for gene delivery in the context of gene therapy. In another non-limiting example, the compositions and methods of the present disclosure can be used in the context of cell-based therapeutic agents. In another non-limiting example, the compositions and methods of the present disclosure can be used extensively to deliver nucleic acids to induce expression of therapeutic proteins, including but not limited to secreted therapeutic proteins. In a non-limiting example, the compositions and methods of the present disclosure can be used broadly in delivering nucleic acids to liver cells in vivo, ex vivo, or in vitro for treating certain liver disorders.
The bioreducable ionizable cationic lipids of the present disclosure are biodegradable, thereby allowing the bioreducable ionizable cationic lipids to be decomposed and metabolized in the animal body. Without wishing to be bound by theory, this bioreduction advantageously reduces cationic lipid-related cytotoxicity.
In some aspects of the compositions and methods of the present disclosure, the bioreducable ionizable cationic lipid used in the LNP composition may be ssPalmO-Ph-P4C2. As understood by the skilled artisan, ssPalmO-Ph-P4C2 has the following structure:
as will be appreciated by those skilled in the art, ssPalmO-Ph-P4C2 may also be referred to asSS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe and ssPalmO-Ph. Thus, ssPalmO-Ph-P4C2、SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph are used interchangeably herein to refer to bioreducable, ionizable cationic lipids having a chemical structure shown in formula I.
Without wishing to be bound by theory, three specific segments of ssPalmO-Ph-P4C2 promote their biodegradation. First, the tertiary amine of each piperidine ring is an acidic pH-responsive cationic charging unit. Upon endocytosis, the tertiary amine moiety becomes positively charged in response to acidic intracellular endosomal compartments. These can now interact and destabilize the membrane, and this leads to endosomal escape. Once inside the cytoplasm, disulfide bonds are easily reduced by glutathione, yielding two free sulfhydryl groups. The resulting high concentration of free thiols further leads to nucleophilic reactions, and the particles undergo self-degradation/collapse by thioesterification and release the payload in the cytoplasm. This is defined as accelerated HyPER or hydrolysis by intra-particle enrichment of the reactants and generally potentially eliminates the potentially toxic side effects of cationic lipids.
Composition of the present disclosure, lipid nanoparticles
The present disclosure provides compositions comprising at least one lipid nanoparticle comprising at least one cationic lipid and at least one nucleic acid molecule. In some aspects, the lipid nanoparticle may further comprise at least one structural lipid. In some aspects, the lipid nanoparticle may further comprise at least one phospholipid. In some aspects, the lipid nanoparticle may further comprise at least one pegylated lipid.
Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid, and at least one pegylated lipid.
Bioreductive ionizable cationic lipids
In some aspects, the cationic lipid may be a bioreducable ionizable cationic lipid. Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducable ionizable cationic lipid.
As used herein, the term "bioreducable ionizable cationic lipid" is used in its broadest sense to mean a composition comprising: at least one tertiary amine, at least one disulfide group, at least one group comprising a bond susceptible to cleavage by thioesterification, and further comprising at least two cationic lipids of saturated or unsaturated hydrocarbon chains. Exemplary bioreductive ionizable cationic lipids include, but are not limited to, those described in Akita et al, (2020) biological and pharmaceutical bulletins (biol. Phar. Bull.) 43:1617-1625, the contents of which are incorporated herein by reference in their entirety.
In some aspects, the bioreducable ionizable cationic lipid may include at least two tertiary amines. In some aspects, at least one tertiary amine may be a substituted piperidinyl group. In some aspects, each tertiary amine can be a substituted piperidinyl group. In some aspects, the bioreducable ionizable cationic lipid may include at least one disulfide bond. In some aspects, the sulfur atom of the disulfide bond is linked to the nitrogen of the piperidinyl ring through an alkylene group, thereby forming two tertiary amine groups flanking the disulfide bond. In some aspects, at least one alkylene is ethylene. In some aspects, each alkylene is ethylene.
In some aspects, at least one group comprising a bond that is susceptible to cleavage by thioesterification may be a phenyl ester group. In some aspects, the bioreducable ionizable cationic lipid may include at least two phenyl ester groups. In some aspects, at least one of the at least two saturated or unsaturated hydrocarbon chains is an unsaturated hydrocarbon chain. In some aspects, each of the at least two saturated or unsaturated hydrocarbon chains is an unsaturated hydrocarbon chain. In some aspects, the unsaturated hydrocarbon chain may be octadecene. In some aspects, the octadecene can be (Z) -octadec-9-ene. In some aspects, the (Z) -octadec-9-enyl group may be linked to a phenyl ester group of a bioreducable ionizable cationic lipid.
Exemplary bioreductive ionizable cationic lipids that can be used in the methods of the present disclosure and methods of making such lipids include those disclosed in international patent application PCT/JP2016/052690 (published as WO/2016/121942) and international patent application PCT/JP 2019/01302 (published as WO/2019/188867), the contents of each of which are incorporated herein by reference in their entirety. Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises any one of the bioreducable ionizable cationic lipids shown in WO/2016/121942 and WO/2019/188867.
Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducable ionizable cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid, and at least one pegylated lipid.
In some aspects, the bioreductive ionizable cationic lipid may be ssPalmO-Ph-P4C2 having a structure represented by formula I (see Akita et al, (2020) biological and pharmaceutical bulletins 43:1617-1625, the contents of which are incorporated by reference in their entirety).
As described herein, LNP compositions of the present disclosure comprising at least one bioreducable ionizable cationic lipid advantageously exhibit significantly reduced toxicity in animals compared to LNP compositions comprising non-bioreducable ionizable cationic lipids. In particular, administration of the LNP compositions of the present disclosure surprisingly did not result in any weight loss. In fact, the LNP compositions of the present disclosure are so nontoxic that the animals administered the LNP actually gain weight even though the amount of LNP administered exceeds the lethal dose of the LNP composition comprising the non-bioreducable ionizable cationic lipid.
LNP component
In some aspects, the LNP of the present disclosure can include about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of the at least one bioreducable, ionizable cationic lipid.
In some aspects, the LNP of the present disclosure can include at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or at least about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one bioreducible lipid of the cations.
In some aspects, the LNP of the present disclosure can include about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of the at least one structuring lipid.
In some aspects, the LNP of the present disclosure can include at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or at least about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one lipid.
In some aspects, the LNP of the present disclosure can include about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of the at least one phospholipid.
In some aspects, the LNP of the present disclosure can include at least one phospholipid at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or at least about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol%.
In some aspects, an LNP of the present disclosure can include about 0.25 mole%, or about 0.5 mole%, or about 0.75 mole%, or about 1.0 mole%, or about 1.25 mole%, or about 1.5 mole%, or about 1.75 mole%, or about 2.0 mole%, or at least about or about 2.5 mole%, or about 5 mole% of at least one pegylated lipid.
In some aspects, the LNP of the present disclosure can comprise at least about 0.25 mole%, or at least about 0.5 mole%, or at least about 0.75 mole%, or at least about 1.0 mole%, or at least about 1.25 mole%, or at least about 1.5 mole%, or at least about 1.75 mole%, or at least about 2.0 mole%, or at least about 2.5 mole%, or at least about 5 mole% of at least one pegylated lipid.
Structural lipids
In some aspects, the structural lipid may be a steroid. In some aspects, the structural lipid may be a sterol. In some aspects, the structural lipid may include cholesterol. In some aspects, the structural lipid may include ergosterol. In some aspects, the structural lipid can be a phytosterol.
Phospholipid
As used herein, the term "phospholipid" is used in its broadest sense to refer to any amphiphilic molecule comprising a polar (hydrophilic) head group comprising a phosphate and two hydrophobic fatty acid chains.
In some aspects of the lipid nanoparticles of the present disclosure, the phospholipid may include dioleoyl phosphatidylethanolamine (DOPE).
In some aspects of the lipid nanoparticles of the present disclosure, the phospholipid may comprise DOPC (1, 2-dioleoyl-sn-glycero-3-phosphorylcholine).
In some aspects of the lipid nanoparticle of the present disclosure, the phospholipid may comprise DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine).
In some aspects, the phospholipid may comprise DDPC (1, 2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1, 2-diglycol-sn-glycero-3-phosphocholine), DEPC (1, 2-diglycol-sn-glycero-3-phosphocholine), DEPE (1, 2-diglycol-sn-glycero-3-phosphoethanolamine), DEPG-NA (1, 2-diglycol-sn-glycero-3 [ phospho-rac- (1-glycero) (sodium salt)), DLOPC (1, 2-dioleoyl-sn-glycero-3-phosphocholine), DLPA-NA (1, 2-dilauroyl-sn-glycero-3-phosphocholine (sodium salt)), DLPC (1, 2-dilauroyl-sn-glycero-3-phosphocholine), DLPE (1, 2-dilauroyl-sn-glycero-3-phosphoethanolamine), DLPG-NA (1, 2-dilauroyl-sn-glycero-3 [ phospho-rac- (1-glycero-sodium salt)), DLOPC (1, 2-dilauroyl-sn-glycero-3-phospho-ammonium salt), DLPA-NA (1, 2-dilauroyl-glycero-3-phospho-sodium salt), DLPS-NA (1, 2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt)), DMPA-NA (1, 2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt)), DMPC (1, 2-dimyristoyl-sn-glycero-3-phosphocholine), DMPE (1, 2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1, 2-dimyristoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (sodium salt)), DMPG-NH4 (1, 2-dimyristoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (ammonium salt)), DMPG-NH4/NA (1, 2-dimyristoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (sodium salt)), DMPA-2-dimyristoyl-sn-glycero-3-phospho-phosphoethanolamine (sodium salt), DMPA-2-dimyristoyl-sn-glycero-3-phospho-ethanolamine (sodium salt), DOS-2-glycero-3-phospho-phosphatidyl-serine (sodium salt), DMPA-NH 4 (1, 2-dimyristo-n-glycero-3-phospho-phosphate (sodium salt), DOPE (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPG-NA (1, 2-dioleoyl-sn-glycero-3 [ phosphoryl-rac- (1-glycero) (sodium salt)), DOPS-NA (1, 2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt)), DPPA-NA (1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DPPC (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPE (1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) DPPG-NA (1, 2-dipalmitoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (sodium salt)), DPPG-NH4 (1, 2-dipalmitoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (ammonium salt)), DPPS-NA (1, 2-dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt)), DSPA-NA (1, 2-distearoyl-sn-glycero-3-phospho-acid (sodium salt)), DSPC (1, 2-distearoyl-sn-glycero-3-phosphocholine), DPPS-NA (1, 2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1, 2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPG-NA (1, 2-distearoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (sodium salt)), DSPG-NH4 (1, 2-distearoyl-sn-glycero-3 [ phospho-rac- (1-glycero) (ammonium salt)), DSPS-NA (1, 2-distearoyl-sn-glycero-3-phosphoserine (sodium salt), EPC (egg PC), HEPC (hydrogenated egg PC), HSPC (hydrogenated soybean PC), LYSOPC MYSTIIC (1-myristoyl-sn-glycero-3-phosphocholine), LYSOPC PALMITIC (1-palmitoyl-sn-glycero-3-phosphocholine), milk sphingomyelin (C; 1-myristoyl-2-palmitoyl-glycero-sn-3-phospho-phosphocholine), MSPC (phospho-palmitoyl-2-phospho-phosphatidylcholine), LYSOPC (phospho-2-phospho-phosphatidyl-3-phospho choline), LYSOPC (1-palmitoyl-sn-glycero-3-phospho-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPG-NA (1-palmitoyl-2-oleoyl-sn-glycero-3 [ phospho-rac- (1-glycero) ] (sodium salt)), PSPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphorylcholine), SMPC (1-stearoyl-2-myristoyl-sn-glycero-3-phosphorylcholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphorylcholine), SPPC (1-stearoyl-2-palmitoyl-sn-glycero-3-phosphorylcholine), or any combination thereof.
PEGylated lipids
As used herein, the term "pegylated lipid" is used to refer to any lipid that is modified by (e.g., covalently linked to) at least one polyethylene glycol molecule. In some aspects, the pegylated lipid may comprise 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000, hereinafter referred to as DMG-PEG2000.
Nucleic acid
In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule. In some aspects, the lipid nanoparticle may include a plurality of nucleic acid molecules. In some aspects, the at least one nucleic acid molecule or the plurality of nucleic acid molecules may be formulated in a lipid nanoparticle.
In some aspects, the nucleic acid molecule may be a synthetic nucleic acid molecule. In some aspects, the nucleic acid molecule may be a non-naturally occurring nucleic acid molecule. In some aspects, a non-naturally occurring nucleic acid molecule can include at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide may be any non-naturally occurring nucleotide known in the art. In some aspects, the nucleic acid molecule may be a modified nucleic acid molecule. In some aspects, the modified nucleic acid molecule may include at least one modified nucleotide. The at least one modified nucleotide may be any modified nucleic acid known in the art.
In some aspects, the lipid nanoparticle may include a specific ratio (weight/weight) of lipid and nucleic acid.
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise a lipid and a nucleic acid in a weight/weight ratio of lipid to nucleic acid of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1, or about 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 105:1 to about 120:1, or about 110:1 to about 120:1, or about 120:1 to about 120:1, or about 110:1 to about 130:1, or about 120:1 to about 120:1, or about 110:1 to about 120:1, or about 120:1 to about 110:1, or about 120:1 to about 120:1.
In some aspects, the lipid nanoparticle may include a lipid and a nucleic acid in a weight/weight ratio of lipid to nucleic acid of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1, or about 200:1.
In some aspects, the lipid nanoparticle may include a lipid and a nucleic acid, the weight to weight ratio of lipid to nucleic acid being about 10:1, or about 17.5:1, or about 25:1.
In some aspects, the nucleic acid molecule may be an RNA molecule. Thus, in some aspects, the lipid nanoparticle may include at least one RNA molecule. In some aspects, the RNA molecule may be an mRNA molecule. In some aspects, the mRNA molecule may include a 5' cap.
In some aspects, the mRNA molecules can be capped using any method and/or capping moiety known in the art. The mRNA molecules may be capped with m7G (5 ') ppp (5') G portions. The m7G (5 ') ppp (5') G portion is also referred to as "Cap0". mRNA molecules can be usedAnd (5) partially capping.The moiety may comprise m7G (5 ') ppp (5 ') (2 ' OMeA) (-A->AG) portion.The part may include m7G (5 ') ppp (5 ') (2 ' OMeG)GG) moiety. mRNA molecules can be encoded by anti-reverse cap analogues +.>And (5) partially capping.The moiety may comprise an m7 (3 ' -O-methyl) G (5 ') ppp (5 ') G moiety. mRNA molecules can be used->3' OMe partCapping.
In some aspects, the mRNA molecule can include at least one modified nucleic acid.
The modified nucleic acid may include, but is not limited to, 5-methoxyuridine (5 moU), N 1 Methyl pseudouridine (me 1 ψ), pseudouridine (ψ), 5-methylcytidine (5-MeC).
In some aspects, the nucleic acid molecule may be a DNA molecule. Thus, in some aspects, the lipid nanoparticle may include at least one DNA molecule. In some aspects, the DNA molecule may be a circular DNA molecule, such as, but not limited to, a DNA plasmid. In some aspects, the lipid nanoparticle may comprise a DNA plasmid. In some aspects, the DNA molecule may be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid. In a certain aspect, the DNA molecule may be a dougybone DNA molecule. In some aspects, the DNA molecule may be a DNA nanoplasmid.
The length of the DNA plasmid may include a length that may be at least about 0.25kb, or at least about 0.5kb, or at least about 0.75kb, or at least about 1.0kb, or at least about 1.25kb, or at least about 1.5kb, or at least about 1.75kb, or at least about 2.0kb, or at least about 2.25kb, or at least about 2.5kb, or at least about 2.75kb, or at least about 3.0kb, or at least about 3.25kb, or at least about 3.5kb, or at least about 3.75kb, or at least about 4.0kb, or at least about 4.25kb, or at least about 4.5kb, or at least about 4.75kb, or at least about 5.0kb, or at least about 5.25kb, or at least about 5.5kb, or at least about 5.75kb, or at least about 6.0kb, or at least about 6.25kb, or at least about 6.5kb, or at least about 6.75kb, or at least about 7.0kb, or at least about 7.25 kb. Or at least about 7.75kb, or at least about 8.0kb, or at least about 8.25kb, or at least about 8.5kb, or at least about 8.75kb, or at least about 9.0kb, or at least about 9.25kb, or at least about 9.5kb, or at least about 9.75kb, or at least about 10.0kb, or at least about 10.25kb, or at least about 10.5kb, or at least about 10.75kb, or at least about 11.0kb, or at least about 11.25kb, or at least about 11.5kb, or at least about 11.75kb, or at least about 12kb, or at least about 12.25kb, or at least about 12.5kb, or at least about 12.75kb, or at least about 13.0kb, or at least about 13.25kb, or at least about 13.5kb, or at least about 13.75kb, or at least about 14.0kb, or at least about 14.25kb, or at least about 14.5kb, or at least about 14.75kb, or at least about 15.0kb.
LNP composition
In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule, at least one bioreducable ionizable cationic lipid, and at least one structural lipid. In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule, at least one bioreducable ionizable cationic lipid, and at least one pegylated lipid. In some aspects, the at least one bioreducable ionizable cationic lipid may be ssPalmO-Ph-P4C2.
In some aspects, the at least one structural lipid may be a mixture of two structural lipids. In some aspects, the at least one pegylated lipid may be a mixture of two pegylated lipids.
In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule, at least one bioreducable ionizable cationic lipid, at least one structural lipid, at least one pegylated lipid, or any combination thereof. In some aspects, the at least one bioreducable ionizable cationic lipid may be ssPalmO-Ph-P4C2.
In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule, at least one bioreducable ionizable cationic lipid, at least one structural lipid, and at least one pegylated lipid. In some aspects, the at least one bioreducable ionizable cationic lipid may be ssPalmO-Ph-P4C2.
In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule, at least one bioreducable ionizable cationic lipid, at least one structural lipid, at least one phospholipid, at least one pegylated lipid, or any combination thereof. In some aspects, the at least one bioreducable ionizable cationic lipid may be ssPalmO-Ph-P4C2.
In some aspects, the lipid nanoparticle may include at least one nucleic acid molecule, at least one bioreducable ionizable cationic lipid, at least one structural lipid, at least one phospholipid, and at least one pegylated lipid. In some aspects, the at least one bioreducable ionizable cationic lipid may be ssPalmO-Ph-P4C2.
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 28 mole% ssPalmO-Ph-P4C2, about 60 mole% cholesterol, about 10 mole% DOPE, and about 2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 10:1 (w/w). In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 29.5 mole% cholesterol, about 10 mole% DOPE, and about 0.5 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 32.92 mole% ssPalmO-Ph-P4C2, about 32.92 mole% cholesterol, about 32.92 mole% DOPE, and about 1.25 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 55:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 10 mole% ssPalmO-Ph-P4C2, about 29.5 mole% cholesterol, about 60 mole% DOPE, and about 0.5 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 60 mole% ssPalmO-Ph-P4C2, about 28 mole% to 29 mole% cholesterol, about 10 mole% DOPE, and about 1.25 mole% to 2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 28 mole% cholesterol, about 10 mole% DOPE, and about 2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 28.75 mole% cholesterol, about 10 mole% DOPE, and about 1.25 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 27.84 mole% ssPalmO-Ph-P4C2, about 56.25 mole% cholesterol, about 13.46 mole% DOPE, and about 2.45 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 88:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 27.9 mole% ssPalmO-Ph-P4C2, about 51.51 mole% cholesterol, about 18.59 mole% DOPE, and about 2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 26.4 mole% cholesterol, about 11.6 mole% DOPE, and about 2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 70:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 30.37 mole% ssPalmO-Ph-P4C2, about 37.27 mole% cholesterol, about 30.36 mole% DOPE, and about 2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 54 to 59 mole% ssPalmO-Ph-P4C2, about 30 to 40 mole% cholesterol, about 5 to 10 mole% DOPC, DSPC, or DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 49 to 60 mole% ssPalmO-Ph-P4C2, about 32 to 44 mole% cholesterol, about 5 mole% DOPC, and about 1.5 to 3.0 mole% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 54 mole% to 59 mole% ssPalmO-Ph-P4C2, about 30 mole% to 40 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the lipid to nucleic acid ratio in the at least one nanoparticle is about 75:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the lipid to nucleic acid ratio in the at least one nanoparticle is about 75:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise
About 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the lipid to nucleic acid ratio in the at least one nanoparticle is about 75:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 49.4 mole% ssPalmO-Ph-P4C2, about 44 mole% cholesterol, about 5 mole% DOPC, and about 1.6 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 60:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 32.8 mole% cholesterol, about 5 mole% DSPC, and about 2.2 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 60:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 34 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 40:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 41.8 mole% ssPalmO-Ph-P4C2, about 52.2 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one mRNA molecule. In some aspects, the mRNA molecule further comprises a 5' cap. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 60:1 (w/w).
In some aspects, the nucleic acid molecule is a DNA molecule. Accordingly, the present disclosure provides a lipid nanoparticle comprising at least one nucleic acid molecule, which may comprise about 22.71 mole% ssPalmO-Ph-P4C2, about 55.21 mole% cholesterol, about 20.89 mole% DOPE, and about 1.25 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA (see WO/2020/154645). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 50 mole% ssPalmO-Ph-P4C2, about 38.6 mole% cholesterol, about 10 mole% DOPE, and about 1.4 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA (see WO/2020/154645). In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 200:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 34.69 mole% ssPalmO-Ph-P4C2, about 39.74 mole% cholesterol, about 24.69 mole% DOPE, and about 1.25 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 50:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 50 mole% ssPalmO-Ph-P4C2, about 20 mole% cholesterol, about 29.5 mole% DOPE, and about 0.5 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 50 mole% ssPalmO-Ph-P4C2, about 20 mole% cholesterol, about 28.7 mole% DOPE, and about 1.3 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 50:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 54 to 59 mole% ssPalmO-Ph-P4C2, about 30 to 40 mole% cholesterol, about 5 to 10 mole% DOPC, DSPC, or DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 to about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DSPC, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 32.5 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 30 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% DOPE, and about 1 mole% DMG-PEG2000, wherein the lipid nanoparticle further comprises at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA molecule. In some aspects, the at least one DNA molecule may be a DNA nanoplasmid. In some aspects, the at least one DNA molecule may be a covalently end-capped DNA. In some aspects, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise from about 48 mol% to about 61 mol% ssPalmO-Ph-P4C2, from about 31 mol% to about 53 mol% cholesterol, from about 4 mol% to about 11 mol% phospholipids, and from about 0.5 mol% to about 3 mol% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise from about 41.8 mol% to about 60 mol% ssPalmO-Ph-P4C2, from about 32.8 mol% to about 52.2 mol% cholesterol, from about 5 mol% to about 10 mol% phospholipids, and from about 1 mol% to about 2.2 mol% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 49.4 mole% ssPalmO-Ph-P4C2, about 44 mole% cholesterol, about 5 mole% phospholipids, and about 1.6 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 32.8 mole% cholesterol, about 5 mole% phospholipids, and about 2.2 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 60 mole% ssPalmO-Ph-P4C2, about 34 mole% cholesterol, about 5 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 41.8 mole% ssPalmO-Ph-P4C2, about 52.2 mole% cholesterol, about 5 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 40:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 60:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise from about 53 mol% to about 60 mol% ssPalmO-Ph-P4C2, from about 34 mol% to about 41 mol% cholesterol, from about 4 mol% to about 11 mol% phospholipids, and from about 0.5 mol% to about 2 mol% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 75:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise from about 54 mole% to about 59 mole% ssPalmO-Ph-P4C2, from about 35 mole% to about 40 mole% cholesterol, from about 5 mole% to about 10 mole% phospholipids, and from about 0.5 mole% to about 1.5 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 75:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 10 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 75:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 59 mole% ssPalmO-Ph-P4C2, about 35 mole% cholesterol, about 5 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 75:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 54 mole% ssPalmO-Ph-P4C2, about 40 mole% cholesterol, about 5 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 75:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 56.5 mole% ssPalmO-Ph-P4C2, about 37.5 mole% cholesterol, about 5 mole% phospholipids, and about 1 mole% DMG-PEG2000. In some aspects, the at least one nucleic acid molecule comprises at least one RNA molecule. In some aspects, the at least one RNA molecule may be mRNA. In some aspects, the mRNA molecule may further include a 5' cap. In some aspects, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some aspects, the at least one DNA molecule may be a dougybone DNA or DNA nanoplasmid. In some aspects, the phospholipid may be DOPE. In some aspects, the phospholipid may be DOPC. In some aspects, the phospholipid may be DSPC. In some aspects, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 75:1 (w/w). In some aspects, the ratio of lipid to nucleic acid can be about 100:1 (w/w).
In some aspects of the disclosure, the lipid nanoparticle or plurality of lipid nanoparticles may be stable at about 2 ℃ to about 6 ℃ or about 4 ℃ for at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In some aspects of the disclosure, the lipid nanoparticle may be stable at about 4 ℃ for at least about 7 days.
In some aspects of the disclosure, the lipid nanoparticle or plurality of lipid nanoparticles may be stable at about 2 ℃ to about 6 ℃ or about 4 ℃ for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days. In some aspects of the disclosure, the lipid nanoparticle or plurality of lipid nanoparticles may be stable at about 4 ℃ for about 7 days.
In some aspects, a lipid nanoparticle or plurality of lipid nanoparticles can be said to be stable if the diameter (in the case of a single lipid nanoparticle) or average diameter (in the case of a plurality of lipid nanoparticles) does not vary by more than about 0.5%, or about 1%, or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%. As will be appreciated by those skilled in the art, the diameter of the lipid nanoparticle or the average diameter of the plurality of nanoparticles may be determined using any standard method known in the art.
In some aspects, a plurality of lipid nanoparticles can be said to be stable if the polydispersity index (PDI) or the plurality of lipid nanoparticles does not vary by more than about 0.5%, or about 1%, or about 5%, or about 10%, or about 15%, or about 20% chance, or about 25%, or about 30%, or about 35%, or about 40%, or about 45% or about 50%. As will be appreciated by those of skill in the art, the PDI of the plurality of lipid nanoparticles may be determined using any standard method known in the art.
C12-200 LNP of the present disclosure
The present disclosure also provides lipid nanoparticles comprising at least one nucleic acid molecule, at least one lipid, at least one structural lipid, at least one phospholipid, at least one pegylated lipid, or any combination thereof.
The lipid may be C12-200, also known as 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol), hereinafter referred to as "C12-200" (see U.S. Pat. Nos. 8,450,298, 8,969,353, 9,556,110 and 10,189,802. See also U.S. patent publication Nos. 2019-0177289).
Thus, a lipid nanoparticle of the present disclosure comprising at least one nucleic acid molecule may comprise from about 25 mole% to about 45 mole% of C12-200, from about 32 mole% to about 52 mole% of at least one structural lipid, from about 10 mole% to about 30 mole% of at least one phospholipid, and from about 0.1 mole% to about 13 mole% of at least one pegylated lipid. In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise from about 30 mole% to about 40 mole% of C12-200, from about 37 mole% to about 47 mole% of at least one structural lipid, from about 15 mole% to about 25 mole% of at least one phospholipid, and from about 0.1 mole% to about 8 mole% of at least one pegylated lipid. In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise from about 32.5 mol% to about 37.5 mol% C12-200, from about 39.5 mol% to about 44.5 mol% at least one structural lipid, from about 17.5 mol% to about 22.5 mol% at least one phospholipid, and from about 0.5 mol% to about 5.5 mol% at least one pegylated lipid. In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 35 mole% C12-200, about 42 mole% of at least one structural lipid, about 20 mole% of at least one phospholipid, and about 3 mole% of at least one pegylated lipid. In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule may comprise about 35 mole% C12-200, about 41.84 mole% of at least one structural lipid, about 20 mole% of at least one phospholipid, and about 3.16 mole% of at least one pegylated lipid. In some aspects, the aforementioned lipid nanoparticle may include a lipid and a nucleic acid, the weight to weight ratio of lipid to nucleic acid being from about 60:1 to about 100:1, or from about 70:1 to about 90:1, or from about 75:1 to about 85:1. In some aspects, the aforementioned lipid nanoparticle may include a lipid and a nucleic acid, the weight/weight ratio of lipid to nucleic acid being about 80:1. The phospholipid may be DOPE. The phospholipid may be DPSC. The phospholipid may be DOPC. The structural lipid may be cholesterol. The pegylated lipid may be DMG-PEG2000.
In some aspects, the at least one nucleic acid molecule is a DNA molecule or an RNA molecule. In some aspects, the nucleic acid molecule is a DNA molecule (e.g., a dougybone DNA molecule). Accordingly, the present disclosure provides a lipid nanoparticle comprising about 35 mole% C12-200, about 42 mole% of at least one structural lipid, about 20 mole% of at least one phospholipid, and about 3 mole% of at least one pegylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. The present disclosure also provides a lipid nanoparticle comprising about 35 mole% C12-200, about 41.84 mole% of at least one structural lipid, about 20 mole% of at least one phospholipid, and about 3.16 mole% of at least one pegylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some aspects, the at least one DNA molecule is a dougybone DNA molecule. In some aspects, the at least one DNA molecule is a DNA nanoplasmid. In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 35 mole% of at least one titratable cationic lipid, about 42 mole% of at least one structural lipid, about 20 mole% of at least one phospholipid, and about 3 mole% of at least one pegylated lipid, wherein the at least one nucleic acid is a DNA molecule (e.g., a dougybone DNA molecule, a DNA nanoplasmlet), wherein the ratio of lipid to nucleic acid in the nanoparticle is about 80:1 (weight/weight). In some aspects, a lipid nanoparticle comprising at least one nucleic acid molecule can comprise about 35 mole% of at least one titratable cationic lipid, about 41.84 mole% of at least one structural lipid, about 20 mole% of at least one phospholipid, and about 3.16 mole% of at least one pegylated lipid, wherein the at least one nucleic acid is a DNA molecule (e.g., a dougybone DNA molecule, a DNA nanoplasmid), wherein the lipid to nucleic acid ratio in the nanoparticle is about 80:1 (weight/weight). In some aspects, the aforementioned lipid nanoparticle may be used to deliver at least one DNA molecule (e.g., a dougybone DNA molecule, a DNA nanoplasmid) to at least one liver cell. The phospholipid may be DOPE. The phospholipid may be DPSC. The phospholipid may be DOPC. The structural lipid may be cholesterol. The pegylated lipid may be DMG-PEG2000.
This publicOpen alternative LNP embodiment
1. A composition comprising lipid nanoparticles comprising at least one of:
about 23 mole% to about 33 mole% ssPalmO-Ph-P4C2;
about 55 mole% to about 65 mole% cholesterol;
about 5 mole% to about 15 mole% DOPE; and
about 0.01 mole% to about 7 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
2. The composition of embodiment 1, wherein the at least one lipid nanoparticle comprises:
about 25.5 mole% to about 30.5 mole% ssPalmO-Ph-P4C2;
about 57.5 mole% to about 62.5 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% DOPE; and
about 0.1 mole% to about 4.5 mole% DMG-PEG2000.
3. The composition of embodiment 1 or embodiment 2, wherein the at least one lipid nanoparticle comprises:
about 27 mole% to about 29 mole% ssPalmO-Ph-P4C2;
about 59 mole% to about 61 mole% cholesterol;
about 9 mole% to about 11 mole% DOPE; and
about 1 mole% to about 3 mole% DMG-PEG2000.
4. The composition of any one of embodiments 1-3, wherein the at least one lipid nanoparticle comprises:
about 28 mole% ssPalmO-Ph-P4C2;
about 60 mole% cholesterol;
about 10 mole% DOPE; and
about 2 mole% DMG-PEG2000.
5. The composition of any one of embodiments 1-4, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 5:1 to about 15:1 (w/w).
6. The composition of any one of embodiments 1-5, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 10:1 (w/w).
7. The composition of any one of embodiments 1-6, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
8. The composition of any one of embodiments 1-7, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
9. The composition of any one of embodiments 1-8, wherein the at least one lipid nanoparticle comprises:
about 28 mole% ssPalmO-Ph-P4C2;
about 60 mole% cholesterol;
about 10 mole% DOPE; and
About 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 10:1 (w/w).
10. The composition of any one of embodiments 1-9, wherein the at least one lipid nanoparticle comprises:
about 28 mole% ssPalmO-Ph-P4C2;
about 60 mole% cholesterol;
about 10 mole% DOPE; and
about 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
11. A composition comprising lipid nanoparticles comprising at least one of:
about 55 mole% to about 65 mole% of ssPalmO-Ph-P4C2;
about 24.5 mole% to about 34.5 mole% cholesterol;
about 5 mole% to about 15 mole% DOPE; and
about 0.01 mole% to about 5.5 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
12. The composition of embodiment 11, wherein the at least one lipid nanoparticle comprises:
about 57.5 mole% to about 62.5 mole% ssPalmO-Ph-P4C2;
about 27 mole% to about 32 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% DOPE; and
about 0.1 mole% to about 3 mole% DMG-PEG2000.
13. The composition of embodiment 11 or embodiment 12, wherein the at least one lipid nanoparticle comprises:
about 59 mole% to about 61 mole% ssPalmO-Ph-P4C2;
about 28.5 mole% to about 30.5 mole% cholesterol;
about 9 mole% to about 11 mole% DOPE; and
about 0.1 mole% to about 1.5 mole% DMG-PEG2000.
14. The composition of any one of embodiments 11-13, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 29.5 mole% cholesterol;
about 10 mole% DOPE; and
about 0.5 mole% DMG-PEG2000.
15. The composition of any one of embodiments 11-14, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
16. The composition of any one of embodiments 11-15, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
17. The composition of any one of embodiments 11 to 16, wherein the at least one nanoparticle comprises:
about 60 mole% of Coatsome SS-OP;
about 29.5 mole% cholesterol;
about 10 mole% DOPE; and
about 0.5 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
18. A composition comprising lipid nanoparticles comprising at least one of:
about 27.92 to about 37.92 mole% ssPalmO-Ph-P4C2;
about 27.92 to about 37.92 mole% cholesterol;
about 27.92 to about 37.92 mole% DOPE; and
about 0.01 mole% to about 6.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
19. The composition of embodiment 18, wherein the at least one lipid nanoparticle comprises:
about 30.42 to about 35.42 mole% ssPalmO-Ph-P4C2;
About 30.42 to about 35.42 mole% cholesterol;
about 30.42 to about 35.42 mole% DOPE; and
about 0.1 mole% to about 3.75 mole% DMG-PEG2000.
20. The composition of embodiment 18 or embodiment 19, wherein the at least one lipid nanoparticle comprises:
about 31.92 to about 32.92 mole% ssPalmO-Ph-P4C2;
about 31.92 to about 32.92 mole% cholesterol;
about 31.92 to about 32.92 mole% DOPE; and
about 0.25 mole% to about 2.25 mole% DMG-PEG2000.
21. The composition of any one of embodiments 18 to 20, wherein the at least one lipid nanoparticle comprises:
about 32.92 mole% ssPalmO-Ph-P4C2;
about 32.92 mole% cholesterol;
about 32.92 mole% DOPE; and
about 1.25 mole% DMG-PEG2000.
22. The composition of any one of embodiments 18-21, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 50:1 to about 60:1 (w/w).
23. The composition of any one of embodiments 18-22, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 55:1 (w/w).
24. The composition of any one of embodiments 18 to 23, wherein the at least one lipid nanoparticle comprises:
about 32.92 mole% ssPalmO-Ph-P4C2;
about 32.92 mole% cholesterol;
about 32.92 mole% DOPE; and
about 1.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 55:1 (w/w).
25. A composition comprising lipid nanoparticles comprising at least one of:
about 5 mole% to about 15 mole% of ssPalmO-Ph-P4C2;
about 24.5 mole% to about 34.5 mole% cholesterol;
about 55 mole% to about 65 mole% DOPE; and
about 0.01 mole% to about 5.5 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
26. The composition of embodiment 25, wherein the at least one lipid nanoparticle comprises:
about 7.5 mole% to about 12.5 mole% ssPalmO-Ph-P4C2;
About 27 mole% to about 32 mole% cholesterol;
about 57.5 mole% to about 62.5 mole% DOPE; and
about 0.1 mole% to about 3 mole% DMG-PEG2000.
27. The composition of embodiment 25 or embodiment 26, wherein the at least one lipid nanoparticle comprises:
about 9 mole% to about 11 mole% ssPalmO-Ph-P4C2;
about 28.5 mole% to about 30.5 mole% cholesterol;
about 59 mole% to about 61 mole% DOPE; and
about 0.1 mole% to about 1.5 mole% DMG-PEG2000.
28. The composition of any one of embodiments 25-27, wherein the at least one lipid nanoparticle comprises:
about 10 mole% ssPalmO-Ph-P4C2;
about 29.5 mole% cholesterol;
about 60 mole% DOPE; and
about 0.5 mole% DMG-PEG2000.
29. The composition of any one of embodiments 25-28, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
30. The composition of any one of embodiments 25-29, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1.
31. The composition of any one of embodiments 25 to 30, wherein the at least one lipid nanoparticle comprises:
About 10 mole% ssPalmO-Ph-P4C2;
about 29.5 mole% cholesterol;
about 60 mole% DOPE; and
about 0.5 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
32. A composition comprising lipid nanoparticles comprising at least one of:
about 55 mole% to about 65 mole% of ssPalmO-Ph-P4C2;
about 23 mole% to about 33 mole% cholesterol;
about 5 mole% to about 15 mole% DOPE; and
about 0.1 mole% to about 7 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
33. The composition of embodiment 32, wherein the at least one lipid nanoparticle comprises:
about 57.5 mole% to about 62.5 mole% ssPalmO-Ph-P4C2;
about 25.5 mole% to about 30.5 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% DOPE; and
About 0.1 mole% to about 4.5 mole% DMG-PEG2000.
34. The composition of embodiment 32 or embodiment 33, wherein the at least one lipid nanoparticle comprises:
about 59 mole% to about 61 mole% ssPalmO-Ph-P4C2;
about 27 mole% to about 29 mole% cholesterol;
about 9 mole% to about 11 mole% DOPE; and
about 1 mole% to about 3 mole% DMG-PEG2000.
35. The composition of any one of embodiments 32 to 34, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 28 mole% cholesterol;
about 10 mole% DOPE; and
about 2 mole% DMG-PEG2000.
36. The composition of any one of embodiments 32-35, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
37. The composition of any one of embodiments 32-36, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
38. The composition of any one of embodiments 32 to 27, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
About 28 mole% cholesterol;
about 10 mole% DOPE; and
about 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
39. A composition comprising lipid nanoparticles comprising at least one of:
about 55 mole% to about 65 mole% of ssPalmO-Ph-P4C2;
about 23.75 mole% to about 33.75 mole% cholesterol;
about 5 mole% to about 15 mole% DOPE; and
about 0.01 mole% to about 6.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
40. The composition of embodiment 39, wherein the at least one nanoparticle comprises:
about 57.5 mole% to about 62.5 mole% ssPalmO-Ph-P4C2;
about 26.25 mole% to about 31.25 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% DOPE; and
about 0.1 mole% to about 3.75 mole% DMG-PEG2000.
41. The composition of embodiment 39 or embodiment 40, wherein the at least one nanoparticle comprises:
about 59 mole% to about 61 mole% ssPalmO-Ph-P4C2;
about 27.75 mole% to about 29.75 mole% cholesterol;
about 9 mole% to about 11 mole% DOPE; and
about 0.25 mole% to about 2.25 mole% DMG-PEG2000.
42. The composition of any one of embodiments 39 to 41, wherein the at least one nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 28.75 mole% cholesterol;
about 10 mole% DOPE; and
about 1.25 mole% DMG-PEG2000.
43. The composition of any one of embodiments 39-42, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 95:1 to about 105:1 (w/w).
44. The composition of any one of embodiments 39-43, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
45. The composition of any one of embodiments 39-44, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 28.75 mole% cholesterol;
about 10 mole% DOPE; and
About 1.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
46. A composition comprising lipid nanoparticles comprising at least one of:
about 17.71 mole% to about 27.71 mole% ssPalmO-Ph-P4C2;
about 50.21 mole% to about 60.21 mole% cholesterol;
about 23.83 to about 33.83 mole% DOPE; and
about 0.01 mole% to about 6.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule.
47. The composition of embodiment 46, wherein the at least one lipid nanoparticle comprises:
about 20.21 mole% to about 25.21 mole% ssPalmO-Ph-P4C2;
about 52.71 to about 57.71 mole% cholesterol;
about 26.33 mole% to about 31.33 mole% DOPE; and
about 0.1 mole% to about 3.75 mole% DMG-PEG2000.
48. The composition of embodiment 46 or embodiment 47, wherein the at least one lipid nanoparticle comprises:
About 21.71 mole% to about 23.71 mole% ssPalmO-Ph-P4C2;
about 54.21 to about 56.21 mole% cholesterol;
about 27.83 mole% to about 29.83 mole% DOPE; and
about 0.25 mole% to about 2.25 mole% DMG-PEG2000.
49. The composition of any one of embodiments 46 to 48, wherein the at least one lipid nanoparticle comprises:
about 22.71 mole% ssPalmO-Ph-P4C2;
about 55.21 mole% cholesterol;
about 28.83 mole% DOPE; and
about 1.25 mole% DMG-PEG2000.
50. The composition of any one of embodiments 46-49, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
51. The composition of any one of embodiments 46-50, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
52. The composition of any one of embodiments 46 to 51, wherein the at least one lipid nanoparticle comprises:
about 22.71 mole% ssPalmO-Ph-P4C2;
about 55.21 mole% cholesterol;
about 28.83 mole% DOPE; and
about 1.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
53. A composition comprising lipid nanoparticles comprising at least one of:
about 45 mole% to about 55 mole% of ssPalmO-Ph-P4C2;
about 33.6 mole% to about 43.6 mole% cholesterol;
about 5 mole% to about 15 mole% DOPE; and
about 0.01 mole% to about 6.4 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule.
54. The composition of embodiment 53, wherein the at least one lipid nanoparticle comprises:
about 47.5 mole% to about 52.5 mole% ssPalmO-Ph-P4C2;
about 36.1 mole% to about 41.1 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% DOPE; and
about 0.1 mole% to about 3.9 mole% DMG-PEG2000.
55. The composition of embodiment 53 or embodiment 54, wherein the at least one lipid nanoparticle comprises:
about 49 mole% to about 51 mole% ssPalmO-Ph-P4C2;
about 37.6 mole% to about 39.6 mole% cholesterol;
about 9 mole% to about 11 mole% DOPE; and
about 0.4 mole% to about 2.4 mole% DMG-PEG2000.
56. The composition of any one of embodiments 53-55, wherein the at least one lipid nanoparticle comprises:
about 50 mole% of ssPalmO-Ph-P4C2;
about 38.6 mole% cholesterol;
about 10 mole% DOPE; and
about 1.4 mole% DMG-PEG2000.
57. The composition of any one of embodiments 53-56, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 195:1 to about 205:1 (w/w).
58. The composition of any one of embodiments 53-57, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 200:1 (w/w).
59. The composition of any one of embodiments 53-58, wherein the at least one lipid nanoparticle comprises:
about 50 mole% of ssPalmO-Ph-P4C2;
about 38.6 mole% cholesterol;
about 10 mole% DOPE; and
about 1.4 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 200:1 (w/w).
60. A composition comprising lipid nanoparticles comprising at least one of:
about 29.69 to about 39.69 mole% ssPalmO-Ph-P4C2;
about 34.74 mole% to about 44.74 mole% cholesterol;
about 19.69 mole% to about 29.69 mole% DOPE; and
about 0.01 mole% to about 6.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule.
61. The composition of embodiment 60, wherein the at least one lipid nanoparticle comprises:
about 32.19 mole% to about 37.19 mole% ssPalmO-Ph-P4C2;
about 37.24 mole% to about 42.24 mole% cholesterol;
about 22.19 mole% to about 27.19 mole% DOPE; and
about 0.1 mole% to about 3.75 mole% DMG-PEG2000.
62. The composition of embodiment 60 or embodiment 61, wherein the at least one lipid nanoparticle comprises:
about 33.69 mole% to about 35.69 mole% ssPalmO-Ph-P4C2;
about 38.74 mole% to about 40.74 mole% cholesterol;
about 23.69 to about 25.69 mole% DOPE; and
about 0.25 mole% to about 2.25 mole% DMG-PEG2000.
63. The composition of any one of embodiments 60 to 62, wherein the at least one lipid nanoparticle comprises:
about 34.69 mole% ssPalmO-Ph-P4C2;
about 39.74 mole% cholesterol;
about 24.69 mole% DOPE; and
about 1.25 mole% DMG-PEG2000.
64. The composition of any one of embodiments 60-63, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 45:1 to about 55:1 (w/w).
65. The composition of any one of embodiments 60-64, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 50:1 (w/w).
66. The composition of any one of embodiments 60 to 65, wherein the at least one lipid nanoparticle comprises:
about 34.69 mole% ssPalmO-Ph-P4C2;
about 39.74 mole% cholesterol;
about 24.69 mole% DOPE; and
about 1.25 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 50:1 (w/w).
67. A composition comprising lipid nanoparticles comprising at least one of:
about 45 mole% to about 55 mole% of ssPalmO-Ph-P4C2;
about 15 mole% to about 25 mole% cholesterol;
about 24.5 mole% to about 34.5 mole% DOPE; and
about 0.01 mole% to about 5.5 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule.
68. The composition of embodiment 67, wherein the at least one lipid nanoparticle comprises:
about 47.5 mole% to about 52.5 mole% ssPalmO-Ph-P4C2;
about 17.5 mole% to about 22.5 mole% cholesterol;
about 27 mole% to about 32 mole% DOPE; and
about 0.1 mole% to about 3 mole% DMG-PEG2000.
69. The composition of embodiment 67 or embodiment 68, wherein the at least one lipid nanoparticle comprises:
about 49 mole% to about 51 mole% ssPalmO-Ph-P4C2;
about 19 mole% to about 21 mole% cholesterol;
about 28.5 mole% to about 30.5 mole% DOPE; and
about 0.25 mole% to about 1.5 mole% DMG-PEG2000.
70. The composition of any one of embodiments 67 to 69, wherein said at least one lipid nanoparticle comprises:
about 50 mole% of ssPalmO-Ph-P4C2;
about 20 mole% cholesterol;
about 29.5 mole% DOPE; and
about 0.5 mole% DMG-PEG2000.
71. The composition of any one of embodiments 67 to 70, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
72. The composition of any one of embodiments 67-71, wherein the ratio of lipid to nucleic acid in said at least one lipid nanoparticle is about 100:1 (w/w).
73. The composition of any one of embodiments 67 to 71, wherein said at least one lipid nanoparticle comprises:
about 50 mole% of ssPalmO-Ph-P4C2;
about 20 mole% cholesterol;
about 29.5 mole% DOPE; and
about 0.5 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
74. A composition comprising lipid nanoparticles comprising at least one of:
About 49 mole% to about 59 mole% ssPalmO-Ph-P4C2;
about 30 mole% to about 40 mole% cholesterol;
about 5 mole% to about 15 mole% DOPE; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
75. The composition of embodiment 74, wherein the at least one lipid nanoparticle comprises:
about 51.5 mole% to about 56.5 mole% ssPalmO-Ph-P4C2;
about 32.5 mole% to about 37.5 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% DOPE; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
76. The composition of embodiment 74 or embodiment 75, wherein the at least one lipid nanoparticle comprises:
about 53 mole% to about 55 mole% ssPalmO-Ph-P4C2;
about 59 mole% to about 61 mole% cholesterol;
about 9 mole% to about 11 mole% DOPE; and
about 0.5 mole% to about 2 mole% DMG-PEG2000.
77. The composition of any one of embodiments 74-76, wherein the at least one lipid nanoparticle comprises:
About 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000.
78. The composition of any one of embodiments 74-77, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
79. The composition of any one of embodiments 74-78, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
80. The composition of any one of embodiments 74-79, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
81. A composition comprising lipid nanoparticles comprising at least one of:
about 22.84 mole% to about 32.84 mole% ssPalmO-Ph-P4C2;
About 51.25 mole% to about 61.25 mole% cholesterol;
about 8.46 mol% to about 18.46 mol% DOPE; and
about 0.01 mole% to about 7.45 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
82. The composition of embodiment 81, wherein the at least one lipid nanoparticle comprises:
about 25.34 mole% to about 30.34 mole% ssPalmO-Ph-P4C2;
about 53.75 to about 58.75 mole% cholesterol;
about 10.96 mole% to about 15.96 mole% DOPE; and
about 0.1 mole% to about 4.95 mole% DMG-PEG2000.
83. The composition of embodiment 81 or embodiment 82, wherein the at least one lipid nanoparticle comprises:
about 26.84 mole% to about 28.84 mole% ssPalmO-Ph-P4C2;
about 55.25 mole% to about 57.25 mole% cholesterol;
about 12.46 mol% to about 13.46 mol% DOPE; and
about 1.45 mole% to about 3.45 mole% DMG-PEG2000.
84. The composition of any one of embodiments 81-83, wherein the at least one lipid nanoparticle comprises:
About 27.84 mole% ssPalmO-Ph-P4C2;
about 56.25 mole% cholesterol;
about 13.46 mole% DOPE; and
about 2.45 mole% DMG-PEG2000.
85. The composition of any one of embodiments 81-84, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 83:1 to about 93:1 (w/w).
86. The composition of any one of embodiments 81-85, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 88:1 (w/w).
87. The composition of any one of embodiments 81-86, wherein the at least one lipid nanoparticle comprises:
about 27.84 mole% ssPalmO-Ph-P4C2;
about 56.25 mole% cholesterol;
about 13.46 mole% DOPE; and
about 2.45 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 88:1 (w/w).
88. A composition comprising lipid nanoparticles comprising at least one of:
about 22.9 mole% to about 32.9 mole% ssPalmO-Ph-P4C2;
About 46.51 to about mole% cholesterol;
about 13.59 mole% to about 23.59 mole% DOPE; and
about 0.01 mole% to about 7 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
89. The composition of embodiment 88, wherein the at least one lipid nanoparticle comprises:
about 25.4 mole% to about 30.4 mole% ssPalmO-Ph-P4C2;
about 49.01 to about mole% cholesterol;
about 16.09 mole% to about 21.09 mole% DOPE; and
about 0.1 mole% to about 4.5 mole% DMG-PEG2000.
90. The composition of embodiment 88 or embodiment 89, wherein the at least one lipid nanoparticle comprises:
about 26.9 mole% to about 28.9 mole% ssPalmO-Ph-P4C2;
about 50.51 mole% to about 52.51 mole% cholesterol;
about 17.59 to about 19.59 mole% DOPE; and
about 1 mole% to about 3 mole% DMG-PEG2000.
91. The composition of any one of embodiments 88 to 90, wherein the at least one lipid nanoparticle comprises:
About 27.9 mole% ssPalmO-Ph-P4C2;
about 51.51 mole% cholesterol;
about 18.59 mole% DOPE; and
about 2 mole% DMG-PEG2000.
92. The composition of any one of embodiments 88 to 91, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
93. The composition of any one of embodiments 88 to 92, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
94. The composition of any one of embodiments 88 to 93, wherein the at least one lipid nanoparticle comprises:
about 27.9 mole% ssPalmO-Ph-P4C2;
about 51.51 mole% cholesterol;
about 18.59 mole% DOPE; and
about 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
95. A composition comprising lipid nanoparticles comprising at least one of:
about 55 mole% to about 65 mole% of ssPalmO-Ph-P4C2;
About 21.4 mole% to about 31.4 mole% cholesterol;
about 6.6 mole% to about 16.6 mole% DOPE; and
about 0.01 mole% to about 7 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
96. The composition of embodiment 95, wherein the at least one lipid nanoparticle comprises:
about 57.5 mole% to about 62.5 mole% ssPalmO-Ph-P4C2;
about 23.9 mole% to about 28.9 mole% cholesterol;
about 9.1 mole% to about 14.1 mole% DOPE; and
about 0.1 mole% to about 4.5 mole% DMG-PEG2000.
97. The composition of embodiment 95 or embodiment 96, wherein the at least one lipid nanoparticle comprises:
about 59 mole% to about 61 mole% ssPalmO-Ph-P4C2;
about 25.4 mole% to about 27.4 mole% cholesterol;
about 10.6 mole% to about 12.6 mole% DOPE; and
about 1 mole% to about 3 mole% DMG-PEG2000.
98. The composition of any one of embodiments 95 to 97, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
About 26.4 mole% cholesterol;
about 11.6 mole% DOPE; and
about 2 mole% DMG-PEG2000.
99. The composition of any one of embodiments 95 to 98, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 65:1 to about 75:1 (w/w).
100. The composition of any one of embodiments 95 to 99, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 70:1 (w/w).
101. The composition of any one of embodiments 95 to 100, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 26.4 mole% cholesterol;
about 11.6 mole% DOPE; and
about 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 70:1 (w/w).
102. A composition comprising lipid nanoparticles comprising at least one of:
about 25.37 mole% to about 35.37 mole% ssPalmO-Ph-P4C2;
about 32.27 mole% to about 42.27 mole% cholesterol;
About 25.36 mole% to about 35.36 mole% DOPE; and
about 0.01 mole% to about 7 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule.
103. The composition of embodiment 102, wherein the at least one lipid nanoparticle comprises:
about 27.87 mole% to about 32.87 mole% ssPalmO-Ph-P4C2;
about 34.77 mole% to about 39.77 mole% cholesterol;
about 27.86 mole% to about 32.86 mole% DOPE; and
about 0.1 mole% to about 4.5 mole% DMG-PEG2000.
104. The composition of embodiment 102 or embodiment 103, wherein the at least one lipid nanoparticle comprises:
about 29.37 mole% to about 31.37 mole% ssPalmO-Ph-P4C2;
about 36.27 to about 38.27 mole% cholesterol;
about 29.36 mole% to about 31.36 mole% DOPE; and
about 1 mole% to about 3 mole% DMG-PEG2000.
105. The composition of any one of embodiments 102-104, wherein the at least one lipid nanoparticle comprises:
about 30.37 mole% ssPalmO-Ph-P4C2;
About 37.27 mole% cholesterol;
about 30.36 mole% DOPE; and
about 2 mole% DMG-PEG2000.
106. The composition of any one of embodiments 102-105, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 95:1 to about 105:1 (w/w).
107. The composition of any one of embodiments 102-106, wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
108. The composition of any one of embodiments 102-107, wherein the at least one lipid nanoparticle comprises:
about 30.37 mole% ssPalmO-Ph-P4C2;
about 37.27 mole% cholesterol;
about 30.36 mole% DOPE; and
about 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
109. A composition comprising lipid nanoparticles comprising at least one of:
about 45 mole% to about 55 mole% of ssPalmO-Ph-P4C2;
about 15 mole% to about 25 mole% cholesterol;
About 23.7 mole% to about 33.7 mole% DOPE; and
about 0.01 mole% to about 6.3 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule.
110. The composition of embodiment 109, wherein the at least one lipid nanoparticle comprises:
about 47.5 mole% to about 52.5 mole% ssPalmO-Ph-P4C2;
about 17.5 mole% to about 22.5 mole% cholesterol;
about 26.2 mole% to about 31.2 mole% DOPE; and
about 0.1 mole% to about 4 mole% DMG-PEG2000.
111. The composition of embodiment 109 or embodiment 110, wherein the at least one lipid nanoparticle comprises:
about 49 mole% to about 51 mole% ssPalmO-Ph-P4C2;
about 19 mole% to about 21 mole% cholesterol;
about 27.7 mole% to about 29.7 mole% DOPE; and
about 0.25 mole% to about 2.3 mole% DMG-PEG2000.
112. The composition of any one of embodiments 109-111, wherein the at least one lipid nanoparticle comprises:
about 50 mole% of ssPalmO-Ph-P4C2;
about 20 mole% cholesterol;
About 28.7 mole% DOPE; and
about 1.3 mole% DMG-PEG2000.
113. The composition of any one of embodiments 109-112, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 45:1 to about 55:1 (w/w).
114. The composition of any one of embodiments 109-113, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 50:1 (w/w).
115. The composition of any one of embodiments 109-114, wherein the at least one lipid nanoparticle comprises:
about 50 mole% of ssPalmO-Ph-P4C2;
about 20 mole% cholesterol;
about 28.7 mole% DOPE; and
about 1.3 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w/w).
116. A composition comprising lipid nanoparticles comprising at least one of:
about 49 mole% to about 59 mole% ssPalmO-Ph-P4C2;
about 30 mole% to about 40 mole% cholesterol;
About 5 mole% to about 15 mole% of a phospholipid; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
117. The composition of embodiment 116, wherein the at least one lipid nanoparticle comprises:
about 51.5 mole% to about 56.5 mole% ssPalmO-Ph-P4C2;
about 32.5 mole% to about 37.5 mole% cholesterol;
about 7.5 mole% to about 12.5 mole% of a phospholipid; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
118. The composition of embodiment 116 or embodiment 117, wherein the at least one lipid nanoparticle comprises:
about 53 mole% to about 55 mole% ssPalmO-Ph-P4C2;
about 34 mole% to about 36 mole% cholesterol;
about 9 to about 11 mole% of a phospholipid; and
about 0.25 mole% to about 2 mole% DMG-PEG2000.
119. The composition of any one of embodiments 116-118, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% of a phospholipid; and
about 1 mole% DMG-PEG2000.
120. A composition comprising lipid nanoparticles comprising at least one of:
about 54 mole% to about 64 mole% ssPalmO-Ph-P4C2;
about 30 mole% to about 40 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
121. The composition of embodiment 120, wherein the at least one lipid nanoparticle comprises:
about 56.5 mole% to about 61.5 mole% ssPalmO-Ph-P4C2;
about 32.5 mole% to about 37.5 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
122. The composition of embodiment 120 or embodiment 121, wherein the at least one lipid nanoparticle comprises:
about 58 mole% to about 60 mole% ssPalmO-Ph-P4C2;
about 34 mole% to about 36 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
about 0.25 mole% to about 2 mole% DMG-PEG2000.
123. The composition of any one of embodiments 120 to 122, wherein the at least one lipid nanoparticle comprises:
About 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% of a phospholipid; and
about 1 mole% DMG-PEG2000.
124. A composition comprising lipid nanoparticles comprising at least one of:
about 49 mole% to about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% to about 45 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
125. The composition of embodiment 124, wherein the at least one lipid nanoparticle comprises:
about 51.5 mole% to about 56.5 mole% ssPalmO-Ph-P4C2;
about 37.5 mole% to about 42.5 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
126. The composition of embodiment 124 or embodiment 125, wherein the at least one lipid nanoparticle comprises:
about 53 mole% to about 55 mole% ssPalmO-Ph-P4C2;
about 39 mole% to about 41 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
About 0.25 mole% to about 2 mole% DMG-PEG2000.
127. The composition of any one of embodiments 124-126, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 40 mole% cholesterol;
about 5 mole% of a phospholipid; and
about 1 mole% DMG-PEG2000.
128. A composition comprising lipid nanoparticles comprising at least one of:
about 51.5 mole% to about 61.5 mole% ssPalmO-Ph-P4C2;
about 32.5 mole% to about 42.5 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
129. The composition of embodiment 128, wherein the at least one lipid nanoparticle comprises:
about 54 mole% to about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% to about 40 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
130. The composition of embodiment 128 or embodiment 129, wherein the at least one lipid nanoparticle comprises:
About 55.5 mole% to about 57.5 mole% ssPalmO-Ph-P4C2;
about 36.5 mole% to about 38.5 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
about 0.25 mole% to about 2 mole% DMG-PEG2000.
131. The composition of any one of embodiments 128 to 130, wherein the at least one lipid nanoparticle comprises:
about 56.5 mole% ssPalmO-Ph-P4C2;
about 37.5 mole% cholesterol;
about 5 mole% of a phospholipid; and
about 1 mole% DMG-PEG2000.
132. A composition comprising lipid nanoparticles comprising at least one of:
about 44.4 mole% to about 54.4 mole% ssPalmO-Ph-P4C2;
about 39 mole% to about 49 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 6.6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
133. The composition of embodiment 132, wherein the at least one lipid nanoparticle comprises:
about 46.9 mole% to about 51.9 mole% ssPalmO-Ph-P4C2;
about 41.5 mole% to about 46.5 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
About 0.1 mole% to about 4.1 mole% DMG-PEG2000.
134. The composition of embodiment 132 or embodiment 133, wherein the at least one lipid nanoparticle comprises:
about 48.4 mole% to about 50.4 mole% ssPalmO-Ph-P4C2;
about 43 mole% to about 45 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
about 0.6 mole% to about 2.6 mole% DMG-PEG2000.
135. The composition of any one of embodiments 132-134, wherein the at least one lipid nanoparticle comprises:
about 49.4 mole% ssPalmO-Ph-P4C2;
about 44 mole% cholesterol;
about 5 mole% of a phospholipid; and
about 1.6 mole% DMG-PEG2000.
136. A composition comprising lipid nanoparticles comprising at least one of:
about 55 mole% to about 65 mole% of ssPalmO-Ph-P4C2;
about 27.8 mole% to about 37.8 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 7.2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
137. The composition of embodiment 136, wherein the at least one lipid nanoparticle comprises:
About 57.5 mole% to about 62.5 mole% ssPalmO-Ph-P4C2;
about 30.3 mole% to about 35.3 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
about 0.1 mole% to about 4.7 mole% DMG-PEG2000.
138. The composition of embodiment 136 or embodiment 137, wherein the at least one lipid nanoparticle comprises:
about 59 mole% to about 61 mole% ssPalmO-Ph-P4C2;
about 31.8 mole% to about 33.8 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
about 1.2 mole% to about 2.2 mole% DMG-PEG2000.
139. The composition of any one of embodiments 136-138, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 32.8 mole% cholesterol;
about 5 mole% of a phospholipid; and
about 2.2 mole% DMG-PEG2000.
140. A composition comprising lipid nanoparticles comprising at least one of:
about 55 mole% to about 65 mole% of ssPalmO-Ph-P4C2;
about 29 mole% to about 39 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
Wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
141. The composition of embodiment 140, wherein the at least one lipid nanoparticle comprises:
about 57.5 mole% to about 62.5 mole% ssPalmO-Ph-P4C2;
about 31.5 mole% to about 36.5 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
142. The composition of embodiment 140 or embodiment 141, wherein the at least one lipid nanoparticle comprises:
about 59 mole% to about 61 mole% ssPalmO-Ph-P4C2;
about 33 mole% to about 35 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
about 0.25 mole% to about 2 mole% DMG-PEG2000.
143. The composition of any one of embodiments 140-142, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 34 mole% cholesterol;
about 5 mole% of a phospholipid; and
about 1 mole% DMG-PEG2000.
144. A composition comprising lipid nanoparticles comprising at least one of:
about 36.8 mole% to about 46.8 mole% ssPalmO-Ph-P4C2;
About 47.2 mole% to about 57.2 mole% cholesterol;
about 0.1 mole% to about 10 mole% of a phospholipid; and
about 0.01 mole% to about 6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
145. The composition of embodiment 144, wherein the at least one lipid nanoparticle comprises:
about 39.3 mole% to about 44.3 mole% ssPalmO-Ph-P4C2;
about 49.7 mole% to about 54.7 mole% cholesterol;
about 2.5 mole% to about 7.5 mole% of a phospholipid; and
about 0.1 mole% to about 3.5 mole% DMG-PEG2000.
146. The composition of embodiment 144 or embodiment 145, wherein the at least one lipid nanoparticle comprises:
about 40.8 mole% to about 42.8 mole% ssPalmO-Ph-P4C2;
about 51.2 mole% to about 53.2 mole% cholesterol;
about 4 to about 6 mole% of a phospholipid; and
about 0.25 mole% to about 2 mole% DMG-PEG2000.
147. The composition of any one of embodiments 144-146, wherein the at least one lipid nanoparticle comprises:
about 41.8 mole% ssPalmO-Ph-P4C2;
about 52.2 mole% cholesterol;
About 5 mole% of a phospholipid; and
about 1 mole% DMG-PEG2000.
148. The composition of any one of embodiments 116-147, wherein the at least one nucleic acid molecule is an RNA molecule.
149. The composition of embodiment 148, wherein the RNA molecule is an mRNA molecule.
150. The composition of embodiment 149, wherein the mRNA molecule further comprises a 5' cap.
151. The composition of any one of embodiments 116-147, wherein the at least one nucleic acid molecule is a DNA molecule.
152. The composition of embodiment 151, wherein the DNA molecule is a dougybone DNA molecule.
153. The composition of embodiment 151, wherein the DNA molecule is a DNA nanoplasmlet.
154. The composition of any one of embodiments 116-153, wherein said phospholipid is DOPE.
155. The composition of any one of embodiments 116-153, wherein said phospholipid is DOPC.
156. The composition of any one of embodiments 116-153, wherein said phospholipid is DSPC.
157. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 55:1 to about 110:1 (w/w).
158. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 20:1 (w/w).
159. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 40:1 (w/w).
160. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 60:1 (w/w).
161. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 75:1 (w/w).
162. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 80:1 (w/w).
163. The composition of any one of embodiments 116-156, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
164. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
About 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
165. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
166. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DSPC; and
about 1 mole% DMG-PEG2000,
Wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
167. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
168. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
169. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 40 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
170. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 40 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
171. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
About 56.5 mole% ssPalmO-Ph-P4C2;
about 37.5 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
172. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 56.5 mole% ssPalmO-Ph-P4C2;
about 37.5 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
173. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPC; and
About 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w/w).
174. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w/w).
175. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w/w).
176. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
177. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w/w).
178. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
About 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w/w).
179. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 49.4 mole% ssPalmO-Ph-P4C2;
about 44 mole% cholesterol;
about 5 mole% DOPC; and
about 1.6 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w/w).
180. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 32.8 mole% cholesterol;
about 5 mole% DSPC; and
About 2.2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w/w).
181. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 60 mole% of ssPalmO-Ph-P4C2;
about 34 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40:1 (w/w).
182. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 41.8 mole% ssPalmO-Ph-P4C2;
about 52.2 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w/w).
183. The composition of any one of the preceding embodiments, wherein the at least one lipid nanoparticle comprises:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w/w).
184. The composition of any one of the preceding embodiments, wherein the mRNA comprises a cytidine residue, i.e., 5-methylcytidine (5-MeC).
185. A composition comprising lipid nanoparticles comprising at least one of:
about 30 mole% to about 40% of C12-200;
about 36.84 to about 46.84 mole% cholesterol;
about 15 mole% to about 25 mole% DOPE; and
about 0.01 mole% to about 8.16 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule.
186. The composition of embodiment 185, wherein the at least one lipid nanoparticle comprises:
About 32.5 mole% to about 37.5 mole% of C12-200;
about 39.34 to about 44.34 mole% cholesterol;
about 17.5 mole% to about 22.5 mole% DOPE; and
about 0.1 mole% to about 5.66 mole% DMG-PEG2000.
187. The composition of embodiment 185 or embodiment 186, wherein the at least one lipid nanoparticle comprises:
about 34 mole% to about 36 mole% of C12-200;
about 40.84 to about 42.84 mole% cholesterol;
about 19 mole% to about 21 mole% DOPE; and
about 2.16 mole% to about 4.16 mole% DMG-PEG2000.
188. The composition of any one of embodiments 185 to 187, wherein the at least one lipid nanoparticle comprises:
about 35 mole% of C12-200;
about 41.84 mole% cholesterol;
about 20 mole% DOPE; and
about 3.16 mole% DMG-PEG2000.
189. The composition of any one of embodiments 185 to 188, wherein said at least one nucleic acid molecule comprises at least one DNA molecule.
190. The composition of embodiment 189, wherein the DNA molecule is a dougybone DNA molecule.
191. The composition of embodiment 190, wherein the DNA molecule is a DNA nanoplasmlet.
192. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is from about 70:1 to about 90:1 (w/w).
193. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 20:1 (w/w).
194. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 40:1 (w/w).
195. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 60:1 (w/w).
196. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 75:1 (w/w).
197. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 80:1 (w/w).
198. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w/w).
Pharmaceutical compositions of the present disclosure
In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure.
In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure. In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the present disclosure and at least one second nanoparticle of the present disclosure, wherein the at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, wherein the at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon.
In some aspects, the present disclosure provides a composition comprising at least one cell that has been contacted with at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified with at least one nanoparticle of the present disclosure. In some aspects, the present disclosure provides a composition comprising at least one cell that has been genetically modified using any of the methods of the present disclosure.
Methods of the present disclosure
The present disclosure provides a method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.
In all methods, compositions and kits of the present disclosure, the at least one cell may be a liver cell. The liver cells may include, but are not limited to, hepatocytes, hepatic stellate cells, kupfu cells, or liver sinusoidal endothelial cells.
In some aspects of any of the methods of the present disclosure, the cells may be in vivo, ex vivo, or in vitro. In some aspects, any of the methods of the present disclosure may be applied in vivo, ex vivo, or in vitro.
The present disclosure provides a method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one composition of the present disclosure. The present disclosure provides a method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one lipid nanoparticle of the present disclosure.
In some aspects, genetically modifying a cell may include delivering at least one exogenous nucleic acid to the cell such that the cell expresses at least one protein that the cell would otherwise normally express, or such that the at least one cell expresses at least one protein at a level that is higher than the cell would otherwise normally express, or such that the cell expresses at least one protein at a level that is lower than the cell would otherwise normally express. In some aspects, genetically modifying the cell may include delivering at least one exogenous nucleic acid to the cell such that the at least one exogenous nucleic acid is integrated into the genome of the at least one cell.
In some aspects, contacting the at least one cell with the at least one lipid nanoparticle of the disclosure may increase the level of expression of the at least one exogenous protein by at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 15-fold, or at least about 20-fold, or at least about 25-fold, or at least about 30-fold, or at least about 50-fold, as compared to the level of expression of the exogenous protein induced by contacting the at least one cell with the at least one control lipid nanoparticle.
In some aspects, the methods of the present disclosure can produce a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells in the plurality of cells express at least one protein encoded in at least one nucleic acid that is delivered to the plurality of cells by the nanoparticles of the present disclosure.
In some aspects, the methods of the present disclosure can produce a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the plurality of cells are hepatocytes, hepatic stellate cells, coulomb cells, or liver sinus endothelial cells.
In some aspects, the nucleic acid molecules formulated in the lipid nanoparticles of the present disclosure may include at least one genome editing composition.
The gene editing composition may comprise at least one nucleic acid molecule comprising at least one nucleic acid sequence encoding a DNA binding domain and a nucleic acid sequence encoding a nuclease protein or nuclease domain thereof. The nucleic acid sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof may comprise a DNA sequence, an RNA sequence, or a combination thereof.
In some aspects, a genome editing composition formulated in a lipid nanoparticle of the present disclosure can include a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises a nuclease-inactivated Cas (dCas) protein or a nuclease domain thereof and an endonuclease protein or a nuclease domain thereof.
In some aspects, a genome editing composition formulated in a lipid nanoparticle of the present disclosure can include a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas 9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof. In some aspects, the fusion protein may further comprise at least one Nuclear Localization Signal (NLS). In some aspects, the fusion protein may further comprise at least two NLS. In some aspects, the nucleic acid molecule may comprise DNA, RNA, or any combination thereof. In some aspects, the nucleic acid molecule may comprise RNA. Exemplary dCas9-Clo051 fusion proteins (referred to in the art as "Cas-clock" proteins) and polynucleotide sequences encoding the dCas9-Clo051 fusion proteins are described in detail in U.S. patent publication No. 2022/0042038, the disclosure of which is incorporated herein by reference in its entirety. Gene editing compositions, including Cas-CLOVER, and methods of gene editing using these compositions are described in detail in U.S. patent publication Nos. 2017/0107541, 2017/014149, 2018/0187185, and U.S. patent No. 10,415,024, the contents of each of which are incorporated herein by reference in their entirety.
In some aspects, the Cas-cycle protein may comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID No. 31.
In some aspects, the Clo051 protein or nuclease domain can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO. 32.
In some aspects, the inactivated Cas9 (dCas 9) protein or the inactivated nuclease domain thereof may comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID No. 33.
In some aspects, the genome editing composition formulated in the lipid nanoparticle of the present disclosure may further comprise at least one guide molecule. In some aspects, the guide molecule may be a guide RNA (gRNA molecule).
Accordingly, the present disclosure provides any of the lipid nanoparticle compositions described herein, wherein the lipid nanoparticle comprises at least one genome editing composition, wherein the at least one genome editing composition comprises: a) A nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas 9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof; and b) at least one gRNA molecule. In some aspects, the fusion protein may further comprise at least one NLS. In some aspects, the at least one genome editing composition can comprise at least two gRNA molecules.
The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure, the at least one composition comprising at least one nucleic acid encoding a therapeutic protein.
The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of a cell, wherein the cell has been contacted with at least one nanoparticle of the present disclosure, the nanoparticle comprising at least one nucleic acid encoding a therapeutic protein. The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of a cell, wherein the cell has been genetically modified using the compositions and/or methods of the present disclosure.
In some aspects, the at least one disease may be a Metabolic Liver Disorder (MLD). MLD may include, but is not limited to, N-acetylglutamate synthase (N-Acetylglutamate Synthetase, NAGS) deficiency, carbamoylphosphate synthase I (Carbamoylphosphate SynthetaseI) deficiency (CPSI deficiency), ornithine carbamoyltransferase (OTC) deficiency (Argininosuccinate Synthetase Deficiency, ASSD) (citrullinemia I (CitrullinemiaI)), schlin protein deficiency (citrin defliciency) (citrullinemia II (Citrullinemia II)), argininosuccinate lyase deficiency (Argininosuccinate Lyase Deficiency) (argininosuccinic acid urea), arginase deficiency (argininasedefliciency) (homoargininemia (Hyperargininemia)), ornithine translocase defliciency (HHH Syndrome)), methylmalonic acid (MMA), progressive familial intrahepatic cholestasis type 1 (progressive familiaintrahepaticcholestasistype, ic1), progressive familial intrahepatic cholestasis type 1 (28, pf1), pf1, 371-type 1, or any combination thereof.
In some aspects, the at least one disease may be hemophilia. In some aspects, the hemophilia is hemophilia a. In some aspects, the hemophilia is hemophilia B. In some aspects, the hemophilia is hemophilia C.
In some aspects, the at least one disease may be a disease and/or disorder characterized by increased LDL-cholesterol. Accordingly, the present disclosure provides methods of reducing LDL-cholesterol in a subject in need thereof.
In some aspects, the nucleic acid molecules formulated in the lipid nanoparticles of the present disclosure may include at least one transgene sequence. In some aspects, the transgene sequence may include a nucleotide sequence encoding at least one therapeutic protein. In some aspects, the transgene sequence may include a nucleotide sequence encoding at least one transposase.
In some aspects, the transgene sequence may include a nucleotide sequence encoding at least one transposon. In some aspects, the transposon may comprise a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a transposon may comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, wherein the at least one therapeutic protein is operably linked to the at least one promoter sequence. In some aspects, the transposon may comprise at least one Inverted Terminal Repeat (ITR). In some aspects, the transposon can include a first ITR and at least a second ITR. In some aspects, the transposon may comprise at least one insulator sequence. In some aspects, the transposon may comprise a first insulator sequence and at least a second insulator sequence. In some aspects, the transposon may comprise at least one sequence encoding at least one therapeutic protein. In some aspects, the transposon can comprise at least one 5' utr sequence. In some aspects, the transposon can comprise at least one 3' utr sequence. In some aspects, the transposon can include a first 3'utr sequence and at least a second 3' utr sequence. In some aspects, the transposon may comprise at least one polyA sequence.
In some aspects, the transposon may comprise at least one sequence encoding a therapeutic protein and a 3' utr sequence. In some aspects, the transposon may include at least one sequence encoding a therapeutic protein in the 5' to 3' direction and a 3' utr sequence. In some aspects, the transposon may comprise at least one sequence encoding a therapeutic protein followed by a 3' utr sequence.
In some aspects, the transposon can include a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a 3' utr sequence, a polyA sequence, a second insulator sequence, and a second ITR. In some aspects, the transposon may include a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a 3' utr sequence, a polyA sequence, a second insulator sequence, and a second ITR in the 5' to 3' direction. In some aspects, the transposon can comprise a first ITR, followed by a first insulator sequence, followed by at least one promoter sequence, followed by at least one sequence encoding at least one therapeutic protein, followed by a 3' utr sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second ITR.
In some aspects of the foregoing transposons, the at least one sequence encoding the at least one therapeutic protein may be a sequence encoding a FVIII-BDD polypeptide, wherein the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO. 21. In some aspects, the sequence encoding a FVIII-BDD polypeptide can include the nucleic acid sequence of SEQ ID NO. 22.
In some aspects of the foregoing transposons, the 3' UTR sequence may comprise the nucleic acid sequence of SEQ ID NO. 27.
Thus, in a non-limiting example, a transposon may comprise at least one therapeutic protein encoding sequence and a 3'UTR sequence, wherein the at least one therapeutic protein encoding sequence is a sequence encoding a FVIII-BDD polypeptide, wherein the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO:21, and wherein the 3' UTR sequence comprises the nucleic acid sequence of SEQ ID NO: 27.
In some aspects, the transposon may include at least one sequence encoding a therapeutic protein, a first 3'utr sequence, and a second 3' utr sequence. In some aspects, the transposon may include at least one therapeutic protein encoding sequence in the 5 'to 3' sequence, a first 3'utr sequence, and a second 3' utr sequence. In some aspects, the transposon may comprise at least one sequence encoding a therapeutic protein, followed by a first 3'utr, followed by a second 3' utr sequence.
In some aspects, the transposon may comprise a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a first 3'utr sequence, a second 3' utr sequence, a polyA sequence, a second insulator sequence, and a second ITR. In some aspects, the transposon may include a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a first 3'utr sequence, a second 3' utr sequence, a polyA sequence, a second insulator sequence, and a second ITR in the 5 'to 3' direction. In some aspects, the transposon may comprise a first ITR, followed by a first insulator sequence, followed by at least one promoter sequence, followed by at least one sequence encoding at least one therapeutic protein, followed by a first 3'utr sequence, followed by a second 3' utr sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second ITR.
In some aspects of the foregoing transposons, the at least one sequence encoding the at least one therapeutic protein may be a sequence encoding a FVIII-BDD polypeptide, wherein the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO. 21. In some aspects, the ta sequence encoding a FVIII-BDD polypeptide can include the nucleic acid sequence of SEQ ID NO. 22.
In some aspects of the foregoing transposons, the first 3' UTR sequence may be an AES 3' UTR sequence, wherein the AES 3' UTR sequence comprises the nucleic acid sequence of SEQ ID No. 25.
In some aspects of the foregoing transposons, the second 3' UTR sequence may be an mtRNR13' UTR sequence, wherein the mtRNR13' UTR sequence comprises the nucleic acid sequence of SEQ ID No. 26.
Thus, in a non-limiting example, a transposon may comprise at least one therapeutic protein encoding sequence, a first 3'UTR sequence, and a second 3' UTR sequence, wherein the at least one therapeutic protein encoding sequence is a FVIII-BDD polypeptide encoding sequence, wherein the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO:21, and wherein the first 3'UTR sequence comprises the nucleic acid sequence of SEQ ID NO:25, and the second 3' UTR sequence comprises the nucleic acid sequence of SEQ ID NO: 26.
In some aspects, a transposon may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID No. 28.
In some aspects, a transposon may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID No. 34.
In some aspects, a transposon may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID No. 35.
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of a methylmalonate monoacyl-coa mutase (MUT 1) polypeptide.
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of an ornithine carbamoyltransferase (OTC) polypeptide.
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of a Factor VIII (FVIII) polypeptide. In some aspects, the FVIII polypeptide can be a B domain-deficient FVIII polypeptide (hereinafter referred to as a FVIII-BDD polypeptide). As will be appreciated by the skilled artisan, factor VIII-BDD polypeptides remain biologically active in vitro and in vivo (see Kessler et al, hemophilia (Haemophilia), 2005,11 (2): 84-91).
In some aspects, the FVIII-BDD polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO. 21. In some aspects, a nucleic acid sequence encoding a FVIII-BDD polypeptide can include, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NO. 22 or 36.
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of a Factor IX (FIX) polypeptide. In some aspects, the FIX polypeptide may include an R338L mutation. As the skilled artisan will appreciate, the R338L mutation may be referred to as a Padua mutation (see VandenDriessche and Chuah, molecular therapy (Molecular Therapy), 2018, volume 26, stage 1, P14-16, the contents of which are incorporated herein by reference in their entirety).
The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
In some aspects of the foregoing methods, the composition comprising a nucleic acid molecule comprising a transposon may be a composition comprising at least one LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. Accordingly, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
In some aspects of the foregoing methods, a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be a composition comprising at least one LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. Accordingly, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of a composition comprising a nucleic acid comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid comprising a nucleotide sequence encoding at least one transposase.
Additionally, the present disclosure also provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
In some aspects of the foregoing methods, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposon may be a composition comprising an adeno-associated virus (AAV) viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. Accordingly, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
Additionally, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
In some aspects of the foregoing methods, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be a composition comprising an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. Accordingly, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of a composition comprising a nucleic acid comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
Additionally, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.
In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises, consists essentially of, or consists of a nucleotide sequence encoding at least one therapeutic protein, wherein the therapeutic protein is OTC, and may comprise, consist of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any of SEQ ID NOs 1-6.
In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises, consists essentially of, or consists of a nucleotide sequence encoding at least one therapeutic protein, wherein the therapeutic protein is factor VIII, and may comprise, consist of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs 8-14.
In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises, consists essentially of, or consists of a nucleotide sequence encoding at least one therapeutic protein, wherein the therapeutic protein is factor IX, and may comprise, consist of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs 15-20.
In non-limiting examples, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding a transposase may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID No. 7.
In some aspects of the foregoing methods, a composition comprising a nucleic acid molecule comprising a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be administered simultaneously, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. In some aspects, a composition comprising a nucleic acid molecule comprising a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be sequentially administered, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. In some aspects, compositions comprising nucleic acid molecules comprising a transposon and compositions comprising nucleic acid molecules comprising nucleotide sequences encoding at least one transposase may be administered in temporal proximity, wherein the transposon comprises nucleotide sequences encoding at least one therapeutic protein.
As used herein, the term "temporally proximate" refers to the administration of one therapeutic composition (including a composition of a transposon) occurring in a time period before or after the administration of another therapeutic composition (e.g., a composition including a transposase) such that the therapeutic effect of one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of one therapeutic agent completely overlaps with the therapeutic effect of another therapeutic agent. In some embodiments, "proximate in time" means that administration of one therapeutic agent occurs within a period of time before or after administration of another therapeutic agent such that there is a synergistic effect between one therapeutic agent and the other therapeutic agent. The "temporal proximity" may vary depending on a variety of factors including, but not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to whom the therapeutic agent is to be administered; a disease or condition to be treated or ameliorated; the therapeutic result to be achieved; dosage, frequency and duration of administration of the therapeutic agent; pharmacokinetics and pharmacodynamics of the therapeutic agent; and the route(s) of administration of the therapeutic agent. In some embodiments, "time proximity" means within 15 minutes, within 30 minutes, within one hour, within two hours, within four hours, within six hours, within eight hours, within 12 hours, within 18 hours, within 24 hours, within 36 hours, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within one week, within 2 weeks, within 3 weeks, within 4 weeks, within 6 weeks, or within 8 weeks. In some embodiments, multiple administrations of one therapeutic agent may occur in close temporal proximity to a single administration of another therapeutic agent. In some embodiments, the temporal proximity may vary during a treatment cycle or within a dosing regimen.
In a non-limiting example, the present disclosure provides a method of treating a metabolic liver disorder in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one DNA molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some aspects, the metabolic liver disorder may be ornithine carbamoyltransferase (OTC) deficiency, and the at least one therapeutic protein may include an ornithine carbamoyltransferase (OTC) polypeptide. In some aspects, the metabolic liver disorder may be methylmalonate (MMA) and the at least one therapeutic protein may comprise a methylmalonate monoacyl-coa mutase (MUT 1) polypeptide.
In a non-limiting example, the present disclosure provides a method of treating hemophilia in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one DNA molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some aspects, the hemophilia can be hemophilia a, and the at least one therapeutic protein can include factor VIII. In some aspects, the hemophilia can be hemophilia B, and the at least one therapeutic protein can include factor IX.
In a non-limiting example, the present disclosure provides a method of treating a metabolic liver disorder in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some aspects, the metabolic liver disorder may be ornithine carbamoyltransferase (OTC) deficiency, and the at least one therapeutic protein may include an ornithine carbamoyltransferase (OTC) polypeptide. In some aspects, the metabolic liver disorder may be methylmalonate (MMA) and the at least one therapeutic protein may comprise a methylmalonate monoacyl-coa mutase (MUT 1) polypeptide.
In a non-limiting example, the present disclosure provides a method of treating hemophilia in a subject, the method comprising administering to the subject: a) At least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some aspects, the hemophilia can be hemophilia a, and the at least one therapeutic protein can include factor VIII. In some aspects, the hemophilia can be hemophilia B, and the at least one therapeutic protein can include factor IX.
The present disclosure provides methods of treating diseases and/or conditions characterized by increased LDL-cholesterol comprising administering to the subject at least one LNP of the disclosure comprising a genome editing composition, wherein the genome editing composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas 9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof. In some aspects, the fusion protein can be a Cas-cycle protein. In some aspects, the genome editing composition may further comprise at least one guide RNA (gRNA) molecule that targets the pcsk9 gene. In some aspects, the genome editing composition may further comprise at least two gRNA molecules targeting the pcsk9 gene. The pcsk9 gene-targeted gRNA molecule may comprise, consist of, or consist essentially of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs 29-30.
The present disclosure provides a method of reducing LDL-cholesterol in a subject in need thereof, the method comprising administering to the subject at least one LNP of the disclosure comprising a genome editing composition, wherein the genome editing composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas 9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof. In some aspects, the fusion protein can be a Cas-cycle protein. In some aspects, the genome editing composition may further comprise at least one guide RNA (gRNA) molecule that targets the pcsk9 gene. In some aspects, the genome editing composition may further comprise at least two gRNA molecules targeting the pcsk9 gene. The pcsk9 gene-targeted gRNA molecule may comprise, consist of, or consist essentially of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs 29-30.
In some aspects of the methods of treatment of the present disclosure, administration of at least one composition and/or nanoparticle of the present disclosure to a subject may cause expression of an exogenous protein (e.g., therapeutic protein, transposase, etc.) in at least one organ and/or tissue of the subject.
In some aspects, at least one composition and/or nanoparticle of the present disclosure is administered such that the exogenous protein is expressed in at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells in the tissue and/or organ.
In some aspects, at least one composition and/or nanoparticle of the present disclosure is administered such that the exogenous protein is expressed in at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of one or more particular cell subsets in the tissue and/or organ.
In some aspects, at least one composition and/or nanoparticle of the present disclosure is administered such that expression of the exogenous protein in the tissue and/or organ lasts at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.
In some aspects, at least one composition and/or nanoparticle of the present disclosure is administered such that expression of the exogenous protein in one or more particular cell subsets in the tissue and/or organ lasts at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.
In some aspects, at least one composition and/or nanoparticle of the present disclosure is administered such that expression of the exogenous protein in the tissue and/or organ lasts no more than about 1 day, or no more than about 2 days, or no more than about 3 days, or no more than about 4 days, or no more than about 5 days, or no more than about 6 days, or no more than about 7 days, or no more than about 8 days, or no more than about 9 days, or no more than about 10 days.
In some aspects, at least one composition and/or nanoparticle of the present disclosure is administered such that expression of the exogenous protein in one or more specific cell subsets in the tissue and/or organ lasts no more than about 1 day, or no more than about 2 days, or no more than about 3 days, or no more than about 4 days, or no more than about 5 days, or no more than about 6 days, or no more than about 7 days, or no more than about 8 days, or no more than about 9 days, or no more than about 10 days.
In some aspects, the percentage of cells expressing an endogenous protein when administered the composition of the present disclosure can be increased by at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 15-fold, or at least about 20-fold, or at least about 25-fold, or at least about 30-fold, or at least about 50-fold, as compared to the percentage of cells expressing an endogenous protein when administered the composition comprising the control nanoparticle.
In some aspects, the tissue and/or organ may be the liver. In some aspects, the one or more specific cell subsets may include, but are not limited to, hepatocytes, hepatic stellate cells, kupfu cells, liver sinusoidal endothelial cells, or any combination thereof.
In some aspects, administration of a composition comprising at least one lipid nanoparticle of the present disclosure is less toxic than administration of a composition comprising at least one control lipid nanoparticle.
In some aspects, the reduced toxicity may be manifested as an increased attenuation of the level of at least one liver enzyme after administration of at least one lipid nanoparticle of the present disclosure. In some aspects, the at least one liver enzyme may be one or more of aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), and alkaline phosphatase (ALP).
In some aspects, the reduced toxicity may be manifested as an increased attenuation of the level of at least one pro-inflammatory cytokine after administration of at least one lipid nanoparticle of the present disclosure. In some aspects, the at least one pro-inflammatory cytokine may be one or more of interleukin-6 (IL-6), interferon gamma (INF-G), and tumor necrosis factor alpha (TNF-a).
In some aspects, the reduction in toxicity may be manifested as an attenuation of weight loss following administration of at least one lipid nanoparticle of the present disclosure.
In some aspects, the control lipid nanoparticle is an otherwise identical lipid nanoparticle except that the cationic lipid is not a bioreducable ionizable cationic lipid.
In some aspects, the control lipid nanoparticle is a lipid nanoparticle that does not include a bioreducable ionizable cationic lipid.
In some aspects, the control lipid nanoparticle is a lipid nanoparticle that does not include ssPalmO-Ph-P4C 2.
In some aspects, the control lipid nanoparticle is an otherwise identical lipid nanoparticle except that the cationic lipid is a lipid that is not ssPalmO-Ph-P4C 2.
In some aspects, the control lipid nanoparticle is a lipid nanoparticle comprising a higher amount of bioreducable ionizable cationic lipid.
In some aspects, the control lipid nanoparticle is a lipid nanoparticle comprising a lower amount of bioreducable ionizable cationic lipid.
In some aspects, the control lipid nanoparticle comprises a lipid nanoparticle composition previously disclosed in the art.
In some aspects, the control lipid nanoparticle is administered to the subject at the same dose as the lipid nanoparticle of the present disclosure.
In some aspects, the lipid nanoparticles of the present disclosure may be produced using a microfluidic mixing platform. In some aspects, the microfluidic mixing platform may be a non-turbulent microfluidic mixing platform.
In some aspects, the microfluidic mixing platform can produce lipid nanoparticles of the present disclosure by combining a miscible solvent phase comprising a lipid component of the nanoparticle and an aqueous phase comprising a lipid nanoparticle cargo (e.g., nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some aspects, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow for immediate mixing of the two phases. When the two phases move under laminar flow in a microfluidic channel, the microfeatures in the channel may allow controlled uniform mixing to produce the lipid nanoparticles of the present disclosure.
In some aspects, the microfluidic mixing platform may include, but is not limited toSpark (precision nanosystems) and +.>Ignite TM (precision nanosystems Co.) A solution,Benchtop (precision nanosystems), ->Blaze (precision nanosystems) or +.>GMP systems (precision nanosystems).
In some aspects, the lipid nanoparticles of the present disclosure may be produced using a microfluidic mixing platform, wherein the microfluidic mixing platform mixes at a rate of at least about 2.5 ml/min, or at least about 5 ml/min, or at least about 7.5 ml/min, or at least about 10 ml/min, or at least about 12.5 ml/min, or at least about 15 ml/min, or at least about 17.5 ml/min, or at least about 20 ml/min, or at least about 22.5 ml/min, or at least about 25 ml/min, or at least about 27.5 ml/min, or at least about 30 ml/min.
In some aspects, the lipid nanoparticles of the present disclosure may be produced using a T-mixer, wherein the T-mixer mixes at a rate of at least about 2.5 ml/min, or at least about 5 ml/min, or at least about 7.5 ml/min, or at least about 10 ml/min, or at least about 12.5 ml/min, or at least about 15 ml/min, or at least about 17.5 ml/min, or at least about 20 ml/min, or at least about 22.5 ml/min, or at least about 25 ml/min, or at least about 27.5 ml/min, or at least about 30 ml/min.
In some aspects, the lipid nanoparticles of the present disclosure may be produced using a microfluidic mixing platform, wherein the microfluidic mixing platform mixes miscible solvent and aqueous phases at a ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10 solvent to water v to v.
In some aspects, lipid nanoparticles of the present disclosure may be produced using a T-mixer, wherein the T-mixer mixes the miscible solvent phase and the aqueous phase in a ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10 solvent to water v to v.
piggyBacITR sequence
In some aspects, the nucleic acid molecule may include a piggyBacITR sequence. In some aspects, the nucleic acid molecule may include a first piggyBac sequence and a second piggyBac ITR sequence.
In some aspects, the piggyBac ITR sequence may include any piggyBac ITR sequence known in the art.
In some aspects of the methods of the present disclosure, the piggyBac ITR sequence, such as the first piggyBac ITR sequence and/or the second piggyBac ITR sequence, may comprise, consist essentially of, or consist of the sleeping beauty transposon ITR, helraiser transposon ITR, the Tol2 transposon ITR, the TcBuster transposon ITR, or any combination thereof.
Insulator sequence
In some aspects, the insulator sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NOS: 146-147.
Promoter sequence
In some aspects, the nucleic acid molecule may include a promoter sequence. In some aspects, the promoter sequence may include any promoter sequence known in the art. In some aspects, the promoter sequence may include any liver-specific promoter sequence known in the art.
In some aspects, the promoter sequence may include a Hybrid Liver Promoter (HLP). In some aspects, the promoter sequence may include an LP1 promoter. In some aspects, the promoter sequence may include leukocyte-specific expression of pp52 (LSP 1) long promoters. In some aspects, the promoter sequence may include a thyroxine-binding globulin (TBG) promoter.
In some aspects, the promoter sequence may include a wTBG promoter. In some aspects, the promoter sequence may include a liver combined beam (HCB) promoter. In some aspects, the promoter sequence may include a 2xApoE-hAAT promoter. In some aspects, the promoter sequence may include leukocyte-specific expression of pp52 (LSP 1) plus the chimeric intron promoter. In some aspects, the promoter sequence may include a Cytomegalovirus (CMV) promoter.
Transgenic sequences
In some aspects, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding a methylmalonate monoacyl-coa mutase (MUT 1) polypeptide. In some aspects, the nucleic acid sequence encoding a MUT1 polypeptide may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NOs 40-51.
In some aspects, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding an ornithine carbamoyltransferase (OTC) polypeptide. In some aspects, the nucleic acid sequence encoding a MUT1 polypeptide may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NOs 56-59.
In some aspects, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding an iCAS9 polypeptide.
In some aspects, the transgenic sequence can comprise, consist essentially of, or consist of a nucleic acid sequence encoding a Factor VIII (FVIII) polypeptide. In some aspects, the FVIII polypeptide can be a B domain-deficient FVIII polypeptide (hereinafter referred to as a FVIII-BDD polypeptide).
In some aspects, the transgene sequence may include a nucleic acid sequence encoding a FVIII-BDD polypeptide. In some aspects, the transgenic sequence can comprise, consist essentially of, or consist of a nucleic acid sequence encoding a FVIII-BDD polypeptide, wherein the FVIII-BDD polypeptide comprises, consists of, or consists of, an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO. 21. In some aspects, the nucleic acid sequence encoding a FVIII-BDD polypeptide can include, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NO. 22.
In some aspects, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding a Factor IX (FIX) polypeptide. In some aspects, the FIX polypeptide may include an R338L mutation. As the skilled artisan will appreciate, the R338L mutation may be referred to as a Padua mutation. In some aspects, the nucleic acid sequence encoding a FIX polypeptide may comprise, consist essentially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 65 nucleic acid sequence.
In some aspects, the transgenic sequences can be codon optimized according to methods known in the art.
In some aspects, at least one transgene sequence may be operably linked to at least one promoter sequence present in the same polynucleotide.
Therapeutic proteins
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of a methylmalonate monoacyl-coa mutase (MUT 1) polypeptide. In some aspects, the MUT1 polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs 52-55.
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of an ornithine carbamoyltransferase (OTC) polypeptide. In some aspects, the OTC polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs 60-63.
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of a Factor VIII (FVIII) polypeptide. In some aspects, the FVIII polypeptide can be a B domain-deficient FVIII polypeptide (hereinafter referred to as a FVIII-BDD polypeptide).
In some aspects, the therapeutic protein may comprise, consist essentially of, or consist of a Factor IX (FIX) polypeptide. In some aspects, the FIX polypeptide may include an R338L mutation. As the skilled artisan will appreciate, the R338L mutation may be referred to as a Padua mutation. In some aspects, a FIX polypeptide comprises, consists essentially of, or consists of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of SEQ ID nos. 64.
3' UTR sequences
In some aspects, the 3'utr sequence may be an AES 3' utr sequence. The AES 3' UTR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO. 25.
In some aspects, the 3'UTR sequence may be an mtRNR13' UTR sequence. The mtRNR13' UTR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO. 26.
In some aspects, the 3' UTR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical to SEQ ID NO 27.
polyA sequences
In some aspects, the nucleic acid molecule may comprise a polyA sequence. In some aspects, the polyA sequence may include any polyA sequence known in the art.
Self-cleaving peptide sequences
In some aspects, the nucleic acid molecule may include a self-cleaving peptide sequence. In some aspects, the self-cleaving peptide sequence may include any self-cleaving peptide sequence known in the art. In some aspects, the self-cleaving peptide sequence may comprise a 2A self-cleaving peptide sequence known in the art. Non-limiting examples of self-cleaving peptides include T2A peptides, GSG-T2A peptides, E2A peptides, GSG-E2A peptides, F2A peptides, GSG-F2A peptides, P2A peptides, or GSG-P2A peptides.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a T2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a GSG-T2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding an E2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a GSG-E2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding an F2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a GSG-F2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a P2A peptide.
In some aspects, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a GSG-P2A peptide.
Swivel mount system
In some aspects, a nucleic acid molecule may include a transposon or a nano-transposon, the transposon or nano-transposon comprising: a first nucleic acid sequence comprising: (a) a first Inverted Terminal Repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence within an ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon.
In some aspects, a nucleic acid molecule may include a transposon or a nano-transposon, the transposon or nano-transposon comprising: a first nucleic acid sequence comprising: (a) a first Inverted Terminal Repeat (ITR) or a sequence encoding a first ITR, (b) a second ITR or a sequence encoding a second ITR, and (c) an intra-ITR sequence or a sequence encoding an intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon; and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding an inter-ITR, wherein the inter-ITR sequence is 700 nucleotides or less in length.
Transposons or nano-transposons of the present disclosure include nucleotide sequences encoding therapeutic proteins. The transposon or nanotransposon may be a plasmid DNA transposon comprising a nucleotide sequence encoding a therapeutic protein flanked by two cis-regulatory insulator elements. Transposons or nanotransposons may further comprise plasmids comprising sequences encoding transposases. The sequence encoding a transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.
The transposon or nanotransposon of the disclosure may be piggyBac TM (PB) transposon. In some aspects, when the transposon is a PB transposon, the transposase is piggyBac TM (PB) transposase, piggyBac-like (PBL) transposase or SuperpiggyBac TM (SPB or sPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.
Non-limiting examples of PB transposons and PB transposases, PBL transposases and SPB transposases are described in detail in U.S. Pat. No. 6,218,182; U.S. patent No. 6,962,810; U.S. patent No. 8,399,643 and PCT publication No. WO 2010/099296.
PB, PBL and SPB transposases recognize transposon-specific Inverted Terminal Repeats (ITRs) at the ends of transposons, and insert the content between ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) or the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal locus. The target sequence of the PB or PBL transposon may comprise or consist of: 5' -CTAA-3', 5' -TTAG-3', 5' -ATAA-3', 5' -TCAA-3', 5' -AGTT-3 ', 5' -ATTA-3', 5' -GTTA-3', 5' -TTGA-3', 5' -TTTA-3', 5' -TTAC-3', 5' -ACTA-3', 5' -AGGG-3', 5' -CTAG-3', 5' -TGAA-3', 5' -AGGT-3', 5' -ATCA-3', 5' -CTCC-3', 5' -TAAA-3', 5' -TCTC-3', 5' -TGAA-3', 5' -AATC-3', 5' -ACA-3 ', 5' -ACAT-3', 5' -ACTC-3', 5' -ATAG-3', 5' -CAAA-3', 5' -CAA-3 ', 5' -CATA-3', 5' -CCAG-3', 5' -CGTA-3', 5' -CTA-3 ', 5' -GTAG-3 ', 5' -TCA-3 ', 5' -TTAG-3', and 5' -TTAG-TTTTT-3 ' TTT-3'. The PB or PBL transposon system has no payload restrictions on the gene of interest contained between ITRs.
Exemplary amino acid sequences of one or more of PB, PBL and SPB transposases are disclosed in U.S. Pat. No. 6,218,185; us patent 6,962,810 and us patent 8,399,643.
The PB or PBL transposase may include or consist of: an amino acid sequence having amino acid substitutions at two or more positions, at three or more positions, or at each of positions 30, 165, 282, or 538. The transposase may be an SPB transposase comprising or consisting of an amino acid sequence, where the amino acid at position 30 may be substituted with valine (V) for isoleucine (I), the amino acid at position 165 may be substituted with serine (S) for glycine (G), the amino acid at position 282 may be substituted with valine (V) for methionine (M), and the amino acid at position 538 may be substituted with lysine (K) for asparagine (N).
In certain aspects, wherein the transposase comprises the mutations described above at positions 30, 165, 282, and/or 538, the PB transposase, the PBL transposase, and the SPB transposase may further comprise amino acid substitutions at one or more of positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591, which positions are described in more detail in PCT publication nos. WO/173636 and PCT/US 2019/042019816.
As described in more detail in PCT publication nos. WO 2019/173636 and PCT/US2019/049816, PB, PBL or SPB transposases may be isolated or derived from insects, vertebrates, crustaceans or tail cord animals. In preferred aspects, the PB, PBL or SPB transposase is isolated or derived from the insect Trichoplusia ni (Trichoplusia ni) (GenBank accession AAA 87375) or from Bombyx mori (Bombyx mori) (GenBank accession BAD 11135).
A highly active PB or PBL transposase is one that is more active than the naturally occurring variant from which it is derived. In a preferred aspect, the highly active PB or PBL transposase is isolated from or derived from Bombyx mori or Xenopus laevis (Xenopus). Examples of superactive PB or PBL transposases are disclosed in U.S. Pat. No. 6,218,185; us patent No. 6,962,810, us patent No. 8,399,643 and WO 2019/173636. A list of highly active amino acid substitutions is disclosed in U.S. patent No. 10,041,077.
In some aspects, the PB or PBL transposase is integration-deficient. An integration-defective PB or PBL transposase is one that can excise its corresponding transposon but integrate the excised transposon less frequently than the corresponding wild type transposase. Examples of integration-defective PB or PBL transposases are disclosed in U.S. patent No. 6,218,185; us patent No. 6,962,810, us patent No. 8,399,643 and WO 2019/173636. An integration defective amino acid substitution list is disclosed in U.S. patent No. 10,041,077.
In some aspects, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposases fused to nuclear localization signals are disclosed in U.S. patent No. 6,218,185; us patent No. 6,962,810, us patent No. 8,399,643 and WO 2019/173636.
In some aspects, the sPB protein can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of SEQ ID NOs 37-39.
The transposon or nanotransposon of the present disclosure may be a sleeping beauty transposon. In some aspects, when the transposon is a sleeping beauty transposon, the transposase is a sleeping beauty transposase (e.g., as disclosed in U.S. patent No. 9,228,180) or an overactive sleeping beauty (SB 100X) transposase.
The transposon or nanotransposon of the disclosure may be a Helraiser transposon. An exemplary Helraiser transposon comprises Helibat1. In some aspects, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO 2019/173636).
The transposon or nanotransposon of the disclosure may be a Tol2 transposon. In some aspects, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO 2019/173636).
The transposon or nanotransposon of the disclosure may be a TcBuster transposon. In some aspects, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a superactive TcBuster transposase (e.g., as disclosed in WO 2019/173636). TcBuster transposases may include or consist of naturally occurring amino acid sequences or non-naturally occurring amino acid sequences. Polynucleotides encoding TcBuster transposases may include or consist of naturally occurring nucleic acid sequences or non-naturally occurring nucleic acid sequences.
In some aspects, the mutant TcBuster transposase includes one or more sequence variations when compared to a wild-type TcBuster transposase, as described in more detail in PCT publication nos. WO 2019/173636 and PCT/US 2019/049816.
The cell delivery compositions (e.g., transposons) disclosed herein can include nucleic acid molecules encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT publication nos. WO 2019/173636 and PCT/US 2019/049816.
Cells and modified cells of the disclosure
The cells and modified cells of the present disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. In one aspect, the cell targeted for modification using the LNP compositions of the present disclosure is a hepatocyte, hepatic stellate cell, kupffer cell or hepatic sinus endothelial cell. In one embodiment, the LNP composition comprises at least one mRNA molecule encoding a transposase, and the modified cell is produced in vivo. In one embodiment, the LNP composition comprises at least one DNA molecule encoding a transposon, and the modified cell is produced in vivo. In one embodiment, the transposon comprises a nucleotide sequence encoding a therapeutic gene operably linked to a liver specific promoter.
The cells and modified cells of the present disclosure may be somatic cells. The cells and modified cells of the present disclosure may be differentiated cells. The cells and modified cells of the present disclosure may be autologous cells or allogeneic cells. Allogeneic cells are engineered to prevent adverse effects of transplantation after administration to a subject. The allogeneic cells may be any type of cell. The allogeneic cells may be stem cells or may be derived from stem cells. The allogeneic cells may be differentiated somatic cells.
Formulation, dosage and mode of administration
The present disclosure provides formulations, dosages, and methods for administering the compositions described herein.
The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable adjuvant such as, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants and the like. Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods of preparing such sterile solutions are well known in the art, such as, but not limited to, gennaro, remington 'sPharmaceutical Sciences, 18 th edition, mark publishing co (mackpubishing co.) (Easton, pa.) 1990 and "Physician's Desk Reference", 52 th edition, medical economy company (Medical Economics) (Montvale, n.j.) 1998. Pharmaceutically acceptable carriers suitable for use in the manner of administration, solubility and/or stability of the compositions known in the art or as described herein may be routinely selected.
For example, the disclosed LNP compositions of the present disclosure may further include a diluent. In some compositions, the diluent may be phosphate buffered saline ("PBS"). In some compositions, the diluent may be sodium acetate.
Non-limiting examples of suitable pharmaceutical excipients and additives include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-oligosaccharides, tri-oligosaccharides, tetra-oligosaccharides, and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, etc., and polysaccharides or sugar polymers), which may be present alone or in combination, in the range of 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/protein components that may also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.
The composition may further comprise a buffer or a pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include salts of organic acids such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris, tromethamine hydrochloride or phosphate buffer. Preferred buffers are organic acid salts, such as citrate.
Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of modes of administration include bolus, oral, infusion, intra-articular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavity, spinal, intracerebellar, intracerebroventricular, colonic, intracervical, intragastric, intrahepatic, intrathecal, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, periosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrathecal, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or intravaginal modes.
The compositions of the present disclosure may be prepared for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid form, such as, but not limited to, creams and suppositories; for buccal or sublingual administration, such as but not limited to in the form of a tablet or capsule; or intranasally, such as, but not limited to, in the form of a powder, nasal drops or aerosol or some pharmaceutical agent; or transdermally, such as but not limited to gels, ointments, lotions, suspensions, or patch delivery systems with chemical enhancers such as dimethylsulfoxide to alter the skin structure or increase the concentration of drug in the transdermal patch (junginer et al, "" drug permeation enhancement ";" Hsieh, d.s. editors, pages 59-90 (makerel de k.er (MarcelDekker, inc.NewYork) 1994) in new york), or to apply an electric field to create a transient transmission pathway, such as electroporation, or to increase the fluidity of a charged drug through the skin, such as iontophoresis, or to apply ultrasound, such as ultrasonic guidance (U.S. patent nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).
For parenteral administration, any of the compositions disclosed herein may be formulated as solutions, suspensions, emulsions, granules, powders, or lyophilized powders associated with or provided separately from a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable-derived oils, hydrogenated naphthalenes and the like as common excipients. Aqueous or oily suspensions for injection may be prepared according to known methods by using suitable emulsifying or moisturizing agents and suspending agents. The agent for injection may be a non-toxic, non-orally administrable diluent, such as an aqueous solution, a sterile injectable solution or a suspension in a solvent. As a vehicle or solvent, water, ringer's solution, isotonic saline, etc. are permissible; common solvents or suspending solvents, sterile, fixed oils may be employed. For this purpose, any kind of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semisynthetic mono-or diglycerides or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, pneumatic needle-free injection devices as described in U.S. patent No. 5,851,198, and laser perforation devices as described in U.S. patent No. 5,839,446.
For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered at a particle size effective to reach the lower airways of the lung or sinuses. The composition or pharmaceutical composition may be delivered by any of a variety of inhalation devices or nasal devices known in the art for administering therapeutic agents by inhalation. These devices capable of depositing an aerosolized formulation in the sinus cavities or alveoli of a patient include metered dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, and the like. All such devices may use a formulation suitable for dispensing the administration of the compositions or pharmaceutical compositions described herein in an aerosol. Such aerosols may comprise solutions (both aqueous and non-aqueous) or solid particles. Alternatively, a spray comprising a composition or pharmaceutical composition described herein may be generated by forcing a suspension or solution of at least one protein scaffold under pressure through a nozzle. In a Metered Dose Inhaler (MDI), the propellant, the composition or pharmaceutical composition described herein and any excipients or other additives are contained in a canister as a mixture comprising liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations and related devices is disclosed in PCT publication No. WO 2019/049816.
For absorption through a mucosal surface, the composition comprises an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which facilitates absorption through the mucosal surface by effecting adhesion of the emulsion particles (U.S. patent No. 5,514,670). Mucous surfaces suitable for use in the emulsion applications of the present disclosure may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain, for example, polyalkylene glycols, petrolatum, cocoa butter, etc., as excipients. Formulations for intranasal administration may be solid and contain, for example, lactose as an excipient, or may be aqueous or oily solutions of nasal drops. For oral administration, excipients include sugar, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. patent No. 5,849,695). A more detailed description of mucosal administration and formulation is disclosed in PCT publication No. WO 2019/049816.
For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as a liposome or polymeric nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise indicated). Many suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids, e.g., polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as natural polymers such as collagen, polyamino acids, albumin and other proteins, alginic acid and other polysaccharides, and combinations thereof (U.S. Pat. No. 5,814,599). A more detailed description of transdermal administration, formulations and suitable devices is disclosed in PCT publication No. WO 2019/049816.
It may be desirable to deliver the disclosed compounds to a subject over an extended period of time, for example, a period of one week to one year from the beginning of a single administration. Various sustained release, storage or implant dosage forms may be utilized.
Suitable dosages are well known in the art. See, e.g., wells et al, handbook for drug treatment (Pharmacotherapy Handbook), 2 nd edition, aprton and lange press (Appleton andLange, stamford, conn.) (2000); the PDR pharmacopoeia-2000 tala Kang Xiuzhen pharmacopoeia (PDR Pharmacopoeia, tarascon Pocket Pharmacopoeia 2000), the essence, talaskan publishing company (Tarascon Publishing, loma Linda, calif.) (2000) of Loma-reach, california; a handbook of medicines for Nursing2001 (Nursing 2001 handbook of drugs), 21 st edition, spring house company of spring house, pennsylvania (spring corp., pa.), 2001; medicine Guide for health professionals (Health Professional's medicine Guide) 2001, editorial, shannon, wilson, stang, preplice-Hall corporation of saddle river, new jersey (preplice-Hall, inc Upper Saddle River, n.j.). Preferred dosages may optionally comprise about 0.1-99 and/or 100-500 mg/kg/administration, or any range, value or fraction thereof, or serum concentrations achieving a serum concentration of about 0.1-5000 μg/ml per single or multiple administration, or any range, value or fraction thereof. The preferred dosage range for the compositions or pharmaceutical compositions disclosed herein is about 1mg/kg, up to about 3, about 6 or about 12mg/kg of the subject's body weight.
Alternatively, the dosage administered may depend on known factors, such as the pharmacodynamic characteristics of the particular agent, and the manner and route of administration thereof; age, health, and weight of the recipient; the nature and extent of the symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect.
As a non-limiting example, treatment of a human or animal may be provided as a disposable or periodic dose of a composition or pharmaceutical composition disclosed herein, which is provided as a single infusion or repeated dose of about 0.1 to 100mg/kg per day, or any range, value or fraction thereof, for at least one day, or alternatively or additionally, at least one week, or alternatively or additionally, at least one year, of 1-52 weeks, or alternatively or additionally, at least one year, of 1-20 days, of the day, which may be on days 1-40.
Where the composition to be administered to a subject in need thereof is a modified cell as disclosed herein, between about 1 x 10 can be administered 3 And 1X 10 15 Between individual cells; between 1X 10 3 And 1X 10 15 Between individual cells, between about 1X 10 4 And 1X 10 12 Between individual cells; between about 1X 10 5 And 1X 10 10 Between individual cells; between about 1X 10 6 And 1X 10 9 Between individual cells; between about 1X 10 6 And 1X 10 8 Between individual cells; between about 1X 10 6 And 1X 10 7 Between individual cells; or between about 1X 10 6 And 25X 10 6 Cells between individual cells. In one aspect, the administration is between about 5X 10 6 And 25X 10 6 Cells between individual cells.
A more detailed description of the disclosed compositions and pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the pharmaceutical compositions is disclosed in PCT publication No. WO 2019/04981.
The present disclosure provides for the use of the disclosed compositions or pharmaceutical compositions for treating a disease or condition as known in the art or as described herein in a cell, tissue, organ, animal or subject, using the disclosed compositions and pharmaceutical compositions, e.g., administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal or subject. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.
Any use or method of the present disclosure may comprise administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal or subject in need of such modulation, treatment or therapy. Such methods may optionally further comprise co-administration or combination therapy for treating such diseases or conditions, wherein administration of any of the compositions or pharmaceutical compositions disclosed herein further comprises administration prior to, concurrent with, and/or subsequent to at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, and radiopharmaceutical).
In some aspects, the subject does not develop graft versus host (GvH) and/or host versus graft (HvG) after administration. In one aspect, administration is systemic. Systemic administration can be in any manner known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or intravenous infusion. In one aspect, the administration is topical. Topical administration may be in any manner known in the art and described in detail herein. Preferably, the topical administration is by intratumoral injection or infusion, intravertebral injection or infusion, intraventricular injection or infusion, intraocular injection or infusion, or intraosseous injection or infusion.
In some aspects, the therapeutically effective dose is a single dose. In some aspects, a single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or any number therebetween manufactured simultaneously. In some aspects, where the composition is autologous or allogeneic, the dose is an amount sufficient to allow cell transplantation and/or for a time sufficient to treat the disease or disorder.
In some aspects of the methods of treatment described herein, the treatment may be modified or terminated. In particular, in aspects where the composition for treatment comprises an inducible pro-apoptotic polypeptide, apoptosis may be selectively induced in cells by contacting the cells with an inducer. Treatment may be modified or terminated in response to, for example, signs of rehabilitation or signs of reduced disease severity/progression, signs of disease remission/cessation, and/or occurrence of adverse events. In some aspects, the methods include the step of administering an inhibitor of the inducer to inhibit the change in the cell therapy, thereby restoring the function and/or efficacy of the cell therapy (e.g., when the sign or symptom of the disease is reproduced or the severity is increased and/or an adverse event is resolved).
Nucleic acid molecules
The nucleic acid molecules encoding the therapeutic proteins of the present disclosure may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or synthetically produced, or any combination thereof. The DNA may be triplex, double or single stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be the coding strand, also referred to as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.
The isolated nucleic acid molecules of the present disclosure may comprise a nucleic acid molecule comprising an Open Reading Frame (ORF), optionally with one or more introns, such as, but not limited to, at least one specific enzymatically active portion of a therapeutic protein; a nucleic acid molecule comprising a coding sequence for a therapeutic protein; and nucleic acid molecules comprising nucleotide sequences substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode a therapeutic protein as described herein and/or known in the art. Of course, the genetic code is well known in the art. Thus, it will be routine for one of skill in the art to produce such degenerate nucleic acid variants encoding specific protein scaffolds of the present disclosure. See, for example, ausubel et al, supra, and such nucleic acid variants are encompassed by the present disclosure.
As indicated herein, the nucleic acid molecules of the present disclosure, including nucleic acid molecules encoding therapeutic proteins, may include, but are not limited to, those that themselves encode the amino acid sequences of enzymatically active fragments of therapeutic proteins; a coding sequence for the entire therapeutic protein or a portion thereof; coding sequences for therapeutic proteins, and additional sequences, such as coding sequences for at least one signal precursor or fusion peptide with or without the additional coding sequences described above, such as at least one intron, and additional non-coding sequences, including but not limited to non-coding 5 'and 3' sequences, such as transcribed non-translated sequences (e.g., ribosome binding and mRNA stability) that function in transcription, mRNA processing (including splicing and polyadenylation signals); additional coding sequences that encode additional amino acids, such as amino acids that provide additional functionality. Thus, the sequence encoding the therapeutic protein may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused therapeutic protein.
Construction of nucleic acids
The isolated nucleic acids of the present disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as are well known in the art.
The nucleic acid may conveniently comprise a sequence other than a polynucleotide of the present disclosure. For example, multiple cloning sites comprising one or more endonuclease restriction sites may be inserted into a nucleic acid to aid in the isolation of a polynucleotide. In addition, translatable sequences may be inserted to aid in isolating the translated polynucleotides of the present disclosure. For example, a hexahistidine tag sequence provides a convenient means of purifying the proteins of the present disclosure. In addition to coding sequences, the nucleic acids of the present disclosure are optionally vectors, adaptors, or linkers for cloning and/or expressing the polynucleotides of the present disclosure.
Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression in order to aid in isolation of the polynucleotide or to improve its introduction into the cell. The use of cloning vectors, expression vectors, adaptors and linkers is well known in the art. (see, e.g., ausubel, supra; or Sambrook, supra).
Recombinant methods for constructing nucleic acids
The isolated nucleic acid compositions of the present disclosure, e.g., RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from a biological source using any number of cloning methods known to those of skill in the art. In some aspects, oligonucleotide probes that selectively hybridize under stringent conditions to polynucleotides of the present disclosure are used to identify a desired sequence in a cDNA or genomic DNA library. Isolation of RNA and construction of cDNA and genomic libraries are well known to those skilled in the art. (see, e.g., ausubel, supra; or Sambrook, supra).
Nucleic acid screening and separation methods
Probes may be used to screen cDNA or genomic libraries based on the sequences of the polynucleotides of the present disclosure. Probes may be used to hybridize to genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that various degrees of hybridization stringency can be employed in the assays; for example, the hybridization or wash medium may be stringent. As hybridization conditions become more stringent, a greater degree of complementarity must exist between the probe and the target for duplex formation to occur. The stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of partially denaturing solvents (e.g., formamide). For example, the stringency of hybridization can be conveniently varied by varying the polarity of the reactant solution by manipulating the concentration of formamide, for example, in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and/or the wash medium. The degree of complementarity will optimally be 100% or 70-100%, or any range or value therein. However, it will be appreciated that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
Methods of amplification of RNA or DNA are well known in the art and, based on the teachings and guidance presented herein, can be used in accordance with the present disclosure without undue experimentation.
Known methods of DNA or RNA amplification include, but are not limited to, polymerase Chain Reaction (PCR) and related amplification methods (see, e.g., U.S. Pat. nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 to Mullis et al, 4,795,699 and 4,921,794 to Tabor et al, 5,142,033 to inis, 5,122,464 to Wilson et al, 5,091,310 to inis, 5,066,584 to gillensten et al, 4,889,818 to Gelfand et al, 4,994,370 to Silver et al, 4,766,067 to biswand, 4,656,134 to Ringold) and RNA-mediated amplification using antisense RNA for target sequence as templates for strand DNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al, which is incorporated herein by reference in its entirety). (see, e.g., ausubel, supra; or Sambrook, supra).
For example, polymerase Chain Reaction (PCR) techniques may be used to amplify the sequences of polynucleotides and related genes of the present disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, cloning nucleic acid sequences encoding a protein to be expressed, using the nucleic acids as probes to detect the presence of a desired mRNA in a sample, nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide the skilled artisan through in vitro amplification methods are found in Berger, supra; sambrook, supra; and Ausubel; the foregoing; and Mullis et al, U.S. patent No. 4,683,202 (1987); innis et al, PCR protocol: methods and application guidelines (PCRProtocolsAGuidetoMethods and Applications), editorial, academic Press inc (Academic Press inc., san Diego, calif.) (1990). Commercially available kits for genomic PCR amplification are known in the art. See, e.g., advantage-GC genomic PCR kit (Clontech). Additionally, for example, the T4 gene 32 protein (BoehringerMannheim) may be used to increase the yield of long PCR products.
Synthetic methods for constructing nucleic acids
The isolated nucleic acids of the present disclosure may also be prepared by direct chemical synthesis by known methods (see, e.g., ausubel et al, supra). Chemical synthesis typically produces single stranded oligonucleotides that can be converted to double stranded DNA by hybridization to complementary sequences or by DNA polymerase polymerization using single strands as templates. Those skilled in the art will recognize that while chemical synthesis of DNA may be limited to sequences of about 100 bases or more, longer sequences may be obtained by ligating shorter sequences.
Recombinant expression cassette
The present disclosure further provides recombinant expression cassettes comprising the nucleic acids of the present disclosure. The nucleic acid sequences of the present disclosure, e.g., cDNA or genomic sequences encoding the protein scaffolds of the present disclosure, can be used to construct recombinant expression cassettes that can be introduced into at least one desired host cell. A recombinant expression cassette will typically comprise a polynucleotide of the present disclosure operably linked to transcriptional initiation regulatory sequences that will direct transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters may be used to direct expression of the nucleic acids of the present disclosure.
In some aspects, an isolated nucleic acid that acts as a promoter, enhancer, or other element can be introduced into a suitable location (upstream, downstream, or within an intron) of a non-heterologous form of a polynucleotide of the present disclosure to up-regulate or down-regulate expression of a polynucleotide of the present disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.
Expression vectors and host cells
The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one therapeutic protein by recombinant techniques well known to those of skill in the art. See, for example, sambrook et al, supra; ausubel et al, each of which is incorporated by reference herein in its entirety.
The polynucleotide may optionally be ligated to a vector containing a selectable marker for propagation in the host. Typically, the plasmid vector is introduced into a precipitate (e.g., calcium phosphate precipitate), or into a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The DNA insert should be operably linked to an appropriate promoter. The expression construct will further contain sites for transcription initiation, termination, and ribosome binding sites for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct will preferably comprise a translation beginning at the beginning and a stop codon (e.g., UAA, UGA or UAG) suitably located at the end of the mRNA to be translated, wherein UAA and UAG are preferably for mammalian or eukaryotic cell expression.
The expression vector will preferably, but optionally, comprise at least one selectable marker. Such markers include, for example, but are not limited to, ampicillin, gecomycin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid or glutamine synthetase (GS, U.S. Pat. No. 5,122,464; no. 5,770,359; no. 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, and ampicillin, bleomycin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418/geneticin (neo gene), kang Mei, spectinomycin, streptomycin, carboxillin, bleomycin, erythromycin, polymyxin B or tetracycline resistance genes for culture in E.coli and other bacteria or prokaryotes (the above patents are hereby incorporated by reference). Suitable media and conditions for the above-described host cells are known in the art. Suitable carriers will be apparent to the skilled person. The vector construct may be introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, e.g., sambrook, supra, chapters 1-4 and 16-18; ausubel, supra, chapters 1, 9, 13, 15, 16.
The expression vector will preferably, but optionally, comprise at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. Selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or proteomes that distinguish a cell or cell subpopulation from another defined cell subpopulation. Preferably, the cell surface markers can be selected to distinguish those cells that are modified by the compositions or methods of the present disclosure from those cells that are not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, but are not limited to, a "name cluster" or "class determinant" protein (commonly abbreviated as "CD"), such as truncated or full length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. The cell surface markers further comprise the suicide gene marker RQR8 (Philipb et al, blood.) (2014, 8/21; 124 (8): 1277-87).
The expression vector will preferably, but optionally, comprise at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. Selectable resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.
At least one protein scaffold of the present disclosure may be expressed in a modified form, such as a fusion protein, and may contain not only secretion signals, but also additional heterologous functional regions. For example, regions of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the protein scaffold to improve stability and persistence in the host cell during purification or during subsequent handling and storage. Likewise, peptide moieties may be added to the protein scaffolds of the present disclosure to facilitate purification. Such regions may be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, chapters 17.29-17.42 and 18.1-18.74; ausubel, supra, chapters 16, 17 and 18.
Those of skill in the art are knowledgeable about the numerous expression systems that may be used to express nucleic acid molecules encoding the proteins of the present disclosure. Alternatively, the nucleic acids of the present disclosure may be expressed in host cells by opening (by manipulation) in host cells containing endogenous DNA encoding the protein scaffold of the present disclosure. Such methods are well known in the art, for example as described in U.S. Pat. nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.
Examples of cell cultures that can be used to produce the protein scaffold, specific portions or variants thereof are bacterial, yeast and mammalian cells known in the art. The mammalian cell system will typically be in the form of a monolayer of cells, although mammalian cell suspensions or bioreactors may also be used. Many suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art and comprise COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, COS-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, heLa cells, etc., which are readily purchased from, for example, the American type culture Collection, manassas, va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2/0-Ag14 cells (ATCC accession number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or SP2/0-Ag14 cell.
Expression vectors for these cells may comprise one or more of the following expression control sequences, such as, but not limited to, an origin of replication; promoters (e.g., late or early SV40 promoter, CMV promoter (U.S. Pat. No. 5,168,062; no. 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1α promoter (U.S. Pat. No. 5,266,491), at least one human promoter, enhancers and/or processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., the SV40 large TAgpolyA addition site), and transcription terminator sequences see, e.g., ausubel et al, supra; sambrook et al, supra other cells useful in producing the nucleic acids or proteins of the present disclosure are known and/or available, e.g., from the American type culture Collection cell line and hybridoma catalog (www.atcc.org) or other known or commercial sources.
When eukaryotic host cells are employed, polyadenylation or transcription terminator sequences are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of transcripts may also be included. Examples of splicing sequences are the VP1 intron from SV40 (Sprague et al J.Virol.) (45:773-781 (1983)). Alternatively, the gene sequences that control replication in the host cell may be incorporated into a vector, as is known in the art.
Amino acid code
Amino acids that make up the protein scaffold of the present disclosure are generally abbreviated. Amino acid names may be indicated by designating the amino acid with its single letter code, its three letter code, name or three nucleotide codon as is well known in the art (see Alberts, b. Et al, cell molecular biology (Molecular BiologyofThe Cell), third edition, garland publishing, inc., new york, 1994). As specified herein, the therapeutic proteins of the present disclosure may comprise one or more amino acid substitutions, deletions or additions from spontaneous or mutations and/or human manipulation. Amino acids essential for function in the therapeutic proteins of the present disclosure may be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., ausubel, supra, chapter 8, 15; cunningham and Wells, science 244:1081-1085 (1989)). The latter procedure introduces a single alanine mutation at each residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites critical for maintaining the activity of therapeutic proteins can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol.) (224:899-904 (1992) and de Vos et al, science 255:306-312 (1992)).
As will be appreciated by the skilled artisan, the present disclosure comprises at least one biologically active therapeutic protein of the present disclosure. The specific activity of the biologically active therapeutic protein is at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90%, or 95% -99% or more of the specific activity of the native (non-synthetic), endogenous or related and known protein scaffolds. Methods for analyzing and quantifying measures of enzyme activity and substrate specificity are well known to those skilled in the art.
In another aspect, the present disclosure relates to therapeutic proteins and fragments as described herein, modified by covalent attachment of an organic moiety. Such modifications can result in protein scaffold fragments with improved pharmacokinetic properties (e.g., increased serum half-life in vivo). The organic moiety may be a linear or branched hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. In particular aspects, the hydrophilic polymeric groups can have a molecular weight of about 800 to about 120,000 daltons and can be polyalkylene glycols (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymers, amino acid polymers, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester groups can include about eight to about forty carbon atoms.
The modified therapeutic proteins and fragments of the present disclosure may include one or more organic moieties that are covalently bound directly or indirectly to an antibody. Each organic moiety bound to a protein scaffold or fragment of the present disclosure may independently be a hydrophilic polymeric group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses both monocarboxylic and dicarboxylic acids. As used herein, the term "hydrophilic polymeric group" refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than octane. Thus, the present disclosure encompasses therapeutic proteins modified by covalent attachment of polylysine. Hydrophilic polymers suitable for modifying the therapeutic proteins of the present disclosure may be linear or branched and include, for example, polyalkylene glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkanoxides (e.g., polyoxyethylene, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymer that modifies the therapeutic proteins of the present disclosure as a separate molecular entity has a molecular weight of about 800 to about 150,000 daltons. For example, PEG5000 and PEG20,000 can be used, where the subscript is the average molecular weight of the polymer in daltons. The hydrophilic polymer groups may be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers substituted with fatty acids or fatty acid ester groups can be prepared by employing suitable methods. For example, a polymer comprising amine groups can be coupled with carboxylate groups of a fatty acid or fatty acid ester, and activated carboxylate groups on the fatty acid or fatty acid ester (e.g., activated with N, N-carbonyldiimidazole) can be coupled with hydroxyl groups on the polymer.
Fatty acids and fatty acid esters suitable for modifying the therapeutic proteins of the present disclosure may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffold of the present disclosure include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, eicosanoate), n-docusate (C22, behenate), n-triacontanoate (C30), n-tetracosanoate (C40), cis- Δ9-octadecanoate (C18, oleate), all cis- Δ5,8,11, 14-eicosatetraenoate (C20, arachidonic acid), suberic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids including straight or branched chain lower alkyl groups. The lower alkyl group may include one to about twelve, preferably one to about six carbon atoms.
Modified therapeutic proteins and fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. The term "modifier" as used herein refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid ester) that includes an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifier and the second chemical group. For example, the amine-reactive activating group comprises an electrophilic group such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimide ester (NHS), and the like. The activating group that can react with the thiol includes, for example, maleimide, iodoacetyl, acryl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. The aldehyde functional group may be coupled with an amine-containing molecule or a hydrazide-containing molecule, and the azide group may react with the trivalent phosphorus group to form a phosphoramidate or a phosphoimide linkage. Suitable methods for introducing activating groups into molecules are known in the art (see, e.g., hermanson, g.t., "bioconjugate techniques" (bioconjugate techniques), academic press company, san diego, california (1996)). The activating group may be directly bound to an organic group (e.g., hydrophilic polymer, fatty acid ester) or through a linker moiety, such as a divalent C1-C12 group, wherein one or more carbon atoms may be substituted with heteroatoms, such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, - (CH 2) 3-, -NH- (CH 2) 6-NH-, -CH 2) 2-NH-, and-CH 2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. For example, a modifier comprising a linker moiety may be produced by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-hexamethylenediamine) with a fatty acid in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate. As described, the Boc protecting group may be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that may be coupled with another carboxylate, or may be reacted with maleic anhydride, and the resulting product cyclized to produce a maleimide derivative of the activated fatty acid. (see, e.g., thompson et al, WO 92/16221, the entire teachings of which are incorporated herein by reference.)
The modified therapeutic proteins of the present disclosure may be produced by reacting a protein scaffold or fragment with a modifying agent. For example, the organic moiety may be bound to the protein scaffold in a non-site specific manner by using an amine reactive modifier, such as a NHS ester of PEG. Modified therapeutic proteins and fragments comprising an organic moiety that binds to a specific site of a protein scaffold of the present disclosure can be prepared using suitable methods, such as reverse proteolysis (Fisch et al, bioconjugate chemistry (Biotechnol. Bioeng.), 3:147-153 (1992), werlen et al, bioconjugate chemistry, 5:411-417 (1994), kumaran et al, protein science 6 (10): 2233-2241 (1997), itoh et al, bioorganic chemistry (Bioorg. Chem.), 24 (1): 59-68 (1996), capella et al, biotechnology and bioengineering (Biotechnol. Bioeng.), 56 (4): 456-463 (1997)), and Herman, G.T., bioconjugate technology, st. George, calif., 1996).
Definition of the definition
As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., limitations of the measurement system. For example, "about" may mean within 1 or more standard deviations. Alternatively, "about" may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times the value, and more preferably within 2 times. When a particular value is described in the application and claims, it should be assumed that the term "about" is intended to be within an acceptable error range for the particular value, unless otherwise indicated.
It is to be understood that while the compounds disclosed herein may be presented without a specified configuration (e.g., without a specified stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, regioisomers and stereoisomers of the compounds. In some embodiments, a compound presented herein without a specified configuration is intended to refer to each of the available isomers, tautomers, regioisomers, and stereoisomers of the compound or any mixture thereof.
It is to be understood that the compounds described herein include the compounds themselves, as well as salts and solvates thereof, if applicable. For example, salts can be formed between anions and positively charged groups (e.g., amino groups) on the substituted compounds disclosed herein. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).
The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or of culture medium when produced by recombinant techniques or of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optionally protein coding sequences) that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide was derived (i.e., sequences located at the 5 'and 3' ends of the polynucleotide). For example, in various aspects, an isolated polynucleotide may contain less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or 0.1kb of nucleotide sequences of the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived, naturally flanked. Proteins that are substantially free of cellular material comprise protein preparations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins. When recombinantly producing a protein of the present disclosure, or a biologically active portion thereof, the optimal medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.
The present disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and the protein encoded thereby. Fragments of DNA sequences that include a coding sequence may encode protein fragments that retain the biological activity of the native protein and thus retain DNA recognition or binding activity to the target DNA sequence as described herein. Alternatively, fragments of DNA sequences useful as hybridization probes typically do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and at most the full length polynucleotides of the present disclosure.
The nucleic acids or proteins of the present disclosure can be constructed by a modular method comprising pre-assembling monomer units and/or repeat units in a target vector, which can then be assembled into a final vector of interest. The polypeptides of the present disclosure may include repeat monomers of the present disclosure and may be constructed by a modular method, i.e., by pre-assembling repeat units in a target vector, which can then be assembled into a final vector of interest. The present disclosure provides polypeptides produced by such methods and nucleic acid sequences encoding such polypeptides. The present disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced by such modular methods.
"binding" refers to sequence-specific non-covalent interactions between macromolecules (e.g., between a protein and a nucleic acid). Not all components of the binding interaction need be sequence specific (e.g., in contact with phosphate residues in the DNA backbone), so long as the interaction as a whole is sequence specific.
The term "comprising" is intended to mean that the compositions and methods comprise the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of …" shall mean excluding other elements of any significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude trace contaminants or inert carriers. "consisting of … …" shall mean the exclusion of other components and a large number of process steps that are larger than trace elements. Aspects defined by each of these transitional terms are within the scope of this disclosure.
The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope may include three amino acids in a spatial conformation, which is unique to the epitope. Typically, an epitope consists of at least 4, 5, 6 or 7 such amino acids, and more typically, at least 8, 9 or 10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may comprise splicing of mRNA in eukaryotic cells.
"Gene expression" refers to the conversion of information contained in a gene into a gene product. The gene product may be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microrna, structural RNA, or any other type of RNA) or a protein produced by mRNA translation. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, tetradecylation and glycosylation.
"Modulation" or "regulation" of gene expression refers to a change in gene activity. Modulation of expression may include, but is not limited to, gene activation and gene suppression.
The term "operatively linked" or an equivalent thereof (e.g., "operatively linked") refers to two or more molecules positioned relative to each other such that they are capable of interacting to affect a function attributed to one or both of the molecules or a combination thereof.
Non-covalently linked components and methods of making and using the non-covalently linked components are disclosed. As described herein, the various components may take a variety of different forms. For example, non-covalently linked (i.e., operatively linked) proteins may be used to allow for temporary interactions, thereby avoiding one or more problems in the art. The ability of non-covalently linked components (e.g., proteins) to associate and dissociate only or primarily to achieve functional association where such association is required for the desired activity. The connection may have a duration sufficient to allow the desired effect.
A method for directing a protein to a specific locus in the genome of an organism is disclosed. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and effector molecule are capable of being operatively linked by a non-covalent linkage.
The term "scFv" refers to a single chain variable fragment. scFv is a fusion protein of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains, linked to a linker peptide. The linker peptide may be about 5 to 40 amino acids in length or about 10 to 30 amino acids or about 5, 10, 15, 20, 25, 30, 35 or 40 amino acids in length. The single chain variable fragment lacks the constant Fc region present in the intact antibody molecule and thus lacks the common binding sites (e.g., protein a/G) for purification of the antibody. The term further comprises scFv, which is an intracellular antibody (an antibody that is stable in the cytoplasm of a cell) and can bind to intracellular proteins.
The term "single domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a particular antigen. Single domain antibodies are typically peptide chains about 110 amino acids long, including the heavy chain antibody or one variable domain (VH) of a normal IgG, which generally have similar affinity for antigen as whole antibodies, but are more thermostable and stable against detergents and high concentrations of urea. Examples are those derived from camelized or fish antibodies. Alternatively, single domain antibodies may be made from a common murine or human IgG having four chains.
The terms "specific binding" (specifically bind) "and" specific binding "as used herein refer to the ability of an antibody, antibody fragment or nanobody to preferentially bind to a particular antigen present in a homogeneous mixture of different antigens. In some aspects, the specific binding interactions will distinguish between desired and undesired antigens in the sample. In some aspects, more than about ten to 100 times or more (e.g., more than about 1000 or 10,000 times). "specificity" refers to the ability of an immunoglobulin or immunoglobulin fragment (e.g., nanobody) to preferentially bind to one antigen target rather than a different antigen target, and does not necessarily mean high affinity.
A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided that sufficient binding conditions exist.
The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides that are covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, the nucleic acid may also encompass the depicted single-stranded complementary strand. The nucleic acids of the present disclosure also encompass substantially identical nucleic acids and complements thereof that retain the same structure or encode the same protein.
Probes of the present disclosure may include single stranded nucleic acids that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure may refer to a probe that hybridizes under stringent hybridization conditions.
The nucleic acids of the present disclosure may be single-stranded or double-stranded. Even when a majority of the molecules are single stranded, the nucleic acids of the present disclosure may contain double stranded sequences. Even when a majority of the molecules are double stranded, the nucleic acids of the present disclosure may contain single stranded sequences. The nucleic acids of the disclosure may comprise a genome DNA, cDNA, RNA or hybrid thereof. The nucleic acids of the present disclosure may contain a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may contain a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids of the present disclosure can be synthesized to include unnatural amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or by recombinant methods.
The nucleic acids of the present disclosure, or the complete sequence thereof, or any portion thereof, may be non-naturally occurring. The nucleic acids of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions such that the entire nucleic acid sequence is non-naturally occurring. The nucleic acids of the present disclosure may contain one or more replicated, inverted, or repeated sequences whose resulting sequence is not naturally occurring, such that the entire nucleic acid sequence is non-naturally occurring. The nucleic acids of the present disclosure may contain non-naturally occurring modified, artificial or synthetic nucleotides such that the entire nucleic acid sequence is non-naturally occurring.
In view of redundancy in the genetic code, multiple nucleotide sequences may encode any particular protein. All such nucleotide sequences are contemplated herein.
As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a promoter to which it is spatially linked. Promoters may be located 5 '(upstream) or 3' (downstream) of a gene under their control. The distance between a promoter and a gene may be about the same as the distance between the promoter and a gene that the promoter controls in the gene from which it originates. The change in distance between the promoter and the gene can be accommodated without losing the function of the promoter.
As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally derived molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. Promoters may include one or more specific transcriptional regulatory sequences to further enhance expression and/or alter spatial and/or temporal expression thereof. Promoters may also include distal enhancer or repressor elements that may be positioned up to several thousand base pairs from the transcription initiation site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters may constitutively or differentially regulate expression of a genomic component relative to the cell, tissue, or organ in which expression occurs, or relative to the developmental stage in which expression occurs, or in response to an external stimulus such as physiological stress, pathogen, metal ion, or inducer. Representative examples of promoters include phage T7 promoter, phage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1 alpha promoter, CAG promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.
As used throughout this disclosure, the term "substantially complementary" means that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.
As used throughout this disclosure, the term "substantially identical" means that the first and second sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or in the case of nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence.
As used throughout this disclosure, the term "variant" when used to describe a nucleic acid refers to: (i) a portion or fragment of the nucleotide sequence recited; (ii) the complement of the cited nucleotide sequence or part thereof; (iii) A nucleic acid substantially identical to the cited nucleic acid or complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.
As used throughout this disclosure, the term "vector" refers to a nucleic acid sequence that contains an origin of replication. The vector may be a viral vector, phage, bacterial artificial chromosome, or yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, and is preferably a DNA plasmid. The vector may include amino acids in combination with DNA sequences, RNA sequences, or both DNA and RNA sequences.
As used throughout this disclosure, the term "variant" when used in reference to a peptide or polypeptide refers to a peptide or polypeptide that differs in amino acid sequence by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity. Variant may also mean a protein having an amino acid sequence that is substantially identical to the referenced protein having an amino acid sequence that retains at least one biological activity.
Conservative substitutions of amino acids, i.e., substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions) are believed in the art to typically involve minor changes. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al, journal of molecular biology 157:105-132 (1982). The hydropathic index of amino acids is based on consideration of their hydrophobicity and charge. Amino acids with similar hydropathic indices may be substituted and still retain protein function. In one aspect, an amino acid having a hydropathic index of + -2 is substituted. The hydrophilicity of amino acids may also be used to reveal substituents that will produce proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of peptides allows calculation of the maximum local average hydrophilicity of the peptides, which is a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated herein by reference in its entirety.
Substitutions of amino acids with similar hydrophilicity values can result in peptides that retain biological activity (e.g., immunogenicity). Amino acids whose hydrophilicity values lie within + -2 of each other may be substituted. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the particular side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological functions are understood to depend on the relative similarity of the amino acids, and in particular the side chains of those amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.
As used herein, "conservative" amino acid substitutions may be defined as set forth in table A, B or C below. In some aspects, fusion polypeptides and/or nucleic acids encoding such fusion polypeptides comprise conservative substitutions introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contributions to secondary and tertiary protein structure. Conservative substitutions are amino acids that are substituted for one another with similar properties. Exemplary conservative substitutions are listed in table 1.
TABLE 1 conservative substitution I
Alternatively, conserved amino acids may be grouped as described by Lehninger (Biochemistry, second edition; woldherers, N.Y. (Worth Publishers, inc. NY, N.Y.) (1975), pages 71-77), as shown in Table 2.
TABLE 2 conservative substitution II
Alternatively, exemplary conservative substitutions are listed in table 3.
TABLE 3 conservative substitution III
| Original residue | Exemplary substitution |
| Ala(A) | ValLeuIleMet |
| Arg(R) | LysHis |
| Asn(N) | Gln |
| Asp(D) | Glu |
| Cys(C) | SerThr |
| Gln(Q) | Asn |
| Glu(E) | Asp |
| Gly(G) | AlaValLeuPro |
| His(H) | LysArg |
| Ile(I) | LeuValMetAlaPhe |
| Leu(L) | IleValMetAlaPhe |
| Lys(K) | ArgHis |
| Met(M) | LeuIleValAla |
| Phe(F) | TrpTyrIle |
| Pro(P) | GlyAlaValLeuIle |
| Ser(S) | Thr |
| Thr(T) | Ser |
| Trp(W) | TyrPheIle |
| Tyr(Y) | TrpPheThrSer |
| Val(V) | IleLeuMetAla |
It will be understood that the polypeptides of the present disclosure are intended to encompass modified polypeptides with insertions, deletions, or substitutions of one or more amino acid residues, or any combination thereof, in addition to insertions, deletions, or substitutions of amino acid residues. The polypeptides or nucleic acids of the present disclosure may contain one or more conservative substitutions.
As used throughout the present disclosure, the term "more than one" of the above amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more of the amino acid substitutions. The term "more than one" may refer to 2, 3, 4 or 5 of said amino acid substitutions.
The polypeptides and proteins of the present disclosure, or the complete sequence thereof, or any portion thereof, may be non-naturally occurring. The polypeptides and proteins of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions such that the entire amino acid sequence is non-naturally occurring. Polypeptides and proteins of the present disclosure may contain one or more replicative, inverted, or repetitive sequences whose resulting sequence is not naturally occurring such that the entire amino acid sequence is non-naturally occurring. The polypeptides and proteins of the present disclosure may contain non-naturally occurring modified, artificial or synthetic amino acids such that the entire amino acid sequence does not occur naturally.
As used throughout this disclosure, "sequence identity" may be determined by using a separate executable BLAST engine program for disrupting two sequences (bl 2 seq), which may be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using default parameters (Tatusova and Madden, joint institute of microbiology letters, FEMS Microbiol letters), 1999,174,247-250; the literature is incorporated herein by reference in its entirety), the term "identical" or "identity" when used in the context of two or more nucleic acid or polypeptide sequences refers to the same percentage of particular residues in a designated region of each sequence. The percentages can be calculated by: optimally aligning the two sequences, comparing the two sequences within the designated region, determining the number of positions in the two sequences where identical residues occur to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the designated region, and multiplying the result by 100 yields the percentage of sequence identity. In the case where two sequences differ in length or an alignment produces one or more staggered ends and the designated comparison region contains only a single sequence, the residues of the single sequence are contained in the denominator but not in the calculated numerator. Thymine (T) and uracil (U) can be considered equivalent when comparing DNA and RNA. Identity can be performed manually or by using a computer sequence algorithm (e.g., BLAST or BLAST 2.0).
As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with a target gene or host cell into which it is introduced.
As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, comprising a non-naturally occurring multiple copies of a naturally occurring nucleic acid, such as a DNA sequence, or a naturally occurring nucleic acid sequence that is located at a non-naturally occurring genomic position.
The present disclosure provides methods of introducing a polynucleotide construct comprising a DNA sequence into a host cell. "introducing" refers to presenting a polynucleotide construct to a cell in such a way that the construct enters the interior of the host cell. The methods of the present disclosure do not depend on the particular method of introducing the polynucleotide construct into the host cell, but merely on the entry of the polynucleotide construct into the interior of the host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
Example 1 mRNA for LNP composition Preparation
DNA plasmid pRT-HA-SPB-CC-AG encodes a SuperpiggyBac transposase comprising a 5' -hemagglutinin tag corresponding to amino acids 98-106 ("HA-SPB"). This plasmid was used as a template for an in vitro transcription reaction to generate an mRNA encoding HA-SPB, which further included a 5' cap.
Briefly, approximately 10ug of supercoiled pRT-HA-SPB-CC-AG was added to a 1.5ml Eppendorf tube containing a total volume of 100. Mu.l of 1 XCutSmart Buffer, 200 units of restriction enzyme SpeI (New England Biolabs (New England Biolabs), catalog number R3133 l). Plasmid DNA was linearized by incubation overnight at 37 ℃ to ensure complete digestion.
The linearized plasmid was purified using a DNA QIAquick PCR purification kit (Qiagen, catalog number 28104) according to the instructions of the manufacturer, and the purified DNA was eluted in 40. Mu.l of nuclease-free water. The DNA concentration of the eluate was determined using a NanoDrop micro-spectrophotometer (ThermoFisher) according to the manufacturer's instructions.
mRNA was produced using the purified plasmid as a DNA template using an in vitro transcription mMESSAGEmMACHINET7 transcription kit (Semer Feishmanic technologies, catalog number AM 1344) according to the manufacturer's instructions. Briefly, a stock solution of 100mM nucleotide GTP, ATP, UTP and 5MeC (5-methylcytidine-5 '-triphosphate) (Trilink company No. N=1014) and CleanCap reagent AG (m 7G (5') ppp (5 ') (2' OMeA) pG; trilink company No. N-7113) was prepared. 15. Mu.l each of ATP, UTP and 5MeC, and 12. Mu.l each of GTP and CleanCap reagent AG were added to make the total volume 100. Mu.l.
Approximately 1.67. Mu.g of linear pRT-HA-SPB-CC-AGDNA, 20. Mu.l of 10XT7 transcription buffer and 20. Mu. l T7 RNA polymerase mixture were added to a 1.5ml Eppendorf tube (final volume 200. Mu.l) and the tubes were incubated at 37℃for 3 hours. An aliquot of 10. Mu.l TURBODNase enzyme (Simer Feishmania technologies) was added and the tube was further incubated for 15 minutes at 37℃to degrade the DNA template.
Using the reagents and procedures provided in the mMESSAGEmMACHINET7 transcription kit (Semer Feishmanic technologies catalog number AM 1344), p was preparedThe oly (A) tail is added to the 5' -The 3' end of the HA-SPB mRNA. />
Purification of 5 '. The RNeasy Mid purification kit (Kaiji, cat. No. 75144) was used according to the manufacturer's instructions-HA-SPB-poly (A) -5MeCmRNA. Briefly, 3.5ml of buffered RLT solution was freshly prepared using 35. Mu.l of 2-mercaptoethanol, combined with 2.5ml of 100% ethanol, and the final mRNA product was eluted from the column using 300. Mu.l of nuclease free water. The average mRNA yield for this process was about 600-800. Mu.g.
Example 2-preparation and in vivo screening of LNPs of the disclosure including mRNA
A. Preparation
The following is a non-limiting example that provides an exemplary method for formulating a variety of multicomponent LNP compositions that include bioreducable ionizable cationic lipids and mRNA.
To formulate LNP, different percentages of the bioreductive ionizable cationic lipid ssPalmO-Ph-P4C2, phospholipid DOPE, structural lipid cholesterol (Chol), and 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (DMG-PEG 2000; a Wen Di polar lipid company of Alabaster, USA) were combined to prepare LNP compositions.
Separate 25mg/ml stock solutions were prepared by dissolving lipids in 200 standard HPLC grade ethanol and stored at-80 ℃ prior to formulation. At the time of formulation, the lipid stock solution was temporarily equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50-55 ℃. Subsequently, the hot lipid stock solutions are combined to produce the desired final mole percent. Tables 4a and 4b show a subset of LNP compositions.
TABLE 4a
TABLE 4b
1mg/ml of a 5' -CleanCap-5 MeC-firefly luciferase mRNA (TriLink Biotech) solution to be incorporated into LNP was added to 150mM sodium acetate buffer (pH 5.2) to form a stock solution, and stored on ice. According to the manufacturer's instructions, useAn instrument (precision nanosystems, vancouver, BC, canada) mixes the lipid phase with the aqueous mRNA phase within the microfluidic chip to form an LNP composition comprising encapsulated mRNA. The nanoassamblr process parameters for mRNA encapsulation are shown in table 5.
TABLE 5
| Total flow rate (ml/min) | Lipid phase Water (RNA) phase (v/v) |
| 20 | 1:3 |
The resulting mRNALNP composition is then transferred to a Repligen flow-A-Lyzer dialysis device (Spectroscopy, calif., USA) (Spectrum Chemical Mfg.Corp, CA, USA) with a molecular weight cut-off (MWCO) of 8-10 kDa) and purified by buffering the saline (PBS) (dialysate) with phosphateDialysis buffer volumes of at least 1:200 v/v), pH 7.4, at 4℃overnight (or alternatively at room temperature for at least 4 hours) to remove 25% ethanol and achieve complete buffer exchange. In some experiments, LNP was spun at about 4100 Xg in an ultracentrifugeThe MWCO-30kDa (Millipore Sigma, USA) was further concentrated in an Ultra-4 centrifugal filtration unit. The mRNALNP is then stored at 4℃until further use.
The mean particle size diameter of the LNP was about 70nm.
B. In vivo screening
Adult female BALB/C mice (n=2/group) were intravenously administered 0.5mg/kg of 5' -clearcap-5 MeC-firefly luciferase mRNA (TriLink biotechnology company) formulated with the LNP compositions shown in table 4. One group of mice was treated with vehicle (PBS, sammer femto technology company (Thermo Fisher Scientific, USA)) as a negative control.
The location and extent of luciferase expression in treated and control mice was determined at 4 hours by bioluminescence imaging (BLI) of anesthetized mice using an IVIS luminea in vivo imaging system (Perkin Elmer) according to manufacturer's instructions. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated table. The mice were then IP-administered with D-fluorescein (Perkin Elmer Corp. No. 122799) and BLI. The results are shown in table 6.
TABLE 6
As shown in table 6, LNP compositions 3, 9, 11, 13, 24, 28 and 29 were able to deliver mRNA in vivo, primarily to cells in the liver, and subsequently express the encoded proteins. Furthermore, administration of LNP compositions 3, 9, 11, 13, 24, 28 and 29 resulted in a significant increase in liver luciferase signal compared to LNP composition 0. LNP composition 0 is the LNP composition most similar to that used in the art (see Tanaka et al, advanced function materials (Advanced FunctionalMaterials), volume 2020: 30, 34). Thus, the LNP compositions of the present disclosure exhibit superior gene delivery activity compared to standard LNP compositions used in the art.
In addition, body weight of mice treated with the LNP composition of table 4a was assessed prior to intravenous administration and twenty-four hours post administration, and baseline and post-treatment body weights were compared. The average percentage of body weight change for each group of mice treated with each LNP composition of table 7 is shown in table 7.
TABLE 7
| LNPID | 24 hours weight change% |
| 3 | +5.9 |
| 9 | +4.6 |
| 11 | +0.25 |
| 13 | +3.6 |
| 24 | +1.1 |
| 28 | -3.15 |
| 29 | +0.6 |
As shown in table 7, the LNP compositions of the present disclosure have good tolerability, with most of the treated mice maintaining original body weight or even slightly gaining weight.
Example 3-delivery of mRNA to in vivo LNP in the liver
A. Delivery of sPB mRNA to liver cells
The following is a non-limiting example demonstrating that the compositions of the present disclosure can be used to deliver mRNA to liver cells, including hepatocytes, in vivo.
In this example, a 5MeC-mRNA molecule comprising a sequence encoding an HA-tagged SPB protein is encapsulated in a lipid nanoparticle of the present disclosure comprising about 28 mole% Coattome SS-OP, about 60 mole% cholesterol, about 10 mole% DOPE, and about 2 mole% DMG-PEG2000. The ratio of lipid to nucleic acid in the nanoparticle was about 100:1 (weight/weight) and the total lipid was 10mM. UsingThe mRNA molecules were further capped. As a negative staining control, mRNA comprising a sequence encoding a sPB protein that is not HA-tagged was used.
Lipid nanoparticles including mRNA were administered to adult female BALB/C mice. The nanoparticles were administered intravenously as a single dose in an amount of 1 mg/kg. Mice were humanly euthanized four hours after treatment and the livers of the mice were processed, blood was removed from the livers, e.g., by portal flushing of about 10mLHBSS+2.5mM EDTA, and analyzed using immunostaining for HA markers, as well as ELISA and western blotting.
HA staining was observed in hepatocytes throughout the liver of treated mice, with approximately 62% of all liver cells of one mouse and 66% of all liver cells of the other mouse tested positive for sPB expression. In addition, in vivo expression of sPB was uniformly detected by each of the main hepatic lobe, the medial lobe, and the left and right lobes. Thus, the nanoparticle compositions of the present disclosure effectively deliver mRNA to hepatocytes throughout the entire liver in vivo, and the delivered mRNA is subsequently translated into protein.
Dose-dependent LNP delivery and tolerability of mRNA to liver cells
The following is a non-limiting example demonstrating that the lipid nanoparticle compositions of the present disclosure can be used to deliver mRNA to liver cells in vivo and express the encoded protein over a wide dosage range and with good tolerability.
Adult female BALB/C mice (n=3/group) were intravenously administered 0.5, 1.0, 2.0, or 3.0mg/kg mrna molecules comprising sequences encoding HA-tagged sPB protein formulated within the LNP composition prepared according to example 1. One group of mice was untreated as a negative control.
Four hours after treatment, a group of mice was sacrificed and the livers of the mice were analyzed using HA-tagged immunostaining, ELISA and western blot. The results are shown in table 8.
TABLE 8
| Dosage (mg/kg) | HA tag sPB protein (ng) |
| 0.5 | 0.8 |
| 1.0 | 1.5 |
| 2.0 | 1.9 |
| 3.0 | 2.4 |
As shown in Table 8, linear dose responses were observed in mice treated with a single dose of 0.5-3mg/kg of HA-tagged sPB mRNA.
In another experiment, the duration of HA-tagged sPB protein expression was measured over time. Adult female BALB/C mice (n=3/group) were intravenously administered 0.5, 1.0, or 3.0mg/kg of mRNA molecules comprising sequences encoding HA-tagged sPB protein formulated within LNP composition 9 prepared according to example 2. One group of mice was untreated as a negative control.
One group of mice at each concentration was sacrificed four hours later, one group of mice at each concentration was sacrificed twenty-four hours later, and one group of mice at each concentration was sacrificed seven days after treatment, and the livers of the mice were analyzed using HA-tagged immunostaining and ELISA. The results are shown in table 9.
TABLE 9
| Dosage (mg/kg) | Time (hours) | HA tag sPB protein (ng) |
| 0.5 | 4 | 0.8 |
| 1.0 | 4 | 1.5 |
| 3.0 | 4 | 2.4 |
| 0.5 | 24 | 0.2 |
| 1.0 | 24 | 0.8 |
| 3.0 | 24 | 1.2 |
| 0.5 | 168 | 0.1 |
| 1.0 | 168 | 0.1 |
| 3.0 | 168 | 0.2 |
As shown in table 9, it was tested that at each concentration tested, the expression of HA-tagged sPB protein decreased over time, and by day 7, sPB expression decreased to near baseline levels.
In addition, the levels of the three liver enzymes present in serum at each concentration tested were evaluated as a measure of potential liver toxicity 24 hours and 7 days after LNP administration. Briefly, blood was withdrawn at 24 hours and 7 days, and each sample was coagulated for 20 minutes and centrifuged at 13Krpm for 3 minutes to remove unwanted cells and debris. The samples were transported on wet ice and stored at-80 ℃ until analysis. Enzyme levels were determined using a standardized test (idex).
Levels of the liver enzymes aspartate Aminotransferase (AST), alanine Aminotransferase (ALT) and alkaline phosphatase (ALP) at 24 hours and 7 days, respectively, are shown in tables 10 a-c.
Table 10a: AST level
| LNP dose (mg/kg) | Time (hours) | AST quantity (Unit/L) |
| 0 | 24 | 49 |
| 0.5 | 24 | 54 |
| 1.0 | 24 | 66 |
| 2.0 | 24 | 78 |
| 3.0 | 24 | 87 |
| 0 | 168 | 93 |
| 0.5 | 168 | 58 |
| 1.0 | 168 | 85 |
| 2.0 | 168 | 69 |
| 3.0 | 168 | 54 |
Table 10b: ALT levels
| LNP dose (mg/kg) | Time (hours) | ALT quantity (Unit/L) |
| 0 | 24 | 29 |
| 0.5 | 24 | 30 |
| 1.0 | 24 | 36 |
| 2.0 | 24 | 46 |
| 3.0 | 24 | 59 |
| 0 | 168 | 44 |
| 0.5 | 168 | 29 |
| 1.0 | 168 | 44 |
| 2.0 | 168 | 29 |
| 3.0 | 168 | 35 |
Table 10c: ALP level
As shown in tables 10a-c, in vivo administration of the LNP compositions of the present disclosure resulted in very slight (< 2X) dose-dependent increases in AST and ALT levels in serum at 24 hours; however, by day 7, all three enzyme levels returned to baseline, demonstrating lower magnitude of liver enzyme elevation.
In addition to serum liver enzymes, for each concentration tested, the levels of the three pro-inflammatory cytokines present in the serum were assessed 4 hours after LNP administration. Briefly, serum samples were prepared as described for liver enzyme analysis, and serum concentrations of each cytokine were determined using commercially available ELISA kits (e.g., the Quantikine ELISA kit of R & D systems). The levels of the pro-inflammatory cytokines interleukin-6 (IL-6), interferon gamma (INF-G) and tumor necrosis factor alpha (TNF-a) at 4 hours are shown in tables 11a-c, respectively.
Table 11a: IL-6 levels
| LNP dose (mg/kg) | Time (hours) | Quantity (pg/mL) |
| 0 | 24 | 0 |
| 0.5 | 24 | 0.1 |
| 1.0 | 24 | 1.0 |
| 2.0 | 24 | 74.8 |
| 3.0 | 24 | 238.7 |
Table 11b: INF-G levels
| LNP dose (mg/kg) | Time (hours) | Quantity (pg/mL) |
| 0 | 24 | 0 |
| 0.5 | 24 | 5.6 |
| 1.0 | 24 | 17.1 |
| 2.0 | 24 | 17.1 |
| 3.0 | 24 | 33.6 |
Table 11c: TNF-a levels
| LNP dose (mg/kg) | Time (hours) | Quantity (pg/mL) |
| 0 | 24 | 0 |
| 0.5 | 24 | 1.3 |
| 1.0 | 24 | 7.4 |
| 2.0 | 24 | 8.7 |
| 3.0 | 24 | 15.4 |
As shown in tables 11a-c, in vivo administration of the LNP compositions of the present disclosure resulted in a dose-dependent increase in serum pro-inflammatory cytokines, although the magnitude of the response was modest considering other non-bioreducable ionizable cationic lipids.
In another experiment, LNP composition 9 of example 2 was compared to LNP particles comprising non-bioreducable ionizable cationic lipid C12-200. Adult female BALB/C mice (n=3/group) were intravenously administered 0.5, 1.0, or 3.0mg/kg mRNA molecules including sequences encoding HA-tagged sPB proteins formulated within LNP compositions. One group of mice was untreated as a negative control. The percentage of SB positive hepatocytes, ALT liver enzyme measurements, and IL-6 cytokine release measurements were compared between treated animals of each group and the value of each at 1mg/kg dose (MTD dose of C12-200 LNP composition) is reported in table 12.
Table 12
As shown in table 12, LNP composition 9 exhibited similar efficacy in delivering SB mRNA to hepatocytes as compared to C12-200 LNP composition, while exhibiting significantly reduced toxicity profile, as indicated by reduced IL-6 cytokine release and reduced ALT liver enzyme as compared to C12-200 LNP.
C. The LNP compositions of the present disclosure deliver RNA to the liver in vivo with high specificity.
In a third experiment, 1mg/kg of HA-tagged sPB mRNA formulated in nanoparticle compositions prepared according to example 1 was administered intravenously to a group of annual female BALB/C mice (n=3/group), and the second group was not treated as a control. Four hours after administration, mice in each group were humanly euthanized and four tissue types were collected: liver, spleen, lung and kidney.
The collected tissue is processed, for example, by flushing about 10mLHBSS+2.5mM EDTA through the portal vein, and blood is removed from the liver. Protein extraction buffers were prepared by adding protease inhibitors (HALT, semerle technology accession No. 78439) to T-PER (Semerle technology accession No. 78510) at a ratio of 1:50 (v: v). The protein extraction buffer is stored at room temperature for up to 1 hour. In an RNAse-free Eppendorf tube (Injetty Corp. No. Am 12425), the tissue samples were added to the protein extraction buffer at a rate of 9mL of tissue per 19 g. One tungsten carbide bead (3 mm; kaijer company No. 69997) was added to the solution and the tube was placed into a pre-chilled adapter block in a tissue disruptor (Kaijer company tissue lyzer II). The sample was lysed by shaking at 25Hz for 5 minutes. The sample was clarified by centrifugation at 14.8k at 4℃for 10 minutes. The supernatant was collected and the precipitate was discarded.
Total protein was measured by BCA assay using a commercially available kit (BCA assay kit, pierce company No. 23225). Clarified liver lysates were diluted 200X, incubated at 37 ℃ for 30 min, and absorbance read at 562 nm. The results are shown in table 13.
TABLE 13
| Tissue of | HA tag sPB protein (ng) |
| Liver | 2.4 |
| Spleen | 0.1 |
| Lung (lung) | 0 |
| Kidneys (kidney) | 0 |
As shown in table 13, HA-tagged sPB expression was detected almost exclusively in the liver of treated animals, with minimal expression in the spleen, and no expression in the lung or kidney, demonstrating the ability of LNP compositions of the present disclosure to preferentially deliver mRNA to the liver in vivo, and subsequent expression of the encoded polypeptide in hepatocytes.
The results presented in example 3 demonstrate that the LNP compositions of the present disclosure are capable of efficiently delivering mRNA in vivo to the liver, which is then expressed in cells of the liver, and protein expression can be controlled by a wide range of administered doses. Furthermore, the LNP compositions of the present disclosure are well tolerated and exhibit low toxicity levels.
Example 4-present disclosure including DNAPreparation of open LNP compositions
The following is a non-limiting example demonstrating that DNA nanoplasmids (circular DNA) can be incorporated into the LNP compositions of the present disclosure.
LNP compositions of the present disclosure include DNA, a nanoplasmid encoding a piggyBac transposon, wherein the piggyBac transposon includes a luciferase under the control of a CMV promoter (referred to herein as pB-nanofluc 2), a coatname SS-OP, a phospholipid DOPE, a structural lipid cholesterol (Chol), and 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (DMG-PEG 2000), the percentages of which are in table 14 as follows:
TABLE 14
1.0mg/kg of total DNA of each of LNP compositions D1-D5 listed in Table 14 (n=4) was administered to adult BALB/C mice. The location and extent of luciferase expression in treated and control mice was determined at 4 hours by bioluminescence imaging (BLI) of anesthetized mice using an IVIS luminea in vivo imaging system (perkin elmer) according to manufacturer's instructions. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated table. The mice were then IP-administered with D-fluorescein (Perkin Elmer Corp. No. 122799) and BLI. The results are shown in table 15.
TABLE 15
As shown in table 15, all LNP compositions were able to deliver DNA to liver cells and express the encoded transgene in the liver cells.
Example 5-LNP of the present disclosure for treatment of hemophilia
The following are non-limiting examples, which demonstrate that the compositions and methods of the present disclosure can be used to treat hemophilia, and more particularly hemophilia a.
Adult mice
Female, adult (8-9 weeks), wild-type BALB/c was first administered 0.5mg/kg FVIII transposon LNP on day 0 of the experiment, and then 3.0mg/kg HA-SPB LNP on day 7. The total injection amount was 200 μl per mouse on both day 0 and day 7. Following administration, the mice were restrained and injected intravenously via the tail vein with a No. 29 insulin syringe.
FVIII transposon LNP is a C12-200 containing LNP comprising a nanoplasmon DNA comprising a transposon, wherein the transposon comprises an expression cassette comprising a first piggyBac Inverted Terminal Repeat (ITR) sequence followed by a first insulator sequence followed by a transthyretin (TTR) enhancer/promoter and a mouse adenovirus (MVM) intron sequence followed by a codon optimized nucleic acid sequence encoding human Factor VIII (FVIII) lacking a B domain (hereinafter referred to as FVIII-BDD), followed by an SV40 polyA sequence followed by a second insulator sequence followed by a second piggyBac ITR. The sequence of the transposon is given in SEQ ID No. 35. FVIII transposon LNP comprises C12-200, DOPE, cholesterol and DMG-PEG2000 in a molar ratio of 0.35:0.2:0.4184:0.0316, and a lipid to DNA ratio of 80:1 (w/w).
HA-SPB LNP is an ssPalmO-Ph-P4C 2-containing LNP of the present disclosure that includes mRNA encoding active SPB. All cytidine residues in mRNA are 5-methylcytidine (5-MeC). HA-SPB LNP includes ssPalmO-Ph-P4C2, DOPE, cholesterol, and DMG-PEG2000 in a molar ratio of 28:10:60:2, and a lipid to RNA ratio of 100:1 (w/w).
On days 6 and 13 after the first 0 day injection, plasma was collected by retroorbital bleeding. Briefly, the post-balloon sinus was destroyed using an uncoated Pasteur pipette and whole blood was collected and mixed with 3.2% buffered sodium citrate at a 9:1 volume ratio. The mixture was centrifuged at 15,000g for 15 min at 22℃and the plasma supernatant was collected and stored at-80 ℃. According to the manufacturer's instructions, use Affinity Biologicals TM Visual of company TM Factor FVIII antigen plus kit the level of human FVIII protein in a sample is analyzed.
The results of this analysis are shown in fig. 1. FIG. 1 shows that on day 13 after administration of HA-SPB LNP on day 6, human FVIII protein levels corresponding to 1-5% of normal human FVIII are observed in the sample.
The results presented in this example demonstrate that LNP of the present disclosure can be used to drive high levels of FVIII expression in vivo, even in adult mice, thereby demonstrating that the compositions and methods of the present disclosure can be used to treat hemophilia a.
Example 6-LNP of the present disclosure for treatment of hemophilia
The following are non-limiting examples, which demonstrate that the compositions and methods of the present disclosure can be used to treat hemophilia, and more particularly hemophilia B.
Young mice
Three week old young C57BL/6 mice were either untreated or were given one of the following two treatments:
process #1:factor IX transposon AAV viral vector particles
Process #2: factor IX transposon AAV viral vector particles are combined with SPB LNP.
Factor IX transposon AAV viral vector particles are AAV viral vector particles comprising a piggyBac transposon, wherein the piggyBac transposon comprises a nucleic acid encoding a human factor IX polypeptide having a R338L mutation.
SPB LNP is an ssPalmO-Ph-P4C 2-containing LNP of the present disclosure that includes mRNA encoding active SPB. SPB LNP includes ssPalmO-Ph-P4C2, DOPE, cholesterol, and DMG-PEG2000 in a molar ratio of 28:10:60:2, and a lipid to RNA ratio of 100:1 (w/w).
Three weeks after administration of the treatment, ELISA experiments were performed to determine the amount of human factor IX polypeptide in mouse plasma. The results of these ELISA experiments are shown in figure 2. Figure 2 shows that the observed human factor IX protein levels in the samples after administration of treatment #2 comprising LNP of the present disclosure are at levels corresponding to about 40-85% of normal human factor IX.
The results presented in this example demonstrate that LNP of the present disclosure can be used to drive high levels of expression of factor IX in vivo, thereby demonstrating that the compositions and methods of the present disclosure can be used to treat hemophilia B. More specifically, the LNP of the present disclosure can be used to drive expression levels of factor IX in vivo, which are in the range of factor IX levels observed in healthy individuals.
Example 7-preparation, in vivo screening and stability test of LNP of the present disclosure including mRNA
A. Preparation
The following is a non-limiting example that provides an exemplary method for formulating a variety of multicomponent LNP compositions that include bioreducable ionizable cationic lipids and mRNA.
To formulate LNP, different percentages of the bioreducable ionizable cationic lipid ssPalmO-Ph-P4C2, phospholipid DOPE, structural lipid cholesterol (Chol), and 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (DMG-PEG 2000; a Wen Di polar lipid company of alabaster, usa) were combined to prepare LNP compositions.
Separate 25mg/ml stock solutions were prepared by dissolving lipids in 200 standard HPLC grade ethanol and stored at-80 ℃ prior to formulation. At the time of formulation, the lipid stock solution was temporarily equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50-55 ℃. Subsequently, the hot lipid stock solutions are combined to produce the desired final mole percent. Table 16 shows a subset of LNP compositions:
Table 16
1mg/ml of 5meC 5' -CleanCap-5MeC-SPB-HA mRNA (prepared as in example 1) to be incorporated into LNP was added to 150mM sodium acetate buffer (pH 5.2) or maleate (pH 5.0) to form a stock solution and stored on ice. mRNA will include nucleic acid sequences encoding the HA-tagged SBP polypeptides. According to the manufacturer's instructions, useThe instrument (precision nanosystems, inc. Of vancomin, columbia, canada) mixes the lipid phase with the aqueous mRNA phase within the microfluidic chip to form an LNP composition comprising the encapsulated mRNA. The nanoasssemblr process parameters for mRNA encapsulation for each LNP composition are shown in table 17, including buffer used, buffer concentration and flow rate:
TABLE 17
The resulting mRNALNP composition is then transferred to a Repligen flow-A-Lyzer dialysis apparatus (spectrochemical Co., calif., U.S.A.) having a molecular weight cut-off (MWCO) of 8-10kDa and treated by dialysis against Phosphate Buffered Saline (PBS) (dialysate: dialysis buffer volume of at least 1:200 v/v), pH7.4 or 6.5 overnight (or alternatively at room temperature for at least 4 hours) at 4℃to remove 25% ethanol and achieve complete buffer exchange. In some experiments, LNP was spun at about 4000 Xg in an ultracentrifuge The mixture was further concentrated in an Ultra-4 centrifugal filtration unit, MWCO-100kDa or 50kDa (Miibo sigma Co., U.S.A.). The mRNALNP is then stored at 4℃until further use.
B. In vivo screening
Adult female BALB/C mice (n=2/group) were intravenously administered 1.0mg/kg of LNP composition comprising HA-SBP mRNA as shown in table 16. One group of mice was treated with vehicle (PBS, sameimer, feeier technologies, usa) as a negative control.
Mice were euthanized 4 hours after dosing and their livers were collected. Expression of HA-SBP was assessed by ELISA. The results of this analysis are shown in table 18:
TABLE 18
As shown in table 18, LNPID2.6, LNPID2.10, LNPID2.11, LNP2.14 and LNP 2.16 are capable of delivering mRNA in vivo, specifically to cells in the liver, and subsequently expressing the encoded protein.
C. Stability test
Aliquots of LNP ID2.6, LNP ID2.10, LNP ID2.11, LNP2.14 and LNP 2.16 were each stored at 0.1mg/mL for 7 days at 4 ℃ to assess the storage stability of the LNP compositions. The mean diameter and polydispersity index (PDI) of each LNP composition was analyzed at the beginning of the 7 day incubation (day 0), and at days 1, 4 and 7 of the incubation. The results of this analysis are shown in fig. 3. As shown in fig. 3, the size and PDI of each of LNPID2.6, LNPID2.10, LNP ID2.11, LNP2.14 and LNP 2.16 were stable during the 7 day incubation period, indicating that these compositions exhibited enhanced storage stability, which is advantageous in clinical and commercial environments.
Taken together, the results described in this example demonstrate that the LNP compositions of the present disclosure can effectively deliver mRNA to cells in vivo, including liver cells, and that these compositions are stable for long periods of time at standard storage temperatures.
Example 8-preparation and in vivo screening of LNPs of the disclosure including mRNA
A. Preparation
The following is a non-limiting example that provides an exemplary method for formulating a variety of multicomponent LNP compositions that include bioreducable ionizable cationic lipids and mRNA.
To formulate LNP, different percentages of the bioreducable ionizable cationic lipid ssPalmO-Ph-P4C2, phospholipids (DOPE, DSPC, or DOPC), structural lipid cholesterol (Chol), and 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (DMG-PEG 2000; angstrom Wen Di polar lipid company of alabaster, usa) were combined to prepare LNP compositions.
Separate 25mg/ml stock solutions were prepared by dissolving lipids in 200 standard HPLC grade ethanol and stored at-80 ℃ prior to formulation. At the time of formulation, the lipid stock solution was temporarily equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50-55 ℃. Subsequently, the hot lipid stock solutions are combined to produce the desired final mole percent. Table 19 shows a subset of LNP compositions.
TABLE 19
1mg/ml of a 5' -CleanCap-5 MeC-firefly luciferase mRNA (TriLink Biotech) solution to be incorporated into LNP was added to 150mM sodium acetate buffer (pH 5.2) to form a stock solution, and stored on ice. According to the manufacturer's instructions, useThe instrument (precision nanosystems, inc. Of vancomin, columbia, canada) mixes the lipid phase with the aqueous mRNA phase within the microfluidic chip to form an LNP composition comprising the encapsulated mRNA. The nanoassamblr process parameters for mRNA encapsulation are shown in table 20.
Table 20
| Total flow rate (ml/min) | Lipid phase Water (RNA) phase (v/v) |
| 20 | 1:3 |
The resulting mRNALNP composition is then transferred to a Repligen flow-A-Lyzer dialysis device (spectrochemical manufacturing company, calif., U.S. Calif.) having a molecular weight cut-off (MWCO) of 8-10kDa and treated by dialysis overnight (or alternatively at room temperature for at least 4 hours) with 25mM sodium acetate (dialysate: dialysis buffer volume of at least 1:200 v/v), pH 5.5, at 4℃to remove 25% ethanol and achieve complete buffer exchange. In some experiments, LNP was spun at about 4100 Xg in an ultracentrifugeThe MWCO-30kDa (Millipore Sigma, USA) was further concentrated in an Ultra-4 centrifugal filtration unit. Sucrose was added to the mRNA LNP at a final concentration of 5% (w/v) and then stored at 4 ℃ or frozen at-80 ℃ until further use.
The mean particle size diameter of the LNP is in the range of about 84-121 nm.
B. In vivo screening
Adult female BALB/C mice (n=2/group) were intravenously administered 0.5mg/kg of 5' -clearcap-5 MeC-firefly luciferase mRNA (TriLink biotechnology company) formulated with the LNP compositions shown in table 19. One group of mice was treated with vehicle (PBS, sameimer, feeier technologies, usa) as a negative control.
The location and extent of luciferase expression in treated and control mice was determined at 4 hours by bioluminescence imaging (BLI) of anesthetized mice using an IVIS luminea in vivo imaging system (perkin elmer) according to manufacturer's instructions. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated table. The mice were then IP-administered with D-fluorescein (Perkin Elmer Corp. No. 122799) and BLI. The results are shown in table 21.
Table 21
As shown in table 21, LNP compositions 3.1-3.10 were able to deliver mRNA in vivo, primarily to cells in the liver, and subsequently express the encoded protein.
Example 9-preparation and in vivo screening of LNPs of the disclosure including mRNA
A. Preparation
The following is a non-limiting example that provides an exemplary method for formulating a variety of multicomponent LNP compositions that include bioreducable ionizable cationic lipids and mRNA.
To formulate LNP, different percentages of the bioreducable ionizable cationic lipid ssPalmO-Ph-P4C2, phospholipids (DOPE, DSPC, or DOPC), structural lipid cholesterol (Chol), and 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (DMG-PEG 2000; angstrom Wen Di polar lipid company of alabaster, usa) were combined to prepare LNP compositions.
Separate 25mg/ml stock solutions were prepared by dissolving lipids in 200 standard HPLC grade ethanol and stored at-80 ℃ prior to formulation. At the time of formulation, the lipid stock solution was temporarily equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50-55 ℃. Subsequently, the hot lipid stock solutions are combined to produce the desired final mole percent. Table 22 shows a subset of LNP compositions.
Table 22
1mg/ml of 5' -CleanCap-5 MeC-firefly luciferase mRNA to be incorporated into LNP (TriLink Biotechnology Co (TriLink Biotec)h) Solution was added to 150mM sodium acetate buffer (pH 5.2) to form a stock solution and stored on ice. According to the manufacturer's instructions, useThe instrument (precision nanosystems, inc. Of vancomin, columbia, canada) mixes the lipid phase with the aqueous mRNA phase within the microfluidic chip to form an LNP composition comprising the encapsulated mRNA. The nanoassamblr process parameters for mRNA encapsulation are shown in table 23.
Table 23
| Total flow rate (ml/min) | Lipid phase Water (RNA) phase (v/v) |
| 20 | 1:3 |
The resulting mRNA LNP composition was then transferred to a Repligen flow-a-Lyzer dialysis apparatus (spectrochemical company, california) with a molecular weight cut-off (MWCO) of 8-10kDa and treated by dialysis overnight (or alternatively at room temperature for at least 4 hours) with 25mM sodium acetate (dialysate: dialysis buffer volume at least 1:200 v/v), pH 5.5 at 4 ℃ to remove 25% ethanol and achieve complete buffer exchange. In some experiments, LNP was spun at about 4100 Xg in an ultracentrifugeThe MWCO-30kDa (Millipore Sigma, USA) was further concentrated in an Ultra-4 centrifugal filtration unit. Sucrose was added to the mRNA LNP at a final concentration of 5% (w/v) and then stored at 4℃orFrozen at-80 ℃ until further use.
The mean particle size diameter of the LNP is in the range of about 80-103 nm.
B. In vivo screening
Adult female BALB/C mice (n=2/group) were intravenously administered 0.5mg/kg of 5' -clearcap-5 MeC-firefly luciferase mRNA (TriLink biotechnology company) formulated with the LNP composition subsets shown in table 22. One group of mice was treated with vehicle (PBS, sameimer, feeier technologies, usa) as a negative control.
In another experiment, adult female BALB/C mice (n=3-4/group) were intravenously administered 1mg/kg of 5' -clearcap-5 MeC-firefly luciferase mRNA (TriLink biotechnology company) formulated with a subset of LNP compositions shown in table 22. One group of mice was treated with vehicle (PBS, sameimer, feeier technologies, usa) as a negative control.
The location and extent of luciferase expression in treated and control mice was determined at 4 hours by bioluminescence imaging (BLI) of anesthetized mice using an IVIS luminea in vivo imaging system (perkin elmer) according to manufacturer's instructions. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated table. The mice were then IP-administered with D-fluorescein (Perkin Elmer Corp. No. 122799) and BLI. The results of the 0.5mg/kg dose experiment are shown in Table 24, and the results of the 1mg/kg dose experiment are shown in Table 25.
Table 24
Table 25
As shown in tables 24 and 25, the LNP compositions of the present disclosure are capable of delivering mRNA in vivo, primarily to cells in the liver, and subsequently expressing the encoded protein.
In addition, body weight of mice treated with the LNP composition subset of table 22 was assessed prior to intravenous administration and twenty-four hours post administration, and baseline and post-treatment body weights were compared. The average percent change in body weight for each group of mice treated with each LNP composition of table 22 is shown in table 26.
Table 26
| LNPID | 24 hours weight change% |
| 4.1 | -7.3 |
| 4.10 | -2.2 |
| 4.2 | +2.7 |
| 4.11 | -1.7 |
| 4.4 | -5.4 |
| 4.12 | -4.7 |
As shown in table 26, the LNP compositions of the present disclosure have good tolerability, with most of the treated mice maintaining original body weight or even slightly gaining weight.
Example 10-LNP compositions of the disclosure reduce immune response and toxicity
A. Immune response to LNP of the present disclosure
The following is a non-limiting example that demonstrates that in vitro administration of certain LNP compositions of the present disclosure results in reduced complement activation as measured by serum levels of C3a in human serum.
LNP compositions were prepared as described in example 8, with the following mole percentages as shown in table 27. The LNP composition encapsulates an RNA molecule comprising a sequence encoding an HA-tagged SPB.
Table 27
Normal Human Serum (NHS) was thawed at 37 ℃ and 100 μl was aliquoted into 1.5mL centrifuge tubes. NHS was then treated with 16. Mu.L of 0.1mg/mL LNP composition and incubated for 30 minutes at 37 ℃. The reaction mixture was then diluted 1:5000 and analyzed using a C3aELISA kit (Quidel Co.).
The C3a levels in samples treated with the LNP compositions of the present disclosure were compared to the C3a levels in samples treated with LNP compositions comprising baseline Phosphoethanolamine (PE) -based phospholipids, and the values are reported in table 28.
Table 28
As shown in table 28, LNP compositions 5.2 and 5.3 of the present disclosure exhibited significantly reduced immune response profiles as shown by reduced C3a serum levels compared to the baseline LNP composition.
B. Toxicity Spectrum
In another evaluation, the levels of four liver enzymes present in the serum were assessed 4 hours and 24 hours after LNP administration as a measure of potential liver toxicity. LNP compositions were prepared as described in example 9, with the following mole percentages as shown in table 29. As described in example 9, the LNP composition encapsulates 5' -clearcap-5 MeC-firefly luciferase mRNA (TriLink biotechnology).
Table 29
Briefly, blood was drawn at 4 hours and 24 hours, and each sample was coagulated for 20 minutes and centrifuged at 13Krpm for 3 minutes to remove unwanted cells and debris. The samples were transported on wet ice and stored at-80 ℃ until analysis. Enzyme levels were determined using the standardized test (idex).
The levels of the liver enzymes aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), alkaline phosphatase (ALP) and Creatine Kinase (CK) at 24 hours are shown in tables 30a-d, respectively. The level of each liver enzyme in the samples treated with the LNP compositions of the present disclosure was compared to the level of each liver enzyme in the samples treated with the LNP compositions including baseline Phosphoethanolamine (PE) -based phospholipids, and the values are reported in tables 30 a-d.
Table 30a: AST level
| LNPID | AST quantity (Unit/L) |
| 5.0 | 193.25 |
| 5.1 | 346.25 |
| 5.2 | 115.0 |
| 5.4 | 110.75 |
| 5.5 | 270.5 |
| 5.6 | 238.25 |
Table 30b: ALT levels
| LNPID | ALT quantity (Unit/L) |
| 5.0 | 120.5 |
| 5.1 | 186.25 |
| 5.2 | 60.25 |
| 5.4 | 70.75 |
| 5.5 | 195.25 |
| 5.6 | 164.5 |
Table 30c: ALP level
Table 30d: CK level
| LNPID | CK quantity (Unit/L) |
| 5.0 | 361.75 |
| 5.1 | 139.0 |
| 5.2 | 85.75 |
| 5.4 | 163.75 |
| 5.5 | 105.5 |
| 5.6 | 116.25 |
As shown in tables 30a-d, the particular LNP compositions of the present disclosure exhibited significantly reduced toxicity profiles, as shown by reduced serum liver enzyme levels, compared to the baseline LNP composition.
EXAMPLE 11 in vivo LNP delivery of DNA into the liver
The following is a non-limiting example demonstrating that the LNP compositions of the present disclosure can be used to deliver DNA to liver cells in vivo and express the encoded protein.
LNP compositions of the present disclosure including DNA encoding firefly luciferase (hereinafter referred to as "Fluc"; triLink) were prepared by combining ssPalmO-Ph-P4C2, phospholipid DOPC, structural lipid cholesterol (Chol), and 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol (DMG-PEG 2000) in the mole percentages presented in Table 31, as described in example 9.
Table 31
Adult BALB/C mice were administered 0.5mg/kg of total DNA of the LNP compositions listed in table 31 (n=4). The location and extent of luciferase expression in treated and control mice was determined at 4 hours by bioluminescence imaging (BLI) of anesthetized mice using an IVIS luminea in vivo imaging system (perkin elmer) according to manufacturer's instructions. Briefly, mice were anesthetized with isoflurane in oxygen and placed supine on a heated table. The mice were then IP-administered with D-fluorescein (Perkin Elmer Corp. No. 122799) and BLI. The results are shown in table 32.
Table 32
As shown in table 32, the LNP compositions of the present disclosure are capable of delivering DNA to liver cells and expressing the encoded transgene in the liver cells.
EXAMPLE 12 LNP of the present disclosure for treatment of hemophilia
The following are non-limiting examples, which demonstrate that the compositions and methods of the present disclosure can be used to treat hemophilia, and more particularly hemophilia a.
The nascent wild-type BALB/c mice (n=5-6) were administered 0.25mg/kg FVIII transposon LNP co-delivered with either 1) 1mg/kg LNP encapsulating functional SPB ("functional SPB") or 2) 0.5mg/kg LNP encapsulating catalytic deficient SPB ("deficient SPB").
FVIII transposon LNP is an ssPalmO-Ph-P4C 2-containing LNP of the present disclosure comprising a nanoplasmic DNA comprising a transposon, wherein the transposon comprises an expression cassette comprising a first piggyBac inverted terminal repeat (right ITR), followed by a first insulator sequence, followed by three tandem copies of the SERPINA1 enhancer, followed by a transthyretin (TTR) enhancer/promoter and a mouse adenovirus (MVM) intron sequence, followed by a codon optimized nucleic acid sequence encoding a modified human Factor VIII (FVIII), followed by an AES untranslated region (UTR), followed by an mTRNR1 UTR, followed by an SV 40-late polyadenylation and cleavage signal sequence, followed by a second insulator sequence, followed by a second piggyBac inverted terminal repeat (left ITR). The sequence of the transposon is given in SEQ ID No. 34. FVIII transposon LNP includes ssPalmO-Ph-P4C2, DOPE, cholesterol and DMG-PEG2000 in a molar ratio of 54:10:35:1, and lipid to DNA ratio of 100:1 (w/w).
Functional SPB LNP is an ssPalmO-Ph-P4C 2-containing LNP of the present disclosure that includes mRNA encoding active SPB, and ssPalmO-Ph-P4C2, DOPE, cholesterol, and DMG-PEG2000 in a molar ratio of 54:10:35:1, and a lipid to RNA ratio of 100:1 (w/w). Defective SPB LNP is a C12-200 containing LNP of the present disclosure that includes mRNA encoding a catalytic defective SPB, and C12-200, DOPE, cholesterol, and DMG-PEG2000 in a molar ratio of 33.5:32:33.5:1. All cytidine residues in the mRNA encoding functional or defective SPB are 5-methylcytidine (5-MeC).
The newborn pups were placed on ice for a short period of time (about 3 minutes) to induce anesthesia prior to LNP administration. For co-delivery administration, DNA-LNP and mRNA-LNP were mixed together in a tube, 30 μl was aspirated into a single No. 29 insulin syringe, and delivered Intravenously (IV) via the facial vein. The pups were allowed to recover normal body temperature on a heating pad at 37 ℃ before being placed back on the mother. Plasma was collected from treated mice at weeks 2, 4, 6 and 8 post-treatment. For plasma collection, the treated mice were anesthetized with isoflurane, approximately 150 μl of whole blood was collected retroorbital, the whole blood was mixed with 10% volume of 3.2% sodium citrate, centrifuged at 15,000g for 15 min at 20 ℃, and the plasma supernatant was collected. Using visual TM Factor VIII antigen increasing kit (Affinity Biologicals) TM Company) measures hFVIII antigen levels.
The results of this analysis are shown in fig. 4. Figure 4 shows that up to 8 weeks after SPB LNP administration, overstreated human FVIII protein levels were observed in the samples.
The results presented in this example demonstrate that LNP of the present disclosure can be used to drive high levels of FVIII expression in vivo, thereby demonstrating that the compositions and methods of the present disclosure can be used to treat hemophilia a.
Example 13-LNP compositions of the disclosure inIn vivo delivery of RNA to the liver with high specificity
This experiment demonstrates the ability of the LNP compositions of the present disclosure to deliver Cas-constituent mrna to the liver, which is targeted by a pair of grnas against the psk9 gene, resulting in subsequent in vivo gene editing of the psk9 gene. The Pcsk9 protein is secreted by hepatocytes and binds to LDL receptors, inducing its internalization and lysosomal degradation, resulting in elevated circulating LDL-cholesterol levels.
In this experiment, mRNA encoding 5' -CleanCap-5MeC-Cas-CLOVER (SEQ ID NO: 31) and a pair of gRNAs targeting the first exon of the mouse pcsk9 gene (SEQ ID NO: 29-30) were co-administered intravenously to each group of annual female BALB/C mice (n=2/group). mRNA and gRNA molecules were formulated in LNP compositions of the present disclosure comprising ssPalmO-Ph-P4C2, DOPE, cholesterol, and DMG-PEG2000 (referred to in tables 33-36 as LNP compositions 6.1, 6.2, 6.3, 6.4, and 6.5) in a molar ratio of 54:10:35:1. All cytidine residues in mRNA are 5-methylcytidine (5-MeC).
LNP compositions of the present disclosure, including the total RNA doses shown in table 33, were administered to mice in each group. A group of mice was administered a dose of Cas-CLOVER mRNA and a pair of pcsk9 grnas, both co-encapsulated in a baseline C12-200 LNP composition. One group of mice was treated with vehicle (PBS, sameimer, feeier technologies, usa) as a negative control.
Table 33
Seven days after administration, DNA was isolated from the following four tissue types of mice in each group: liver, spleen, lung and kidney. Briefly, tissues were excised after euthanasia, flash frozen in liquid nitrogen, mixed with lysis buffer (15 mg of tissue in 200uL lysis buffer+10 uL proteinase K) and crushed in TissueLyser II (kejie company) using triple pure zirconium beads (sameire feishier technologies). The homogenized tissue was then incubated at 56C for 30 minutes and column purified using the Monarch genomic DNA purification kit (Monarch Genomic DNA Purification kit) from new england biology laboratory according to the manufacturer's instructions. The final DNA elution was performed in 50uL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 and 280nm using Nanodrop. In addition, blood samples were withdrawn for LDL-C quantification. Briefly, after euthanasia, 500uL of blood was collected by cardiac puncture using a 2ml syringe and 25G needle, transferred into a microcentrifuge tube, incubated for 1 hour at room temperature, and centrifuged at 1500G for 15 minutes to separate the cell fraction from serum. The serum fraction (200 uL) was transferred to a new tube and stored at-80C until further analysis.
In addition, the body weight of mice treated with the LNP composition of table 33 was assessed during seven days post-administration and baseline and post-treatment body weights were compared. The average percentage of body weight change after seven days post-treatment for each group of mice treated with each LNP composition of table 33 is shown in table 34.
Watch 34
| LNPID | 7 days body weight change% |
| Benchmark C12-200LNP | 3.25％ |
| 6.1 | 4.11％ |
| 6.2 | 0.51％ |
| 6.3 | 4.38％ |
| 6.4 | 3.09％ |
| 6.5 | 3.86％ |
As shown in table 34, the LNP compositions of the present disclosure were well tolerated, with most of the treated mice maintaining original body weight or even slightly gaining weight.
Gene editing by Cas-cycle mRNA delivered to mice was measured by Next Generation Sequencing (NGS). Briefly, genomic DNA samples were first amplified with primers flanking Pcsk9 exon 1 and containing Illumina partial adaptors. The resulting amplicons were subjected to a second PCR reaction using primers containing the Illumina P5 and P7 sequences and unique index sequences (New England Biolabs). The final amplicons were pooled at equimolar concentration, loaded in a Miseq mini-kit v2300 cycle (Illumina) and run in a Miseq bench-top sequencer according to the standard Illumina procedure for amplicon-sequence. Sequencing data was then analyzed using CRISPResso2 to determine the frequency of insertions/deletions (indels) in each sample. The results of NGS are provided in table 35 as the percent indels found in the pcsk9 gene.
Table 35
| LNPID | Pcsk9 indels% |
| C12-200 | 45.11％ |
| 6.1 | 12.38％ |
| 6.2 | 20.66％ |
| 6.3 | 49.19％ |
| 6.4 | 63.27％ |
| 6.5 | 63.78％ |
As shown in table 35, the LNP compositions of the present disclosure successfully delivered Cas-clsplice mrna to the liver, as shown by the subsequent gene editing of the pcsk9 gene, with insertion loss rates equal to or better than the baseline C12-200 composition for total RNA doses of 1.5mg/kg or higher.
Serum levels of pcsk9 protein in mice were also measured 7 days after administration and the results are shown in table 36. Briefly, the mouse Pcsk9 ELISA kit (Biolegend) was used to determine Pcsk9 in each serum sample according to the manufacturer's instructions. All serum samples were assayed in triplicate and the results were expressed as a percentage decrease in Pcsk9 levels compared to Pcsk9 levels of PBS-treated mice.
Table 36
| LNPID | Pcsk9 reduction |
| C12-200 | 78.23％ |
| 6.1 | 11.47％ |
| 6.2 | 38.73％ |
| 6.3 | 75.96％ |
| 6.4 | 82.66％ |
| 6.5 | 81.09％ |
Taken together, the results of tables 35 and 36 demonstrate that Cas-clomermrna delivered by the LNP compositions of the present disclosure is effective in editing the pcsk9 gene in the liver in vivo. Gene editing efficacy was maximal at 2mg/kg total RNA, as shown by the high% indels ratio compared to baseline and low pcsk9 protein expression level compared to baseline.
Example 14-LNP of the disclosure delivers mRNA to non-human primate
The following is a non-limiting example, which demonstrates that the compositions of the present disclosure can be used to deliver mRNA to a non-human primate (NHP) in vivo, and that the delivered mRNA is well tolerated in the NHP.
In a first study, mRNA molecules comprising a sequence encoding human erythropoietin (hEPO) protein were encapsulated in lipid nanoparticles of the present disclosure comprising about 54 mole% ssPalmO-Ph-P4C2 SS-OP, about 35 mole% cholesterol, about 10 mole% DOPE, and about 1 mole% DMG-PEG2000 (referred to as bosai dada mRNA LNP in fig. 5).
LNP of the present disclosure encapsulating hEPO mRNA was administered to two monkeys, and the baseline lipid nanoparticle composition MC3 was administered to one monkey. The monkeys were administered increasing doses of LNP, with 0.25mg/kg dose administered on day 1 and 0.5mg/kg dose administered on day 21. Within one week after each infusion, blood was sampled at the following times: 0 hours, 1 hour, 4 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 168 hours. All blood draws were non-fasted and the volume of each collection was 2mL.
After administration on day 1 (0.25 mg/kg dose), the level of human erythropoietin (hEPO) in the withdrawn blood sample was determined using a standardized assay. Briefly, whole blood is processed into serum using standardized methods. hEPO was detected in serum with an ELISA kit (R & D systems) optimized for NHP. Fig. 5 shows that hEPO levels in samples from monkeys treated with LNP of the present disclosure were higher than hEPO levels in samples from monkeys treated with the baseline composition within 168 hours after infusion. As shown in fig. 5, peak EPO levels were reached about 12 hours after infusion after administration of the LNP of the present disclosure; also shown in fig. 5, EPO peak levels were up to 3 times the hEPO peak levels after administration of baseline LNP over the course of 168 hours.
The results of this study demonstrate that mRNA is delivered to NHPs in vivo as evidenced by increased hEPO levels measured in serum.
In another study, the levels of two liver enzymes in serum of rats and NHPs treated with LNP of the present disclosure were measured as a measure of potential liver toxicity. In the experiments of this study, a 5MeC-mRNA molecule comprising a sequence encoding an HA-tagged SPB was encapsulated in lipid nanoparticles of the present disclosure comprising about 54 mole% of ssPalmO-Ph-P4C2 SS-OP, about 35 mole% of cholesterol, about 10 mole% of DOPE, and about 1 mole% of DMG-PEG2000.
In the first experiment, levels of liver enzymes aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) present in serum of adult rats were assessed 4 hours, 24 hours and 7 days after administration of LNPs of the present disclosure at concentrations of 0mg/kg, 0.25mg/kg, 0.5mg/kg and 1 mg/kg. Rats (n=3) were intravenously injected with LNP or vehicle (PBS) of the present disclosure via the tail vein and blood was withdrawn at 4 hours, 24 hours and 7 days.
In a separate experiment, levels of AST and ALT present in serum of female cynomolgus monkeys were assessed 0 hours, 24 hours, and 7 days after administration of LNP of the present disclosure at concentrations of 0mg/kg, 0.1mg/kg, and 0.25 mg/kg. Monkeys (n=2-3) were injected with LNP or vehicle (PBS) of the present disclosure and blood was drawn at 0 hours, 24 hours, and 7 days.
After the serum samples were drawn, each sample was coagulated for 20 minutes and centrifuged at 13K rpm for 3 minutes to remove unwanted cells and debris. The samples were transported on wet ice and stored at-80 ℃ until analysis. Enzyme levels were determined using standardized tests.
Fig. 6 shows AST and ALT levels after 7 days in test subjects for two different experiments in this study. As shown in fig. 6, in vivo administration of LNP compositions of the present disclosure did not result in a significant increase in AST and ALT levels in serum after 7 days in both rats and NHPs.
Example 15-LNP of the disclosure for treating ornithine carbamoyltransferase (OTC) deficiency
The following are non-limiting examples demonstrating that the compositions and methods of the present disclosure can be used to treat OTC deficiency.
The following treatments were applied to 1 day old B6EiC3Sn male OTCD puppies (n=4-9):
process #1:human OTC (htoc) transposon AAV viral vector particles
Process #2: human OTC (htoc) transposon AAV viral vector particles are combined with SPB LNP.
The human OTC (httc) transposon AAV viral vector particle is an AAV viral vector particle comprising a piggyBac transposon, wherein the piggyBac transposon comprises a nucleic acid encoding a human OTC polypeptide.
SPB LNP is an ssPalmO-Ph-P4C 2-containing LNP of the present disclosure that includes mRNA encoding active SPB. SPB LNP includes ssPalmO-Ph-P4C2, DOPE, cholesterol, and DMG-PEG2000 in a molar ratio of 54:10:35:1, and a lipid to RNA ratio of 100:1 (w/w).
Inducing severe disease in OTCD models by removing residual endogenous mouse OTC using individual AAV8 viral vector particles comprising sequences encoding shRNA targeting the endogenous mouse OTC; in this model, only successful gene therapy with human OTC transgenes can rescue severe OTCD.
For both treatments, mice were administered increasing doses of the htoc transposon AAV viral vector; mice in treatment #2 group were also dosed with 0.5mg/kg SPB LNP. Figure 7 shows that 100% OTC deficient mice survival was observed in the low dose range AAV treated #2 group 47 days after administration of the treatment, in contrast to severe OTCD morbidity over the entire dose range following severe OTC disease induction with AAV treatment alone.
Further, the levels of integrated Viral Copy Number (VCN) and httcmrna in the mouse liver in treatment #2 group were measured by drop digital PCR (ddPCR) and real-time qPCR, respectively. Briefly, DNA was isolated from powdered liver tissue using dnaapidlysise kit (Ma Qielei-interior green-Nagel) and vector copies were analyzed using QX200 automated DG droplet digital PCR system (Biorad), which utilizes separate primers targeting the AAV backbone outside the 3' utr and piggyBac ITR of the htoc transgene to distinguish between episomal and integrative vector copies. Primers to the HMBS (hydroxymethyl dioxane synthase) gene were used to normalize the number of mouse cells. For mRNA quantification, mRNA was isolated from powdered liver tissue using the rneasy mini kit (qijie corporation) and after conversion to cDNA (high capacity RNA to cDNA kit, zemer feier technology corporation), gene expression was analyzed by real-time quantitative qPCR (applied biosystems quantsudio 6) using specific primer sets targeting both htoc and mActb (β -actin). Figure 8 shows AAV dose-dependent increases in both integrated VCN and htoc mRNA levels demonstrating that administration of htoc transposon AAV viral vector particles in combination with the SPB LNP of the present disclosure successfully integrated functional OTC genes into the liver.
In another study, the level of orotic acid, a biomarker of increase in OTCD, was measured in urine of treated mice. Briefly, a combination of 2E13vg/kg dose of httc transposon AAV viral vector particles with increasing doses of SPB LNP was administered to 1 day old B6EiC3Sn male pups (genotype confirmed at 21 days old only) (lowest dose group n=3, remaining groups 6-8). Orotic acid levels were measured by liquid chromatography-mass spectrometry (LC-MS/MS). Briefly, diluted urine samples were analyzed with a reverse phase UPLC column followed by MS/MS detection with Micromass Quattro in negative ion mode. Results were normalized by measuring creatinine levels in urine using hydrophilic interaction LC-MS/MS and data was collected in positive ion mode. Figure 9 shows that 100% survival of mice was observed over the full dose range of SPB LNP at 47 days post-administration treatment and orotic acid dose-dependent reduction after severe OTC disease induction.
The results presented in this example demonstrate that the LNP of the present disclosure can be used to drive high levels of human OTC polypeptide expression in vivo, thereby demonstrating that the compositions and methods of the present disclosure can be used to treat OTCD. Furthermore, the LNP of the present disclosure can be used to successfully integrate OTC genes into the liver and address disease phenotypes as measured by orotic acid.
Sequence listing
<110> Bosaida treatment company (Poseida Therapeutics, inc.)
<120> compositions and methods for delivering nucleic acids
<130> POTH-066/001WO 325002-2570
<150> US 63/152,517
<151> 2021-02-23
<150> US 63/164,174
<151> 2021-03-22
<150> US 63/197,946
<151> 2021-06-07
<150> US 63/156,649
<151> 2021-03-04
<160> 65
<170> patent In version 3.5
<210> 1
<211> 4596
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
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gcaccgagac aggctttgcc ctgctcggag gacacccctg ctttctgaca acccaggaca 1320
tccacctggg cgtgaacgag agcctgaccg atacagccag agtgctgtcc tctatggccg 1380
atgccgtgct ggctagagtg tataagcaga gcgacctgga caccctggct aaagaggcca 1440
gcattcccat catcaacggc ctgtccgacc tgtatcaccc catccagatc ctggccgact 1500
acctgacact gcaagagcac tacagcagcc tgaagggact gaccctgtct tggatcggcg 1560
acggcaacaa catcctgcac agcattatga tgagcgccgc caagttcgga atgcacctcc 1620
aggccgctac acccaagggc tatgaacctg atgccagcgt gacaaagctg gccgagcagt 1680
acgccaaaga gaacggcaca aagctgctgc tgaccaacga tcccctggaa gctgctcacg 1740
gcggcaatgt gctgatcacc gatacctgga tcagcatggg ccaagaggaa gagaagaaga 1800
agcggctgca agccttccag ggctaccaag tgaccatgaa gacagccaag gtggccgcca 1860
gcgattggac ctttctgcac tgcctgcctc ggaagcctga agaggtggac gacgaggtgt 1920
tctacagccc tagaagcctg gtgttccccg aggccgagaa cagaaagtgg accatcatgg 1980
ctgtgatggt gtctctgctg accgactact cccctcagct ccagaagcct aagttctaat 2040
gaagatctca tatgccttta attaaacact agttctatag tgtcacctaa attcccttta 2100
gtgagggtta atggccgtag gccgccagaa ttgggtccag acatgataag atacattgat 2160
gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt 2220
gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat 2280
tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggttttttc ggactctagg 2340
acctgcgcat gcgcttggcg taatcatggt catagctgtt tcctgttttc cccgtatccc 2400
cccaggtgtc tgcaggctca aagagcagcg agaagcgttc agaggaaagc gatcccgtgc 2460
caccttcccc gtgcccgggc tgtccccgca cgctgccggc tcggggatgc ggggggagcg 2520
ccggaccgga gcggagcccc gggcggctcg ctgctgcccc ctagcggggg agggacgtaa 2580
ttacatccct gggggctttg ggggggggct gtccctctca ccgcggtgga gctccagctt 2640
ttgttcgaat tggggccccc cctcgagggt atcgatgata tctataacaa gaaaatatat 2700
atataataag ttatcacgta agtagaacat gaaataacaa tataattatc gtatgagtta 2760
aatcttaaaa gtcacgtaaa agataatcat gcgtcatttt gactcacgcg gtcgttatag 2820
ttcaaaatca gtgacactta ccgcattgac aagcacgcct cacgggagct ccaagcggcg 2880
actgagatgt cctaaatgca cagcgacgga ttcgcgctat ttagaaagag agagcaatat 2940
ttcaagaatg catgcgtcaa ttttacgcag actatctttc tagggttaat ctagctagcc 3000
ttaagggcgc tttcctggac tacttcagac gaacttcgta gggcgcataa gtctcgacca 3060
cgcaatgacg cagcgatgct tgaaaaaaac accgctttca aggcggctgg acgaagaccg 3120
caagacaccc tccacctcac ctagcctgta tctctgcaat agcctaatta cttcggaatc 3180
tcctgtcgta attccttaga taaacggcaa ttaggtgaca ctctaaatct gtgtggaacc 3240
ggcttccaaa cactctacca cctctattag tgacacagag agaatccttg agtggcttct 3300
aggtatattg aacaacttca tcacgaattg agcagtgatc atggttctac gtatcaacca 3360
atattaacca ctgtgctctg tagcattgct aaatcgggct gtctgtttca ccatagatcg 3420
tgaggccatg cccacgggca ttagaactta gcctgtttag cgataatccc aacaatgagc 3480
tgggatatga cgagaagtat ttagcaacct tttcgtgatc ggctacgtaa aacctcatat 3540
tacggcatgt acctgatcat tgacctcagg ccatacgcat gtgggagaat agagggaata 3600
gcacgatata ctgcctccct tatcttcctc aggttgaggc gcaatgatta tcactgctac 3660
gggcacagtg ttactatcgg gatcagggac tgtgtctgta cccggttcta ccacgccgta 3720
gctcccacat tgtcgccgta ctggtagtaa cctaccagta ctacagggac aggtcagatt 3780
attcttagaa tgcctgtgta tagtttagag ccatagattg ccgagaaacg aagagagtgt 3840
atcacggcga tttacggagt caagatagca acacatagga gtaccaaatc aataagtgta 3900
gtagagttac aagtcgctcc cgagaatcag gtgatacttc tcaatcgtcg gaaacctgta 3960
acgattagac cgcgtattag ttccatctaa tgatattgcc aagtactaaa cctcaatcac 4020
ataacaacgc atcaggctag cgacatcata ggagtcctgc aacatacgca ggcgtccagc 4080
gtcataaacg caggtgatga gtgccttatg caatctgggg cacaccaata cgagaatacc 4140
acgtcgttag ggccagcctg cgcttggtac gtgatcgagg agtatcgtct tgtgcacaca 4200
aacaaaatag actgggtccc aagcgcactt agacaaggca ttgtactact caagtgttgg 4260
atgtgaggaa tgatacaatc gaaccccgtc gttcggtcaa caatttggct agagctgtgt 4320
tgatacacag cggacgggag ttacggggac agtcctccga gctggccagc aagatgcagc 4380
tgacgcctgc accggaagta atccggaggc cggccaggcc tcctgcgagg gggcgcctcg 4440
agaccttgcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 4500
gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 4560
cctcagtgag cgagcgagcg cgcagctgcc tgcagg 4596
<210> 2
<211> 4790
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 2
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc aaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgag tactcctagg cgcgccccta aaatgggcaa acattgcaag cagcaaacag 720
caaacacaca gccctccctg cctgctgacc ttggagctgg ggcagaggtc agagacctct 780
ctgggcccat gccacctcca acatccactc gaccccttgg aatttcggtg gagaggagca 840
gaggttgtcc tggcgtggtt taggtagtgt gagaggggaa tgactccttt cggtaagtgc 900
agtggaagct gtacactgcc caggcaaagc gtccgggcag cgtaggcggg cgactcagat 960
cccagccagt ggacttagcc cctgtttgct cctccgataa ctggggtgac cttggttaat 1020
attcaccagc agcctccccc gttgcccctc tggatccact gcttaaatac ggacgaggac 1080
agggccctgt ctcctcagct tcaggcacca ccactgacct gggacagtga atcaagctta 1140
ttggacgtcg cttagcggta ccgccaccat gctgttcaac ctgcgcatcc tgctgaacaa 1200
cgccgccttc agaaacggcc acaacttcat ggttcgaaac ttcagatgcg gccagcctct 1260
ccagaacaag gtgcagctga aaggcaggga cctgctgacc ctgaagaact tcaccggcga 1320
agagatcaag tacatgctgt ggctgtccgc cgacctgaag ttcagaatca agcagaaggg 1380
cgagtacctg cctctgctcc agggaaagtc tctgggcatg atcttcgaga agcggagcac 1440
cagaaccaga ctgagcaccg agacaggctt tgccctgctc ggaggacacc cctgctttct 1500
gacaacccag gacatccacc tgggcgtgaa cgagagcctg accgatacag ccagagtgct 1560
gtcctctatg gccgatgccg tgctggctag agtgtataag cagagcgacc tggacaccct 1620
ggctaaagag gccagcattc ccatcatcaa cggcctgtcc gacctgtatc accccatcca 1680
gatcctggcc gactacctga cactgcaaga gcactacagc agcctgaagg gactgaccct 1740
gtcttggatc ggcgacggca acaacatcct gcacagcatt atgatgagcg ccgccaagtt 1800
cggaatgcac ctccaggccg ctacacccaa gggctatgaa cctgatgcca gcgtgacaaa 1860
gctggccgag cagtacgcca aagagaacgg cacaaagctg ctgctgacca acgatcccct 1920
ggaagctgct cacggcggca atgtgctgat caccgatacc tggatcagca tgggccaaga 1980
ggaagagaag aagaagcggc tgcaagcctt ccagggctac caagtgacca tgaagacagc 2040
caaggtggcc gccagcgatt ggacctttct gcactgcctg cctcggaagc ctgaagaggt 2100
ggacgacgag gtgttctaca gccctagaag cctggtgttc cccgaggccg agaacagaaa 2160
gtggaccatc atggctgtga tggtgtctct gctgaccgac tactcccctc agctccagaa 2220
gcctaagttc taatgaagat ctcatatgcc tttaattaaa cactagttct atagtgtcac 2280
ctaaattccc tttagtgagg gttaatggcc gtaggccgcc agaattgggt ccagacatga 2340
taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 2400
tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 2460
ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt 2520
tttcggactc taggacctgc gcatgcgctt ggcgtaatca tggtcatagc tgtttcctgt 2580
tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga 2640
aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg 2700
atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg 2760
ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct ctcaccgcgg 2820
tggagctcca gcttttgttc gaattggggc cccccctcga gggtatcgat gatatctata 2880
acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat 2940
tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca 3000
cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg 3060
agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa 3120
agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt 3180
taatctagct agccttaagg gcgctttcct ggactacttc agacgaactt cgtagggcgc 3240
ataagtctcg accacgcaat gacgcagcga tgcttgaaaa aaaccccgct ttcaaggcgg 3300
ctggacgaag accgcaagac accctccacc tcacctagcc tgtatctctg caatagccta 3360
attacttcgg aatctcctgt cgtaattcct tagataaacg gcaattaggt gacactctaa 3420
atctgtgtgg aaccggcttc caaacactct accacctcta ttagtgacac agagagaatc 3480
cttgagtggc ttctaggtat attgaacaac ttcatcacga attgagcagt gatcatggtt 3540
ctacgtatca accaatatta accactgtgc tctgtagcat tgctaaatcg ggctgtctgt 3600
ttcaccatag atcgtgaggc catgcccacg ggcattagaa cttagcctgt ttagcgataa 3660
tcccaacaat gagctgggat atgacgagaa gtatttagca accttttcgt gatcggctac 3720
gtaaaacctc atattacggc atgtacctga tcattgacct caggccatac gcatgtggga 3780
gaatagaggg aatagcacga tatactgcct cccttatctt cctcaggttg aggcgcaatg 3840
attatcactg ctacgggcac agtgttacta tcgggatcag ggactgtgtc tgtacccggt 3900
tctaccacgc cgtagctccc acattgtcgc cgtactggta gtaacctacc agtactacag 3960
ggacaggtca gattattctt agaatgcctg tgtatagttt agagccatag attgccgaga 4020
aacgaagaga gtgtatcacg gcgatttacg gagtcaagat agcaacacat aggagtacca 4080
aatcaataag tgtagtagag ttacaagtcg ctcccgagaa tcaggtgata cttctcaatc 4140
gtcggaaacc tgtaacgatt agaccgcgta ttagttccat ctaatgatat tgccaagtac 4200
taaacctcaa tcacataaca acgcatcagg ctagcgacat cataggagtc ctgcaacata 4260
cgcaggcgtc cagcgtcata aacgcaggtg atgagtgcct tatgcaatct ggggcacacc 4320
aatacgagaa taccacgtcg ttagggccag cctgcgcttg gtacgtgatc gaggagtatc 4380
gtcttgtgca cacaaacaaa atagactggg tcccaagcgc acttagacaa ggcattgtac 4440
tactcaagtg ttggatgtga ggaatgatac aatcgaaccc cgtcgttcgg tcaacaattt 4500
ggctagagct gtgttgatac acagcggacg ggagttacgg ggacagtcct ccgagctggc 4560
cagcaagatg cagctgacgc ctgcaccgga agtaatccgg aggccggcca ggcctcctgc 4620
gagggggcgc ctcgagacct tgcggccgca ggaaccccta gtgatggagt tggccactcc 4680
ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg 4740
ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg 4790
<210> 3
<211> 4700
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 3
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc aaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgag tactcctagg agctagcagg ttaattttta aaaagcagtc aaaagtccaa 720
gtggcccttg gcagcattta ctctctctgt ttgctctggt taataatctc aggagcacaa 780
acattccaga tccaggttaa tttttaaaaa gcagtcaaaa gtccaagtgg cccttggcag 840
catttactct ctctgtttgc tctggttaat aatctcagga gcacaaacat tccagatccg 900
gcgcgccagg gctggaagct acctttgaca tcatttcctc tgcgaatgca tgtataattt 960
ctacagaacc tattagaaag gatcacccag cctctgcttt tgtacaactt tcccttaaaa 1020
aactgccaat tccactgctg tttggcccaa tagtgagaac tttttcctgc tgcctcttgg 1080
tgcttttgcc tatggcccct attctgcctg ctgaagacac tcttgccagc atggacttaa 1140
acccctccag ctctgacaat cctctttctc ttttgtttta catgaagggt ctggcagcca 1200
aagcaatcac tcaaagttca aaccttatca ttttttgctt tgttcctctt ggccttggtt 1260
ttgtacatca gctttgaaaa taccatccca gggttaatgc tggggttaat ttataactaa 1320
gagtgctcta gttttgcaat acaggacatg ctataaaaat ggaaagatgt tgctttctga 1380
gagactgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt acaagacagg 1440
tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat 1500
aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gtccagtggc 1560
aaagcttatt ggacgtcgct tagcggtacc gccaccatgc tgttcaacct gcgcatcctg 1620
ctgaacaacg ccgccttcag aaacggccac aacttcatgg ttcgaaactt cagatgcggc 1680
cagcctctcc agaacaaggt gcagctgaaa ggcagggacc tgctgaccct gaagaacttc 1740
accggcgaag agatcaagta catgctgtgg ctgtccgccg acctgaagtt cagaatcaag 1800
cagaagggcg agtacctgcc tctgctccag ggaaagtctc tgggcatgat cttcgagaag 1860
cggagcacca gaaccagact gagcaccgag acaggctttg ccctgctcgg aggacacccc 1920
tgctttctga caacccagga catccacctg ggcgtgaacg agagcctgac cgatacagcc 1980
agagtgctgt cctctatggc cgatgccgtg ctggctagag tgtataagca gagcgacctg 2040
gacaccctgg ctaaagaggc cagcattccc atcatcaacg gcctgtccga cctgtatcac 2100
cccatccaga tcctggccga ctacctgaca ctgcaagagc actacagcag cctgaaggga 2160
ctgaccctgt cttggatcgg cgacggcaac aacatcctgc acagcattat gatgagcgcc 2220
gccaagttcg gaatgcacct ccaggccgct acacccaagg gctatgaacc tgatgccagc 2280
gtgacaaagc tggccgagca gtacgccaaa gagaacggca caaagctgct gctgaccaac 2340
gatcccctgg aagctgctca cggcggcaat gtgctgatca ccgatacctg gatcagcatg 2400
ggccaagagg aagagaagaa gaagcggctg caagccttcc agggctacca agtgaccatg 2460
aagacagcca aggtggccgc cagcgattgg acctttctgc actgcctgcc tcggaagcct 2520
gaagaggtgg acgacgaggt gttctacagc cctagaagcc tggtgttccc cgaggccgag 2580
aacagaaagt ggaccatcat ggctgtgatg gtgtctctgc tgaccgacta ctcccctcag 2640
ctccagaagc ctaagttcta atgaagatct catatgcctt taattaaaca ctagttctat 2700
agtgtcacct aaattccctt tagtgagggt taatggccgt aggccgccag aattgggtcc 2760
agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 2820
atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2880
taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2940
ggaggttttt tcggactcta ggacctgcgc atgcgcttgg cgtaatcatg gtcatagctg 3000
tttcctgttt tccccgtatc cccccaggtg tctgcaggct caaagagcag cgagaagcgt 3060
tcagaggaaa gcgatcccgt gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg 3120
gctcggggat gcggggggag cgccggaccg gagcggagcc ccgggcggct cgctgctgcc 3180
ccctagcggg ggagggacgt aattacatcc ctgggggctt tggggggggg ctgtccctct 3240
caccgcggtg gagctccagc ttttgttcga attggggccc cccctcgagg gtatcgatga 3300
tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac 3360
aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt 3420
ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc 3480
ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct 3540
atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt 3600
tctagggtta atctagctag ccttaagggc gctttcctgt cgacggaggc atgtacctga 3660
tcattgacct caggccatac gcatgtggga gaatagaggg aatagcacga tatactgcct 3720
cccttatctt cctcaggttg aggcgcaatg attatcactg ctacgggcac agtgttacta 3780
tcgggatcag ggactgtgtc tgtacccggt tctaccacgc cgtagctccc acattgtcgc 3840
cgtactggta gtaacctacc agtactacag ggacaggtca gattattctt agaatgcctg 3900
tgtatagttt agagccatag attgccgaga aacgaagaga gtgtatcacg gcgatttacg 3960
gagtcaagat agcaacacat aggagtacca aatcaataag tgtagtagag ttacaagtcg 4020
ctcccgagaa tcaggtgata cttctcaatc gtcggaaacc tgtaacgatt agaccgcgta 4080
ttagttccat ctaatgatat tgccaagtac taaacctcaa tcacataaca acgcatcagg 4140
ctagcgacat cataggagtc ctgcaacata cgcaggcgtc cagcgtcata aacgcaggtg 4200
atgagtgcct tatgcaatct ggggcacacc aatacgagaa taccacgtcg ttagggccag 4260
cctgcgcttg gtacgtgatc gaggagtatc gtcttgtgca cacaaacaaa atagactggg 4320
tcccaagcgc acttagacaa ggcattgtac tactcaagtg ttggatgtga ggaatgatac 4380
aatcgaaccc cgtcgttcgg tcaacaattt ggctagagct gtgttgatac acagcggacg 4440
ggagttacgg ggacagtcct ccgagctggc cagcaagatg cagctgacgc ctgcaccgga 4500
agtaatccgg aggccggcca ggcctcctgc gagggggcgc ctcgagacct tgcggccgca 4560
ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 4620
cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 4680
agcgcgcagc tgcctgcagg 4700
<210> 4
<211> 4531
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 4
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc aaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgag tactcctagg cgcgtgtttg ctgcttgcaa tgtttgccca ttttagggtg 720
gacacaggac gctgtggttt ctgagccagg gggcgactca gatcccagcc agtggactta 780
gcccctgttt gctcctccga taactggggt gaccttggtt aatattcacc agcagcctcc 840
cccgttgccc ctctggatcc actgcttaaa tacggacgag gacagggccc tgtctcctca 900
gcttcaggca ccaccactga cctgggacag tgaatcgcaa agcttattgg acgtcgctta 960
gcggtaccgc caccatgctg ttcaacctgc gcatcctgct gaacaacgcc gccttcagaa 1020
acggccacaa cttcatggtt cgaaacttca gatgcggcca gcctctccag aacaaggtgc 1080
agctgaaagg cagggacctg ctgaccctga agaacttcac cggcgaagag atcaagtaca 1140
tgctgtggct gtccgccgac ctgaagttca gaatcaagca gaagggcgag tacctgcctc 1200
tgctccaggg aaagtctctg ggcatgatct tcgagaagcg gagcaccaga accagactga 1260
gcaccgagac aggctttgcc ctgctcggag gacacccctg ctttctgaca acccaggaca 1320
tccacctggg cgtgaacgag agcctgaccg atacagccag agtgctgtcc tctatggccg 1380
atgccgtgct ggctagagtg tataagcaga gcgacctgga caccctggct aaagaggcca 1440
gcattcccat catcaacggc ctgtccgacc tgtatcaccc catccagatc ctggccgact 1500
acctgacact gcaagagcac tacagcagcc tgaagggact gaccctgtct tggatcggcg 1560
acggcaacaa catcctgcac agcattatga tgagcgccgc caagttcgga atgcacctcc 1620
aggccgctac acccaagggc tatgaacctg atgccagcgt gacaaagctg gccgagcagt 1680
acgccaaaga gaacggcaca aagctgctgc tgaccaacga tcccctggaa gctgctcacg 1740
gcggcaatgt gctgatcacc gatacctgga tcagcatggg ccaagaggaa gagaagaaga 1800
agcggctgca agccttccag ggctaccaag tgaccatgaa gacagccaag gtggccgcca 1860
gcgattggac ctttctgcac tgcctgcctc ggaagcctga agaggtggac gacgaggtgt 1920
tctacagccc tagaagcctg gtgttccccg aggccgagaa cagaaagtgg accatcatgg 1980
ctgtgatggt gtctctgctg accgactact cccctcagct ccagaagcct aagttcggat 2040
ccggcgaagg cagaggctca ctgcttactt gtggcgacgt ggaggagaac cccggaccta 2100
tggtttccaa gggcgaagaa ctgtttaccg gcgtggtgcc catcctggtg gaactggatg 2160
gcgacgttaa cggacacaag ttcagcgtca gcggagaagg cgaaggcgac gccacatacg 2220
gaaagctgac actgaagttt atctgcacca ccggcaagct gcccgtgcct tggcctacac 2280
tggtcaccac actgacatac ggcgtgcagt gcttcagcag ataccccgac cacatgaagc 2340
agcacgattt cttcaagagc gccatgcctg agggctacgt gcaagagcgg accatcttct 2400
tcaaggacga cgggaactac aagaccagag ccgaagtgaa gttcgagggc gacaccctcg 2460
tgaaccggat cgagctgaag ggcatcgact tcaaagagga cggaaacatc ctgggccaca 2520
aacttgagta caactacaac agccacaacg tctacatcat ggccgacaag cagaaaaacg 2580
gcatcaaagt gaacttcaag atccggcaca acatcgagga cggctctgtg cagctggctg 2640
accactacca gcagaacaca cccatcggag atggccctgt gctgctgccc gataaccact 2700
acctgagcac acagagcgcc ctgagcaagg accccaacga gaagagggat cacatggtgc 2760
tgctggaatt tgtgaccgct gccggcatca ccctcggcat ggatgaactg tacaagggct 2820
ccggagaagg acggggaagc ctgcttacat gcggagatgt ggaggagaat cctggtccca 2880
tggtctttac cctggaagat ttcgtcggcg actggcggca gacagccggc tataatctgg 2940
accaggtgct ggaacaaggc ggagtgtcca gcctgttcca gaatctggga gtgtccgtga 3000
cacccatcca gcggattgtg ctgtctggcg agaacggcct gaagatcgac atccacgtga 3060
tcatccctta cgagggcctg agcggcgatc agatgggaca gatcgagaag attttcaagg 3120
tggtgtaccc cgtggacgac caccacttca aagtgatcct gcactacggc accctggtca 3180
tcgatggcgt gacccctaac atgatcgact acttcggcag accctacgag ggaatcgccg 3240
tgttcgacgg caagaaaatc accgtgaccg gcacactgtg gaacgggaac aagatcatcg 3300
acgagcggct gatcaacccc gatggcagcc tgctgttcag agtgaccatt aacggcgtga 3360
caggctggcg gctgtgcgaa aggattctgg cctgatgatc tagagatctc atatgccttt 3420
aattaaacac tagttctata gtgtcaccta aattcccttt agtgagggtt aatggccgta 3480
ggccgccaga attgggtcca gacatgataa gatacattga tgagtttgga caaaccacaa 3540
ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg 3600
taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 3660
aggttcaggg ggaggtgtgg gaggtttttt cggactctag gacctgcgca tgcgcttggc 3720
gtaatcatgg tcatagctgt ttcctgtttt ccccgtatcc ccccaggtgt ctgcaggctc 3780
aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg 3840
ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc 3900
cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt 3960
gggggggggc tgtccctctc accgcggtgg agctccagct tttgttcgaa ttggggcccc 4020
ccctcgaggg tatcgatgat atctataaca agaaaatata tatataataa gttatcacgt 4080
aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa 4140
aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt 4200
accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc 4260
acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca 4320
attttacgca gactatcttt ctagggttaa tctagctagc cttaagggcg cctcgagacc 4380
ttgcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4440
ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 4500
gtgagcgagc gagcgcgcag ctgcctgcag g 4531
<210> 5
<211> 4725
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 5
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc aaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgag tactcctagg cgcgccccta aaatgggcaa acattgcaag cagcaaacag 720
caaacacaca gccctccctg cctgctgacc ttggagctgg ggcagaggtc agagacctct 780
ctgggcccat gccacctcca acatccactc gaccccttgg aatttcggtg gagaggagca 840
gaggttgtcc tggcgtggtt taggtagtgt gagaggggaa tgactccttt cggtaagtgc 900
agtggaagct gtacactgcc caggcaaagc gtccgggcag cgtaggcggg cgactcagat 960
cccagccagt ggacttagcc cctgtttgct cctccgataa ctggggtgac cttggttaat 1020
attcaccagc agcctccccc gttgcccctc tggatccact gcttaaatac ggacgaggac 1080
agggccctgt ctcctcagct tcaggcacca ccactgacct gggacagtga atcaagctta 1140
ttggacgtcg cttagcggta ccgccaccat gctgttcaac ctgcgcatcc tgctgaacaa 1200
cgccgccttc agaaacggcc acaacttcat ggttcgaaac ttcagatgcg gccagcctct 1260
ccagaacaag gtgcagctga aaggcaggga cctgctgacc ctgaagaact tcaccggcga 1320
agagatcaag tacatgctgt ggctgtccgc cgacctgaag ttcagaatca agcagaaggg 1380
cgagtacctg cctctgctcc agggaaagtc tctgggcatg atcttcgaga agcggagcac 1440
cagaaccaga ctgagcaccg agacaggctt tgccctgctc ggaggacacc cctgctttct 1500
gacaacccag gacatccacc tgggcgtgaa cgagagcctg accgatacag ccagagtgct 1560
gtcctctatg gccgatgccg tgctggctag agtgtataag cagagcgacc tggacaccct 1620
ggctaaagag gccagcattc ccatcatcaa cggcctgtcc gacctgtatc accccatcca 1680
gatcctggcc gactacctga cactgcaaga gcactacagc agcctgaagg gactgaccct 1740
gtcttggatc ggcgacggca acaacatcct gcacagcatt atgatgagcg ccgccaagtt 1800
cggaatgcac ctccaggccg ctacacccaa gggctatgaa cctgatgcca gcgtgacaaa 1860
gctggccgag cagtacgcca aagagaacgg cacaaagctg ctgctgacca acgatcccct 1920
ggaagctgct cacggcggca atgtgctgat caccgatacc tggatcagca tgggccaaga 1980
ggaagagaag aagaagcggc tgcaagcctt ccagggctac caagtgacca tgaagacagc 2040
caaggtggcc gccagcgatt ggacctttct gcactgcctg cctcggaagc ctgaagaggt 2100
ggacgacgag gtgttctaca gccctagaag cctggtgttc cccgaggccg agaacagaaa 2160
gtggaccatc atggctgtga tggtgtctct gctgaccgac tactcccctc agctccagaa 2220
gcctaagttc ggatccggcg aaggcagagg ctcactgctt acttgtggcg acgtggagga 2280
gaaccccgga cctatggttt ccaagggcga agaactgttt accggcgtgg tgcccatcct 2340
ggtggaactg gatggcgacg ttaacggaca caagttcagc gtcagcggag aaggcgaagg 2400
cgacgccaca tacggaaagc tgacactgaa gtttatctgc accaccggca agctgcccgt 2460
gccttggcct acactggtca ccacactgac atacggcgtg cagtgcttca gcagataccc 2520
cgaccacatg aagcagcacg atttcttcaa gagcgccatg cctgagggct acgtgcaaga 2580
gcggaccatc ttcttcaagg acgacgggaa ctacaagacc agagccgaag tgaagttcga 2640
gggcgacacc ctcgtgaacc ggatcgagct gaagggcatc gacttcaaag aggacggaaa 2700
catcctgggc cacaaacttg agtacaacta caacagccac aacgtctaca tcatggccga 2760
caagcagaaa aacggcatca aagtgaactt caagatccgg cacaacatcg aggacggctc 2820
tgtgcagctg gctgaccact accagcagaa cacacccatc ggagatggcc ctgtgctgct 2880
gcccgataac cactacctga gcacacagag cgccctgagc aaggacccca acgagaagag 2940
ggatcacatg gtgctgctgg aatttgtgac cgctgccggc atcaccctcg gcatggatga 3000
actgtacaag ggctccggag aaggacgggg aagcctgctt acatgcggag atgtggagga 3060
gaatcctggt cccatggtct ttaccctgga agatttcgtc ggcgactggc ggcagacagc 3120
cggctataat ctggaccagg tgctggaaca aggcggagtg tccagcctgt tccagaatct 3180
gggagtgtcc gtgacaccca tccagcggat tgtgctgtct ggcgagaacg gcctgaagat 3240
cgacatccac gtgatcatcc cttacgaggg cctgagcggc gatcagatgg gacagatcga 3300
gaagattttc aaggtggtgt accccgtgga cgaccaccac ttcaaagtga tcctgcacta 3360
cggcaccctg gtcatcgatg gcgtgacccc taacatgatc gactacttcg gcagacccta 3420
cgagggaatc gccgtgttcg acggcaagaa aatcaccgtg accggcacac tgtggaacgg 3480
gaacaagatc atcgacgagc ggctgatcaa ccccgatggc agcctgctgt tcagagtgac 3540
cattaacggc gtgacaggct ggcggctgtg cgaaaggatt ctggcctgat gatctagaga 3600
tctcatatgc ctttaattaa acactagttc tatagtgtca cctaaattcc ctttagtgag 3660
ggttaatggc cgtaggccgc cagaattggg tccagacatg ataagataca ttgatgagtt 3720
tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc 3780
tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat 3840
tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttcggact ctaggacctg 3900
cgcatgcgct tggcgtaatc atggtcatag ctgtttcctg ttttccccgt atccccccag 3960
gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct 4020
tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga 4080
ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca 4140
tccctggggg ctttgggggg gggctgtccc tctcaccgcg gtggagctcc agcttttgtt 4200
cgaattgggg ccccccctcg agggtatcga tgatatctat aacaagaaaa tatatatata 4260
ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg agttaaatct 4320
taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt tatagttcaa 4380
aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag cggcgactga 4440
gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc aatatttcaa 4500
gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaatctagc tagccttaag 4560
ggcgcctcga gaccttgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 4620
tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 4680
cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcagg 4725
<210> 6
<211> 4363
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 6
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc aaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgag tactcctagg agctagcagg ttaattttta aaaagcagtc aaaagtccaa 720
gtggcccttg gcagcattta ctctctctgt ttgctctggt taataatctc aggagcacaa 780
acattccaga tccaggttaa tttttaaaaa gcagtcaaaa gtccaagtgg cccttggcag 840
catttactct ctctgtttgc tctggttaat aatctcagga gcacaaacat tccagatccg 900
gcgcgccagg gctggaagct acctttgaca tcatttcctc tgcgaatgca tgtataattt 960
ctacagaacc tattagaaag gatcacccag cctctgcttt tgtacaactt tcccttaaaa 1020
aactgccaat tccactgctg tttggcccaa tagtgagaac tttttcctgc tgcctcttgg 1080
tgcttttgcc tatggcccct attctgcctg ctgaagacac tcttgccagc atggacttaa 1140
acccctccag ctctgacaat cctctttctc ttttgtttta catgaagggt ctggcagcca 1200
aagcaatcac tcaaagttca aaccttatca ttttttgctt tgttcctctt ggccttggtt 1260
ttgtacatca gctttgaaaa taccatccca gggttaatgc tggggttaat ttataactaa 1320
gagtgctcta gttttgcaat acaggacatg ctataaaaat ggaaagatgt tgctttctga 1380
gagactgcag aagttggtcg tgaggcactg ggcaggtaag tatcaaggtt acaagacagg 1440
tttaaggaga ccaatagaaa ctgggcttgt cgagacagag aagactcttg cgtttctgat 1500
aggcacctat tggtcttact gacatccact ttgcctttct ctccacaggt gaagcttatt 1560
ggacgtcgct tagcggtacc gccaccatgc tgttcaacct gcgcatcctg ctgaacaacg 1620
ccgccttcag aaacggccac aacttcatgg ttcgaaactt cagatgcggc cagcctctcc 1680
agaacaaggt gcagctgaaa ggcagggacc tgctgaccct gaagaacttc accggcgaag 1740
agatcaagta catgctgtgg ctgtccgccg acctgaagtt cagaatcaag cagaagggcg 1800
agtacctgcc tctgctccag ggaaagtctc tgggcatgat cttcgagaag cggagcacca 1860
gaaccagact gagcaccgag acaggctttg ccctgctcgg aggacacccc tgctttctga 1920
caacccagga catccacctg ggcgtgaacg agagcctgac cgatacagcc agagtgctgt 1980
cctctatggc cgatgccgtg ctggctagag tgtataagca gagcgacctg gacaccctgg 2040
ctaaagaggc cagcattccc atcatcaacg gcctgtccga cctgtatcac cccatccaga 2100
tcctggccga ctacctgaca ctgcaagagc actacagcag cctgaaggga ctgaccctgt 2160
cttggatcgg cgacggcaac aacatcctgc acagcattat gatgagcgcc gccaagttcg 2220
gaatgcacct ccaggccgct acacccaagg gctatgaacc tgatgccagc gtgacaaagc 2280
tggccgagca gtacgccaaa gagaacggca caaagctgct gctgaccaac gatcccctgg 2340
aagctgctca cggcggcaat gtgctgatca ccgatacctg gatcagcatg ggccaagagg 2400
aagagaagaa gaagcggctg caagccttcc agggctacca agtgaccatg aagacagcca 2460
aggtggccgc cagcgattgg acctttctgc actgcctgcc tcggaagcct gaagaggtgg 2520
acgacgaggt gttctacagc cctagaagcc tggtgttccc cgaggccgag aacagaaagt 2580
ggaccatcat ggctgtgatg gtgtctctgc tgaccgacta ctcccctcag ctccagaagc 2640
ctaagttcgg atccggagaa ggacggggaa gcctgcttac atgcggagat gtggaggaga 2700
atcctggtcc catggtcttt accctggaag atttcgtcgg cgactggcgg cagacagccg 2760
gctataatct ggaccaggtg ctggaacaag gcggagtgtc cagcctgttc cagaatctgg 2820
gagtgtccgt gacacccatc cagcggattg tgctgtctgg cgagaacggc ctgaagatcg 2880
acatccacgt gatcatccct tacgagggcc tgagcggcga tcagatggga cagatcgaga 2940
agattttcaa ggtggtgtac cccgtggacg accaccactt caaagtgatc ctgcactacg 3000
gcaccctggt catcgatggc gtgaccccta acatgatcga ctacttcggc agaccctacg 3060
agggaatcgc cgtgttcgac ggcaagaaaa tcaccgtgac cggcacactg tggaacggga 3120
acaagatcat cgacgagcgg ctgatcaacc ccgatggcag cctgctgttc agagtgacca 3180
ttaacggcgt gacaggctgg cggctgtgcg aaaggattct ggcctgatga tctagagatc 3240
tcatatgcct ttaattaaac actagttcta tagtgtcacc taaattccct ttagtgaggg 3300
ttaatggccg taggccgcca gaattgggtc cagacatgat aagatacatt gatgagtttg 3360
gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 3420
ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 3480
attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttcggactct aggacctgcg 3540
catgcgcttg gcgtaatcat ggtcatagct gtttcctgtt ttccccgtat ccccccaggt 3600
gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc 3660
cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc 3720
ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc 3780
cctgggggct ttgggggggg gctgtccctc tcaccgcggt ggagctccag cttttgttcg 3840
aattggggcc ccccctcgag ggtatcgatg atatctataa caagaaaata tatatataat 3900
aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta 3960
aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa 4020
tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga 4080
tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga 4140
atgcatgcgt caattttacg cagactatct ttctagggtt aatctagcta gccttaaggg 4200
cgcctcgaga ccttgcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg 4260
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc 4320
cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc agg 4363
<210> 7
<211> 4651
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 7
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tatcgatggc gcgcctccag atatcataca ctcgagcaac 180
tttgtataga aaagttggcg cgtgtttgct gcttgcaatg tttgcccatt ttagggtgga 240
cacaggacgc tgtggtttct gagccagggg gcgactcaga tcccagccag tggacttagc 300
ccctgtttgc tcctccgata actggggtga ccttggttaa tattcaccag cagcctcccc 360
cgttgcccct ctggatccac tgcttaaata cggacgagga cagggccctg tctcctcagc 420
ttcaggcacc accactgacc tgggacagtg aatcgcaaga attcacgcgt caattgctcg 480
aggccaccat ggctcccaag aagaagcgga aagttggcgg cggaggcagc agcctggatg 540
atgagcatat tctgagcgcc ctgctgcaga gcgacgatga actcgtgggc gaagatagcg 600
acagcgaggt gtccgatcac gtgtccgagg atgacgtgca gtccgatacc gaggaagcct 660
tcatcgacga ggtgcacgaa gtgcagccta caagcagcgg cagcgagatc ctggacgagc 720
agaatgtgat cgagcagcca ggatctagcc tggccagcaa cagaatcctg acactgcccc 780
agagaaccat ccggggcaag aacaagcact gctggtccac cagcaagagc accagacggt 840
ctagagtgtc tgccctgaac atcgtgcgaa gccagagggg ccctaccaga atgtgccgga 900
acatctacga ccctctgctg tgcttcaagc tgttcttcac cgacgagatc atctccgaga 960
tcgtgaagtg gaccaacgcc gagatcagcc tgaagcggag agaatccatg accagcgcca 1020
ccttcagaga caccaacgag gacgagatct acgccttctt cggcatcctg gtcatgacag 1080
ccgtgcggaa ggacaaccac atgagcaccg acgacctgtt cgaccgcagc ctgtctatgg 1140
tgtacgtgtc cgtgatgagc cgggacagat tcgacttcct gatccggtgc ctgcggatgg 1200
acgacaagtc catcagaccc acactgcgcg agaacgacgt gttcacacct gtgcggaaga 1260
tctgggacct gttcatccac cagtgcatcc agaactacac ccctggcgct cacctgacca 1320
tcgacgaaca gctgctgggc ttcagaggca gatgcccctt cagagtgtac atccccaaca 1380
agccctctaa gtacggcatc aagatcctga tgatgtgcga cagcggcacc aagtacatga 1440
tcaacggcat gccctacctc ggcagaggca cccaaacaaa tggcgtgcca ctgggcgagt 1500
actacgtgaa agaactgagc aagcctgtgc acggcagctg cagaaacatc acctgtgaca 1560
actggtttac cagcattccc ctggccaaga acctgctgca agaaccctac aagctgacaa 1620
tcgtgggcac cgtgcggagc aacaagaggg aaattcccga ggtgctgaag aactctcgga 1680
gcagacctgt gggcaccagc atgttctgct tcgacggacc tctgacactg gtgtcctaca 1740
agcccaagcc tgccaagatg gtgtacctgc tgagcagctg tgacgaggac gccagcatca 1800
atgagagcac cggcaagccc cagatggtca tgtactacaa ccagaccaaa ggcggcgtgg 1860
acaccctgga tcagatgtgc agcgtgatga cctgcagcag aaagaccaac agatggccca 1920
tggctctgct gtacggcatg atcaatatcg cctgcatcaa cagcttcatc atctacagcc 1980
acaacgtgtc cagcaagggc gagaaggtgc agagccggaa gaaattcatg cggaacctgt 2040
acatgagcct gaccagcagc ttcatgagaa agcggctgga agcccctaca ctgaagagat 2100
acctgcggga caacatcagc aacatcctgc ctaaagaggt gcccggcacc agcgacgata 2160
gcacagagga acccgtgatg aagaagagga cctactgcac ctactgtccc agcaagatcc 2220
ggcggaaggc caacgccagc tgcaaaaagt gcaagaaagt gatctgccgc gagcacaaca 2280
tcgatatgtg ccagagctgc ttctgatgag atgcattcga agcggccgcg agctcaagct 2340
tgcaattccg ataacttgtt tattgcagct tataatggtt acaaataaag caatagcatc 2400
acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc 2460
atcaatgtat cttatcatgt ctggcgctag cagcacaagt ttgtacaaaa aagcaggctc 2520
ctcgcaggag gcctggccgg cctccggatt acttccggtg caggcgtcag ctgcatcttg 2580
ctggccagct cggaggactg tccccgtaac tcccgtccgc tgtgtatcaa cacagctcta 2640
gccaaattgt tgaccgaacg acggggttcg attgtatcat tcctcacatc caacacttga 2700
gtagtacaat gccttgtcta agtgcgcttg ggacccagtc tattttgttt gtgtgcacaa 2760
gacgatactc ctcgatcacg taccaagcgc aggctggccc taacgacgtg gtattctcgt 2820
attggtgtgc cccagattgc ataaggcact catcacctgc gtttatgacg ctggacgcct 2880
gcgtatgttg caggactcct atgatgtcgc tagcctgatg cgttgttatg tgattgaggt 2940
ttagtacttg gcaatatcat tagatggaac taatacgcgg tctaatcgtt acaggtttcc 3000
gacgattgag aagtatcacc tgattctcgg gagcgacttg taactctact acacttattg 3060
atttggtact cctatgtgtt gctatcttga ctccgtaaat cgccgtgata cactctcttc 3120
gtttctcggc aatctatggc tctaaactat acacaggcat tctaagaata atctgacctg 3180
tccctgtagt actggtaggt tactaccagt acggcgacaa tgtgggagct acggcgtggt 3240
agaaccgggt acagacacag tccctgatcc cgatagtaac actgtgcccg tagcagtgat 3300
aatcattgcg cctcaacctg aggaagataa gggaggcagt atatcgtgct attccctcta 3360
ttctcccaca tgcgtatggc ctgaggtcaa tgatcaggta catgccgtaa tatgaggttt 3420
tacgtagccg atcacgaaaa ggttgctaaa tacttctcgt catatcccag ctcattgttg 3480
ggattatcgc taaacaggct aagttctaat gcccgtgggc atggcctcac gatctatggt 3540
gaaacagaca gcccgattta gcaatgctac agagcacagt ggttaatatt ggttgatacg 3600
tagaaccatg atcactgctc aattcgtgat gaagttgttc aatataccta gaagccactc 3660
aaggattctc tctgtgtcac taatagaggt ggtagagtgt ttggaagccg gttccacaca 3720
gatttagagt gtcacctaat tgccgtttat ctaaggaatt acgacaggag attccgaagt 3780
aattaggcta ttgcagagat acaggctagg tgaggtggag ggtgtcttgc ggtcttcgtc 3840
cagccgcctt gaaagcgggg tttttttcaa gcatcgctgc gtcattgcgt ggtcgagact 3900
tatgcgccct acgaagttcg tctgaagtag tccaggaaag acctactttg cagttatctt 3960
cgcattccca cactcaccac tacaactact cttccctcaa tttcccggtt agtttcgcta 4020
agctccgacc ttgggttact gtgttgcatc cgactcgctg cggctttcta gtacgctgta 4080
ctgtttcatt cttctgtagg tctggttccg taagtccgaa tttccaggcc gtggtctagt 4140
cctaattatt ttctgtcccg gtagctatat ttagccgagg gtttgtccat ttgcccggcg 4200
tagagcgccg cgtttgcgaa catttgcgcc cgtaatacgt agggacaccg tcgggtaatg 4260
gatggcaaaa gccgaaaacg gcgtcttccg gcgcttggat tcagcgctct tgagccataa 4320
accgcgttgc ttctttggtt aattcgtatt aatgatccta agcgccagct tattcgttaa 4380
gaggcactag gcgcgccgcg gcatgcgatc gccagcatgg ctacgaccca gctttcttgt 4440
acaaagtggt gatggccggc cgcttcgagt taattaatcc aaccggttac cgcctaggat 4500
cgatagatct aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4560
ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 4620
gtgagcgagc gagcgcgcag ctgcctgcag g 4651
<210> 8
<211> 6724
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 8
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg gaattaacca 540
ggctcagagg cacacaggag tttctgggct caccctgccc ccttccaacc cctcagttcc 600
catcctccag cagctgtttg tgtgctgcct ctgaagtcca cactgaacaa acttcagcct 660
actcatgtcc ctaaaatggg caaacattgc aagcagcaaa cagcaaacac acagccctcc 720
ctgcctgctg accttggagc tggggcagag gtcagagacc tctctgggcc catgccacct 780
ccaacatcca ctcgacccct tggaatttcg gtggagagga gcagaggttg tcctggcgtg 840
gtttaggtag tgtgagaggg gtacccgggg atcttgctac cagtggaaca gccactaagg 900
attctgcagt gagagcagag ggccagctaa gtggtactct cccagagact gtctgactca 960
cgccaccccc tccaccttgg acacaggacg ctgtggtttc tgagccaggt acaatgactc 1020
ctttcggtaa gtgcagtgga agctgtacac tgcccaggca aagcgtccgg gcagcgtagg 1080
cgggcgactc agatcccagc cagtggactt agcccctgtt tgctcctccg ataactgggg 1140
tgaccttggt taatattcac cagcagcctc ccccgttgcc cctctggatc cactgcttaa 1200
atacggacga ggacagggcc ctgtctcctc agcttcaggc accaccactg acctgggaca 1260
gtgaatccgc ggtctagaaa gaggtaaggg tttaagggat ggttggttgg tggggtatta 1320
atgtttaatt acctggagca cctgcctgaa atcacttttt ttcaggttgg acgcgtcgct 1380
agcatgcaga ttgagctgag cacctgcttc ttcctgtgcc tgctgaggtt ctgcttctct 1440
gccaccagga gatactacct gggggctgtg gagctgagct gggactacat gcagtctgac 1500
ctgggggagc tgcctgtgga tgccaggttc ccccccagag tgcccaagag cttccccttc 1560
aacacctctg tggtgtacaa gaagaccctg tttgtggagt tcactgacca cctgttcaac 1620
attgccaagc ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggctgaggtg 1680
tatgacactg tggtgatcac cctgaagaac atggccagcc accctgtgag cctgcatgct 1740
gtgggggtga gctactggaa ggcctctgag ggggctgagt atgatgacca gaccagccag 1800
agggagaagg aggatgacaa ggtgttccct gggggcagcc acacctatgt gtggcaggtg 1860
ctgaaggaga atggccccat ggcctctgac cccctgtgcc tgacctacag ctacctgagc 1920
catgtggacc tggtgaagga cctgaactct ggcctgattg gggccctgct ggtgtgcagg 1980
gagggcagcc tggccaagga gaagacccag accctgcaca agttcatcct gctgtttgct 2040
gtgtttgatg agggcaagag ctggcactct gaaaccaaga acagcctgat gcaggacagg 2100
gatgctgcct ctgccagggc ctggcccaag atgcacactg tgaatggcta tgtgaacagg 2160
agcctgcctg gcctgattgg ctgccacagg aagtctgtgt actggcatgt gattggcatg 2220
ggcaccaccc ctgaggtgca cagcatcttc ctggagggcc acaccttcct ggtcaggaac 2280
cacaggcagg ccagcctgga gatcagcccc atcaccttcc tgactgccca gaccctgctg 2340
atggacctgg gccagttcct gctgttctgc cacatcagca gccaccagca tgatggcatg 2400
gaggcctatg tgaaggtgga cagctgccct gaggagcccc agctgaggat gaagaacaat 2460
gaggaggctg aggactatga tgatgacctg actgactctg agatggatgt ggtgaggttt 2520
gatgatgaca acagccccag cttcatccag atcaggtctg tggccaagaa gcaccccaag 2580
acctgggtgc actacattgc tgctgaggag gaggactggg actatgcccc cctggtgctg 2640
gcccctgatg acaggagcta caagagccag tacctgaaca atggccccca gaggattggc 2700
aggaagtaca agaaggtcag gttcatggcc tacactgatg aaaccttcaa gaccagggag 2760
gccatccagc atgagtctgg catcctgggc cccctgctgt atggggaggt gggggacacc 2820
ctgctgatca tcttcaagaa ccaggccagc aggccctaca acatctaccc ccatggcatc 2880
actgatgtga ggcccctgta cagcaggagg ctgcccaagg gggtgaagca cctgaaggac 2940
ttccccatcc tgcctgggga gatcttcaag tacaagtgga ctgtgactgt ggaggatggc 3000
cccaccaagt ctgaccccag gtgcctgacc agatactaca gcagctttgt gaacatggag 3060
agggacctgg cctctggcct gattggcccc ctgctgatct gctacaagga gtctgtggac 3120
cagaggggca accagatcat gtctgacaag aggaatgtga tcctgttctc tgtgtttgat 3180
gagaacagga gctggtacct gactgagaac atccagaggt tcctgcccaa ccctgctggg 3240
gtgcagctgg aggaccctga gttccaggcc agcaacatca tgcacagcat caatggctat 3300
gtgtttgaca gcctgcagct gtctgtgtgc ctgcatgagg tggcctactg gtacatcctg 3360
agcattgggg cccagactga cttcctgtct gtgttcttct ctggctacac cttcaagcac 3420
aagatggtgt atgaggacac cctgaccctg ttccccttct ctggggagac tgtgttcatg 3480
agcatggaga accctggcct gtggattctg ggctgccaca actctgactt caggaacagg 3540
ggcatgactg ccctgctgaa agtctccagc tgtgacaaga acactgggga ctactatgag 3600
gacagctatg aggacatctc tgcctacctg ctgagcaaga acaatgccat tgagcccagg 3660
agcttcagcc agaacccccc agtgctgaag aggcaccaga gggagatcac caggaccacc 3720
ctgcagtctg accaggagga gattgactat gatgacacca tctctgtgga gatgaagaag 3780
gaggactttg acatctacga cgaggacgag aaccagagcc ccaggagctt ccagaagaag 3840
accaggcact acttcattgc tgctgtggag aggctgtggg actatggcat gagcagcagc 3900
ccccatgtgc tgaggaacag ggcccagtct ggctctgtgc cccagttcaa gaaggtggtg 3960
ttccaggagt tcactgatgg cagcttcacc cagcccctgt acagagggga gctgaatgag 4020
cacctgggcc tgctgggccc ctacatcagg gctgaggtgg aggacaacat catggtgacc 4080
ttcaggaacc aggccagcag gccctacagc ttctacagca gcctgatcag ctatgaggag 4140
gaccagaggc agggggctga gcccaggaag aactttgtga agcccaatga aaccaagacc 4200
tacttctgga aggtgcagca ccacatggcc cccaccaagg atgagtttga ctgcaaggcc 4260
tgggcctact tctctgatgt ggacctggag aaggatgtgc actctggcct gattggcccc 4320
ctgctggtgt gccacaccaa caccctgaac cctgcccatg gcaggcaggt gactgtgcag 4380
gagtttgccc tgttcttcac catctttgat gaaaccaaga gctggtactt cactgagaac 4440
atggagagga actgcagggc cccctgcaac atccagatgg aggaccccac cttcaaggag 4500
aactacaggt tccatgccat caatggctac atcatggaca ccctgcctgg cctggtgatg 4560
gcccaggacc agaggatcag gtggtacctg ctgagcatgg gcagcaatga gaacatccac 4620
agcatccact tctctggcca tgtgttcact gtgaggaaga aggaggagta caagatggcc 4680
ctgtacaacc tgtaccctgg ggtgtttgag actgtggaga tgctgcccag caaggctggc 4740
atctggaggg tggagtgcct gattggggag cacctgcatg ctggcatgag caccctgttc 4800
ctggtgtaca gcaacaagtg ccagaccccc ctgggcatgg cctctggcca catcagggac 4860
ttccagatca ctgcctctgg ccagtatggc cagtgggccc ccaagctggc caggctgcac 4920
tactctggca gcatcaatgc ctggagcacc aaggagccct tcagctggat caaggtggac 4980
ctgctggccc ccatgatcat ccatggcatc aagacccagg gggccaggca gaagttcagc 5040
agcctgtaca tcagccagtt catcatcatg tacagcctgg atggcaagaa gtggcagacc 5100
tacaggggca acagcactgg caccctgatg gtgttctttg gcaatgtgga cagctctggc 5160
atcaagcaca acatcttcaa cccccccatc attgccagat acatcaggct gcaccccacc 5220
cactacagca tcaggagcac cctgaggatg gagctgatgg gctgtgacct gaacagctgc 5280
agcatgcccc tgggcatgga gagcaaggcc atctctgatg cccagatcac tgccagcagc 5340
tacttcacca acatgtttgc cacctggagc cccagcaagg ccaggctgca cctgcagggc 5400
aggagcaatg cctggaggcc ccaggtcaac aaccccaagg agtggctgca ggtggacttc 5460
cagaagacca tgaaggtgac tggggtgacc acccaggggg tgaagagcct gctgaccagc 5520
atgtatgtga aggagttcct gatcagcagc agccaggatg gccaccagtg gaccctgttc 5580
ttccagaatg gcaaggtgaa ggtgttccag ggcaaccagg acagcttcac ccctgtggtg 5640
aacagcctgg acccccccct gctgaccaga tacctgagga ttcaccccca gagctgggtg 5700
caccagattg ccctgaggat ggaggtgctg ggctgtgagg cccaggacct gtactgatga 5760
aacgttagat ctggtaccga tcacatatgc ctttaattaa acactagttc tatagtgtca 5820
cctaaattcc ctttagtgag ggttaatggc cgtaggccgc cagaattggg tccagacatg 5880
ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt 5940
atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa 6000
gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt 6060
ttttcggact ctaggacctg cgcatgcgct tggcgtaatc atggtcatag ctgtttcctg 6120
ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 6180
aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 6240
gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 6300
gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc tctcaccgcg 6360
gtggagctcc agcttttgtt cgaattgggg ccccccctcg agggtatcga tgatatctat 6420
aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa 6480
ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc 6540
acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg 6600
gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga 6660
aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg 6720
ttaa 6724
<210> 9
<211> 6550
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 9
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg gaattgtacc 540
gcgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 600
ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 660
ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 720
caccccagtt atcggaggag caaacagggg ctaagtccac ggatcccact gggaggatgt 780
tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgtttga 840
acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg cacatttcgt 900
agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt gtaggttact 960
tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc 1020
agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc aggagaagcc 1080
gtcacacaga tccacaagct cctgaagagg taagggttta agggatggtt ggttggtggg 1140
gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggacgc 1200
gtcgccacca tgcagattga gctgagcacc tgcttcttcc tgtgcctgct gaggttctgc 1260
ttctctgcca ccaggagata ctacctgggg gctgtggagc tgagctggga ctacatgcag 1320
tctgacctgg gggagctgcc tgtggatgcc aggttccccc ccagagtgcc caagagcttc 1380
cccttcaaca cctctgtggt gtacaagaag accctgtttg tggagttcac tgaccacctg 1440
ttcaacattg ccaagcccag gcccccctgg atgggcctgc tgggccccac catccaggct 1500
gaggtgtatg acactgtggt gatcaccctg aagaacatgg ccagccaccc tgtgagcctg 1560
catgctgtgg gggtgagcta ctggaaggcc tctgaggggg ctgagtatga tgaccagacc 1620
agccagaggg agaaggagga tgacaaggtg ttccctgggg gcagccacac ctatgtgtgg 1680
caggtgctga aggagaatgg ccccatggcc tctgaccccc tgtgcctgac ctacagctac 1740
ctgagccatg tggacctggt gaaggacctg aactctggcc tgattggggc cctgctggtg 1800
tgcagggagg gcagcctggc caaggagaag acccagaccc tgcacaagtt catcctgctg 1860
tttgctgtgt ttgatgaggg caagagctgg cactctgaaa ccaagaacag cctgatgcag 1920
gacagggatg ctgcctctgc cagggcctgg cccaagatgc acactgtgaa tggctatgtg 1980
aacaggagcc tgcctggcct gattggctgc cacaggaagt ctgtgtactg gcatgtgatt 2040
ggcatgggca ccacccctga ggtgcacagc atcttcctgg agggccacac cttcctggtc 2100
aggaaccaca ggcaggccag cctggagatc agccccatca ccttcctgac tgcccagacc 2160
ctgctgatgg acctgggcca gttcctgctg ttctgccaca tcagcagcca ccagcatgat 2220
ggcatggagg cctatgtgaa ggtggacagc tgccctgagg agccccagct gaggatgaag 2280
aacaatgagg aggctgagga ctatgatgat gacctgactg actctgagat ggatgtggtg 2340
aggtttgatg atgacaacag ccccagcttc atccagatca ggtctgtggc caagaagcac 2400
cccaagacct gggtgcacta cattgctgct gaggaggagg actgggacta tgcccccctg 2460
gtgctggccc ctgatgacag gagctacaag agccagtacc tgaacaatgg cccccagagg 2520
attggcagga agtacaagaa ggtcaggttc atggcctaca ctgatgaaac cttcaagacc 2580
agggaggcca tccagcatga gtctggcatc ctgggccccc tgctgtatgg ggaggtgggg 2640
gacaccctgc tgatcatctt caagaaccag gccagcaggc cctacaacat ctacccccat 2700
ggcatcactg atgtgaggcc cctgtacagc aggaggctgc ccaagggggt gaagcacctg 2760
aaggacttcc ccatcctgcc tggggagatc ttcaagtaca agtggactgt gactgtggag 2820
gatggcccca ccaagtctga ccccaggtgc ctgaccagat actacagcag ctttgtgaac 2880
atggagaggg acctggcctc tggcctgatt ggccccctgc tgatctgcta caaggagtct 2940
gtggaccaga ggggcaacca gatcatgtct gacaagagga atgtgatcct gttctctgtg 3000
tttgatgaga acaggagctg gtacctgact gagaacatcc agaggttcct gcccaaccct 3060
gctggggtgc agctggagga ccctgagttc caggccagca acatcatgca cagcatcaat 3120
ggctatgtgt ttgacagcct gcagctgtct gtgtgcctgc atgaggtggc ctactggtac 3180
atcctgagca ttggggccca gactgacttc ctgtctgtgt tcttctctgg ctacaccttc 3240
aagcacaaga tggtgtatga ggacaccctg accctgttcc ccttctctgg ggagactgtg 3300
ttcatgagca tggagaaccc tggcctgtgg attctgggct gccacaactc tgacttcagg 3360
aacaggggca tgactgccct gctgaaagtc tccagctgtg acaagaacac tggggactac 3420
tatgaggaca gctatgagga catctctgcc tacctgctga gcaagaacaa tgccattgag 3480
cccaggagct tcagccagaa ccccccagtg ctgaagaggc accagaggga gatcaccagg 3540
accaccctgc agtctgacca ggaggagatt gactatgatg acaccatctc tgtggagatg 3600
aagaaggagg actttgacat ctacgacgag gacgagaacc agagccccag gagcttccag 3660
aagaagacca ggcactactt cattgctgct gtggagaggc tgtgggacta tggcatgagc 3720
agcagccccc atgtgctgag gaacagggcc cagtctggct ctgtgcccca gttcaagaag 3780
gtggtgttcc aggagttcac tgatggcagc ttcacccagc ccctgtacag aggggagctg 3840
aatgagcacc tgggcctgct gggcccctac atcagggctg aggtggagga caacatcatg 3900
gtgaccttca ggaaccaggc cagcaggccc tacagcttct acagcagcct gatcagctat 3960
gaggaggacc agaggcaggg ggctgagccc aggaagaact ttgtgaagcc caatgaaacc 4020
aagacctact tctggaaggt gcagcaccac atggccccca ccaaggatga gtttgactgc 4080
aaggcctggg cctacttctc tgatgtggac ctggagaagg atgtgcactc tggcctgatt 4140
ggccccctgc tggtgtgcca caccaacacc ctgaaccctg cccatggcag gcaggtgact 4200
gtgcaggagt ttgccctgtt cttcaccatc tttgatgaaa ccaagagctg gtacttcact 4260
gagaacatgg agaggaactg cagggccccc tgcaacatcc agatggagga ccccaccttc 4320
aaggagaact acaggttcca tgccatcaat ggctacatca tggacaccct gcctggcctg 4380
gtgatggccc aggaccagag gatcaggtgg tacctgctga gcatgggcag caatgagaac 4440
atccacagca tccacttctc tggccatgtg ttcactgtga ggaagaagga ggagtacaag 4500
atggccctgt acaacctgta ccctggggtg tttgagactg tggagatgct gcccagcaag 4560
gctggcatct ggagggtgga gtgcctgatt ggggagcacc tgcatgctgg catgagcacc 4620
ctgttcctgg tgtacagcaa caagtgccag acccccctgg gcatggcctc tggccacatc 4680
agggacttcc agatcactgc ctctggccag tatggccagt gggcccccaa gctggccagg 4740
ctgcactact ctggcagcat caatgcctgg agcaccaagg agcccttcag ctggatcaag 4800
gtggacctgc tggcccccat gatcatccat ggcatcaaga cccagggggc caggcagaag 4860
ttcagcagcc tgtacatcag ccagttcatc atcatgtaca gcctggatgg caagaagtgg 4920
cagacctaca ggggcaacag cactggcacc ctgatggtgt tctttggcaa tgtggacagc 4980
tctggcatca agcacaacat cttcaacccc cccatcattg ccagatacat caggctgcac 5040
cccacccact acagcatcag gagcaccctg aggatggagc tgatgggctg tgacctgaac 5100
agctgcagca tgcccctggg catggagagc aaggccatct ctgatgccca gatcactgcc 5160
agcagctact tcaccaacat gtttgccacc tggagcccca gcaaggccag gctgcacctg 5220
cagggcagga gcaatgcctg gaggccccag gtcaacaacc ccaaggagtg gctgcaggtg 5280
gacttccaga agaccatgaa ggtgactggg gtgaccaccc agggggtgaa gagcctgctg 5340
accagcatgt atgtgaagga gttcctgatc agcagcagcc aggatggcca ccagtggacc 5400
ctgttcttcc agaatggcaa ggtgaaggtg ttccagggca accaggacag cttcacccct 5460
gtggtgaaca gcctggaccc ccccctgctg accagatacc tgaggattca cccccagagc 5520
tgggtgcacc agattgccct gaggatggag gtgctgggct gtgaggccca ggacctgtac 5580
tgatgaaacg ttagatctgg taccgatcac atatgccttt aattaaacac tagttctata 5640
gtgtcaccta aattcccttt agtgagggtt aatggccgta ggccgccaga attgggtcca 5700
gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 5760
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 5820
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 5880
gaggtttttt cggactctag gacctgcgca tgcgcttggc gtaatcatgg tcatagctgt 5940
ttcctgtttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt 6000
cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg 6060
ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc 6120
cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccctctc 6180
accgcggtgg agctccagct tttgttcgaa ttggggcccc ccctcgaggg tatcgatgat 6240
atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca 6300
atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt 6360
tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc 6420
tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta 6480
tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt 6540
ctagggttaa 6550
<210> 10
<211> 6550
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 10
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg gaattgtacc 540
gcgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 600
ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 660
ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 720
caccccagtt atcggaggag caaacagggg ctaagtccac ggatcccact gggaggatgt 780
tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgtttga 840
acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg cacatttcgt 900
agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt gtaggttact 960
tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc 1020
agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc aggagaagcc 1080
gtcacacaga tccacaagct cctgaagagg taagggttta agggatggtt ggttggtggg 1140
gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggacgc 1200
gtcgctagca tgcagattga gctgagcacc tgcttcttcc tgtgcctgct gaggttctgc 1260
ttctctgcca ccaggagata ctacctgggg gctgtggagc tgagctggga ctacatgcag 1320
tctgacctgg gggagctgcc tgtggatgcc aggttccccc ccagagtgcc caagagcttc 1380
cccttcaaca cctctgtggt gtacaagaag accctgtttg tggagttcac tgaccacctg 1440
ttcaacattg ccaagcccag gcccccctgg atgggcctgc tgggccccac catccaggct 1500
gaggtgtatg acactgtggt gatcaccctg aagaacatgg ccagccaccc tgtgagcctg 1560
catgctgtgg gggtgagcta ctggaaggcc tctgaggggg ctgagtatga tgaccagacc 1620
agccagaggg agaaggagga tgacaaggtg ttccctgggg gcagccacac ctatgtgtgg 1680
caggtgctga aggagaatgg ccccatggcc tctgaccccc tgtgcctgac ctacagctac 1740
ctgagccatg tggacctggt gaaggacctg aactctggcc tgattggggc cctgctggtg 1800
tgcagggagg gcagcctggc caaggagaag acccagaccc tgcacaagtt catcctgctg 1860
tttgctgtgt ttgatgaggg caagagctgg cactctgaaa ccaagaacag cctgatgcag 1920
gacagggatg ctgcctctgc cagggcctgg cccaagatgc acactgtgaa tggctatgtg 1980
aacaggagcc tgcctggcct gattggctgc cacaggaagt ctgtgtactg gcatgtgatt 2040
ggcatgggca ccacccctga ggtgcacagc atcttcctgg agggccacac cttcctggtc 2100
aggaaccaca ggcaggccag cctggagatc agccccatca ccttcctgac tgcccagacc 2160
ctgctgatgg acctgggcca gttcctgctg ttctgccaca tcagcagcca ccagcatgat 2220
ggcatggagg cctatgtgaa ggtggacagc tgccctgagg agccccagct gaggatgaag 2280
aacaatgagg aggctgagga ctatgatgat gacctgactg actctgagat ggatgtggtg 2340
aggtttgatg atgacaacag ccccagcttc atccagatca ggtctgtggc caagaagcac 2400
cccaagacct gggtgcacta cattgctgct gaggaggagg actgggacta tgcccccctg 2460
gtgctggccc ctgatgacag gagctacaag agccagtacc tgaacaatgg cccccagagg 2520
attggcagga agtacaagaa ggtcaggttc atggcctaca ctgatgaaac cttcaagacc 2580
agggaggcca tccagcatga gtctggcatc ctgggccccc tgctgtatgg ggaggtgggg 2640
gacaccctgc tgatcatctt caagaaccag gccagcaggc cctacaacat ctacccccat 2700
ggcatcactg atgtgaggcc cctgtacagc aggaggctgc ccaagggggt gaagcacctg 2760
aaggacttcc ccatcctgcc tggggagatc ttcaagtaca agtggactgt gactgtggag 2820
gatggcccca ccaagtctga ccccaggtgc ctgaccagat actacagcag ctttgtgaac 2880
atggagaggg acctggcctc tggcctgatt ggccccctgc tgatctgcta caaggagtct 2940
gtggaccaga ggggcaacca gatcatgtct gacaagagga atgtgatcct gttctctgtg 3000
tttgatgaga acaggagctg gtacctgact gagaacatcc agaggttcct gcccaaccct 3060
gctggggtgc agctggagga ccctgagttc caggccagca acatcatgca cagcatcaat 3120
ggctatgtgt ttgacagcct gcagctgtct gtgtgcctgc atgaggtggc ctactggtac 3180
atcctgagca ttggggccca gactgacttc ctgtctgtgt tcttctctgg ctacaccttc 3240
aagcacaaga tggtgtatga ggacaccctg accctgttcc ccttctctgg ggagactgtg 3300
ttcatgagca tggagaaccc tggcctgtgg attctgggct gccacaactc tgacttcagg 3360
aacaggggca tgactgccct gctgaaagtc tccagctgtg acaagaacac tggggactac 3420
tatgaggaca gctatgagga catctctgcc tacctgctga gcaagaacaa tgccattgag 3480
cccaggagct tcagccagaa ccccccagtg ctgaagaggc accagaggga gatcaccagg 3540
accaccctgc agtctgacca ggaggagatt gactatgatg acaccatctc tgtggagatg 3600
aagaaggagg actttgacat ctacgacgag gacgagaacc agagccccag gagcttccag 3660
aagaagacca ggcactactt cattgctgct gtggagaggc tgtgggacta tggcatgagc 3720
agcagccccc atgtgctgag gaacagggcc cagtctggct ctgtgcccca gttcaagaag 3780
gtggtgttcc aggagttcac tgatggcagc ttcacccagc ccctgtacag aggggagctg 3840
aatgagcacc tgggcctgct gggcccctac atcagggctg aggtggagga caacatcatg 3900
gtgaccttca ggaaccaggc cagcaggccc tacagcttct acagcagcct gatcagctat 3960
gaggaggacc agaggcaggg ggctgagccc aggaagaact ttgtgaagcc caatgaaacc 4020
aagacctact tctggaaggt gcagcaccac atggccccca ccaaggatga gtttgactgc 4080
aaggcctggg cctacttctc tgatgtggac ctggagaagg atgtgcactc tggcctgatt 4140
ggccccctgc tggtgtgcca caccaacacc ctgaaccctg cccatggcag gcaggtgact 4200
gtgcaggagt ttgccctgtt cttcaccatc tttgatgaaa ccaagagctg gtacttcact 4260
gagaacatgg agaggaactg cagggccccc tgcaacatcc agatggagga ccccaccttc 4320
aaggagaact acaggttcca tgccatcaat ggctacatca tggacaccct gcctggcctg 4380
gtgatggccc aggaccagag gatcaggtgg tacctgctga gcatgggcag caatgagaac 4440
atccacagca tccacttctc tggccatgtg ttcactgtga ggaagaagga ggagtacaag 4500
atggccctgt acaacctgta ccctggggtg tttgagactg tggagatgct gcccagcaag 4560
gctggcatct ggagggtgga gtgcctgatt ggggagcacc tgcatgctgg catgagcacc 4620
ctgttcctgg tgtacagcaa caagtgccag acccccctgg gcatggcctc tggccacatc 4680
agggacttcc agatcactgc ctctggccag tatggccagt gggcccccaa gctggccagg 4740
ctgcactact ctggcagcat caatgcctgg agcaccaagg agcccttcag ctggatcaag 4800
gtggacctgc tggcccccat gatcatccat ggcatcaaga cccagggggc caggcagaag 4860
ttcagcagcc tgtacatcag ccagttcatc atcatgtaca gcctggatgg caagaagtgg 4920
cagacctaca ggggcaacag cactggcacc ctgatggtgt tctttggcaa tgtggacagc 4980
tctggcatca agcacaacat cttcaacccc cccatcattg ccagatacat caggctgcac 5040
cccacccact acagcatcag gagcaccctg aggatggagc tgatgggctg tgacctgaac 5100
agctgcagca tgcccctggg catggagagc aaggccatct ctgatgccca gatcactgcc 5160
agcagctact tcaccaacat gtttgccacc tggagcccca gcaaggccag gctgcacctg 5220
cagggcagga gcaatgcctg gaggccccag gtcaacaacc ccaaggagtg gctgcaggtg 5280
gacttccaga agaccatgaa ggtgactggg gtgaccaccc agggggtgaa gagcctgctg 5340
accagcatgt atgtgaagga gttcctgatc agcagcagcc aggatggcca ccagtggacc 5400
ctgttcttcc agaatggcaa ggtgaaggtg ttccagggca accaggacag cttcacccct 5460
gtggtgaaca gcctggaccc ccccctgctg accagatacc tgaggattca cccccagagc 5520
tgggtgcacc agattgccct gaggatggag gtgctgggct gtgaggccca ggacctgtac 5580
tgatgaaacg ttagatctgg taccgatcac atatgccttt aattaaacac tagttctata 5640
gtgtcaccta aattcccttt agtgagggtt aatggccgta ggccgccaga attgggtcca 5700
gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 5760
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat 5820
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 5880
gaggtttttt cggactctag gacctgcgca tgcgcttggc gtaatcatgg tcatagctgt 5940
ttcctgtttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc gagaagcgtt 6000
cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc acgctgccgg 6060
ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc gctgctgccc 6120
cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc tgtccctctc 6180
accgcggtgg agctccagct tttgttcgaa ttggggcccc ccctcgaggg tatcgatgat 6240
atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca tgaaataaca 6300
atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca tgcgtcattt 6360
tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga caagcacgcc 6420
tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg attcgcgcta 6480
tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca gactatcttt 6540
ctagggttaa 6550
<210> 11
<211> 6172
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 11
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
gtgtttgctg cttgcaatgt ttgcccattt tagggtggac acaggacgct gtggtttctg 600
agccaggggg cgactcagat cccagccagt ggacttagcc cctgtttgct cctccgataa 660
ctggggtgac cttggttaat attcaccagc agcctccccc gttgcccctc tggatccact 720
gcttaaatac ggacgaggac agggccctgt ctcctcagct tcaggcacca ccactgacct 780
gggacagtga atcgcaagaa ttctaatacg acgtctctga cctaggccac catgcagatt 840
gagctgagca cctgcttctt cctgtgcctg ctgaggttct gcttctctgc caccaggaga 900
tactacctgg gggctgtgga gctgagctgg gactacatgc agtctgacct gggggagctg 960
cctgtggatg ccaggttccc ccccagagtg cccaagagct tccccttcaa cacctctgtg 1020
gtgtacaaga agaccctgtt tgtggagttc actgaccacc tgttcaacat tgccaagccc 1080
aggcccccct ggatgggcct gctgggcccc accatccagg ctgaggtgta tgacactgtg 1140
gtgatcaccc tgaagaacat ggccagccac cctgtgagcc tgcatgctgt gggggtgagc 1200
tactggaagg cctctgaggg ggctgagtat gatgaccaga ccagccagag ggagaaggag 1260
gatgacaagg tgttccctgg gggcagccac acctatgtgt ggcaggtgct gaaggagaat 1320
ggccccatgg cctctgaccc cctgtgcctg acctacagct acctgagcca tgtggacctg 1380
gtgaaggacc tgaactctgg cctgattggg gccctgctgg tgtgcaggga gggcagcctg 1440
gccaaggaga agacccagac cctgcacaag ttcatcctgc tgtttgctgt gtttgatgag 1500
ggcaagagct ggcactctga aaccaagaac agcctgatgc aggacaggga tgctgcctct 1560
gccagggcct ggcccaagat gcacactgtg aatggctatg tgaacaggag cctgcctggc 1620
ctgattggct gccacaggaa gtctgtgtac tggcatgtga ttggcatggg caccacccct 1680
gaggtgcaca gcatcttcct ggagggccac accttcctgg tcaggaacca caggcaggcc 1740
agcctggaga tcagccccat caccttcctg actgcccaga ccctgctgat ggacctgggc 1800
cagttcctgc tgttctgcca catcagcagc caccagcatg atggcatgga ggcctatgtg 1860
aaggtggaca gctgccctga ggagccccag ctgaggatga agaacaatga ggaggctgag 1920
gactatgatg atgacctgac tgactctgag atggatgtgg tgaggtttga tgatgacaac 1980
agccccagct tcatccagat caggtctgtg gccaagaagc accccaagac ctgggtgcac 2040
tacattgctg ctgaggagga ggactgggac tatgcccccc tggtgctggc ccctgatgac 2100
aggagctaca agagccagta cctgaacaat ggcccccaga ggattggcag gaagtacaag 2160
aaggtcaggt tcatggccta cactgatgaa accttcaaga ccagggaggc catccagcat 2220
gagtctggca tcctgggccc cctgctgtat ggggaggtgg gggacaccct gctgatcatc 2280
ttcaagaacc aggccagcag gccctacaac atctaccccc atggcatcac tgatgtgagg 2340
cccctgtaca gcaggaggct gcccaagggg gtgaagcacc tgaaggactt ccccatcctg 2400
cctggggaga tcttcaagta caagtggact gtgactgtgg aggatggccc caccaagtct 2460
gaccccaggt gcctgaccag atactacagc agctttgtga acatggagag ggacctggcc 2520
tctggcctga ttggccccct gctgatctgc tacaaggagt ctgtggacca gaggggcaac 2580
cagatcatgt ctgacaagag gaatgtgatc ctgttctctg tgtttgatga gaacaggagc 2640
tggtacctga ctgagaacat ccagaggttc ctgcccaacc ctgctggggt gcagctggag 2700
gaccctgagt tccaggccag caacatcatg cacagcatca atggctatgt gtttgacagc 2760
ctgcagctgt ctgtgtgcct gcatgaggtg gcctactggt acatcctgag cattggggcc 2820
cagactgact tcctgtctgt gttcttctct ggctacacct tcaagcacaa gatggtgtat 2880
gaggacaccc tgaccctgtt ccccttctct ggggagactg tgttcatgag catggagaac 2940
cctggcctgt ggattctggg ctgccacaac tctgacttca ggaacagggg catgactgcc 3000
ctgctgaaag tctccagctg tgacaagaac actggggact actatgagga cagctatgag 3060
gacatctctg cctacctgct gagcaagaac aatgccattg agcccaggag cttcagccag 3120
aaccccccag tgctgaagag gcaccagagg gagatcacca ggaccaccct gcagtctgac 3180
caggaggaga ttgactatga tgacaccatc tctgtggaga tgaagaagga ggactttgac 3240
atctacgacg aggacgagaa ccagagcccc aggagcttcc agaagaagac caggcactac 3300
ttcattgctg ctgtggagag gctgtgggac tatggcatga gcagcagccc ccatgtgctg 3360
aggaacaggg cccagtctgg ctctgtgccc cagttcaaga aggtggtgtt ccaggagttc 3420
actgatggca gcttcaccca gcccctgtac agaggggagc tgaatgagca cctgggcctg 3480
ctgggcccct acatcagggc tgaggtggag gacaacatca tggtgacctt caggaaccag 3540
gccagcaggc cctacagctt ctacagcagc ctgatcagct atgaggagga ccagaggcag 3600
ggggctgagc ccaggaagaa ctttgtgaag cccaatgaaa ccaagaccta cttctggaag 3660
gtgcagcacc acatggcccc caccaaggat gagtttgact gcaaggcctg ggcctacttc 3720
tctgatgtgg acctggagaa ggatgtgcac tctggcctga ttggccccct gctggtgtgc 3780
cacaccaaca ccctgaaccc tgcccatggc aggcaggtga ctgtgcagga gtttgccctg 3840
ttcttcacca tctttgatga aaccaagagc tggtacttca ctgagaacat ggagaggaac 3900
tgcagggccc cctgcaacat ccagatggag gaccccacct tcaaggagaa ctacaggttc 3960
catgccatca atggctacat catggacacc ctgcctggcc tggtgatggc ccaggaccag 4020
aggatcaggt ggtacctgct gagcatgggc agcaatgaga acatccacag catccacttc 4080
tctggccatg tgttcactgt gaggaagaag gaggagtaca agatggccct gtacaacctg 4140
taccctgggg tgtttgagac tgtggagatg ctgcccagca aggctggcat ctggagggtg 4200
gagtgcctga ttggggagca cctgcatgct ggcatgagca ccctgttcct ggtgtacagc 4260
aacaagtgcc agacccccct gggcatggcc tctggccaca tcagggactt ccagatcact 4320
gcctctggcc agtatggcca gtgggccccc aagctggcca ggctgcacta ctctggcagc 4380
atcaatgcct ggagcaccaa ggagcccttc agctggatca aggtggacct gctggccccc 4440
atgatcatcc atggcatcaa gacccagggg gccaggcaga agttcagcag cctgtacatc 4500
agccagttca tcatcatgta cagcctggat ggcaagaagt ggcagaccta caggggcaac 4560
agcactggca ccctgatggt gttctttggc aatgtggaca gctctggcat caagcacaac 4620
atcttcaacc cccccatcat tgccagatac atcaggctgc accccaccca ctacagcatc 4680
aggagcaccc tgaggatgga gctgatgggc tgtgacctga acagctgcag catgcccctg 4740
ggcatggaga gcaaggccat ctctgatgcc cagatcactg ccagcagcta cttcaccaac 4800
atgtttgcca cctggagccc cagcaaggcc aggctgcacc tgcagggcag gagcaatgcc 4860
tggaggcccc aggtcaacaa ccccaaggag tggctgcagg tggacttcca gaagaccatg 4920
aaggtgactg gggtgaccac ccagggggtg aagagcctgc tgaccagcat gtatgtgaag 4980
gagttcctga tcagcagcag ccaggatggc caccagtgga ccctgttctt ccagaatggc 5040
aaggtgaagg tgttccaggg caaccaggac agcttcaccc ctgtggtgaa cagcctggac 5100
ccccccctgc tgaccagata cctgaggatt cacccccaga gctgggtgca ccagattgcc 5160
ctgaggatgg aggtgctggg ctgtgaggcc caggacctgt actgatgaaa cgttagatct 5220
ggtaccgatc acatatgcct ttaattaaac actagttcta tagtgtcacc taaattccct 5280
ttagtgaggg ttaatggccg taggccgcca gaattgggtc cagacatgat aagatacatt 5340
gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 5400
tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac 5460
aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttcggactct 5520
aggacctgcg catgcgcttg gcgtaatcat ggtcatagct gtttcctgtt ttccccgtat 5580
ccccccaggt gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg 5640
tgccaccttc cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga 5700
gcgccggacc ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg 5760
taattacatc cctgggggct ttgggggggg gctgtccctc tcaccgcggt ggagctccag 5820
cttttgttcg aattggggcc ccccctcgag ggtatcgatg atatctataa caagaaaata 5880
tatatataat aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag 5940
ttaaatctta aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta 6000
tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg 6060
gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa 6120
tatttcaaga atgcatgcgt caattttacg cagactatct ttctagggtt aa 6172
<210> 12
<211> 6234
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 12
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
ggtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt 600
tcatattgac ttaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc 660
aggtttggag tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag 720
ccccttcacc aggagaagcc gtcacacaga tccacaagct cctgctagca ggtaagtgcc 780
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 840
actgacactg acatccactt tttctttttc tccacaggaa ttcctaggcc accatgcaga 900
ttgagctgag cacctgcttc ttcctgtgcc tgctgaggtt ctgcttctct gccaccagga 960
gatactacct gggggctgtg gagctgagct gggactacat gcagtctgac ctgggggagc 1020
tgcctgtgga tgccaggttc ccccccagag tgcccaagag cttccccttc aacacctctg 1080
tggtgtacaa gaagaccctg tttgtggagt tcactgacca cctgttcaac attgccaagc 1140
ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggctgaggtg tatgacactg 1200
tggtgatcac cctgaagaac atggccagcc accctgtgag cctgcatgct gtgggggtga 1260
gctactggaa ggcctctgag ggggctgagt atgatgacca gaccagccag agggagaagg 1320
aggatgacaa ggtgttccct gggggcagcc acacctatgt gtggcaggtg ctgaaggaga 1380
atggccccat ggcctctgac cccctgtgcc tgacctacag ctacctgagc catgtggacc 1440
tggtgaagga cctgaactct ggcctgattg gggccctgct ggtgtgcagg gagggcagcc 1500
tggccaagga gaagacccag accctgcaca agttcatcct gctgtttgct gtgtttgatg 1560
agggcaagag ctggcactct gaaaccaaga acagcctgat gcaggacagg gatgctgcct 1620
ctgccagggc ctggcccaag atgcacactg tgaatggcta tgtgaacagg agcctgcctg 1680
gcctgattgg ctgccacagg aagtctgtgt actggcatgt gattggcatg ggcaccaccc 1740
ctgaggtgca cagcatcttc ctggagggcc acaccttcct ggtcaggaac cacaggcagg 1800
ccagcctgga gatcagcccc atcaccttcc tgactgccca gaccctgctg atggacctgg 1860
gccagttcct gctgttctgc cacatcagca gccaccagca tgatggcatg gaggcctatg 1920
tgaaggtgga cagctgccct gaggagcccc agctgaggat gaagaacaat gaggaggctg 1980
aggactatga tgatgacctg actgactctg agatggatgt ggtgaggttt gatgatgaca 2040
acagccccag cttcatccag atcaggtctg tggccaagaa gcaccccaag acctgggtgc 2100
actacattgc tgctgaggag gaggactggg actatgcccc cctggtgctg gcccctgatg 2160
acaggagcta caagagccag tacctgaaca atggccccca gaggattggc aggaagtaca 2220
agaaggtcag gttcatggcc tacactgatg aaaccttcaa gaccagggag gccatccagc 2280
atgagtctgg catcctgggc cccctgctgt atggggaggt gggggacacc ctgctgatca 2340
tcttcaagaa ccaggccagc aggccctaca acatctaccc ccatggcatc actgatgtga 2400
ggcccctgta cagcaggagg ctgcccaagg gggtgaagca cctgaaggac ttccccatcc 2460
tgcctgggga gatcttcaag tacaagtgga ctgtgactgt ggaggatggc cccaccaagt 2520
ctgaccccag gtgcctgacc agatactaca gcagctttgt gaacatggag agggacctgg 2580
cctctggcct gattggcccc ctgctgatct gctacaagga gtctgtggac cagaggggca 2640
accagatcat gtctgacaag aggaatgtga tcctgttctc tgtgtttgat gagaacagga 2700
gctggtacct gactgagaac atccagaggt tcctgcccaa ccctgctggg gtgcagctgg 2760
aggaccctga gttccaggcc agcaacatca tgcacagcat caatggctat gtgtttgaca 2820
gcctgcagct gtctgtgtgc ctgcatgagg tggcctactg gtacatcctg agcattgggg 2880
cccagactga cttcctgtct gtgttcttct ctggctacac cttcaagcac aagatggtgt 2940
atgaggacac cctgaccctg ttccccttct ctggggagac tgtgttcatg agcatggaga 3000
accctggcct gtggattctg ggctgccaca actctgactt caggaacagg ggcatgactg 3060
ccctgctgaa agtctccagc tgtgacaaga acactgggga ctactatgag gacagctatg 3120
aggacatctc tgcctacctg ctgagcaaga acaatgccat tgagcccagg agcttcagcc 3180
agaacccccc agtgctgaag aggcaccaga gggagatcac caggaccacc ctgcagtctg 3240
accaggagga gattgactat gatgacacca tctctgtgga gatgaagaag gaggactttg 3300
acatctacga cgaggacgag aaccagagcc ccaggagctt ccagaagaag accaggcact 3360
acttcattgc tgctgtggag aggctgtggg actatggcat gagcagcagc ccccatgtgc 3420
tgaggaacag ggcccagtct ggctctgtgc cccagttcaa gaaggtggtg ttccaggagt 3480
tcactgatgg cagcttcacc cagcccctgt acagagggga gctgaatgag cacctgggcc 3540
tgctgggccc ctacatcagg gctgaggtgg aggacaacat catggtgacc ttcaggaacc 3600
aggccagcag gccctacagc ttctacagca gcctgatcag ctatgaggag gaccagaggc 3660
agggggctga gcccaggaag aactttgtga agcccaatga aaccaagacc tacttctgga 3720
aggtgcagca ccacatggcc cccaccaagg atgagtttga ctgcaaggcc tgggcctact 3780
tctctgatgt ggacctggag aaggatgtgc actctggcct gattggcccc ctgctggtgt 3840
gccacaccaa caccctgaac cctgcccatg gcaggcaggt gactgtgcag gagtttgccc 3900
tgttcttcac catctttgat gaaaccaaga gctggtactt cactgagaac atggagagga 3960
actgcagggc cccctgcaac atccagatgg aggaccccac cttcaaggag aactacaggt 4020
tccatgccat caatggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 4080
agaggatcag gtggtacctg ctgagcatgg gcagcaatga gaacatccac agcatccact 4140
tctctggcca tgtgttcact gtgaggaaga aggaggagta caagatggcc ctgtacaacc 4200
tgtaccctgg ggtgtttgag actgtggaga tgctgcccag caaggctggc atctggaggg 4260
tggagtgcct gattggggag cacctgcatg ctggcatgag caccctgttc ctggtgtaca 4320
gcaacaagtg ccagaccccc ctgggcatgg cctctggcca catcagggac ttccagatca 4380
ctgcctctgg ccagtatggc cagtgggccc ccaagctggc caggctgcac tactctggca 4440
gcatcaatgc ctggagcacc aaggagccct tcagctggat caaggtggac ctgctggccc 4500
ccatgatcat ccatggcatc aagacccagg gggccaggca gaagttcagc agcctgtaca 4560
tcagccagtt catcatcatg tacagcctgg atggcaagaa gtggcagacc tacaggggca 4620
acagcactgg caccctgatg gtgttctttg gcaatgtgga cagctctggc atcaagcaca 4680
acatcttcaa cccccccatc attgccagat acatcaggct gcaccccacc cactacagca 4740
tcaggagcac cctgaggatg gagctgatgg gctgtgacct gaacagctgc agcatgcccc 4800
tgggcatgga gagcaaggcc atctctgatg cccagatcac tgccagcagc tacttcacca 4860
acatgtttgc cacctggagc cccagcaagg ccaggctgca cctgcagggc aggagcaatg 4920
cctggaggcc ccaggtcaac aaccccaagg agtggctgca ggtggacttc cagaagacca 4980
tgaaggtgac tggggtgacc acccaggggg tgaagagcct gctgaccagc atgtatgtga 5040
aggagttcct gatcagcagc agccaggatg gccaccagtg gaccctgttc ttccagaatg 5100
gcaaggtgaa ggtgttccag ggcaaccagg acagcttcac ccctgtggtg aacagcctgg 5160
acccccccct gctgaccaga tacctgagga ttcaccccca gagctgggtg caccagattg 5220
ccctgaggat ggaggtgctg ggctgtgagg cccaggacct gtactgatga aacgttagat 5280
ctggtaccga tcacatatgc ctttaattaa acactagttc tatagtgtca cctaaattcc 5340
ctttagtgag ggttaatggc cgtaggccgc cagaattggg tccagacatg ataagataca 5400
ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa 5460
tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca 5520
acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttcggact 5580
ctaggacctg cgcatgcgct tggcgtaatc atggtcatag ctgtttcctg ttttccccgt 5640
atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg aaagcgatcc 5700
cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg 5760
gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc gggggaggga 5820
cgtaattaca tccctggggg ctttgggggg gggctgtccc tctcaccgcg gtggagctcc 5880
agcttttgtt cgaattgggg ccccccctcg agggtatcga tgatatctat aacaagaaaa 5940
tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa ttatcgtatg 6000
agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc acgcggtcgt 6060
tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg gagctccaag 6120
cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga aagagagagc 6180
aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg ttaa 6234
<210> 13
<211> 6748
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 13
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
gtgtttgctg cttgcaatgt ttgcccattt tagggtggac acaggacgct gtggtttctg 600
agccaggggg cgactcagat cccagccagt ggacttagcc cctgtttgct cctccgataa 660
ctggggtgac cttggttaat attcaccagc agcctccccc gttgcccctc tggatccact 720
gcttaaatac ggacgaggac agggccctgt ctcctcagct tcaggcacca ccactgacct 780
gggacagtga atcgcaagaa ttctaatacg acgtctctga cctaggccac catgcagatt 840
gagctgagca cctgcttctt cctgtgcctg ctgaggttct gcttctctgc caccaggaga 900
tactacctgg gggctgtgga gctgagctgg gactacatgc agtctgacct gggggagctg 960
cctgtggatg ccaggttccc ccccagagtg cccaagagct tccccttcaa cacctctgtg 1020
gtgtacaaga agaccctgtt tgtggagttc actgaccacc tgttcaacat tgccaagccc 1080
aggcccccct ggatgggcct gctgggcccc accatccagg ctgaggtgta tgacactgtg 1140
gtgatcaccc tgaagaacat ggccagccac cctgtgagcc tgcatgctgt gggggtgagc 1200
tactggaagg cctctgaggg ggctgagtat gatgaccaga ccagccagag ggagaaggag 1260
gatgacaagg tgttccctgg gggcagccac acctatgtgt ggcaggtgct gaaggagaat 1320
ggccccatgg cctctgaccc cctgtgcctg acctacagct acctgagcca tgtggacctg 1380
gtgaaggacc tgaactctgg cctgattggg gccctgctgg tgtgcaggga gggcagcctg 1440
gccaaggaga agacccagac cctgcacaag ttcatcctgc tgtttgctgt gtttgatgag 1500
ggcaagagct ggcactctga aaccaagaac agcctgatgc aggacaggga tgctgcctct 1560
gccagggcct ggcccaagat gcacactgtg aatggctatg tgaacaggag cctgcctggc 1620
ctgattggct gccacaggaa gtctgtgtac tggcatgtga ttggcatggg caccacccct 1680
gaggtgcaca gcatcttcct ggagggccac accttcctgg tcaggaacca caggcaggcc 1740
agcctggaga tcagccccat caccttcctg actgcccaga ccctgctgat ggacctgggc 1800
cagttcctgc tgttctgcca catcagcagc caccagcatg atggcatgga ggcctatgtg 1860
aaggtggaca gctgccctga ggagccccag ctgaggatga agaacaatga ggaggctgag 1920
gactatgatg atgacctgac tgactctgag atggatgtgg tgaggtttga tgatgacaac 1980
agccccagct tcatccagat caggtctgtg gccaagaagc accccaagac ctgggtgcac 2040
tacattgctg ctgaggagga ggactgggac tatgcccccc tggtgctggc ccctgatgac 2100
aggagctaca agagccagta cctgaacaat ggcccccaga ggattggcag gaagtacaag 2160
aaggtcaggt tcatggccta cactgatgaa accttcaaga ccagggaggc catccagcat 2220
gagtctggca tcctgggccc cctgctgtat ggggaggtgg gggacaccct gctgatcatc 2280
ttcaagaacc aggccagcag gccctacaac atctaccccc atggcatcac tgatgtgagg 2340
cccctgtaca gcaggaggct gcccaagggg gtgaagcacc tgaaggactt ccccatcctg 2400
cctggggaga tcttcaagta caagtggact gtgactgtgg aggatggccc caccaagtct 2460
gaccccaggt gcctgaccag atactacagc agctttgtga acatggagag ggacctggcc 2520
tctggcctga ttggccccct gctgatctgc tacaaggagt ctgtggacca gaggggcaac 2580
cagatcatgt ctgacaagag gaatgtgatc ctgttctctg tgtttgatga gaacaggagc 2640
tggtacctga ctgagaacat ccagaggttc ctgcccaacc ctgctggggt gcagctggag 2700
gaccctgagt tccaggccag caacatcatg cacagcatca atggctatgt gtttgacagc 2760
ctgcagctgt ctgtgtgcct gcatgaggtg gcctactggt acatcctgag cattggggcc 2820
cagactgact tcctgtctgt gttcttctct ggctacacct tcaagcacaa gatggtgtat 2880
gaggacaccc tgaccctgtt ccccttctct ggggagactg tgttcatgag catggagaac 2940
cctggcctgt ggattctggg ctgccacaac tctgacttca ggaacagggg catgactgcc 3000
ctgctgaaag tctccagctg tgacaagaac actggggact actatgagga cagctatgag 3060
gacatctctg cctacctgct gagcaagaac aatgccattg agcccaggag cttcagccag 3120
aaccccccag tgctgaagag gcaccagagg gagatcacca ggaccaccct gcagtctgac 3180
caggaggaga ttgactatga tgacaccatc tctgtggaga tgaagaagga ggactttgac 3240
atctacgacg aggacgagaa ccagagcccc aggagcttcc agaagaagac caggcactac 3300
ttcattgctg ctgtggagag gctgtgggac tatggcatga gcagcagccc ccatgtgctg 3360
aggaacaggg cccagtctgg ctctgtgccc cagttcaaga aggtggtgtt ccaggagttc 3420
actgatggca gcttcaccca gcccctgtac agaggggagc tgaatgagca cctgggcctg 3480
ctgggcccct acatcagggc tgaggtggag gacaacatca tggtgacctt caggaaccag 3540
gccagcaggc cctacagctt ctacagcagc ctgatcagct atgaggagga ccagaggcag 3600
ggggctgagc ccaggaagaa ctttgtgaag cccaatgaaa ccaagaccta cttctggaag 3660
gtgcagcacc acatggcccc caccaaggat gagtttgact gcaaggcctg ggcctacttc 3720
tctgatgtgg acctggagaa ggatgtgcac tctggcctga ttggccccct gctggtgtgc 3780
cacaccaaca ccctgaaccc tgcccatggc aggcaggtga ctgtgcagga gtttgccctg 3840
ttcttcacca tctttgatga aaccaagagc tggtacttca ctgagaacat ggagaggaac 3900
tgcagggccc cctgcaacat ccagatggag gaccccacct tcaaggagaa ctacaggttc 3960
catgccatca atggctacat catggacacc ctgcctggcc tggtgatggc ccaggaccag 4020
aggatcaggt ggtacctgct gagcatgggc agcaatgaga acatccacag catccacttc 4080
tctggccatg tgttcactgt gaggaagaag gaggagtaca agatggccct gtacaacctg 4140
taccctgggg tgtttgagac tgtggagatg ctgcccagca aggctggcat ctggagggtg 4200
gagtgcctga ttggggagca cctgcatgct ggcatgagca ccctgttcct ggtgtacagc 4260
aacaagtgcc agacccccct gggcatggcc tctggccaca tcagggactt ccagatcact 4320
gcctctggcc agtatggcca gtgggccccc aagctggcca ggctgcacta ctctggcagc 4380
atcaatgcct ggagcaccaa ggagcccttc agctggatca aggtggacct gctggccccc 4440
atgatcatcc atggcatcaa gacccagggg gccaggcaga agttcagcag cctgtacatc 4500
agccagttca tcatcatgta cagcctggat ggcaagaagt ggcagaccta caggggcaac 4560
agcactggca ccctgatggt gttctttggc aatgtggaca gctctggcat caagcacaac 4620
atcttcaacc cccccatcat tgccagatac atcaggctgc accccaccca ctacagcatc 4680
aggagcaccc tgaggatgga gctgatgggc tgtgacctga acagctgcag catgcccctg 4740
ggcatggaga gcaaggccat ctctgatgcc cagatcactg ccagcagcta cttcaccaac 4800
atgtttgcca cctggagccc cagcaaggcc aggctgcacc tgcagggcag gagcaatgcc 4860
tggaggcccc aggtcaacaa ccccaaggag tggctgcagg tggacttcca gaagaccatg 4920
aaggtgactg gggtgaccac ccagggggtg aagagcctgc tgaccagcat gtatgtgaag 4980
gagttcctga tcagcagcag ccaggatggc caccagtgga ccctgttctt ccagaatggc 5040
aaggtgaagg tgttccaggg caaccaggac agcttcaccc ctgtggtgaa cagcctggac 5100
ccccccctgc tgaccagata cctgaggatt cacccccaga gctgggtgca ccagattgcc 5160
ctgaggatgg aggtgctggg ctgtgaggcc caggacctgt acggctccgg agaaggacgg 5220
ggaagcctgc ttacatgcgg agatgtggag gagaatcctg gtcccatggt ctttaccctg 5280
gaagatttcg tcggcgactg gcggcagaca gccggctata atctggacca ggtgctggaa 5340
caaggcggag tgtccagcct gttccagaat ctgggagtgt ccgtgacacc catccagcgg 5400
attgtgctgt ctggcgagaa cggcctgaag atcgacatcc acgtgatcat cccttacgag 5460
ggcctgagcg gcgatcagat gggacagatc gagaagattt tcaaggtggt gtaccccgtg 5520
gacgaccacc acttcaaagt gatcctgcac tacggcaccc tggtcatcga tggcgtgacc 5580
cctaacatga tcgactactt cggcagaccc tacgagggaa tcgccgtgtt cgacggcaag 5640
aaaatcaccg tgaccggcac actgtggaac gggaacaaga tcatcgacga gcggctgatc 5700
aaccccgatg gcagcctgct gttcagagtg accattaacg gcgtgacagg ctggcggctg 5760
tgcgaaagga ttctggcctg atgaaacgtt agatctggta ccgatcacat atgcctttaa 5820
ttaaacacta gttctatagt gtcacctaaa ttccctttag tgagggttaa tggccgtagg 5880
ccgccagaat tgggtccaga catgataaga tacattgatg agtttggaca aaccacaact 5940
agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 6000
accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 6060
gttcaggggg aggtgtggga ggttttttcg gactctagga cctgcgcatg cgcttggcgt 6120
aatcatggtc atagctgttt cctgttttcc ccgtatcccc ccaggtgtct gcaggctcaa 6180
agagcagcga gaagcgttca gaggaaagcg atcccgtgcc accttccccg tgcccgggct 6240
gtccccgcac gctgccggct cggggatgcg gggggagcgc cggaccggag cggagccccg 6300
ggcggctcgc tgctgccccc tagcggggga gggacgtaat tacatccctg ggggctttgg 6360
gggggggctg tccctctcac cgcggtggag ctccagcttt tgttcgaatt ggggcccccc 6420
ctcgagggta tcgatgatat ctataacaag aaaatatata tataataagt tatcacgtaa 6480
gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag tcacgtaaaa 6540
gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag tgacacttac 6600
cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc ctaaatgcac 6660
agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc atgcgtcaat 6720
tttacgcaga ctatctttct agggttaa 6748
<210> 14
<211> 6810
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 14
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
ggtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt 600
tcatattgac ttaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc 660
aggtttggag tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag 720
ccccttcacc aggagaagcc gtcacacaga tccacaagct cctgctagca ggtaagtgcc 780
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 840
actgacactg acatccactt tttctttttc tccacaggaa ttcctaggcc accatgcaga 900
ttgagctgag cacctgcttc ttcctgtgcc tgctgaggtt ctgcttctct gccaccagga 960
gatactacct gggggctgtg gagctgagct gggactacat gcagtctgac ctgggggagc 1020
tgcctgtgga tgccaggttc ccccccagag tgcccaagag cttccccttc aacacctctg 1080
tggtgtacaa gaagaccctg tttgtggagt tcactgacca cctgttcaac attgccaagc 1140
ccaggccccc ctggatgggc ctgctgggcc ccaccatcca ggctgaggtg tatgacactg 1200
tggtgatcac cctgaagaac atggccagcc accctgtgag cctgcatgct gtgggggtga 1260
gctactggaa ggcctctgag ggggctgagt atgatgacca gaccagccag agggagaagg 1320
aggatgacaa ggtgttccct gggggcagcc acacctatgt gtggcaggtg ctgaaggaga 1380
atggccccat ggcctctgac cccctgtgcc tgacctacag ctacctgagc catgtggacc 1440
tggtgaagga cctgaactct ggcctgattg gggccctgct ggtgtgcagg gagggcagcc 1500
tggccaagga gaagacccag accctgcaca agttcatcct gctgtttgct gtgtttgatg 1560
agggcaagag ctggcactct gaaaccaaga acagcctgat gcaggacagg gatgctgcct 1620
ctgccagggc ctggcccaag atgcacactg tgaatggcta tgtgaacagg agcctgcctg 1680
gcctgattgg ctgccacagg aagtctgtgt actggcatgt gattggcatg ggcaccaccc 1740
ctgaggtgca cagcatcttc ctggagggcc acaccttcct ggtcaggaac cacaggcagg 1800
ccagcctgga gatcagcccc atcaccttcc tgactgccca gaccctgctg atggacctgg 1860
gccagttcct gctgttctgc cacatcagca gccaccagca tgatggcatg gaggcctatg 1920
tgaaggtgga cagctgccct gaggagcccc agctgaggat gaagaacaat gaggaggctg 1980
aggactatga tgatgacctg actgactctg agatggatgt ggtgaggttt gatgatgaca 2040
acagccccag cttcatccag atcaggtctg tggccaagaa gcaccccaag acctgggtgc 2100
actacattgc tgctgaggag gaggactggg actatgcccc cctggtgctg gcccctgatg 2160
acaggagcta caagagccag tacctgaaca atggccccca gaggattggc aggaagtaca 2220
agaaggtcag gttcatggcc tacactgatg aaaccttcaa gaccagggag gccatccagc 2280
atgagtctgg catcctgggc cccctgctgt atggggaggt gggggacacc ctgctgatca 2340
tcttcaagaa ccaggccagc aggccctaca acatctaccc ccatggcatc actgatgtga 2400
ggcccctgta cagcaggagg ctgcccaagg gggtgaagca cctgaaggac ttccccatcc 2460
tgcctgggga gatcttcaag tacaagtgga ctgtgactgt ggaggatggc cccaccaagt 2520
ctgaccccag gtgcctgacc agatactaca gcagctttgt gaacatggag agggacctgg 2580
cctctggcct gattggcccc ctgctgatct gctacaagga gtctgtggac cagaggggca 2640
accagatcat gtctgacaag aggaatgtga tcctgttctc tgtgtttgat gagaacagga 2700
gctggtacct gactgagaac atccagaggt tcctgcccaa ccctgctggg gtgcagctgg 2760
aggaccctga gttccaggcc agcaacatca tgcacagcat caatggctat gtgtttgaca 2820
gcctgcagct gtctgtgtgc ctgcatgagg tggcctactg gtacatcctg agcattgggg 2880
cccagactga cttcctgtct gtgttcttct ctggctacac cttcaagcac aagatggtgt 2940
atgaggacac cctgaccctg ttccccttct ctggggagac tgtgttcatg agcatggaga 3000
accctggcct gtggattctg ggctgccaca actctgactt caggaacagg ggcatgactg 3060
ccctgctgaa agtctccagc tgtgacaaga acactgggga ctactatgag gacagctatg 3120
aggacatctc tgcctacctg ctgagcaaga acaatgccat tgagcccagg agcttcagcc 3180
agaacccccc agtgctgaag aggcaccaga gggagatcac caggaccacc ctgcagtctg 3240
accaggagga gattgactat gatgacacca tctctgtgga gatgaagaag gaggactttg 3300
acatctacga cgaggacgag aaccagagcc ccaggagctt ccagaagaag accaggcact 3360
acttcattgc tgctgtggag aggctgtggg actatggcat gagcagcagc ccccatgtgc 3420
tgaggaacag ggcccagtct ggctctgtgc cccagttcaa gaaggtggtg ttccaggagt 3480
tcactgatgg cagcttcacc cagcccctgt acagagggga gctgaatgag cacctgggcc 3540
tgctgggccc ctacatcagg gctgaggtgg aggacaacat catggtgacc ttcaggaacc 3600
aggccagcag gccctacagc ttctacagca gcctgatcag ctatgaggag gaccagaggc 3660
agggggctga gcccaggaag aactttgtga agcccaatga aaccaagacc tacttctgga 3720
aggtgcagca ccacatggcc cccaccaagg atgagtttga ctgcaaggcc tgggcctact 3780
tctctgatgt ggacctggag aaggatgtgc actctggcct gattggcccc ctgctggtgt 3840
gccacaccaa caccctgaac cctgcccatg gcaggcaggt gactgtgcag gagtttgccc 3900
tgttcttcac catctttgat gaaaccaaga gctggtactt cactgagaac atggagagga 3960
actgcagggc cccctgcaac atccagatgg aggaccccac cttcaaggag aactacaggt 4020
tccatgccat caatggctac atcatggaca ccctgcctgg cctggtgatg gcccaggacc 4080
agaggatcag gtggtacctg ctgagcatgg gcagcaatga gaacatccac agcatccact 4140
tctctggcca tgtgttcact gtgaggaaga aggaggagta caagatggcc ctgtacaacc 4200
tgtaccctgg ggtgtttgag actgtggaga tgctgcccag caaggctggc atctggaggg 4260
tggagtgcct gattggggag cacctgcatg ctggcatgag caccctgttc ctggtgtaca 4320
gcaacaagtg ccagaccccc ctgggcatgg cctctggcca catcagggac ttccagatca 4380
ctgcctctgg ccagtatggc cagtgggccc ccaagctggc caggctgcac tactctggca 4440
gcatcaatgc ctggagcacc aaggagccct tcagctggat caaggtggac ctgctggccc 4500
ccatgatcat ccatggcatc aagacccagg gggccaggca gaagttcagc agcctgtaca 4560
tcagccagtt catcatcatg tacagcctgg atggcaagaa gtggcagacc tacaggggca 4620
acagcactgg caccctgatg gtgttctttg gcaatgtgga cagctctggc atcaagcaca 4680
acatcttcaa cccccccatc attgccagat acatcaggct gcaccccacc cactacagca 4740
tcaggagcac cctgaggatg gagctgatgg gctgtgacct gaacagctgc agcatgcccc 4800
tgggcatgga gagcaaggcc atctctgatg cccagatcac tgccagcagc tacttcacca 4860
acatgtttgc cacctggagc cccagcaagg ccaggctgca cctgcagggc aggagcaatg 4920
cctggaggcc ccaggtcaac aaccccaagg agtggctgca ggtggacttc cagaagacca 4980
tgaaggtgac tggggtgacc acccaggggg tgaagagcct gctgaccagc atgtatgtga 5040
aggagttcct gatcagcagc agccaggatg gccaccagtg gaccctgttc ttccagaatg 5100
gcaaggtgaa ggtgttccag ggcaaccagg acagcttcac ccctgtggtg aacagcctgg 5160
acccccccct gctgaccaga tacctgagga ttcaccccca gagctgggtg caccagattg 5220
ccctgaggat ggaggtgctg ggctgtgagg cccaggacct gtacggctcc ggagaaggac 5280
ggggaagcct gcttacatgc ggagatgtgg aggagaatcc tggtcccatg gtctttaccc 5340
tggaagattt cgtcggcgac tggcggcaga cagccggcta taatctggac caggtgctgg 5400
aacaaggcgg agtgtccagc ctgttccaga atctgggagt gtccgtgaca cccatccagc 5460
ggattgtgct gtctggcgag aacggcctga agatcgacat ccacgtgatc atcccttacg 5520
agggcctgag cggcgatcag atgggacaga tcgagaagat tttcaaggtg gtgtaccccg 5580
tggacgacca ccacttcaaa gtgatcctgc actacggcac cctggtcatc gatggcgtga 5640
cccctaacat gatcgactac ttcggcagac cctacgaggg aatcgccgtg ttcgacggca 5700
agaaaatcac cgtgaccggc acactgtgga acgggaacaa gatcatcgac gagcggctga 5760
tcaaccccga tggcagcctg ctgttcagag tgaccattaa cggcgtgaca ggctggcggc 5820
tgtgcgaaag gattctggcc tgatgaaacg ttagatctgg taccgatcac atatgccttt 5880
aattaaacac tagttctata gtgtcaccta aattcccttt agtgagggtt aatggccgta 5940
ggccgccaga attgggtcca gacatgataa gatacattga tgagtttgga caaaccacaa 6000
ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg 6060
taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 6120
aggttcaggg ggaggtgtgg gaggtttttt cggactctag gacctgcgca tgcgcttggc 6180
gtaatcatgg tcatagctgt ttcctgtttt ccccgtatcc ccccaggtgt ctgcaggctc 6240
aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg 6300
ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc 6360
cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt 6420
gggggggggc tgtccctctc accgcggtgg agctccagct tttgttcgaa ttggggcccc 6480
ccctcgaggg tatcgatgat atctataaca agaaaatata tatataataa gttatcacgt 6540
aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa 6600
aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt 6660
accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc 6720
acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca 6780
attttacgca gactatcttt ctagggttaa 6810
<210> 15
<211> 3246
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 15
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
ggtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt 600
tcatattgac ttaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc 660
aggtttggag tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag 720
ccccttcacc aggagaagcc gtcacacaga tccacaagct cctgctagca ggtaagtgcc 780
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 840
actgacactg acatccactt tttctttttc tccacaggaa ttcctaggcc accatgcagc 900
gcgtgaacat gattatggcc gagtctcccg gcctgatcac catctgtctg ctgggctatc 960
tgctgagcgc cgagtgcacc gtgtttctgg atcacgagaa cgccaacaag atcctgaaca 1020
gacccaagcg gtacaacagc ggcaaactgg aagagttcgt gcagggtaac ctggaacgcg 1080
agtgcatgga agagaagtgc agcttcgaag aggccagaga ggtgttcgag aacaccgaga 1140
gaaccaccga gttctggaag cagtacgtgg acggcgatca gtgcgagagc aacccttgtc 1200
tgaatggcgg cagctgcaag gatgacatca acagctacga gtgctggtgc cccttcggct 1260
tcgagggcaa gaattgcgag ctggatgtga cctgcaacat caagaacggc agatgcgagc 1320
agttctgcaa gaacagcgcc gacaacaagg tcgtgtgctc ctgcacagag ggctacagac 1380
tggccgagaa tcagaagtcc tgcgagcccg ctgtgccctt tccatgtggc agagtgtctg 1440
tgtcccagac cagcaagctg accagagccg agacagtgtt ccccgatgtg gactacgtga 1500
acagcaccga ggccgagaca atcctggaca acatcaccca gagcacccag tccttcaacg 1560
acttcaccag agtcgtcggc ggcgaggatg ctaagcctgg acagtttcct tggcaagtgg 1620
tgctgaacgg caaggtggac gctttttgtg gcggctccat cgtgaacgag aagtggatcg 1680
tgaccgccgc tcactgtgtg gaaaccggcg tgaagattac agtggtggcc ggggagcaca 1740
acatcgagga aacagagcac accgagcaga aacggaacgt gatcagaatc atccctcacc 1800
acaactacaa cgccgccatc aacaagtaca accacgacat tgccctgctc gagctggacg 1860
aacccctggt cctgaactct tacgtgaccc ctatctgtat cgccgacaaa gagtacacca 1920
acatctttct gaagttcggc agcggctacg tgtccggctg gggaagagtt ttccacaagg 1980
gcagatcagc cctggtgctg cagtacctga gagtgcccct ggtggataga gccacatgcc 2040
tgctgagcac caagttcacc atctacaaca acatgttctg cgccggcttc cacgaaggcg 2100
gcagagattc ttgtcagggc gattctggcg gccctcacgt gacagaagtc gagggcacat 2160
cttttctgac cggcatcatc agctggggcg aagagtgtgc catgaagggg aagtacggca 2220
tctataccaa ggtgtccaga tacgtgaact ggatcaaaga aaagaccaag ctcacctgat 2280
gaaacgttag atctggtacc gatcacatat gcctttaatt aaacactagt tctatagtgt 2340
cacctaaatt ccctttagtg agggttaatg gccgtaggcc gccagaattg ggtccagaca 2400
tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 2460
ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 2520
aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg 2580
ttttttcgga ctctaggacc tgcgcatgcg cttggcgtaa tcatggtcat agctgtttcc 2640
tgttttcccc gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga 2700
ggaaagcgat cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg 2760
gggatgcggg gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta 2820
gcgggggagg gacgtaatta catccctggg ggctttgggg gggggctgtc cctctcaccg 2880
cggtggagct ccagcttttg ttcgaattgg ggccccccct cgagggtatc gatgatatct 2940
ataacaagaa aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat 3000
aattatcgta tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac 3060
tcacgcggtc gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac 3120
gggagctcca agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta 3180
gaaagagaga gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag 3240
ggttaa 3246
<210> 16
<211> 3184
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 16
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
gtgtttgctg cttgcaatgt ttgcccattt tagggtggac acaggacgct gtggtttctg 600
agccaggggg cgactcagat cccagccagt ggacttagcc cctgtttgct cctccgataa 660
ctggggtgac cttggttaat attcaccagc agcctccccc gttgcccctc tggatccact 720
gcttaaatac ggacgaggac agggccctgt ctcctcagct tcaggcacca ccactgacct 780
gggacagtga atcgcaagaa ttctaatacg acgtctctga cctaggccac catgcagcgc 840
gtgaacatga ttatggccga gtctcccggc ctgatcacca tctgtctgct gggctatctg 900
ctgagcgccg agtgcaccgt gtttctggat cacgagaacg ccaacaagat cctgaacaga 960
cccaagcggt acaacagcgg caaactggaa gagttcgtgc agggtaacct ggaacgcgag 1020
tgcatggaag agaagtgcag cttcgaagag gccagagagg tgttcgagaa caccgagaga 1080
accaccgagt tctggaagca gtacgtggac ggcgatcagt gcgagagcaa cccttgtctg 1140
aatggcggca gctgcaagga tgacatcaac agctacgagt gctggtgccc cttcggcttc 1200
gagggcaaga attgcgagct ggatgtgacc tgcaacatca agaacggcag atgcgagcag 1260
ttctgcaaga acagcgccga caacaaggtc gtgtgctcct gcacagaggg ctacagactg 1320
gccgagaatc agaagtcctg cgagcccgct gtgccctttc catgtggcag agtgtctgtg 1380
tcccagacca gcaagctgac cagagccgag acagtgttcc ccgatgtgga ctacgtgaac 1440
agcaccgagg ccgagacaat cctggacaac atcacccaga gcacccagtc cttcaacgac 1500
ttcaccagag tcgtcggcgg cgaggatgct aagcctggac agtttccttg gcaagtggtg 1560
ctgaacggca aggtggacgc tttttgtggc ggctccatcg tgaacgagaa gtggatcgtg 1620
accgccgctc actgtgtgga aaccggcgtg aagattacag tggtggccgg ggagcacaac 1680
atcgaggaaa cagagcacac cgagcagaaa cggaacgtga tcagaatcat ccctcaccac 1740
aactacaacg ccgccatcaa caagtacaac cacgacattg ccctgctcga gctggacgaa 1800
cccctggtcc tgaactctta cgtgacccct atctgtatcg ccgacaaaga gtacaccaac 1860
atctttctga agttcggcag cggctacgtg tccggctggg gaagagtttt ccacaagggc 1920
agatcagccc tggtgctgca gtacctgaga gtgcccctgg tggatagagc cacatgcctg 1980
ctgagcacca agttcaccat ctacaacaac atgttctgcg ccggcttcca cgaaggcggc 2040
agagattctt gtcagggcga ttctggcggc cctcacgtga cagaagtcga gggcacatct 2100
tttctgaccg gcatcatcag ctggggcgaa gagtgtgcca tgaaggggaa gtacggcatc 2160
tataccaagg tgtccagata cgtgaactgg atcaaagaaa agaccaagct cacctgatga 2220
aacgttagat ctggtaccga tcacatatgc ctttaattaa acactagttc tatagtgtca 2280
cctaaattcc ctttagtgag ggttaatggc cgtaggccgc cagaattggg tccagacatg 2340
ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt 2400
atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa 2460
gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt 2520
ttttcggact ctaggacctg cgcatgcgct tggcgtaatc atggtcatag ctgtttcctg 2580
ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 2640
aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 2700
gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 2760
gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc tctcaccgcg 2820
gtggagctcc agcttttgtt cgaattgggg ccccccctcg agggtatcga tgatatctat 2880
aacaagaaaa tatatatata ataagttatc acgtaagtag aacatgaaat aacaatataa 2940
ttatcgtatg agttaaatct taaaagtcac gtaaaagata atcatgcgtc attttgactc 3000
acgcggtcgt tatagttcaa aatcagtgac acttaccgca ttgacaagca cgcctcacgg 3060
gagctccaag cggcgactga gatgtcctaa atgcacagcg acggattcgc gctatttaga 3120
aagagagagc aatatttcaa gaatgcatgc gtcaatttta cgcagactat ctttctaggg 3180
ttaa 3184
<210> 17
<211> 3760
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 17
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
gtgtttgctg cttgcaatgt ttgcccattt tagggtggac acaggacgct gtggtttctg 600
agccaggggg cgactcagat cccagccagt ggacttagcc cctgtttgct cctccgataa 660
ctggggtgac cttggttaat attcaccagc agcctccccc gttgcccctc tggatccact 720
gcttaaatac ggacgaggac agggccctgt ctcctcagct tcaggcacca ccactgacct 780
gggacagtga atcgcaagaa ttctaatacg acgtctctga cctaggccac catgcagcgc 840
gtgaacatga ttatggccga gtctcccggc ctgatcacca tctgtctgct gggctatctg 900
ctgagcgccg agtgcaccgt gtttctggat cacgagaacg ccaacaagat cctgaacaga 960
cccaagcggt acaacagcgg caaactggaa gagttcgtgc agggtaacct ggaacgcgag 1020
tgcatggaag agaagtgcag cttcgaagag gccagagagg tgttcgagaa caccgagaga 1080
accaccgagt tctggaagca gtacgtggac ggcgatcagt gcgagagcaa cccttgtctg 1140
aatggcggca gctgcaagga tgacatcaac agctacgagt gctggtgccc cttcggcttc 1200
gagggcaaga attgcgagct ggatgtgacc tgcaacatca agaacggcag atgcgagcag 1260
ttctgcaaga acagcgccga caacaaggtc gtgtgctcct gcacagaggg ctacagactg 1320
gccgagaatc agaagtcctg cgagcccgct gtgccctttc catgtggcag agtgtctgtg 1380
tcccagacca gcaagctgac cagagccgag acagtgttcc ccgatgtgga ctacgtgaac 1440
agcaccgagg ccgagacaat cctggacaac atcacccaga gcacccagtc cttcaacgac 1500
ttcaccagag tcgtcggcgg cgaggatgct aagcctggac agtttccttg gcaagtggtg 1560
ctgaacggca aggtggacgc tttttgtggc ggctccatcg tgaacgagaa gtggatcgtg 1620
accgccgctc actgtgtgga aaccggcgtg aagattacag tggtggccgg ggagcacaac 1680
atcgaggaaa cagagcacac cgagcagaaa cggaacgtga tcagaatcat ccctcaccac 1740
aactacaacg ccgccatcaa caagtacaac cacgacattg ccctgctcga gctggacgaa 1800
cccctggtcc tgaactctta cgtgacccct atctgtatcg ccgacaaaga gtacaccaac 1860
atctttctga agttcggcag cggctacgtg tccggctggg gaagagtttt ccacaagggc 1920
agatcagccc tggtgctgca gtacctgaga gtgcccctgg tggatagagc cacatgcctg 1980
ctgagcacca agttcaccat ctacaacaac atgttctgcg ccggcttcca cgaaggcggc 2040
agagattctt gtcagggcga ttctggcggc cctcacgtga cagaagtcga gggcacatct 2100
tttctgaccg gcatcatcag ctggggcgaa gagtgtgcca tgaaggggaa gtacggcatc 2160
tataccaagg tgtccagata cgtgaactgg atcaaagaaa agaccaagct caccggctcc 2220
ggagaaggac ggggaagcct gcttacatgc ggagatgtgg aggagaatcc tggtcccatg 2280
gtctttaccc tggaagattt cgtcggcgac tggcggcaga cagccggcta taatctggac 2340
caggtgctgg aacaaggcgg agtgtccagc ctgttccaga atctgggagt gtccgtgaca 2400
cccatccagc ggattgtgct gtctggcgag aacggcctga agatcgacat ccacgtgatc 2460
atcccttacg agggcctgag cggcgatcag atgggacaga tcgagaagat tttcaaggtg 2520
gtgtaccccg tggacgacca ccacttcaaa gtgatcctgc actacggcac cctggtcatc 2580
gatggcgtga cccctaacat gatcgactac ttcggcagac cctacgaggg aatcgccgtg 2640
ttcgacggca agaaaatcac cgtgaccggc acactgtgga acgggaacaa gatcatcgac 2700
gagcggctga tcaaccccga tggcagcctg ctgttcagag tgaccattaa cggcgtgaca 2760
ggctggcggc tgtgcgaaag gattctggcc tgatgaaacg ttagatctgg taccgatcac 2820
atatgccttt aattaaacac tagttctata gtgtcaccta aattcccttt agtgagggtt 2880
aatggccgta ggccgccaga attgggtcca gacatgataa gatacattga tgagtttgga 2940
caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt 3000
gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat 3060
tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt cggactctag gacctgcgca 3120
tgcgcttggc gtaatcatgg tcatagctgt ttcctgtttt ccccgtatcc ccccaggtgt 3180
ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc 3240
cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg 3300
agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc 3360
tgggggcttt gggggggggc tgtccctctc accgcggtgg agctccagct tttgttcgaa 3420
ttggggcccc ccctcgaggg tatcgatgat atctataaca agaaaatata tatataataa 3480
gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt aaatcttaaa 3540
agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc 3600
agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc gactgagatg 3660
tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata tttcaagaat 3720
gcatgcgtca attttacgca gactatcttt ctagggttaa 3760
<210> 18
<211> 3822
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 18
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaactcgtc gactaggcgc 540
ggtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt 600
tcatattgac ttaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc 660
aggtttggag tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag 720
ccccttcacc aggagaagcc gtcacacaga tccacaagct cctgctagca ggtaagtgcc 780
gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 840
actgacactg acatccactt tttctttttc tccacaggaa ttcctaggcc accatgcagc 900
gcgtgaacat gattatggcc gagtctcccg gcctgatcac catctgtctg ctgggctatc 960
tgctgagcgc cgagtgcacc gtgtttctgg atcacgagaa cgccaacaag atcctgaaca 1020
gacccaagcg gtacaacagc ggcaaactgg aagagttcgt gcagggtaac ctggaacgcg 1080
agtgcatgga agagaagtgc agcttcgaag aggccagaga ggtgttcgag aacaccgaga 1140
gaaccaccga gttctggaag cagtacgtgg acggcgatca gtgcgagagc aacccttgtc 1200
tgaatggcgg cagctgcaag gatgacatca acagctacga gtgctggtgc cccttcggct 1260
tcgagggcaa gaattgcgag ctggatgtga cctgcaacat caagaacggc agatgcgagc 1320
agttctgcaa gaacagcgcc gacaacaagg tcgtgtgctc ctgcacagag ggctacagac 1380
tggccgagaa tcagaagtcc tgcgagcccg ctgtgccctt tccatgtggc agagtgtctg 1440
tgtcccagac cagcaagctg accagagccg agacagtgtt ccccgatgtg gactacgtga 1500
acagcaccga ggccgagaca atcctggaca acatcaccca gagcacccag tccttcaacg 1560
acttcaccag agtcgtcggc ggcgaggatg ctaagcctgg acagtttcct tggcaagtgg 1620
tgctgaacgg caaggtggac gctttttgtg gcggctccat cgtgaacgag aagtggatcg 1680
tgaccgccgc tcactgtgtg gaaaccggcg tgaagattac agtggtggcc ggggagcaca 1740
acatcgagga aacagagcac accgagcaga aacggaacgt gatcagaatc atccctcacc 1800
acaactacaa cgccgccatc aacaagtaca accacgacat tgccctgctc gagctggacg 1860
aacccctggt cctgaactct tacgtgaccc ctatctgtat cgccgacaaa gagtacacca 1920
acatctttct gaagttcggc agcggctacg tgtccggctg gggaagagtt ttccacaagg 1980
gcagatcagc cctggtgctg cagtacctga gagtgcccct ggtggataga gccacatgcc 2040
tgctgagcac caagttcacc atctacaaca acatgttctg cgccggcttc cacgaaggcg 2100
gcagagattc ttgtcagggc gattctggcg gccctcacgt gacagaagtc gagggcacat 2160
cttttctgac cggcatcatc agctggggcg aagagtgtgc catgaagggg aagtacggca 2220
tctataccaa ggtgtccaga tacgtgaact ggatcaaaga aaagaccaag ctcaccggct 2280
ccggagaagg acggggaagc ctgcttacat gcggagatgt ggaggagaat cctggtccca 2340
tggtctttac cctggaagat ttcgtcggcg actggcggca gacagccggc tataatctgg 2400
accaggtgct ggaacaaggc ggagtgtcca gcctgttcca gaatctggga gtgtccgtga 2460
cacccatcca gcggattgtg ctgtctggcg agaacggcct gaagatcgac atccacgtga 2520
tcatccctta cgagggcctg agcggcgatc agatgggaca gatcgagaag attttcaagg 2580
tggtgtaccc cgtggacgac caccacttca aagtgatcct gcactacggc accctggtca 2640
tcgatggcgt gacccctaac atgatcgact acttcggcag accctacgag ggaatcgccg 2700
tgttcgacgg caagaaaatc accgtgaccg gcacactgtg gaacgggaac aagatcatcg 2760
acgagcggct gatcaacccc gatggcagcc tgctgttcag agtgaccatt aacggcgtga 2820
caggctggcg gctgtgcgaa aggattctgg cctgatgaaa cgttagatct ggtaccgatc 2880
acatatgcct ttaattaaac actagttcta tagtgtcacc taaattccct ttagtgaggg 2940
ttaatggccg taggccgcca gaattgggtc cagacatgat aagatacatt gatgagtttg 3000
gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 3060
ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 3120
attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttcggactct aggacctgcg 3180
catgcgcttg gcgtaatcat ggtcatagct gtttcctgtt ttccccgtat ccccccaggt 3240
gtctgcaggc tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc 3300
cccgtgcccg ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc 3360
ggagcggagc cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc 3420
cctgggggct ttgggggggg gctgtccctc tcaccgcggt ggagctccag cttttgttcg 3480
aattggggcc ccccctcgag ggtatcgatg atatctataa caagaaaata tatatataat 3540
aagttatcac gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta 3600
aaagtcacgt aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa 3660
tcagtgacac ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga 3720
tgtcctaaat gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga 3780
atgcatgcgt caattttacg cagactatct ttctagggtt aa 3822
<210> 19
<211> 4582
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 19
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc aaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgaa ctcgtcgact aggcgcggtc tgtctgcaca tttcgtagag cgagtgttcc 720
gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt 780
tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc 840
tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca 900
caagctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 960
gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca 1020
caggaattcc taggccacca tgcagcgcgt gaacatgatt atggccgagt ctcccggcct 1080
gatcaccatc tgtctgctgg gctatctgct gagcgccgag tgcaccgtgt ttctggatca 1140
cgagaacgcc aacaagatcc tgaacagacc caagcggtac aacagcggca aactggaaga 1200
gttcgtgcag ggtaacctgg aacgcgagtg catggaagag aagtgcagct tcgaagaggc 1260
cagagaggtg ttcgagaaca ccgagagaac caccgagttc tggaagcagt acgtggacgg 1320
cgatcagtgc gagagcaacc cttgtctgaa tggcggcagc tgcaaggatg acatcaacag 1380
ctacgagtgc tggtgcccct tcggcttcga gggcaagaat tgcgagctgg atgtgacctg 1440
caacatcaag aacggcagat gcgagcagtt ctgcaagaac agcgccgaca acaaggtcgt 1500
gtgctcctgc acagagggct acagactggc cgagaatcag aagtcctgcg agcccgctgt 1560
gccctttcca tgtggcagag tgtctgtgtc ccagaccagc aagctgacca gagccgagac 1620
agtgttcccc gatgtggact acgtgaacag caccgaggcc gagacaatcc tggacaacat 1680
cacccagagc acccagtcct tcaacgactt caccagagtc gtcggcggcg aggatgctaa 1740
gcctggacag tttccttggc aagtggtgct gaacggcaag gtggacgctt tttgtggcgg 1800
ctccatcgtg aacgagaagt ggatcgtgac cgccgctcac tgtgtggaaa ccggcgtgaa 1860
gattacagtg gtggccgggg agcacaacat cgaggaaaca gagcacaccg agcagaaacg 1920
gaacgtgatc agaatcatcc ctcaccacaa ctacaacgcc gccatcaaca agtacaacca 1980
cgacattgcc ctgctcgagc tggacgaacc cctggtcctg aactcttacg tgacccctat 2040
ctgtatcgcc gacaaagagt acaccaacat ctttctgaag ttcggcagcg gctacgtgtc 2100
cggctgggga agagttttcc acaagggcag atcagccctg gtgctgcagt acctgagagt 2160
gcccctggtg gatagagcca catgcctgct gagcaccaag ttcaccatct acaacaacat 2220
gttctgcgcc ggcttccacg aaggcggcag agattcttgt cagggcgatt ctggcggccc 2280
tcacgtgaca gaagtcgagg gcacatcttt tctgaccggc atcatcagct ggggcgaaga 2340
gtgtgccatg aaggggaagt acggcatcta taccaaggtg tccagatacg tgaactggat 2400
caaagaaaag accaagctca cctaatgaag atctcatatg cctttaatta aacactagtt 2460
ctatagtgtc acctaaattc cctttagtga gggttaatgg ccgtaggccg ccagaattgg 2520
gtccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 2580
aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 2640
gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 2700
tgtgggaggt tttttcggac tctaggacct gcgcatgcgc ttggcgtaat catggtcata 2760
gctgtttcct gttttccccg tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa 2820
gcgttcagag gaaagcgatc ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct 2880
gccggctcgg ggatgcgggg ggagcgccgg accggagcgg agccccgggc ggctcgctgc 2940
tgccccctag cgggggaggg acgtaattac atccctgggg gctttggggg ggggctgtcc 3000
ctctcaccgc ggtggagctc cagcttttgt tcgaattggg gccccccctc gagggtatcg 3060
atgatatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa 3120
taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt 3180
cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc 3240
acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg 3300
cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta 3360
tctttctagg gttaatctag ctagccttaa gggcgctttc ctggactact tcagacgaac 3420
ttcgtagggc gcataagtct cgaccacgca atgacgcagc gatgcttgaa aaaaacaccg 3480
ctttcaaggc ggctggacga agaccgcaag acaccctcca cctcacctag cctgtatctc 3540
tgcaatagcc taattacttc ggaatctcct gtcgtaattc cttagataaa cggcaattag 3600
gtgacactct aaatctgtgt ggaaccggct tccaaacact ctaccacctc tattagtgac 3660
acagagagaa tccttgagtg gcttctaggt atattgaaca acttcatcac gaattgagca 3720
gtgatcatgg ttctacgtat caaccaatat taaccactgt gctctgtagc attgctaaat 3780
cgggctgtct gtttcaccat agatcgtgag gccatgccca cgggcattag aacttagcct 3840
gtttagcgat aatcccaaca atgagctggg atatgacgag aagtatttag caaccttttc 3900
gtgatcggct acgtaaaacc tcatattacg gcatgtacct gatcattgac ctcaggccat 3960
acgcatgtgg gagaatagag ggaatagcac gatatactgc ctcccttatc ttcctcaggt 4020
tgaggcgcaa tgattatcac tgctacgggc acagtgttac tatcgggatc agggactgtg 4080
tctgtacccg gttctaccac gccgtagctc ccacattgtc gccgtcacgt cgttagggcc 4140
agcctgcgct tggtacgtga tcgaggagta tcgtcttgtg cacacaaaca aaatagactg 4200
ggtcccaagc gcacttagac aaggcattgt actactcaag tgttggatgt gaggaatgat 4260
acaatcgaac cccgtcgttc ggtcaacaat ttggctagag ctgtgttgat acacagcgga 4320
cgggagttac ggggacagtc ctccgagctg gccagcaaga tgcagctgac gcctgcaccg 4380
gaagtaatcc ggaggccggc caggcctcct gcgagggggc gcctcgagac cttgcggccg 4440
caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag 4500
gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag 4560
cgagcgcgca gctgcctgca gg 4582
<210> 20
<211> 4137
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 20
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60
ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120
aggggttcct gcggccgcga agactcttaa ccctagaaag ataatcatat tgtgacgtac 180
gttaaagata atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt 240
tatttattaa tttgaataga tattaagttt tattatattt acacttacat actaataata 300
aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa 360
tttcttctat aaagtaacaa aacttttatc gaatacctgc agcccggggg atgcagaggg 420
acagcccccc cccaaagccc ccagggatgt aattacgtcc ctcccccgct agggggcagc 480
agcgagccgc ccggggctcc gctccggtcc ggcgctcccc ccgcatcccc gagccggcag 540
cgtgcgggga cagcccgggc acggggaagg tggcacggga tcgctttcct ctgaacgctt 600
ctcgctgctc tttgagcctg cagacacctg gggggatacg gggaaaagtt gactgtgcct 660
ttcgatcgaa ctcgtcgact aggcgcggtc tgtctgcaca tttcgtagag cgagtgttcc 720
gatactctaa tctccctagg caaggttcat attgacttag gttacttatt ctccttttgt 780
tgactaagtc aataatcaga atcagcaggt ttggagtcag cttggcaggg atcagcagcc 840
tgggttggaa ggagggggta taaaagcccc ttcaccagga gaagccgtca cacagatcca 900
caagctcctg ctagcaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 960
gttatggccc ttgcgtgcct tgaattactg acactgacat ccactttttc tttttctcca 1020
caggaattcc taggccacca tgcagcgcgt gaacatgatt atggccgagt ctcccggcct 1080
gatcaccatc tgtctgctgg gctatctgct gagcgccgag tgcaccgtgt ttctggatca 1140
cgagaacgcc aacaagatcc tgaacagacc caagcggtac aacagcggca aactggaaga 1200
gttcgtgcag ggtaacctgg aacgcgagtg catggaagag aagtgcagct tcgaagaggc 1260
cagagaggtg ttcgagaaca ccgagagaac caccgagttc tggaagcagt acgtggacgg 1320
cgatcagtgc gagagcaacc cttgtctgaa tggcggcagc tgcaaggatg acatcaacag 1380
ctacgagtgc tggtgcccct tcggcttcga gggcaagaat tgcgagctgg atgtgacctg 1440
caacatcaag aacggcagat gcgagcagtt ctgcaagaac agcgccgaca acaaggtcgt 1500
gtgctcctgc acagagggct acagactggc cgagaatcag aagtcctgcg agcccgctgt 1560
gccctttcca tgtggcagag tgtctgtgtc ccagaccagc aagctgacca gagccgagac 1620
agtgttcccc gatgtggact acgtgaacag caccgaggcc gagacaatcc tggacaacat 1680
cacccagagc acccagtcct tcaacgactt caccagagtc gtcggcggcg aggatgctaa 1740
gcctggacag tttccttggc aagtggtgct gaacggcaag gtggacgctt tttgtggcgg 1800
ctccatcgtg aacgagaagt ggatcgtgac cgccgctcac tgtgtggaaa ccggcgtgaa 1860
gattacagtg gtggccgggg agcacaacat cgaggaaaca gagcacaccg agcagaaacg 1920
gaacgtgatc agaatcatcc ctcaccacaa ctacaacgcc gccatcaaca agtacaacca 1980
cgacattgcc ctgctcgagc tggacgaacc cctggtcctg aactcttacg tgacccctat 2040
ctgtatcgcc gacaaagagt acaccaacat ctttctgaag ttcggcagcg gctacgtgtc 2100
cggctgggga agagttttcc acaagggcag atcagccctg gtgctgcagt acctgagagt 2160
gcccctggtg gatagagcca catgcctgct gagcaccaag ttcaccatct acaacaacat 2220
gttctgcgcc ggcttccacg aaggcggcag agattcttgt cagggcgatt ctggcggccc 2280
tcacgtgaca gaagtcgagg gcacatcttt tctgaccggc atcatcagct ggggcgaaga 2340
gtgtgccatg aaggggaagt acggcatcta taccaaggtg tccagatacg tgaactggat 2400
caaagaaaag accaagctca ccggatccgg agaaggacgg ggaagcctgc ttacatgcgg 2460
agatgtggag gagaatcctg gtcccatggt ctttaccctg gaagatttcg tcggcgactg 2520
gcggcagaca gccggctata atctggacca ggtgctggaa caaggcggag tgtccagcct 2580
gttccagaat ctgggagtgt ccgtgacacc catccagcgg attgtgctgt ctggcgagaa 2640
cggcctgaag atcgacatcc acgtgatcat cccttacgag ggcctgagcg gcgatcagat 2700
gggacagatc gagaagattt tcaaggtggt gtaccccgtg gacgaccacc acttcaaagt 2760
gatcctgcac tacggcaccc tggtcatcga tggcgtgacc cctaacatga tcgactactt 2820
cggcagaccc tacgagggaa tcgccgtgtt cgacggcaag aaaatcaccg tgaccggcac 2880
actgtggaac gggaacaaga tcatcgacga gcggctgatc aaccccgatg gcagcctgct 2940
gttcagagtg accattaacg gcgtgacagg ctggcggctg tgcgaaagga ttctggcctg 3000
atgatctaga gatctcatat gcctttaatt aaacactagt tctatagtgt cacctaaatt 3060
ccctttagtg agggttaatg gccgtaggcc gccagaattg ggtccagaca tgataagata 3120
cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 3180
aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 3240
caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttcgga 3300
ctctaggacc tgcgcatgcg cttggcgtaa tcatggtcat agctgtttcc tgttttcccc 3360
gtatcccccc aggtgtctgc aggctcaaag agcagcgaga agcgttcaga ggaaagcgat 3420
cccgtgccac cttccccgtg cccgggctgt ccccgcacgc tgccggctcg gggatgcggg 3480
gggagcgccg gaccggagcg gagccccggg cggctcgctg ctgcccccta gcgggggagg 3540
gacgtaatta catccctggg ggctttgggg gggggctgtc cctctcaccg cggtggagct 3600
ccagcttttg ttcgaattgg ggccccccct cgagggtatc gatgatatct ataacaagaa 3660
aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta 3720
tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc 3780
gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca 3840
agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga 3900
gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaatcta 3960
gctagcctta agggcgcctc gagaccttgc ggccgcagga acccctagtg atggagttgg 4020
ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac 4080
gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcagg 4137
<210> 21
<211> 1457
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 21
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
1 5 10 15
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg
35 40 45
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
50 55 60
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile
65 70 75 80
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln
85 90 95
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
100 105 110
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser
115 120 125
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
130 135 140
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu
145 150 155 160
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
165 170 175
Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
180 185 190
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr
195 200 205
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly
210 215 220
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp
225 230 235 240
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
245 250 255
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
260 265 270
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
275 280 285
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser
290 295 300
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
305 310 315 320
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His
325 330 335
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
340 345 350
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
355 360 365
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
370 375 380
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
385 390 395 400
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
420 425 430
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met
435 440 445
Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu
450 455 460
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu
465 470 475 480
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
500 505 510
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
515 520 525
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg
545 550 555 560
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
580 585 590
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu
595 600 605
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
610 615 620
Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
625 630 635 640
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
705 710 715 720
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
725 730 735
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys
740 745 750
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu
755 760 765
Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln
770 775 780
Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu
785 790 795 800
Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe
805 810 815
Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp
820 825 830
Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln
835 840 845
Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr
850 855 860
Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His
865 870 875 880
Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile
885 890 895
Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser
900 905 910
Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg
915 920 925
Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val
930 935 940
Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp
945 950 955 960
Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu
965 970 975
Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His
980 985 990
Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe
995 1000 1005
Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn
1010 1015 1020
Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys
1025 1030 1035
Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr
1040 1045 1050
Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr
1055 1060 1065
Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe
1070 1075 1080
Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met
1085 1090 1095
Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met
1100 1105 1110
Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly
1115 1120 1125
Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser
1130 1135 1140
Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg
1145 1150 1155
Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro
1160 1165 1170
Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser
1175 1180 1185
Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro
1190 1195 1200
Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe
1205 1210 1215
Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp
1220 1225 1230
Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu
1235 1240 1245
Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn
1250 1255 1260
Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro
1265 1270 1275
Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly
1280 1285 1290
Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys
1295 1300 1305
Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn
1310 1315 1320
Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln
1325 1330 1335
Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu
1340 1345 1350
Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val
1355 1360 1365
Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys
1370 1375 1380
Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu
1385 1390 1395
Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp
1400 1405 1410
Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr
1415 1420 1425
Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala
1430 1435 1440
Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
1445 1450 1455
<210> 22
<211> 4371
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 22
atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60
accaggagat actacctggg ggctgtggag ctgagctggg actacatgca gtctgacctg 120
ggggagctgc ctgtggatgc caggttcccc cccagagtgc ccaagagctt ccccttcaac 180
acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240
gccaagccca ggcccccctg gatgggcctg ctgggcccca ccatccaggc tgaggtgtat 300
gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360
ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420
gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg 480
aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta cctgagccat 540
gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600
ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg 660
tttgatgagg gcaagagctg gcactctgaa accaagaaca gcctgatgca ggacagggat 720
gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc 780
ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840
accacccctg aggtgcacag catcttcctg gagggccaca ccttcctggt caggaaccac 900
aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg 960
gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag 1020
gcctatgtga aggtggacag ctgccctgag gagccccagc tgaggatgaa gaacaatgag 1080
gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140
gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc 1200
tgggtgcact acattgctgc tgaggaggag gactgggact atgcccccct ggtgctggcc 1260
cctgatgaca ggagctacaa gagccagtac ctgaacaatg gcccccagag gattggcagg 1320
aagtacaaga aggtcaggtt catggcctac actgatgaaa ccttcaagac cagggaggcc 1380
atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440
ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctaccccca tggcatcact 1500
gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggacttc 1560
cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620
accaagtctg accccaggtg cctgaccaga tactacagca gctttgtgaa catggagagg 1680
gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740
aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800
aacaggagct ggtacctgac tgagaacatc cagaggttcc tgcccaaccc tgctggggtg 1860
cagctggagg accctgagtt ccaggccagc aacatcatgc acagcatcaa tggctatgtg 1920
tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980
attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag 2040
atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gttcatgagc 2100
atggagaacc ctggcctgtg gattctgggc tgccacaact ctgacttcag gaacaggggc 2160
atgactgccc tgctgaaagt ctccagctgt gacaagaaca ctggggacta ctatgaggac 2220
agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc 2280
ttcagccaga accccccagt gctgaagagg caccagaggg agatcaccag gaccaccctg 2340
cagtctgacc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag 2400
gactttgaca tctacgacga ggacgagaac cagagcccca ggagcttcca gaagaagacc 2460
aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc 2520
catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc 2580
caggagttca ctgatggcag cttcacccag cccctgtaca gaggggagct gaatgagcac 2640
ctgggcctgc tgggccccta catcagggct gaggtggagg acaacatcat ggtgaccttc 2700
aggaaccagg ccagcaggcc ctacagcttc tacagcagcc tgatcagcta tgaggaggac 2760
cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgaaac caagacctac 2820
ttctggaagg tgcagcacca catggccccc accaaggatg agtttgactg caaggcctgg 2880
gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg 2940
ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000
tttgccctgt tcttcaccat ctttgatgaa accaagagct ggtacttcac tgagaacatg 3060
gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaac 3120
tacaggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180
caggaccaga ggatcaggtg gtacctgctg agcatgggca gcaatgagaa catccacagc 3240
atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300
tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360
tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420
gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc 3480
cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac 3540
tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg 3600
ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gttcagcagc 3660
ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaagtg gcagacctac 3720
aggggcaaca gcactggcac cctgatggtg ttctttggca atgtggacag ctctggcatc 3780
aagcacaaca tcttcaaccc ccccatcatt gccagataca tcaggctgca ccccacccac 3840
tacagcatca ggagcaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc 3900
atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagcagctac 3960
ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg 4020
agcaatgcct ggaggcccca ggtcaacaac cccaaggagt ggctgcaggt ggacttccag 4080
aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gaccagcatg 4140
tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac cctgttcttc 4200
cagaatggca aggtgaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac 4260
agcctggacc cccccctgct gaccagatac ctgaggattc acccccagag ctgggtgcac 4320
cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta c 4371
<210> 23
<211> 232
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 23
gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 60
gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tccccgagcc 120
ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 180
cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aa 232
<210> 24
<211> 231
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 24
ttttccccgt atccccccag gtgtctgcag gctcaaagag cagcgagaag cgttcagagg 60
aaagcgatcc cgtgccacct tccccgtgcc cgggctgtcc ccgcacgctg ccggctcggg 120
gatgcggggg gagcgccgga ccggagcgga gccccgggcg gctcgctgct gccccctagc 180
gggggaggga cgtaattaca tccctggggg ctttgggggg gggctgtccc t 231
<210> 25
<211> 136
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 25
ctggtactgc atgcacgcaa tgctagctgc ccctttcccg tcctgggcac cccgagtctc 60
ccccgacccc gggtcccagg tatgctccca cctccacctg ccccactcac cacctctgct 120
agttccagac acctcc 136
<210> 26
<211> 139
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 26
caagcacgca gcaatgcagc tcaaaacgct tagcctagcc acacccccac gggaaacagc 60
agtgattaac ctttagcaat aaacgaaagt ttaactaagc tatactaacc ccagggttgg 120
tcaatttcgt gccagccac 139
<210> 27
<211> 278
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 27
ctggtactgc atgcacgcaa tgctagctgc ccctttcccg tcctgggcac cccgagtctc 60
ccccgacccc gggtcccagg tatgctccca cctccacctg ccccactcac cacctctgct 120
agttccagac acctcccaag cacgcagcaa tgcagctcaa aacgcttagc ctagccacac 180
ccccacggga aacagcagtg attaaccttt agcaataaac gaaagtttaa ctaagctata 240
ctaaccccag ggttggtcaa tttcgtgcca gccacacc 278
<210> 28
<211> 6828
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 28
ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg cgtaaaattg 60
acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa tagatattaa 120
gttttattat atttacactt acatactaat aataaattca acaaacaatt tatttatgtt 180
tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta acaaaacttt 240
tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa gcccccaggg 300
atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg ctccgctccg 360
gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc gggcacgggg 420
aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag cctgcagaca 480
cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg gaattgtacc 540
gcgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga ggagcaaaca 600
ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac cccagttatc 660
ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat taaccaaggt 720
caccccagtt atcggaggag caaacagggg ctaagtccac ggatcccact gggaggatgt 780
tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg acatgtttga 840
acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg cacatttcgt 900
agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt gtaggttact 960
tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc 1020
agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc aggagaagcc 1080
gtcacacaga tccacaagct cctgaagagg taagggttta agggatggtt ggttggtggg 1140
gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca ggttggacgc 1200
gtcgccacca tgcagattga gctgagcacc tgcttcttcc tgtgcctgct gaggttctgc 1260
ttctctgcca ccaggagata ctacctgggg gctgtggagc tgagctggga ctacatgcag 1320
tctgacctgg gggagctgcc tgtggatgcc aggttccccc ccagagtgcc caagagcttc 1380
cccttcaaca cctctgtggt gtacaagaag accctgtttg tggagttcac tgaccacctg 1440
ttcaacattg ccaagcccag gcccccctgg atgggcctgc tgggccccac catccaggct 1500
gaggtgtatg acactgtggt gatcaccctg aagaacatgg ccagccaccc tgtgagcctg 1560
catgctgtgg gggtgagcta ctggaaggcc tctgaggggg ctgagtatga tgaccagacc 1620
agccagaggg agaaggagga tgacaaggtg ttccctgggg gcagccacac ctatgtgtgg 1680
caggtgctga aggagaatgg ccccatggcc tctgaccccc tgtgcctgac ctacagctac 1740
ctgagccatg tggacctggt gaaggacctg aactctggcc tgattggggc cctgctggtg 1800
tgcagggagg gcagcctggc caaggagaag acccagaccc tgcacaagtt catcctgctg 1860
tttgctgtgt ttgatgaggg caagagctgg cactctgaaa ccaagaacag cctgatgcag 1920
gacagggatg ctgcctctgc cagggcctgg cccaagatgc acactgtgaa tggctatgtg 1980
aacaggagcc tgcctggcct gattggctgc cacaggaagt ctgtgtactg gcatgtgatt 2040
ggcatgggca ccacccctga ggtgcacagc atcttcctgg agggccacac cttcctggtc 2100
aggaaccaca ggcaggccag cctggagatc agccccatca ccttcctgac tgcccagacc 2160
ctgctgatgg acctgggcca gttcctgctg ttctgccaca tcagcagcca ccagcatgat 2220
ggcatggagg cctatgtgaa ggtggacagc tgccctgagg agccccagct gaggatgaag 2280
aacaatgagg aggctgagga ctatgatgat gacctgactg actctgagat ggatgtggtg 2340
aggtttgatg atgacaacag ccccagcttc atccagatca ggtctgtggc caagaagcac 2400
cccaagacct gggtgcacta cattgctgct gaggaggagg actgggacta tgcccccctg 2460
gtgctggccc ctgatgacag gagctacaag agccagtacc tgaacaatgg cccccagagg 2520
attggcagga agtacaagaa ggtcaggttc atggcctaca ctgatgaaac cttcaagacc 2580
agggaggcca tccagcatga gtctggcatc ctgggccccc tgctgtatgg ggaggtgggg 2640
gacaccctgc tgatcatctt caagaaccag gccagcaggc cctacaacat ctacccccat 2700
ggcatcactg atgtgaggcc cctgtacagc aggaggctgc ccaagggggt gaagcacctg 2760
aaggacttcc ccatcctgcc tggggagatc ttcaagtaca agtggactgt gactgtggag 2820
gatggcccca ccaagtctga ccccaggtgc ctgaccagat actacagcag ctttgtgaac 2880
atggagaggg acctggcctc tggcctgatt ggccccctgc tgatctgcta caaggagtct 2940
gtggaccaga ggggcaacca gatcatgtct gacaagagga atgtgatcct gttctctgtg 3000
tttgatgaga acaggagctg gtacctgact gagaacatcc agaggttcct gcccaaccct 3060
gctggggtgc agctggagga ccctgagttc caggccagca acatcatgca cagcatcaat 3120
ggctatgtgt ttgacagcct gcagctgtct gtgtgcctgc atgaggtggc ctactggtac 3180
atcctgagca ttggggccca gactgacttc ctgtctgtgt tcttctctgg ctacaccttc 3240
aagcacaaga tggtgtatga ggacaccctg accctgttcc ccttctctgg ggagactgtg 3300
ttcatgagca tggagaaccc tggcctgtgg attctgggct gccacaactc tgacttcagg 3360
aacaggggca tgactgccct gctgaaagtc tccagctgtg acaagaacac tggggactac 3420
tatgaggaca gctatgagga catctctgcc tacctgctga gcaagaacaa tgccattgag 3480
cccaggagct tcagccagaa ccccccagtg ctgaagaggc accagaggga gatcaccagg 3540
accaccctgc agtctgacca ggaggagatt gactatgatg acaccatctc tgtggagatg 3600
aagaaggagg actttgacat ctacgacgag gacgagaacc agagccccag gagcttccag 3660
aagaagacca ggcactactt cattgctgct gtggagaggc tgtgggacta tggcatgagc 3720
agcagccccc atgtgctgag gaacagggcc cagtctggct ctgtgcccca gttcaagaag 3780
gtggtgttcc aggagttcac tgatggcagc ttcacccagc ccctgtacag aggggagctg 3840
aatgagcacc tgggcctgct gggcccctac atcagggctg aggtggagga caacatcatg 3900
gtgaccttca ggaaccaggc cagcaggccc tacagcttct acagcagcct gatcagctat 3960
gaggaggacc agaggcaggg ggctgagccc aggaagaact ttgtgaagcc caatgaaacc 4020
aagacctact tctggaaggt gcagcaccac atggccccca ccaaggatga gtttgactgc 4080
aaggcctggg cctacttctc tgatgtggac ctggagaagg atgtgcactc tggcctgatt 4140
ggccccctgc tggtgtgcca caccaacacc ctgaaccctg cccatggcag gcaggtgact 4200
gtgcaggagt ttgccctgtt cttcaccatc tttgatgaaa ccaagagctg gtacttcact 4260
gagaacatgg agaggaactg cagggccccc tgcaacatcc agatggagga ccccaccttc 4320
aaggagaact acaggttcca tgccatcaat ggctacatca tggacaccct gcctggcctg 4380
gtgatggccc aggaccagag gatcaggtgg tacctgctga gcatgggcag caatgagaac 4440
atccacagca tccacttctc tggccatgtg ttcactgtga ggaagaagga ggagtacaag 4500
atggccctgt acaacctgta ccctggggtg tttgagactg tggagatgct gcccagcaag 4560
gctggcatct ggagggtgga gtgcctgatt ggggagcacc tgcatgctgg catgagcacc 4620
ctgttcctgg tgtacagcaa caagtgccag acccccctgg gcatggcctc tggccacatc 4680
agggacttcc agatcactgc ctctggccag tatggccagt gggcccccaa gctggccagg 4740
ctgcactact ctggcagcat caatgcctgg agcaccaagg agcccttcag ctggatcaag 4800
gtggacctgc tggcccccat gatcatccat ggcatcaaga cccagggggc caggcagaag 4860
ttcagcagcc tgtacatcag ccagttcatc atcatgtaca gcctggatgg caagaagtgg 4920
cagacctaca ggggcaacag cactggcacc ctgatggtgt tctttggcaa tgtggacagc 4980
tctggcatca agcacaacat cttcaacccc cccatcattg ccagatacat caggctgcac 5040
cccacccact acagcatcag gagcaccctg aggatggagc tgatgggctg tgacctgaac 5100
agctgcagca tgcccctggg catggagagc aaggccatct ctgatgccca gatcactgcc 5160
agcagctact tcaccaacat gtttgccacc tggagcccca gcaaggccag gctgcacctg 5220
cagggcagga gcaatgcctg gaggccccag gtcaacaacc ccaaggagtg gctgcaggtg 5280
gacttccaga agaccatgaa ggtgactggg gtgaccaccc agggggtgaa gagcctgctg 5340
accagcatgt atgtgaagga gttcctgatc agcagcagcc aggatggcca ccagtggacc 5400
ctgttcttcc agaatggcaa ggtgaaggtg ttccagggca accaggacag cttcacccct 5460
gtggtgaaca gcctggaccc ccccctgctg accagatacc tgaggattca cccccagagc 5520
tgggtgcacc agattgccct gaggatggag gtgctgggct gtgaggccca ggacctgtac 5580
tgatgaaacg ttagatctgg taccgatcac atatgccttt aattaaacac tggtactgca 5640
tgcacgcaat gctagctgcc cctttcccgt cctgggcacc ccgagtctcc cccgaccccg 5700
ggtcccaggt atgctcccac ctccacctgc cccactcacc acctctgcta gttccagaca 5760
cctcccaagc acgcagcaat gcagctcaaa acgcttagcc tagccacacc cccacgggaa 5820
acagcagtga ttaaccttta gcaataaacg aaagtttaac taagctatac taaccccagg 5880
gttggtcaat ttcgtgccag ccacacccta gttctatagt gtcacctaaa ttccctttag 5940
tgagggttaa tggccgtagg ccgccagaat tgggtccaga catgataaga tacattgatg 6000
agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg 6060
atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt 6120
gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttcg gactctagga 6180
cctgcgcatg cgcttggcgt aatcatggtc atagctgttt cctgttttcc ccgtatcccc 6240
ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg atcccgtgcc 6300
accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg gggggagcgc 6360
cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga gggacgtaat 6420
tacatccctg ggggctttgg gggggggctg tccctctcac cgcggtggag ctccagcttt 6480
tgttcgaatt ggggcccccc ctcgagggta tcgatgatat ctataacaag aaaatatata 6540
tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa 6600
atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt 6660
tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga 6720
ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt 6780
tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaa 6828
<210> 29
<211> 100
<212> RNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 29
ccugggcacc agcgccggug guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 30
<211> 100
<212> RNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 30
gaugcucgcc cucccguccc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> 31
<211> 1591
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 31
Met Ala Pro Lys Lys Lys Arg Lys Val Glu Gly Ile Lys Ser Asn Ile
1 5 10 15
Ser Leu Leu Lys Asp Glu Leu Arg Gly Gln Ile Ser His Ile Ser His
20 25 30
Glu Tyr Leu Ser Leu Ile Asp Leu Ala Phe Asp Ser Lys Gln Asn Arg
35 40 45
Leu Phe Glu Met Lys Val Leu Glu Leu Leu Val Asn Glu Tyr Gly Phe
50 55 60
Lys Gly Arg His Leu Gly Gly Ser Arg Lys Pro Asp Gly Ile Val Tyr
65 70 75 80
Ser Thr Thr Leu Glu Asp Asn Phe Gly Ile Ile Val Asp Thr Lys Ala
85 90 95
Tyr Ser Glu Gly Tyr Ser Leu Pro Ile Ser Gln Ala Asp Glu Met Glu
100 105 110
Arg Tyr Val Arg Glu Asn Ser Asn Arg Asp Glu Glu Val Asn Pro Asn
115 120 125
Lys Trp Trp Glu Asn Phe Ser Glu Glu Val Lys Lys Tyr Tyr Phe Val
130 135 140
Phe Ile Ser Gly Ser Phe Lys Gly Lys Phe Glu Glu Gln Leu Arg Arg
145 150 155 160
Leu Ser Met Thr Thr Gly Val Asn Gly Ser Ala Val Asn Val Val Asn
165 170 175
Leu Leu Leu Gly Ala Glu Lys Ile Arg Ser Gly Glu Met Thr Ile Glu
180 185 190
Glu Leu Glu Arg Ala Met Phe Asn Asn Ser Glu Phe Ile Leu Lys Tyr
195 200 205
Gly Gly Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly
210 215 220
Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro
225 230 235 240
Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
245 250 255
Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu
260 265 270
Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys
275 280 285
Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys
290 295 300
Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
305 310 315 320
Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp
325 330 335
Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys
340 345 350
Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu
355 360 365
Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly
370 375 380
Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
385 390 395 400
Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser
405 410 415
Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg
420 425 430
Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly
435 440 445
Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe
450 455 460
Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys
465 470 475 480
Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp
485 490 495
Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile
500 505 510
Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro
515 520 525
Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu
530 535 540
Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
545 550 555 560
Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp
565 570 575
Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu
580 585 590
Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu
595 600 605
Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His
610 615 620
Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
625 630 635 640
Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu
645 650 655
Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser
660 665 670
Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp
675 680 685
Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile
690 695 700
Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu
705 710 715 720
Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu
725 730 735
Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu
740 745 750
Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn
755 760 765
Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile
770 775 780
Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
785 790 795 800
Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys
805 810 815
Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val
820 825 830
Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu
835 840 845
Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys
850 855 860
Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn
865 870 875 880
Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys
885 890 895
Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp
900 905 910
Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln
915 920 925
Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala
930 935 940
Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val
945 950 955 960
Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala
965 970 975
Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg
980 985 990
Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu
995 1000 1005
Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu
1010 1015 1020
Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln
1025 1030 1035
Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp Ala Ile
1040 1045 1050
Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
1055 1060 1065
Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro
1070 1075 1080
Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu
1085 1090 1095
Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr
1100 1105 1110
Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe
1115 1120 1125
Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val
1130 1135 1140
Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn
1145 1150 1155
Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
1160 1165 1170
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
1175 1180 1185
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala
1190 1195 1200
Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser
1205 1210 1215
Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met
1220 1225 1230
Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr
1235 1240 1245
Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr
1250 1255 1260
Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn
1265 1270 1275
Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala
1280 1285 1290
Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys
1295 1300 1305
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu
1310 1315 1320
Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp
1325 1330 1335
Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr
1340 1345 1350
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys
1355 1360 1365
Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg
1370 1375 1380
Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly
1385 1390 1395
Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr
1400 1405 1410
Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1415 1420 1425
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys
1430 1435 1440
Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys
1445 1450 1455
Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln
1460 1465 1470
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe
1475 1480 1485
Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu
1490 1495 1500
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala
1505 1510 1515
Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro
1520 1525 1530
Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr
1535 1540 1545
Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser
1550 1555 1560
Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly
1565 1570 1575
Gly Asp Gly Ser Pro Lys Lys Lys Arg Lys Val Ser Ser
1580 1585 1590
<210> 32
<211> 199
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 32
Glu Gly Ile Lys Ser Asn Ile Ser Leu Leu Lys Asp Glu Leu Arg Gly
1 5 10 15
Gln Ile Ser His Ile Ser His Glu Tyr Leu Ser Leu Ile Asp Leu Ala
20 25 30
Phe Asp Ser Lys Gln Asn Arg Leu Phe Glu Met Lys Val Leu Glu Leu
35 40 45
Leu Val Asn Glu Tyr Gly Phe Lys Gly Arg His Leu Gly Gly Ser Arg
50 55 60
Lys Pro Asp Gly Ile Val Tyr Ser Thr Thr Leu Glu Asp Asn Phe Gly
65 70 75 80
Ile Ile Val Asp Thr Lys Ala Tyr Ser Glu Gly Tyr Ser Leu Pro Ile
85 90 95
Ser Gln Ala Asp Glu Met Glu Arg Tyr Val Arg Glu Asn Ser Asn Arg
100 105 110
Asp Glu Glu Val Asn Pro Asn Lys Trp Trp Glu Asn Phe Ser Glu Glu
115 120 125
Val Lys Lys Tyr Tyr Phe Val Phe Ile Ser Gly Ser Phe Lys Gly Lys
130 135 140
Phe Glu Glu Gln Leu Arg Arg Leu Ser Met Thr Thr Gly Val Asn Gly
145 150 155 160
Ser Ala Val Asn Val Val Asn Leu Leu Leu Gly Ala Glu Lys Ile Arg
165 170 175
Ser Gly Glu Met Thr Ile Glu Glu Leu Glu Arg Ala Met Phe Asn Asn
180 185 190
Ser Glu Phe Ile Leu Lys Tyr
195
<210> 33
<211> 1369
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 33
Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly
1 5 10 15
Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys
20 25 30
Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly
35 40 45
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys
50 55 60
Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr
65 70 75 80
Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe
85 90 95
Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His
100 105 110
Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His
115 120 125
Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser
130 135 140
Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met
145 150 155 160
Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp
165 170 175
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn
180 185 190
Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys
195 200 205
Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu
210 215 220
Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu
225 230 235 240
Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp
245 250 255
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp
260 265 270
Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu
275 280 285
Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile
290 295 300
Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met
305 310 315 320
Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala
325 330 335
Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp
340 345 350
Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln
355 360 365
Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly
370 375 380
Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys
385 390 395 400
Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly
405 410 415
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu
420 425 430
Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro
435 440 445
Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met
450 455 460
Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val
465 470 475 480
Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn
485 490 495
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu
500 505 510
Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr
515 520 525
Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys
530 535 540
Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val
545 550 555 560
Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser
565 570 575
Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr
580 585 590
Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn
595 600 605
Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu
610 615 620
Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His
625 630 635 640
Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr
645 650 655
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys
660 665 670
Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala
675 680 685
Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys
690 695 700
Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His
705 710 715 720
Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile
725 730 735
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg
740 745 750
His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr
755 760 765
Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu
770 775 780
Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val
785 790 795 800
Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln
805 810 815
Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu
820 825 830
Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys Asp
835 840 845
Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly
850 855 860
Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn
865 870 875 880
Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe
885 890 895
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys
900 905 910
Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys
915 920 925
His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu
930 935 940
Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys
945 950 955 960
Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu
965 970 975
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val
980 985 990
Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val
995 1000 1005
Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr
1025 1030 1035
Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn
1040 1045 1050
Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr
1055 1060 1065
Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg
1070 1075 1080
Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu
1085 1090 1095
Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg
1100 1105 1110
Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys
1115 1120 1125
Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu
1130 1135 1140
Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser
1145 1150 1155
Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe
1160 1165 1170
Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu
1175 1180 1185
Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe
1190 1195 1200
Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu
1205 1210 1215
Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn
1220 1225 1230
Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro
1235 1240 1245
Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg
1265 1270 1275
Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr
1280 1285 1290
Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile
1295 1300 1305
Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe
1310 1315 1320
Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr
1325 1330 1335
Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly
1340 1345 1350
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Gly
1355 1360 1365
Ser
<210> 34
<211> 7336
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 34
tgtacataga ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg 60
cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa 120
tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt 180
tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta 240
acaaaacttt tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa 300
gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg 360
ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc 420
gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag 480
cctgcagaca cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg 540
gaattgtacc gcgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga 600
ggagcaaaca ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac 660
cccagttatc ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat 720
taaccaaggt caccccagtt atcggaggag caaacagggg ctaagtccac ggatcccact 780
gggaggatgt tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg 840
acatgtttga acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg 900
cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt 960
gtaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag 1020
tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc 1080
aggagaagcc gtcacacaga tccacaagct cctgaagagg taagggttta agggatggtt 1140
ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca 1200
ggttggacgc gtcgccacca tgcagattga gctgagcacc tgcttcttcc tgtgcctgct 1260
gaggttctgc ttctctgcca ccaggagata ctacctgggg gctgtggagc tgagctggga 1320
ctacatgcag tctgacctgg gggagctgcc tgtggatgcc aggttccccc ccagagtgcc 1380
caagagcttc cccttcaaca cctctgtggt gtacaagaag accctgtttg tggagttcac 1440
tgaccacctg ttcaacattg ccaagcccag gcccccctgg atgggcctgc tgggccccac 1500
catccaggct gaggtgtatg acactgtggt gatcaccctg aagaacatgg ccagccaccc 1560
tgtgagcctg catgctgtgg gggtgagcta ctggaaggcc tctgaggggg ctgagtatga 1620
tgaccagacc agccagaggg agaaggagga tgacaaggtg ttccctgggg gcagccacac 1680
ctatgtgtgg caggtgctga aggagaatgg ccccatggcc tctgaccccc tgtgcctgac 1740
ctacagctac ctgagccatg tggacctggt gaaggacctg aactctggcc tgattggggc 1800
cctgctggtg tgcagggagg gcagcctggc caaggagaag acccagaccc tgcacaagtt 1860
catcctgctg tttgctgtgt ttgatgaggg caagagctgg cactctgaaa ccaagaacag 1920
cctgatgcag gacagggatg ctgcctctgc cagggcctgg cccaagatgc acactgtgaa 1980
tggctatgtg aacaggagcc tgcctggcct gattggctgc cacaggaagt ctgtgtactg 2040
gcatgtgatt ggcatgggca ccacccctga ggtgcacagc atcttcctgg agggccacac 2100
cttcctggtc aggaaccaca ggcaggccag cctggagatc agccccatca ccttcctgac 2160
tgcccagacc ctgctgatgg acctgggcca gttcctgctg ttctgccaca tcagcagcca 2220
ccagcatgat ggcatggagg cctatgtgaa ggtggacagc tgccctgagg agccccagct 2280
gaggatgaag aacaatgagg aggctgagga ctatgatgat gacctgactg actctgagat 2340
ggatgtggtg aggtttgatg atgacaacag ccccagcttc atccagatca ggtctgtggc 2400
caagaagcac cccaagacct gggtgcacta cattgctgct gaggaggagg actgggacta 2460
tgcccccctg gtgctggccc ctgatgacag gagctacaag agccagtacc tgaacaatgg 2520
cccccagagg attggcagga agtacaagaa ggtcaggttc atggcctaca ctgatgaaac 2580
cttcaagacc agggaggcca tccagcatga gtctggcatc ctgggccccc tgctgtatgg 2640
ggaggtgggg gacaccctgc tgatcatctt caagaaccag gccagcaggc cctacaacat 2700
ctacccccat ggcatcactg atgtgaggcc cctgtacagc aggaggctgc ccaagggggt 2760
gaagcacctg aaggacttcc ccatcctgcc tggggagatc ttcaagtaca agtggactgt 2820
gactgtggag gatggcccca ccaagtctga ccccaggtgc ctgaccagat actacagcag 2880
ctttgtgaac atggagaggg acctggcctc tggcctgatt ggccccctgc tgatctgcta 2940
caaggagtct gtggaccaga ggggcaacca gatcatgtct gacaagagga atgtgatcct 3000
gttctctgtg tttgatgaga acaggagctg gtacctgact gagaacatcc agaggttcct 3060
gcccaaccct gctggggtgc agctggagga ccctgagttc caggccagca acatcatgca 3120
cagcatcaat ggctatgtgt ttgacagcct gcagctgtct gtgtgcctgc atgaggtggc 3180
ctactggtac atcctgagca ttggggccca gactgacttc ctgtctgtgt tcttctctgg 3240
ctacaccttc aagcacaaga tggtgtatga ggacaccctg accctgttcc ccttctctgg 3300
ggagactgtg ttcatgagca tggagaaccc tggcctgtgg attctgggct gccacaactc 3360
tgacttcagg aacaggggca tgactgccct gctgaaagtc tccagctgtg acaagaacac 3420
tggggactac tatgaggaca gctatgagga catctctgcc tacctgctga gcaagaacaa 3480
tgccattgag cccaggagct tcagccagaa ccccccagtg ctgaagaggc accagaggga 3540
gatcaccagg accaccctgc agtctgacca ggaggagatt gactatgatg acaccatctc 3600
tgtggagatg aagaaggagg actttgacat ctacgacgag gacgagaacc agagccccag 3660
gagcttccag aagaagacca ggcactactt cattgctgct gtggagaggc tgtgggacta 3720
tggcatgagc agcagccccc atgtgctgag gaacagggcc cagtctggct ctgtgcccca 3780
gttcaagaag gtggtgttcc aggagttcac tgatggcagc ttcacccagc ccctgtacag 3840
aggggagctg aatgagcacc tgggcctgct gggcccctac atcagggctg aggtggagga 3900
caacatcatg gtgaccttca ggaaccaggc cagcaggccc tacagcttct acagcagcct 3960
gatcagctat gaggaggacc agaggcaggg ggctgagccc aggaagaact ttgtgaagcc 4020
caatgaaacc aagacctact tctggaaggt gcagcaccac atggccccca ccaaggatga 4080
gtttgactgc aaggcctggg cctacttctc tgatgtggac ctggagaagg atgtgcactc 4140
tggcctgatt ggccccctgc tggtgtgcca caccaacacc ctgaaccctg cccatggcag 4200
gcaggtgact gtgcaggagt ttgccctgtt cttcaccatc tttgatgaaa ccaagagctg 4260
gtacttcact gagaacatgg agaggaactg cagggccccc tgcaacatcc agatggagga 4320
ccccaccttc aaggagaact acaggttcca tgccatcaat ggctacatca tggacaccct 4380
gcctggcctg gtgatggccc aggaccagag gatcaggtgg tacctgctga gcatgggcag 4440
caatgagaac atccacagca tccacttctc tggccatgtg ttcactgtga ggaagaagga 4500
ggagtacaag atggccctgt acaacctgta ccctggggtg tttgagactg tggagatgct 4560
gcccagcaag gctggcatct ggagggtgga gtgcctgatt ggggagcacc tgcatgctgg 4620
catgagcacc ctgttcctgg tgtacagcaa caagtgccag acccccctgg gcatggcctc 4680
tggccacatc agggacttcc agatcactgc ctctggccag tatggccagt gggcccccaa 4740
gctggccagg ctgcactact ctggcagcat caatgcctgg agcaccaagg agcccttcag 4800
ctggatcaag gtggacctgc tggcccccat gatcatccat ggcatcaaga cccagggggc 4860
caggcagaag ttcagcagcc tgtacatcag ccagttcatc atcatgtaca gcctggatgg 4920
caagaagtgg cagacctaca ggggcaacag cactggcacc ctgatggtgt tctttggcaa 4980
tgtggacagc tctggcatca agcacaacat cttcaacccc cccatcattg ccagatacat 5040
caggctgcac cccacccact acagcatcag gagcaccctg aggatggagc tgatgggctg 5100
tgacctgaac agctgcagca tgcccctggg catggagagc aaggccatct ctgatgccca 5160
gatcactgcc agcagctact tcaccaacat gtttgccacc tggagcccca gcaaggccag 5220
gctgcacctg cagggcagga gcaatgcctg gaggccccag gtcaacaacc ccaaggagtg 5280
gctgcaggtg gacttccaga agaccatgaa ggtgactggg gtgaccaccc agggggtgaa 5340
gagcctgctg accagcatgt atgtgaagga gttcctgatc agcagcagcc aggatggcca 5400
ccagtggacc ctgttcttcc agaatggcaa ggtgaaggtg ttccagggca accaggacag 5460
cttcacccct gtggtgaaca gcctggaccc ccccctgctg accagatacc tgaggattca 5520
cccccagagc tgggtgcacc agattgccct gaggatggag gtgctgggct gtgaggccca 5580
ggacctgtac tgatgaaacg ttagatctgg taccgatcac atatgccttt aattaaacac 5640
tggtactgca tgcacgcaat gctagctgcc cctttcccgt cctgggcacc ccgagtctcc 5700
cccgaccccg ggtcccaggt atgctcccac ctccacctgc cccactcacc acctctgcta 5760
gttccagaca cctcccaagc acgcagcaat gcagctcaaa acgcttagcc tagccacacc 5820
cccacgggaa acagcagtga ttaaccttta gcaataaacg aaagtttaac taagctatac 5880
taaccccagg gttggtcaat ttcgtgccag ccacacccta gttctatagt gtcacctaaa 5940
ttccctttag tgagggttaa tggccgtagg ccgccagaat tgggtccaga catgataaga 6000
tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 6060
gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 6120
aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttcg 6180
gactctagga cctgcgcatg cgcttggcgt aatcatggtc atagctgttt cctgttttcc 6240
ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca gaggaaagcg 6300
atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct cggggatgcg 6360
gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc tagcggggga 6420
gggacgtaat tacatccctg ggggctttgg gggggggctg tccctctcac cgcggtggag 6480
ctccagcttt tgttcgaatt ggggcccccc ctcgagggta tcgatgatat ctataacaag 6540
aaaatatata tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg 6600
tatgagttaa atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg 6660
tcgttatagt tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc 6720
caagcggcga ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga 6780
gagcaatatt tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaatc 6840
tagctagcct taagggcgct ggtctcatga cgcgtagccc gcctaatgag cgggcttttt 6900
tttggcttgt tgtccacaac cgttaaacct taaaagcttt aaaagcctta tatattcttt 6960
tttttcttat aaaacttaaa accttagagg ctatttaagt tgctgattta tattaatttt 7020
attgttcaaa catgagagct tagtacgtga aacatgagag cttagtacgt tagccatgag 7080
agcttagtac gttagccatg agggtttagt tcgttaaaca tgagagctta gtacgttaaa 7140
catgagagct tagtacgtac tatcaacagg ttgaactgct gatccacgtt gtggtagaat 7200
tggtaaagag agtcgtgtaa aatatcgagt tcgcacatct tgttgtctga ttattgattt 7260
ttggcgaaac catttgatca tatgacaaga tgtgtatcta ccttaactta atgattttga 7320
taaaaatcat taggta 7336
<210> 35
<211> 7058
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 35
tgtacataga ttaaccctag aaagataatc atattgtgac gtacgttaaa gataatcatg 60
cgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta ttaatttgaa 120
tagatattaa gttttattat atttacactt acatactaat aataaattca acaaacaatt 180
tatttatgtt tatttattta ttaaaaaaaa acaaaaactc aaaatttctt ctataaagta 240
acaaaacttt tatcgaatac ctgcagcccg ggggatgcag agggacagcc cccccccaaa 300
gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg 360
ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc 420
gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag 480
cctgcagaca cctgggggga tacggggaaa agttgactgt gcctttcgat cgaaccatgg 540
gaattgtacc gcgggggagg ctgctggtga atattaacca aggtcacccc agttatcgga 600
ggagcaaaca ggggctaagt ccaccggggg aggctgctgg tgaatattaa ccaaggtcac 660
cccagttatc ggaggagcaa acaggggcta agtccaccgg gggaggctgc tggtgaatat 720
taaccaaggt caccccagtt atcggaggag caaacagggg ctaagtccac ggatcccact 780
gggaggatgt tgagtaagat ggaaaactac tgatgaccct tgcagagaca gagtattagg 840
acatgtttga acaggggccg ggcgatcagc aggtagctct agaggatccc cgtctgtctg 900
cacatttcgt agagcgagtg ttccgatact ctaatctccc taggcaaggt tcatatttgt 960
gtaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag 1020
tcagcttggc agggatcagc agcctgggtt ggaaggaggg ggtataaaag ccccttcacc 1080
aggagaagcc gtcacacaga tccacaagct cctgaagagg taagggttta agggatggtt 1140
ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca ctttttttca 1200
ggttggacgc gtcgccacca tgcagattga gctgagcacc tgcttcttcc tgtgcctgct 1260
gaggttctgc ttctctgcca ccaggagata ctacctgggg gctgtggagc tgagctggga 1320
ctacatgcag tctgacctgg gggagctgcc tgtggatgcc aggttccccc ccagagtgcc 1380
caagagcttc cccttcaaca cctctgtggt gtacaagaag accctgtttg tggagttcac 1440
tgaccacctg ttcaacattg ccaagcccag gcccccctgg atgggcctgc tgggccccac 1500
catccaggct gaggtgtatg acactgtggt gatcaccctg aagaacatgg ccagccaccc 1560
tgtgagcctg catgctgtgg gggtgagcta ctggaaggcc tctgaggggg ctgagtatga 1620
tgaccagacc agccagaggg agaaggagga tgacaaggtg ttccctgggg gcagccacac 1680
ctatgtgtgg caggtgctga aggagaatgg ccccatggcc tctgaccccc tgtgcctgac 1740
ctacagctac ctgagccatg tggacctggt gaaggacctg aactctggcc tgattggggc 1800
cctgctggtg tgcagggagg gcagcctggc caaggagaag acccagaccc tgcacaagtt 1860
catcctgctg tttgctgtgt ttgatgaggg caagagctgg cactctgaaa ccaagaacag 1920
cctgatgcag gacagggatg ctgcctctgc cagggcctgg cccaagatgc acactgtgaa 1980
tggctatgtg aacaggagcc tgcctggcct gattggctgc cacaggaagt ctgtgtactg 2040
gcatgtgatt ggcatgggca ccacccctga ggtgcacagc atcttcctgg agggccacac 2100
cttcctggtc aggaaccaca ggcaggccag cctggagatc agccccatca ccttcctgac 2160
tgcccagacc ctgctgatgg acctgggcca gttcctgctg ttctgccaca tcagcagcca 2220
ccagcatgat ggcatggagg cctatgtgaa ggtggacagc tgccctgagg agccccagct 2280
gaggatgaag aacaatgagg aggctgagga ctatgatgat gacctgactg actctgagat 2340
ggatgtggtg aggtttgatg atgacaacag ccccagcttc atccagatca ggtctgtggc 2400
caagaagcac cccaagacct gggtgcacta cattgctgct gaggaggagg actgggacta 2460
tgcccccctg gtgctggccc ctgatgacag gagctacaag agccagtacc tgaacaatgg 2520
cccccagagg attggcagga agtacaagaa ggtcaggttc atggcctaca ctgatgaaac 2580
cttcaagacc agggaggcca tccagcatga gtctggcatc ctgggccccc tgctgtatgg 2640
ggaggtgggg gacaccctgc tgatcatctt caagaaccag gccagcaggc cctacaacat 2700
ctacccccat ggcatcactg atgtgaggcc cctgtacagc aggaggctgc ccaagggggt 2760
gaagcacctg aaggacttcc ccatcctgcc tggggagatc ttcaagtaca agtggactgt 2820
gactgtggag gatggcccca ccaagtctga ccccaggtgc ctgaccagat actacagcag 2880
ctttgtgaac atggagaggg acctggcctc tggcctgatt ggccccctgc tgatctgcta 2940
caaggagtct gtggaccaga ggggcaacca gatcatgtct gacaagagga atgtgatcct 3000
gttctctgtg tttgatgaga acaggagctg gtacctgact gagaacatcc agaggttcct 3060
gcccaaccct gctggggtgc agctggagga ccctgagttc caggccagca acatcatgca 3120
cagcatcaat ggctatgtgt ttgacagcct gcagctgtct gtgtgcctgc atgaggtggc 3180
ctactggtac atcctgagca ttggggccca gactgacttc ctgtctgtgt tcttctctgg 3240
ctacaccttc aagcacaaga tggtgtatga ggacaccctg accctgttcc ccttctctgg 3300
ggagactgtg ttcatgagca tggagaaccc tggcctgtgg attctgggct gccacaactc 3360
tgacttcagg aacaggggca tgactgccct gctgaaagtc tccagctgtg acaagaacac 3420
tggggactac tatgaggaca gctatgagga catctctgcc tacctgctga gcaagaacaa 3480
tgccattgag cccaggagct tcagccagaa ccccccagtg ctgaagaggc accagaggga 3540
gatcaccagg accaccctgc agtctgacca ggaggagatt gactatgatg acaccatctc 3600
tgtggagatg aagaaggagg actttgacat ctacgacgag gacgagaacc agagccccag 3660
gagcttccag aagaagacca ggcactactt cattgctgct gtggagaggc tgtgggacta 3720
tggcatgagc agcagccccc atgtgctgag gaacagggcc cagtctggct ctgtgcccca 3780
gttcaagaag gtggtgttcc aggagttcac tgatggcagc ttcacccagc ccctgtacag 3840
aggggagctg aatgagcacc tgggcctgct gggcccctac atcagggctg aggtggagga 3900
caacatcatg gtgaccttca ggaaccaggc cagcaggccc tacagcttct acagcagcct 3960
gatcagctat gaggaggacc agaggcaggg ggctgagccc aggaagaact ttgtgaagcc 4020
caatgaaacc aagacctact tctggaaggt gcagcaccac atggccccca ccaaggatga 4080
gtttgactgc aaggcctggg cctacttctc tgatgtggac ctggagaagg atgtgcactc 4140
tggcctgatt ggccccctgc tggtgtgcca caccaacacc ctgaaccctg cccatggcag 4200
gcaggtgact gtgcaggagt ttgccctgtt cttcaccatc tttgatgaaa ccaagagctg 4260
gtacttcact gagaacatgg agaggaactg cagggccccc tgcaacatcc agatggagga 4320
ccccaccttc aaggagaact acaggttcca tgccatcaat ggctacatca tggacaccct 4380
gcctggcctg gtgatggccc aggaccagag gatcaggtgg tacctgctga gcatgggcag 4440
caatgagaac atccacagca tccacttctc tggccatgtg ttcactgtga ggaagaagga 4500
ggagtacaag atggccctgt acaacctgta ccctggggtg tttgagactg tggagatgct 4560
gcccagcaag gctggcatct ggagggtgga gtgcctgatt ggggagcacc tgcatgctgg 4620
catgagcacc ctgttcctgg tgtacagcaa caagtgccag acccccctgg gcatggcctc 4680
tggccacatc agggacttcc agatcactgc ctctggccag tatggccagt gggcccccaa 4740
gctggccagg ctgcactact ctggcagcat caatgcctgg agcaccaagg agcccttcag 4800
ctggatcaag gtggacctgc tggcccccat gatcatccat ggcatcaaga cccagggggc 4860
caggcagaag ttcagcagcc tgtacatcag ccagttcatc atcatgtaca gcctggatgg 4920
caagaagtgg cagacctaca ggggcaacag cactggcacc ctgatggtgt tctttggcaa 4980
tgtggacagc tctggcatca agcacaacat cttcaacccc cccatcattg ccagatacat 5040
caggctgcac cccacccact acagcatcag gagcaccctg aggatggagc tgatgggctg 5100
tgacctgaac agctgcagca tgcccctggg catggagagc aaggccatct ctgatgccca 5160
gatcactgcc agcagctact tcaccaacat gtttgccacc tggagcccca gcaaggccag 5220
gctgcacctg cagggcagga gcaatgcctg gaggccccag gtcaacaacc ccaaggagtg 5280
gctgcaggtg gacttccaga agaccatgaa ggtgactggg gtgaccaccc agggggtgaa 5340
gagcctgctg accagcatgt atgtgaagga gttcctgatc agcagcagcc aggatggcca 5400
ccagtggacc ctgttcttcc agaatggcaa ggtgaaggtg ttccagggca accaggacag 5460
cttcacccct gtggtgaaca gcctggaccc ccccctgctg accagatacc tgaggattca 5520
cccccagagc tgggtgcacc agattgccct gaggatggag gtgctgggct gtgaggccca 5580
ggacctgtac tgatgaaacg ttagatctgg taccgatcac atatgccttt aattaaacac 5640
tagttctata gtgtcaccta aattcccttt agtgagggtt aatggccgta ggccgccaga 5700
attgggtcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 5760
gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 5820
aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 5880
ggaggtgtgg gaggtttttt cggactctag gacctgcgca tgcgcttggc gtaatcatgg 5940
tcatagctgt ttcctgtttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc 6000
gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc 6060
acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc 6120
gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc 6180
tgtccctctc accgcggtgg agctccagct tttgttcgaa ttggggcccc ccctcgaggg 6240
tatcgatgat atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca 6300
tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca 6360
tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga 6420
caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg 6480
attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca 6540
gactatcttt ctagggttaa tctagctagc cttaagggcg ctggtctcat gacgcgtagc 6600
ccgcctaatg agcgggcttt tttttggctt gttgtccaca accgttaaac cttaaaagct 6660
ttaaaagcct tatatattct tttttttctt ataaaactta aaaccttaga ggctatttaa 6720
gttgctgatt tatattaatt ttattgttca aacatgagag cttagtacgt gaaacatgag 6780
agcttagtac gttagccatg agagcttagt acgttagcca tgagggttta gttcgttaaa 6840
catgagagct tagtacgtta aacatgagag cttagtacgt actatcaaca ggttgaactg 6900
ctgatccacg ttgtggtaga attggtaaag agagtcgtgt aaaatatcga gttcgcacat 6960
cttgttgtct gattattgat ttttggcgaa accatttgat catatgacaa gatgtgtatc 7020
taccttaact taatgatttt gataaaaatc attaggta 7058
<210> 36
<211> 4374
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 36
atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60
accaggagat actacctggg ggctgtggag ctgagctggg actacatgca gtctgacctg 120
ggggagctgc ctgtggatgc caggttcccc cccagagtgc ccaagagctt ccccttcaac 180
acctctgtgg tgtacaagaa gaccctgttt gtggagttca ctgaccacct gttcaacatt 240
gccaagccca ggcccccctg gatgggcctg ctgggcccca ccatccaggc tgaggtgtat 300
gacactgtgg tgatcaccct gaagaacatg gccagccacc ctgtgagcct gcatgctgtg 360
ggggtgagct actggaaggc ctctgagggg gctgagtatg atgaccagac cagccagagg 420
gagaaggagg atgacaaggt gttccctggg ggcagccaca cctatgtgtg gcaggtgctg 480
aaggagaatg gccccatggc ctctgacccc ctgtgcctga cctacagcta cctgagccat 540
gtggacctgg tgaaggacct gaactctggc ctgattgggg ccctgctggt gtgcagggag 600
ggcagcctgg ccaaggagaa gacccagacc ctgcacaagt tcatcctgct gtttgctgtg 660
tttgatgagg gcaagagctg gcactctgaa accaagaaca gcctgatgca ggacagggat 720
gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc 780
ctgcctggcc tgattggctg ccacaggaag tctgtgtact ggcatgtgat tggcatgggc 840
accacccctg aggtgcacag catcttcctg gagggccaca ccttcctggt caggaaccac 900
aggcaggcca gcctggagat cagccccatc accttcctga ctgcccagac cctgctgatg 960
gacctgggcc agttcctgct gttctgccac atcagcagcc accagcatga tggcatggag 1020
gcctatgtga aggtggacag ctgccctgag gagccccagc tgaggatgaa gaacaatgag 1080
gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt gaggtttgat 1140
gatgacaaca gccccagctt catccagatc aggtctgtgg ccaagaagca ccccaagacc 1200
tgggtgcact acattgctgc tgaggaggag gactgggact atgcccccct ggtgctggcc 1260
cctgatgaca ggagctacaa gagccagtac ctgaacaatg gcccccagag gattggcagg 1320
aagtacaaga aggtcaggtt catggcctac actgatgaaa ccttcaagac cagggaggcc 1380
atccagcatg agtctggcat cctgggcccc ctgctgtatg gggaggtggg ggacaccctg 1440
ctgatcatct tcaagaacca ggccagcagg ccctacaaca tctaccccca tggcatcact 1500
gatgtgaggc ccctgtacag caggaggctg cccaaggggg tgaagcacct gaaggacttc 1560
cccatcctgc ctggggagat cttcaagtac aagtggactg tgactgtgga ggatggcccc 1620
accaagtctg accccaggtg cctgaccaga tactacagca gctttgtgaa catggagagg 1680
gacctggcct ctggcctgat tggccccctg ctgatctgct acaaggagtc tgtggaccag 1740
aggggcaacc agatcatgtc tgacaagagg aatgtgatcc tgttctctgt gtttgatgag 1800
aacaggagct ggtacctgac tgagaacatc cagaggttcc tgcccaaccc tgctggggtg 1860
cagctggagg accctgagtt ccaggccagc aacatcatgc acagcatcaa tggctatgtg 1920
tttgacagcc tgcagctgtc tgtgtgcctg catgaggtgg cctactggta catcctgagc 1980
attggggccc agactgactt cctgtctgtg ttcttctctg gctacacctt caagcacaag 2040
atggtgtatg aggacaccct gaccctgttc cccttctctg gggagactgt gttcatgagc 2100
atggagaacc ctggcctgtg gattctgggc tgccacaact ctgacttcag gaacaggggc 2160
atgactgccc tgctgaaagt ctccagctgt gacaagaaca ctggggacta ctatgaggac 2220
agctatgagg acatctctgc ctacctgctg agcaagaaca atgccattga gcccaggagc 2280
ttcagccaga accccccagt gctgaagagg caccagaggg agatcaccag gaccaccctg 2340
cagtctgacc aggaggagat tgactatgat gacaccatct ctgtggagat gaagaaggag 2400
gactttgaca tctacgacga ggacgagaac cagagcccca ggagcttcca gaagaagacc 2460
aggcactact tcattgctgc tgtggagagg ctgtgggact atggcatgag cagcagcccc 2520
catgtgctga ggaacagggc ccagtctggc tctgtgcccc agttcaagaa ggtggtgttc 2580
caggagttca ctgatggcag cttcacccag cccctgtaca gaggggagct gaatgagcac 2640
ctgggcctgc tgggccccta catcagggct gaggtggagg acaacatcat ggtgaccttc 2700
aggaaccagg ccagcaggcc ctacagcttc tacagcagcc tgatcagcta tgaggaggac 2760
cagaggcagg gggctgagcc caggaagaac tttgtgaagc ccaatgaaac caagacctac 2820
ttctggaagg tgcagcacca catggccccc accaaggatg agtttgactg caaggcctgg 2880
gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggccccctg 2940
ctggtgtgcc acaccaacac cctgaaccct gcccatggca ggcaggtgac tgtgcaggag 3000
tttgccctgt tcttcaccat ctttgatgaa accaagagct ggtacttcac tgagaacatg 3060
gagaggaact gcagggcccc ctgcaacatc cagatggagg accccacctt caaggagaac 3120
tacaggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct ggtgatggcc 3180
caggaccaga ggatcaggtg gtacctgctg agcatgggca gcaatgagaa catccacagc 3240
atccacttct ctggccatgt gttcactgtg aggaagaagg aggagtacaa gatggccctg 3300
tacaacctgt accctggggt gtttgagact gtggagatgc tgcccagcaa ggctggcatc 3360
tggagggtgg agtgcctgat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420
gtgtacagca acaagtgcca gacccccctg ggcatggcct ctggccacat cagggacttc 3480
cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctgcactac 3540
tctggcagca tcaatgcctg gagcaccaag gagcccttca gctggatcaa ggtggacctg 3600
ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gttcagcagc 3660
ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaagtg gcagacctac 3720
aggggcaaca gcactggcac cctgatggtg ttctttggca atgtggacag ctctggcatc 3780
aagcacaaca tcttcaaccc ccccatcatt gccagataca tcaggctgca ccccacccac 3840
tacagcatca ggagcaccct gaggatggag ctgatgggct gtgacctgaa cagctgcagc 3900
atgcccctgg gcatggagag caaggccatc tctgatgccc agatcactgc cagcagctac 3960
ttcaccaaca tgtttgccac ctggagcccc agcaaggcca ggctgcacct gcagggcagg 4020
agcaatgcct ggaggcccca ggtcaacaac cccaaggagt ggctgcaggt ggacttccag 4080
aagaccatga aggtgactgg ggtgaccacc cagggggtga agagcctgct gaccagcatg 4140
tatgtgaagg agttcctgat cagcagcagc caggatggcc accagtggac cctgttcttc 4200
cagaatggca aggtgaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac 4260
agcctggacc cccccctgct gaccagatac ctgaggattc acccccagag ctgggtgcac 4320
cagattgccc tgaggatgga ggtgctgggc tgtgaggccc aggacctgta ctga 4374
<210> 37
<211> 593
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 37
Gly Ser Ser Leu Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln Ser
1 5 10 15
Asp Asp Glu Leu Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp His
20 25 30
Val Ser Glu Asp Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile Asp
35 40 45
Glu Val His Glu Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu Asp
50 55 60
Glu Gln Asn Val Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn Arg
65 70 75 80
Ile Leu Thr Leu Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His Cys
85 90 95
Trp Ser Thr Ser Lys Ser Thr Arg Arg Ser Arg Val Ser Ala Leu Asn
100 105 110
Ile Val Arg Ser Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile Tyr
115 120 125
Asp Pro Leu Leu Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile Ser
130 135 140
Glu Ile Val Lys Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg Glu
145 150 155 160
Ser Met Thr Ser Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile Tyr
165 170 175
Ala Phe Phe Gly Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn His
180 185 190
Met Ser Thr Asp Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr Val
195 200 205
Ser Val Met Ser Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu Arg
210 215 220
Met Asp Asp Lys Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val Phe
225 230 235 240
Thr Pro Val Arg Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile Gln
245 250 255
Asn Tyr Thr Pro Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu Gly
260 265 270
Phe Arg Gly Arg Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro Ser
275 280 285
Lys Tyr Gly Ile Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys Tyr
290 295 300
Met Ile Asn Gly Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn Gly
305 310 315 320
Val Pro Leu Gly Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val His
325 330 335
Gly Ser Cys Arg Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile Pro
340 345 350
Leu Ala Lys Asn Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val Gly
355 360 365
Thr Val Arg Ser Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn Ser
370 375 380
Arg Ser Arg Pro Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro Leu
385 390 395 400
Thr Leu Val Ser Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu Leu
405 410 415
Ser Ser Cys Asp Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys Pro
420 425 430
Gln Met Val Met Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr Leu
435 440 445
Asp Gln Met Cys Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg Trp
450 455 460
Pro Met Ala Leu Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn Ser
465 470 475 480
Phe Ile Ile Tyr Ser His Asn Val Ser Ser Lys Gly Glu Lys Val Gln
485 490 495
Ser Arg Lys Lys Phe Met Arg Asn Leu Tyr Met Ser Leu Thr Ser Ser
500 505 510
Phe Met Arg Lys Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu Arg
515 520 525
Asp Asn Ile Ser Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser Asp
530 535 540
Asp Ser Thr Glu Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr Tyr
545 550 555 560
Cys Pro Ser Lys Ile Arg Arg Lys Ala Asn Ala Ser Cys Lys Lys Cys
565 570 575
Lys Lys Val Ile Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser Cys
580 585 590
Phe
<210> 38
<211> 605
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 38
Met Ala Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Ser Leu
1 5 10 15
Asp Asp Glu His Ile Leu Ser Ala Leu Leu Gln Ser Asp Asp Glu Leu
20 25 30
Val Gly Glu Asp Ser Asp Ser Glu Val Ser Asp His Val Ser Glu Asp
35 40 45
Asp Val Gln Ser Asp Thr Glu Glu Ala Phe Ile Asp Glu Val His Glu
50 55 60
Val Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu Asp Glu Gln Asn Val
65 70 75 80
Ile Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn Arg Ile Leu Thr Leu
85 90 95
Pro Gln Arg Thr Ile Arg Gly Lys Asn Lys His Cys Trp Ser Thr Ser
100 105 110
Lys Ser Thr Arg Arg Ser Arg Val Ser Ala Leu Asn Ile Val Arg Ser
115 120 125
Gln Arg Gly Pro Thr Arg Met Cys Arg Asn Ile Tyr Asp Pro Leu Leu
130 135 140
Cys Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile Ser Glu Ile Val Lys
145 150 155 160
Trp Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg Glu Ser Met Thr Ser
165 170 175
Ala Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile Tyr Ala Phe Phe Gly
180 185 190
Ile Leu Val Met Thr Ala Val Arg Lys Asp Asn His Met Ser Thr Asp
195 200 205
Asp Leu Phe Asp Arg Ser Leu Ser Met Val Tyr Val Ser Val Met Ser
210 215 220
Arg Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu Arg Met Asp Asp Lys
225 230 235 240
Ser Ile Arg Pro Thr Leu Arg Glu Asn Asp Val Phe Thr Pro Val Arg
245 250 255
Lys Ile Trp Asp Leu Phe Ile His Gln Cys Ile Gln Asn Tyr Thr Pro
260 265 270
Gly Ala His Leu Thr Ile Asp Glu Gln Leu Leu Gly Phe Arg Gly Arg
275 280 285
Cys Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro Ser Lys Tyr Gly Ile
290 295 300
Lys Ile Leu Met Met Cys Asp Ser Gly Thr Lys Tyr Met Ile Asn Gly
305 310 315 320
Met Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn Gly Val Pro Leu Gly
325 330 335
Glu Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val His Gly Ser Cys Arg
340 345 350
Asn Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile Pro Leu Ala Lys Asn
355 360 365
Leu Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val Gly Thr Val Arg Ser
370 375 380
Asn Lys Arg Glu Ile Pro Glu Val Leu Lys Asn Ser Arg Ser Arg Pro
385 390 395 400
Val Gly Thr Ser Met Phe Cys Phe Asp Gly Pro Leu Thr Leu Val Ser
405 410 415
Tyr Lys Pro Lys Pro Ala Lys Met Val Tyr Leu Leu Ser Ser Cys Asp
420 425 430
Glu Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys Pro Gln Met Val Met
435 440 445
Tyr Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr Leu Asp Gln Met Cys
450 455 460
Ser Val Met Thr Cys Ser Arg Lys Thr Asn Arg Trp Pro Met Ala Leu
465 470 475 480
Leu Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn Ser Phe Ile Ile Tyr
485 490 495
Ser His Asn Val Ser Ser Lys Gly Glu Lys Val Gln Ser Arg Lys Lys
500 505 510
Phe Met Arg Asn Leu Tyr Met Ser Leu Thr Ser Ser Phe Met Arg Lys
515 520 525
Arg Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu Arg Asp Asn Ile Ser
530 535 540
Asn Ile Leu Pro Lys Glu Val Pro Gly Thr Ser Asp Asp Ser Thr Glu
545 550 555 560
Glu Pro Val Met Lys Lys Arg Thr Tyr Cys Thr Tyr Cys Pro Ser Lys
565 570 575
Ile Arg Arg Lys Ala Asn Ala Ser Cys Lys Lys Cys Lys Lys Val Ile
580 585 590
Cys Arg Glu His Asn Ile Asp Met Cys Gln Ser Cys Phe
595 600 605
<210> 39
<211> 604
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 39
Ala Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Ser Leu Asp
1 5 10 15
Asp Glu His Ile Leu Ser Ala Leu Leu Gln Ser Asp Asp Glu Leu Val
20 25 30
Gly Glu Asp Ser Asp Ser Glu Val Ser Asp His Val Ser Glu Asp Asp
35 40 45
Val Gln Ser Asp Thr Glu Glu Ala Phe Ile Asp Glu Val His Glu Val
50 55 60
Gln Pro Thr Ser Ser Gly Ser Glu Ile Leu Asp Glu Gln Asn Val Ile
65 70 75 80
Glu Gln Pro Gly Ser Ser Leu Ala Ser Asn Arg Ile Leu Thr Leu Pro
85 90 95
Gln Arg Thr Ile Arg Gly Lys Asn Lys His Cys Trp Ser Thr Ser Lys
100 105 110
Ser Thr Arg Arg Ser Arg Val Ser Ala Leu Asn Ile Val Arg Ser Gln
115 120 125
Arg Gly Pro Thr Arg Met Cys Arg Asn Ile Tyr Asp Pro Leu Leu Cys
130 135 140
Phe Lys Leu Phe Phe Thr Asp Glu Ile Ile Ser Glu Ile Val Lys Trp
145 150 155 160
Thr Asn Ala Glu Ile Ser Leu Lys Arg Arg Glu Ser Met Thr Ser Ala
165 170 175
Thr Phe Arg Asp Thr Asn Glu Asp Glu Ile Tyr Ala Phe Phe Gly Ile
180 185 190
Leu Val Met Thr Ala Val Arg Lys Asp Asn His Met Ser Thr Asp Asp
195 200 205
Leu Phe Asp Arg Ser Leu Ser Met Val Tyr Val Ser Val Met Ser Arg
210 215 220
Asp Arg Phe Asp Phe Leu Ile Arg Cys Leu Arg Met Asp Asp Lys Ser
225 230 235 240
Ile Arg Pro Thr Leu Arg Glu Asn Asp Val Phe Thr Pro Val Arg Lys
245 250 255
Ile Trp Asp Leu Phe Ile His Gln Cys Ile Gln Asn Tyr Thr Pro Gly
260 265 270
Ala His Leu Thr Ile Asp Glu Gln Leu Leu Gly Phe Arg Gly Arg Cys
275 280 285
Pro Phe Arg Val Tyr Ile Pro Asn Lys Pro Ser Lys Tyr Gly Ile Lys
290 295 300
Ile Leu Met Met Cys Asp Ser Gly Thr Lys Tyr Met Ile Asn Gly Met
305 310 315 320
Pro Tyr Leu Gly Arg Gly Thr Gln Thr Asn Gly Val Pro Leu Gly Glu
325 330 335
Tyr Tyr Val Lys Glu Leu Ser Lys Pro Val His Gly Ser Cys Arg Asn
340 345 350
Ile Thr Cys Asp Asn Trp Phe Thr Ser Ile Pro Leu Ala Lys Asn Leu
355 360 365
Leu Gln Glu Pro Tyr Lys Leu Thr Ile Val Gly Thr Val Arg Ser Asn
370 375 380
Lys Arg Glu Ile Pro Glu Val Leu Lys Asn Ser Arg Ser Arg Pro Val
385 390 395 400
Gly Thr Ser Met Phe Cys Phe Asp Gly Pro Leu Thr Leu Val Ser Tyr
405 410 415
Lys Pro Lys Pro Ala Lys Met Val Tyr Leu Leu Ser Ser Cys Asp Glu
420 425 430
Asp Ala Ser Ile Asn Glu Ser Thr Gly Lys Pro Gln Met Val Met Tyr
435 440 445
Tyr Asn Gln Thr Lys Gly Gly Val Asp Thr Leu Asp Gln Met Cys Ser
450 455 460
Val Met Thr Cys Ser Arg Lys Thr Asn Arg Trp Pro Met Ala Leu Leu
465 470 475 480
Tyr Gly Met Ile Asn Ile Ala Cys Ile Asn Ser Phe Ile Ile Tyr Ser
485 490 495
His Asn Val Ser Ser Lys Gly Glu Lys Val Gln Ser Arg Lys Lys Phe
500 505 510
Met Arg Asn Leu Tyr Met Ser Leu Thr Ser Ser Phe Met Arg Lys Arg
515 520 525
Leu Glu Ala Pro Thr Leu Lys Arg Tyr Leu Arg Asp Asn Ile Ser Asn
530 535 540
Ile Leu Pro Lys Glu Val Pro Gly Thr Ser Asp Asp Ser Thr Glu Glu
545 550 555 560
Pro Val Met Lys Lys Arg Thr Tyr Cys Thr Tyr Cys Pro Ser Lys Ile
565 570 575
Arg Arg Lys Ala Asn Ala Ser Cys Lys Lys Cys Lys Lys Val Ile Cys
580 585 590
Arg Glu His Asn Ile Asp Met Cys Gln Ser Cys Phe
595 600
<210> 40
<211> 2250
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 40
atgctgcggg ccaagaatca actgttcctg ctgtcccctc actacctgcg gcaagtgaaa 60
gagagcagcg gcagcagact gatccagcag agactgctgc atcagcagca gccactgcac 120
cctgaatggg ccgctctggc taagaagcag ctcaagggca agaaccccga ggacctgatc 180
tggcacacac cagagggcat cagcatcaag cccctgtact ccaagcggga cacaatggat 240
ctgcccgagg aactgcctgg cgtgaagcct tttacaagag gcccctatcc taccatgtat 300
accttcagac cctggaccat ccggcagtac gccggctttt ctaccgtgga agagagcaac 360
aagttctaca aggacaacat caaggccggc cagcagggac tgagcgtggc atttgatctg 420
gctacccaca ggggctacga cagcgacaac cctagagtgc ggggagatgt tggaatggcc 480
ggcgtggcaa tcgacacagt ggaagatacc aagatcctgt tcgacggcat ccctctggaa 540
aagatgagcg tgtccatgac catgaacggc gctgtgatcc ccgtgctggc taactttatt 600
gtgaccggcg aggaacaggg cgtgcccaaa gaaaagctga ccggcaccat ccagaacgac 660
atcctgaaag agttcatggt tcgaaacacc tacatcttcc cacctgagcc gagcatgaag 720
atcattgccg acatcttcga gtacaccgcc aagcacatgc ccaagttcaa cagcatctcc 780
atcagcggct accacatgca agaggctggc gccgatgcca tcctggaact ggcttataca 840
ctggccgacg gcctggaata ctccagaaca ggactgcaag ccggcctgac catcgatgag 900
tttgccccta gactgagctt cttctggggc atcggcatga acttctacat ggaaatcgcc 960
aagatgagag ccggcagacg gctgtgggct cacctgatcg agaagatgtt ccagcctaag 1020
aacagcaaga gcctgctcct gagagcccac tgtcagacaa gtggctggtc cctgactgag 1080
caggacccct acaacaacat cgtgcgcaca gccatcgaag ctatggccgc cgtgtttggc 1140
ggaacacaga gcctgcacac caacagcttt gacgaggctc tgggcctgcc taccgtgaag 1200
tctgccagaa tcgcccggaa cacccagatc atcatccaag aggaaagcgg catccccaag 1260
gtggcagatc cttggggcgg cagctacatg atggaatgcc tgaccaacga cgtgtacgac 1320
gccgctctga agctgatcaa cgagatcgaa gagatgggcg gcatggctaa ggctgtggcc 1380
gagggaatcc ccaagctgag aatcgaggaa tgcgccgcca gacggcaggc cagaattgat 1440
agcggaagcg aagtgatcgt gggcgtgaac aagtaccagc tcgaaaaaga ggacgccgtc 1500
gaggtcctgg ctatcgacaa taccagcgtg cggaaccggc agattgagaa gctgaagaag 1560
atcaagagca gccgcgatca ggccctggcc gaaagatgtc ttgctgccct gacagagtgt 1620
gccgccagcg gcgacggaaa tattctggct ctggccgtgg atgccagccg ggctagatgt 1680
accgtgggcg agattacaga cgccctgaag aaggtgttcg gcgagcacaa ggccaacgac 1740
agaatggtgt ctggcgccta cagacaagag tttggcgaga gcaaagagat caccagcgcc 1800
atcaagcggg tccacaagtt catggaaaga gaaggcaggc ggcccagact gctggtggct 1860
aagatgggac aagacggcca tgacagaggc gccaaagtga tcgccacagg ctttgccgat 1920
ctgggcttcg acgtggacat cggccctctg tttcagaccc ctagagaggt ggcacagcag 1980
gccgttgatg ccgatgttca cgctgtgggc atctctacac tggctgccgg acacaagaca 2040
ctggtgcccg aactgatcaa agagctgaac agcctgggca gacccgacat ccttgtgatg 2100
tgtggcggag tgatcccacc gcaggactac gagttcctgt ttgaagtggg cgtgtccaac 2160
gtgttcggcc ctggcacaag aatccctaaa gccgccgtgc aggttctgga cgacatcgag 2220
aagtgcctgg aaaaaaagca gcagagcgtg 2250
<210> 41
<211> 2247
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 41
ctgcgggcca agaatcaact gttcctgctg tcccctcact acctgcggca agtgaaagag 60
agcagcggca gcagactgat ccagcagaga ctgctgcatc agcagcagcc actgcaccct 120
gaatgggccg ctctggctaa gaagcagctc aagggcaaga accccgagga cctgatctgg 180
cacacaccag agggcatcag catcaagccc ctgtactcca agcgggacac aatggatctg 240
cccgaggaac tgcctggcgt gaagcctttt acaagaggcc cctatcctac catgtatacc 300
ttcagaccct ggaccatccg gcagtacgcc ggcttttcta ccgtggaaga gagcaacaag 360
ttctacaagg acaacatcaa ggccggccag cagggactga gcgtggcatt tgatctggct 420
acccacaggg gctacgacag cgacaaccct agagtgcggg gagatgttgg aatggccggc 480
gtggcaatcg acacagtgga agataccaag atcctgttcg acggcatccc tctggaaaag 540
atgagcgtgt ccatgaccat gaacggcgct gtgatccccg tgctggctaa ctttattgtg 600
accggcgagg aacagggcgt gcccaaagaa aagctgaccg gcaccatcca gaacgacatc 660
ctgaaagagt tcatggttcg aaacacctac atcttcccac ctgagccgag catgaagatc 720
attgccgaca tcttcgagta caccgccaag cacatgccca agttcaacag catctccatc 780
agcggctacc acatgcaaga ggctggcgcc gatgccatcc tggaactggc ttatacactg 840
gccgacggcc tggaatactc cagaacagga ctgcaagccg gcctgaccat cgatgagttt 900
gcccctagac tgagcttctt ctggggcatc ggcatgaact tctacatgga aatcgccaag 960
atgagagccg gcagacggct gtgggctcac ctgatcgaga agatgttcca gcctaagaac 1020
agcaagagcc tgctcctgag agcccactgt cagacaagtg gctggtccct gactgagcag 1080
gacccctaca acaacatcgt gcgcacagcc atcgaagcta tggccgccgt gtttggcgga 1140
acacagagcc tgcacaccaa cagctttgac gaggctctgg gcctgcctac cgtgaagtct 1200
gccagaatcg cccggaacac ccagatcatc atccaagagg aaagcggcat ccccaaggtg 1260
gcagatcctt ggggcggcag ctacatgatg gaatgcctga ccaacgacgt gtacgacgcc 1320
gctctgaagc tgatcaacga gatcgaagag atgggcggca tggctaaggc tgtggccgag 1380
ggaatcccca agctgagaat cgaggaatgc gccgccagac ggcaggccag aattgatagc 1440
ggaagcgaag tgatcgtggg cgtgaacaag taccagctcg aaaaagagga cgccgtcgag 1500
gtcctggcta tcgacaatac cagcgtgcgg aaccggcaga ttgagaagct gaagaagatc 1560
aagagcagcc gcgatcaggc cctggccgaa agatgtcttg ctgccctgac agagtgtgcc 1620
gccagcggcg acggaaatat tctggctctg gccgtggatg ccagccgggc tagatgtacc 1680
gtgggcgaga ttacagacgc cctgaagaag gtgttcggcg agcacaaggc caacgacaga 1740
atggtgtctg gcgcctacag acaagagttt ggcgagagca aagagatcac cagcgccatc 1800
aagcgggtcc acaagttcat ggaaagagaa ggcaggcggc ccagactgct ggtggctaag 1860
atgggacaag acggccatga cagaggcgcc aaagtgatcg ccacaggctt tgccgatctg 1920
ggcttcgacg tggacatcgg ccctctgttt cagaccccta gagaggtggc acagcaggcc 1980
gttgatgccg atgttcacgc tgtgggcatc tctacactgg ctgccggaca caagacactg 2040
gtgcccgaac tgatcaaaga gctgaacagc ctgggcagac ccgacatcct tgtgatgtgt 2100
ggcggagtga tcccaccgca ggactacgag ttcctgtttg aagtgggcgt gtccaacgtg 2160
ttcggccctg gcacaagaat ccctaaagcc gccgtgcagg ttctggacga catcgagaag 2220
tgcctggaaa aaaagcagca gagcgtg 2247
<210> 42
<211> 2256
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 42
atgctgcggg ccaagaatca actgttcctg ctgtcccctc actacctgcg gcaagtgaaa 60
gagagcagcg gcagcagact gatccagcag agactgctgc atcagcagca gccactgcac 120
cctgaatggg ccgctctggc taagaagcag ctcaagggca agaaccccga ggacctgatc 180
tggcacacac cagagggcat cagcatcaag cccctgtact ccaagcggga cacaatggat 240
ctgcccgagg aactgcctgg cgtgaagcct tttacaagag gcccctatcc taccatgtat 300
accttcagac cctggaccat ccggcagtac gccggctttt ctaccgtgga agagagcaac 360
aagttctaca aggacaacat caaggccggc cagcagggac tgagcgtggc atttgatctg 420
gctacccaca ggggctacga cagcgacaac cctagagtgc ggggagatgt tggaatggcc 480
ggcgtggcaa tcgacacagt ggaagatacc aagatcctgt tcgacggcat ccctctggaa 540
aagatgagcg tgtccatgac catgaacggc gctgtgatcc ccgtgctggc taactttatt 600
gtgaccggcg aggaacaggg cgtgcccaaa gaaaagctga ccggcaccat ccagaacgac 660
atcctgaaag agttcatggt tcgaaacacc tacatcttcc cacctgagcc gagcatgaag 720
atcattgccg acatcttcga gtacaccgcc aagcacatgc ccaagttcaa cagcatctcc 780
atcagcggct accacatgca agaggctggc gccgatgcca tcctggaact ggcttataca 840
ctggccgacg gcctggaata ctccagaaca ggactgcaag ccggcctgac catcgatgag 900
tttgccccta gactgagctt cttctggggc atcggcatga acttctacat ggaaatcgcc 960
aagatgagag ccggcagacg gctgtgggct cacctgatcg agaagatgtt ccagcctaag 1020
aacagcaaga gcctgctcct gagagcccac tgtcagacaa gtggctggtc cctgactgag 1080
caggacccct acaacaacat cgtgcgcaca gccatcgaag ctatggccgc cgtgtttggc 1140
ggaacacaga gcctgcacac caacagcttt gacgaggctc tgggcctgcc taccgtgaag 1200
tctgccagaa tcgcccggaa cacccagatc atcatccaag aggaaagcgg catccccaag 1260
gtggcagatc cttggggcgg cagctacatg atggaatgcc tgaccaacga cgtgtacgac 1320
gccgctctga agctgatcaa cgagatcgaa gagatgggcg gcatggctaa ggctgtggcc 1380
gagggaatcc ccaagctgag aatcgaggaa tgcgccgcca gacggcaggc cagaattgat 1440
agcggaagcg aagtgatcgt gggcgtgaac aagtaccagc tcgaaaaaga ggacgccgtc 1500
gaggtcctgg ctatcgacaa taccagcgtg cggaaccggc agattgagaa gctgaagaag 1560
atcaagagca gccgcgatca ggccctggcc gaaagatgtc ttgctgccct gacagagtgt 1620
gccgccagcg gcgacggaaa tattctggct ctggccgtgg atgccagccg ggctagatgt 1680
accgtgggcg agattacaga cgccctgaag aaggtgttcg gcgagcacaa ggccaacgac 1740
agaatggtgt ctggcgccta cagacaagag tttggcgaga gcaaagagat caccagcgcc 1800
atcaagcggg tccacaagtt catggaaaga gaaggcaggc ggcccagact gctggtggct 1860
aagatgggac aagacggcca tgacagaggc gccaaagtga tcgccacagg ctttgccgat 1920
ctgggcttcg acgtggacat cggccctctg tttcagaccc ctagagaggt ggcacagcag 1980
gccgttgatg ccgatgttca cgctgtgggc gtgtctacac tggctgccgg acacaagaca 2040
ctggtgcccg aactgatcaa agagctgaac agcctgggca gacccgacat ccttgtgatg 2100
tgtggcggag tgatcccacc gcaggactac gagttcctgt ttgaagtggg cgtgtccaac 2160
gtgttcggcc ctggcacaag aatccctaaa gccgccgtgc aggttctgga cgacatcgag 2220
aagtgcctgg aaaaaaagca gcagagcgtg ggatcc 2256
<210> 43
<211> 2253
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 43
ctgcgggcca agaatcaact gttcctgctg tcccctcact acctgcggca agtgaaagag 60
agcagcggca gcagactgat ccagcagaga ctgctgcatc agcagcagcc actgcaccct 120
gaatgggccg ctctggctaa gaagcagctc aagggcaaga accccgagga cctgatctgg 180
cacacaccag agggcatcag catcaagccc ctgtactcca agcgggacac aatggatctg 240
cccgaggaac tgcctggcgt gaagcctttt acaagaggcc cctatcctac catgtatacc 300
ttcagaccct ggaccatccg gcagtacgcc ggcttttcta ccgtggaaga gagcaacaag 360
ttctacaagg acaacatcaa ggccggccag cagggactga gcgtggcatt tgatctggct 420
acccacaggg gctacgacag cgacaaccct agagtgcggg gagatgttgg aatggccggc 480
gtggcaatcg acacagtgga agataccaag atcctgttcg acggcatccc tctggaaaag 540
atgagcgtgt ccatgaccat gaacggcgct gtgatccccg tgctggctaa ctttattgtg 600
accggcgagg aacagggcgt gcccaaagaa aagctgaccg gcaccatcca gaacgacatc 660
ctgaaagagt tcatggttcg aaacacctac atcttcccac ctgagccgag catgaagatc 720
attgccgaca tcttcgagta caccgccaag cacatgccca agttcaacag catctccatc 780
agcggctacc acatgcaaga ggctggcgcc gatgccatcc tggaactggc ttatacactg 840
gccgacggcc tggaatactc cagaacagga ctgcaagccg gcctgaccat cgatgagttt 900
gcccctagac tgagcttctt ctggggcatc ggcatgaact tctacatgga aatcgccaag 960
atgagagccg gcagacggct gtgggctcac ctgatcgaga agatgttcca gcctaagaac 1020
agcaagagcc tgctcctgag agcccactgt cagacaagtg gctggtccct gactgagcag 1080
gacccctaca acaacatcgt gcgcacagcc atcgaagcta tggccgccgt gtttggcgga 1140
acacagagcc tgcacaccaa cagctttgac gaggctctgg gcctgcctac cgtgaagtct 1200
gccagaatcg cccggaacac ccagatcatc atccaagagg aaagcggcat ccccaaggtg 1260
gcagatcctt ggggcggcag ctacatgatg gaatgcctga ccaacgacgt gtacgacgcc 1320
gctctgaagc tgatcaacga gatcgaagag atgggcggca tggctaaggc tgtggccgag 1380
ggaatcccca agctgagaat cgaggaatgc gccgccagac ggcaggccag aattgatagc 1440
ggaagcgaag tgatcgtggg cgtgaacaag taccagctcg aaaaagagga cgccgtcgag 1500
gtcctggcta tcgacaatac cagcgtgcgg aaccggcaga ttgagaagct gaagaagatc 1560
aagagcagcc gcgatcaggc cctggccgaa agatgtcttg ctgccctgac agagtgtgcc 1620
gccagcggcg acggaaatat tctggctctg gccgtggatg ccagccgggc tagatgtacc 1680
gtgggcgaga ttacagacgc cctgaagaag gtgttcggcg agcacaaggc caacgacaga 1740
atggtgtctg gcgcctacag acaagagttt ggcgagagca aagagatcac cagcgccatc 1800
aagcgggtcc acaagttcat ggaaagagaa ggcaggcggc ccagactgct ggtggctaag 1860
atgggacaag acggccatga cagaggcgcc aaagtgatcg ccacaggctt tgccgatctg 1920
ggcttcgacg tggacatcgg ccctctgttt cagaccccta gagaggtggc acagcaggcc 1980
gttgatgccg atgttcacgc tgtgggcgtg tctacactgg ctgccggaca caagacactg 2040
gtgcccgaac tgatcaaaga gctgaacagc ctgggcagac ccgacatcct tgtgatgtgt 2100
ggcggagtga tcccaccgca ggactacgag ttcctgtttg aagtgggcgt gtccaacgtg 2160
ttcggccctg gcacaagaat ccctaaagcc gccgtgcagg ttctggacga catcgagaag 2220
tgcctggaaa aaaagcagca gagcgtggga tcc 2253
<210> 44
<211> 2250
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 44
atgctgcggg ccaagaatca actgttcctg ctgtcccctc actacctgcg gcaagtgaaa 60
gagagcagcg gcagcagact gatccagcag agactgctgc atcagcagca gccactgcac 120
cctgaatggg ccgctctggc taagaagcag ctcaagggca agaaccccga ggacctgatc 180
tggcacacac cagagggcat cagcatcaag cccctgtact ccaagcggga cacaatggat 240
ctgcccgagg aactgcctgg cgtgaagcct tttacaagag gcccctatcc taccatgtat 300
accttcagac cctggaccat ccggcagtac gccggctttt ctaccgtgga agagagcaac 360
aagttctaca aggacaacat caaggccggc cagcagggac tgagcgtggc atttgatctg 420
gctacccaca ggggctacga cagcgacaac cctagagtgc ggggagatgt tggaatggcc 480
ggcgtggcaa tcgacacagt ggaagatacc aagatcctgt tcgacggcat ccctctggaa 540
aagatgagcg tgtccatgac catgaacggc gctgtgatcc ccgtgctggc taactttatt 600
gtgaccggcg aggaacaggg cgtgcccaaa gaaaagctga ccggcaccat ccagaacgac 660
atcctgaaag agttcatggt tcgaaacacc tacatcttcc cacctgagcc gagcatgaag 720
atcattgccg acatcttcga gtacaccgcc aagcacatgc ccaagttcaa cagcatctcc 780
atcagcggct accacatgca agaggctggc gccgatgcca tcctggaact ggcttataca 840
ctggccgacg gcctggaata ctccagaaca ggactgcaag ccggcctgac catcgatgag 900
tttgccccta gactgagctt cttctggggc atcggcatga acttctacat ggaaatcgcc 960
aagatgagag ccggcagacg gctgtgggct cacctgatcg agaagatgtt ccagcctaag 1020
aacagcaaga gcctgctcct gagagcccac tgtcagacaa gtggctggtc cctgactgag 1080
caggacccct acaacaacat cgtgcgcaca gccatcgaag ctatggccgc cgtgtttggc 1140
ggaacacaga gcctgcacac caacagcttt gacgaggctc tgggcctgcc taccgtgaag 1200
tctgccagaa tcgcccggaa cacccagatc atcatccaag aggaaagcgg catccccaag 1260
gtggcagatc cttggggcgg cagctacatg atggaatgcc tgaccaacga cgtgtacgac 1320
gccgctctga agctgatcaa cgagatcgaa gagatgggcg gcatggctaa ggctgtggcc 1380
gagggaatcc ccaagctgag aatcgaggaa tgcgccgcca gacggcaggc cagaattgat 1440
agcggaagcg aagtgatcgt gggcgtgaac aagtaccagc tcgaaaaaga ggacgccgtc 1500
gaggtcctgg ctatcgacaa taccagcgtg cggaaccggc agattgagaa gctgaagaag 1560
atcaagagca gccgcgatca ggccctggcc gaaagatgtc ttgctgccct gacagagtgt 1620
gccgccagcg gcgacggaaa tattctggct ctggccgtgg atgccagccg ggctagatgt 1680
accgtgggcg agattacaga cgccctgaag aaggtgttcg gcgagcacaa ggccaacgac 1740
agaatggtgt ctggcgccta cagacaagag tttggcgaga gcaaagagat caccagcgcc 1800
atcaagcggg tccacaagtt catggaaaga gaaggcaggc ggcccagact gctggtggct 1860
aagatgggac aagacggcca tgacagaggc gccaaagtga tcgccacagg ctttgccgat 1920
ctgggcttcg acgtggacat cggccctctg tttcagaccc ctagagaggt ggcacagcag 1980
gccgttgatg ccgatgttca cgctgtgggc atctctacac tggctgccgg acacaagaca 2040
ctggtgcccg aactgatcaa agagctgaac agcctgggca gacccgacat ccttgtgatg 2100
tgtggcggag tgatcccacc gcaggactac gagttcctgt ttgaagtggg cgtgtccaac 2160
gtgttcggcc ctggcacaag aatccctaaa gccgccgtgc aggttctgga cgacatcgag 2220
aagtgcctgg aaaaaaagca gcagagcgtg 2250
<210> 45
<211> 2247
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 45
ctgcgggcca agaatcaact gttcctgctg tcccctcact acctgcggca agtgaaagag 60
agcagcggca gcagactgat ccagcagaga ctgctgcatc agcagcagcc actgcaccct 120
gaatgggccg ctctggctaa gaagcagctc aagggcaaga accccgagga cctgatctgg 180
cacacaccag agggcatcag catcaagccc ctgtactcca agcgggacac aatggatctg 240
cccgaggaac tgcctggcgt gaagcctttt acaagaggcc cctatcctac catgtatacc 300
ttcagaccct ggaccatccg gcagtacgcc ggcttttcta ccgtggaaga gagcaacaag 360
ttctacaagg acaacatcaa ggccggccag cagggactga gcgtggcatt tgatctggct 420
acccacaggg gctacgacag cgacaaccct agagtgcggg gagatgttgg aatggccggc 480
gtggcaatcg acacagtgga agataccaag atcctgttcg acggcatccc tctggaaaag 540
atgagcgtgt ccatgaccat gaacggcgct gtgatccccg tgctggctaa ctttattgtg 600
accggcgagg aacagggcgt gcccaaagaa aagctgaccg gcaccatcca gaacgacatc 660
ctgaaagagt tcatggttcg aaacacctac atcttcccac ctgagccgag catgaagatc 720
attgccgaca tcttcgagta caccgccaag cacatgccca agttcaacag catctccatc 780
agcggctacc acatgcaaga ggctggcgcc gatgccatcc tggaactggc ttatacactg 840
gccgacggcc tggaatactc cagaacagga ctgcaagccg gcctgaccat cgatgagttt 900
gcccctagac tgagcttctt ctggggcatc ggcatgaact tctacatgga aatcgccaag 960
atgagagccg gcagacggct gtgggctcac ctgatcgaga agatgttcca gcctaagaac 1020
agcaagagcc tgctcctgag agcccactgt cagacaagtg gctggtccct gactgagcag 1080
gacccctaca acaacatcgt gcgcacagcc atcgaagcta tggccgccgt gtttggcgga 1140
acacagagcc tgcacaccaa cagctttgac gaggctctgg gcctgcctac cgtgaagtct 1200
gccagaatcg cccggaacac ccagatcatc atccaagagg aaagcggcat ccccaaggtg 1260
gcagatcctt ggggcggcag ctacatgatg gaatgcctga ccaacgacgt gtacgacgcc 1320
gctctgaagc tgatcaacga gatcgaagag atgggcggca tggctaaggc tgtggccgag 1380
ggaatcccca agctgagaat cgaggaatgc gccgccagac ggcaggccag aattgatagc 1440
ggaagcgaag tgatcgtggg cgtgaacaag taccagctcg aaaaagagga cgccgtcgag 1500
gtcctggcta tcgacaatac cagcgtgcgg aaccggcaga ttgagaagct gaagaagatc 1560
aagagcagcc gcgatcaggc cctggccgaa agatgtcttg ctgccctgac agagtgtgcc 1620
gccagcggcg acggaaatat tctggctctg gccgtggatg ccagccgggc tagatgtacc 1680
gtgggcgaga ttacagacgc cctgaagaag gtgttcggcg agcacaaggc caacgacaga 1740
atggtgtctg gcgcctacag acaagagttt ggcgagagca aagagatcac cagcgccatc 1800
aagcgggtcc acaagttcat ggaaagagaa ggcaggcggc ccagactgct ggtggctaag 1860
atgggacaag acggccatga cagaggcgcc aaagtgatcg ccacaggctt tgccgatctg 1920
ggcttcgacg tggacatcgg ccctctgttt cagaccccta gagaggtggc acagcaggcc 1980
gttgatgccg atgttcacgc tgtgggcatc tctacactgg ctgccggaca caagacactg 2040
gtgcccgaac tgatcaaaga gctgaacagc ctgggcagac ccgacatcct tgtgatgtgt 2100
ggcggagtga tcccaccgca ggactacgag ttcctgtttg aagtgggcgt gtccaacgtg 2160
ttcggccctg gcacaagaat ccctaaagcc gccgtgcagg ttctggacga catcgagaag 2220
tgcctggaaa aaaagcagca gagcgtg 2247
<210> 46
<211> 2253
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 46
atgctgagag ccaaaaacca gctgttcctg ctgagccccc actatctgag acaggtcaaa 60
gaaagttccg ggagtagact gatccagcag agactgctgc accagcagca gccactgcat 120
cctgagtggg ccgctctggc caagaaacag ctgaagggca aaaacccaga agacctgatc 180
tggcacactc cagaggggat ttcaatcaag cccctgtaca gcaaaaggga cactatggat 240
ctgccagagg aactgccagg agtgaagcct ttcacccgcg gaccttaccc aactatgtat 300
acctttcgac cctggacaat tcggcagtac gccggcttca gtactgtgga ggaatcaaac 360
aagttttata aggacaacat caaggctgga cagcagggcc tgagtgtggc attcgatctg 420
gccacacatc gcggctatga ctcagataat cccagagtca ggggggacgt gggaatggca 480
ggagtcgcta tcgacacagt ggaagatact aagattctgt tcgatggaat ccctctggag 540
aaaatgtctg tgagtatgac aatgaacggc gctgtcattc ccgtgctggc aaacttcatc 600
gtcactggcg aggaacaggg ggtgcctaag gaaaaactga ccggcacaat tcagaacgac 660
atcctgaagg agttcatggt gcggaatact tacatttttc cccctgaacc atccatgaaa 720
atcattgccg atatcttcga gtacaccgct aagcacatgc ccaagttcaa ctcaattagc 780
atctccgggt atcatatgca ggaagcagga gccgacgcta ttctggagct ggcttacacc 840
ctggcagatg gcctggaata ttctcgaacc ggactgcagg caggcctgac aatcgacgag 900
ttcgctccta gactgagttt cttttgggga attggcatga acttttacat ggagatcgcc 960
aagatgaggg ctggccggag actgtgggca cacctgatcg agaagatgtt ccagcctaag 1020
aactctaaga gtctgctgct gcgggcccat tgccagacat ccggctggtc tctgactgaa 1080
caggacccat ataacaatat tgtcagaacc gcaatcgagg caatggcagc cgtgttcgga 1140
ggaacccaga gcctgcacac aaactccttt gatgaggccc tggggctgcc taccgtgaag 1200
tctgctagga ttgcacgcaa tacacagatc attatccagg aggaatccgg aatcccaaag 1260
gtggccgatc cctggggagg ctcttacatg atggagtgcc tgacaaacga cgtgtatgat 1320
gctgcactga agctgattaa tgaaatcgag gaaatggggg gaatggcaaa ggccgtggct 1380
gagggcattc caaaactgag gatcgaggaa tgtgcagcta ggcgccaggc acgaattgac 1440
tcaggaagcg aagtgatcgt cggggtgaat aagtaccagc tggagaaaga agacgcagtc 1500
gaagtgctgg ccatcgataa cacaagcgtg cgcaatcgac agattgagaa gctgaagaaa 1560
atcaaaagct cccgcgatca ggcactggcc gaacgatgcc tggcagccct gactgagtgt 1620
gctgcaagcg gggacggaaa cattctggct ctggcagtcg atgcctcccg ggctagatgc 1680
actgtggggg aaatcaccga cgccctgaag aaagtcttcg gagagcacaa ggccaatgat 1740
cggatggtga gcggcgctta tagacaggag ttcggggaat ctaaagagat taccagtgcc 1800
atcaagaggg tgcacaagtt catggagaga gaagggcgac ggcccaggct gctggtggca 1860
aagatgggac aggacggaca tgatcgcgga gcaaaagtca ttgccaccgg gttcgctgac 1920
ctgggatttg acgtggatat cggccctctg ttccagacac cacgagaggt cgcacagcag 1980
gcagtcgacg ctgatgtgca cgcagtcgga gtgtccactc tggcagctgg ccataagacc 2040
ctggtgcctg aactgatcaa agagctgaac tctctgggca gaccagacat cctggtcatg 2100
tgcggcggcg tgatcccacc ccaggattac gaattcctgt ttgaggtcgg ggtgagcaac 2160
gtgttcggac caggaaccag gatccctaag gccgcagtgc aggtcctgga tgatattgaa 2220
aagtgtctgg aaaagaaaca gcagtcagtg taa 2253
<210> 47
<211> 2250
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 47
ctgagagcca aaaaccagct gttcctgctg agcccccact atctgagaca ggtcaaagaa 60
agttccggga gtagactgat ccagcagaga ctgctgcacc agcagcagcc actgcatcct 120
gagtgggccg ctctggccaa gaaacagctg aagggcaaaa acccagaaga cctgatctgg 180
cacactccag aggggatttc aatcaagccc ctgtacagca aaagggacac tatggatctg 240
ccagaggaac tgccaggagt gaagcctttc acccgcggac cttacccaac tatgtatacc 300
tttcgaccct ggacaattcg gcagtacgcc ggcttcagta ctgtggagga atcaaacaag 360
ttttataagg acaacatcaa ggctggacag cagggcctga gtgtggcatt cgatctggcc 420
acacatcgcg gctatgactc agataatccc agagtcaggg gggacgtggg aatggcagga 480
gtcgctatcg acacagtgga agatactaag attctgttcg atggaatccc tctggagaaa 540
atgtctgtga gtatgacaat gaacggcgct gtcattcccg tgctggcaaa cttcatcgtc 600
actggcgagg aacagggggt gcctaaggaa aaactgaccg gcacaattca gaacgacatc 660
ctgaaggagt tcatggtgcg gaatacttac atttttcccc ctgaaccatc catgaaaatc 720
attgccgata tcttcgagta caccgctaag cacatgccca agttcaactc aattagcatc 780
tccgggtatc atatgcagga agcaggagcc gacgctattc tggagctggc ttacaccctg 840
gcagatggcc tggaatattc tcgaaccgga ctgcaggcag gcctgacaat cgacgagttc 900
gctcctagac tgagtttctt ttggggaatt ggcatgaact tttacatgga gatcgccaag 960
atgagggctg gccggagact gtgggcacac ctgatcgaga agatgttcca gcctaagaac 1020
tctaagagtc tgctgctgcg ggcccattgc cagacatccg gctggtctct gactgaacag 1080
gacccatata acaatattgt cagaaccgca atcgaggcaa tggcagccgt gttcggagga 1140
acccagagcc tgcacacaaa ctcctttgat gaggccctgg ggctgcctac cgtgaagtct 1200
gctaggattg cacgcaatac acagatcatt atccaggagg aatccggaat cccaaaggtg 1260
gccgatccct ggggaggctc ttacatgatg gagtgcctga caaacgacgt gtatgatgct 1320
gcactgaagc tgattaatga aatcgaggaa atggggggaa tggcaaaggc cgtggctgag 1380
ggcattccaa aactgaggat cgaggaatgt gcagctaggc gccaggcacg aattgactca 1440
ggaagcgaag tgatcgtcgg ggtgaataag taccagctgg agaaagaaga cgcagtcgaa 1500
gtgctggcca tcgataacac aagcgtgcgc aatcgacaga ttgagaagct gaagaaaatc 1560
aaaagctccc gcgatcaggc actggccgaa cgatgcctgg cagccctgac tgagtgtgct 1620
gcaagcgggg acggaaacat tctggctctg gcagtcgatg cctcccgggc tagatgcact 1680
gtgggggaaa tcaccgacgc cctgaagaaa gtcttcggag agcacaaggc caatgatcgg 1740
atggtgagcg gcgcttatag acaggagttc ggggaatcta aagagattac cagtgccatc 1800
aagagggtgc acaagttcat ggagagagaa gggcgacggc ccaggctgct ggtggcaaag 1860
atgggacagg acggacatga tcgcggagca aaagtcattg ccaccgggtt cgctgacctg 1920
ggatttgacg tggatatcgg ccctctgttc cagacaccac gagaggtcgc acagcaggca 1980
gtcgacgctg atgtgcacgc agtcggagtg tccactctgg cagctggcca taagaccctg 2040
gtgcctgaac tgatcaaaga gctgaactct ctgggcagac cagacatcct ggtcatgtgc 2100
ggcggcgtga tcccacccca ggattacgaa ttcctgtttg aggtcggggt gagcaacgtg 2160
ttcggaccag gaaccaggat ccctaaggcc gcagtgcagg tcctggatga tattgaaaag 2220
tgtctggaaa agaaacagca gtcagtgtaa 2250
<210> 48
<211> 2253
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 48
atgttaagag ctaagaatca gcttttttta ctttcacctc attacctgag gcaggtaaaa 60
gaatcatcag gctccaggct catacagcaa cgacttctac accagcaaca gccccttcac 120
ccagaatggg ctgccctggc taaaaagcag ctgaaaggca aaaacccaga agacctaata 180
tggcacaccc cggaagggat ctctataaaa cccttgtatt ccaagagaga tactatggac 240
ttacctgaag aacttccagg agtgaagcca ttcacacgtg gaccatatcc taccatgtat 300
acctttaggc cctggaccat ccgccagtat gctggtttta gtactgtgga agaaagcaat 360
aagttctata aggacaacat taaggctggt cagcagggat tatcagttgc ctttgatctg 420
gcgacacatc gtggctatga ttcagacaac cctcgagttc gtggtgatgt tggaatggct 480
ggagttgcta ttgacactgt ggaagatacc aaaattcttt ttgatggaat tcctttagaa 540
aaaatgtcag tttccatgac tatgaatgga gcagttattc cagttcttgc aaattttata 600
gtaactggag aagaacaagg tgtacctaaa gagaagctta ctggtaccat ccaaaatgat 660
atactaaagg aatttatggt tcgaaataca tacatttttc ctccagaacc atccatgaaa 720
attattgctg acatatttga atatacagca aagcacatgc caaaatttaa ttcaatttca 780
attagtggat accatatgca ggaagcaggg gctgatgcca ttctggagct ggcctatact 840
ttagcagatg gattggagta ctctagaact ggactccagg ctggcctgac aattgatgaa 900
tttgcaccaa ggttgtcttt cttctgggga attggaatga atttctatat ggaaatagca 960
aagatgagag ctggtagaag actctgggct cacttaatag agaaaatgtt tcagcctaaa 1020
aactcaaaat ctcttcttct aagagcacac tgtcagacat ctggatggtc acttactgag 1080
caggatccct acaataatat tgtccgtact gcaatagaag caatggcagc agtatttgga 1140
gggactcagt ctttgcacac aaattctttt gatgaagctt tgggtttgcc aactgtgaaa 1200
agtgctcgaa ttgccaggaa cacacaaatc atcattcaag aagaatctgg gattcccaaa 1260
gtggctgatc cttggggagg ttcttacatg atggaatgtc tcacaaatga tgtttatgat 1320
gctgctttaa agctcattaa tgaaattgaa gaaatgggtg gaatggccaa agctgtagct 1380
gagggaatac ctaaacttcg aattgaagaa tgtgctgccc gaagacaagc tagaatagat 1440
tctggttctg aagtaattgt tggagtaaat aagtaccagt tggaaaaaga agacgctgta 1500
gaagttctgg caattgataa tacttcagtg cgaaacaggc agattgaaaa acttaagaag 1560
atcaaatcca gcagggatca agctttggct gaacgttgtc ttgctgcact aaccgaatgt 1620
gctgctagcg gagatggaaa tatcctggct cttgcagtgg atgcatctcg ggcaagatgt 1680
acagtgggag aaatcacaga tgccctgaaa aaggtatttg gtgaacataa agcgaatgat 1740
cgaatggtga gtggagcata tcgccaggaa tttggagaaa gtaaagagat aacatctgct 1800
atcaagaggg ttcataaatt catggaacgt gaaggtcgca gacctcgtct tcttgtagca 1860
aaaatgggac aagatggcca tgacagagga gcaaaagtta ttgctacagg atttgctgat 1920
cttggttttg atgtggacat aggccctctt ttccagactc ctcgtgaagt ggcccagcag 1980
gctgtggatg cggatgtgca tgctgtgggc ataagcaccc tcgctgctgg tcataaaacc 2040
ctagttcctg aactcatcaa agaacttaac tcccttggac ggccagatat tcttgtcatg 2100
tgtggagggg tgataccacc tcaggattat gaatttctgt ttgaagttgg tgtttccaat 2160
gtatttggtc ctgggactcg aattccaaag gctgccgttc aggtgcttga tgatattgag 2220
aagtgtttgg aaaagaagca gcaatctgta taa 2253
<210> 49
<211> 2250
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 49
ttaagagcta agaatcagct ttttttactt tcacctcatt acctgaggca ggtaaaagaa 60
tcatcaggct ccaggctcat acagcaacga cttctacacc agcaacagcc ccttcaccca 120
gaatgggctg ccctggctaa aaagcagctg aaaggcaaaa acccagaaga cctaatatgg 180
cacaccccgg aagggatctc tataaaaccc ttgtattcca agagagatac tatggactta 240
cctgaagaac ttccaggagt gaagccattc acacgtggac catatcctac catgtatacc 300
tttaggccct ggaccatccg ccagtatgct ggttttagta ctgtggaaga aagcaataag 360
ttctataagg acaacattaa ggctggtcag cagggattat cagttgcctt tgatctggcg 420
acacatcgtg gctatgattc agacaaccct cgagttcgtg gtgatgttgg aatggctgga 480
gttgctattg acactgtgga agataccaaa attctttttg atggaattcc tttagaaaaa 540
atgtcagttt ccatgactat gaatggagca gttattccag ttcttgcaaa ttttatagta 600
actggagaag aacaaggtgt acctaaagag aagcttactg gtaccatcca aaatgatata 660
ctaaaggaat ttatggttcg aaatacatac atttttcctc cagaaccatc catgaaaatt 720
attgctgaca tatttgaata tacagcaaag cacatgccaa aatttaattc aatttcaatt 780
agtggatacc atatgcagga agcaggggct gatgccattc tggagctggc ctatacttta 840
gcagatggat tggagtactc tagaactgga ctccaggctg gcctgacaat tgatgaattt 900
gcaccaaggt tgtctttctt ctggggaatt ggaatgaatt tctatatgga aatagcaaag 960
atgagagctg gtagaagact ctgggctcac ttaatagaga aaatgtttca gcctaaaaac 1020
tcaaaatctc ttcttctaag agcacactgt cagacatctg gatggtcact tactgagcag 1080
gatccctaca ataatattgt ccgtactgca atagaagcaa tggcagcagt atttggaggg 1140
actcagtctt tgcacacaaa ttcttttgat gaagctttgg gtttgccaac tgtgaaaagt 1200
gctcgaattg ccaggaacac acaaatcatc attcaagaag aatctgggat tcccaaagtg 1260
gctgatcctt ggggaggttc ttacatgatg gaatgtctca caaatgatgt ttatgatgct 1320
gctttaaagc tcattaatga aattgaagaa atgggtggaa tggccaaagc tgtagctgag 1380
ggaataccta aacttcgaat tgaagaatgt gctgcccgaa gacaagctag aatagattct 1440
ggttctgaag taattgttgg agtaaataag taccagttgg aaaaagaaga cgctgtagaa 1500
gttctggcaa ttgataatac ttcagtgcga aacaggcaga ttgaaaaact taagaagatc 1560
aaatccagca gggatcaagc tttggctgaa cgttgtcttg ctgcactaac cgaatgtgct 1620
gctagcggag atggaaatat cctggctctt gcagtggatg catctcgggc aagatgtaca 1680
gtgggagaaa tcacagatgc cctgaaaaag gtatttggtg aacataaagc gaatgatcga 1740
atggtgagtg gagcatatcg ccaggaattt ggagaaagta aagagataac atctgctatc 1800
aagagggttc ataaattcat ggaacgtgaa ggtcgcagac ctcgtcttct tgtagcaaaa 1860
atgggacaag atggccatga cagaggagca aaagttattg ctacaggatt tgctgatctt 1920
ggttttgatg tggacatagg ccctcttttc cagactcctc gtgaagtggc ccagcaggct 1980
gtggatgcgg atgtgcatgc tgtgggcata agcaccctcg ctgctggtca taaaacccta 2040
gttcctgaac tcatcaaaga acttaactcc cttggacggc cagatattct tgtcatgtgt 2100
ggaggggtga taccacctca ggattatgaa tttctgtttg aagttggtgt ttccaatgta 2160
tttggtcctg ggactcgaat tccaaaggct gccgttcagg tgcttgatga tattgagaag 2220
tgtttggaaa agaagcagca atctgtataa 2250
<210> 50
<211> 2250
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 50
atgctgcggg ccaagaacca gctgttcctg ctgagccctc actacctgcg gcaggtgaag 60
gagagcagcg gcagccggct gatccagcag cggctgctgc accagcagca gcccctgcac 120
cccgagtggg ccgccctggc caagaagcag ctgaagggca agaaccccga ggacctgatc 180
tggcacacgc ccgagggcat cagcatcaag cccctgtaca gcaagcggga caccatggac 240
ctgcccgagg agctgcccgg cgtgaagccc ttcacccggg gcccctaccc caccatgtac 300
accttccggc cctggaccat ccggcagtac gccggcttca gcaccgtgga ggagagcaac 360
aagttctaca aggacaacat caaggccggc cagcagggcc tgagcgtggc cttcgacctg 420
gccacccacc ggggctacga cagcgacaac ccacgggtgc ggggcgacgt gggcatggcc 480
ggcgtggcca tcgacaccgt ggaggacacc aagatcctgt tcgacggcat ccctctggag 540
aagatgagcg tgagcatgac catgaacggc gccgtgatcc ccgtgctggc caacttcatc 600
gtgaccggcg aggagcaggg cgtgcccaag gagaagctga ccggcaccat ccagaacgac 660
atcctgaagg agttcatggt gcggaacacc tacatcttcc ctcccgagcc cagcatgaag 720
atcatcgccg acatcttcga gtacaccgcc aagcacatgc ccaagttcaa cagcatcagc 780
atcagcggct accacatgca ggaggccggc gccgacgcca tcctggagct ggcctacacc 840
ctggccgacg gcctggagta cagccggacc ggcctgcagg ccggcctgac catcgacgag 900
ttcgcgcccc ggctgagctt cttctggggc atcggcatga acttctacat ggagatcgcc 960
aagatgcggg ccggccggcg gctgtgggcc cacctgatcg agaagatgtt ccagcccaag 1020
aacagcaaga gcctgctgct gcgggcccac tgccagacca gcggctggag cctgaccgag 1080
caggacccct acaacaacat cgtgcggacc gccatcgagg ccatggccgc cgtgttcggc 1140
ggcacccaga gcctgcacac caacagcttc gacgaggccc tgggcctgcc caccgtgaag 1200
agcgcccgga tcgcccggaa cacccagatc atcatccagg aggagagcgg catccccaag 1260
gtggccgacc cctggggcgg cagctacatg atggagtgcc tgaccaacga cgtgtacgac 1320
gccgccctga agctgatcaa cgagatcgag gagatgggcg gcatggccaa ggccgtggcc 1380
gagggcatcc ccaagctgcg gatcgaggag tgcgccgccc ggcggcaggc ccggatcgac 1440
agcggcagcg aggtgatcgt gggcgtgaac aagtaccagc tggagaagga ggacgccgtg 1500
gaggtgctgg ccatcgacaa caccagcgtg cggaaccggc agatcgagaa gctgaagaag 1560
atcaagagca gccgggacca ggccctggcc gagcggtgcc tggccgccct gaccgagtgc 1620
gccgccagcg gcgacggcaa catcctggcc ctggccgtgg acgccagccg ggcccggtgc 1680
accgtgggcg agatcaccga cgccctgaag aaggtgttcg gcgagcacaa ggccaacgac 1740
cggatggtga gcggcgccta ccggcaggag ttcggcgaga gcaaggagat caccagcgcc 1800
atcaagcggg tgcacaagtt catggagcgg gagggccggc ggccccggct gctggtggcc 1860
aagatgggcc aggacggcca cgaccggggc gccaaggtga tcgccaccgg cttcgccgac 1920
ctgggcttcg acgtggacat cggcccactg ttccagacgc cccgggaggt ggcccagcag 1980
gccgtggacg ccgacgtgca cgccgtgggc gtgagcaccc tggccgccgg ccacaagacc 2040
ctggtgcccg agctgatcaa ggagctgaac agcctgggcc ggcccgacat cctggtgatg 2100
tgcggcggcg tgatcccgcc ccaggactac gagttcctgt tcgaggtggg cgtgagcaac 2160
gtgttcggcc ccggcacccg gatccccaag gccgccgtgc aggtgctgga cgacatcgag 2220
aagtgcctgg agaagaagca gcagagcgtg 2250
<210> 51
<211> 2247
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 51
ctgcgggcca agaaccagct gttcctgctg agccctcact acctgcggca ggtgaaggag 60
agcagcggca gccggctgat ccagcagcgg ctgctgcacc agcagcagcc cctgcacccc 120
gagtgggccg ccctggccaa gaagcagctg aagggcaaga accccgagga cctgatctgg 180
cacacgcccg agggcatcag catcaagccc ctgtacagca agcgggacac catggacctg 240
cccgaggagc tgcccggcgt gaagcccttc acccggggcc cctaccccac catgtacacc 300
ttccggccct ggaccatccg gcagtacgcc ggcttcagca ccgtggagga gagcaacaag 360
ttctacaagg acaacatcaa ggccggccag cagggcctga gcgtggcctt cgacctggcc 420
acccaccggg gctacgacag cgacaaccca cgggtgcggg gcgacgtggg catggccggc 480
gtggccatcg acaccgtgga ggacaccaag atcctgttcg acggcatccc tctggagaag 540
atgagcgtga gcatgaccat gaacggcgcc gtgatccccg tgctggccaa cttcatcgtg 600
accggcgagg agcagggcgt gcccaaggag aagctgaccg gcaccatcca gaacgacatc 660
ctgaaggagt tcatggtgcg gaacacctac atcttccctc ccgagcccag catgaagatc 720
atcgccgaca tcttcgagta caccgccaag cacatgccca agttcaacag catcagcatc 780
agcggctacc acatgcagga ggccggcgcc gacgccatcc tggagctggc ctacaccctg 840
gccgacggcc tggagtacag ccggaccggc ctgcaggccg gcctgaccat cgacgagttc 900
gcgccccggc tgagcttctt ctggggcatc ggcatgaact tctacatgga gatcgccaag 960
atgcgggccg gccggcggct gtgggcccac ctgatcgaga agatgttcca gcccaagaac 1020
agcaagagcc tgctgctgcg ggcccactgc cagaccagcg gctggagcct gaccgagcag 1080
gacccctaca acaacatcgt gcggaccgcc atcgaggcca tggccgccgt gttcggcggc 1140
acccagagcc tgcacaccaa cagcttcgac gaggccctgg gcctgcccac cgtgaagagc 1200
gcccggatcg cccggaacac ccagatcatc atccaggagg agagcggcat ccccaaggtg 1260
gccgacccct ggggcggcag ctacatgatg gagtgcctga ccaacgacgt gtacgacgcc 1320
gccctgaagc tgatcaacga gatcgaggag atgggcggca tggccaaggc cgtggccgag 1380
ggcatcccca agctgcggat cgaggagtgc gccgcccggc ggcaggcccg gatcgacagc 1440
ggcagcgagg tgatcgtggg cgtgaacaag taccagctgg agaaggagga cgccgtggag 1500
gtgctggcca tcgacaacac cagcgtgcgg aaccggcaga tcgagaagct gaagaagatc 1560
aagagcagcc gggaccaggc cctggccgag cggtgcctgg ccgccctgac cgagtgcgcc 1620
gccagcggcg acggcaacat cctggccctg gccgtggacg ccagccgggc ccggtgcacc 1680
gtgggcgaga tcaccgacgc cctgaagaag gtgttcggcg agcacaaggc caacgaccgg 1740
atggtgagcg gcgcctaccg gcaggagttc ggcgagagca aggagatcac cagcgccatc 1800
aagcgggtgc acaagttcat ggagcgggag ggccggcggc cccggctgct ggtggccaag 1860
atgggccagg acggccacga ccggggcgcc aaggtgatcg ccaccggctt cgccgacctg 1920
ggcttcgacg tggacatcgg cccactgttc cagacgcccc gggaggtggc ccagcaggcc 1980
gtggacgccg acgtgcacgc cgtgggcgtg agcaccctgg ccgccggcca caagaccctg 2040
gtgcccgagc tgatcaagga gctgaacagc ctgggccggc ccgacatcct ggtgatgtgc 2100
ggcggcgtga tcccgcccca ggactacgag ttcctgttcg aggtgggcgt gagcaacgtg 2160
ttcggccccg gcacccggat ccccaaggcc gccgtgcagg tgctggacga catcgagaag 2220
tgcctggaga agaagcagca gagcgtg 2247
<210> 52
<211> 750
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 52
Met Leu Arg Ala Lys Asn Gln Leu Phe Leu Leu Ser Pro His Tyr Leu
1 5 10 15
Arg Gln Val Lys Glu Ser Ser Gly Ser Arg Leu Ile Gln Gln Arg Leu
20 25 30
Leu His Gln Gln Gln Pro Leu His Pro Glu Trp Ala Ala Leu Ala Lys
35 40 45
Lys Gln Leu Lys Gly Lys Asn Pro Glu Asp Leu Ile Trp His Thr Pro
50 55 60
Glu Gly Ile Ser Ile Lys Pro Leu Tyr Ser Lys Arg Asp Thr Met Asp
65 70 75 80
Leu Pro Glu Glu Leu Pro Gly Val Lys Pro Phe Thr Arg Gly Pro Tyr
85 90 95
Pro Thr Met Tyr Thr Phe Arg Pro Trp Thr Ile Arg Gln Tyr Ala Gly
100 105 110
Phe Ser Thr Val Glu Glu Ser Asn Lys Phe Tyr Lys Asp Asn Ile Lys
115 120 125
Ala Gly Gln Gln Gly Leu Ser Val Ala Phe Asp Leu Ala Thr His Arg
130 135 140
Gly Tyr Asp Ser Asp Asn Pro Arg Val Arg Gly Asp Val Gly Met Ala
145 150 155 160
Gly Val Ala Ile Asp Thr Val Glu Asp Thr Lys Ile Leu Phe Asp Gly
165 170 175
Ile Pro Leu Glu Lys Met Ser Val Ser Met Thr Met Asn Gly Ala Val
180 185 190
Ile Pro Val Leu Ala Asn Phe Ile Val Thr Gly Glu Glu Gln Gly Val
195 200 205
Pro Lys Glu Lys Leu Thr Gly Thr Ile Gln Asn Asp Ile Leu Lys Glu
210 215 220
Phe Met Val Arg Asn Thr Tyr Ile Phe Pro Pro Glu Pro Ser Met Lys
225 230 235 240
Ile Ile Ala Asp Ile Phe Glu Tyr Thr Ala Lys His Met Pro Lys Phe
245 250 255
Asn Ser Ile Ser Ile Ser Gly Tyr His Met Gln Glu Ala Gly Ala Asp
260 265 270
Ala Ile Leu Glu Leu Ala Tyr Thr Leu Ala Asp Gly Leu Glu Tyr Ser
275 280 285
Arg Thr Gly Leu Gln Ala Gly Leu Thr Ile Asp Glu Phe Ala Pro Arg
290 295 300
Leu Ser Phe Phe Trp Gly Ile Gly Met Asn Phe Tyr Met Glu Ile Ala
305 310 315 320
Lys Met Arg Ala Gly Arg Arg Leu Trp Ala His Leu Ile Glu Lys Met
325 330 335
Phe Gln Pro Lys Asn Ser Lys Ser Leu Leu Leu Arg Ala His Cys Gln
340 345 350
Thr Ser Gly Trp Ser Leu Thr Glu Gln Asp Pro Tyr Asn Asn Ile Val
355 360 365
Arg Thr Ala Ile Glu Ala Met Ala Ala Val Phe Gly Gly Thr Gln Ser
370 375 380
Leu His Thr Asn Ser Phe Asp Glu Ala Leu Gly Leu Pro Thr Val Lys
385 390 395 400
Ser Ala Arg Ile Ala Arg Asn Thr Gln Ile Ile Ile Gln Glu Glu Ser
405 410 415
Gly Ile Pro Lys Val Ala Asp Pro Trp Gly Gly Ser Tyr Met Met Glu
420 425 430
Cys Leu Thr Asn Asp Val Tyr Asp Ala Ala Leu Lys Leu Ile Asn Glu
435 440 445
Ile Glu Glu Met Gly Gly Met Ala Lys Ala Val Ala Glu Gly Ile Pro
450 455 460
Lys Leu Arg Ile Glu Glu Cys Ala Ala Arg Arg Gln Ala Arg Ile Asp
465 470 475 480
Ser Gly Ser Glu Val Ile Val Gly Val Asn Lys Tyr Gln Leu Glu Lys
485 490 495
Glu Asp Ala Val Glu Val Leu Ala Ile Asp Asn Thr Ser Val Arg Asn
500 505 510
Arg Gln Ile Glu Lys Leu Lys Lys Ile Lys Ser Ser Arg Asp Gln Ala
515 520 525
Leu Ala Glu Arg Cys Leu Ala Ala Leu Thr Glu Cys Ala Ala Ser Gly
530 535 540
Asp Gly Asn Ile Leu Ala Leu Ala Val Asp Ala Ser Arg Ala Arg Cys
545 550 555 560
Thr Val Gly Glu Ile Thr Asp Ala Leu Lys Lys Val Phe Gly Glu His
565 570 575
Lys Ala Asn Asp Arg Met Val Ser Gly Ala Tyr Arg Gln Glu Phe Gly
580 585 590
Glu Ser Lys Glu Ile Thr Ser Ala Ile Lys Arg Val His Lys Phe Met
595 600 605
Glu Arg Glu Gly Arg Arg Pro Arg Leu Leu Val Ala Lys Met Gly Gln
610 615 620
Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Thr Gly Phe Ala Asp
625 630 635 640
Leu Gly Phe Asp Val Asp Ile Gly Pro Leu Phe Gln Thr Pro Arg Glu
645 650 655
Val Ala Gln Gln Ala Val Asp Ala Asp Val His Ala Val Gly Ile Ser
660 665 670
Thr Leu Ala Ala Gly His Lys Thr Leu Val Pro Glu Leu Ile Lys Glu
675 680 685
Leu Asn Ser Leu Gly Arg Pro Asp Ile Leu Val Met Cys Gly Gly Val
690 695 700
Ile Pro Pro Gln Asp Tyr Glu Phe Leu Phe Glu Val Gly Val Ser Asn
705 710 715 720
Val Phe Gly Pro Gly Thr Arg Ile Pro Lys Ala Ala Val Gln Val Leu
725 730 735
Asp Asp Ile Glu Lys Cys Leu Glu Lys Lys Gln Gln Ser Val
740 745 750
<210> 53
<211> 749
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 53
Leu Arg Ala Lys Asn Gln Leu Phe Leu Leu Ser Pro His Tyr Leu Arg
1 5 10 15
Gln Val Lys Glu Ser Ser Gly Ser Arg Leu Ile Gln Gln Arg Leu Leu
20 25 30
His Gln Gln Gln Pro Leu His Pro Glu Trp Ala Ala Leu Ala Lys Lys
35 40 45
Gln Leu Lys Gly Lys Asn Pro Glu Asp Leu Ile Trp His Thr Pro Glu
50 55 60
Gly Ile Ser Ile Lys Pro Leu Tyr Ser Lys Arg Asp Thr Met Asp Leu
65 70 75 80
Pro Glu Glu Leu Pro Gly Val Lys Pro Phe Thr Arg Gly Pro Tyr Pro
85 90 95
Thr Met Tyr Thr Phe Arg Pro Trp Thr Ile Arg Gln Tyr Ala Gly Phe
100 105 110
Ser Thr Val Glu Glu Ser Asn Lys Phe Tyr Lys Asp Asn Ile Lys Ala
115 120 125
Gly Gln Gln Gly Leu Ser Val Ala Phe Asp Leu Ala Thr His Arg Gly
130 135 140
Tyr Asp Ser Asp Asn Pro Arg Val Arg Gly Asp Val Gly Met Ala Gly
145 150 155 160
Val Ala Ile Asp Thr Val Glu Asp Thr Lys Ile Leu Phe Asp Gly Ile
165 170 175
Pro Leu Glu Lys Met Ser Val Ser Met Thr Met Asn Gly Ala Val Ile
180 185 190
Pro Val Leu Ala Asn Phe Ile Val Thr Gly Glu Glu Gln Gly Val Pro
195 200 205
Lys Glu Lys Leu Thr Gly Thr Ile Gln Asn Asp Ile Leu Lys Glu Phe
210 215 220
Met Val Arg Asn Thr Tyr Ile Phe Pro Pro Glu Pro Ser Met Lys Ile
225 230 235 240
Ile Ala Asp Ile Phe Glu Tyr Thr Ala Lys His Met Pro Lys Phe Asn
245 250 255
Ser Ile Ser Ile Ser Gly Tyr His Met Gln Glu Ala Gly Ala Asp Ala
260 265 270
Ile Leu Glu Leu Ala Tyr Thr Leu Ala Asp Gly Leu Glu Tyr Ser Arg
275 280 285
Thr Gly Leu Gln Ala Gly Leu Thr Ile Asp Glu Phe Ala Pro Arg Leu
290 295 300
Ser Phe Phe Trp Gly Ile Gly Met Asn Phe Tyr Met Glu Ile Ala Lys
305 310 315 320
Met Arg Ala Gly Arg Arg Leu Trp Ala His Leu Ile Glu Lys Met Phe
325 330 335
Gln Pro Lys Asn Ser Lys Ser Leu Leu Leu Arg Ala His Cys Gln Thr
340 345 350
Ser Gly Trp Ser Leu Thr Glu Gln Asp Pro Tyr Asn Asn Ile Val Arg
355 360 365
Thr Ala Ile Glu Ala Met Ala Ala Val Phe Gly Gly Thr Gln Ser Leu
370 375 380
His Thr Asn Ser Phe Asp Glu Ala Leu Gly Leu Pro Thr Val Lys Ser
385 390 395 400
Ala Arg Ile Ala Arg Asn Thr Gln Ile Ile Ile Gln Glu Glu Ser Gly
405 410 415
Ile Pro Lys Val Ala Asp Pro Trp Gly Gly Ser Tyr Met Met Glu Cys
420 425 430
Leu Thr Asn Asp Val Tyr Asp Ala Ala Leu Lys Leu Ile Asn Glu Ile
435 440 445
Glu Glu Met Gly Gly Met Ala Lys Ala Val Ala Glu Gly Ile Pro Lys
450 455 460
Leu Arg Ile Glu Glu Cys Ala Ala Arg Arg Gln Ala Arg Ile Asp Ser
465 470 475 480
Gly Ser Glu Val Ile Val Gly Val Asn Lys Tyr Gln Leu Glu Lys Glu
485 490 495
Asp Ala Val Glu Val Leu Ala Ile Asp Asn Thr Ser Val Arg Asn Arg
500 505 510
Gln Ile Glu Lys Leu Lys Lys Ile Lys Ser Ser Arg Asp Gln Ala Leu
515 520 525
Ala Glu Arg Cys Leu Ala Ala Leu Thr Glu Cys Ala Ala Ser Gly Asp
530 535 540
Gly Asn Ile Leu Ala Leu Ala Val Asp Ala Ser Arg Ala Arg Cys Thr
545 550 555 560
Val Gly Glu Ile Thr Asp Ala Leu Lys Lys Val Phe Gly Glu His Lys
565 570 575
Ala Asn Asp Arg Met Val Ser Gly Ala Tyr Arg Gln Glu Phe Gly Glu
580 585 590
Ser Lys Glu Ile Thr Ser Ala Ile Lys Arg Val His Lys Phe Met Glu
595 600 605
Arg Glu Gly Arg Arg Pro Arg Leu Leu Val Ala Lys Met Gly Gln Asp
610 615 620
Gly His Asp Arg Gly Ala Lys Val Ile Ala Thr Gly Phe Ala Asp Leu
625 630 635 640
Gly Phe Asp Val Asp Ile Gly Pro Leu Phe Gln Thr Pro Arg Glu Val
645 650 655
Ala Gln Gln Ala Val Asp Ala Asp Val His Ala Val Gly Ile Ser Thr
660 665 670
Leu Ala Ala Gly His Lys Thr Leu Val Pro Glu Leu Ile Lys Glu Leu
675 680 685
Asn Ser Leu Gly Arg Pro Asp Ile Leu Val Met Cys Gly Gly Val Ile
690 695 700
Pro Pro Gln Asp Tyr Glu Phe Leu Phe Glu Val Gly Val Ser Asn Val
705 710 715 720
Phe Gly Pro Gly Thr Arg Ile Pro Lys Ala Ala Val Gln Val Leu Asp
725 730 735
Asp Ile Glu Lys Cys Leu Glu Lys Lys Gln Gln Ser Val
740 745
<210> 54
<211> 750
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 54
Met Leu Arg Ala Lys Asn Gln Leu Phe Leu Leu Ser Pro His Tyr Leu
1 5 10 15
Arg Gln Val Lys Glu Ser Ser Gly Ser Arg Leu Ile Gln Gln Arg Leu
20 25 30
Leu His Gln Gln Gln Pro Leu His Pro Glu Trp Ala Ala Leu Ala Lys
35 40 45
Lys Gln Leu Lys Gly Lys Asn Pro Glu Asp Leu Ile Trp His Thr Pro
50 55 60
Glu Gly Ile Ser Ile Lys Pro Leu Tyr Ser Lys Arg Asp Thr Met Asp
65 70 75 80
Leu Pro Glu Glu Leu Pro Gly Val Lys Pro Phe Thr Arg Gly Pro Tyr
85 90 95
Pro Thr Met Tyr Thr Phe Arg Pro Trp Thr Ile Arg Gln Tyr Ala Gly
100 105 110
Phe Ser Thr Val Glu Glu Ser Asn Lys Phe Tyr Lys Asp Asn Ile Lys
115 120 125
Ala Gly Gln Gln Gly Leu Ser Val Ala Phe Asp Leu Ala Thr His Arg
130 135 140
Gly Tyr Asp Ser Asp Asn Pro Arg Val Arg Gly Asp Val Gly Met Ala
145 150 155 160
Gly Val Ala Ile Asp Thr Val Glu Asp Thr Lys Ile Leu Phe Asp Gly
165 170 175
Ile Pro Leu Glu Lys Met Ser Val Ser Met Thr Met Asn Gly Ala Val
180 185 190
Ile Pro Val Leu Ala Asn Phe Ile Val Thr Gly Glu Glu Gln Gly Val
195 200 205
Pro Lys Glu Lys Leu Thr Gly Thr Ile Gln Asn Asp Ile Leu Lys Glu
210 215 220
Phe Met Val Arg Asn Thr Tyr Ile Phe Pro Pro Glu Pro Ser Met Lys
225 230 235 240
Ile Ile Ala Asp Ile Phe Glu Tyr Thr Ala Lys His Met Pro Lys Phe
245 250 255
Asn Ser Ile Ser Ile Ser Gly Tyr His Met Gln Glu Ala Gly Ala Asp
260 265 270
Ala Ile Leu Glu Leu Ala Tyr Thr Leu Ala Asp Gly Leu Glu Tyr Ser
275 280 285
Arg Thr Gly Leu Gln Ala Gly Leu Thr Ile Asp Glu Phe Ala Pro Arg
290 295 300
Leu Ser Phe Phe Trp Gly Ile Gly Met Asn Phe Tyr Met Glu Ile Ala
305 310 315 320
Lys Met Arg Ala Gly Arg Arg Leu Trp Ala His Leu Ile Glu Lys Met
325 330 335
Phe Gln Pro Lys Asn Ser Lys Ser Leu Leu Leu Arg Ala His Cys Gln
340 345 350
Thr Ser Gly Trp Ser Leu Thr Glu Gln Asp Pro Tyr Asn Asn Ile Val
355 360 365
Arg Thr Ala Ile Glu Ala Met Ala Ala Val Phe Gly Gly Thr Gln Ser
370 375 380
Leu His Thr Asn Ser Phe Asp Glu Ala Leu Gly Leu Pro Thr Val Lys
385 390 395 400
Ser Ala Arg Ile Ala Arg Asn Thr Gln Ile Ile Ile Gln Glu Glu Ser
405 410 415
Gly Ile Pro Lys Val Ala Asp Pro Trp Gly Gly Ser Tyr Met Met Glu
420 425 430
Cys Leu Thr Asn Asp Val Tyr Asp Ala Ala Leu Lys Leu Ile Asn Glu
435 440 445
Ile Glu Glu Met Gly Gly Met Ala Lys Ala Val Ala Glu Gly Ile Pro
450 455 460
Lys Leu Arg Ile Glu Glu Cys Ala Ala Arg Arg Gln Ala Arg Ile Asp
465 470 475 480
Ser Gly Ser Glu Val Ile Val Gly Val Asn Lys Tyr Gln Leu Glu Lys
485 490 495
Glu Asp Ala Val Glu Val Leu Ala Ile Asp Asn Thr Ser Val Arg Asn
500 505 510
Arg Gln Ile Glu Lys Leu Lys Lys Ile Lys Ser Ser Arg Asp Gln Ala
515 520 525
Leu Ala Glu Arg Cys Leu Ala Ala Leu Thr Glu Cys Ala Ala Ser Gly
530 535 540
Asp Gly Asn Ile Leu Ala Leu Ala Val Asp Ala Ser Arg Ala Arg Cys
545 550 555 560
Thr Val Gly Glu Ile Thr Asp Ala Leu Lys Lys Val Phe Gly Glu His
565 570 575
Lys Ala Asn Asp Arg Met Val Ser Gly Ala Tyr Arg Gln Glu Phe Gly
580 585 590
Glu Ser Lys Glu Ile Thr Ser Ala Ile Lys Arg Val His Lys Phe Met
595 600 605
Glu Arg Glu Gly Arg Arg Pro Arg Leu Leu Val Ala Lys Met Gly Gln
610 615 620
Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Thr Gly Phe Ala Asp
625 630 635 640
Leu Gly Phe Asp Val Asp Ile Gly Pro Leu Phe Gln Thr Pro Arg Glu
645 650 655
Val Ala Gln Gln Ala Val Asp Ala Asp Val His Ala Val Gly Val Ser
660 665 670
Thr Leu Ala Ala Gly His Lys Thr Leu Val Pro Glu Leu Ile Lys Glu
675 680 685
Leu Asn Ser Leu Gly Arg Pro Asp Ile Leu Val Met Cys Gly Gly Val
690 695 700
Ile Pro Pro Gln Asp Tyr Glu Phe Leu Phe Glu Val Gly Val Ser Asn
705 710 715 720
Val Phe Gly Pro Gly Thr Arg Ile Pro Lys Ala Ala Val Gln Val Leu
725 730 735
Asp Asp Ile Glu Lys Cys Leu Glu Lys Lys Gln Gln Ser Val
740 745 750
<210> 55
<211> 749
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 55
Leu Arg Ala Lys Asn Gln Leu Phe Leu Leu Ser Pro His Tyr Leu Arg
1 5 10 15
Gln Val Lys Glu Ser Ser Gly Ser Arg Leu Ile Gln Gln Arg Leu Leu
20 25 30
His Gln Gln Gln Pro Leu His Pro Glu Trp Ala Ala Leu Ala Lys Lys
35 40 45
Gln Leu Lys Gly Lys Asn Pro Glu Asp Leu Ile Trp His Thr Pro Glu
50 55 60
Gly Ile Ser Ile Lys Pro Leu Tyr Ser Lys Arg Asp Thr Met Asp Leu
65 70 75 80
Pro Glu Glu Leu Pro Gly Val Lys Pro Phe Thr Arg Gly Pro Tyr Pro
85 90 95
Thr Met Tyr Thr Phe Arg Pro Trp Thr Ile Arg Gln Tyr Ala Gly Phe
100 105 110
Ser Thr Val Glu Glu Ser Asn Lys Phe Tyr Lys Asp Asn Ile Lys Ala
115 120 125
Gly Gln Gln Gly Leu Ser Val Ala Phe Asp Leu Ala Thr His Arg Gly
130 135 140
Tyr Asp Ser Asp Asn Pro Arg Val Arg Gly Asp Val Gly Met Ala Gly
145 150 155 160
Val Ala Ile Asp Thr Val Glu Asp Thr Lys Ile Leu Phe Asp Gly Ile
165 170 175
Pro Leu Glu Lys Met Ser Val Ser Met Thr Met Asn Gly Ala Val Ile
180 185 190
Pro Val Leu Ala Asn Phe Ile Val Thr Gly Glu Glu Gln Gly Val Pro
195 200 205
Lys Glu Lys Leu Thr Gly Thr Ile Gln Asn Asp Ile Leu Lys Glu Phe
210 215 220
Met Val Arg Asn Thr Tyr Ile Phe Pro Pro Glu Pro Ser Met Lys Ile
225 230 235 240
Ile Ala Asp Ile Phe Glu Tyr Thr Ala Lys His Met Pro Lys Phe Asn
245 250 255
Ser Ile Ser Ile Ser Gly Tyr His Met Gln Glu Ala Gly Ala Asp Ala
260 265 270
Ile Leu Glu Leu Ala Tyr Thr Leu Ala Asp Gly Leu Glu Tyr Ser Arg
275 280 285
Thr Gly Leu Gln Ala Gly Leu Thr Ile Asp Glu Phe Ala Pro Arg Leu
290 295 300
Ser Phe Phe Trp Gly Ile Gly Met Asn Phe Tyr Met Glu Ile Ala Lys
305 310 315 320
Met Arg Ala Gly Arg Arg Leu Trp Ala His Leu Ile Glu Lys Met Phe
325 330 335
Gln Pro Lys Asn Ser Lys Ser Leu Leu Leu Arg Ala His Cys Gln Thr
340 345 350
Ser Gly Trp Ser Leu Thr Glu Gln Asp Pro Tyr Asn Asn Ile Val Arg
355 360 365
Thr Ala Ile Glu Ala Met Ala Ala Val Phe Gly Gly Thr Gln Ser Leu
370 375 380
His Thr Asn Ser Phe Asp Glu Ala Leu Gly Leu Pro Thr Val Lys Ser
385 390 395 400
Ala Arg Ile Ala Arg Asn Thr Gln Ile Ile Ile Gln Glu Glu Ser Gly
405 410 415
Ile Pro Lys Val Ala Asp Pro Trp Gly Gly Ser Tyr Met Met Glu Cys
420 425 430
Leu Thr Asn Asp Val Tyr Asp Ala Ala Leu Lys Leu Ile Asn Glu Ile
435 440 445
Glu Glu Met Gly Gly Met Ala Lys Ala Val Ala Glu Gly Ile Pro Lys
450 455 460
Leu Arg Ile Glu Glu Cys Ala Ala Arg Arg Gln Ala Arg Ile Asp Ser
465 470 475 480
Gly Ser Glu Val Ile Val Gly Val Asn Lys Tyr Gln Leu Glu Lys Glu
485 490 495
Asp Ala Val Glu Val Leu Ala Ile Asp Asn Thr Ser Val Arg Asn Arg
500 505 510
Gln Ile Glu Lys Leu Lys Lys Ile Lys Ser Ser Arg Asp Gln Ala Leu
515 520 525
Ala Glu Arg Cys Leu Ala Ala Leu Thr Glu Cys Ala Ala Ser Gly Asp
530 535 540
Gly Asn Ile Leu Ala Leu Ala Val Asp Ala Ser Arg Ala Arg Cys Thr
545 550 555 560
Val Gly Glu Ile Thr Asp Ala Leu Lys Lys Val Phe Gly Glu His Lys
565 570 575
Ala Asn Asp Arg Met Val Ser Gly Ala Tyr Arg Gln Glu Phe Gly Glu
580 585 590
Ser Lys Glu Ile Thr Ser Ala Ile Lys Arg Val His Lys Phe Met Glu
595 600 605
Arg Glu Gly Arg Arg Pro Arg Leu Leu Val Ala Lys Met Gly Gln Asp
610 615 620
Gly His Asp Arg Gly Ala Lys Val Ile Ala Thr Gly Phe Ala Asp Leu
625 630 635 640
Gly Phe Asp Val Asp Ile Gly Pro Leu Phe Gln Thr Pro Arg Glu Val
645 650 655
Ala Gln Gln Ala Val Asp Ala Asp Val His Ala Val Gly Val Ser Thr
660 665 670
Leu Ala Ala Gly His Lys Thr Leu Val Pro Glu Leu Ile Lys Glu Leu
675 680 685
Asn Ser Leu Gly Arg Pro Asp Ile Leu Val Met Cys Gly Gly Val Ile
690 695 700
Pro Pro Gln Asp Tyr Glu Phe Leu Phe Glu Val Gly Val Ser Asn Val
705 710 715 720
Phe Gly Pro Gly Thr Arg Ile Pro Lys Ala Ala Val Gln Val Leu Asp
725 730 735
Asp Ile Glu Lys Cys Leu Glu Lys Lys Gln Gln Ser Val
740 745
<210> 56
<211> 1062
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 56
atgctgttca acctgcgcat cctgctgaac aacgccgcct tcagaaacgg ccacaacttc 60
atggttcgaa acttcagatg cggccagcct ctccagaaca aggtgcagct gaaaggcagg 120
gacctgctga ccctgaagaa cttcaccggc gaagagatca agtacatgct gtggctgtcc 180
gccgacctga agttcagaat caagcagaag ggcgagtacc tgcctctgct ccagggaaag 240
tctctgggca tgatcttcga gaagcggagc accagaacca gactgagcac cgagacaggc 300
tttgccctgc tcggaggaca cccctgcttt ctgacaaccc aggacatcca cctgggcgtg 360
aacgagagcc tgaccgatac agccagagtg ctgtcctcta tggccgatgc cgtgctggct 420
agagtgtata agcagagcga cctggacacc ctggctaaag aggccagcat tcccatcatc 480
aacggcctgt ccgacctgta tcaccccatc cagatcctgg ccgactacct gacactgcaa 540
gagcactaca gcagcctgaa gggactgacc ctgtcttgga tcggcgacgg caacaacatc 600
ctgcacagca ttatgatgag cgccgccaag ttcggaatgc acctccaggc cgctacaccc 660
aagggctatg aacctgatgc cagcgtgaca aagctggccg agcagtacgc caaagagaac 720
ggcacaaagc tgctgctgac caacgatccc ctggaagctg ctcacggcgg caatgtgctg 780
atcaccgata cctggatcag catgggccaa gaggaagaga agaagaagcg gctgcaagcc 840
ttccagggct accaagtgac catgaagaca gccaaggtgg ccgccagcga ttggaccttt 900
ctgcactgcc tgcctcggaa gcctgaagag gtggacgacg aggtgttcta cagccctaga 960
agcctggtgt tccccgaggc cgagaacaga aagtggacca tcatggctgt gatggtgtct 1020
ctgctgaccg actactcccc tcagctccag aagcctaagt tc 1062
<210> 57
<211> 1062
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 57
atgctgttca acctgcgaat cctgctgaac aatgccgctt ttcggaacgg gcacaatttc 60
atggtgagga actttcgctg cggacagccc ctccagaaca aggtccagct gaagggcagg 120
gacctgctga ccctgaaaaa tttcacaggg gaggaaatca agtacatgct gtggctgtca 180
gccgatctga agttccggat caagcagaag ggcgaatatc tgcctctgct ccagggcaaa 240
agcctgggga tgatcttcga aaagcgcagt actcggacca gactgtcaac agagactgga 300
ttcgcactgc tgggaggaca cccatgtttt ctgaccacac aggacattca tctgggagtg 360
aacgagtccc tgaccgacac agcacgcgtc ctgagctcca tggctgatgc agtgctggct 420
cgagtctaca aacagtctga cctggatacc ctggccaagg aagcttctat cccaatcatt 480
aatggcctga gtgacctgta tcaccccatc cagattctgg ccgattacct gaccctccag 540
gagcattatt ctagtctgaa agggctgaca ctgagctgga ttggggacgg aaacaatatc 600
ctgcactcca ttatgatgag cgccgccaag tttggaatgc acctccaggc tgcaacccca 660
aaaggctacg aacccgatgc ctccgtgaca aagctggcag aacagtatgc caaagagaac 720
ggcactaagc tgctgctcac caatgaccct ctggaggccg ctcacggagg caacgtgctg 780
atcactgata cctggattag tatgggacag gaggaagaga agaagaagcg gctccaggcc 840
ttccagggct accaggtgac aatgaaaact gctaaggtcg cagccagcga ctggaccttt 900
ctgcattgcc tgcccagaaa gcctgaagag gtggacgatg aggtcttcta ctcacccaga 960
agcctggtgt ttcctgaagc tgagaatagg aagtggacaa tcatggcagt gatggtcagc 1020
ctgctgactg attattcccc tcagctccag aaaccaaagt tc 1062
<210> 58
<211> 1059
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 58
ctgttcaacc tgcgcatcct gctgaacaac gccgccttca gaaacggcca caacttcatg 60
gttcgaaact tcagatgcgg ccagcctctc cagaacaagg tgcagctgaa aggcagggac 120
ctgctgaccc tgaagaactt caccggcgaa gagatcaagt acatgctgtg gctgtccgcc 180
gacctgaagt tcagaatcaa gcagaagggc gagtacctgc ctctgctcca gggaaagtct 240
ctgggcatga tcttcgagaa gcggagcacc agaaccagac tgagcaccga gacaggcttt 300
gccctgctcg gaggacaccc ctgctttctg acaacccagg acatccacct gggcgtgaac 360
gagagcctga ccgatacagc cagagtgctg tcctctatgg ccgatgccgt gctggctaga 420
gtgtataagc agagcgacct ggacaccctg gctaaagagg ccagcattcc catcatcaac 480
ggcctgtccg acctgtatca ccccatccag atcctggccg actacctgac actgcaagag 540
cactacagca gcctgaaggg actgaccctg tcttggatcg gcgacggcaa caacatcctg 600
cacagcatta tgatgagcgc cgccaagttc ggaatgcacc tccaggccgc tacacccaag 660
ggctatgaac ctgatgccag cgtgacaaag ctggccgagc agtacgccaa agagaacggc 720
acaaagctgc tgctgaccaa cgatcccctg gaagctgctc acggcggcaa tgtgctgatc 780
accgatacct ggatcagcat gggccaagag gaagagaaga agaagcggct gcaagccttc 840
cagggctacc aagtgaccat gaagacagcc aaggtggccg ccagcgattg gacctttctg 900
cactgcctgc ctcggaagcc tgaagaggtg gacgacgagg tgttctacag ccctagaagc 960
ctggtgttcc ccgaggccga gaacagaaag tggaccatca tggctgtgat ggtgtctctg 1020
ctgaccgact actcccctca gctccagaag cctaagttc 1059
<210> 59
<211> 1059
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 59
ctgttcaacc tgcgaatcct gctgaacaat gccgcttttc ggaacgggca caatttcatg 60
gtgaggaact ttcgctgcgg acagcccctc cagaacaagg tccagctgaa gggcagggac 120
ctgctgaccc tgaaaaattt cacaggggag gaaatcaagt acatgctgtg gctgtcagcc 180
gatctgaagt tccggatcaa gcagaagggc gaatatctgc ctctgctcca gggcaaaagc 240
ctggggatga tcttcgaaaa gcgcagtact cggaccagac tgtcaacaga gactggattc 300
gcactgctgg gaggacaccc atgttttctg accacacagg acattcatct gggagtgaac 360
gagtccctga ccgacacagc acgcgtcctg agctccatgg ctgatgcagt gctggctcga 420
gtctacaaac agtctgacct ggataccctg gccaaggaag cttctatccc aatcattaat 480
ggcctgagtg acctgtatca ccccatccag attctggccg attacctgac cctccaggag 540
cattattcta gtctgaaagg gctgacactg agctggattg gggacggaaa caatatcctg 600
cactccatta tgatgagcgc cgccaagttt ggaatgcacc tccaggctgc aaccccaaaa 660
ggctacgaac ccgatgcctc cgtgacaaag ctggcagaac agtatgccaa agagaacggc 720
actaagctgc tgctcaccaa tgaccctctg gaggccgctc acggaggcaa cgtgctgatc 780
actgatacct ggattagtat gggacaggag gaagagaaga agaagcggct ccaggccttc 840
cagggctacc aggtgacaat gaaaactgct aaggtcgcag ccagcgactg gacctttctg 900
cattgcctgc ccagaaagcc tgaagaggtg gacgatgagg tcttctactc acccagaagc 960
ctggtgtttc ctgaagctga gaataggaag tggacaatca tggcagtgat ggtcagcctg 1020
ctgactgatt attcccctca gctccagaaa ccaaagttc 1059
<210> 60
<211> 354
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 60
Met Leu Phe Asn Leu Arg Ile Leu Leu Asn Asn Ala Ala Phe Arg Asn
1 5 10 15
Gly His Asn Phe Met Val Arg Asn Phe Arg Cys Gly Gln Pro Leu Gln
20 25 30
Asn Lys Val Gln Leu Lys Gly Arg Asp Leu Leu Thr Leu Lys Asn Phe
35 40 45
Thr Gly Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys
50 55 60
Phe Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys
65 70 75 80
Ser Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser
85 90 95
Thr Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr
100 105 110
Thr Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala
115 120 125
Arg Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg Val Tyr Lys
130 135 140
Gln Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile
145 150 155 160
Asn Gly Leu Ser Asp Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr
165 170 175
Leu Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser
180 185 190
Trp Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met Ser Ala
195 200 205
Ala Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu
210 215 220
Pro Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn
225 230 235 240
Gly Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala His Gly
245 250 255
Gly Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met Gly Gln Glu Glu
260 265 270
Glu Lys Lys Lys Arg Leu Gln Ala Phe Gln Gly Tyr Gln Val Thr Met
275 280 285
Lys Thr Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu
290 295 300
Pro Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg
305 310 315 320
Ser Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala
325 330 335
Val Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro
340 345 350
Lys Phe
<210> 61
<211> 353
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 61
Leu Phe Asn Leu Arg Ile Leu Leu Asn Asn Ala Ala Phe Arg Asn Gly
1 5 10 15
His Asn Phe Met Val Arg Asn Phe Arg Cys Gly Gln Pro Leu Gln Asn
20 25 30
Lys Val Gln Leu Lys Gly Arg Asp Leu Leu Thr Leu Lys Asn Phe Thr
35 40 45
Gly Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys Phe
50 55 60
Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser
65 70 75 80
Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser Thr
85 90 95
Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr Thr
100 105 110
Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala Arg
115 120 125
Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg Val Tyr Lys Gln
130 135 140
Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile Asn
145 150 155 160
Gly Leu Ser Asp Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr Leu
165 170 175
Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp
180 185 190
Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met Ser Ala Ala
195 200 205
Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu Pro
210 215 220
Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn Gly
225 230 235 240
Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala His Gly Gly
245 250 255
Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met Gly Gln Glu Glu Glu
260 265 270
Lys Lys Lys Arg Leu Gln Ala Phe Gln Gly Tyr Gln Val Thr Met Lys
275 280 285
Thr Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro
290 295 300
Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg Ser
305 310 315 320
Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala Val
325 330 335
Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro Lys
340 345 350
Phe
<210> 62
<211> 354
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 62
Met Leu Ser Asn Leu Arg Ile Leu Leu Asn Asn Ala Ala Leu Arg Lys
1 5 10 15
Gly His Thr Ser Val Val Arg His Phe Trp Cys Gly Lys Pro Val Gln
20 25 30
Asn Lys Val Gln Leu Lys Gly Arg Asp Leu Leu Thr Leu Lys Asn Phe
35 40 45
Thr Gly Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys
50 55 60
Phe Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys
65 70 75 80
Ser Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser
85 90 95
Thr Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr
100 105 110
Thr Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala
115 120 125
Arg Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg Val Tyr Lys
130 135 140
Gln Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile
145 150 155 160
Asn Gly Leu Ser Asp Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr
165 170 175
Leu Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser
180 185 190
Trp Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met Ser Ala
195 200 205
Ala Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu
210 215 220
Pro Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn
225 230 235 240
Gly Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala His Gly
245 250 255
Gly Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met Gly Gln Glu Glu
260 265 270
Glu Lys Lys Lys Arg Leu Gln Ala Phe Gln Gly Tyr Gln Val Thr Met
275 280 285
Lys Thr Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu
290 295 300
Pro Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg
305 310 315 320
Ser Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala
325 330 335
Val Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro
340 345 350
Lys Phe
<210> 63
<211> 353
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 63
Leu Ser Asn Leu Arg Ile Leu Leu Asn Asn Ala Ala Leu Arg Lys Gly
1 5 10 15
His Thr Ser Val Val Arg His Phe Trp Cys Gly Lys Pro Val Gln Asn
20 25 30
Lys Val Gln Leu Lys Gly Arg Asp Leu Leu Thr Leu Lys Asn Phe Thr
35 40 45
Gly Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys Phe
50 55 60
Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser
65 70 75 80
Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser Thr
85 90 95
Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr Thr
100 105 110
Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala Arg
115 120 125
Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg Val Tyr Lys Gln
130 135 140
Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile Asn
145 150 155 160
Gly Leu Ser Asp Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr Leu
165 170 175
Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp
180 185 190
Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met Ser Ala Ala
195 200 205
Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu Pro
210 215 220
Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn Gly
225 230 235 240
Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala His Gly Gly
245 250 255
Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met Gly Gln Glu Glu Glu
260 265 270
Lys Lys Lys Arg Leu Gln Ala Phe Gln Gly Tyr Gln Val Thr Met Lys
275 280 285
Thr Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro
290 295 300
Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg Ser
305 310 315 320
Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala Val
325 330 335
Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro Lys
340 345 350
Phe
<210> 64
<211> 461
<212> PRT
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 64
Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr
1 5 10 15
Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu
20 25 30
Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn
35 40 45
Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys
50 55 60
Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn
65 70 75 80
Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln
85 90 95
Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile
100 105 110
Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys
115 120 125
Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
130 135 140
Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly
145 150 155 160
Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe
165 170 175
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala
180 185 190
Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu
195 200 205
Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe
210 215 220
Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp
225 230 235 240
Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile
245 250 255
Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly
260 265 270
Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu
275 280 285
His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn
290 295 300
Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu
305 310 315 320
Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile
325 330 335
Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr
340 345 350
Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val
355 360 365
Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Leu
370 375 380
Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His
385 390 395 400
Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val
405 410 415
Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly
420 425 430
Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser
435 440 445
Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr
450 455 460
<210> 65
<211> 1383
<212> DNA
<213> artificial sequence
<220>
<223> synthetic sequence
<400> 65
atgcagcgcg tgaacatgat tatggccgag tctcccggcc tgatcaccat ctgtctgctg 60
ggctatctgc tgagcgccga gtgcaccgtg tttctggatc acgagaacgc caacaagatc 120
ctgaacagac ccaagcggta caacagcggc aaactggaag agttcgtgca gggtaacctg 180
gaacgcgagt gcatggaaga gaagtgcagc ttcgaagagg ccagagaggt gttcgagaac 240
accgagagaa ccaccgagtt ctggaagcag tacgtggacg gcgatcagtg cgagagcaac 300
ccttgtctga atggcggcag ctgcaaggat gacatcaaca gctacgagtg ctggtgcccc 360
ttcggcttcg agggcaagaa ttgcgagctg gatgtgacct gcaacatcaa gaacggcaga 420
tgcgagcagt tctgcaagaa cagcgccgac aacaaggtcg tgtgctcctg cacagagggc 480
tacagactgg ccgagaatca gaagtcctgc gagcccgctg tgccctttcc atgtggcaga 540
gtgtctgtgt cccagaccag caagctgacc agagccgaga cagtgttccc cgatgtggac 600
tacgtgaaca gcaccgaggc cgagacaatc ctggacaaca tcacccagag cacccagtcc 660
ttcaacgact tcaccagagt cgtcggcggc gaggatgcta agcctggaca gtttccttgg 720
caagtggtgc tgaacggcaa ggtggacgct ttttgtggcg gctccatcgt gaacgagaag 780
tggatcgtga ccgccgctca ctgtgtggaa accggcgtga agattacagt ggtggccggg 840
gagcacaaca tcgaggaaac agagcacacc gagcagaaac ggaacgtgat cagaatcatc 900
cctcaccaca actacaacgc cgccatcaac aagtacaacc acgacattgc cctgctcgag 960
ctggacgaac ccctggtcct gaactcttac gtgaccccta tctgtatcgc cgacaaagag 1020
tacaccaaca tctttctgaa gttcggcagc ggctacgtgt ccggctgggg aagagttttc 1080
cacaagggca gatcagccct ggtgctgcag tacctgagag tgcccctggt ggatagagcc 1140
acatgcctgc tgagcaccaa gttcaccatc tacaacaaca tgttctgcgc cggcttccac 1200
gaaggcggca gagattcttg tcagggcgat tctggcggcc ctcacgtgac agaagtcgag 1260
ggcacatctt ttctgaccgg catcatcagc tggggcgaag agtgtgccat gaaggggaag 1320
tacggcatct ataccaaggt gtccagatac gtgaactgga tcaaagaaaa gaccaagctc 1380
acc 1383
Claims (29)
1. A composition comprising lipid nanoparticles comprising at least one of:
about 53 mole% to about 60 mole% ssPalmO-Ph-P4C2;
about 34 mole% to about 41 mole% cholesterol;
about 4 mole% to about 11 mole% DOPC, DSPC or DOPE; and
about 0.5 mole% to about 2 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is from about 70:1 to about 105:1 (w/w).
2. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
3. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w/w).
4. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
About 40 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
5. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 56.5 mole% ssPalmO-Ph-P4C2;
about 37.5 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
6. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
7. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% DOPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w/w).
8. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
9. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
About 40 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
10. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 56.5 mole% ssPalmO-Ph-P4C2;
about 37.5 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
11. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 59 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 5 mole% DSPC; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
12. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
13. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
about 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w/w).
14. The composition of claim 1, comprising at least one lipid nanoparticle comprising:
about 54 mole% ssPalmO-Ph-P4C2;
About 35 mole% cholesterol;
about 10 mole% DOPE; and
about 1 mole% DMG-PEG2000,
wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one DNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w/w).
15. The composition of any one of claims 1 to 13, wherein the RNA molecule is an mRNA molecule, preferably wherein the mRNA molecule further comprises a 5' cap.
16. The composition of any one of claims 1 to 13 or 15, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding at least one transposase, preferably wherein the transposase is piggyBac TM (PB) transposase, piggyBac-like (PBL) transposase, super piggyBac TM (SPB) transposase polypeptide, sleeping beauty transposase (Sleeping Beauty transposase), hyperactive sleeping beauty (Hyperactive Sleeping Beauty) (SB 100X) transposase, heliron transposase, tol2 transposase, tcBuster transposase, or mutant TcBuster transposase.
17. The composition of claim 14, wherein the DNA molecule is a circular DNA molecule, a dougybone DNA molecule, a DNA plasmid, a DNA nanoplasmid, or a linearized DNA molecule, preferably wherein the DNA molecule is a dougybone DNA molecule or a DNA nanoplasmid.
18. The composition of claim 14 or claim 17, wherein the at least one DNA molecule comprises a nucleic acid sequence encoding at least one transposon.
19. The composition of any one of the preceding claims, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one therapeutic protein.
20. The composition of any one of claims 14, 17 and 18, wherein the at least one nucleic acid molecule comprises a nucleic acid sequence encoding at least one transposon, wherein the transposon comprises a nucleic acid sequence encoding at least one therapeutic protein.
21. The composition of claim 19 or claim 20, wherein the at least one therapeutic protein is ornithine carbamoyltransferase (ornithine transcarbamylase, OTC), methylmalonate monoacyl-CoA mutase (MUT 1), factor VIII (Factor VIII), or Factor IX (Factor IX).
22. A method of delivering at least one nucleic acid to at least one cell, the method comprising contacting the at least one cell with at least one composition according to any one of the preceding claims.
23. A method of genetically modifying at least one cell, the method comprising contacting the at least one cell with at least one composition according to any one of the preceding claims.
24. The method of claim 22 or claim 23, wherein the at least one cell is at least one liver cell, preferably wherein the at least one liver cell is a hepatocyte, hepatic stellate cell, kupffer cell (Kupffer cell), or liver sinus endothelial cell.
25. A method of treating at least one disease or disorder in a subject in need thereof, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition according to any one of claims 1 to 21.
26. The method of claim 25, wherein the at least one disease or disorder is a liver disease or disorder, preferably wherein the liver disease or disorder is a metabolic liver disorder, preferably wherein the metabolic liver disorder is:
i) Urea cycle disorders; or (b)
ii) N-acetylglutamate synthase (N-Acetylglutamate Synthetase, NAGS) deficiency, carbamyl phosphate synthase I (Carbamoylphosphate SynthetaseI) deficiency (CPSI deficiency), ornithine carbamoyltransferase (OTC) deficiency, argininosuccinate synthase deficiency (Argininosuccinate Synthetase Deficiency, ASSD) (citrullinemia I (Citrullinemia I)), hitachi protein deficiency (Citrin Deficiency) (citrullinemia II (Citrullinemia II)), argininosuccinate lyase deficiency (Argininosuccinate Lyase Deficiency) (argininosuccinia (Argininosuccinic Aciduria)), arginase deficiency (Arginase Deficiency) (homoargininmia), ornithine translocase deficiency (Ornithine Translocase Deficiency) (HHHH Syndrome (HHH Syndome)), progressive familial intrahepatic cholestasis type 1 (progressive familia intrahepatic cholestasis type, PFIC1), progressive familial intrahepatic cholestasis type 1 (progressive familia intrahepatic cholestasis type, PFIC2), progressive familial intrahepatic cholestasis type 1 (progressive familia intrahepatic cholestasis type, PFIC3), or any combination thereof.
27. The method of claim 25, wherein the at least one disease or disorder is hemophilia, preferably wherein the hemophilia is hemophilia a, hemophilia B, hemophilia C, or any combination thereof.
28. The composition of any one of claims 1 to 13, 15 and 16, wherein the at least one RNA molecule comprises a nucleic acid sequence encoding a fusion protein, wherein the fusion protein comprises (i) an inactivated Cas9 (dCas 9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof.
29. The composition of claim 28, wherein the composition further comprises at least one guide RNA molecule.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63/152517 | 2021-02-23 | ||
| US63/156649 | 2021-03-04 | ||
| US63/164174 | 2021-03-22 | ||
| US202163197946P | 2021-06-07 | 2021-06-07 | |
| US63/197946 | 2021-06-07 | ||
| PCT/US2022/017570 WO2022182792A1 (en) | 2021-02-23 | 2022-02-23 | Compositions and methods for delivery of nucleic acids |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117377464A true CN117377464A (en) | 2024-01-09 |
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ID=89393298
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202280016755.3A Pending CN117377464A (en) | 2021-02-23 | 2022-02-23 | Compositions and methods for delivering nucleic acids |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117377464A (en) |
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2022
- 2022-02-23 CN CN202280016755.3A patent/CN117377464A/en active Pending
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