METHOD FOR mRNA-MEDIATED IMMUNIZATION

Abstract

To provide methods of immunization and methods for generating antibodies using compositions comprising cationic lipids and polynucleotide molecules, such as polyribonucleotide molecules, e.g., mRNA, which code for immunogens (e.g., a target protein or a fragment thereof).SOLUTION: A method for producing monoclonal antibodies against a target protein is provided, comprising the steps of: (a) mixing at least one cationic lipid with a polyribonucleotide such as a messenger RNA (mRNA) coding for the target protein or a fragment thereof, thereby forming a cationic lipid-polyribonucleotide complex; (b) administering the lipid-polyribonucleotide complex to a non-human animal; and (c) obtaining antibodies that specifically bind to the target protein from the animal.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is a continuation of U.S. Provisional Application No. 62 / 371,834, filed August 7, 2016. This application claims the benefit of U.S. Provisional Application No. 62 / 399,544, filed September 26, Each is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference. The entirety of the sequence listing ASC created on August 3, 2017 is incorporated herein by reference. The second copy is named PAT057169-WO-PCT_SL.txt and is 1 It is 46,992 bytes.

[0003] The present disclosure is in the field of immunology. In particular, the present disclosure relates to cationic lipids and poly Ribonucleotide molecules, e.g., a nucleic acid sequence that encodes an immunogen (e.g., a target protein or a fragment thereof) The present invention relates to a method of immunization using a composition containing a polynucleotide molecule, such as an mRNA encoding a The present disclosure also provides methods for immunizing animals (e.g., non-human animals) for the purpose of generating therapeutic antibodies. ), as well as methods for producing antibodies (e.g., monoclonal antibodies) from the do. [Background technology]

[0004] In vivo therapeutic monoclonal antibody development often involves the use of antibodies against a host cell that is used for immunization. The current approach is limited by the ability to generate high quality antigens that can be used in a wide variety of applications. Ideally, antigens would be fully It should be a highly purified protein with complete structural conformation. and sufficient sequences with the animal host strain to break immune tolerance and induce a robust humoral response However, many target proteins intended for use as antigens Regarding quality, meeting these requirements is the key to producing proteins that do not require overexpression / purification. Poor biophysical properties, cytotoxicity in host production cells, and target protein This is not possible due to problems such as poor immunogenicity of quality amino acid sequences.

[0005] Traditional animal immunization methods have employed two general strategies for producing antibodies: The first strategy involves the administration of the antibody in a purified form in the presence of an adjuvant to enhance the immune response. This involves repeated injections of full-length protein antigens in small to medium sized cells. For soluble proteins, this treatment inhibits the binding of the antigen to its native conformation. This may be a successful method for the production of monoclonal antibodies. Proteins, transmembrane proteins, proteins with unique post-translational modifications, or proteins with low solubility For proteins, this method is of very limited practical use because This is because it is difficult to obtain the necessary amounts of pure, native full-length protein for this purpose. requires immunization of animals with a DNA construct encoding the antigen of interest. This strategy allows the in situ expression of proteins that are difficult to purify in their native state. However, it has the disadvantage that it produces relatively low antibody titers, which ultimately leads to However, this is correlated with the low yield of monoclonal hybridomas. ard et al.Making and using antibodies:A Practical Handbook, 2 nd Edition CRC Press , 2013). Summary of the Invention [Means for solving the problem]

[0006] The present disclosure relates to a method for eliciting an immune response in an animal (e.g., a non-human animal). (a) attaching at least one cationic lipid to a polyribonucleic acid molecule encoding an antigenic determinant; and a polynucleotide such as a nucleic acid fragment (e.g., mRNA), thereby forming a cationic lipid. (b) forming a lipid-polynucleotide complex; and administering the complex to an animal. The present invention further relates to a method for genetic immunization. The method of the present invention further comprises the steps of: The present invention relates to a method in which the nucleic acid sequence is a polyribonucleotide molecule, such as an mRNA molecule, that encodes the nucleic acid sequence of the present invention. Maki further relates to the use of the genetic immunization methods described herein and the isolation and generation of antibodies from immunized animals. and isolating an antibody (e.g., polyclonal or monoclonal). The present invention relates to a method for producing a human ovarian tumor suppressor antibody (an antibody).

[0007] The present invention also provides a method for producing a monoclonal antibody, comprising: (a) at least One cationic lipid is mixed with the polyribonucleotide, thereby forming a lipid-polyribonucleotide complex. forming a nucleotide complex, the polynucleotide comprising an mRNA encoding an immunogen; (b) injecting the lipid-polynucleotide complex into at least one mammalian cell line; (c) administering to a mouse a lymphocyte (e.g., a B lymphocyte) or a splenocyte. (d) removing antibody-producing cells from the immunized mouse; The step of fusing the derived B lymphocytes with myeloma cells, thereby producing hybridomas. (e) cloning the hybridomas; and (f) producing anti-immunogen antibodies. (g) selecting positive clones that produce anti-immunogen antibodies; and (g) culturing the clones that produce anti-immunogen antibodies. and (h) isolating the anti-immunogen antibodies from the culture medium. In some embodiments, the methods provided herein for generating antibodies further include the step of: Amino acid sequences of antibody heavy and light chain variable regions and corresponding encoding nucleic acids In certain aspects, the method includes determining the sequence of the nucleic acid sequence as provided herein for generating antibodies. The method further includes the step of producing a chimeric or humanized antibody of the anti-immunogen antibody. Includes

[0008] The present disclosure also provides methods for treating immune tissue with cationic lipid nanoparticles (LNPs) containing mRNA. The present invention relates to a method for selectively isolating B cells harvested from an immunized animal. The antibodies are then directly screened for production of antibodies having the desired properties, and the antibodies are recombinantly produced. The vectors can be directly cloned and expressed, avoiding the need for hybridoma production.

[0009] The mRNA encapsulated LNPs of the present disclosure can also be administered to immunized host animals (e.g., rodents (e.g., For example, mice and rats), rabbits, chickens, cows, camelids, pigs, sheep Recombinant antibody libraries are generated from immune tissues of various mammals, including mice, goats, sharks, and non-human primates. This library may then be used to generate phage or yeast vectors. The desired properties can be screened for in heterologous host systems such as sprays. do.

[0010] The polynucleotide-based, e.g., mRNA-based, immunization methods of the present disclosure include the administration of antigens. The present invention addresses many of the problems associated with the above-mentioned obstacles inherent in the generation and / or production of antibodies. Among other things, the method obviates the need to directly express and purify the target protein antigen. Uses the host's own cellular machinery to produce target proteins and present them to the immune system For eukaryotic target proteins, this means eukaryote-specific post-translational modifications and It has the added advantage of allowing additional protein processing. The unpurified mRNA obtained is due in part to the presence of double-stranded RNA elements in the preparation. It has highly inflammatory properties. Double-stranded RNA binds to toll-like receptors and other receptors in the innate immune system. The pathogen-associated molecular patterns recognized by the receptors including Without being bound to any particular theory, this is believed to result in humoral binding to the target protein. It acts as an adjuvant to boost the response, resulting in high titer antibody production. It is thought that.

[0011] The generation of an immunogen, e.g., a monoclonal antibody against a target protein or a fragment thereof, In particular, immunization of animals with mRNA encoding said target protein or a fragment thereof. This method can be facilitated through the heterologous production and purification of the target protein. Therefore, transmembrane proteins (e.g., multi-transmembrane proteins), e.g., G proteins, Historically, there have been technical challenges in developing specific antibodies against GPCRs, Without being bound to any particular theory, Although not all cases are expected to be caused by the adjuvant-like properties of mRNA, host defense mechanisms elicited by the adjuvant properties of mRNA may be , resulting in rapid development of serum titers and being comparable to DNA immunization or other conventional immunization methods ( It is believed to be a better choice than immunization with recombinant proteins (e.g., immunization with recombinant proteins).

[0012] Non-limiting embodiments of the present disclosure are described in the following aspects.

[0013] Embodiment 1. To generate antibodies (e.g., monoclonal antibodies) against a target protein (a) attaching at least one cationic lipid to a target protein or Mix with polyribonucleotides such as messenger RNA (mRNA) that encode the fragment and thereby forming cationic lipid-polyribonucleotide complexes (e.g., mRNA-L (b) forming a lipid-polyribonucleotide complex; and (b) forming a lipid-polyribonucleotide complex into a non-human NP complex. (c) obtaining from the animal an antibody that specifically binds to the target protein. and

[0014] 2. To generate antibodies (e.g., monoclonal antibodies) against a target protein (a) a lipid-polyribonucleotide complex (e.g., mRNA-LNP) The complex is administered to a non-human animal, thereby inducing an immune response against the target protein. wherein the complex is an mRNA encoding a target protein or a fragment thereof. which comprises at least one cationic lipid having a polyribonucleotide; (b) obtaining an antibody that specifically binds to the target protein produced by the animal; A method comprising:

[0015] Embodiment 3. The method of embodiment 1 or 2, wherein the target protein is a transmembrane protein.

[0016] Aspect 4. The transmembrane protein is selected from the group consisting of: (i) G protein-coupled receptors (GPCRs); (ii) single-pass transmembrane protein receptors; (iii) tumor necrosis factor receptor superfamily (TNFRSF) members; (iv) interleukin (IL) receptor; (v) ion channels; (vi) solute transporters; (vii) an immune receptor; and (viii) Multi-transmembrane proteins The method of embodiment 3, wherein the

[0017] 5. The transmembrane protein is a multi-membrane protein, such as a G protein-coupled receptor (GPCR). The method of embodiment 3 or 4, wherein the protein is a transmembrane protein.

[0018] 6. The GPCR is RXFP1, TSHR, APJ, GPR40, GPR64, GP R4, or GPR15.

[0019] 7. The transmembrane protein is a single-pass transmembrane protein receptor protein, such as GP130. 3. The method of claim 2, wherein the IL-11 is a IL-11 protein receptor or a multi-transmembrane protein such as SLC52A2. 4 ways.

[0020] 8. The transmembrane protein is selected from the group consisting of IL-1 receptor, IL-2 receptor, IL-3 receptor, and I I L-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, I IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, I IL-18 receptor, IL-19 receptor, IL-20 receptor, IL-21 receptor, IL-22 Receptor, IL-23 receptor, IL-24 receptor, IL-25 receptor, IL-26 receptor, IL-27 receptor, IL-28 receptor, IL-29 receptor, IL-30 receptor, IL-3 1 receptor, IL-32 receptor, IL-33 receptor, IL-35 receptor, or IL-36 The method of embodiment 3 or 4, wherein the receptor is an interleukin (IL) receptor, such as a receptor.

[0021] Aspect 9. The transmembrane protein is selected from the group consisting of TNFRSF1A, TNFRSF1B, TNFR SF3, TNFRSF4, TNFRSF5, TNFRSF6, TNFRSF6B, TNF RSF7, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B , TNFRSF10C, TNFRSF10D, TNFRSF11A, TNFRSF11B , TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF16, TNFRSF17, TNFRSF18, TNFRSF19, TNFR TNFRSF21, TNFRSF25, and TNFRSF27 The subject of embodiment 3 or 4, which is a member of the death factor receptor superfamily (TNFRSF) Law.

[0022] Embodiment 10. The method of embodiment 3, wherein the transmembrane protein is an ion channel, such as TMEM16A. Or 4 ways.

[0023] Embodiment 11 The method of embodiment 3 or 4, wherein the transmembrane protein is a solute carrier.

[0024] 12. The target protein is one of the following: ACKR1, ACKR2, ACKR3, ACKR 4, ADCYAP1R1, ADGRA1, ADGRA2, ADGRA3, ADGRB1, ADGRB2、ADGRB3、ADGRD1、ADGRD2、ADGRE1、ADGRE 2、ADGRE3、ADGRE4P、ADGRE5、ADGRF1、ADGRF2、AD GRF3、ADGRF4、ADGRF5、ADGRG1、ADGRG2、ADGRG3、 ADGRG4、ADGRG5、ADGRG6、ADGRG7、ADGRL1、ADGRL 2、ADGRL3、ADGRL4、ADGRV1、ADORA1、ADORA2A、AD ORA2B、ADORA3、ADRA1A、ADRA1B、ADRA1D、ADRA2A 、ADRA2B、ADRA2C、ADRB1、ADRB2、ADRB3、AGTR1、A GTR2、APLNR / APJ、ASGR1、ASGR2、AVPR1A、AVPR1B 、AVPR2、BDKRB1、BDKRB2、BRS3、BRS3、C3AR1、C5A R1、C5AR2、CALCR、CALCRL、CASR、CCKAR、CCKBR、C CR1、CCR10、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、 CCR8、CCR9、CCRL2、CELSR1、CELSR2、CELSR3、CHR M1、CHRM2、CHRM3、CHRM4、CHRM5、CMKLR1、CNR1、C NR2、CRHR1、CRHR2、CX3CR1、CXCR1、CXCR2、CXCR3 、CXCR4、CXCR5、CXCR6、CYSLTR1、CYSLTR2、DRD1、 DRD2、DRD3、DRD4、DRD5、EDNRA、EDNRB、F2R、F2RL 1、F2RL2、F2RL3、FFAR1、FFAR2、FFAR3、FFAR4、FP R1、FPR2、FPR2、FPR3、FSHR、FZD1、FZD10、FZD2、F ZD3、FZD4、FZD5、FZD6、FZD7、FZD8、FZD9、GABBR1 、GABBR2、GALR1、GALR2、GALR3、GCGR、GHRHR、GHS R、GIPR、GLP1R、GLP2R、GNRHR、GNRHR2、GPBAR1、G PER1、GPR1、GPR4、GPR12、GPR15、GPR17、GPR18、G PR19、GPR20、GPR21、GPR22、GPR25、GPR26、GPR27 、GPR3、GPR31、GPR32、GPR33、GPR34、GPR35、GPR3 7、GPR37L1、GPR39、GPR40、GPR42、GPR42、GPR45、 GPR50、GPR52、GPR55、GPR6、GPR61、GPR62、GPR63 、GPR65、GPR68、GPR75、GPR78、GPR79、GPR82、GPR 83、GPR84、GPR85、GPR87、GPR88、GPR101、GPR107 、GPR132、GPR135、GPR137、GPR139、GPR141、GPR1 42、GPR143、GPR146、GPR148、GPR149、GPR15、GPR 150、GPR151、GPR152、GPR153、GPR156、GPR157、G PR158、GPR160、GPR161、GPR162、GPR171、GPR173 、GPR174、GPR176、GPR179、GPR182、GPR183、GPRC 5A、GPRC5B、GPRC5C、GPRC5D、GPRC6A、GRM1、GRM2 、GRM3、GRM4、GRM5、GRM6、GRM7、GRM8、GRPR、HCAR 1、HCAR2、HCAR3、HCRTR1、HCRTR2、HRH1、HRH2、HR H3、HRH4、HTR1A、HTR1B、HTR1D、HTR1E、HTR1F、HT R2A、HTR2B、HTR2C、HTR4、HTR5A、HTR5BP、HTR6、H TR7、KISS1R、LGR3、LGR4、LGR5、LGR6、LHCGR、LPA R1、LPAR2、LPAR3、LPAR4、LPAR5、LPAR6、LTB4R、L TB4R2、MAS1、MAS1L、MC1R、MC2R、MC3R、MC4R、MC5 R、MCHR1、MCHR2、MLNR、MRGPRD、MRGPRE、MRGPRF、 MRGPRG、MRGPRX1、MRGPRX2、MRGPRX3、MRGPRX4、M TNR1A、MTNR1B、NMBR、NMUR1、NMUR2、NPBWR1、NPB WR2、NPFFR1、NPFFR2、NPSR1、NPY1R、NPY2R、NPY4 R、NPY5R、NPY6R、NTSR1、NTSR2、OPN3、OPN4、OPN5 、OPRD1、OPRK1、OPRL1、OPRM1、OR51E1、OXER1、OX GR1、OXTR、P2RY1、P2RY10、P2RY11、P2RY12、P2RY 13、P2RY14、P2RY2、P2RY4、P2RY6、P2RY8、PRLHR、 PROKR1、PROKR2、PTAFR、PTGDR、PTGDR2、PTGER1、 PTGER2、PTGER3、PTGER4、PTGFR、PTGIR、PTH1R、P TH2R、QRFPR、RXFP1、RXFP2、RXFP3、RXFP4、S1PR1 、S1PR2、S1PR3、S1PR4、S1PR5、SCTR、SMO、SSTR1、 SSTR2、SSTR3、SSTR4、SSTR5、SUCNR1、TAAR1、TAA R2、TAAR3、TAAR4P、TAAR5、TAAR6、TAAR8、TAAR9、 TACR1、TACR2、TACR3、TAS1R1、TAS1R2、TAS1R3、T AS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TA S2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS 2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2 R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R 50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TBXA2R, T PRA1, TRHR, TSHR, UTS2R, VIPR1, VIPR2, XCR1, TC R-α, TCR-β, CD3, ζ-chain accessory, CD4, CD8, SIGIRR (single containing a single Ig and TIR domain), mannose receptor (MR), asialoglycoprotein Protein receptor family (e.g., asialoglycoprotein receptor macrophage galactose receptor type lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K ), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), Dectin-1 / CL EC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CL EC12A), CLEC2 (also called CLEC1B), CLEC12B, DCIR / CLEC4A, Dectin 2 / CLEC6A, Blood DC Antigen 2 (BDCA2) (CLEC4 C), macrophage-inducible C-type lectin (CLEC4E), TLR1, TLR2, TL R3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, T LR11, TLR12, TLR13, FcγRI(CD64), FcγRIIA(CD3 2), FcγRIIB1(CD32), FcγRIIB2(CD32), FcγRIII A(CD16a), FcγRIIIB(CD16b), FcεRI, FcεRII(CD 23), FcαR1 (CD89), Fcα / μR, FcRn, CD27, CD40, OX 40, GITR, CD137, PD-1, CTLA-4, PD-L1, TIGIT, T-thin Transcellular immunoglobulin and mucin domain 3 (TIM3), a V-domain involved in T cell activation Main Ig inhibitor (VISTA), CD28, CD122, ICOS, A2AR, B7 -H3, B7-H4, B and T lymphocyte attenuator (BTLA), indole amine Indole 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR) , lymphocyte activation gene-3 (LAG3), FAM159B, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB 1. HLA-DRA, HLA-DRB1, gp130, IL-1 receptor, IL-2 receptor , IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor , IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12 Receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor, IL-19 receptor, IL-20 receptor, IL-2 1 receptor, IL-22 receptor, IL-23 receptor, IL-24 receptor, IL-25 receptor , IL-26 receptor, IL-27 receptor, IL-28 receptor, IL-29 receptor, IL- IL-30 receptor, IL-31 receptor, IL-32 receptor, IL-33 receptor, IL-35 receptor Body, IL-36 receptor, FGFR1, FGFR2, FGFR3, FGFR4, TNFRS F1A, TNFRSF1B, TNFRSF3, TNFRSF4, TNFRSF5, TNF RSF6、TNFRSF6B、TNFRSF7、TNFRSF8、TNFRSF9、TN FRSF10A、TNFRSF10B、TNFRSF10C、TNFRSF10D、TN FRSF11A、TNFRSF11B、TNFRSF12A、TNFRSF13B、TN FRSF13C、TNFRSF14、TNFRSF16、TNFRSF17、TNFRS F18、TNFRSF19、TNFRSF21、TNFRSF25、TNFRSF27、 SCN1A、SCN1B、SCN2A、SCN2B、SCN3A、SCN3B、SCN4 A、SCN5A、SCN7A、SCN8A、SCN9A、SCN10A、SCN11A、 CACNA1A、CACNA1B、CACNA1C、CACNA1D、CACNA1E、 CACNA1F、CACNA1G、CACNA1H、CACNA1I、CACNA1S、 TRPA1、TRPC1、TRPC2、TRPC3、TRPC4、TRPC5、TRPC 6、TRPC7、TRPM1、TRPM2、TRPM3、TRPM4、TRPM5、TR PM6、TRPM7、TRPM8、MCOLN1、MCOLN2、MCOLN3、PKD 1、PKD2、PKD2L1、PKD2L2、TRPV1、TRPV2、TRPV3、T RPV4、TRPV5、TRPV6、CATSPER1、CATSPER2、CATSP ER3、CATSPER4、TPCN1、TPCN2、CNGA1、CNGA2、CNG A3、CNGA4、CNGB1、CNGB3、HCN1、HCN2、HCN3、HCN4 、KCNMA1、KCNN1、KCNN2、KCNN3、KCNN4、KCNT1、KC NT2、KCNU1、KCNA1、KCNA2、KCNA3、KCNA4、KCNA5、 KCNA6、KCNA7、KCNA10、KCNB1、KCNB2、KCNC1、KCN C2、KCNC3、KCNC4、KCND1、KCND2、KCND3、KCNF1、K CNG1、KCNG2、KCNG3、KCNG4、KCNH1、KCNH2、KCNH3 、KCNH4、KCNH5、KCNH6、KCNH7、KCNH8、KCNQ1、Industrial park Q2、KCNQ3、KCNQ4、KCNQ5、KCNS1、KCNS2、KCNS3、K CNV1、KCNV2、KCNJ1、KCNJ2、KCNJ3、KCNJ4、KCNJ5 、KCNJ6、KCNJ8、KCNJ9、KCNJ10、KCNJ11、KCNJ12、 KCNJ13、KCNJ14、KCNJ15、KCNJ16、KCNJ18、KCNK1 、KCNK2、KCNK3、KCNK4、KCNK5、KCNK6、KCNK7、KCN K9、KCNK10、KCNK12、KCNK13、KCNK15、KCNK16、KC NK17、KCNK18、HVCN1、HTR3A、HTR3B、HTR3C、HTR3 D、HTR3E、CHRNA1、CHRNA2、CHRNA3、CHRNA4、CHRN A5、CHRNA6、CHRNA7、CHRNA9、CHRNA10、CHRNB1、C HRNB2、CHRNB3、CHRNB4、CHRND、CHRNE、CHRNG、GA BRA1、GABRA2、GABRA3、GABRA4、GABRA5、GABRA6、 G ABRB1、GABRB2、GABRB3、GABRD、GABRE、GABRG1、G ABRG2、GABRG3、GABRP、GABRQ、GABRR1、GABRR2、G ABRR3、GRIA1、GRIA2、GRIA3、GRIA4、GRID1、GRID 2, GRIK1, GRIK2, GRIK3, GRIK4, GRIK5, GRIN1, GR IN2A, GRIN2B, GRIN2C, GRIN2D, GRIN3A, GRIN3B, GLRA1, GLRA2, GLRA3, GLRA4, P2RX1, P2RX2, P2RX 3, P2RX4, P2RX5, P2RX6, P2RX7, ZACN, ASIC1, ASI C2, ASIC3, ASIC4, AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP9, AQP10, AQP11, AQP12A, A QP12B, MIP, CLCN1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, CLCNKB, cystic fibrosis transmembrane conductance Regulatory factors (CFTR), ANO1, ANO2, ANO3, ANO4, ANO5, ANO6 , ANO7, ANO8, ANO9, ANO10, BEST1, BEST2, BEST3, BEST4, CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, CLIC 6, GJA1, GJA3, GJA4, GJA5, GJA6P, GJA8, GJA9, GJ A10, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, G JC1, GJC2, GJC3, GJD2, GJD3, GJD4, GJE1, ITPR1, ITPR2, ITPR3, PANX1, PANX2, PANX3, RYR1, RYR2, RYR3, NALCN, SCNN1A, SCNN1B, SCNN1D, SCNN1G, T EM16A, ADAMTS7, ANGPTL3, ANGPTL4, ANGPTL8, LP L, GDF15, galectin-1, galectin-2, galectin-3, galectin-4, Galectin-7, Galectin-8, Galectin-9, Galectin-10, Galectin-1 2. Galectin-13, matrix gla protein (MGP), PRNP, DGAT 1, GPAT3, DMC1, BLM, BRCA2, human endogenous retrovirus type K ( A member of the HERV-K family, an ectonucleoside triphosphate diphosphohydrolase ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), ectonucleoside triphosphate diphosphohydrolase 2 (ENTP D2), SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, S LC1A6, SLC1A7, SLC2A1, SLC2A2, SLC2A3, SLC2A4 , SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2 A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14, SLC3 A1, SLC3A2, SLC4A1, SLC4A2, SLC4A3, SLC4A4, SL C4A5, SLC4A6, SLC4A7, SLC4A8, SLC4A9, SLC4A10 , SLC4A11, SLC5A1, SLC5A2, SLC5A3, SLC5A4, SLC 5A5, SLC5A6, SLC5A7, SLC5A8, SLC5A9, SLC5A10, SLC5A11, SLC5A12, SLC6A1, SLC6A2, SLC6A3, SLC 6A4, SLC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, S LC6A10, SLC6A11, SLC6A12, SLC6A13, SLC6A14, S LC6A15, SLC6A16, SLC6A17, SLC6A18, SLC6A19, S LC6A20, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A 9, SLC7A10, SLC7A11, SLC7A13, SLC7A14, SLC8A1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC8A2, SLC8A3, SLC9A1, SLC9A2, SLC9A3, SLC9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C9A10、SLC9A11、SLC9B1、SLC9B2、SLC10A1、SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0A2、SLC10A3、SLC10A4、SLC10A5、SLC10A6、SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0A7、SLC11A1、SLC11A2、SLC12A1、SLC12A2、SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2A3, SLC12A4, SLC12A5, SLC12A6, SLC12A7, SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2A8, SLC12A9, SLC13A1, SLC13A2, SLC13A3, SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3A4, SLC13A5, SLC14A1, SLC14A2, SLC15A1, SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5A2、SLC15A3、SLC15A4、SLC16A1、SLC16A2、SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6A3、SLC16A4、SLC16A5、SLC16A6、SLC16A7、SLC1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6A8、SLC16A9、SLC16A10、SLC16A11、SLC16A12、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC16A13, SLC16A14, SLC17A1, SLC17A2, SLC17A3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC17A4, SLC17A5, SLC17A6, SLC17A7, SLC17A8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC17A9, SLC18A1, SLC18A2, SLC18A3, SLC19A1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC19A2, SLC19A3, SLC20A1, SLC20A2, SLCO1A2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLCO1B1、SLCO1B3、SLCO1C1、SLCO2A1、SLCO2B1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLCO3A1、SLCO4A1、SLCO4C1、SLCO5A1、SLCO6A1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC22A1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 0、SLC22A11、SLC22A12、SLC22A13、SLC22A14、SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C22A15, SLC22A16, SLC22A17, SLC22A18, SLC22A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 18AS, SLC22A19, SLC22A20, SLC22A23, SLC22A24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC22A25, SLC22A31, SLC23A1, SLC23A2, SLC23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC23, A4, SLC24, A1, SLC24, A2, SLC24, A3, SLC24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A4、SLC24A5、SLC24A6、SLC25A1、SLC25A2、SLC25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC25A4, SLC25A5, SLC25A6, SLC25A7, SLC25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A8, SLC25A9, SLC25A10, SLC25A11, SLC25A12, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C25A13, SLC25A14, SLC25A15, SLC25A16, SLC25A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 17、SLC25A18、SLC25A19、SLC25A20、SLC25A21、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC25A22, SLC25A23, SLC25A24, SLC25A25, SLC25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A26, SLC25A27, SLC25A28, SLC25A29, SLC25A30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC25A31, SLC25A32, SLC25A33, SLC25A34, SLC2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5A35, SLC25A36, SLC25A37, SLC25A38, SLC25A39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLC25A40、SLC25A41、SLC25A42、SLC25A43、SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 25A44, SLC25A45, SLC25A46, SLC26A1, SLC26A2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC26A3, SLC26A4, SLC26A5, SLC26A6, SLC26A7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC26A8, SLC26A9, SLC26A10, SLC26A11, SLC27A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1, SLC27A2, SLC27A3, SLC27A4, SLC27A5, SLC27A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6、SLC28A1、SLC28A2、SLC28A3、SLC29A1、SLC29A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2、SLC29A3、SLC29A4、SLC30A1、SLC30A2、SLC30A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3, SLC30A4, SLC30A5, SLC30A6, SLC30A7, SLC30A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8, SLC30A9, SLC30A10, SLC31A1, SLC31A2, SLC32<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A1, SLC33A1, SLC34A1, SLC34A2, SLC34A3, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A1, SLC35A2, SLC35A3, SLC35A4, SLC35A5, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> B1, SLC35B2, SLC35B3, SLC35B4, SLC35C1, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C2, SLC35D1, SLC35D2, SLC35D3, SLC35E1, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> E2, SLC35E3, SLC35E4, SLC35F1, SLC35F2, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F3, SLC35F4, SLC35F5, SLC35G1, SLC35G3, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G4、SLC35G5、SLC35G6、SLC36A1、SLC36A2、SLC36<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC36A4, SLC37A1, SLC37A2, SLC37A3, SLC37<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A4、SLC38A1、SLC38A2、SLC38A3、SLC38A4、SLC38<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A5, SLC38A6, SLC38A7, SLC38A8, SLC38A9, SLC38<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A10, SLC38A11, SLC39A1, SLC39A2, SLC39A3, SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A8, SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39A9, SLC39A10, SLC39A11, SLC39A12, SLC39A13<h2 style=";text-align:left;direction:ltr"> , SLC39A14, SLC40A1, SLC41A1, SLC41A2, SLC41A 3, RhAG, RhBG, RhCG, SLC43A1, SLC43A2, SLC43A3 , SLC44A1, SLC44A2, SLC44A3, SLC44A4, SLC44A5 , SLC45A1, SLC45A2, SLC45A3, SLC45A4, SLC46A1 , SLC46A2, SLC46A3, SLC47A1, SLC47A2, HCP-1, M FSD5, MFSD10, SLC50A1, OSTα, OSTβ, SLC52A1, SL The method of embodiment 1 or 2, wherein the polypeptide is selected from the group consisting of C52A2, and SLC52A3.

[0025] 13. The target antigen is difficult to express or to produce antibodies against. 3. The method of embodiment 1 or 2, wherein the method is difficult to produce.

[0026] 14. The expression of the target protein results in cytotoxicity in the host production cell, or increasing cytotoxicity.

[0027] 15. When the target protein is recombinantly expressed and / or purified, it is The method of embodiment 13, which exhibits sufficient yield, stability, solubility, and / or functional activity.

[0028] 16. The polynucleotide of the complex comprises the following: a consensus Kozak sequence; 7-methylguanosine cap on the 5' end of NA; found at the 3' end of mRNA transcripts a polyadenosine (polyA) tail; and 5'- and 3'-untranslated regions (UTRs). The method of any one of embodiments 1-15, comprising one or more of:

[0029] Embodiment 17 The method of any one of embodiments 1 to 16, wherein said administration is parenteral.

[0030] Embodiment 18 The method of any one of embodiments 1 to 17, wherein said administering is intravenous.

[0031] Embodiment 19 The method of any one of embodiments 1 to 18, wherein said administration is intramuscular.

[0032] Embodiment 20 The method of any one of embodiments 1 to 19, wherein said administration is subcutaneous.

[0033] Embodiment 21 The method of any one of embodiments 1 to 20, wherein said administration is intranasal.

[0034] 22. The method of claim 1, wherein the target protein is RXFP1 or a fragment thereof. Either one way.

[0035] 23. The complex is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 2, 4 and 37. 23. Any one of embodiments 1 to 22, comprising a polyribonucleotide comprising one nucleotide sequence. How to.

[0036] 24. The method of claim 1, wherein the target protein is SLC52A2 or a fragment thereof. Either one of the methods.

[0037] 25. The complex is selected from the group consisting of SEQ ID NO: 7, or any one of SEQ ID NOs: 5, 7, and 40. 25. Any one of embodiments 1 to 24, comprising a polyribonucleotide comprising one nucleotide sequence. How to.

[0038] 26. The method of claim 1, wherein the target protein is ANGPTL8 or a fragment thereof. Either one of the methods.

[0039] 27. The complex is selected from the group consisting of SEQ ID NO: 10, or SEQ ID NOs: 8, 10, and 43. 27. Any of embodiments 1 to 26, comprising a polyribonucleotide comprising one or more nucleotide sequences. One way.

[0040] 28. The method of any one of 1 to 27, wherein the target protein is TSHR or a fragment thereof. One way.

[0041] 29. The complex is selected from the group consisting of SEQ ID NO: 16, or SEQ ID NO: 14, 16, and 46. Any one of embodiments 1 to 28, comprising a polyribonucleotide comprising any one of the nucleotide sequences. Or one way.

[0042] 30. The method of any one of claims 1 to 29, wherein the target protein is APJ or a fragment thereof. Or one way.

[0043] 31. The complex is selected from the group consisting of SEQ ID NO: 19, or SEQ ID NOs: 17, 19, and 49. Any one of embodiments 1 to 30, comprising a polyribonucleotide having any one of the nucleotide sequences. Or one way.

[0044] 32. The method of claim 1, wherein the target protein is gp130 or a fragment thereof. Either one way.

[0045] 33. The complex is selected from the group consisting of SEQ ID NO: 22, or any of SEQ ID NOs: 20, 22, and 52. Any one of embodiments 1 to 32, comprising a polyribonucleotide having any one of the nucleotide sequences. Or one way.

[0046] 34. The method of claim 1, wherein the target protein is galectin 3 or a fragment thereof. Either one of the methods.

[0047] 35. The complex is selected from the group consisting of SEQ ID NO: 55, or SEQ ID NOs: 26, 55, and 56. Any one of embodiments 1 to 34, comprising a polyribonucleotide having any one of the nucleotide sequences. Or one way.

[0048] Embodiment 36. Any one of embodiments 1 to 35, wherein the complex has a diameter of about 30 to 150 nm. Or one way.

[0049] Embodiment 37 The method of any one of embodiments 1 to 35, wherein the complex comprises a helper lipid.

[0050] 38. A complex comprising (i) a cationic lipid, (ii) a helper lipid, such as a cholesterol lipid, (iii) neutral lipids, e.g., DSPC, and (iv) stealth lipids, e.g., For example, any combination of S010, S024, S027, S031 or S033. , The method of any one of aspects 1 to 35.

[0051] 39. The animal is administered 5 μg, 10 μg, 12.5 μg, 20 μg, 25 μg, 30 μg , 40μg, 50μg, 60μg, 70μg, 80μg, 90μg, 100μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg of polyribonucleotide The method of any one of embodiments 1-38, wherein the

[0052] 40. The cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride. DODAC, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-tetradecane Dimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium Ammonium-propane (DODAP), N-(1-(2,3-dioleyloxy)propionate (Dimethylammonium chloride)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleo Dimethylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-diphenylcarbamyl Neoyl-3-dimethylammonium-propane (DLINDAP), dilauryl (C1 2:0 ) Trimethylammonium propane (DLTAP), dioctadecyl glyceryl amido Dioleoyloxy-N-[2-spermine (DOGS)], DC-Chol, N,N-dimethyl-1-propaneaminium trifluoro Acetate (DOSPA), 1,2-Dimyristyloxypropyl-3-dimethyl- Dimethyloxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholesteryl) cis,cis-9,12-o-5-en-3-β-oxybutane-4-oxy)-1- N,N-dimethyl-2,3-diole (Cutadecadienoxy)propane (CLinDMA) 2-[5'-(cholest-5-ene-3β-yloxy)propylamine (DODMA), -oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12 '-Octadecadienoxy)propane (CpLinDMA) and N,N-dimethyl-3 ,4-Dioleyloxybenzylamine (DMOBA), and 1,2-N,N'-di From the group consisting of oleylcarbamyl-3-dimethylaminopropane (DOcarbDAP) The method of any one of embodiments 1 to 39, wherein the method is selected from the group consisting of

[0053] Embodiment 41 The method of embodiment 40, wherein the cationic lipid is DOTAP or DLTAP.

[0054] 42. Producing hybridomas that produce antibodies that specifically bind to a target protein The method of any one of aspects 1 to 41, further comprising the step of:

[0055] Embodiment 43. The method further comprises purifying an antibody that specifically binds to the target protein. The method of any one of embodiments 1 to 42.

[0056] 44. A chimeric or The method of any one of aspects 1-43, further comprising the step of generating a humanized antibody.

[0057] 45. The method comprises the step of: compared with methods involving immunization with whole cells, The method of any one of embodiments 1-44, which results in higher antibody titers in the serum.

[0058] 46. ​​The method comprises the step of: Rather than methods involving immunization with whole cells, the method uses recombinant human immunized cells that produce antibodies specific to a target protein. The method of any one of embodiments 1-44, wherein the method produces more hybridomas.

[0059] 47. The target protein is a human target protein and the non-human animal is a mouse, a rat, rat, rabbit, sheep, cat, dog, camelid, shark, monkey, pig, or horse. , The method of any one of aspects 1 to 46.

[0060] 48. A hybridoma producing an antibody that specifically binds to a target protein. The hybridoma is obtained by any one of the methods of embodiments 1 to 47. Ma.

[0061] 49. A mixture of polyclonal antibodies that specifically bind to a target protein, comprising: A mixture, wherein the mixture is obtainable by the method of any one of embodiments 1 to 47.

[0062] 50. An isolated monoclonal antibody that specifically binds to a target protein, and the monoclonal antibody is obtained by the method of any one of aspects 1 to 47. Monoclonal antibodies.

[0063] 51. A method for inducing an immune response to a target protein in a non-human animal. administering to the animal a lipid-polynucleotide complex, The polynucleotide complex contains cationic lipids and the mRNA encoding the target protein. wherein the target protein is a protein of a species different from that of the animal.

[0064] 52. The complex comprises: a consensus Kozak sequence; a 5′-terminal end of the mRNA; 7-methylguanosine cap; the polyadenosine ( a poly(A) tail; and one or more 5'- and 3'-untranslated regions (UTRs), The method of embodiment 51.

[0065] The method of embodiment 51, wherein said administration is parenteral.

[0066] Embodiment 54 The method of embodiment 51, wherein said administering is intravenous.

[0067] Embodiment 55 The method of embodiment 51, wherein said administration is intramuscular.

[0068] Embodiment 56 The method of embodiment 51, wherein said administration is subcutaneous.

[0069] Embodiment 57 The method of embodiment 51, wherein said administration is intranasal.

[0070] 58. The method of any one of 51 to 57, wherein the target protein is RXFP1. method.

[0071] 59. The complex is selected from the group consisting of SEQ ID NO: 4, or any one of SEQ ID NOs: 2, 4, and 37. 58. A method according to any one of claims 51 to 57, comprising a polyribonucleotide comprising one nucleotide sequence. There are two ways.

[0072] 60. Any one of embodiments 51 to 57, wherein the target protein is SLC52A2. There are two ways.

[0073] 61. The complex is selected from the group consisting of SEQ ID NO: 7, or any one of SEQ ID NOs: 5, 7, and 40. 58. A method according to any one of claims 51 to 57, comprising a polyribonucleotide comprising one nucleotide sequence. There are two ways.

[0074] 62. Any one of embodiments 51 to 57, wherein the target protein is ANGPTL8. There are two ways.

[0075] 63. The complex is selected from the group consisting of SEQ ID NO: 10, or SEQ ID NOs: 8, 10, and 43. 58. Any of embodiments 51 to 57, comprising a polyribonucleotide comprising one or more nucleotide sequences. Or one way.

[0076] 64. The method of any one of 51 to 57, wherein the target protein is TSHR. Law.

[0077] 65. The complex is selected from the group consisting of SEQ ID NO: 16, or SEQ ID NOs: 14, 16, and 46. Any one of embodiments 51 to 57, comprising a polyribonucleotide comprising any one of the nucleotide sequences. One way.

[0078] 66. The method of any one of claims 51 to 57, wherein the target protein is APJ. .

[0079] 67. The complex is selected from the group consisting of SEQ ID NO: 19, or SEQ ID NOs: 17, 19, and 49. Any one of embodiments 51 to 57, comprising a polyribonucleotide comprising any one of the nucleotide sequences. One way.

[0080] 68. The method of any one of 51 to 57, wherein the target protein is GP130. method.

[0081] 69. The complex comprises SEQ ID NO: 22, or any of SEQ ID NOs: 20, 22, and 52. Any one of embodiments 51 to 57, comprising a polyribonucleotide comprising any one of the nucleotide sequences. One way.

[0082] Embodiment 70 The method of embodiment 51, wherein said target protein is galectin 3.

[0083] 71. The complex is selected from the group consisting of SEQ ID NO: 55, or SEQ ID NOs: 26, 55, and 56. 52. The method of embodiment 51, comprising any one of the following:

[0084] 72. Obtaining antibodies or antibody-producing cells that specifically bind to a target protein from an animal The method of any one of aspects 51-71, further comprising the steps of:

[0085] 73. The target protein is a human target protein and the non-human animal is a mouse, a rat, rat, rabbit, sheep, cat, dog, camelid, shark, monkey, pig, or horse. , The method of any one of aspects 51 to 72.

[0086] Embodiment 74. A complex comprising (i) a cationic lipid, (ii) a helper lipid, such as a cholesterol lipid, (iii) neutral lipids, e.g., DSPC, and (iv) stealth lipids, e.g., For example, any combination of S010, S024, S027, S031 or S033. , any one of embodiments 51 to 73.

[0087] 75. The animal is administered 5 μg, 10 μg, 12.5 μg, 20 μg, 25 μg, 30 μg , 40μg, 50μg, 60μg, 70μg, 80μg, 90μg, 100μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg of polyribonucleotide The method of any one of embodiments 51-74, wherein the patient is administered an

[0088] 76. The cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride. DODAC, N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-tetradecane Dimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium Ammonium-propane (DODAP), N-(1-(2,3-dioleyloxy)propionate (Dimethylammonium chloride)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleo Dimethylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-diphenylcarbamyl Neoyl-3-dimethylammonium-propane (DLINDAP), dilauryl (C1 2:0 ) Trimethylammonium propane (DLTAP), dioctadecyl glyceryl amido Dioleoyloxy-N-[2-spermine (DOGS)], DC-Chol, N,N-dimethyl-1-propaneaminium trifluoro Acetate (DOSPA), 1,2-Dimyristyloxypropyl-3-dimethyl- Dimethyloxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholesteryl) cis,cis-9,12-o-5-en-3-β-oxybutane-4-oxy)-1- N,N-dimethyl-2,3-diole (Cutadecadienoxy)propane (CLinDMA) 2-[5'-(cholest-5-ene-3β-yloxy)propylamine (DODMA), -oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12 '-Octadecadienoxy)propane (CpLinDMA) and N,N-dimethyl-3 ,4-Dioleyloxybenzylamine (DMOBA), and 1,2-N,N'-di From the group consisting of oleylcarbamyl-3-dimethylaminopropane (DOcarbDAP) The method of any one of embodiments 51 to 75, wherein the method is selected from the following:

[0089] Embodiment 77 The method of embodiment 76, wherein the cationic lipid is DOTAP or DLTAP.

[0090] 78. The target protein is one of the following: ACKR1, ACKR2, ACKR3, ACKR 4, ADCYAP1R1, ADGRA1, ADGRA2, ADGRA3, ADGRB1, ADGRB2, ADGRB3, ADGRD1, ADGRD2, ADGRE1, ADGRE 2, ADGRE3, ADGRE4P, ADGRE5, ADGRF1, ADGRF2, AD GRF3, ADGRF4, ADGRF5, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRG6, ADGRG7, ADGRL1, ADGRL 2、ADGRL3、ADGRL4、ADGRV1、ADORA1、ADORA2A、AD ORA2B、ADORA3、ADRA1A、ADRA1B、ADRA1D、ADRA2A 、ADRA2B、ADRA2C、ADRB1、ADRB2、ADRB3、AGTR1、A GTR2、APLNR / APJ、ASGR1、ASGR2、AVPR1A、AVPR1B 、AVPR2、BDKRB1、BDKRB2、BRS3、BRS3、C3AR1、C5A R1、C5AR2、CALCR、CALCRL、CASR、CCKAR、CCKBR、C CR1、CCR10、CCR2、CCR3、CCR4、CCR5、CCR6、CCR7、 CCR8、CCR9、CCRL2、CELSR1、CELSR2、CELSR3、CHR M1、CHRM2、CHRM3、CHRM4、CHRM5、CMKLR1、CNR1、C NR2、CRHR1、CRHR2、CX3CR1、CXCR1、CXCR2、CXCR3 、CXCR4、CXCR5、CXCR6、CYSLTR1、CYSLTR2、DRD1、 DRD2、DRD3、DRD4、DRD5、EDNRA、EDNRB、F2R、F2RL 1、F2RL2、F2RL3、FFAR1、FFAR2、FFAR3、FFAR4、FP R1、FPR2、FPR2、FPR3、FSHR、FZD1、FZD10、FZD2、F ZD3、FZD4、FZD5、FZD6、FZD7、FZD8、FZD9、GABBR1 、GABBR2、GALR1、GALR2、GALR3、GCGR、GHRHR、GHS R、GIPR、GLP1R、GLP2R、GNRHR、GNRHR2、GPBAR1、G PER1、GPR1、GPR4、GPR12、GPR15、GPR17、GPR18、G PR19、GPR20、GPR21、GPR22、GPR25、GPR26、GPR27 、GPR3、GPR31、GPR32、GPR33、GPR34、GPR35、GPR3 7、GPR37L1、GPR39、GPR40、GPR42、GPR42、GPR45、 GPR50、GPR52、GPR55、GPR6、GPR61、GPR62、GPR63 、GPR65、GPR68、GPR75、GPR78、GPR79、GPR82、GPR 83、GPR84、GPR85、GPR87、GPR88、GPR101、GPR107 、GPR132、GPR135、GPR137、GPR139、GPR141、GPR1 42、GPR143、GPR146、GPR148、GPR149、GPR15、GPR 150、GPR151、GPR152、GPR153、GPR156、GPR157、G PR158、GPR160、GPR161、GPR162、GPR171、GPR173 、GPR174、GPR176、GPR179、GPR182、GPR183、GPRC 5A、GPRC5B、GPRC5C、GPRC5D、GPRC6A、GRM1、GRM2 、GRM3、GRM4、GRM5、GRM6、GRM7、GRM8、GRPR、HCAR 1、HCAR2、HCAR3、HCRTR1、HCRTR2、HRH1、HRH2、HR H3、HRH4、HTR1A、HTR1B、HTR1D、HTR1E、HTR1F、HT R2A、HTR2B、HTR2C、HTR4、HTR5A、HTR5BP、HTR6、H TR7、KISS1R、LGR3、LGR4、LGR5、LGR6、LHCGR、LPA R1、LPAR2、LPAR3、LPAR4、LPAR5、LPAR6、LTB4R、L TB4R2、MAS1、MAS1L、MC1R、MC2R、MC3R、MC4R、MC5 R、MCHR1、MCHR2、MLNR、MRGPRD、MRGPRE、MRGPRF、 MRGPRG、MRGPRX1、MRGPRX2、MRGPRX3、MRGPRX4、M TNR1A、MTNR1B、NMBR、NMUR1、NMUR2、NPBWR1、NPB WR2、NPFFR1、NPFFR2、NPSR1、NPY1R、NPY2R、NPY4 R、NPY5R、NPY6R、NTSR1、NTSR2、OPN3、OPN4、OPN5 、OPRD1、OPRK1、OPRL1、OPRM1、OR51E1、OXER1、OX GR1、OXTR、P2RY1、P2RY10、P2RY11、P2RY12、P2RY 13、P2RY14、P2RY2、P2RY4、P2RY6、P2RY8、PRLHR、 PROKR1、PROKR2、PTAFR、PTGDR、PTGDR2、PTGER1、 PTGER2、PTGER3、PTGER4、PTGFR、PTGIR、PTH1R、P TH2R、QRFPR、RXFP1、RXFP2、RXFP3、RXFP4、S1PR1 、S1PR2、S1PR3、S1PR4、S1PR5、SCTR、SMO、SSTR1、 SSTR2、SSTR3、SSTR4、SSTR5、SUCNR1、TAAR1、TAA R2、TAAR3、TAAR4P、TAAR5、TAAR6、TAAR8、TAAR9、 TACR1、TACR2、TACR3、TAS1R1、TAS1R2、TAS1R3、T AS2R1、TAS2R10、TAS2R13、TAS2R14、TAS2R16、TA S2R19、TAS2R20、TAS2R3、TAS2R30、TAS2R31、TAS 2R38、TAS2R39、TAS2R4、TAS2R40、TAS2R41、TAS2 R42、TAS2R43、TAS2R45、TAS2R46、TAS2R5、TAS2R 50, TAS2R60, TAS2R7, TAS2R8, TAS2R9, TBXA2R, T PRA1, TRHR, TSHR, UTS2R, VIPR1, VIPR2, XCR1, TC R-α, TCR-β, CD3, ζ-chain accessory, CD4, CD8, SIGIRR (single containing a single Ig and TIR domain), mannose receptor (MR), asialoglycoprotein Protein receptor family (e.g., asialoglycoprotein receptor macrophage galactose receptor type lectin (MGL), DC-SIGN (CLEC4L), Langerin (CLEC4K ), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), Dectin-1 / CL EC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CL EC12A), CLEC2 (also called CLEC1B), CLEC12B, DCIR / CLEC4A, Dectin 2 / CLEC6A, Blood DC Antigen 2 (BDCA2) (CLEC4 C), macrophage-inducible C-type lectin (CLEC4E), TLR1, TLR2, TL R3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, T LR11, TLR12, TLR13, FcγRI(CD64), FcγRIIA(CD3 2), FcγRIIB1(CD32), FcγRIIB2(CD32), FcγRIII A(CD16a), FcγRIIIB(CD16b), FcεRI, FcεRII(CD 23), FcαR1 (CD89), Fcα / μR, FcRn, CD27, CD40, OX 40, GITR, CD137, PD-1, CTLA-4, PD-L1, TIGIT, T-thin Transcellular immunoglobulin and mucin domain 3 (TIM3), a V-domain involved in T cell activation Main Ig inhibitor (VISTA), CD28, CD122, ICOS, A2AR, B7 -H3, B7-H4, B and T lymphocyte attenuator (BTLA), indole amine Indole 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR) , lymphocyte activation gene-3 (LAG3), FAM159B, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB 1. HLA-DRA, HLA-DRB1, gp130, IL-1 receptor, IL-2 receptor , IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor , IL-8 receptor, IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12 Receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor, IL-19 receptor, IL-20 receptor, IL-2 1 receptor, IL-22 receptor, IL-23 receptor, IL-24 receptor, IL-25 receptor , IL-26 receptor, IL-27 receptor, IL-28 receptor, IL-29 receptor, IL- IL-30 receptor, IL-31 receptor, IL-32 receptor, IL-33 receptor, IL-35 receptor Body, IL-36 receptor, FGFR1, FGFR2, FGFR3, FGFR4, TNFRS F1A, TNFRSF1B, TNFRSF3, TNFRSF4, TNFRSF5, TNF RSF6, TNFRSF6B, TNFRSF7, TNFRSF8, TNFRSF9, TN FRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TN FRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TN FRSF13C, TNFRSF14, TNFRSF16, TNFRSF17, TNFRS F18、TNFRSF19、TNFRSF21、TNFRSF25、TNFRSF27、 SCN1A、SCN1B、SCN2A、SCN2B、SCN3A、SCN3B、SCN4 A、SCN5A、SCN7A、SCN8A、SCN9A、SCN10A、SCN11A、 CACNA1A、CACNA1B、CACNA1C、CACNA1D、CACNA1E、 CACNA1F、CACNA1G、CACNA1H、CACNA1I、CACNA1S、 TRPA1、TRPC1、TRPC2、TRPC3、TRPC4、TRPC5、TRPC 6、TRPC7、TRPM1、TRPM2、TRPM3、TRPM4、TRPM5、TR PM6、TRPM7、TRPM8、MCOLN1、MCOLN2、MCOLN3、PKD 1、PKD2、PKD2L1、PKD2L2、TRPV1、TRPV2、TRPV3、T RPV4、TRPV5、TRPV6、CATSPER1、CATSPER2、CATSP ER3、CATSPER4、TPCN1、TPCN2、CNGA1、CNGA2、CNG A3、CNGA4、CNGB1、CNGB3、HCN1、HCN2、HCN3、HCN4 、KCNMA1、KCNN1、KCNN2、KCNN3、KCNN4、KCNN1、KC NT2、KCNU1、KCNA1、KCNA2、KCNA3、KCNA4、KCNA5、 KCNA6、KCNA7、KCNA10、KCNB1、KCNB2、KCNC1、KCN C2、KCNC3、KCNC4、KCND1、KCND2、KCND3、KCNF1、K CNG1、KCNG2、KCNG3、KCNG4、KCNH1、KCNH2、KCNH3 、KCNH4、KCNH5、KCNH6、KCNH7、KCNH8、KCNQ1、Industrial park Q2、KCNQ3、KCNQ4、KCNQ5、KCNS1、KCNS2、KCNS3、K CNV1、KCNV2、KCNJ1、KCNJ2、KCNJ3、KCNJ4、KCNJ5 、KCNJ6、KCNJ8、KCNJ9、KCNJ10、KCNJ11、KCNJ12、 KCNJ13、KCNJ14、KCNJ15、KCNJ16、KCNJ18、KCNK1 、KCNK2、KCNK3、KCNK4、KCNK5、KCNK6、KCNK7、KCN K9、KCNK10、KCNK12、KCNK13、KCNK15、KCNK16、KC NK17、KCNK18、HVCN1、HTR3A、HTR3B、HTR3C、HTR3 D、HTR3E、CHRNA1、CHRNA2、CHRNA3、CHRNA4、CHRN A5、CHRNA6、CHRNA7、CHRNA9、CHRNA10、CHRNB1、C HRNB2、CHRNB3、CHRNB4、CHRND、CHRNE、CHRNG、GA BRA1、GABRA2、GABRA3、GABRA4、GABRA5、GABRA6、 G ABRB1、GABRB2、GABRB3、GABRD、GABRE、GABRG1、G ABRG2、GABRG3、GABRP、GABRQ、GABRR1、GABRR2、G ABRR3、GRIA1、GRIA2、GRIA3、GRIA4、GRID1、GRID 2、GRIK1、GRIK2、GRIK3、GRIK4、GRIK5、GRIN1、GR IN2A、GRIN2B、GRIN2C、GRIN2D、GRIN3A、GRIN3B、 GLRA1、GLRA2、GLRA3、GLRA4、P2RX1、P2RX2、P2RX 3、P2RX4、P2RX5、P2RX6、P2RX7、ZACN、ASIC1、ASI C2, ASIC3, ASIC4, AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP9, AQP10, AQP11, AQP12A, A QP12B, MIP, CLCN1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, CLCNKB, cystic fibrosis transmembrane conductance Regulatory factors (CFTR), ANO1, ANO2, ANO3, ANO4, ANO5, ANO6 , ANO7, ANO8, ANO9, ANO10, BEST1, BEST2, BEST3, BEST4, CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, CLIC 6, GJA1, GJA3, GJA4, GJA5, GJA6P, GJA8, GJA9, GJ A10, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, G JC1, GJC2, GJC3, GJD2, GJD3, GJD4, GJE1, ITPR1, ITPR2, ITPR3, PANX1, PANX2, PANX3, RYR1, RYR2, RYR3, NALCN, SCNN1A, SCNN1B, SCNN1D, SCNN1G, T EM16A, ADAMTS7, ANGPTL3, ANGPTL4, ANGPTL8, LP L, GDF15, galectin-1, galectin-2, galectin-3, galectin-4, Galectin-7, Galectin-8, Galectin-9, Galectin-10, Galectin-1 2. Galectin-13, matrix gla protein (MGP), PRNP, DGAT 1, GPAT3, DMC1, BLM, BRCA2, human endogenous retrovirus type K ( A member of the HERV-K family, an ectonucleoside triphosphate diphosphohydrolase Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), Ectonucleoside triphosphate diphosphohydrolase 2 (ENTP D2), SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, S LC1A6, SLC1A7, SLC2A1, SLC2A2, SLC2A3, SLC2A4 、SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2 A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14, SLC3 A1, SLC3A2, SLC4A1, SLC4A2, SLC4A3, SLC4A4, SL C4A5, SLC4A6, SLC4A7, SLC4A8, SLC4A9, SLC4A10 、SLC4A11, SLC5A1, SLC5A2, SLC5A3, SLC5A4, SLC 5A5, SLC5A6, SLC5A7, SLC5A8, SLC5A9, SLC5A10, SLC5A11, SLC5A12, SLC6A1, SLC6A2, SLC6A3, SLC 6A4, SLC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, S LC6A10, SLC6A11, SLC6A12, SLC6A13, SLC6A14, S LC6A15, SLC6A16, SLC6A17, SLC6A18, SLC6A19, S LC6A20, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A 9, SLC7A10, SLC7A11, SLC7A13, SLC7A14, SLC8A1 、SLC8A2, SLC8A3, SLC9A1, SLC9A2, SLC9A3, SLC9 A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SL C9A10, SLC9A11, SLC9B1, SLC9B2, SLC10A1, SLC1 0A2, SLC10A3, SLC10A4, SLC10A5, SLC10A6, SLC1 0A7、SLC11A1、SLC11A2、SLC12A1、SLC12A2、SLC1 2A3、SLC12A4、SLC12A5、SLC12A6、SLC12A7、SLC1 2A8、SLC12A9、SLC13A1、SLC13A2、SLC13A3、SLC1 3A4、SLC13A5、SLC14A1、SLC14A2、SLC15A1、SLC1 5A2、SLC15A3、SLC15A4、SLC16A1、SLC16A2、SLC1 6A3、SLC16A4、SLC16A5、SLC16A6、SLC16A7、SLC1 6A8、SLC16A9、SLC16A10、SLC16A11、SLC16A12、S LC16A13、SLC16A14、SLC17A1、SLC17A2、SLC17A3 、SLC17A4、SLC17A5、SLC17A6、SLC17A7、SLC17A8 、SLC17A9、SLC18A1、SLC18A2、SLC18A3、SLC19A1 、SLC19A2、SLC19A3、SLC20A1、SLC20A2、SLCO1A2 、SLCO1B1、SLCO1B3、SLCO1C1、SLCO2A1、SLCO2B1 、SLCO3A1、SLCO4A1、SLCO4C1、SLCO5A1、SLCO6A1 、SLC22A1、SLC22A2、SLC22A3、SLC22A4、SLC22A5 、SLC22A6、SLC22A7、SLC22A8、SLC22A9、SLC22A1 0、SLC22A11、SLC22A12、SLC22A13、SLC22A14、SL C22A15、SLC22A16、SLC22A17、SLC22A18、SLC22A 18AS,SLC22A19、SLC22A20、SLC22A23、SLC22A24 <h2 style=";text-align:left;direction:ltr">SLC22A25, SLC22A31, SLC23A1, SLC23A2, SLC23<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC23, A4, SLC24, A1, SLC24, A2, SLC24, A3, SLC24<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A4、SLC24A5、SLC24A6、SLC25A1、SLC25A2、SLC25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC25A4, SLC25A5, SLC25A6, SLC25A7, SLC25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A8, SLC25A9, SLC25A10, SLC25A11, SLC25A12, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C25A13, SLC25A14, SLC25A15, SLC25A16, SLC25A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 17、SLC25A18、SLC25A19、SLC25A20、SLC25A21、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC25A22, SLC25A23, SLC25A24, SLC25A25, SLC25<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A26, SLC25A27, SLC25A28, SLC25A29, SLC25A30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC25A31, SLC25A32, SLC25A33, SLC25A34, SLC2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5A35, SLC25A36, SLC25A37, SLC25A38, SLC25A39<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLC25A40、SLC25A41、SLC25A42、SLC25A43、SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 25A44, SLC25A45, SLC25A46, SLC26A1, SLC26A2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC26A3, SLC26A4, SLC26A5, SLC26A6, SLC26A7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC26A8, SLC26A9, SLC26A10, SLC26A11, SLC27A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1, SLC27A2, SLC27A3, SLC27A4, SLC27A5, SLC27A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6、SLC28A1、SLC28A2、SLC28A3、SLC29A1、SLC29A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 2、SLC29A3、SLC29A4、SLC30A1、SLC30A2、SLC30A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3, SLC30A4, SLC30A5, SLC30A6, SLC30A7, SLC30A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 8, SLC30A9, SLC30A10, SLC31A1, SLC31A2, SLC32<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A1, SLC33A1, SLC34A1, SLC34A2, SLC34A3, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A1, SLC35A2, SLC35A3, SLC35A4, SLC35A5, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> B1, SLC35B2, SLC35B3, SLC35B4, SLC35C1, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C2, SLC35D1, SLC35D2, SLC35D3, SLC35E1, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> E2, SLC35E3, SLC35E4, SLC35F1, SLC35F2, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F3, SLC35F4, SLC35F5, SLC35G1, SLC35G3, SLC35<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G4、SLC35G5、SLC35G6、SLC36A1、SLC36A2、SLC36<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC36A4, SLC37A1, SLC37A2, SLC37A3, SLC37<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A4、SLC38A1、SLC38A2、SLC38A3、SLC38A4、SLC38<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A5, SLC38A6, SLC38A7, SLC38A8, SLC38A9, SLC38<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A10, SLC38A11, SLC39A1, SLC39A2, SLC39A3, SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39A4, SLC39A5, SLC39A6, SLC39A7, SLC39A8, SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 39A9, SLC39A10, SLC39A11, SLC39A12, SLC39A13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLC39A14、SLC40A1、SLC41A1、SLC41A2、SLC41A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 3、RhAG、RhBG、RhCG、SLC43A1、SLC43A2、SLC43A3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLC44A1、SLC44A2、SLC44A3、SLC44A4、SLC44A5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 、SLC45A1、SLC45A2、SLC45A3、SLC45A4、SLC46A1<h2 style=";text-align:left;direction:ltr"> , SLC46A2, SLC46A3, SLC47A1, SLC47A2, HCP-1, M FSD5, MFSD10, SLC50A1, OSTα, OSTβ, SLC52A1, SL 78. The method of any one of embodiments 51 to 77, wherein the IL-11 is selected from the group consisting of CLC52A2, and SLC52A3. method.

[0091] 79. The step of obtaining an antibody that specifically binds to a target protein comprises: Obtaining live cells and producing hybridomas having antibody-producing cells; Selecting hybridomas that produce antibodies that specifically bind to the target protein; and isolating the antibody produced by the hybridoma. Either one of the methods.

[0092] 80. A method for determining a nucleic acid sequence encoding an antibody that specifically binds to a target protein. The method of embodiment 79, further comprising the step of:

[0093] 81. The antibody-producing cells are lymphocytes, splenocytes, or peripheral blood mononuclear cells (PBMCs). The method of embodiment 72, 79, or 80,

[0094] 82. A chimeric or humanized antibody based on an antibody that specifically binds to a target protein. generating antibodies, the chimeric or humanized antibodies binding to the target protein with equivalent affinity; 82. The method of any one of aspects 1 to 81, further comprising the step of: method.

[0095] 83. The method of any one of 1 to 82, wherein the animal is genetically modified to produce human antibodies. Either one way.

[0096] Embodiment 84. The method according to any one of embodiments 1 to 83, wherein the polyribonucleotide of the complex is pseudouridine. Either one of the methods.

[0097] 85. The polyribonucleotide of the complex is (a) The following modified nucleotides for cytidine: 5-formylcytidine, 5-methylcytidine Cytidine, 5-methoxycytidine, 5-hydroxycytidine, and 5-hydroxymethylcytidine one or more cytidines; (b) The following modified nucleotides for uridine: 5-formyluridine, 5-methyluridine Lysine, 5-methoxyuridine, 5-carboxymethyl ester uridine, pseudouridine dine, and one or more of N1-methylpseudouridine; (c) N6-methyladenosine as a modified nucleotide for adenosine: and / or or (d) The modified nucleotide for guanosine is thienoguanosine, Any one of 84 ways.

[0098] 86. The complex comprises two or more different target proteins that can bind to each other. 85. The method of claim 1, further comprising: Either one of the methods.

[0099] 87. The conjugate comprises a sequence of Tables 1-7, e.g., SEQ ID NOs: 2, 4, 37, 5, 7 , 40, 8, 10, 43, 14, 16, 46, 17, 19, 49, 20, 22, 52, 2 6, 55, or 56 and at least 80%, 85%, 90%, 91%, A sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical 87. The method of any one of embodiments 1-86, comprising a polyribonucleotide comprising a sequence. [Brief description of the drawings]

[0100] [Figure 1A] Figure 1: Figures 1A-1D show an exemplary RXFP1 immunization strategy and the resulting FACS-based serum responses. Figure 1A is a schematic of an exemplary immunization strategy for human RXFP1. Figure 1B shows FACS-based serum responses from animals 10 days after priming immunization, illustrating the rapid induction of target-specific titers with mRNA immunization compared to more conventional forms of immunization, whole cell overexpressed human RXFP1 (BaF / 3) or virus-like particle overexpressed human RXFP1 (VLP-300.19). Figure 1C shows the final serum responses of immunized mice before the final boost and initiation of hybridoma fusion. Eight mice were selected for fusion and boosted with 100 μg of the indicated immunogen. Figure 1D shows sample FACS profiles of three anti-human RXFP1 hybridoma clones obtained from the mass immunization. In total, 207 RXFP1-specific clones were obtained. [Figure 1B] (As stated above.) [Figure 1C] (As stated above.) [Figure 1D] (As stated above.) [Diagram 2] An exemplary SLC52A2 immunization strategy and the resulting FACS-based serum response are shown. The immunization strategy employed for the production of anti-SLC52A antibodies and corresponding serum titers is shown. Conventional immunogens such as overexpressing cells, virus-like particles and peptides encoding extracellular loops (EC2) failed to induce significant target-specific titers. A total of 228 hybridomas capable of producing antibodies specific to SLC52A2 were generated from 8 fusion mice. [Diagram 3] 1 shows an exemplary galectin-3 immunization strategy and the resulting ELISA serological responses. [Figure 4]Bioanalyzer traces for purified human RXFP1 mRNA. The total amount of mRNA loaded per well is shown. The sample synthesized with pseudouridine shows a molecular weight closer to the predicted size (2687 bases) than the transcript synthesized with uridine. [Diagram 5] Western blotting of plasma membrane fractions prepared from HEK293 cells transiently transfected with increasing concentrations of human RXFP1 mRNA. As control samples, untransfected cells and cells transfected with a DNA plasmid encoding human RXFP1 were also loaded. The amount of nucleic acid used per six transfected wells is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0101] The present disclosure relates to cationic lipids and polyribonucleotide molecules, such as immunogens (e.g. A polynucleotide molecule, such as an mRNA, that encodes a target protein or a fragment thereof. The present disclosure also relates to a method of immunization using a composition comprising a genetically immunized animal. Methods for producing polyclonal and monoclonal antibodies from the subject Immunization methods provided herein include chimeric and humanized variants of such antibodies. The present disclosure also relates to antibodies produced by the immunization methods provided herein. For example, the hybridoma obtained by the immunization method using mRNA-LNP .

[0102] The present disclosure relates to methods for detecting inflammatory bowel disease in animals (e.g., non-human animals such as mice, rats, or rabbits). 1. A method for eliciting an immune response comprising: (a) administering at least one cationic lipid to an antibody or antibody complex; and a polynucleotide encoding the protoplast, thereby forming a cationic lipid-polynucleotide. (b) forming a lipid complex (e.g., an mRNA-LNP complex); - administering the polynucleotide complex to an animal. Additionally, the polynucleotide may be used to target an immunogen, such as a GPCR (e.g., a transmembrane protein (e.g., A method for genetic immunization using mRNA molecules encoding multiple transmembrane proteins mRNA is challenged and multiplexed to target complex protein targets (e.g., GPCRs, etc.). It is an excellent polynucleotide for rapid production of antibodies against transmembrane proteins. It has been found that.

[0103] The present disclosure further relates to the use of the genetic immunization methods described above and the isolation of polyclonal antibodies from immunized animals. and isolating the polyclonal antibodies. Regarding.

[0104] The present disclosure also provides a method for producing a monoclonal antibody, comprising: (a) administering to a non-human animal (e.g., a mouse) is administered at least one cationic lipid and a polynucleotide (e.g., Administering a composition comprising a polyribonucleotide, (b) a gene encoding an mRNA sequence that specifically binds to the immunogen; and obtaining an antibody that specifically binds to an immunogen. The step of obtaining an antibody comprising the steps of: (a) detecting a lymphocyte (e.g., a B lymphocyte) or (b) obtaining antibody-producing cells, such as splenocytes, from the immunized animal; and (c) obtaining myeloma cells. Fusing antibody-producing cells from immunized animals with the antibody, thereby producing hybridomas. (c) cloning the hybridomas; and (d) isolating the anti-immunogen. A process for selecting positive clones that produce antibodies (i.e., antibodies that specifically bind to the immunogen) (e) culturing the anti-immunogen antibody-producing clone; and (f) isolating the anti-immunogen antibody. from the culture medium.

[0105] The present disclosure also provides a method for producing a monoclonal antibody, comprising: (a) at least One cationic lipid is mixed with a polyribonucleotide, e.g. combining the polynucleotide with the lipid-polynucleotide complex, thereby forming a lipid-polynucleotide complex; (b) the lipid-polynucleotide comprises an mRNA sequence encoding an immunogen; (c) administering the otide conjugate to at least one mouse; and (b) detecting lymphocytes (e.g., A step to remove antibody-producing cells, such as B lymphocytes or splenocytes, from immunized mice. (d) using B lymphocytes from immunized mice with myeloma cells, (e) a step of cloning the hybridomas. (f) selecting positive clones that produce anti-immunogen antibodies; and (g) selecting the positive clones that produce anti-immunogen antibodies. (h) culturing the anti-immunogen antibody-producing clones; and (h) isolating anti-immunogen antibodies from the culture medium. The present invention relates to a method comprising the steps of:

[0106] Various formulations of cationic lipids are used to transfect cells in vitro (see, for example, International Publication No. WO 91 / 17424; International Publication No. WO 91 / 1 No. 6024; U.S. Pat. No. 4,897,355; U.S. Pat. No. 4,94 No. 6,787; U.S. Pat. No. 5,049,386; and U.S. Pat. Cationic lipids have also been shown to be effective in frog and rat cells in vivo. have been used to introduce exogenous polynucleotides (see, e.g., Holt et al. al.,Neuron 4:203-214(1990);Hazinski et a l.,Am.J.Respr.Cell.Mol.Biol.4:206-209(19 91). In certain embodiments provided herein, the cationic lipid is Generally, they are used to deliver or introduce biologically active substances (e.g. , International Publication No. 91 / 17424 Brochure; International Publication No. 91 / 16024 Brochure and WO 93 / 03709). In certain embodiments described herein, the cationic liposomes are comprised of polyribonucleotides (e.g., Introduction of an exogenous polynucleotide, such as a nucleic acid sequence (RNA) into a host cell for genetic immunization It is possible to provide an efficient carrier for the

[0107] A variety of cationic lipids well known in the art may be used in the compositions and compositions provided herein. One well-known cationic lipid is N -[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium DOTMA alone or dioleoylphosphatidylcholine chloride (DOTMA) is In a 1:1 combination with diethyl ethanolamine (DOPE), the The compound can be formulated into a liposome. gner et al.(Proc.Natl.Acad.Sci.USA84: 7413-7417 (1987) reported that such liposomes could effectively deliver nucleic acids to cultured cells. The DOTMA:DOPE (1:1) formulation was shown to provide efficient delivery of LIPO FECTIN(TM)(GIBCO / BRL:Life Technologies,I It is sold under the name of NC State, Gaithersburg, Md. The cationic lipid used is 1,2-bis(oleoyloxy)-3-(trimethylaniline). dimethylaminopropane (DOTAP), which is a diamine in which the oleoyl moiety is substituted with propylamine. It differs from DOTMA in that it is bonded via an ester bond rather than a di-ester bond. DOTAP is believed to be more readily degraded by target cells.

[0108] A related group of known compounds is the trimethylammonium group, in which one of the methyl groups is hydroxyl. It differs from DOTMA and DOTAP in that it is substituted with an ethyl group. The compounds are Rosenthal inhibitors of phospholipase A Similar to t al., J. Biol. Chem. 235:2202-2206 (1960) R The dioleoyl analogue of the osenthal inhibitor (RI) is a propylamine derivative of the fatty acid moiety. Depending on the bond to the core, they are commonly abbreviated as DORI ethers and DORI esters. The hydroxy group can be used as a site for further functionalization, e.g., carboxysper It can be used by esterification to the amine.

[0109] Another class of known compounds is described by Behr et al. (Proc. Natl. Acad. Sci. U SA 86:6982-6986(1989);EPO Publication 0 394 111), in which carboxyspermine is a component of two lipid Dipalmitoylphosphatidylethanolamine 5-carboxylate is bound to the substrate. This results in permylamide (DDPES).

[0110] Efficient transfection using both DOGS and DPPES to coat the plasmid These compounds form lipid aggregate complexes that result in the activation of certain cell lines. It is more efficient and less toxic than DOTMA for transfection. DOGS is a product of TRANSFECTAM (Promega, Massachusetts Institute of Technology) Dison, Wis.)

[0111] A cationic cholesterol derivative (DC-Chol) has been synthesized and combined with DOPE. It has been combined and formulated into liposomes (Gao et al., Biochim. Biol. iophys.Res.Comm.179:280-285(1991)). DC-Ch Liposomes formulated with ol were shown to be more effective than DOTMA-containing liposomes for certain cell lines. It offers more efficient transfection and lower toxicity than the IgG1 genome.

[0112] Lipopolylysine is formed by binding polylysine to DOPE. The compound has been reported to be particularly efficient for transfection in the presence of serum. (Zhou et al., Biochim. Biophys. Res. Comm. 1 65:8-14 (1991)). Thus, lipopolylysine is an efficient antibody for immunization. It can be a carrier.

[0113] Lipids that can be used in the methods and compositions provided herein (e.g., Other non-limiting examples of cationic lipids, helper lipids, and stealth lipids include those listed in the US Pat. International Publication No. 2015 / 095346 Brochure, International Publication No. 2015 / 095340 Brochure, International Publication No. 2016 / 037053 Brochure, International Publication No. 2014 / 136086 Brochure and International Publication No. 2011 / 076807 Brochure No. 6,399,433, each of which is incorporated herein by reference in its entirety.

[0114] In certain embodiments, cationic lipids for the compositions and methods described herein include is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N, N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-( 2,3-Dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride DOTAP, 1,2-dioleoyl-3-dimethylammonium-propane (DO DAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl Dioleoyl ammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3-dimethyl Dimethylammonium-propanediol (DOCDAP), 1,2-diaminetetraacetic acid monium-propane (DLINDAP), dilauryl (C 12:0 ) Trimethylammonium DLTAP, dioctadecylamidoglycylspermine (DOGS), DC-Chol, Dioleoyloxy-N-[2-sperminecarboxamido)ethyl} -N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide 3-Dimethylamino-2-(cholest-5-ene-3-β-oxidase) Cibutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane CLinDMA, N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 2-[5'-(cholest-5-en-3β-oxy)-3'-oxapeptide 9',12'-octadecadienoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy) Propane (CpLinDMA) and N,N-dimethyl-3,4-dioleyloxybene Dioleylamine (DMOBA), and 1,2-N,N'-dioleylcarbamyl-3-di These include, but are not limited to, methylaminopropane (DOcarbDAP). In one embodiment, the cationic lipid for the compositions and methods provided herein is D These compounds may be used alone or in other lipid aggregate forms. Lipoproteins, either in combination with other components (such as DOPE or cholesterol), These are useful for formulation into cationic lipid aggregates. It is anionic and can form complexes with anionic macromolecules such as DNA or RNA. do.

[0115] The methods of mRNA-based immunization provided herein include the generation of antigens and / or the administration of antibodies. The present invention addresses many of the problems associated with the above-mentioned obstacles inherent in somatic production. This eliminates the need to directly express and purify target protein antigens. It is used to create target proteins and present them to the immune system. For proteins, this implies eukaryote-specific post-translational modifications and protein processing. Without wishing to be bound by any particular theory, However, the unpurified mRNA used for immunization may contain a double-stranded RNA element in the preparation. In certain embodiments, this is due to the presence of adjuvants. It functions as a stimulant to boost the humoral response to the target protein.

[0116] In certain aspects provided herein, a monoclonal antibody for a target protein is Development is accelerated through immunization of animals with mRNA encoding the target protein. This method does not require heterologous production and purification of the target protein, To develop specific antibodies against G protein-coupled receptors, mRNA offers significant advantages over proteins that have historically been used as adjuvants. The host defense mechanisms triggered by the trait result in the rapid development of serum titers.

[0117] In certain embodiments, immunization using the encapsulated mRNA-based immunization methods provided herein Hybridoma fusions using antibody-producing cells obtained from immunized animals have been highly amplified. The encapsulated mRNA is potent and highly immunogenic, allowing for rapid replication on the day of immunization. The process can be shortened and fewer animals required. Proteins of all types, e.g., soluble, membrane-bound, complex / heteromeric, etc., can be classified into complex Expression allows the antigen to be produced in its native conformation regardless of the degree of expression. This should be a solution.

[0118] The methods provided herein also include methods for detecting complexed proteins (e.g., IgG, receptor complexes, etc.) The mRNAs provided herein can result in co-expression of multiple chains for the purposes of In certain embodiments of the antibody-based immunization method, the expressed antigens are immunogenic because they are synthetic. Not epigenically contaminated, i.e., does not alter humoral responses or the specificity of infectious agents It does not have any contaminants that can cause

[0119] In a particular embodiment, the present invention describes a stable method of antigen production and reagent storage, The lipid-encapsulated mRNA of the present invention can be kept stable for several months at 4°C. Furthermore, because the mRNA is entirely synthetic, it does not have the required IMPACT (infection There is no pathogen testing (PCR amplification test for infectious microorganisms).

[0120] term A "cloning vector" is a plasmid or phage DNA or other DNA A sequence means a sequence capable of autonomously replicating in a host cell and having one or more Characterized by a small number of restriction endonuclease recognition sites, where such DNA The A sequence can be determinably cut without destroying the essential biological functions of the vector, and DNA can be spliced ​​into it to effect cloning. The researchers further demonstrated that the cloning vectors are useful in identifying cells transfected with the vector. It may also contain a marker suitable for use. The marker may be, for example, tetracycline resistance. or ampicillin resistance.

[0121] An "expression vector" is a vector similar to a cloning vector, but which is used to deliver a gene to a host. After transformation, it has the ability to enhance the expression of genes cloned into the expression vector. The cloned gene is usually placed under the control of certain regulatory sequences, such as a promoter sequence. (i.e., operably linked). Promoter sequences can be either constitutive or inducible. It could be either.

[0122] "Expression" is the cellular process by which a polypeptide is produced from a structural gene. The process involves the transcription of genes into messenger RNA (mRNA) and the synthesis of such mRNAs. "Expression" also includes, where applicable, transcription, translation, These processes may include, but are not limited to, folding, modification, and processing. "Expression product" refers to the RNA transcribed from a gene and the mRNA transcribed from a gene. In certain embodiments, "expression" of a nucleic acid sequence includes a polypeptide obtained by translation of the nucleic acid. The following events occur: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) Processing of RNA transcripts (e.g., splicing, editing, 5' capping, and (3) the synthesis of a polypeptide or protein from an RNA (4) one or more post-translational modifications of a polypeptide or protein. vinegar.

[0123] "Exogenous" nucleic acids are those derived from nucleic acids introduced into a biological system, such as a cell or organism, through a process that involves human intervention. Nucleic acids not normally found therein or found in lower amounts, introduced by The factor (e.g., a modified synthetic mRNA as described herein) may be a factor (e.g., a modified synthetic mRNA as described herein). Modified synthetic mRNA is a compound that is introduced into direct progenitor or descendant cells that inherit the compound. In contrast, "endogenous" means something that is natural to a living system or cell. A gene is a naturally occurring factor or expression product (e.g., endogenous expression of a gene).

[0124] "Isolated," in the case of a nucleic acid or polypeptide, refers to a substance that is not found in its natural source. present with a nucleic acid or polypeptide and / or expressed in a cell such that In some cases, it may be present with a nucleic acid or polypeptide, or may be present with a secreted polypeptide. In the case of a peptide, at least one other component (e.g., a nucleic acid or polypeptide) that is secreted It means a nucleic acid or polypeptide that has been isolated from a nucleic acid or polypeptide that has been chemically synthesized. Polypeptides or those synthesized using in vitro transcription / translation are considered "isolated." It is possible.

[0125] An "isolated cell" is a cell that has been removed from the organism in which it is naturally found or Optionally, the cell is a descendant of such a cell. Optionally, the cells are later introduced into a second organism or or when it (or the cell or population of cells from which it was derived) is reintroduced into the organism from which it was isolated. can be.

[0126] "Modified" refers to an altered state or structure of a molecule described herein. The may be modified in a variety of ways, including chemically, structurally, and functionally. In some forms, the mRNA described herein may contain natural and non-natural nucleosides and / or A sequence is modified by the introduction of a nucleotide. Modification also refers to any change that differs from the wild type. "Modified" RNA is an RNA molecule that has been produced in vitro and has at least It also contains one modified nucleoside.

[0127] "Modified nucleosides" are nucleosides that are similar to the standard guanine (G), adenine (A), and cytidine ( C) and uridine (U) nucleosides. Such modifications, as known to those skilled in the art, include, for example, modifications that are usually post-transcriptional in mammals. These include modifications introduced into mRNA in living cells, and artificial chemical modifications. In one embodiment, the following are non-limiting examples of modified nucleotides: 5-formylcysteine cytidine, 5-methylcytidine, 5-methoxycytidine, 5-hydroxycytidine, 5-hydroxycytidine 5-Methoxyuridine, 5-methyluridine, 5-methoxyuridine Uridine, 5-carboxymethyl ester uridine, pseudouridine, N1-methylpseudouridine Douridine, N6-methyladenosine, and thienoguanosine.

[0128] "Co-transcriptionally added" refers to the addition of a modified synthetic mRNA of the present invention during transcription of an RNA molecule. features, such as the addition of a 5' methylguanosine cap or other modified nucleosides. This means that the modified RNA is not fully transcribed prior to the addition of the 5' cap. do not have).

[0129] "Contacting" a cell refers to contacting a cell with a factor (e.g., a modified synthetic mRNA described herein). means to contact the cells with one another, optionally including exposing the cells to a transfection system. When such a cell is in vivo, the cell is transfected with the modified synthetic mRNA described herein. Contacting can refer to administering a compound in a formulation, such as a pharmaceutical composition, such that the compound contacts a cell in vivo. The method includes administering the modified synthetic mRNA described herein to a subject by a suitable route of administration. nothing.

[0130] Generally, a "recombinant host" refers to a host that contains the desired clone in an expression or cloning vector. The gene may be any prokaryotic or eukaryotic microorganism or cell that contains the cloned gene. The term also refers to a genetically modified organism that contains a desired gene in a chromosome or genome of an organism. The term "microorganisms" is meant to include those microorganisms that have been genetically engineered.

[0131] A "recombinant vector" is any cloning vector containing a desired cloned gene. The vector is a coding vector or an expression vector.

[0132] A "host" is a recipient of a replicable expression or cloning vector. A host also refers to any prokaryotic or eukaryotic microorganism or cell that is capable of expressing or integrating a chromosome or a chromosome-specific gene. or genetically engineered by well-known techniques to contain the desired gene on the genome. Examples of such hosts include prokaryotic or eukaryotic microorganisms or cells capable of Maniatis et al., Molecular Cloning:A La boratory manual, Cold Spring Harbor Lab Ratory, Cold Spring Harbor, NY (1982). I want to.

[0133] A "promoter" is generally defined as the 5' region of a gene, located adjacent to the start codon. The DNA sequence illustrated is that transcription of adjacent genes is initiated from the promoter region. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, if a promoter is a constitutive promoter, the rate of transcription is increased by an inducing agent. is not adjusted.

[0134] A "gene" contains the information necessary to express a polypeptide or protein. It is a DNA sequence.

[0135] A "structural gene" is a set of nucleotides, e.g., amino acids, that are subsequently encoded by a specific polypeptide. A DNA sequence that is transcribed into messenger RNA (mRNA) which is translated into a sequence.

[0136] "Transfection" refers to the process of transfection of a cell with a polynucleotide, e.g., DNA or RNA. Transformation of a host cell refers to a recombinant host cell that contains a transfected polynucleotide. The gene expresses a protein encoded by a specific DNA or RNA. In embodiments, "transfection" refers to the transfer of an exogenous gene, such as a modified synthetic mRNA, as described herein. By recombinant vectors is meant the use of methods, including chemical methods, to introduce recombinant nucleic acid into a host cell, such as a eukaryotic cell. As used herein, the term "transfection" refers to the introduction of an exogenous nucleic acid into a cell. Non-limiting methods of transfection include physical transfection. treatment (e.g., electroporation, nanoparticles, magnetofection), and Chemical-based transfection methods are included. Methods of dissolution include cyclodextrins, polymers, liposomes, and nanoparticles. These include, but are not limited to:

[0137] An "epitope" is the portion of a non-immunoglobulin antigen that is bound by the variable region of an antibody. An "antigenic determinant" is a protein or peptide that contains one or more epitopes. An "immunogen" is a protein that is capable of eliciting an immune response due to the presence of one or more epitopes. The terms "antigen," "antigenic determinant," and "immunogen" are used herein in In the specification, these terms are used interchangeably. In certain aspects, epitopes are generally defined as amino acids or sugar moieties. It consists of chemically active surface groups of molecules, such as chains, and has specific three-dimensional structural characteristics and specific In certain embodiments, conformational and non-conformational epitopes may have charge characteristics. The epitope is characterized by the fact that the binding to the former but not the latter is lost in the presence of denaturing solvents. In certain embodiments, an epitope is a continuous amino acid sequence of a fragment or portion of an antigen. In certain embodiments, the epitope may be a linear epitope comprising a sequence of It may be a conformational epitope comprising a non-contiguous amino acid sequence of the antigen.

[0138] A "transfection agent" is a method for the delivery of a polynucleotide, such as DNA or RNA, to a host. In certain embodiments, the cellular uptake is induced in the absence of such a reagent. For example, at least 50-90% of the modified synthetic mRNA described herein is In one embodiment, the preparation of a pharmaceutical composition, if or cationic or non-cationic compounds useful for co-administration of the modified synthetic mRNAs described herein. In another embodiment, a lipid molecule is used as a transfection reagent. The modified synthetic mRNAs described herein may be modified with, for example, ligands, peptide groups, lipophilic groups, targeting moieties, etc. In another embodiment, the transfection reagent comprises a chemical bond that binds to the target protein or the like. Charged lipids, emulsions, liposomes, etc., as known in the art or as described herein; These include cationic or non-cationic lipids, anionic lipids, or penetration enhancers.

[0139] The "innate immune response" or "interferon response" is a response to foreign organisms, e.g. viruses. or bacteria, or products of such organisms, e.g., RNA produced in the cells of interest. Cellular defenses elicited by cells responding to the recognition of infection by RNA lacking specific modifications The innate immune response acts by inducing the death of cells that detect exogenous nucleic acid. to protect against viral and bacterial infections.

[0140] A "therapeutically effective amount" or "effective amount" is a quantity sought by a researcher, veterinarian, physician, or other clinician. by inducing a biological or medical response in a tissue, system, animal or human for which The amount of the target compound or combination is

[0141] A "primer" is also a nucleic acid sequence. PCR primers are usually polymerase chain reaction The oligonucleotides used in the reaction are fairly short in length (e.g., 8-30 nucleotides). PCR primers and hybridization probes are nucleotides that are specific to the target sequence. Using the sequence information from the array, these can be readily developed and produced by one of skill in the art. brook et al.(1989)Molecular Cloning:A La boratory Manual(Cold Spring Harbor Labs See Press.

[0142] "Selectively binds" or "specifically binds" means that one protein binds to another. A protein (e.g., an antibody, a fragment thereof, or a binding partner to a target protein) By "specific binding" we mean specific binding to the target protein, and the level of binding can be measured using any standard assay (e.g., immunoassay). Statistically significant compared to the background control for the assay, as measured by Very arrogant.

[0143] "Conserved" nucleotides or amino acids are those that are at the same position in two or more sequences being compared. are residues of a polynucleotide or polypeptide sequence, respectively, that occur without variance in Relatively conserved nucleotides or amino acids are those that occur elsewhere in the sequence. It is something that is conserved among related sequences rather than nucleotides or amino acids. Two or more sequences are "completely conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are identified if they are at least 90% identical to each other. are "highly conserved." In some embodiments, Conservation of sequences may occur in the form of oligonucleotides or polynucleotides. It may be applied to the entire length of a polypeptide, or to a portion, region or feature thereof. For polypeptides, the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine ( K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6 ) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S) , threonine (T); and 8) cysteine ​​(C), methionine (M) (e.g., Cre (see Lighton, Proteins (1984)).

[0144] "Delivery" means the act or action of delivering a compound, substance, entity, moiety, cargo or payload. means style.

[0145] A "delivery agent" is any agent that facilitates, at least in part, the in vivo delivery of a nucleic acid molecule to a target cell. It is a substance.

[0146] The "formulation" includes at least the modified nucleic acid molecule and a delivery agent.

[0147] "Homology" refers to the degree of homology between polymeric molecules, e.g., nucleic acid molecules (e.g., DNA molecules and / or The term refers to the overall relationship between polypeptide molecules (or RNA molecules) and / or between polypeptide molecules. In some embodiments, the polymer molecules are at least 25% identical in their sequence. The term "homologous" always refers to at least two It refers to a comparison between sequences (polynucleotide or polypeptide sequences) of two polynucleotides. Nucleotide sequences are intended to ensure that the polypeptides they encode have at least about 20 amino acids. Homologous sequences are considered to be at least about 50% identical over at least one contiguous sequence. In some embodiments, the homologous polynucleotide sequence comprises at least 4 to 5 It is characterized by its ability to code for a uniquely specified sequence of amino acids. For polynucleotide sequences less than 1 nucleotide in length, homology is defined as at least 4-5 nucleotides. It is determined by its ability to code for a uniquely specified sequence of amino acids. Based on this, the two protein sequences are similar in that the proteins have at least about 20 amino acids. At least about 50% identical, at least about 60% identical in at least one contiguous sequence. are considered to be homologous.

[0148] "Identity" can occur between polymer molecules, for example, oligonucleotide molecules (e.g., DNA The overall relationship between the polypeptide molecules (and / or RNA molecules) For example, calculation of the percent identity of two polynucleotide sequences is based on optimal comparison. This can be done by aligning two sequences for the purpose (e.g., optimal Introducing gaps into one or both of the first and second nucleic acid sequences for proper alignment. (The corresponding sequences can then be compared and non-identical sequences can be ignored for comparison purposes.) The nucleotides at corresponding nucleotide positions in the first sequence are compared. If the corresponding position in is occupied by the same nucleotide, then these molecules The percent identity between two sequences is determined by optimal alignment of the two sequences. Consider the number of gaps that need to be introduced and the length of each gap to It is a function of the number of identical positions shared by the sequences. Determining the percent identity can be accomplished using a mathematical algorithm. Two non-limiting examples of algorithms suitable for determining sequence identity and sequence similarity: are the BLAST and BLAST2.0 algorithms, which are tschul et al.,Nuc.Acids Res.25:3389-3402 , 1977; and Altschul et al., J. Mol. Biol. 215: 403-410, 1990. The software is supported by the National Center for Biotechnology This information is publicly available through the National Institute of Standards and Technology (NIS) at:

[0149] The term "cationic liposome" or "cationic lipid" refers to a liposome made from positively charged lipids. It is a construct that can interact with negatively charged DNA and cell membranes.

[0150] The term "lipid nanoparticle" or "LNP" refers to lipid nanoparticles that are physically associated with each other through intermolecular forces. It refers to a particle that contains multiple (i.e., more than one) lipid molecules. Lipid nanoparticles are, for example, , microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), emulsions The polymer may be a dispersed phase within the polymer, a micelle, or an internal phase within the suspension.

[0151] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide." xylose (DNA) or ribonucleotides (RNA), or messenger Polyribonucleotides, including mRNA, and single- or double-stranded forms The term refers to polymers thereof containing either known nucleotide analogs or modified nucleotides. Nucleic acids containing modified backbone residues or bonds, including synthetic, naturally occurring, and non-naturally occurring nucleic acids that have similar properties to the reference nucleic acid and can be used to Examples of such analogs include nucleic acids that are metabolized in a manner similar to that of nucleic acids. Phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O -methyl ribonucleotides, peptide nucleic acids (PNAs), but are not limited to these.

[0152] Unless otherwise indicated, a particular nucleic acid sequence also refers to a sequence that is not explicitly indicated, but is instead a conserved sequence. Substantially modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences are also implicitly included. In particular, as described in detail below, degenerate codon substitutions include one or more selected ( or all) codons with mixed base and / or deoxyinosine residues at the third position This may be achieved by creating sequences that are leic Acid Res.19:5081,1991;Ohtsuka et al. ., J. Biol. Chem. 260:2605-2608, 1985; and Ross olini et al.,Mol.Cell.Probes 8:91-98,199 4).

[0153] The term "pharmaceutically acceptable" is used herein to mean any compound that, within the scope of sound medical judgment, is acceptable. in humans and animals without undue toxicity, irritation, allergic responses or other problems or complications. Compounds, materials, and methods suitable for use in contact with tissue and commensurate with a reasonable benefit / risk ratio It is taken to refer to the composition, the dosage form, and / or the

[0154] A "sample" is a subset of that tissue, cell, or component part (e.g., bodily fluid). The material may be a whole organism or a subset or fraction of its tissues, cells or components. or portions thereof, for example, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, skin Prepared from external, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, The sample may further comprise a homogenate, lysate or extract from which the protein is isolated. Refers to a medium, such as a nutrient broth or gel, that may contain cellular components such as proteins or nucleic acid molecules. .

[0155] "Synthetic" means produced, prepared, and / or manufactured through human intervention The synthesis of polynucleotides or polypeptides or other molecules described herein may be carried out by chemical synthesis. The enzyme may be chemical or enzymatic.

[0156] As used herein, "pseudouridine" refers to the C-substituted uridine derivative of the nucleoside uridine. -Refers to glycoside isomers.

[0157] As used herein, "purify," "purified," and "purified" refer to a substance that is substantially pure. means to remove unwanted ingredients, material contaminants, mixtures or impurities. do.

[0158] As used herein, the term "antibody" refers to both full length antibodies and any antigen-binding fragments (i.e. A full-length antibody is a single chain of a polypeptide that is bound to a disulfide bond. A glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by Each heavy chain is a protein that contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light The light chain constant region consists of one domain, CL. The VH and VL regions are composed of more conserved regions called framework regions (FR). These are further subdivided into hypervariable regions called complementarity determining regions (CDRs) into which regions that VH and VL can be arranged from the amino terminus to the carboxy terminus in the following order: It is composed of three CDRs and four FRs, arranged as follows: FR1, CDR1, FR2, C DR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains interact with antigens. The constant region of an antibody contains a binding domain. Immunity to host tissues or host factors, including the first component of the classical complement system (Clq) and the second component of the classical complement system (CLq). It can mediate the binding of globulins.

[0159] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. The antigen-binding function of an antibody can be performed by fragments of a complete antibody. Examples of binding fragments encompassed within the term antigen-binding fragment include Fab fragments, VL domain fragments, and the like. a monovalent fragment consisting of the VH domain, the CL domain, and the CH1 domain; a hinge region F( ab) 2 fragments; Fd fragment consisting of the VH domain and the CH1 domain; a single arch of the antibody Fv fragment consisting of the VL and VH domains of a gene; Single domain antibody (dAb) fragments (Ward et al., 1989 Na ture 341:544-546); and isolated complementarity determining regions (CDRs). In certain embodiments, the CDRs of the antibody are numbered according to (i) the Kabat numbering system ( abat et al.(1971)Ann.NY Acad.Sci.190:382 -391 and Kabat et al. (1991) Sequences of Pr oteins of Immunological Interest Fifth E dition,USDepartment of Health and Huma NIH Publication No. 91-3242); or (ii) the Chothia numbering scheme (e.g., Chothia and Le sk,1987,J.Mol.Biol.,196:901-917;Al-Lazik ani et al,1997,J.Mol.Biol.,273:927-948;C hothia et al.,1992,J.Mol.Biol.,227:799-8 17;Tramontano A et al.,1990,J.Mol.Biol.2 15(1):175-82; and U.S. Pat. No. 7,709,226. ); or (iii) see, for example, Lefranc, M.-P., 1999, The Immunotherapy for Cancer. unologist, 7:132-136 and Lefranc, M.-P. et al. l,1999,Nucleic Acids Res.,27:209-212(“IM The ImMunoGeneTics (IMGT) numbering system is shown in stem; or (iv) MacCallum et al, 1996, J. Mol. Biology ol., 262:732-745. tin, A., “Protein Sequence and Structure A analysis of Antibody Variable Domains,”in Antibody Engineering, Kontermann and Diibe l,eds.,Chapter 31,pp.422-439,Springer-Ve See rlag, Berlin (2001).

[0160] Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes. However, they are represented by a single molecule in which the VL and VH regions pair to form a monovalent molecule. The artificial peptide linker allows the synthesis of a protein chain of the The Fv can be linked using methods known as single-chain Fv (scFv); e.g. For example, Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies may comprise one or more antigen-binding These antibody fragments can be produced by conventional techniques known to those skilled in the art. The fragments obtained using the antibodies are screened for utility in the same manner as are intact antibodies.

[0161] Antigen-binding fragments also include single domain antibodies, maxibodies, minibodies, intrabodies, , diabodies, triabodies, tetrabodies, v-NAR, and bis-scFv (e.g., Hollinger and Hudson, 2005, Na (see Nature Biotechnology, 23, 9, 1126-1136) The antigen-binding portion of the antibody is based on a polypeptide such as fibronectin type III (Fn3). The fibronectin polypeptide monobody can be implanted in a scaffold made of fibronectin. (See U.S. Patent No. 6,703,199, incorporated herein by reference).

[0162] An antigen-binding fragment is a polypeptide that, together with a complementary light chain polypeptide, forms a pair of antigen-binding regions. The recombinant Fv segments are then assembled into a single chain molecule comprising a pair of tandem Fv segments (VH-CH1-VH-CH1). (Zapata et al. (1995) Protein Eng. 8(1 0):1057-1062; and U.S. Pat. No. 5,641,870.

[0163] As used herein, the term "affinity" refers to the affinity of an antibody to an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" are multiply bound to each other by the strength of the interaction between the antigenic site and the variable region. The stronger the interaction, the greater the binding of the antigen. As used herein, an antibody or antigen-binding fragment thereof (e.g., The term "high affinity" for a given antibody (e.g., Fab fragment) is usually D 10 -9 Antibodies below M or Refers to an antigen-binding fragment.

[0164] As used herein, the term "human antibody" refers to a human antibody that is The present invention is intended to include antibodies having variable regions derived from sequences of human origin. In some cases, if the antibody contains a constant region, the constant region may also contain such a human sequence, e.g., human Derived from germline sequences or mutated versions of human germline sequences. Human antibodies, such as human monoclonal antibodies, can be produced by the synthesis of human heavy chain transgenes and light chain transgenes. Transgenic non-human animals having genomes that include offspring, e.g. It is produced by a hybridoma that contains immortalized cells fused to B cells obtained from a mouse. This is also fine.

[0165] "Humanized" antibodies are less immunogenic in humans but retain the reactivity of non-human antibodies. These are antibodies that, for example, retain the non-human CDR regions and replace the remainder of the antibody with By replacing the constant and variable regions of For example, Morrison et al., Proc. Natl.Acad.Sci.USA,81:6851-6855,1984;Morr ison and Oi, Adv. Immunol., 44:65-92, 1988; hoeyen et al.,Science,239:1534-1536,1988 ;Padlan,Molec.Immun.,28:489-498,1991;and See Padlan, Molec. Immun., 31:169-217, 1994. Another example of a human-operated technique is disclosed in U.S. Pat. No. 5,766,886. This includes, but is not limited to, XOMA technology.

[0166] The term "hybridoma" refers to the fusion of antibody-producing cells, such as B lymphoblasts, with myeloma cells, etc. Hybridomas refer to immortalized cells derived from fusion with a fusion partner. In certain embodiments, hybridomas are The antibody-producing cells used to generate the antibodies may be those immunized with an antigen, e.g., The antibodies are obtained from animals immunized according to the mRNA-based immunization methods described herein.

[0167] The term "isolated antibody" refers to an antibody that is an antibody having a different antigen specificity and / or a different amino acid sequence. In certain embodiments, however, the term "antigen-specific antibody" refers to an antibody that is substantially free of antibodies of the antigen. The isolated antibody that binds may have cross-reactivity to other antigens. The body may be substantially free of other cellular material and / or chemicals.

[0168] The term "isotype" refers to the antibody class provided by the heavy chain constant region genes (e.g. IgM, IgE, IgG, such as IgG1, IgG2, or IgG4. A type also includes modified forms of one of these classes, e.g., Fc receptor binding, such as enhancing or reducing effector function or binding to Fc receptors The term "monoclonal antibody" refers to a specific antibody that is specific to a particular epitope. Antibodies obtained from a homogeneous or substantially homogeneous population of antibodies that exhibit binding specificity and affinity to The term "monoclonal" refers to a cell that produces antibodies. The present invention is not limited to any particular method for the production of monoclonal antibodies. Generally, a population of monoclonal antibodies is produced by the addition of cells, The antibodies can be produced by a population of cells, or a cell line. The monoclonal antibody may be a chimeric or humanized antibody. The antibody may be a monovalent antibody or a multivalent (eg, bivalent) antibody.

[0169] As used herein, the term "polyclonal antibody" refers to a polyclonal antibody that reacts with a specific antigen. It refers to a heterogeneous antibody population that contains a variety of different antibodies that target different epitopes in an antigen. Recognize the group.

[0170] As used herein, the term "recombinant human antibody" refers to an antibody produced using human immunoglobulin genes. isolated from transgenic or transgenic animals (e.g., mice) or an antibody isolated from a hybridoma produced therefrom, expressing a human antibody. Antibodies isolated from host cells transformed to produce the desired product, e.g., from transfectomas. Antibodies isolated from recombinant combinatorial human antibody libraries, and human immunoglobulins Any of the above methods may be used, including splicing all or part of the sequence of the globin gene to another DNA sequence. may be produced, expressed, created, or isolated by any other means. Antibodies, etc., may be produced, expressed, engineered, or isolated by recombinant means. Such recombinant human antibodies include all human antibodies that are isolated from the human genome. The CDR regions have variable regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are produced by in vitro mutagenesis ( Alternatively, in vivo somatic cell proliferation may be achieved when animals transgenic for human Ig sequences are used. Thus, the VH and VL regions of the recombinant antibody can be subjected to mutagenesis (mutagenesis). The amino acid sequences are derived from and related to human germline VH and VL sequences, but These are sequences that cannot naturally occur within the human antibody germline repertoire in vivo.

[0171] As used herein, the term "optimized" refers to a nucleotide sequence that is optimized for use in a production cell or or organism, usually a eukaryotic cell, that encodes an amino acid sequence that uses preferred codons. In certain embodiments, the optimized nucleotide sequence is The sequence is originally encoded by a starting nucleotide sequence, also known as the "parent" sequence. The amino acid sequence is engineered to be preserved entirely, or as much as possible. In particular, the optimized sequences described herein are preferred in mammalian cells, such as mouse cells. In other embodiments, the gene is engineered to contain new codons. Optimized expression of sequences in is also contemplated herein.

[0172] I. Target Protein / Antigenic Determinant Provided herein are methods for detecting target proteins (e.g., nucleotides) in animals (e.g., non-human animals). human proteins, such as human transmembrane proteins, e.g., human GPCRs) or A method for inducing an immune response to a fragment of a lipid comprising administering to an animal a lipid and a target protein. A polyribonucleotide, such as a polyribonucleotide (e.g., mRNA), that encodes a protein or a fragment thereof. Methods for administering compositions containing complexes containing oligonucleotides and target proteins thereof The methods provided herein include the step of generating antibodies against any desired target protein. Antibodies (e.g., monoclonal antibodies) against target proteins (e.g., transmembrane proteins) of the It is useful for generating or inducing an immune response against certain In an embodiment, the target protein is a human target protein, and in the methods described herein The immunized animal used herein is a non-human animal.

[0173] In certain aspects, provided herein are methods for detecting targets in animals (e.g., non-human animals). target proteins (e.g., human proteins such as human transmembrane proteins, e.g., human GPCRs) The antibody against the target protein or a fragment thereof is coated with the lipid and the target protein or a fragment thereof. The composition includes a complex containing a polyribonucleotide such as an mRNA that encodes a The method for detecting a target protein, comprising: (i) detecting a G protein-coupled receptor (GPCR) (ii) single-pass transmembrane protein receptors; (iii) tumor necrosis factor receptor superfamily (iv) interleukin (IL) receptors; (v) (vi) an ion channel; (vii) a solute transporter; (viii) an immunoreceptor; or It is a transmembrane protein.

[0174] In certain aspects, provided herein are methods for detecting targets in animals (e.g., non-human animals). target proteins (e.g., human proteins such as human transmembrane proteins, e.g., human GPCRs) The antibody against the target protein or a fragment thereof is coated with the lipid and the target protein or a fragment thereof. The composition includes a complex containing a polyribonucleotide such as an mRNA that encodes a The method for detecting ACKR1, ACKR2, ACKR3, or the like, wherein the target protein is selected from the group consisting of ACKR1, ACKR2, ACKR3, and the like. ,ACKR4,ADCYAP1R1,ADGRA1,ADGRA2,ADGRA3,AD GRB1, ADGRB2, ADGRB3, ADGRD1, ADGRD2, ADGRE1, ADGRE2, ADGRE3, ADGRE4P, ADGRE5, ADGRF1, ADGR F2, ADGRF3, ADGRF4, ADGRF5, ADGRG1, ADGRG2, AD GRG3, ADGRG4, ADGRG5, ADGRG6, ADGRG7, ADGRL1, ADGRL2, ADGRL3, ADGRL4, ADGRV1, ADORA1, ADORA 2A, ADORA2B, ADORA3, ADRA1A, ADRA1B, ADRA1D, A DRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AG TR1, AGTR2, APLNR / APJ, ASGR1, ASGR2, AVPR1A, A VPR1B, AVPR2, BDKRB1, BDKRB2, BRS3, BRS3, C3AR 1, C5AR1, C5AR2, CALCR, CALCRL, CASR, CCKAR, CC KBR、CCR1、CCR10、CCR2、CCR3、CCR4、CCR5、CCR6、 CCR7、CCR8、CCR9、CCRL2、CELSR1、CELSR2、CELSR 3、CHRM1、CHRM2、CHRM3、CHRM4、CHRM5、CMKLR1、C NR1、CNR2、CRHR1、CRHR2、CX3CR1、CXCR1、CXCR2、 CXCR3、CXCR4、CXCR5、CXCR6、CYSLTR1、CYSLTR2、 DRD1、DRD2、DRD3、DRD4、DRD5、EDNRA、EDNRB、F2R 、F2RL1、F2RL2、F2RL3、FFAR1、FFAR2、FFAR3、FFA R4、FPR1、FPR2、FPR2、FPR3、FSHR、FZD1、FZD10、F ZD2、FZD3、FZD4、FZD5、FZD6、FZD7、FZD8、FZD9、G ABBR1、GABBR2、GALR1、GALR2、GALR3、GCGR、GHRH R、GHSR、GIPR、GLP1R、GLP2R、GNRHR、GNRHR2、GPB AR1、GPER1、GPR1、GPR4、GPR12、GPR15、GPR17、GP R18、GPR19、GPR20、GPR21、GPR22、GPR25、GPR26、 GPR27、GPR3、GPR31、GPR32、GPR33、GPR34、GPR35 、GPR37、GPR37L1、GPR39、GPR40、GPR42、GPR42、G PR45、GPR50、GPR52、GPR55、GPR55、GPR6、GPR61、 GPR62、GPR63、GPR65、GPR68、GPR75、GPR78、GPR7 9、GPR82、GPR83、GPR84、GPR85、GPR87、GPR88、GP R101、GPR107、GPR132、GPR135、GPR137、GPR139、 GPR141、GPR142、GPR143、GPR146、GPR148、GPR14 9、GPR15、GPR150、GPR151、GPR152、GPR153、GPR1 56、GPR157、GPR158、GPR160、GPR161、GPR162、GP R171、GPR173、GPR174、GPR176、GPR179、GPR182、 GPR183、GPRC5A、GPRC5B、GPRC5C、GPRC5D、GPRC6 A、GRM1、GRM2、GRM3、GRM4、GRM5、GRM6、GRM7、GRM 8、GRPR、HCAR1、HCAR2、HCAR3、HCRTR1、HCRTR2、H RH1、HRH2、HRH3、HRH4、HTR1A、HTR1B、HTR1D、HTR 1E、HTR1F、HTR2A、HTR2B、HTR2C、HTR4、HTR5A、HT R5BP、HTR6、HTR7、KISS1R、LGR3、LGR4、LGR5、LGR 6、LHCGR、LPAR1、LPAR2、LPAR3、LPAR4、LPAR5、LP AR6、LTB4R、LTB4R2、MAS1、MAS1L、MC1R、MC2R、MC 3R、MC4R、MC5R、MCHR1、MCHR2、MLNR、MRGPRD、MRG PRE、MRGPRF、MRGPRG、MRGPRX1、MRGPRX2、MRGPRX 3、MRGPRX4、MTNR1A、MTNR1B、NMBR、NMUR1、NMUR2 、NPBWR1、NPBWR2、NPFFR1、NPFFR2、NPSR1、NPY1R 、NPY2R、NPY4R、NPY5R、NPY6R、NTSR1、NTSR2、OPN 3、OPN4、OPN5、OPRD1、OPRK1、OPRL1、OPRM1、OR51 E1, OXER1, OXGR1, OXTR, P2RY1, P2RY10, P2RY11, P2RY12, P2RY13, P2RY14, P2RY2, P2RY4, P2RY6, P 2RY8, PRLHR, PROKR1, PROKR2, PTAFR, PTGDR, PTG DR2, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PT GIR, PTH1R, PTH2R, QRFPR, RXFP1, RXFP2, RXFP3, RXFP4, S1PR1, S1PR2, S1PR3, S1PR4, S1PR5, SCTR , SMO, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, SUCNR 1, TAAR1, TAAR2, TAAR3, TAAR4P, TAAR5, TAAR6, T AAR8, TAAR9, TACR1, TACR2, TACR3, TAS1R1, TAS1 R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14 , TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, T AS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, T AS2R5, TAS2R50, TAS2R60, TAS2R7, TAS2R8, TAS2 R9, TBXA2R, TPRA1, TRHR, TSHR, UTS2R, VIPR1, VI PR2, XCR1, TCR-α, TCR-β, CD3, ζ-chain accessory, CD4 and and CD8, mannose receptor (MR), and asialoglycoprotein receptor family (e.g. , asialoglycoprotein receptor macrophage galactose-type lectin (MGL), D C-SIGN (CLEC4L), Langerin (CLEC4K), myeloid DAP12-associated receptor Dectin (MDL)-1 (CLEC5A), Dectin 1 / CLEC7A, DNGR1 / CL EC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 ( CLEC1B, CLEC12B, DCIR / CLEC4A, Dectin 2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), macrophage-inducible C-type lectin (CLEC4E), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TL R13, FcγRI(CD64), FcγRIIA(CD32), FcγRIIB1(C D32), FcγRIIB2 (CD32), FcγRIIIA (CD16a), FcγR IIIB (CD16b), FcεRI, FcεRII (CD23), FcαR1 (CD8 9), Fcα / μR, FcRn, CD27, CD40, OX40, GITR, CD137 , PD-1, CTLA-4, PD-L1, TIGIT, T cell immunoglobulin domain and and mucin domain 3 (TIM3), V-domain Ig inhibitor of T cell activation (VIS TA), CD28, CD122, ICOS, A2AR, B7-H3, B7-H4, B and and T-lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), FAM159B, HLA-A, HLA-B, HLA-C, HLA-DPA 1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA -DRB1, gp130, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4 Receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 Receptor, IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-1 8 receptor, IL-19 receptor, IL-20 receptor, IL-21 receptor, IL-22 receptor , IL-23 receptor, IL-24 receptor, IL-25 receptor, IL-26 receptor, IL- 27 receptor, IL-28 receptor, IL-29 receptor, IL-30 receptor, IL-31 receptor Body, IL-32 receptor, IL-33 receptor, IL-35 receptor, IL-36 receptor, FG FR1, FGFR2, FGFR3, FGFR4, TNFRSF1A, TNFRSF1B, TNFRSF3, TNFRSF4, TNFRSF5, TNFRSF6, TNFRSF6B , TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRS F10B, TNFRSF10C, TNFRSF10D, TNFRSF11A, TNFRS F11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRS F14, TNFRSF16, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, TNFRSF25, TNFRSF27, SCN1A, SCN1B, S CN2A, SCN2B, SCN3A, SCN3B, SCN4A, SCN5A, SCN7A , SCN8A, SCN9A, SCN10A, SCN11A, CACNA1A, CACNA 1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA 1G, CACNA1H, CACNA1I, CACNA1S, TRPA1, TRPC1, T RPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPM1 、TRPM2、TRPM3、TRPM4、TRPM5、TRPM6、TRPM7、TRP M8、MCOLN1、MCOLN2、MCOLN3、PKD1、PKD2、PKD2L1 、PKD2L2、TRPV1、TRPV2、TRPV3、TRPV4、TRPV5、TR PV6、CATSPER1、CATSPER2、CATSPER3、CATSPER4、 TPCN1、TPCN2、CNGA1、CNGA2、CNGA3、CNGA4、CNGB 1、CNGB3、HCN1、HCN2、HCN3、HCN4、KCNMA1、KCNN1 、KCNN2、KCNN3、KCNN4、KCNT1、KCNT2、KCNU1、KCN A1、KCNA2、KCNA3、KCNA4、KCNA5、KCNA6、KCNA7、K CNA10、KCNB1、KCNB2、KCNC1、KCNC2、KCNC3、KCNC 4、KCND1、KCND2、KCND3、KCNF1、KCNG1、KCNG2、KC NG3、KCNG4、KCNH1、KCNH2、KCNH3、KCNH4、KCNH5、 KCNH6、KCNH7、KCNH8、KCNQ1、KCNQ2、KCNQ3、KCNQ 4、KCNQ5、KCNS1、KCNS2、KCNS3、KCNV1、KCNV2、KC NJ1、KCNJ2、KCNJ3、KCNJ4、KCNJ5、KCNJ6、KCNJ8、 KCNJ9、KCNJ10、KCNJ11、KCNJ12、KCNJ13、KCNJ14 、KCNJ15、KCNJ16、KCNJ18、KCNK1、KCNK2、KCNK3、 KCNK4、KCNK5、KCNK6、KCNK7、KCNK9、KCNK10、KCN K12、KCNK13、KCNK15、KCNK16、KCNK17、KCNK18、H VCN1, HTR3A, HTR3B, HTR3C, HTR3D, HTR3E, CHRNA 1 CHRNA2, CHRNA3, CHRNA4, CHRNA5, CHRNA6, CHRN A7, CHRNA9, CHRNA10, CHRNB1, CHRNB2, CHRNB3.C HRNB4, CHRND, CHRNE, CHRNG, GABRA1, GABRA2, GA BRA3, GABRA4, GABRA5, GABRA6, GABRB1, GABRB2 GABRB3, GABRD, GABRE, GABRG1, GABRG2, GABRG3 GABRP、GABRQ、GABRR1、GABRR2、GABRR3、GRIA1、G RIA2、GRIA3、GRIA4、GRID1、GRID2、GRIK1、GRIK2 、GREEK3、GREEK4、GREEK5、GREEN1、GREEN2A、GREEN2B、G RIN2C、GRIN2D、GRIN3A、GRIN3B、GLRA1、GLRA2、G LRA3, GLRA4, P2RX1, P2RX2, P2RX3, P2RX4, P2RX5 P2RX6, P2RX7, ZACN, ASIC1, ASIC2, ASIC3, ASIC 4, AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP 8, AQP9, AQP10, AQP11, AQP12A, AQP12B, MIP, CLC N1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7C LCNKA, CLCNKB, National Consumer Protection Agency (CFTR) AN O1 / TMEM16a、ANO2、ANO3、ANO4、ANO5、ANO6、ANO7 、ANO8、ANO9、ANO10、BEST1、BEST2、BEST3、BEST4 , CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, CLIC6, GJA 1, GJA3, GJA4, GJA5, GJA6P, GJA8, GJA9, GJA10, G JB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC1, G JC2, GJC3, GJD2, GJD3, GJD4, GJE1, ITPR1, ITPR2 , ITPR3, PANX1, PANX2, PANX3, RYR1, RYR2, RYR3, NALCN, SCNN1A, SCNN1B, SCNN1D, SCNN1G, ADAMTS 7, ANGPTL3, ANGPTL4, ANGPTL8, LPL, GDF15, Galecti Galectin-1, Galectin-2, Galectin-3, Galectin-4, Galectin-7, Galectin Galectin-8, Galectin-9, Galectin-10, Galectin-12, Galectin-13, Trix gla protein (MGP), PRNP, DGAT1, GPAT3, DMC1 , BLM, BRCA2, human endogenous retrovirus type K (HERV-K) family A member of the ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), Entonucleoside triphosphate diphosphohydrolase 2 (ENTPD2), SLC1A1, S LC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7 , SLC2A1, SLC2A2, SLC2A3, SLC2A4, SLC2A5, SLC2 A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14, SLC3A1, SLC3A2, SL C4A1, SLC4A2, SLC4A3, SLC4A4, SLC4A5, SLC4A6, <h2 style=";text-align:left;direction:ltr">SLC4A7、SLC4A8、SLC4A9、SLC4A10、SLC4A11、SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5A1、SLC5A2、SLC5A3、SLC5A4、SLC5A5、SLC5A6、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC5A7, SLC5A8, SLC5A9, SLC5A10, SLC5A11, SLC5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A12、SLC6A1、SLC6A2、SLC6A3、SLC6A4、SLC6A5、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC6A6、SLC6A7、SLC6A8、SLC6A9、SLC6A10、SLC6A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 11、SLC6A12、SLC6A13、SLC6A14、SLC6A15、SLC6A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 16, SLC6A17, SLC6A18, SLC6A19, SLC6A20, SLC7A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5, SLC7A6, SLC7A7, SLC7A8, SLC7A9, SLC7A10, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C7A11, SLC7A13, SLC7A14, SLC8A1, SLC8A2, SLC8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A3, SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C9A6, SLC9A7, SLC9A8, SLC9A9, SLC9A10, SLC9A1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1、SLC9B1、SLC9B2、SLC10A1、SLC10A2、SLC10A3、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC10A4、SLC10A5、SLC10A6、SLC10A7、SLC11A1、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC11A2, SLC12A1, SLC12A2, SLC12A3, SLC12A4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC12A5, SLC12A6, SLC12A7, SLC12A8, SLC12A9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC13A1, SLC13A2, SLC13A3, SLC13A4, SLC13A5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC14A1, SLC14A2, SLC15A1, SLC15A2, SLC15A3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC15A4、SLC16A1、SLC16A2、SLC16A3、SLC16A4、<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC16A5、SLC16A6、SLC16A7、SLC16A8、SLC16A9、 SLC16A10、SLC16A11、SLC16A12、SLC16A13、SLC1 6A14、SLC17A1、SLC17A2、SLC17A3、SLC17A4、SLC 17A5、SLC17A6、SLC17A7、SLC17A8、SLC17A9、SLC 18A1、SLC18A2、SLC18A3、SLC19A1、SLC19A2、SLC 19A3、SLC20A1、SLC20A2、SLCO1A2、SLCO1B1、SLC O1B3、SLCO1C1、SLCO2A1、SLCO2B1、SLCO3A1、SLC O4A1、SLCO4C1、SLCO5A1、SLCO6A1、SLC22A1、SLC 22A2、SLC22A3、SLC22A4、SLC22A5、SLC22A6、SLC 22A7、SLC22A8、SLC22A9、SLC22A10、SLC22A11、S LC22A12、SLC22A13、SLC22A14、SLC22A15、SLC22 A16、SLC22A17、SLC22A18、SLC22A18AS,SLC22A1 9、SLC22A20、SLC22A23、SLC22A24、SLC22A25、SL C22A31、SLC23A1、SLC23A2、SLC23A3、SLC23A4、S LC24A1、SLC24A2、SLC24A3、SLC24A4、SLC24A5、S LC24A6、SLC25A1、SLC25A2、SLC25A3、SLC25A4、S LC25A5、SLC25A6、SLC25A7、SLC25A8、SLC25A9、S LC25A10、SLC25A11、SLC25A12、SLC25A13、SLC25 A14、SLC25A15、SLC25A16、SLC25A17、SLC25A18、 <h2 style=";text-align:left;direction:ltr">SLC25A19, SLC25A20, SLC25A21, SLC25A22, SLC2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 5A23, SLC25A24, SLC25A25, SLC25A26, SLC25A27<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> SLC25A28, SLC25A29, SLC25A30, SLC25A31, SLC<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 25A32, SLC25A33, SLC25A34, SLC25A35, SLC25A3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6, SLC25A37, SLC25A38, SLC25A39, SLC25A40, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C25A41、SLC25A42、SLC25A43、SLC25A44、SLC25A<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 45, SLC25A46, SLC26A1, SLC26A2, SLC26A3, SLC2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6A4, SLC26A5, SLC26A6, SLC26A7, SLC26A8, SLC2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 6A9, SLC26A10, SLC26A11, SLC27A1, SLC27A2, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C27A3, SLC27A4, SLC27A5, SLC27A6, SLC28A1, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C28A2, SLC28A3, SLC29A1, SLC29A2, SLC29A3, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C29A4, SLC30A1, SLC30A2, SLC30A3, SLC30A4, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C30A5, SLC30A6, SLC30A7, SLC30A8, SLC30A9, SL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> C30A10、SLC31A1、SLC31A2、SLC32A1、SLC33A1、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC34A1, SLC34A2, SLC34A3, SLC35A1, SLC35A2, S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC35A3, SLC35A4, SLC35A5, SLC35B1, SLC35B2, S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC35B3、SLC35B4、SLC35C1、SLC35C2、SLC35D1、S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC35D2, SLC35D3, SLC35E1, SLC35E2, SLC35E3, S<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> LC35E4、SLC35F1、SLC35F2、SLC35F3、SLC35F4、S LC35F5, SLC35G1, SLC35G3, SLC35G4, SLC35G5, S LC35G6, SLC36A1, SLC36A2, SLC36A3, SLC36A4, S LC37A1, SLC37A2, SLC37A3, SLC37A4, SLC38A1, S LC38A2, SLC38A3, SLC38A4, SLC38A5, SLC38A6, S LC38A7, SLC38A8, SLC38A9, SLC38A10, SLC38A11 , SLC39A1, SLC39A2, SLC39A3, SLC39A4, SLC39A5 , SLC39A6, SLC39A7, SLC39A8, SLC39A9, SLC39A1 0, SLC39A11, SLC39A12, SLC39A13, SLC39A14, SL C40A1, SLC41A1, SLC41A2, SLC41A3, RhAG, RhBG, RhCG, SLC43A1, SLC43A2, SLC43A3, SLC44A1, SLC 44A2, SLC44A3, SLC44A4, SLC44A5, SLC45A1, SLC 45A2, SLC45A3, SLC45A4, SLC46A1, SLC46A2, SLC 46A3, SLC47A1, SLC47A2, HCP-1, MFSD5, MFSD10, SLC50A1, OSTα, OSTβ, SLC52A1, SLC52A2, and SL C52A3.

[0175] In certain aspects, the methods provided herein involve the analysis of transmembrane proteins, such as transmembrane receptors. Target proteins that are difficult to express or for which antibodies are difficult to produce The target protein may be modified to improve, for example, cytotoxicity, yield, solubility, aggregation, and / or stability. This is useful for generating antibodies against difficult target proteins due to problems.

[0176] In certain embodiments, a target protein described herein (e.g., a human target protein) are classified into type I, type II, type II single anchor, C-terminal anchor, and polytopic types (e.g. For example, Ott and Lingappa, 2002, J. Cell Sci., 115 (P t10):2003-2009) In certain embodiments, the target proteins described herein (e.g., human target proteins) are included. In certain embodiments, the target proteins described herein are The protein (eg, the human target protein) is a multi-transmembrane protein.

[0177] G protein-coupled receptors (GPCRs) In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, The protein (e.g., a human target protein) is a GPCR (e.g., a human GPCR). As seven-transmembrane (7TM) domain receptors and G protein-coupled receptors (GPLRs) The two main signaling pathways involving GPCRs are cAMP signaling and These are the signaling pathway and the phosphatidylinositol signaling pathway.

[0178] There are two classes of GPCR: class A (rhodopsin-like), class B (secretin receptor), family), Class C (metabotropic glutamate / pheromone), Class D (fungal mating factor Class E (cyclic AMP receptors), and Class F (Friz zled / Smoothened).

[0179] GPCRs (classes A, B, C, Frizzled, and other seven-transmembrane proteins) Non-limiting examples of target proteins that are cleavable proteins include ACKR1, ACKR2, ACKR3, ACKR4, ADCYAP1R1, ADGRA1, ADGRA2, ADGRA3, ADG RB1, ADGRB2, ADGRB3, ADGRD1, ADGRD2, ADGRE1, A DGRE2, ADGRE3, ADGRE4P, ADGRE5, ADGRF1, ADGRF 2, ADGRF3, ADGRF4, ADGRF5, ADGRG1, ADGRG2, ADG RG3, ADGRG4, ADGRG5, ADGRG6, ADGRG7, ADGRL1, A DGRL2, ADGRL3, ADGRL4, ADGRV1, ADORA1, ADORA2 A, ADORA2B, ADORA3, ADRA1A, ADRA1B, ADRA1D, AD RA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AGT R1, AGTR2, APLNR / APJ, AVPR1A, AVPR1B, AVPR2, B DKRB1, BDKRB2, BRS3, BRS3, C3AR1, C5AR1, C5AR2 , CALCR, CALCRL, CASR, CCKAR, CCKBR, CCR1, CCR1 0, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR 9, CCRL2, CELSR1, CELSR2, CELSR3, CHRM1, CHRM2 , CHRM3, CHRM4, CHRM5, CMKLR1, CNR1, CNR2, CRHR 1, CRHR2, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, C XCR5, CXCR6, CYSLTR1, CYSLTR2, DRD1, DRD2, DRD 3、DRD4、DRD5、EDNRA、EDNRB、F2R、F2RL1、F2RL2、 F2RL3、FFAR1、FFAR2、FFAR3、FFAR4、FPR1、FPR2、 FPR2、FPR3、FSHR、FZD1、FZD10、FZD2、FZD3、FZD4 、FZD5、FZD6、FZD7、FZD8、FZD9、GABBR1、GABBR2、 GALR1、GALR2、GALR3、GCGR、GHRHR、GHSR、GIPR、G LP1R、GLP2R、GNRHR、GNRHR2、GPBAR1、GPER1、GPR 1、GPR4、GPR12、GPR15、GPR17、GPR18、GPR19、GPR 20、GPR21、GPR22、GPR25、GPR26、GPR27、GPR3、GP R31、GPR32、GPR33、GPR34、GPR35、GPR37、GPR37L 1、GPR39、GPR40、GPR42、GPR42、GPR45、GPR50、GP R52、GPR55、GPR6、GPR61、GPR62、GPR63、GPR65、G PR68、GPR75、GPR78、GPR79、GPR82、GPR83、GPR84 、GPR85、GPR87、GPR88、GPR101、GPR107、GPR132、 GPR135、GPR137、GPR139、GPR141、GPR142、GPR14 3、GPR146、GPR148、GPR149、GPR15、GPR150、GPR1 51、GPR152、GPR153、GPR156、GPR157、GPR158、GP R160、GPR161、GPR162、GPR171、GPR173、GPR174、 GPR176、GPR179、GPR182、GPR183、GPRC5A、GPRC5 B、GPRC5C、GPRC5D、GPRC6A、GRM1、GRM2、GRM3、GR M4、GRM5、GRM6、GRM7、GRM8、GRPR、HCAR1、HCAR2、 HCAR3、HCRTR1、HCRTR2、HRH1、HRH2、HRH3、HRH4、 HTR1A、HTR1B、HTR1D、HTR1E、HTR1F、HTR2A、HTR2 B、HTR2C、HTR4、HTR5A、HTR5BP、HTR6、HTR7、KISS 1R、LGR4、LGR5、LGR6、LHCGR、LPAR1、LPAR2、LPAR 3、LPAR4、LPAR5、LPAR6、LTB4R、LTB4R2、MAS1、MA S1L、MC1R、MC2R、MC3R、MC4R、MC5R、MCHR1、MCHR2 、MLNR、MRGPRD、MRGPRE、MRGPRF、MRGPRG、MRGPRX 1、MRGPRX2、MRGPRX3、MRGPRX4、MTNR1A、MTNR1B、 NMBR、NMUR1、NMUR2、NPBWR1、NPBWR2、NPFFR1、NP FFR2、NPSR1、NPY1R、NPY2R、NPY4R、NPY5R、NPY6R 、NTSR1、NTSR2、OPN3、OPN4、OPN5、OPRD1、OPRK1、 OPRL1、OPRM1、OR51E1、OXER1、OXGR1、OXTR、P2RY 1、P2RY10、P2RY11、P2RY12、P2RY13、P2RY14、P2R Y2、P2RY4、P2RY6、P2RY8、PRLHR、PROKR1、PROKR2 、PTAFR、PTGDR、PTGDR2、PTGER1、PTGER2、PTGER3 、PTGER4、PTGFR、PTGIR、PTH1R、PTH2R、QRFPR、RX FP1、RXFP2、RXFP3、RXFP4、S1PR1、S1PR2、S1PR3、 S1PR4, S1PR5, SCTR, SMO, SSTR1, SSTR2, SSTR3, S STR4, SSTR5, SUCNR1, TAAR1, TAAR2, TAAR3, TAAR 4P, TAAR5, TAAR6, TAAR8, TAAR9, TACR1, TACR2, T ACR3, TAS1R1, TAS1R2, TAS1R3, TAS2R1, TAS2R10 , TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20 , TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, T AS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60, TA S2R7, TAS2R8, TAS2R9, TBXA2R, TPRA1, TRHR, TSH R, UTS2R, VIPR1, VIPR2, and XCR1.

[0180] Transmembrane and membrane-bound immune receptors: In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins act as pattern recognition receptors (PRRs), toll-like receptors (TLRs), killer activation receptors, and and killer inhibitory receptors (KAR and KIR), complement receptors, Fc receptors, and B cell receptors , and T cell receptors (e.g., TCR-α, TCR-β, CD3, ζ-accessory chain , CD4, and CD8) and major histocompatibility complexes.

[0181] Non-limiting examples of pattern recognition receptors (PRRs) include the mannose receptor (MR), the asiatic receptor (ASR), and the mitochondrial receptor (MCR). The asialoglycoprotein receptor family (e.g., asialoglycoprotein receptor macrophage Galactose-type lectin (MGL)), DC-SIGN (CLEC4L), Langerin ( CLEC4K), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), Dec 1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MI CL) (CLEC12A), CLEC2 (also called CLEC1B), CLEC12B , and the DC immune receptor (DCIR) subfamily (e.g., DCIR / CLEC4A , Dectin2 / CLEC6A, Blood DC antigen 2 (BDCA2) (CLEC4C), and Macrophage-inducible C-type lectin 4E (CLEC4E).

[0182] In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins are Toll-like receptors (TLRs). TLRs play an important role in the innate immune system. They are a class of proteins that structurally bind conserved molecules of microbial origin. It is normally expressed in cells such as macrophages and dendritic cells that recognize and activate immune responses. Non-limiting examples of TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, TLR14, TLR15, TLR16, TLR17, TLR18, TLR19 ...2, TLR13, T R2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TL R10, TLR11, TLR12, and TLR13.

[0183] Non-limiting examples of Fc receptors (e.g., Fcγ receptors, Fcα receptors, and Fcε receptors) Examples include the polymeric immunoglobulin receptor (plgR), FcγRI (CD64), and FcγR IIA(CD32), FcγRIIB1(CD32), FcγRIIB2(CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI, Fc Includes εRII (CD23), FcαR1 (CD89), Fcα / μR, and FcRn .

[0184] In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins include: CD27, CD40, OX40, GITR, CD137, PD-1, C TLA-4, PD-L1, TIGIT, T cell immunoglobulin domains and mucin domains TIM3, V-domain Ig inhibitor of T cell activation (VISTA), CD2 8, CD122, ICOS, A2AR, B7-H3, B7-H4, B and T lymphocyte Tenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), Lar cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3) The immune receptor is selected from the group consisting of:

[0185] In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins are either major histocompatibility complex I or II (MHC I or MHC II) or or part of the MHC in a complex with peptide fragments. MHC proteins are involved in adaptive immunity. It is part of the IL-1 system, which mediates the presentation of processed peptide fragments to T cells and activation of the humoral immune system. Non-limiting examples of MHC proteins include HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F ... LA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HL A-DQB1, HLA-DRA, and HLA-DRB1.

[0186] Cytokine Receptors: In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins bind to cytokine receptors (e.g., interleukin (IL) receptors or fibroblast A non-limiting example of a cytokine receptor is a fibroblast growth factor (FGF) receptor. Growth factor (NGF), myostatin (GDF-8), growth differentiation factor (GDF), granulocyte macrophage globulin colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) , platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin ( TPO), epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), tissue inhibitors of metalloproteinases (TIMPs), matrix metalloproteinases Membrane proteinase (MMP), macrophage stimulating factor (MSF), ciliary neurotrophic factor (CNT F), cardiotrophin, oncostatin M, leukemia inhibitory factor (LIF), transformation enhancement Transforming growth factor (TGF)-α and -β, interferon (IFN)-β and -γ, and In certain embodiments, the present invention relates to a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of the present invention. The complexes for use in the methods provided herein include a lipid and a target protein encoding the target protein is gp130; These include IL-6, IL-11, IL-27, leukemia inhibitory factor (LIF), oncostatin, Osteoarthritis M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) , and several related sites, including cardiotrophin-like cytokines (CLCs). It is a shared receptor utilized by kine.

[0187] In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, The protein is an interleukin (IL) receptor. A non-limiting example of an IL receptor is an IL -1 receptor, IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL -6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, IL-10 receptor, I IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14 receptor, IL-15 Receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor, IL-19 receptor, IL-20 receptor, IL-21 receptor, IL-22 receptor, IL-23 receptor, IL-2 4 receptor, IL-25 receptor, IL-26 receptor, IL-27 receptor, IL-28 receptor , IL-29 receptor, IL-30 receptor, IL-31 receptor, IL-32 receptor, IL- These include the IL-33 receptor, the IL-35 receptor, and the IL-36 receptor.

[0188] In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins are fibroblast growth factor (FGF) receptors (FGFRs). Non-limiting examples include FGFR1, FGFR2, FGFR3, and FGFR4.

[0189] In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins are members of the tumor necrosis factor receptor superfamily (TNFRSF). Non-limiting examples of NFRSF members include TNFRSF1A, TNFRSF1B, TNFR SF3, TNFRSF4, TNFRSF5, TNFRSF6, TNFRSF6B, TNF RSF7, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B , TNFRSF10C, TNFRSF10D, TNFRSF11A, TNFRSF11B , TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF16, TNFRSF17, TNFRSF18, TNFRSF19, TNFR SF21, TNFRSF25, and TNFRSF27.

[0190] Ion channels; In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Proteins are ion channels.

[0191] There are over 300 types of ion channels, each with different gating properties, These gates are classified according to the ion species passing through them, the number of gates (pores), and the localization of proteins. For example, voltage-gated ion channels can be classified into voltage-gated sodium channels. channel, voltage-gated calcium channel, voltage-gated potassium channel (K v ), hyperpolarization activity These include activated cyclic nucleotide-gated channels and voltage-gated proton channels. Ligand-gated ion channels include, but are not limited to, cation-permeable "nicotinic" acetylcholine channels. Alcohol receptors, ionotropic glutamate-dependent receptors and ATP-dependent P2X receptors receptors, as well as anion-permeable γ-aminobutyric acid-dependent GABA receptors. Classification of ion channels by ion species includes chloride channels, potassium channels, etc. ATP-sensitive potassium ion channels, sodium channels For example, NaVs, ENaCs, CaVs), calcium channels, proton channels, and and nonselective cation channels.

[0192] Non-limiting examples of voltage-gated sodium channels include SCN1A, SCN1B, SCN2 A, SCN2B, SCN3A, SCN3B, SCN4A, SCN5A, SCN7A, SC Includes N8A, SCN9A, SCN10A, and SCN11A.

[0193] Non-limiting examples of voltage-gated calcium channels include CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, Includes CACNA1H, CACNA1I, and CACNA1S.

[0194] Non-limiting examples of transient receptor potential cation channels include TRPA1, TRPC1, TR PC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPM1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM 8, MCOLN1, MCOLN2, MCOLN3, PKD1, PKD2, PKD2L1, PKD2L2, TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, and Contains TRPV6.

[0195] Non-limiting examples of CatSper channels include CATSPER1, CATSPER2, C Includes ATSPER3 and CATSPER4.

[0196] Non-limiting examples of two-pore channels include TPCN1 and TPCN2.

[0197] Non-limiting examples of cyclic nucleotide-gated channels include CNGA1, CNGA2, CNG A3, CNGA4, CNGB1, CNGB3, HCN1, HCN2, HCN3, and H Includes CN4.

[0198] Non-limiting examples of calcium-activated potassium channels include KCNMA1, KCNN1, K Includes CNN2, KCNN3, KCNN4, KCNT1, KCNT2, and KCNU1 .

[0199] Non-limiting examples of voltage-gated potassium channels include KCNA1, KCNA2, KCNA3. , KCNA4, KCNA5, KCNA6, KCNA7, KCNA10, KCNB1, KC NB2, KCNC1, KCNC2, KCNC3, KCNC4, KCND1, KCND2, KCND3, KCNF1, KCNG1, KCNG2, KCNG3, KCNG4, KCNH 1, KCNH2, KCNH3, KCNH4, KCNH5, KCNH6, KCNH7, KC NH8, KCNQ1, KCNQ2, KCNQ3, KCNQ4, KCNQ5, KCNS1, Includes KCNS2, KCNS3, KCNV1, and KCNV2.

[0200] Non-limiting examples of inwardly rectifying potassium channels include KCNJ1, KCNJ2, KCNJ 3, KCNJ4, KCNJ5, KCNJ6, KCNJ8, KCNJ9, KCNJ10, K CNJ11, KCNJ12, KCNJ13, KCNJ14, KCNJ15, KCNJ16 , and KCNJ18.

[0201] Non-limiting examples of 2P potassium channels include KCNK1, KCNK2, KCNK3, KCNK4, KCNK5, KCNK6, KCNK7, KCNK8, KCNK9, KCNK10, KCNK11, KCNK22, KCNK33, KCNK12, KCNK13, KCNK14, KCNK15, KCNK16, KCNK17, KCNK18, KCNK1 NK4, KCNK5, KCNK6, KCNK7, KCNK9, KCNK10, KCNK1 2, KCNK13, KCNK15, KCNK16, KCNK17, and KCNK18 include.

[0202] Non-limiting examples of hydrogen voltage-gated ion channels include HVCN1.

[0203] Non-limiting examples of ionotropic 5-HT (serotonin) receptors include HTR3A, HT Includes R3B, HTR3C, HTR3D, and HTR3E.

[0204] Non-limiting examples of nicotinic acetylcholine receptors include CHRNA1, CHRNA2, C HRNA3, CHRNA4, CHRNA5, CHRNA6, CHRNA7, CHRNA9 , CHRNA10, CHRNB1, CHRNB2, CHRNB3, CHRNB4, CHR Includes ND, CHRNE, and CHRNG.

[0205] Non-limiting examples of GABA(A) receptors include GABRA1, GABRA2, and GABRA3. , GABRA4, GABRA5, GABRA6, GABRB1, GABRB2, GABR B3, GABRD, GABRE, GABRG1, GABRG2, GABRG3, GABR P, GABRQ, GABRR1, GABRR2, and GABRR3.

[0206] Non-limiting examples of ionotropic glutamate receptors include GRIA1, GRIA2, G RIA3, GRIA4, GRID1, GRID2, GRIK1, GRIK2, GRIK3 , GRIK4, GRIK5, GRIN1, GRIN2A, GRIN2B, GRIN2C, Includes GRIN2D, GRIN3A, and GRIN3B.

[0207] Non-limiting examples of glycine receptors include GLRA1, GLRA2, GLRA3, and GL Includes RA4.

[0208] Non-limiting examples of ionotropic purinergic receptors include P2RX1, P2RX2, P2RX 3, including P2RX4, P2RX5, P2RX6, and P2RX7.

[0209] A non-limiting example of a zinc-activated channel includes ZACN.

[0210] Non-limiting examples of acid-sensitive (proton-gated) ion channels include ASIC1, ASIC 2, including ASIC3 and ASIC4.

[0211] Non-limiting examples of aquaporins include AQP1, AQP2, AQP3, AQP4, AQP5. , AQP6, AQP7, AQP8, AQP9, AQP10, AQP11, AQP12A, AQP12B, and MIP.

[0212] Non-limiting examples of voltage-sensitive chloride channels include CLCN1, CLCN2, CLCN 3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, and CLCN Includes KB.

[0213] Non-limiting examples of cystic fibrosis transmembrane conductance regulators include CFTR.

[0214] Non-limiting examples of calcium-activated chloride channels (CaCCs) include ANO1, AN O2, ANO3, ANO4, ANO5, ANO6, ANO7, ANO8, ANO9, AN Includes O10, BEST1, BEST2, BEST3, and BEST4.

[0215] Non-limiting examples of chloride intracellular channels include CLIC1, CLIC2, CLIC3, Includes CLIC4, CLIC5, and CLIC6.

[0216] Non-limiting examples of gap junction proteins (connexins) include GJA1, GJA3, GJA4, GJA5, GJA6, GJA7, GJA8, GJA9, GJA10, GJA11, GJA12, GJA13, GJA14, GJA15, GJA16, GJA17, GJA18, GJA19, GJA110, GJA111, GJA122, GJA133, GJA1 A4, GJA5, GJA6P, GJA8, GJA9, GJA10, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC1, GJC2, GJC3, These include GJD2, GJD3, GJD4, and GJE1.

[0217] Non-limiting examples of IP3 receptors include ITPR1, ITPR2, and ITPR3.

[0218] Non-limiting examples of pannexins include PANX1, PANX2, and PANX3.

[0219] Non-limiting examples of ryanodine receptors include RYR1, RYR2, and RYR3.

[0220] A non-limiting example of a non-selective sodium leak channel includes NALCN.

[0221] Non-limiting examples of non-voltage-gated sodium channels include SCNN1A, SCNN1B, SCNN1C, SCNN1D, SCNN1E, SCNN1F, SCNN1G, SCNN1H, SCNN1I, SCNN1I, SCNN1IH ... Includes CNN1D, and SCNN1G.

[0222] Solute carrier proteins: In certain aspects, the complexes for use in the methods provided herein comprise a lipid and and a polynucleotide (e.g., mRNA) encoding the target protein, Solute carrier proteins transport solutes from one side of a lipid membrane to the other. These are integral membrane proteins characterized by their ability to transport , secondary active transporters that move solutes against their electrochemical gradients, and There are over 300 types of solute transporters, including facilitative transporters that move solutes in the direction of their electrochemical gradient. The 52 families, which contain proteins that Non-limiting example members include: (1) The high affinity glutamate and neutral amino acid transporter family, including but not limited to Examples are SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SL including C1A6, and SLC1A7; (2) The facilitative glucose transporter (GLUT) family, a non-limiting example of which is SL C2A1, SLC2A2, SLC2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC including 2A12, SLC2A13, and SLC2A14; (3) Large subunits of heterodimeric amino acid families, a non-limiting example being S Contains LC3A1 and SLC3A2; (4) The bicarbonate family, examples of which include SLC4A1, SLC4A2, SLC4A3 , SLC4A4, SLC4A5, SLC4A6, SLC4A7, SLC4A8, SLC4 A9, SLC4A10, and SLC4A11; (5) The sodium-glucose cotransporter family, examples of which include SLC5A1, SLC5A2, and SLC5A3. C5A2, SLC5A3, SLC5A4, SLC5A5, SLC5A6, SLC5A7, SLC5A8, SLC5A9, SLC5A10, SLC5A11, and SLC5A12 Includes; (6) Sodium- and chloride-dependent sodium:neurotransmitter cotransporter family Examples are SLC6A1, SLC6A2, SLC6A3, SLC6A4, SLC6 A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, SLC6A10, S LC6A11, SLC6A12, SLC6A13, SLC6A14, SLC6A15, S LC6A16, SLC6A17, SLC6A18, SLC6A19, and SLC6A2 Includes 0; (7) Cationic amino acid transporter / glycoprotein-related family, including, but not limited to, (i) cationic amino acid transporters (SLC7A1, SLC7A2, SLC7A3, S LC7A4) and (ii) glycoprotein-associated / small or heterodimeric amino acid transporters Catalytic subunits (SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC 7A9, SLC7A10, SLC7A11, SLC7A13, SLC7A14); (8) The Na+ / Ca2+ exchanger family, a non-limiting example of which is SLC8A1 , SLC8A2, and SLC8A3; (9) The Na+ / H+ exchanger family, non-limiting examples of which include SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SLC9A10, SLC9A11, SLC9A12, SLC9A13, SLC9A14, SLC9A15, SLC9A16, SLC9 LC9A2, SLC9A3, SLC9A4, SLC9A5, SLC9A6, SLC9A7 , SLC9A8, SLC9A9, SLC9A10, SLC9A11, SLC9B1, and and SLC9B2; (10) The sodium bile salt cotransporter family, a non-limiting example of which is SLC10A 1, SLC10A2, SLC10A3, SLC10A4, SLC10A5, SLC10A 6, and SLC10A7; (11) The proton-coupled metal ion transporter family, a non-limiting example of which is SLC including 11A1 and SLC11A2; (12) The electroneutral cation-Cl cotransporter family, a non-limiting example of which is SL C12A1, SLC12A1, SLC12A2, SLC12A3, SLC12A4, SL C12A5, SLC12A6, SLC12A7, SLC12A8, and SLC12A9 Includes; (13) The Na+-SO42- / carboxylate cotransporter family, non-limiting examples of which are , SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC1 Including 3A5; (14) The urea transporter family. Non-limiting examples include SLC14A1 and SLC Includes 14A2; (15) The proton oligopeptide cotransporter family, a non-limiting example of which is SLC including SLC15A1, SLC15A2, SLC15A3, and SLC15A4; (16) The monocarboxylate transporter family, non-limiting examples of which include SLC16A1, SLC16A2, SLC16A3, SLC16A4, SLC16A5, SLC16A6, SLC16A7, SLC16A8, SLC16A9, SLC16A10, SLC16A1 1, containing SLC16A12, SLC16A13, and SLC16A14; (17) The vesicular glutamate transporter family, a non-limiting example of which is SLC17A1 , SLC17A2, SLC17A3, SLC17A4, SLC17A5, SLC17A6 , including SLC17A7, SLC17A8, and SLC17A9; (18) The vesicular amine transporter family, non-limiting examples of which include SLC18A1, SL including C18A2, and SLC18A3; (19) The folate / thiamine transporter family, non-limiting examples include SLC19A1, including SLC19A2, and SLC19A3; (20) The type III Na+-phosphate cotransporter family, a non-limiting example of which is SLC Includes 20A1 and SLC20A2; (21) The organic anion transporter family, non-limiting examples of which include: (i) the subfamily -1 SLCO1A2, SLCO1B1, SLCO1B3, and SLCO1C1; (i) subfamily 2, which includes SLCO2A1 and SLCO2B1; (iii) subfamily Family 3, SLCO3A1; (iv) Subfamily 4, SLCO4A1, SLCO4 C1; (v) subfamily 5, SLCO5A1; and (vi) subfamily 6, Contains SLCO6A1; (22) The organic cation / anion / zwitterion transporter family, including, but not limited to, are SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A 5, SLC22A6, SLC22A7, SLC22A8, SLC22A9, SLC22A 10, SLC22A11, SLC22A12, SLC22A13, SLC22A14, S LC22A15, SLC22A16, SLC22A17, SLC22A18, SLC22 A18AS, SLC22A19, SLC22A20, SLC22A23, SLC22A2 4, including SLC22A25, and SLC22A31; (23) The Na+-dependent ascorbic acid transporter family, a non-limiting example of which is SL including C23A1, SLC23A2, SLC23A3, and SLC23A4; (24) The Na+ / (Ca2+-K+) exchanger family, non-limiting examples of which include: SLC24A1, SLC24A2, SLC24A3, SLC24A4, SLC24A5, and SLC24A6; (25) The mitochondrial carrier family, a non-limiting example of which is SLC25A1 , SLC25A2, SLC25A3, SLC25A4, SLC25A5, SLC25A6 , SLC25A7, SLC25A8, SLC25A9, SLC25A10, SLC25A 11, SLC25A12, SLC25A13, SLC25A14, SLC25A15, S LC25A16, SLC25A17, SLC25A18, SLC25A19, SLC25 A20, SLC25A21, SLC25A22, SLC25A23, SLC25A24, SLC25A25, SLC25A26, SLC25A27, SLC25A28, SLC2 5A29, SLC25A30, SLC25A31, SLC25A32, SLC25A33 , SLC25A34, SLC25A35, SLC25A36, SLC25A37, SLC 25A38, SLC25A39, SLC25A40, SLC25A41, SLC25A4 2, SLC25A43, SLC25A44, SLC25A45, and SLC25A46 Includes; (26) The multifunctional anion exchanger family, a non-limiting example of which is SLC26 A1, SLC26A2, SLC26A3, SLC26A4, SLC26A5, SLC26 A6, SLC26A7, SLC26A8, SLC26A9, SLC26A10, and S Contains LC26A11; (27) The fatty acid transport protein family, non-limiting examples of which include SLC27A1, SLC27A2, SLC27A3, SLC27A4, SLC27A5, and SLC27 Including A6; (28) The Na+-coupled nucleoside transporter family, a non-limiting example of which is SLC2 including 8A1, SLC28A2, and SLC28A3; (29) The facilitative nucleoside transporter family, a non-limiting example of which is SLC29A 1, containing SLC29A2, SLC29A3, and SLC29A4; (30) Zinc efflux family. Non-limiting examples include SLC30A1, SLC30A 2, SLC30A3, SLC30A4, SLC30A5, SLC30A6, SLC30A 7, including SLC30A8, SLC30A9, and SLC30A10; (31) The copper transporter family, non-limiting examples of which include SLC31A1 and SLC3 Includes 1A2; (32) The vesicular inhibitory amino acid transporter family, a non-limiting example of which is SLC32A Includes 1; (33) The acetyl-CoA transporter family, a non-limiting example of which is SLC33A1 Includes; (34) The type II Na+-phosphate cotransporter family, a non-limiting example of which is SLC3 including 4A1, SLC34A2, and SLC34A3; (35) The nucleoside-sugar transporter family, non-limiting examples of which include: (i) subfamily Millie A, SLC35A1, SLC35A2, SLC35A3, SLC35A4, SLC 35A5; (ii) Subfamily B, SLC35B1, SLC35B2, SLC35B 3, SLC35B4; (iii) subfamily C, SLC35C1, SLC35C2; (iv) Subfamily D, SLC35D1, SLC35D2, SLC35D3; (v) Subfamily E, SLC35E1, SLC35E2, SLC35E3, SLC35E4 (vi) Subfamily F, SLC35F1, SLC35F2, SLC35F3, SL C35F4, SLC35F5; (vii) subfamily G, SLC35G1, SLC3 Including 5G3, SLC35G4, SLC35G5, SLC35G6; (36) The proton-coupled amino acid transporter family, a non-limiting example of which is SLC3 including 6A1, SLC36A2, SLC36A3, and SLC36A4; (37) The sugar-phosphate / phosphate exchanger family, a non-limiting example of which is SLC3 including SLC37A1, SLC37A2, SLC37A3, and SLC37A4; (38) System A and N sodium-coupled neutral amino acid transporter family, Non-limiting examples include SLC38A1, SLC38A2, SLC38A3, SLC38A4, SLC38A5, SLC38A6, SLC38A7, SLC38A8, SLC38A9, Including SLC38A10, and SLC38A11; (39) The metal ion transporter family, non-limiting examples of which include SLC39A1, SL C39A2, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SL C39A7, SLC39A8, SLC39A9, SLC39A10, SLC39A11, Contains SLC39A12, SLC39A13, and SLC39A14; (40) The basolateral iron transporter family, a non-limiting example includes SLC40A1; (41) The MgtE-like magnesium transporter family, a non-limiting example of which is SLC4 including 1A1, SLC41A2, and SLC41A3; (42) The ammonia transporter family, non-limiting examples of which include RhAG, RhBG and and RhCG; (43) The Na+-dependent system L-like amino acid transporter family, non-limiting examples of which include , including SLC43A1, SLC43A2, and SLC43A3; (44) The choline-like transporter family, non-limiting examples of which include SLC44A1, SLC including SLC44A2, SLC44A3, SLC44A4, and SLC44A5; (45) The putative sugar transporter family, non-limiting examples of which include SLC45A1, SL including C45A2, SLC45A3, and SLC45A4; (46) The folate transporter family, non-limiting examples of which include SLC46A1, SLC46 A2, and SLC46A3; (47) The multidrug and toxin efflux family, non-limiting examples of which include SLC47A1 and and SLC47A2; (48) The heme transporter family, a non-limiting example includes HCP-1; (49) The major facilitator transporter superfamily, a non-limiting example of which is MFSD 5 and MFSD10; (50) The SWEET family of sugar efflux transporters, a non-limiting example of which is SLC50A Includes 1; (51) Transporters of steroid-derived molecules, non-limiting examples include OSTα and OS Contains Tβ; (52) Riboflavin transporter family RFVT / SLC52, non-limiting examples of which include , SLC52A1, SLC52A2, and SLC52A3.

[0223] Difficult to express target proteins: In certain embodiments, antibodies can be produced using conventional methods, such as purified recombinant proteins. Immunizing animals with proteins is not effective for target proteins that are difficult to express. Many factors can cause a host production cell to fail to overexpress / purify a protein. Cytotoxicity in the presence of cytotoxic agents; inherently poor biophysical properties (e.g., size, solubility, Conversion, post-translational modifications (e.g., glycosylation), etc., can contribute to expression problems. Non-limiting examples of difficult protein characteristics include large proteins (e.g., molecular weight ≥ 150k). proteins with specific post-translational modifications, or poorly soluble proteins, unstable proteins, and secreted proteins that do not contain signal peptides. Proteins, membrane-bound proteins, intrinsically disordered proteins, and proteins with short half-lives Including, but not limited to:

[0224] Soluble targets, which may be difficult to express, for use in the methods provided herein Non-limiting examples of target proteins include ADAMTS7, ANGPTL3, , ANGPTL4, ANGPTL8, LPL, GDF15, Galectin-1, Galectin -2, galectin-3, matrix gla protein (MGP), PRNP, DGAT 1, GPAT3, DMC1, BLM, and BRCA2.

[0225] Characteristics of difficult-to-express proteins can be characterized using methods described in the art, e.g., solubility, Evaluation methods (e.g., dynamic light scattering, liquid chromatography mass spectrometry), stability evaluation methods ( For example, differential scanning fluorimetry, differential scanning calorimetry, circular dichroism, NMR, and chromatographic methods. In certain embodiments, difficult to express proteins can be evaluated using chromatographic techniques. Proteins may be stored in solution at room temperature or at 4°C for more than a week, several weeks, a month, or several months. For a period of more than one month (e.g., 3, 4 or 5 months), or more than 6 months or one year If present, more aggregation (e.g., at least 5% aggregation, at least 10% aggregation, At least 20% aggregation, at least 30% aggregation, at least 40% aggregation, at least The aggregates may be more susceptible to aggregation (at least 50%, or at least 60%).

[0226] In certain embodiments, the difficult to express protein has a positive charge. In certain embodiments, the difficult to express protein has a negative charge. It is water-based.

[0227] In certain embodiments, difficult to express proteins have a short half-life, e.g., less than 24 hours. Less than 20 hours, Less than 15 hours, Less than 12 hours, Less than 10 hours, Less than 8 hours, Less than 6 hours, It has a half-life of less than 4 hours, less than 2 hours, or less than 1 hour.

[0228] In certain embodiments, the target proteins described herein are positively charged proteins. , negatively charged proteins, hydrophobic proteins, and glycoproteins.

[0229] In certain embodiments, the target proteins described herein are secreted enzymes and membrane-bound. Contains enzymes such as enzymes.

[0230] II. Cationic Liposomes Any of the cationic lipids known in the prior art may be used in the practice of the claimed invention. For example, Felgner et al. (Proc. Natl. Acad.Sci.USA84:7413-7417(1987));Felgn er et al.(Focus 11:21-25(1989));Felgner( “Cationic Liposome-Mediated Transfection with Lipofectin™ Reagent,” in Gene Trans Answer and Expression Protocols Vol.7,Mu rray,EJ,Ed.,Humana Press,New Jersey,pp. .81-89(1991);International Publication No. 91 / 17424 Pamphlet;International Publication No. 91 / 16024; U.S. Pat. No. 4,897,355; U.S. Pat. No. 4,946,787; U.S. Pat. No. 5,049,386; U.S. Pat. No. 208,036; Behr et al. (Proc.Natl.Acad.S ci.USA 86:6982-6986(1989);EPO Publicatio n 0 394 111);Gao et al.(Biochim.Biophys. Res.Comm.179:280-285(1991));Zhou et al., (Biochim. Biophys. Res. Comm. 165:8-14(1991) ); and Gebeychu et al. (co-owned U.S. Patent Application No. 07 / 937,50 No. 8, filed August 28, 1992, the entire contents of which are incorporated by reference. is embedded in a surface area.

[0231] Lipids that can be used in the methods and compositions provided herein (e.g., Non-limiting examples of lipids include cationic lipids, neutral lipids, helper lipids, and stealth lipids. are disclosed in International Publication No. 2016 / 037053 and International Publication No. 2016 / 0108 Brochure No. 40, International Publication No. 2015 / 095346, International Publication No. Brochure No. 015 / 095340, Brochure No. WO 2016 / 037053 , International Publication No. 2014 / 136086, and International Publication No. 2011 / 0 No. 76807, each of which is incorporated herein by reference in its entirety. In certain aspects, cationic lipids suitable for the methods described herein are contains lipid A, lipid B and lipid C, which have the following chemical structures (see International Publication No. 2015 / 095346 and International Publication No. 2015 / 095340 (This is described in further detail in the brochure no. [ka]

[0232] In certain embodiments, cationic lipids for the compositions and methods described herein include is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N, N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-( 2,3-Dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride DOTAP, 1,2-dioleoyl-3-dimethylammonium-propane (DO DAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl Dioleoyl ammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3-dimethyl Dimethylammonium-propanediol (DOCDAP), 1,2-diaminetetraacetic acid monium-propane (DLINDAP), dilauryl (C 12:0 ) Trimethylammonium DLTAP, dioctadecylamidoglycylspermine (DOGS), DC-Chol, Dioleoyloxy-N-[2-sperminecarboxamido)ethyl} -N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide 3-Dimethylamino-2-(cholest-5-ene-3-β-oxidase) Cibutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane CLinDMA, N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 2-[5'-(cholest-5-en-3[β]-oxy)-3'-oxy sapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoside CpLinDMA and N,N-dimethyl-3,4-dioleyloxy Benzylamine (DMOBA) and 1,2-N,N'-dioleylcarbamyl-3 -Dimethylaminopropane (DOcarbDAP) In one embodiment, the cationic lipid for the compositions and methods provided herein is , DOTAP or DLTAP.

[0233] In certain embodiments, the cationic lipid for the compositions and methods described herein is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium DOTMA alone or dioleoylphosphatidyl phosphate (DOTMA) is also used. In a 1:1 combination with diethyl ethanolamine (DOPE), using standard techniques It can be formulated into liposomes. DOTMA:DOPE (1:1) formulations are suitable for LIP OFECTIN(TM)(GIBCO / BRL:Life Technologies, The product is sold under the name of Epson Corporation, Gaithersburg, Md. In this case, the commercially available cationic lipid is 1,2-bis(oleoyloxy)-3-(2-hydroxyethyl)-2,3-dimethyl-2,4-diphenyl-2,4-diol. -(trimethylammonia)propane (DOTAP), which is D in that it is bonded to the propylamine via an ester bond rather than an ether bond. Different from OTMA.

[0234] In certain embodiments, the cationic lipids for the compositions and methods described herein are The linked group is a trimethylammonium group in which one of the methyl groups is substituted by a hydroxyethyl group. This class of compounds differs from DOTMA and DOTAP in that they are substituted. Rosenthal Inhibitor of Spholipase A (Rosenthal et al., supra) which has a stearoyl ester attached to a propylamine core. The dioleoyl analogue of the Rosenthal inhibitor (RI) is a propyl aryl group in the fatty acid moiety. Depending on the bond to the amine core, they are commonly referred to as DORI ethers and DORI esters, abbreviated as DORI ethers and DORI esters. The hydroxy group can be used as a site for further functionalization, e.g., carboxyl group. It can be used by esterification to permine.

[0235] In certain embodiments, the cationic lipids for the compositions and methods described herein are Another class contains carboxyspermine bound to two lipids, 5-carboxylspermine, This results in permylglycine octadecylamide (DOGS). DOGS is a TRANS It is commercially available as FECTAM™ (Promega, Madison, Wis.). It is possible to use.

[0236] Another class of known compounds is described by Behr et al. (Proc. Natl. Acad. Sci. U SA 86:6982-6986(1989);EPO Publication 0 394 111), in which carboxyspermine is a component of two lipid Dipalmitoylphosphatidylethanolamine 5-carboxylate is bound to the substrate. This results in permylamide (DDPES).

[0237] In certain aspects, another cationic lipid for the compositions and methods described herein is Cholesterol derivatives synthesized in combination with DOPE and formulated into liposomes ( In another specific embodiment, the compositions and methods described herein are The cationic lipid for is lipopolylysine, which is polylysine bound to DOPE. It is formed by:

[0238] Further non-limiting examples of cationic lipids for the compositions and methods provided herein include: Examples include those described below and in WO 2015 / 095346 : 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa- 3-Azaoctadecan-18-yl)propane-1,3-diyldioctanoate; 2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2 -Azaheptadecan-17-yl)propane-1,3-diyldioctanoate; 2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2 -Azapentadecan-15-yl)propane-1,3-diyldioctanoate; 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa- 3-Azahexadecan-16-yl)propane-1,3-diyldioctanoate; 2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2 -Azaheptadecan-17-yl)propane-1,3-diyldioctanoate; 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa- 3-Azanonadecan-19-yl)propane-1,3-diyldioctanoate; 2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2 -Azaoctadecan-18-yl)propane-1,3-diyldioctanoate; 2-(8-dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2 -Azaoctadecan-18-yl)propane-1,3-diyldioctanoate; 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa- 3-Azaicosan-20-yl)propane-1,3-diyldioctanoate; 2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2 -Azanonadecan-19-yl)propane-1,3-diyldioctanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 4, 4-Bis(octyloxy)butanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 4, 4-Bis((2-ethylhexyl)oxy)butanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 4, 4-Bis((2-propylpentyl)oxy)butanoate; 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadeca Sil 4,4-bis((2-propylpentyl)oxy)butanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 4, 4-Bis((2-propylpentyl)oxy)butanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6, 6-Bis(octyloxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6, 6-Bis(hexyloxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6, 6-Bis((2-ethylhexyl)oxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 8, 8-Bis(hexyloxy)octanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 8, 8-Dibutoxyoctanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 8, 8-Bis((2-propylpentyl)oxy)octanoate; 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadeca Sil 8,8-bis((2-propylpentyl)oxy)octanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 8, 8-Bis((2-propylpentyl)oxy)octanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 3- Octylundecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 3- Octylundec-2-enoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 7- Hexyltridec-6-enoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 9- Pentyltetradecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 9- Pentyltetradec-8-enoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 5- heptyldodecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)tridecyl 5-hexyl Petit Dodecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)undecyl 5-hexyl Petit Dodecanoate; 1,3-Bis(octanoyloxy)propan-2-yl(3-(((2-(dimethyla Amino)ethoxy)carbonyl)oxy)pentadecyl)succinate; 1,3-Bis(octanoyloxy)propan-2-yl(3-(((3-(dimethyla Amino)propoxy)carbonyl)oxy)pentadecyl)succinate; 1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl le) 10-octyldecanediate; 1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl le) 10-octyldecanediate; 1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pe 10-octyldecyldecanediate; 1-(3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl l)10-(2-ethylhexyl) decanedioate; 1-(3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pe 10-(2-ethylhexyl)decane diester; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 10 -(octanoyloxy)decanoate; 8-Dodecyl-2-methyl-6,12-dioxo-5,7,11-trioxa-2-aza Nonadecane-19-yldecanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 10 -(octanoyloxy)decanoate; 3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadeca Sil 10-(octanoyloxy)decanoate; (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy )Pentadecyl octadeca-9,12-dienoate; (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy )Pentadecyl octadeca-9,12-dienoate; (9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl )oxy)pentadecyl octadeca-9,12-dienoate; (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy) Pentadecyl octadec-9,12-dienoate; 1-((9Z,12Z)-Octadeca-9,12-dienoyloxy)pentadecane-3 -yl 1,4-dimethylpiperidine-4-carboxylate; 2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl 4, 4-Bis((2-ethylhexyl)oxy)butanoate; (9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propanoyl )Oxy)henicosa-12,15-dien-1-yloctadeca-9,12-dienyl rt; (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa -12,15-dien-1-yl 3-octylundecanoate; (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa -12,15-dien-1-yl 5-heptyldodecanoate; (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa -12,15-dien-1-yl 7-hexyltridecanoate; (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa -12,15-dien-1-yl 9-pentyltetradecanoate; (12Z,15Z)-1-((((9Z,12Z)-Octadeca-9,12-diene-1 -yloxy)carbonyl)oxy)henicosa-12,15-dien-3-yl 3-( Dimethylamino)propanoate; (13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy )docosa-13,16-dien-1-yl 2,2-bis(heptyloxy)acetate; (13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy Docosa-13,16-dien-1-yl 2,2-bis(heptyloxy)acetate ; 2,2-Bis(heptyloxy)ethyl 3-((3-ethyl-10-((9Z,12Z) -Octadeca-9,12-dien-1-yl)-8,15-dioxo-7,9,14- Rioxa-3-azaheptadecan-17-yl)disulfanyl)propanoate; (13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy C) docosa-13,16-dien-1-ylheptadecan-9-yl succinate; (9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propane) 11,14-dien-1-yl)oxy)ethyloctadeca-1-yl)oxy -9,12-dienoate; (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy )-13-(octanoyloxy)tridecyl octadeca-9,12-dienoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octa (noyloxy)tridecyl 3-octylundecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxy Citridecyl 5-heptyldodecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octa (noyloxy)tridecyl 5-heptyldodecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octa (noyloxy)tridecyl 7-hexyltridecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxy cytridecyl 9-pentyltetradecanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octa (noyloxy)tridecyl 9-pentyltetradecanoate; 1-(3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-( Octanoyloxy)tridecyl)10-octyldecane dioate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octa 10-(octanoyloxy)tridecyl 10-(octanoyloxy)decanoate; (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy )-5-Octyltridecyl octadeca-9,12-dienoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltrate Redecyldecanoate; 5-(((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octyl Ntadecyl octanoart; (9Z,12Z)-5-(((3-(dimethylamino)propoxy)carbonyl)oxy )-7-Octylpentadecyloctadeca-9,12-dienoate; 9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octyl nonadecyl octanoate; 9-(((3-(dimethylamino)propoxy)carbonyl)oxy)-11-octyl Nonadecyldecanoate; (9Z,12Z)-9-(((3-(dimethylamino)propoxy)carbonyl)oxy ) nonadecyl octadeca-9,12-dienoate;9-(((3-(dimethylamino) Propoxy)carbonyl)oxy)nonadecylhexanoate; 9-(((3-(dimethylamino)propoxy)carbonyl)oxy)nonadecyl 3-oxy Ctylundecanoate; 9-((4-(dimethylamino)butanoyl)oxy)nonadecylhexanoate; 9-((4-(dimethylamino)butanoyl)oxy)nonadecyl 3-octylundeca Noart; (9Z,9'Z,12Z,12'Z)-2-((4-(((3-(dimethylamino)propionate (oxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl S(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((4-(((3-(diethylamino)propionate (oxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl S(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z,15Z,15'Z)-2-((4-(((3-(di Methylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane -1,3-diylbis(octadeca-9,12,15-trienoate); (Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy) Hexadecanoyl)oxy)propane-1,3-diyl dioleate; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexade (oxy)propane-1,3-diylditetradecanoate; 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexade (oxy)propane-1,3-diylditetradecanoate; 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy) Hexadecanoyl)oxy)propane-1,3-diylditetradecanoate; 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexade (canoyl)oxy)propane-1,3-diyldidodecanoate; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexade (canoyl)oxy)propane-1,3-diyldidodecanoate; 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy) Hexadecanoyl)oxy)propane-1,3-diyldidodecanoate; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexade (canoyl)oxy)propane-1,3-diylbis(decanoate); 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy) Hexadecanoyl)oxy)propane-1,3-diylbis(decanoate); 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexade (canoyl)oxy)propane-1,3-diyldioctanoate; 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy) Hexadecanoyl)oxy)propane-1,3-diyldioctanoate; 2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl 1,3-Dimethyloxy)docosa-13,16-dienoyloxy)propane-1,3-diyldioxy Kutano Art; 2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl 1,3-Dimethyloxy)docosa-13,16-dienoyloxy)propane-1,3-diyldioxy Kutano Art; (9Z,9'Z,12Z,12'Z)-2-((2-(((3-(diethylamino)propionate (oxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diylbis S(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((2-(((3-(dimethylamino)propionate (oxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diylbis( octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((2-(((3-(dimethylamino)propionate (oxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diylbis S(octadeca-9,12-dienoate); (9Z,9'Z,12Z,12'Z)-2-((2-(((3-(diethylamino)propionate (oxy)carbonyl)oxy)dodecanoyl)oxy)propane-1,3-diylbis( octadeca-9,12-dienoate); 2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecane (canoyl)oxy)propane-1,3-diyldioctanoate; 4.4-Bis(octyloxy)butyl 4-(((3-(dimethylamino)propoxy) Carbonyl)oxy)hexadecanoate; 4,4-Bis(octyloxy)butyl 2-(((3-(diethylamino)propoxy) Carbonyl)oxy)dodecanoate; (9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-o CUTADECA-9,12-DIENOYLOXY)ETHYL)-8,13-DIOXO-7,9-DIOXO 3,14-diazahexadecan-16-yl octadec-9,12-dienoate ; 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)-11- (Octanoyloxy)undecanoyl)oxy)propane-1,3-diyldioctanoyl art; (9Z,9'Z,12Z,12'Z)-2-(9-dodecyl-2-methyl-7,12-di Oxo-6,8,13-trioxa-2-azatetradecane-14-yl)propane-1 ,3-diylbis(octadeca-9,12-dienoate); 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 4, 4-Bis(octyloxy)butanoate; 3-(((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate; 3-(((3-(piperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate; 3-(((4-(diethylamino)butoxy)carbonyl)oxy)pentadecyl 6,6 -Bis(octyloxy)hexanoate; 3-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy) Pentadecyl 6,6-bis(octyloxy)hexanoate; 3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)penta Decyl 6,6-bis(octyloxy)hexanoate; 3-(((3-morpholinopropoxy)carbonyl)oxy)pentadecyl 6,6-bis (Octyloxy)hexanoate; 3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecyl 6,6 -Bis(octyloxy)hexanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 6, 6-Bis(octyloxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6, 6-Bis((2-propylpentyl)oxy)hexanoate; 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 6, 6-Bis((2-propylpentyl)oxy)hexanoate LXR420:3-(( (3-(Dimethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6-bis ((3-ethylpentyl)oxy)hexanoate; (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadeca 1-methylpyrrolidine-2-carboxylate; (2S)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadeca 1-methylpyrrolidine-2-carboxylate; (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadeca benzoylpyrrolidine-2-carboxylate; 1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1,3-dimethylpyrrolidine-3-carboxylate; 3-((3-(1-methylpiperidin-4-yl)propanoyl)oxy)pentadecyl 6,6-Bis(octyloxy)hexanoate; 1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1,4-dimethylpiperidine-4-carboxylate; 3-((5-(diethylamino)pentanoyl)oxy)pentadecyl 6,6-bis(oxy) octyloxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 5- (4,6-diheptyl-1,3-dioxan-2-yl)pentanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl 6,6 -Bis(octyloxy)hexanoate; 3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl 6,6 -Bis(octyloxy)hexanoate; (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy Henicosa-12,15-dien-1-yl 6,6-bis(octyloxy)hexa Noart; 6-((6,6-bis(octyloxy)hexanoyl)oxy)-4-(((3-(di Ethylamino)propoxy)carbonyl)oxy)hexyl octanoate; 4,4-Bis(octyloxy)butyl 5-(((3-(diethylamino)propoxy) Carbonyl)oxy)heptadecanoate; 4,4-Bis(octyloxy)butyl(3-(diethylamino)propyl)pentadeca 1,3-diyl dicarbonate; 2-(5-((4-((1,4-dimethylpiperidine-4-carbonyl)oxy)hexa Decyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate; 2-(5-((4-((1,3-dimethylpyrrolidine-3-carbonyl)oxy)hexa Decyl)oxy)-5-oxopentyl)propane-1,3-diyldioctanoate; 2-(5-oxo-5-((4-(((S)-pyrrolidine-2-carbonyl)oxy)he (xadecyl)oxy)pentyl)propane-1,3-diyldioctanoate; 2-(5-((4-(((((S)-1-methylpyrrolidin-3-yl)oxy)carbo 1,3-diphenyloxy)hexadecyloxy)-5-oxopentylpropane-1,3-diyl Ludio Kutano Art; 2-(5-((4-(((((R)-1-methylpyrrolidin-3-yl)oxy)carbo 1,3-diphenyloxy)hexadecyloxy)-5-oxopentylpropane-1,3-diyl Ludio Kutano Art; 2-(5-((4-((((1-ethylpiperidin-3-yl)methoxy)carbonyl) Oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyldioxy Kutano Art; 2-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy 5-Oxopentyl)propane-1,3-diyldioxadecyloxy Tanoart; 2-(10-dodecyl-3-ethyl-8,15-dioxo-7,9,14-trioxa- 3-Azanonadecan-19-yl)propane-1,3-diyldioctanoate; 2-(11-dodecyl-3-ethyl-9,15-dioxo-8,10,14-trioxa -3-Azanonadecan-19-yl)propane-1,3-diyldioctanoate; 2-(5-((3-(((3-(1H-imidazol-yl)propoxy)carbonyl) Oxy)pentadecyl)oxy)-5-oxopentyl)propane-1,3-diyldi Kutano Art; 2-(5-oxo-5-((3-(((3-(piperidin-1-yl)propoxy)carbamoyl (oxy)pentadecyl)oxy)pentyl)propane-1,3-diyldioctadecyl and 2-(12-dodecyl-3-ethyl-8,14-dioxo-7,9,1 3-Trioxa-3-azaoctadecan-18-yl)propane-1,3-diyldioxa Tanoart.

[0239] In certain embodiments, these cationic lipid compounds may be present alone or in other lipid aggregate forms. Lipoproteins, either in combination with other components (such as DOPE or cholesterol), These are useful for formulation into cationic lipid aggregates. It is anionic and can form complexes with anionic macromolecules such as DNA or RNA. do.

[0240] "Neutral lipids" suitable for use in the lipid compositions and methods described herein include, for example, Suitable lipids for use in the present invention include a variety of neutral lipids, uncharged lipids, or zwitterionic lipids. An example of a neutral phospholipid is 5-heptadecylbenzene-1,3-diol (resorcinol). dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), Dimyristoyl phosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoyl phosphatidylcholine DMPC, phosphatidylcholine (PLPC), 1,2-distearoyl-sn- Glycerol-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), Egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), Dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl Phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine 1-palmitoyl-2-stearoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine ( PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC) , 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-di Eicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyl oleoyl lysophosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylcholine DOPE, dilinoleoylphosphatidylcholine, disodium DSPE, dimyristoyl phosphatidylethanolamine Dipalmitoylethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (D PPE), palmitoyloleoylphosphatidylethanolamine (POPE), lyso Phosphatidylethanolamine, and combinations thereof. In one embodiment, the neutral phospholipid is distearoylphosphatidylcholine (DSPC). ) and dimyristoylphosphatidylethanolamine (DMPE). It is selected.

[0241] "Helper lipids" act to some degree to facilitate transfection (e.g., delivery of biologically active agents). The helper lipid is a lipid that enhances the transfection of nanoparticles containing Mechanisms that enhance infection include, for example, increasing particle stability and / or membrane fusion. Helper lipids may include steroids and alkylresorcinols. Suitable helper lipids for the compositions and methods described herein include cholesterol. , 5-heptadecylresorcinol, and cholesterol hemisuccinate, Non-limiting examples of helper lipids for the compositions and methods described herein include: Examples of such methods include International Publication No. 2015 / 095346 and International Publication No. / 095340 Brochure, International Publication No. 2016 / 037053 Brochure, Country International Publication No. 2014 / 136086 and International Publication No. 2011 / 0768 No. 5,970,633, each of which is incorporated herein by reference in its entirety. Be absorbed.

[0242] The stealth lipids allow the nanoparticles to persist in vivo (e.g., in the blood) for a certain period of time. Stealth lipids suitable for the compositions and methods described herein are: Stealth lipids include, but are not limited to, stealth lipids having a hydrophobic head group linked to a lipid moiety. Non-limiting examples of stealth lipids for the compositions and methods described herein include the following: International Publication No. 2015 / 095346 Brochure, International Publication No. 2015 / 095340 Brochure, International Publication No. 2016 / 037053 Brochure, International Publication No. 2014 / 136086 Brochure and International Publication No. 2011 / 076807 Brochure , each of which is incorporated herein by reference in its entirety. In certain embodiments, exemplary stealth lipids are described in WO 2011 / 076807. and the compound of formula (XI) according to WO 2016 / 010840 In certain embodiments, the lipid compositions described herein include those listed in Table 1. Other stealth lipids suitable for use, and information on the biochemistry of such lipids, are available from Ro mberg et al.,Pharmaceutical Research,Vol. 25, No. 1, 2008, pp. 55-71 and Hoekstra et al. Biochimica et Biophysica Acta 1660(2004) 41-52.

[0243] In one aspect, a suitable stealth lipid is PEG, also known as poly(ethylene oxide). In some cases, poly(oxazoline), poly(vinyl alcohol), poly(glycerol), etc. rol), poly(N-vinylpyrrolidone), polyamino acids and poly[N-(2-hydropyrrolidone)] and polymers based on dimethylaminopropyl (dimethylaminopropyl)methacrylamide, as well as polymers based on dimethylaminopropyl (dimethylaminopropyl)methacrylamide, as described, for example, in WO 2004 / 023666. Further suitable PEG lipids are selected from those disclosed in US Pat. No. 5,977,333. This includes groups that can be

[0244] In certain embodiments, non-limiting examples of suitable stealth lipids include those having a molecular weight of about C4 to about C 40 Saturation or Dialkylglycerols or dialkylglycerols having independently alkyl chain lengths containing unsaturated carbon atoms. Polyethylene glycol-diacylglycerols, including those containing alkylglycamide groups or polyethylene glycol-diacylglycamide (PEG-DAG) conjugates In a further embodiment, the dialkyl glycerol or dialkyl glycamide group is Additionally, it may include one or more substituted alkyl groups. Further embodiments described herein In this study, the PEG conjugates were PEG-dilaurylglycerol, PEG-dimyristyl PEG-DMG (NOF catalog number GM-020, Tokyo, Japan) , PEG-dipalmitoyl glycerol, PEG-distearyl glycerol, PEG- Dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide glycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'- (Cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaoxa dimethyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N-[Methoxy(polyethylene glycol)-2000] (Avanti Polar Lipids Catalog Number 880150P, Alabaster, Alabama, USA SA). In one embodiment, the stealth lipid can be selected from S010, S024, S027, S031, or S033 (International Publication No. 2016 / 037053) In another embodiment, the stealth lipid is S024 (International Publication No. 2003 / 0133662, e.g., as described in Table 1). No. 2016 / 037053, for example as described in Table 1).

[0245] III. Composition of mRNA Molecules In certain embodiments, the polynucleotide used for the immunization method of the invention is , polyribonucleotide-based, e.g., mRNA-based. This mRNA molecule directly codes for the translation of a target protein to which an antibody response is desired, and Therefore, the molecule must contain several components. In certain embodiments, the first component is a protein, e.g., a human target protein. It is an open reading frame corresponding to the amino acid sequence of a protein or a fragment thereof. Natural codon sequences may be used, or alternatively, the efficiency of translation and the ultimate target protein may be improved. Codon-optimized for the host species (e.g., mouse or rabbit) to increase expression levels Further modifications may be made to the open reading frame to For secreted or membrane proteins, this may enhance protein expression / transport. This is because the secretion signal from a foreign signal peptide, such as the secretion signal from interleukin 2 (IL-2), In the specific example for a secreted protein, the mRNA molecule may include the use of a human The signal peptides may include heterologous signal peptides such as those of human IL-2 or IgG kappa. That's fine too.

[0246] In certain embodiments, the second component is a consensus Kozak sequence. The DNA sequence provided is GCCACCATG (SEQ ID NO: 1), with the nucleotides AT G represents the initiator methionine. Exemplary Kozak RNA sequences include: GCCACCAUG (SEQ ID NO: 11) is provided, in which the nucleotides AUG represent the start methionine. Other non-limiting examples of sequences include those encoded by either RNA or DNA: (GCC)GCCRCCAUGG (SEQ ID NO: 12), AGNNAUGN (SEQ ID NO: 13) , ANNAUGG (SEQ ID NO: 23), ACCAUGG (SEQ ID NO: 24), GACACCA UGG (SEQ ID NO: 25), GCCRCCATGG (SEQ ID NO: 57), CAAACATG ( SEQ ID NO:58), AAAAAATGTCT (SEQ ID NO:28), AAAAAAATGRNA (SEQ ID NO: 29), NTAAAAATGRCT (SEQ ID NO: 30), TAAAAAATGA AN (SEQ ID NO: 31), GNCAAAATGG (SEQ ID NO: 32), NNNANNATGN C (SEQ ID NO:33), and AACAATGGC (SEQ ID NO:34), " refers to any nucleotide (e.g., A, G, C, or T in the context of DNA, and and in relation to RNA, A, G, C, or U), and "R" stands for A or G. The inclusion of a Kozak sequence 5' of the open reading frame identifies a eukaryotic It is widely known that translation in a biological host is enhanced.

[0247] In certain embodiments, the third component comprises a 7-methylguanosine capsid on the 5' end of the mRNA. This cap is required for the recruitment of eukaryotic initiation factor eIF4E and maturation of the ribosome. Following production of the mRNA transcript, it is enzymatically or chemically methylglucose (MG)-dependent An anosine cap can be added.

[0248] In certain embodiments, the fourth component is a polyadenosyltransferase found on the 3' end of an mRNA transcript. The poly(A) tail extends the half-life of mRNA in the cell. It is known to promote efficient ribosome assembly and protein translation. In certain embodiments, the mRNA for the compositions and methods described herein is In certain embodiments, the polyA tail of nucleotides (SEQ ID NO:59) is The mRNA for the compositions and methods described in is a polynucleotide having 60 to 120 nucleotides. In certain embodiments, the mR NA is 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, PolyA tails of 100, 110, or 120 nucleotides The inclusion of a polyA tract can be achieved via in vitro transcription or by the addition of poly(A) polymerase. This can be achieved by enzymatic polyadenylation using enzymes.

[0249] In certain embodiments, the fifth component of the mRNA molecule comprises 5'- and 3'-untranslated regions (UT In certain embodiments, the 5'UTR is a 5'UTR of the Tobacco Etch Virus (Tob acco etch virus), and the 3'UTR is found in human β-globin. Tandem repeats of the 3'UTR. The presence of UTRs enhances translation of mRNA, It is widely accepted that the half-life of a cytoplasmic agonist can be increased in cells (e.g., For example, R.L. Tanguay and DR Gallie Molecular and d Cellular Biology 1996 vol 16 no1 pp 14 (See 6-156.)

[0250] Sufficient quantities of such RNA molecules can be prepared by in vitro transcription followed by purification of the RNA. It is also possible to use DNA-dependent RNA polymerase to synthesize the cloned DNA sequence. In vitro transcription techniques are well known in the art (e.g., Sambroo k et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor L (See Aboratory Press, 1989.) Uracil, guanine, cytosine Naturally occurring ribonucleotides such as synthase, adenine, pseudouracil, or modified Any of the ribonucleotides that are added will still result in proper codon recognition and protein synthesis. As long as it corresponds to translation, it may be used for mRNA synthesis. Guanine, cytosine, adenine, and pseudouracil were used. By using pseudouracil instead of uracil, the BioAnalyzer This allows for more accurate estimation of mRNA size during quality control evaluation (described below). do.

[0251] IV. Use of Lipid / Polynucleotide Complexes According to the present disclosure, in certain embodiments, lipid / polynucleotide complexes are used to perform in vivo Transfected cells are then transfected with a polynucleotide (e.g. For example, a polyribonucleotide (e.g., mRNA) is used to express a protein encoded by the gene. In this way, the foreign protein can be expressed or displayed, for example, on the cell surface. As a result, the host animal The host animal (e.g., a non-human host animal) mounts an immune response to the foreign protein, or immunogen. Start.

[0252] Synthetic mRNA was synthesized using a plasmid DNA template, rNTPs and T7 RNA polymerase. The 7-methylguanosine cap structure (Cap1) is added to the mR Capped mRNAs are enzymatically added to the 5' end of mRNA to facilitate efficient translation. A was formulated into cationic lipid nanoparticles (LNPs) to protect mRNA from degradation, Enhanced cytoplasmic delivery. mRNA LNPs are stable for 3-4 months at 4°C and are suitable for immunization. In certain embodiments, the cationic lipid-polynucleotide The cationic lipid complexes were prepared by mixing an equal volume of the cationic lipid solution with the polynucleotide solution. The cationic lipids and polynucleotides are dissolved in sterile, physiologically compatible In certain embodiments, the cationic lipid and The polynucleotide is dissolved in sterile saline (150 mM NaCl). are mixed at ambient temperature. In certain embodiments, the solutions are mixed at 25° C. After mixing The cationic lipid-polynucleotide complex is then incubated at room temperature for, for example, 15 to 45 minutes. will be invited.

[0253] Administration of the lipid / polynucleotide complexes of the methods described herein can be parenteral, intravenous, intramuscular, In mice, the administration may be intravenously, subcutaneously, intranasally, or by any other suitable means. Intravenous administration of RNA LNPs was found to be superior to subcutaneous delivery (see Figure 1B). The specific dose to be administered will depend on the age, weight, type of current treatment, if any, and the nature of the immunogen to be expressed. Immunization with mRNA LNPs may also enhance the immunogenic response of other immune cells. The pathogenic morphology may vary (see Figure 1C).

[0254] Since immunization induces the production of immunogen-specific antibodies in the host, the present disclosure also provides The present invention relates to a method for producing an immunogen-specific antibody. Polyclonal antibodies are known in the art. The host cells can be isolated and purified from the host animal using well known methods (e.g., Harvard Medical School, 1999). low et al.,Antibodies:A Laboratory Manual l,Cold Spring Harbor Laboratory Press,19 (See 88.)

[0255] The present disclosure also provides methods for mRNA LNP-based immunization to generate monoclonal antibodies. According to this method, a non-human animal (e.g., a mouse) is subjected to the administration of a lipid / mRNA complex. The body of the immunized animal is then injected with antibodies, producing cells (e.g., B lymphocytes or splenocytes) that produce antibodies. Monoclonal antibodies can be isolated from any of the following known in the art: Any method, such as that described by Kohler and Milstein (Nature 256:49 5-497 (1975) (see, e.g., Harlow et al., supra). In summary, monoclonal antibodies are produced in animals (e.g., mice) according to the method of ) with the cationic lipid-mRNA complex and serum samples were taken to obtain antibodies. The presence of production is confirmed, the spleen is removed to obtain B lymphocytes, and the B lymphocytes are then separated into myeloma cells and The hybridomas are fused to produce hybridomas, which are then cloned to produce anti-immunogen antibodies. Positive clones that produce anti-immunogen antibodies are selected, and the clones that produce anti-immunogen antibodies are cultured. The antibody can be prepared by isolating the antibody from a hybridoma culture.

[0256] V. RNA Modifications Polyribonucleotides, such as mRNA, for the compositions and methods described herein include: Prevents rapid degradation by endonucleases and exonucleases and protects RNA Modifications to avoid or reduce the cellular innate immune or interferon response to Modifications can include, for example, (a) terminal modifications, such as 5' terminal modifications (phosphate 3'-terminal modification (conjugation, dephosphorylation, conjugation, reverse ligation, etc.), ion, DNA nucleotides, reverse bond, etc.), (b) base modifications, e.g. Base pairing with a broad repertoire of bases, stabilizing bases, destabilizing bases, or partners (c) sugar modifications (e.g., 2' (d) modification of the phosphodiester bond or These include, but are not limited to, modifications of the internucleoside linkages, including substitutions.

[0257] In certain embodiments, the polyribonucleotides, such as the mRNAs described herein, further comprise: In some embodiments of the aspects described herein, the modification Synthetic mRNA is a modified form of a nucleic acid sequence that is linked to the 5' end of an RNA molecule using a 5'-5' triphosphate linkage. The term "5' cap" also refers to a 5' cap that contains a guanine nucleotide modified with a 5' cap. , e.g., a 5' diguanosine cap, a tetramer having a methylene-bis(phosphonate) moiety Phosphate cap analogs (e.g., Rydzik, A. M. et al. (2009) O rg Biomol Chem 7(22):4763-76), phosphoro Dinucleotide cap analogs with thioate modifications (e.g., Kowalska, J. et al. (2008) RNA 14(6):1119-1131) , cap analogs with sulfur substitutions for the non-bridging oxygens (e.g., Grudzien -Nogalska, E. et al., (2007) RNA 13(10):1745 -1755), N7-benzylated dinucleoside tetraphosphate analogs (e.g., Grudzien, E. et al. (2004) RNA 10(9):1479-14 87), or anti-reverse cap analogs (e.g., Jemieli ty, J. et al., (2003) RNA 9(9):1108-1122 and S tepinski, J. et al. (2001) RNA 7(10):1486-14 95). In such embodiments, the 5' cap analog is a 5' diguanosine cap. In some embodiments, the modified synthetic mRNA of the invention does not contain a 5' triphosphate.

[0258] The 5' cap is important in the recognition and binding of mRNA to ribosomes to initiate translation. The 5' cap also prevents the modified synthetic mRNA described herein from being 5' exonucleic acid. Protects against ATPase-mediated degradation.

[0259] Polyribonucleotides, such as the mRNAs described herein, may further comprise 5' and / or It can contain a 3' untranslated region (UTR). The untranslated region is located immediately above the initiation codon (5'). The region of RNA downstream of the stop codon (3') and thus the translational apparatus The untranslated regions are involved in ribonucleases and RNA degradation, as well as other The modification of an RNA molecule by one or more untranslated regions can interfere with the function of a protein. In addition, 5' and / or 3' modifications can improve the stability of the mRNA. 'The modification of RNA by the non-translated region results in altered ribosome binding to mRNA. The 3'UTR enhances the translation efficiency of the fusion protein. can be used to maintain the cytoplasmic localization of RNA, thereby In one embodiment, the modified synthetic mRNA of the present invention is provided with a 5' In another embodiment, the modified synthetic mRNA of the present invention does not include a 5' or 3' UTR. In another embodiment, the modified synthetic mRNA of the present invention comprises either a 5' UTR or a 3' UTR. In one embodiment, the 5' and / or 3' UTRs include mRNA known to have high stability in cells (e.g., mouse α-globin In some embodiments, the 5'UTR, 3'UTR, Or both contain one or more modified nucleosides.

[0260] In some embodiments, the polyribonucleotides, such as the mRNAs described herein, In addition, it contains the Kozak sequence. The "Kozak sequence" is the consensus (gcc) gccRcc AUGG (SEQ ID NO: 12), where R is It is a purine (adenine or guanine) three bases upstream of the start codon (AUG). The Kozak consensus sequence is recognized by the ribosome. Translation of a polypeptide is initiated at the ribozyme, usually located at a translation site proximal to the 5' end of the transcript. This occurs at the first AUG codon encountered by the translation apparatus. The G codon can be bypassed in a process called leaky scanning. The presence of a Kozak sequence adjacent to an AUG codon enhances that codon as the initiation site for translation. This results in the translation of the appropriate polypeptide. Even if there is no ambiguity regarding the addition of a Kozak sequence to the modified synthetic mRNA described herein, Addition of the nucleotide sequence can facilitate more efficient translation. In addition, the modified synthetic RNAs described herein can be used to produce polypeptides of appropriate length. Contains a Kozak consensus sequence at the desired site for initiation of translation. In such embodiments, the Kozak sequence comprises one or more modified nucleosides.

[0261] In some embodiments, the modified synthetic mRNA described herein further comprises a "poly(A) nucleotide sequence." The term "tail" refers to a 3' homopolymeric tail of adenine nucleotides, the length of which is can vary (e.g., at least five adenine nucleotides) and may contain hundreds of adenine nucleotides. The inclusion of a 3' poly(A) tail enhances the It can protect the bright modified synthetic mRNA from degradation in cells and promote its extranuclear localization, In some embodiments, the poly(A) tail comprises 1 to 500 amino acids. (SEQ ID NO:60); in other embodiments, the poly(A) tail comprises At least five adenine nucleotides. In one embodiment, the poly(A) tail In one embodiment, the poly(A) tail comprises 1 to 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises 60 to 120 adenine nucleotides. In some such embodiments, the amino acid sequence includes 90 to 120 adenine nucleotides. , the poly(A) tail comprises one or more modified nucleosides.

[0262] The following applies to production and expression according to the mRNA immunization methods provided herein: This is a typical example of a target protein antigen that can be used to Immunization of an individual as described herein demonstrates proof of concept for the method.

[0263] I.RXFP1 RXFP1, or relaxin / insulin-like family peptide receptor 1, is a leukocyte-associated A 757 amino acid class AG protein containing a synrich repeat N-terminal extracellular domain Phylogenetically, it is a GPCR that is a member of the follicle-stimulating hormone receptor, the luteinizing hormone receptor, and the luteinizing hormone receptor. It is a member of the same receptor subfamily that includes the thyrotropin receptor and the thyrotropin receptor. The endogenous ligand for RXFP1 is the protein hormone relaxin. RXFP1 and its ligands mediate physiological responses associated with menstruation, pregnancy, and parturition In patients with acute decompensated heart failure, The Phase III clinical trial (RELAX-AHF) is evaluating recombinant RELAX for 48 hours during hospitalization. These results show that injection of 1,2-dioxin significantly reduced the mortality rate after 6 months.

[0264] Establishing cell lines with high levels of RXFP1 expression is difficult due to cytotoxicity. As with many GPCRs, expression of purified full-length recombinant proteins is also technically feasible. I can't get it out.

[0265] To generate human RXFP1 mRNA, the open reading frame of RXFP1 was cloned. The naturally occurring human nucleotide sequence for 4.3 / NP_067647.2) is the GeneArt® mouse codon Codon optimization was performed using the optimization algorithm (see Table 1). In addition to changing the codon sequence based on the sequence, the sequence was modified so that these were followed by subcloning. and mRNA synthesis, so BamHI, RsrII, and The cDNA was modified to remove the CsDNA3 and BspQI restriction enzyme sites.

[0266] [Table 1]

[0267] [Table 2]

[0268] [Table 3]

[0269] [Table 4]

[0270] [Table 5]

[0271] [Table 6]

[0272] [Table 7]

[0273] [Table 8]

[0274] II.SLC52A2 SLC52A2 (GPR172A) contains 10 or 11 putative transmembrane helices. It is a 445 amino acid multi-spanning membrane protein predicted to have a ribosomal domain. It has been shown to mediate the intracellular uptake of flavin and inhibits the porcine endogenous retrovirus sarcopenia. It has been reported that SLC52A2 is a receptor for group A subunits. Genetic variants are associated with motor, sensory, and cranial neuropathies.

[0275] To generate mRNA for human SLC52A2, we cloned the open reading frame for this protein. The native human nucleotide sequence in the corresponding frame (e.g., accession number NM_0 01253816.1 / NP_001240745) is a registered trademark of GeneArt® Codon optimization was performed using a mouse codon optimization algorithm (see Table 2 In addition to the codon sequence changes based on mouse bias, the sequence is BamH will be used for subcloning and mRNA synthesis. The primers were modified to remove the I, RsrII, and BspQI restriction enzyme sites.

[0276] [Table 9]

[0277] [Table 10]

[0278] [Table 11]

[0279] [Table 12]

[0280] [Table 13]

[0281] III.ANGPTL8 ANGPTL8 is a secreted protein involved in lipid metabolism and is expressed in human plasma as a ng / mL The protein can be found at concentrations as low as 1 / ml. However, in heterologous strains, However, its expression in its native soluble form is difficult. Furthermore, the biochemical function of this protein is unclear. Therefore, it is not possible to measure the activity of recombinantly produced proteins. There is no in vitro functional assay that can be used to determine the function of the antibody. Given the difficulty in verifying the quality of recombinant ANGPTL8 for use in Proteins are good candidates for mRNA-mediated immunization to generate monoclonal antibodies. He was a supplementary member.

[0282] The full length coding sequence of human ANGPTL8 (e.g., accession number NP_06115 7) was codon-optimized for expression in human cells and purified using T7 polymerase. 3) Can maintain RNA transcription and aid in mRNA stability and translatability; The sequences were cloned into a vector containing both the 5′ untranslated region and the 5′ nucleotide sequence (see Table 3 for sequences). (See references). The mRNA was transcribed in vitro as described above and encapsulated in lipid nanoparticles. .

[0283] [Table 14]

[0284] [Table 15]

[0285] [Table 16]

[0286] [Table 17]

[0287] IV.TSHR The thyroid-stimulating hormone receptor (TSHR) regulates thyroid development and thyroid hormone production. It is an essential G protein-coupled receptor. It is also an autoantigen in Graves' disease. Prolonged activation of the TSHR by TSHR-specific antibodies may be the underlying cause of Graves' disease. This is one of the main underlying causes (Davies TF (2015) Expert Op in Ther Targets;19:835-47).

[0288] TSHR has a large extracellular domain (ECD) and a transmembrane domain (TMD). The ECD contains 11 leucine-rich residues that contain the binding sites for TSH and many autoantibodies. TSHR has a repeat domain (LRD). It undergoes extensive post-translational modification and forms homodimers. and can form multimers. TSHR has a low baseline constitutive activity. The signal transduction is mediated by Gs, Gi / o, Gq / 11 or G12 / 13. TSHR is the luteinizing hormone and chorionic gonadotropin receptor. (LHCGR) and 48% identical to the follicle-stimulating hormone receptor (FSHR). It is.

[0289] In the normal thyroid gland, TSH activates the TSHR, which promotes thyroid cell proliferation and thyroid hormone production. In Graves' disease, agonist autoantibodies are produced in patients. These replace TSH and overactivate the receptor in an unregulated manner. That is, the thyroid gland becomes enlarged and levels of T3 and T4 increase.

[0290] The full length coding sequence of human TSHR (e.g., protein accession number NP_000 360.2) was codon-optimized for expression in human cells and engineered with T7 polymerase It can maintain mRNA transcription by the endonuclease and promote mRNA stability and translatability. The sequences were cloned into a vector containing both the 3' and 5' untranslated regions of (See Table 4 for details.) The mRNA was in vitro transcribed as described above and loaded onto lipid nanoparticles. Encapsulated.

[0291] [Table 18]

[0292] [Table 19]

[0293] [Table 20]

[0294] [Table 21]

[0295] [Table 22]

[0296] [Table 23]

[0297] [Table 24]

[0298] V.APJ APJ, also known as angiotensin-like 1 receptor, angiotensin II-like 1 receptor, etc. The apelin receptor, which is cognate to the endogenous ligand apelin, is a previously orphan The apelin / APJ pathway is a G protein-coupled receptor (GPCR) that mediates cardiovascular Apelin is widely expressed in the vascular system and has been shown to play a beneficial role in preclinical models. Acute apelin administration in humans has been shown to have potent cardiovascular effects on peripheral and coronary vasculature. This causes dilation and increases cardiac output (Circulation. 2010;121 :1818-1827). As a result, APJ agonism is of great importance for patients with heart failure. Activation of the apelin receptor APJ has emerged as a promising therapeutic target. It is believed to increase cardiac contractility and provide cardioprotection without the need for vasopressin.

[0299] APJ affects not only the heart but also other organs, including blood vessels, kidneys, liver, adipose tissue, and the brain. It is widely distributed in tissues.

[0300] The full length coding sequence of human APJ (e.g., protein accession number NP_0051 52.1) was codon-optimized for expression in human cells and engineered with T7 polymerase. This allows the maintenance of mRNA transcription by the ribozyme and aids in mRNA stability and translatability. The sequences were cloned into a vector containing both the 3' and 5' untranslated regions (see sequence references). (See Table 5.) The mRNA was in vitro transcribed as described above and attached to lipid nanoparticles. Wrapped it.

[0301] [Table 25]

[0302] [Table 26]

[0303] [Table 27]

[0304] [Table 28]

[0305] [Table 29]

[0306] [Table 30]

[0307] VI.GP130 Glycoprotein 130 (GP130) is a protein containing 918 amino acids. It is a member of the type I single-pass transmembrane protein receptor family. It is involved in the regulation of interleukin-1 (IL-1) receptors. Interleukin-6, interleukin-11, ciliary neurotrophic factor, leukemia inhibitory factor, and oncolytic factor A central component of the signaling complex used by many cytokines, including tatin M. In the case of interleukin 6 (IL6), GP130 binds to the IL6 / IL6R (α chain ) complex, causing the formation of high affinity IL6 binding sites and initiating signal transduction. GP130 contains five type III fibronectin domains and one Ig-like C2 domain. Contains a type domain.

[0308] The full length coding sequence of human GP130 (e.g., protein accession number NP_00 2175.2 or AAI17405) was codon-optimized for expression in human cells, and can maintain mRNA transcription by T7 polymerase and stabilize mRNA. Cloned into a vector that contains both 3 and 5' untranslated regions to aid in quality and translatability. The mRNA was cloned into 100 kDa DNA (see Table 6 for sequences). and encapsulated in lipid nanoparticles.

[0309] [Table 31]

[0310] [Table 32]

[0311] [Table 33]

[0312] [Table 34]

[0313] [Table 35]

[0314] [Table 36]

[0315] [Table 37]

[0316] [Table 38]

[0317] VII. Galectin-3 Galectin-3 is a 26 kDa protein and a β-galactoside-binding lectin. It is a member of the galectin family. It has a collagen-like N-terminal domain and It contains a C-terminal carbohydrate recognition domain that confers the ability to bind the -3 carbohydrate. Through these interactions, galectin-3 can form higher order oligomers. -3 plays a role in cell adhesion, differentiation, metastasis, embryogenesis, inflammation, and fibrogenesis It has been suggested that...

[0318] The full length coding sequence of human galectin-3 (e.g., protein accession number NP_ 002297) was codon-optimized for expression in human cells and expressed using the T7 polymerase It is possible to maintain mRNA transcription by the enzyme and to enhance mRNA stability and translatability. The sequences were cloned into a vector containing both the 3' and 5' untranslated regions (see sequence description). (See Table 7 for details.) The mRNA was transcribed in vitro as described above and injected into lipid nanoparticles. was encapsulated.

[0319] [Table 39]

[0320] [Table 40]

[0321] [Table 41]

[0322] [Table 42]

[0323] V. Encapsulated nucleic acid nanoparticles The term "lipid nanoparticle" or "LNP" or "LNPs" refers to a group of molecules that are mutually interacting due to intermolecular forces. A plurality of (i.e., covalently or non-covalently) physically associated (i.e., A lipid nanoparticle refers to a particle that contains two or more lipid molecules. A lipid nanoparticle is, for example, a microsphere ( including unilamellar and multilamellar vesicles (e.g., liposomes), the dispersed phase in emulsions, It may be the internal phase in the cell or in suspension.

[0324] The term "lipid nanoparticle host" refers to lipid nanoparticles that are physically associated with each other through intermolecular forces / electrostatic interactions. By this term, it refers to multiple lipid molecules that encapsulate one or more nucleic acid molecules, such as mRNA.

[0325] Certain embodiments include a encapsulation method comprising administering to the subject a nucleic acid that is encapsulated in a medicament comprising a pharma- ceutically acceptable carrier and an encapsulated nucleic acid nanoparticle. The present invention provides an encapsulated nucleic acid nanoparticle composition comprising a lipid nanoparticle host, and Nucleic acids, e.g., polyribonucleotides such as mRNA, that are encapsulated within a lipid nanoparticle host. Includes.

[0326] The term "pharmaceutically acceptable carrier" as used herein refers to a non-toxic, inert diluent. Materials that can function as pharma- ceutically acceptable carriers include those that are pyrogen- Free water, deionized water, isotonic saline, Ringer's solution, and phosphate buffer, but In a preferred embodiment, the encapsulated nucleic acid nanoparticles have an average diameter of about 40 to about 70 nm. and dynamic light scattering, e.g., Malvern Zetasizer Nan o Having a polydispersity index of less than about 0.1 as determined using ZS. The degradable cationic lipids, lipidated polyethylene, as described elsewhere herein, Glycol, cholesterol, and 1,2-distearoyl-sn-glycero-3-hydroxybutyric acid Contains the ingredient sukolin.

[0327] Provided herein are encapsulated nucleic acid nanoparticles that include a cationic lipid and another lipid component. Another embodiment is a method of making a nanoparticle composition comprising a cationic lipid and a helper lipid. Another embodiment provides a method of using a cationic lipid, such as cholesterol. , helper lipids such as cholesterol, and neutral lipids such as DSPC. Another embodiment of the present invention provides a method for the preparation of a cationic lipid, a helper lipid, e.g. esterol, neutral lipids such as DSPC, and stealth lipids such as S010, Methods of using S024, S027, S031 or S033 are provided. Embodiments provide a method of encapsulating a nucleic acid in a lipid nanoparticle host, the nanoparticle comprising a cationic Neutral lipids, such as DSPC, esters, etc. lipids, e.g., S010, S024, S027, S031 or S033; The cationic lipid is, for example, RNA or DNA. lipids, such as cholesterol, neutral lipids, such as DSPC, and stealth lipids. The present invention provides a method for using a substance such as S010, S024, S027, S031 or S033. The nucleic acid may be, for example, mRNA, mRNA or DNA.

[0328] In some embodiments, the lipid solution / flow comprises a cationic lipid compound, a helper lipid (cobalt lipid), esterol), an optional neutral lipid (DSPC), and a stealth lipid (e.g., SO 10, S024, S027, or S031). The formulation contains four lipid components. When using a lipid-based ester, the molar ratio of lipids should be 20-70, with a target of 40-60 for cationic lipids. The mole percent of the helper lipid may range from about 30 to about 50. The mole percent of the target lipids ranged from 20 to 70, and the mole percent of the neutral lipids ranged from 10 to 30. Alternatively, the mole percent of PEG lipids may range from 1-6 with a target of 2-5.

[0329] In some embodiments, the lipid solution / stream contains 30-60% of a compound of formula (III), 3 0-60% cholesterol / 5-10% DSPC and 1-5% PEG-DMG , S010, S011 or S024.

[0330] Another embodiment of the present disclosure is a glycerol-based lipid composition comprising about 40-55 cationic lipids / about 40-55 helper lipids. In the lipid molar ratio of the quality, cationic lipid and helper lipid, e.g., cholesterol Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using about Cationic lipids of 40-55 / Helper lipids of about 40-55 / Neutral lipids of about 5-15 In a qualitative molar ratio, cationic lipid, helper lipid, e.g., cholesterol, and neutral lipid are Another embodiment provides a method using a soluble lipid, such as DSPC, having a molecular weight of about 40 to 55. Cationic lipid / about 40-55 Helper lipid / about 5-15 Neutral lipid / about 1-10 In the lipid molar ratio of the telth lipid, cationic lipid, helper lipid, e.g., cholesterol oles, neutral lipids such as DSPC, and stealth lipids such as S010, S024 , S027, S031 or S033.

[0331] Another embodiment of the present disclosure is a lipid composition comprising about 40-50 cationic lipids / about 40-50 helper lipids. In the lipid molar ratio of the quality, cationic lipid and helper lipid, e.g., cholesterol Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using about 40-50 cationic lipids / about 40-50 helper lipids / about 5-15 neutral lipids In a qualitative molar ratio, cationic lipid, helper lipid, e.g., cholesterol, and neutral lipid are Another embodiment provides a method using a soluble lipid, such as DSPC, having about 40-50 Cationic lipid / about 40-50 Helper lipid / about 5-15 Neutral lipid / about 1-5 Steroid lipid In the lipid molar ratio of the lipids, cationic lipid, helper lipid, e.g., cholesterol ole, neutral lipids such as DSPC, and stealth lipids such as S010, S024, A method of using S027, S031 or S033 is provided.

[0332] Another embodiment of the present disclosure is a 43-47 cationic lipid / 43-47 helper lipid mixture. In the lipid molar ratio of the quality, cationic lipid and helper lipid, e.g., cholesterol Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using about Cationic lipids of 43-47 / Helper lipids of about 43-47 / Neutral lipids of about 7-12 In a qualitative molar ratio, cationic lipid, helper lipid, e.g., cholesterol, and neutral lipid are Another embodiment provides a method using a soluble lipid, such as DSPC, having a solubility of about 43-47 Cationic lipids / about 43-47 Helper lipids / about 7-12 Neutral lipids / about 1-4 Steroids In the lipid molar ratio of the lipids, cationic lipid, helper lipid, e.g., cholesterol ole, neutral lipids such as DSPC, and stealth lipids such as S010, S024, A method of using S027, S031 or S033 is provided.

[0333] Another embodiment of the present disclosure is a lipid composition comprising about 45% cationic lipid and about 44% helper lipid. In the lipid molar ratio, cationic lipid and helper lipid, e.g., cholesterol, are used. Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using a method comprising the steps of: % cationic lipid, about 44% helper lipid, and about 9% neutral lipid in the lipid molar ratio. In the present invention, cationic lipids, helper lipids, e.g., cholesterol, and neutral lipids, e.g. For example, a method is provided that uses DSPC. Another embodiment is a method that uses about 45% cationic lipid. , lipid molarity of about 44% helper lipids, about 9% neutral lipids, and about 2% stealth lipids. In the ratio, cationic lipid, helper lipid, e.g., cholesterol, neutral lipid, e.g. For example, DSPC, and stealth lipids, e.g., S010, S024, S027, S031 Or provide a method for using S033.

[0334] One embodiment of the present disclosure is an encapsulated nucleic acid nanoparticle comprising a cationic lipid and another lipid component. Another embodiment provides a method for producing a composition comprising a compound of formula (I) and a helper The present invention provides a method using a lipid, such as cholesterol. Another embodiment uses a cationic lipid. The enzyme is used in the synthesis of proteins, helper lipids such as cholesterol, and neutral lipids such as DSPC. Another embodiment of the present disclosure provides a method for the preparation of a lipid-binding protein comprising the steps of: Cholesterol, neutral lipids, such as DSPC, and stealth lipids, such as S010 Methods of using the method of any one of claims 1 to 4, S024, S027, S031, or S033 of the present disclosure are provided.

[0013] An embodiment provides a method of encapsulating a nucleic acid in a lipid nanoparticle host, the nanoparticle comprising a cationic helper lipids, e.g., cholesterol; neutral lipids, e.g., DSPC; lipids, such as S010, S024, S027, S031 or S033, The acid is, for example, RNA or DNA. Another embodiment of the present disclosure is a cationic lipid, Helper lipids, such as cholesterol, neutral lipids, such as DSPC, and stealth lipids. Methods using lipids, e.g., S010, S024, S027, S031 or S033 The nucleic acid is, for example, mRNA, mRNA or DNA.

[0335] Another embodiment of the present disclosure is a method for the preparation of a composition comprising about 40-55 compounds of formula (I) / about 40-55 helper In the lipid molar ratio of the lipids, cationic lipids and helper lipids, e.g., cholesterol In another embodiment, a method for encapsulating a nucleic acid in a lipid nanoparticle host is provided, comprising: About 40-55 cationic lipids / about 40-55 helper lipids / about 5-15 neutral lipids In the lipid molar ratio, cationic lipid, helper lipid, e.g., cholesterol, and The method further includes using a neutral lipid, such as DSPC. Compound of formula (I) / about 40-55 helper lipid / about 5-15 neutral lipid / about 1-10 In the lipid molar ratio of the stealth lipid, cationic lipid, helper lipid, e.g., cholesterol terols, neutral lipids such as DSPC, and stealth lipids such as S010, S0 24, S027, S031 or S033.

[0336] Another embodiment of the present disclosure is a lipid composition comprising about 40-50 cationic lipids / about 40-50 helper lipids. In the lipid molar ratio of the quality, cationic lipid and helper lipid, e.g., cholesterol Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using about 40-50 cationic lipids / about 40-50 helper lipids / about 5-15 neutral lipids In a qualitative molar ratio, cationic lipid, helper lipid, e.g., cholesterol, and neutral lipid are Another embodiment provides a method using a soluble lipid, such as DSPC, having about 40-50 Cationic lipid / about 40-50 Helper lipid / about 5-15 Neutral lipid / about 1-5 Steroid lipid In the lipid molar ratio of the lipids, cationic lipid, helper lipid, e.g., cholesterol ole, neutral lipids such as DSPC, and stealth lipids such as S010, S024, A method of using S027, S031 or S033 is provided.

[0337] Another embodiment of the present disclosure is a 43-47 cationic lipid / 43-47 helper lipid mixture. In the lipid molar ratio of the quality, cationic lipid and helper lipid, e.g., cholesterol Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using about Cationic lipids of 43-47 / Helper lipids of about 43-47 / Neutral lipids of about 7-12 In a qualitative molar ratio, cationic lipid, helper lipid, e.g., cholesterol, and neutral lipid are Another embodiment provides a method using a soluble lipid, such as DSPC, having a solubility of about 43-47 Cationic lipids / about 43-47 Helper lipids / about 7-12 Neutral lipids / about 1-4 Steroids In the lipid molar ratio of the lipids, cationic lipid, helper lipid, e.g., cholesterol ole, neutral lipids such as DSPC, and stealth lipids such as S010, S024, A method of using S027, S031 or S033 is provided.

[0338] Another embodiment of the present disclosure is a lipid composition comprising about 45% cationic lipid and about 44% helper lipid. In the lipid molar ratio, cationic lipid and helper lipid, e.g., cholesterol, are used. Another embodiment provides a method for encapsulating a nucleic acid in a lipid nanoparticle host using a method comprising the steps of: % cationic lipid, about 44% helper lipid, and about 9% neutral lipid in the lipid molar ratio. In the present invention, cationic lipids, helper lipids, e.g., cholesterol, and neutral lipids, e.g. For example, a method is provided that uses DSPC. Another embodiment is a method that uses about 45% cationic lipid. , lipid molarity of about 44% helper lipids, about 9% neutral lipids, and about 2% stealth lipids. In the ratio, cationic lipid, helper lipid, e.g., cholesterol, neutral lipid, e.g. For example, DSPC, and stealth lipids, e.g., S010, S024, S027, S031 Or provide a method for using S033.

[0339] Lipids in the process of the present disclosure: Nucleic acids (e.g., polyribonucleotides such as mRNA) ) may be about 15-20:1 (wt / wt). In certain embodiments, the ratio of In another embodiment, the lipid:nucleic acid ratio is about 17-19:1. In other embodiments, the lipid:nucleic acid ratio is at least about 30:1, 25:8. :1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17 :1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1 (wt / wt).

[0340] In certain embodiments, the nanoparticles produced by the processes of the present disclosure have an average The grains have a size distribution around the mean (age / mean) diameter and a narrower range of grain sizes. , which corresponds to a more uniform distribution of particle sizes. The particle sizes change after an incubation period or at nanometers. After thorough processing of the particle formulation (e.g., dilution, filtration, dialysis, etc.), the nanoparticles were measured upon recovery. For example, particle size measurements are typically performed after a 60 minute incubation period and / or or after extensive sample processing. The average particle size is reported as either the Z-average or the number average. Z-average was measured by dynamic light scattering on a Malvern Zetasizer. The nanoparticle sample was prepared in phosphate buffer so that the counting rate was about 200 to 400 kcts. The data are presented as the weighted average of the intensity measures. Dynamic light scattering also provides a polydispersity index (PDI) that quantifies the width of the particle size distribution. An increase in the particle size distribution correlates with an increase in the number average, and vice versa. It can be determined by measurement under a microscope.

[0341] In some embodiments, the encapsulated nucleic acid nanoparticles produced by the processes of the present disclosure include In other embodiments, the particles have an average particle size of about 30 to about 150 nm. In another embodiment, the particles have an average particle size of about 40 to about 70 nm. In another embodiment, the particles have an average particle size of about 65 to about 80 nm. In embodiments, the particles have a Z-average of about 50 to about 80 nm and / or a Z-average of about 40 to about 80 nm. In yet another embodiment, the particles have a number average Z average of about 50 to about 70 nm. and / or has a number average of about 40 to about 65 nm. The resulting nanoparticles have a Z-average of about 70 to about 80 nm and / or a number average of about 60 to about 80 nm. The size of the particles obtained depends on the linear velocity of the nucleic acid and lipid flows, the use of an optional dilution step, and The increase in linear velocity and the amount of the first outlet solution may depend on the specific nucleic acid or lipid used. Maintaining the organic solvent concentration below 33% tends to produce smaller particle sizes.

[0342] In some embodiments, the encapsulated mRNA nanoparticles produced by the processes of the present disclosure The particles have an average particle size of about 30 to about 150 nm. In another embodiment, the particles have an average particle size of about 40 to about 70 nm. In another embodiment, the particles have an average particle size of about 65 to about 80 nm. In this embodiment, the particles have a Z-average of about 50 to about 80 nm, and / or a Z-average of about 40 to about 80 nm. In yet another embodiment, the particles have a number average Z-average of about 50 to about 70 nm. and / or has a number average of about 40 to about 65 nm. has a Z average of about 70 to about 80 nm and / or a number average of about 60 to about 80 nm In yet other embodiments, encapsulated mRNA nanoparticles produced by the processes of the present disclosure. The children are about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, and about It may have an average particle size of about 60 nm, about 65 nm, about 70 nm, about 75 nm, or about 80 nm. In yet another embodiment, the encapsulated mRNA nanoparticles produced by the process of the present disclosure. The particles are at least about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about Average of 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, or about 80 nm In yet another embodiment, the encapsulated particles produced by the process of the present disclosure may have a particle size. The mRNA nanoparticles may be less than about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, m, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, or about 80 nm The particle size may be

[0343] Using dynamic light scattering (e.g., Malvern Zetasizer NanoZS) The polydispersity index (PDI) may range from 0 to 1.0. The PDI is less than about 0.2. In other embodiments, the PDI is less than about 0.1. In some embodiments, the PDI is less than 1.5, less than 1.4, less than 1.3, less than 1.2, less than 1. Less than 1, Less than 1.0, Less than 0.9, Less than 0.8, Less than 0.7, Less than 0.6, Less than 0.5, It is less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

[0344] The process of the present disclosure provides a method for preparing cationic lipids in the desired pKa range, stealth lipids, helper lipids, and In combination with lipids, and neutral lipids, for delivery to specific cells and tissues in vivo To this end, those skilled in the art can easily incorporate the above-mentioned methods into formulations, including, for example, liposome formulations, lipid nanoparticle (LNP) formulations, and the like. In one embodiment, the further optimization may be performed by In one embodiment, the lipid molar ratio between the different lipid types is adjusted. Optimization involves adjusting one or more of the desired particle size, N / P ratio, and / or process parameters. Various optimization techniques known to those skilled in the art for the above-listed embodiments are obtained by selecting The optimization techniques are considered part of the present invention.

[0345] Process for encapsulating nucleic acids in lipid nanoparticle hosts The lipid nanoparticles provided herein can be made using the following methods. Non-limiting examples of making polymeric nanoparticles are described in, for example, PCT International Patent Application Publication No. International Publication No. 2016 / 010840 and International Publication No. 2016 / 037 Pamphlet No. 053, Pamphlet No. WO 2015 / 095346, Pamphlet No. WO 2015 / 095346 Brochure No. 2015 / 095340, Brochure No. WO 2014 / 136086 and WO 2011 / 076807, each of which is incorporated herein by reference in its entirety. , which is incorporated herein by reference in its entirety. In order to increase the uniformity of the size of the particles, the skilled artisan may use the techniques presented below. Various combinations of method steps may be experimented with. Sonication, filtration or other sizing methods used in somatic preparations can be employed. Cut.

[0346] The process for making the compositions provided herein typically involves placing a nucleic acid in a first reservoir. and preparing an aqueous solution of an organic alcohol, e.g., ethanol. Providing a second reservoir containing an organic solution of lipids, such as lipid sorbent, and then dissolving the aqueous solution in the organic solution. and mixing the lipid solution with the lipid solution. The first reservoir is optionally mixed with the lipid solution in the second reservoir. The mixing step is optionally followed by an incubation step. A tapping, filtration or dialysis step, as well as a dilution and / or concentration step, are then performed. The incubation step is performed by incubating the solution from the mixing step at about room temperature for about 0 to about 24 hours. Allow to stand in the container for an hour (preferably about 1 hour) and optionally protected from light. In one embodiment, a dilution step follows the incubation step. The dilution step may be, for example, by diluting the aqueous solution using a pumping device (e.g., a peristaltic pump). The filtration step may include dilution with a buffer (e.g., citrate buffer or pure water). Ultrafiltration may be performed by ultrafiltration or dialysis. Ultrafiltration may be performed by using a suitable ultrafiltration membrane (e.g., GE's H Suitable pumping with low-fiber cartridge or equivalent A pumping system (e.g., a peristaltic pump or equivalent) is used to pump the diluted Dialysis involves concentrating the diluted solution, followed by diafiltration. This involves solvent (buffer) exchange through a 10,000mwc snakeskin membrane.

[0347] In one embodiment, the mixing step results in a transparent single phase. After the flip, the organic solvent is removed to give a suspension of particles, with the nucleic acid being encapsulated by the lipids. .

[0348] The choice of organic solvent usually depends on the polarity of the solvent and the ability to remove the solvent at a later stage of particle formation. It is necessary to consider the ease with which the solvent can be dissolved. , preferably in an amount sufficient to obtain a clear, single-phase mixture of nucleic acid and lipids. Regarding its use as a solvent and as a co-solvent for injectable formulations, see Strickley, P. Described by Harmaceutical Res. (2004), 21, 201-230 For example, organic solvents include ethanol, propylene glycol, polyethylene Polyethylene glycol 300, polyethylene glycol 400, glycerin, dimethylacetate Dimethyl ether (DMA), N-methyl-2-pyrrolidone (NMP), and dimethyl sulfoxide (DMSO). The organic solvent is ethanol.

[0349] Disclosed herein are devices for making the compositions of the present disclosure. The devices typically comprise a nucleic acid at least one reservoir for holding an aqueous solution comprising the organic lipid solution; The device also typically contains one or more separate reservoirs for dispensing aqueous and organic lipid solutions. The mixing region or chamber may include a pump mechanism configured to pump the mixture into the mixing region or chamber. In some embodiments, the mixing region or chamber is configured such that the aqueous and organic liquid flows cross-sectionally. Allow the inputs to mix into the connector, and the resulting mixed aqueous and organic solution allows the fluid to exit the cross connector and flow into a collection reservoir or equivalent. In another embodiment, the mixing region or mixing channel may include a cross joint or its equivalent. The member allows aqueous and organic liquid streams to mix as inputs to the T-connector. The resulting mixed aqueous and organic solution exits the T-connector and is collected in a collection reservoir. or its equivalent.

[0350] In certain embodiments, the concentration of nucleic acid in one or more nucleic acid streams is about 0.1 to about 1.5 mg / mL and the concentration of lipid in the one or more lipid streams is from about 10 to about 25 mg / mL. In other embodiments, the concentration of nucleic acid in one or more nucleic acid streams is about 0.2 to about 0.9 mg / mL. and the concentration of lipid in the one or more lipid streams is about 15 to about 20 mg / mL. In embodiments, the concentration of nucleic acid in one or more nucleic acid streams is about 0.225, 0.3, 0.33, or or 0.45 to about 0.675 mg / mL, and the concentration of lipid in the one or more lipid streams is about In another embodiment, the concentration of the nucleic acid in the one or more nucleic acid streams is: Approximately 0.225, 0.3, 0.33, 0.45, or 0.675 mg / mL, The concentration of lipid in the above lipid stream is approximately 16.7 mg / mL.

[0351] The lipid stream comprises a mixture of one or more lipids in an organic solvent. The one or more lipids may be cationic. The lipids may be a mixture of active lipids, neutral lipids, helper lipids, and stealth lipids. Each of the above is described elsewhere herein with respect to the final encapsulated nucleic acid nanoparticles. The organic solvent used in the lipid stream may be present in approximately the same relative amounts as those used in the lipid stream to solubilize the lipids. Suitable organic solvents are those which are capable of being dissolved in water and which are also miscible with aqueous media. Alcohol, propylene glycol, polyethylene glycol 300, polyethylene glycol 400, glycerin, dimethylacetamide (DMA), N-methyl-2-pyrrolidone In one embodiment, the organic solvent includes dimethyl sulfoxide (DMSO), dimethyl ether (DMSO), and methyl ether (Methyl ether). In certain embodiments, the organic solvent comprises about 90% or more ethanol. In certain embodiments, the organic solvent is ethanol. In the form, the lipid stream is buffered in any suitable solution, such as sodium citrate (e.g., 25 mM). Contains a buffer solution of your choice.

[0352] The nucleic acid stream contains a mixture of suitable nucleic acids in a first aqueous solution. The first aqueous solution does not contain salt. For example, the first aqueous solution may be free of added salts or may contain at least one salt. Suitable nucleic acids may be contained in deionized or distilled water without salt. In this embodiment, the first aqueous solution may contain, for example, sodium chloride and / or sodium citrate. The first aqueous solution is a first buffer solution containing at least one salt. The chloride may be present at a concentration ranging from about 0 to about 300 mM. The concentration of sodium is about 50, 66, 75, 100, or 150 mM. The solution may contain sodium citrate at a concentration of about 0 mM to about 100 mM. The solution preferably has a pH of about 4 to about 6.5, more preferably about 4.5 to 5.5. In some embodiments, the pH of the first buffer solution is about 5 and contains a sodium citrate concentration. In another embodiment, the pH of the first buffer solution is about 6. The concentration of sodium enoate is about 100 mM. In certain embodiments, the first buffer solution has a pH below the pKa of the cationic lipid. In terms of morphology, the lipid stream may optionally include a buffer solution. In the absence of a salt (eg, sodium citrate) in the encapsulation does not occur.

[0353] Other possible buffers include sodium acetate / acetic acid, Na2HPO4 / citric acid, and phthalic acid. Potassium hydrogen phosphate / sodium hydroxide, disodium hydrogen phthalate / sodium dihydrogen orthophosphate Thorium, dipotassium hydrogen phthalate / potassium dihydrogen orthophosphate, dihydrogen orthophosphate Including but not limited to potassium / sodium hydroxide.

[0354] In certain embodiments, the organic solvent comprises ethanol and the first outlet solution is about 20 to 20% by weight. 5% ethanol, approximately 0.15-0.25 mg / mL nucleic acid, and approximately 3-4.5 mg / mL mL of lipid. In another embodiment, the organic solvent comprises ethanol and the first outlet solution is , about 20% ethanol, about 0.15 to 0.2 mg / mL of nucleic acid, and about 3 to 3.5 mg / mL of nucleic acid. In yet another embodiment, the organic solvent comprises ethanol, and the first The outlet solution contains approximately 20% ethanol, approximately 0.18 mg / mL nucleic acid, and approximately 3.3 mg In another embodiment, the organic solvent comprises ethanol and the first outlet solution comprises lipid. contains approximately 25% ethanol, approximately 0.2-0.25 mg / mL of nucleic acid, and approximately 4-4.5 In yet another embodiment, the organic solvent comprises ethanol, and the first The outlet solution was approximately 25% ethanol, approximately 0.23 mg / mL nucleic acid, and approximately 4.2 ml Contains g / mL lipid.

[0355] In some embodiments of the present disclosure, the concentrations of nucleic acids and lipids are both decreased or increased. For example, to keep the concentration as high as possible for a more efficient process. Generally, it is desirable to keep the nucleic acid concentration at about 0.045 mg / mL and the lipid concentration at about It is possible to reduce the concentration to 1.67 mg / mL. However, at even lower concentrations , particle aggregation tends to increase.

[0356] In certain embodiments, the concentration of nucleic acid in one or more nucleic acid streams is about 0.1 to about 1.5 mg / mL and the concentration of lipid in the one or more lipid streams is from about 10 to about 25 mg / mL. In other embodiments, the concentration of nucleic acid in one or more nucleic acid streams is about 0.2 to about 0.9 mg / mL. and the concentration of lipid in the one or more lipid streams is about 15 to about 20 mg / mL. In embodiments, the concentration of nucleic acid in one or more nucleic acid streams is about 0.225, 0.3, 0.33, or or 0.45 to about 0.675 mg / mL, and the concentration of lipid in the one or more lipid streams is about In another embodiment, the concentration of the nucleic acid in the one or more nucleic acid streams is: Approximately 0.225, 0.3, 0.33, 0.45, or 0.675 mg / mL, The lipid concentration in the above lipid stream is approximately 16.7 mg / mL. The concentration is in the first outlet stream 60 in a preferred range (e.g., about 0.15 to 0.25 mg / mL). The level of dilution from dilution stream 50 was correspondingly increased to maintain the nucleic acid concentration at It will be necessary to do so.

[0357] In certain embodiments, the lipid:nucleic acid mass ratio is about 15-20:1 or about 17-19: 1, and the concentration of the organic solvent in the outlet solution is about 20-25%. In the elution solution, the lipid:nucleic acid mass ratio is about 18.5:1, and the concentration of organic solvent in the outlet solution is It's about 25%.

[0358] In certain embodiments, the lipid:nucleic acid mass ratio is about 17-19:1. In an embodiment, the lipid:nucleic acid mass ratio is about 18.5:1.

[0359] In certain embodiments, the lipid:nucleic acid mass ratio is about 15-20:1 or about 17-19: 1, and the concentration of the organic solvent in the outlet solution is about 20-25%. In the elution solution, the lipid:nucleic acid mass ratio is about 18.5:1, and the concentration of organic solvent in the outlet solution is It's about 20% or 25%.

[0360] In certain embodiments, the lipid:nucleic acid mass ratio is about 17-19:1. In embodiments, the lipid:nucleic acid mass ratio is about 18.5:1. The mass ratio of the organic compound to the nucleic acid in the outlet solution is about 15-20:1 or about 17-19:1. The concentration of the solvent is about 20-25%. In another particular embodiment, the lipid:nucleic acid mass ratio is , about 18.5:1, and the concentration of organic solvent in the outlet solution is about 20%.

[0361] In certain embodiments, the lipid:nucleic acid mass ratio is about 17-19:1. In an embodiment, the lipid:nucleic acid mass ratio is about 18.5:1.

[0362] In certain embodiments, the encapsulation rate is greater than 60%. In certain embodiments, the encapsulation rate is greater than 65%. In certain aspects, the encapsulation rate is greater than 70%. In some embodiments of the present disclosure, 75% or more of the nucleic acid is encapsulated. In other embodiments, 80% or 85% of the nucleic acid is encapsulated. In yet other embodiments, 90% or more of the nucleic acid is encapsulated. 91%, approx. 92%, approx. 93%, approx. 94%, approx. 95%, approx. 96%, approx. 97%, approx. 98%, approx. 99%, or about 100%, of the nucleic acid is encapsulated.

[0363] In certain embodiments, after formation of the encapsulated nucleic acid nanoparticles described herein, a first outlet solution The solution may be incubated at room temperature for about 60 minutes. After incubation, the solution is diluted The first outlet solution may be diluted approximately two-fold by mixing with a dilution solvent to provide a second outlet solution. The second dilution solvent can be a third buffer solution or water. The dilution step can be, for example, In the U-shaped connector, the incubated first outlet solution is mixed with the second dilution solvent (water). The first outlet solution and the second dilution solvent are incubated. The T-shaped connector is connected to the casing at any suitable flow rate or velocity, such as, for example, about 0.5 to 1 meter / second. After the dilution step, the concentration of the organic solvent in the second outlet solution may be Therefore, in some embodiments, the second outlet solution The concentration of organic solvent (e.g., ethanol) in the liquid is less than 16.5%. In the second outlet solution, the concentration of the organic solvent (e.g., ethanol) is about 10-15%, The second outlet solution is about 10-12.5%, about 12.5%, or about 10%. The cells were concentrated by rapid flow filtration and diluted with phosphate buffered saline (PBS) for 15×. The starting buffer and ethanol may be removed by filtration. The buffer and ethanol are replaced with PBS. After tangential flow filtration, The concentrated pool of encapsulated nucleic acid nanoparticles in PBS may be collected and sterile filtered. The encapsulated nucleic acid nanoparticles present in the formulation produced by the further process steps described above It can be stably stored at 4°C for more than 6 months.

[0364] According to each of the embodiments disclosed herein, the nucleic acid is a polyribonucleic acid, such as an mRNA. There are further embodiments which are otides. For example, according to the embodiments described herein wherein the nucleic acid stream comprises a mixture of one or more mRNA molecules in a buffer solution and is There are further embodiments in which the mRNA stream has a linear velocity that is controlled by the flow rate.

[0365] VI. Animal Immunization The host animal used for immunization is capable of mounting a humoral (antibody) mediated immune response Non-limiting examples of host animals, such as non-human animals, used for immunization include: Mice, rats, rabbits, goats, sheep, camelids, horses, chickens, dogs, cats, Includes pigs, donkeys, cows, monkeys, and sharks.

[0366] In certain aspects of the present disclosure for generating human antibodies against a target protein, mouse Transgenic mice or transgenic mice that carry parts of the human immune system rather than the human immune system. Suchromosomic mice were immunized with the mRNA-LNP complexes described herein. These transgenic mice and / or transgenic mice may be used as host animals. Non-limiting examples of transchromosomic mice are referred to herein as HuMAb mice, respectively. and KM mice, collectively referred to herein as "human Ig mice." can be.

[0367] HuMAb Mouse® (Medarex, Inc.) expresses endogenous μ and κ chains. The unrearranged human heavy chain (μ and A small human immunoglobulin gene encoding the γ and κ light chain immunoglobulin sequences (e.g., Lonberg, et al., 1994 Nature 36 8(6474):856-859). Thus, mice can express mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes The offspring undergoes class switching and somatic mutation to produce a high-affinity human IgGκ monoclonal (Lonberg, N. et al., 1994 supra; Lonberg, N., 1994 Handbook of Experimental Pharmacolo gy 113:49-101; Lonberg, N. and Husz ar, D., 1995 Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N., 1995 Ann. NY. Acad.Sci.764:536-546). For the generation and use of HuMAb mice The genomic alterations carried by such mice are described in Taylor, L. et al. ,1992 Nucleic Acids Research 20:6287-629 5;Chen,J.et at.,1993 International Immun ology 5:647-656;Tuaillon et al.,1993 Pro c.Natl.Acad.Sci.USA 94:3720-3724;Choi et al.,1993 Nature Genetics 4:117-123;Chen ,J.et al.,1993 EMBO J.12:821-830;Tuaillo n et al.,1994 J.Immunol.152:2912-2920;Ta ylor, L. et al., 1994 International Immunol ogy 579-591; and Fishwild, D. et al., 1996 Na and is further described in Nature Biotechnology 14:845-851. No. 6,399,623, the entire contents of which are specifically incorporated herein by reference in their entirety. In addition, U.S. Patent No. 5,545, all by Lonberg and Kay, is incorporated herein by reference. ,806 specification; 5,569,825 specification; 5,625,126 specification ; Specification No. 5,633,425; Specification No. 5,789,650; No. 5,877 ,397 specification; 5,661,016 specification; 5,814,318 specification 5,874,299; and 5,770,429, Sura No. 5,545,807 to Lonberg et al.; ay, PCT Publication No. WO 92103918, PCT Publication No. 93 / 1 Brochure No. 2227, Brochure No. 94 / 25585, Brochure No. 97113852 Brochure No. 98 / 24884 and Brochure No. 99 / 45962 brochure; and PCT Publication No. WO 01 / 1442 by Korman et al. Please also refer to pamphlet No. 4.

[0368] In another embodiment, the human antibody comprises a human heavy chain transgene and a human light chain transgene. Transgenes and transchromosomally human immunoglobulins, such as mice carrying the Mice carrying the purine sequence were used to immunize mice with the mRNA immunization method provided herein. Such mice, referred to herein as "KM mice," can be produced by This is described in detail in PCT Publication WO 02 / 43478 by Ishida et al. Additionally, alternative transgenic animal systems expressing human immunoglobulin genes are Hosts for the mRNA-immunization methods available in the art and provided herein For example, Xenomouse (Abgenix, In Alternative transgenic systems, called mouse genomic DNA (c.), can be used. For example, see U.S. Pat. No. 5,939,598 to Kucherlapati et al. Specification; Specification No. 6,075,181; Specification No. 6,114,598; Specification No. 6,1 Nos. 50,584 and 6,162,963.

[0369] In addition, alternative transchromosomic animal systems expressing human immunoglobulin genes are , which are available in the art and provide host cells for the mRNA-immunization methods provided herein. For example, a human heavy chain transgenic mouse, called a "TC mouse," can be used as the host animal. Using mice carrying both the human light chain transchromosome and the human light chain transchromosome, Such mice can be generated by the method described in Tomizuka et al., 2000. Natl. Acad. Sci. USA 97:722-727 Additionally, the art has described cows carrying human heavy and light chain transchromosomes. It has also been described (Kuroiwa et al., 2002 Nature Biot echnology 20:889-894), mRNA-immunization provided herein The animal may be used as a host animal for the method.

[0370] Following encapsulated mRNA-mediated immunization, antibody-secreting cells (e.g., lymphocytes) from the host animal are , bone marrow cells, plasma cells, or splenocytes) and extracting proteins containing the desired properties. One may screen for antibodies raised against the target. Use of a chromosome-based approach, direct screening of antibody-producing B cells, followed by cloning and recombinant antibody production, or generation of a recombinant antibody library from B cells, followed by Expression of the nucleotide sequence ... This may be done via screening.

[0371] In certain embodiments, antibody-secreting cells (e.g., lymphocytes, myeloid cells, cytoplasmic cells) from the host animal are cultured. stromal cells, or splenocytes) were cultured with F0 cells (ATCC®, CRL-1646) and SP2 / 0 myeloma cells (ATCC®, CRL-1581). The cells are then fused with a donor cell (eg, an immortalized B cell cancer cell, such as an immortalized myeloma cell). Cell fusion can be achieved by electrofusion or by chemical protocols, e.g., using polyethylene glycol. This can be done by a variety of methods, such as by

[0372] In the case of immunization in mice as host animals, hybridoma-based antibody production followed by FAC S or ELISA-based screening for efficient antibody expression and screening The present invention provides a platform for measuring circulating levels (i.e., serum titers) of target-specific antibodies. Monitoring throughout hybridoma-based herd immunization to assess efficacy of humoral responses Given the high degree of target specificity associated with mRNA-based immunization, Considering this, serum titers for integral or membrane proteins can be calculated using the Soluble proteins can be efficiently monitored by FACS using cells that are The titer for IgG4 can also be assessed by ELISA. Depending on the serum titer, the dose is adjusted. to reach a level deemed suitable for initiating B cell isolation and myeloma fusion. The route of mRNA administration (e.g., intravenous, subcutaneous, intramuscular, etc.) can be changed to improve immunization. The magnitude and perhaps diversity of the response can also be altered. has proven to be a particularly effective route for generating rapid target-specific titers.

[0373] One generalizable immunization schedule is outlined below as an example: Day 0: Bleed to define baseline titers in immunologically naïve mice . Day 1 (first immunization): 5-100 μg, e.g., 25-50 μg, of encapsulated mRNA is injected subcutaneously into 4 mice and intravenously into 4 mice. Day 10: Blood is drawn to monitor serum titers. Day 21 (second immunization): 5-100 μg, e.g., 25-50 μg, of encapsulated mRNA A is injected subcutaneously in 4 mice and intravenously in 4 mice. Day 31: Blood is drawn to monitor serum titers. Day 42 (last immunization): 5-100 μg, e.g., 25-50 μg, of encapsulated mRNA is injected intravenously into mice. Day 45: Harvest spleens for splenocyte isolation and hybridoma fusion.

[0374] In certain embodiments, a generalizable immunization schedule includes immunization with encapsulated mRNA. and immunization with recombinant proteins or whole cells / whole cell extracts. For example, the first immunization may be with an encapsulated m Immunization with RNA followed by immunization with recombinant protein or whole cell / A second immunization is performed using conventional immunization methods, such as immunization with whole cell extracts. In certain embodiments, the steps of immunization, blood sampling to monitor serum titers, and subsequent immunizations are The number of days between rounds varies from 1, 2, 3, 4, 5, 6, or 7 days. Good too.

[0375] VIII. Antibody Production Generation of monoclonal antibodies Monoclonal antibodies (mAbs) can be produced using conventional monoclonal antibody methodologies, e.g., Standards from Heller and Milstein, 1975 Nature 256:495 They can be produced by a variety of techniques, including somatic cell hybridization techniques. Viral or oncogenic transformation of lymphocytes, etc. to produce monoclonal antibodies Many techniques can be employed to achieve this.

[0376] Animal systems for producing hybridomas include mouse, rat, and rabbit systems. Hybridoma production in mice is a well-established procedure. Coll is described herein and is used to isolate immunized splenocytes for fusion. Techniques are known in the art. Fusion partners (e.g., mouse myeloma cells) ) and fusion procedures are also known and described.

[0377] Chimeric or humanized antibodies of the present disclosure are produced as described herein, and are not intended to be construed as limiting the scope of the invention. For example, it can be prepared based on the sequence of a mouse monoclonal antibody. DNA encoding the two chain immunoglobulins can be obtained from the hybridoma of interest. , containing non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. For example, techniques known in the art for creating chimeric antibodies can be used to engineer The mouse variable regions can be linked to human constant regions using known methods (e.g., (See U.S. Patent No. 4,816,567 to Cabilly et al.) Humanized Antibodies To create the CDR sequences, the murine CDR regions are cloned into the human CDR sequences using methods known in the art. For example, US Patent No. 522 by Winter No. 5,539; and U.S. Pat. No. 5,530,101 to Queen et al.; Nos. 5,585,089; 5,693,762; and 6,180,370 Please refer to the specification.

[0378] A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules. For example, chimeric antibodies are made by fusion of a mouse or rat monoclonal antibody with a human antibody constant region. Methods for producing chimeric antibodies are known in the art. For example, Morrison, 1985, Science 229: 1202;Oi et al.,1986,BioTechniques 4:214; See Gillies et al., 1989, J. Immunol.

[0379] In certain aspects, the antibodies of the present disclosure are human monoclonal antibodies. Monoclonal antibodies are made using transgenic antibodies that carry parts of the human immune system rather than the mouse system. These can be generated using transchromosomic mice or transchromosomic mice. Transgenic and / or transchromosomic mice are referred to herein as These include mice designated HuMAb mice and KM mice, respectively, and are referred to herein as " They are collectively referred to as "human Ig mice."

[0380] Immunology 579-591; and Fishwild, D. et al. ,1996 Nature Biotechnology 14:845-851 The contents of all of these are specifically incorporated herein by reference in their entirety. Additionally, U.S. Patent No. 5,545,806, all by Lonberg and Kay. Specification; Specification No. 5,569,825; Specification No. 5,625,126; Specification No. 5, Specification No. 633,425; Specification No. 5,789,650; Specification No. 5,877,397 Specification; Specification No. 5,661,016; Specification No. 5,814,318; Specification No. 5, Nos. 874,299; and 5,770,429, by Surani et al. No. 5,545,807, all by Lonberg and Kay; , PCT Publication No. International Publication No. 92103918, PCT Publication No. 93 / 12227 Brochure, Brochure No. 94 / 25585, Brochure No. 97113852 , Brochure No. 98 / 24884, and Brochure No. 99 / 45962 and Korman et al., PCT Publication No. WO 01 / 14424. See also Lett.

[0381] Antibodies or antigen-binding fragments generated using techniques such as those described herein can be used to identify targets. The antibodies can be isolated using standard well-known techniques, such as antibody or antigen binding. Fragments can be obtained, for example, from culture medium, ascites fluid, serum, cell lysates, synthetic reaction materials, etc., e.g. Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis By conventional immunoglobulin purification methods such as filtration, dialysis, or affinity chromatography. and can be conveniently isolated. EXAMPLES

[0382] The following examples are offered by way of illustration and, unless otherwise specified, are not intended to be limiting of the invention. This document does not intend to determine the scope of the specification.

[0383] Example 1 Encapsulated mRNA Production Workflow Step 1: Designing cDNA and cloning into an in vitro transcription vector Design of cDNA constructs The natural cDNA sequence may be used in a subcloning strategy or as the final Any consensus sites for the restriction enzymes used in linearizing the construct are In this particular example, the subcloning The restriction enzymes used for cloning were BamHI and RsrII, and The restriction enzyme used is BspQI. However, any suitable restriction enzyme and its corresponding restriction enzyme may be used. For example, a specific cDNA encoding a target protein of interest can be used. Certain restriction enzyme sites not present in NA were added for the subcloning strategy and linearization. can be selected.

[0384] The native cDNA sequence may also be modified for expression in non-human animals such as mice or rabbits. For example, GeneOptimizer® software (ThermoFix Codon-optimized genomic DNA was obtained using conventional methods using the Sigma-Aldrich Co., Ltd. The process of codon optimization can be: (i) to increase the stability of the RNA; (ii) removal of cryptic splice sites and RNA destabilizing sequence elements; (iii) codon optimization for specific expression systems and the addition of G stabilizing sequence elements; (iv) intron removal; and (v) avoidance of stable RNA secondary structures. The GeneOptimizer® software is used to In certain embodiments, the codon optimization settings preserve the 5' / 3' restriction and separate it from the rest of the molecule. The BspQ1 consensus sequence (forward [GCTCT Both the reverse [TC] and reverse [GAAGAGC], BspQ1 is a restriction enzyme for palindromic sequences. (because it is not prime) must also be eliminated.

[0385] [Table 43]

[0386] Vector Description cDNA encoding the target protein (see, for example, Tables 1-7) was transformed into T7 R to drive RNA polymerase-mediated transcription from the RNA polymerase promoter The vector was designed to clone the T7 promoter into the tobacco etchant vector. The 5' untranslated region (UT) of the Tobacco etch virus (TEV) The UTR is a sequence that encodes the nucleotide sequence (RNA) of the target gene, which is known to improve translation efficiency in eukaryotic cells. The cDNA for the target protein is placed downstream of the TEV UTR. The Kozak consensus sequence (ccgccacc) was inserted into the initiator to enhance translation. Two stop codons were inserted upstream of the methionine / start codon. The 3'UTR of human β-globin was placed at the end of the cDNA. This element enhances the stability of the mRNA in the cell. The C-terminal element of the transcription-related component of the vector is a polyA tail. In certain embodiments, the polyA of the mRNA encoding the target protein or a fragment thereof is The tail is about 50bps to 120bps or 60bps to 120bps. In embodiments, the polyA tail of the mRNA encoding the target protein or fragment thereof is In certain embodiments, the target protein or The poly(A) tails of the mRNAs encoding the fragments are approximately 70 bps, 80 bps, and 90 bps. s, 100bps, or 110bps.

[0387] Cloning of cDNA into an in vitro transcription vector The cDNA construct was digested together with the vector using the restriction enzymes BamHI / RsrII. 2. Transforming the DNA into a DNA fragment by agarose gel electrophoresis to produce a compatible fragment that is purified by agarose gel electrophoresis; The purified cDNA is then ligated into the digested vector to produce the desired transcription vector construct. Got the strut.

[0388] The ligation mixture was transformed into stbl3 competent bacterial cells and transfected with ampicillin. The plates were incubated at 37°C overnight. Prior to subcloning, select colonies by digestion with restriction enzymes and select appropriate The inserts and backbones were sized according to the sequence. Colonies were also cloned in parallel. RsrII and SapI (isoschizomers of BspQ1 that cleave efficiently at 37°C) to establish the integrity of the polyA tail. The plasmid was expanded and used for mRNA production.

[0389] Step 2: Transcript linearization, in vitro transcription and capping Circular plasmid DNA was prepared according to conventional methods. Purified plasmid DNA The plasmid DNA was digested with BspQI restriction endonuclease. Reaction buffer (Buffer 4, New England Biolabs, 10x stock solution) and B The mixture was mixed with spQI enzyme (1,250 U per mg of DNA). The reaction was incubated at 50°C for 2 h. The reaction was incubated for 1 h and then placed on ice or at 4°C. A small aliquot of the reaction was Complete linearization of the circular plasmid DNA was confirmed by running it on a gel electrophoresis column. The NA template was purified by ethanol precipitation. The DNA pellet from the ethanol precipitation step was , and dissolved in nuclease-free water to a concentration of greater than 0.5 mg / ml.

[0390] In vitro transcription and capping of modified synthetic mRNA The modified synthetic mRNA in this example was produced by in vitro transcription (IVT) and was synthesized using lithium chloride. The antibodies were purified by LiCl purification and purchased from New England Biolabs (registered trademark). The tubes were capped using a commercially available kit from Eppendorf (Beverly, MA USA). Materials and reagents are shown in Table 9.

[0391] [Table 44]

[0392] Transcription reactions are constructed, for example, as listed in Table 10, and RNase-free tubulin is used. Attention was paid to the use of tools, chips and implementation.

[0393] [Table 45]

[0394] The procedure in this example for making modified synthetic mRNA was carried out as follows: 1. The above materials were incubated at 30°C for 2 hours while monitoring the temperature. The NA template was digested by adding 0.04 U / μL of DNase, and the reaction mixture was Incubated at 37°C for 30 minutes. 2. LiCl is added to a final concentration of 2.81 M, the reaction mixture is mixed thoroughly, and - The mixture was then incubated at 20° C. for more than 1 hour. The mixture was then incubated at 4° C. for 15 minutes, approximately 20 minutes, The supernatant was removed and the pellet was placed in 1 mL of PBS. Washed with 70% ethanol. Preparations were centrifuged for 10 min as described immediately above. The supernatant was then removed and the remaining pellet was centrifuged again for less than 1 min as above. 3. The residual ethanol was removed and the pellet was resuspended in nuclease-free water. The concentration was measured and adjusted to approximately 1 μg / μL. 4. Add 10% by volume of 3M sodium acetate, pH 5.5, to the preparation and thoroughly mix. Then, one volume of room temperature isopropanol was added to the preparation and mixed well. The mixture was incubated overnight at -20°C. The preparation was then incubated at 4°C for 15 min for approximately 20,000 Centrifuge at maximum speed of 10 x g (max speed), remove the supernatant and suspend the remaining pellet in 1 mL of 7 The preparation was centrifuged again for 10 min as described immediately above. The supernatant was then removed and a centrifugation step was performed again for less than 1 minute as above. 5. Residual ethanol was removed and the pellet was resuspended in nuclease-free water. The concentration was measured and adjusted to approximately 4 μg / μL.

[0395] The modified synthetic mRNA can then be stored at -80 °C until capping and upon thawing The concentration is measured again.

[0396] For capping, the procedure used was as described by New England BioLabs. The synthetic mRNA and water mixture was heat denatured at 65°C for 10 min and then cooled. The plate was then transferred to a block and quenched for 5 minutes. Immediately before use, S-adenosylmethionine (SA Dilute the stock solution of 1:8 HO in water to 4 mM and then mix the remaining reaction components as shown in Table 1. 1 in the specified order.

[0397] [Table 46]

[0398] The mixture was then incubated for 1 h at 37° C. The samples were then LiCl precipitated as above. and then stored at -80°C.

[0399] Step 3: Determining mRNA quality and functionality After the initial in vitro transcription reaction and / or capping reaction, Agilent Synthetic mRNA was analyzed for quality and integrity using the 2100 Bioanalyzer. The Agilent 2100 BioAnalyzer was used to analyze DNA, RNase A, and A nanofluidic device for size fractionation and quantification of small samples of proteins. Agilent RNA 6000 Nano Kit (Cat. No. 5067-1 511) was used to conduct the analysis.

[0400] All kit reagents were equilibrated to room temperature for 30 minutes before use. The ladder was stored on ice.

[0401] Preparation of gels, gel / dye mixtures, and samples The gel matrix (550 μL) was pipetted onto the spin filter and incubated at room temperature for 10 min. The mixture was centrifuged at 1,500 g for 1 h and then stored at 4°C (for use within 1 month). The solution (1 μl) was added to a 65 μl aliquot of the filtered gel matrix. The gel matrix mixture was vortexed and then centrifuged at 13,000 g for 10 min at room temperature. The mRNA samples, ladders and kit standards were centrifuged (for use within 1 day). Heat denaturation at 0°C for 3–5 min was used to destroy any higher order structures, followed by quenching on ice prior to analysis. I punched him.

[0402] Purification of Bioanalyzer Electrodes Clean the Bioanalyzer electrodes with 350 μl of RNaseZap electrode cleaner and Nu Purified with creatine-free water.

[0403] Loading the gel / dye mixture onto the chip Place a new tip on the priming station (position C) of the Bioanalyzer. Place on the platform (clip in top position) and add 9 μl of gel / dye mixture The mixture was added to the wells.

[0404] Loading the sample onto the chip A volume of 5 μl of marker was added to the ladder wells and the sample wells, and a volume of 1 μl of mRNP A sample (<1 μg) was added to the sample well. The samples were then vortexed at 400 rpm for 1 min. The samples were then analyzed using the mRNA assay method. Measured on a Gilent 2100 Bioanalyzer. Adenine, guanine, Human R synthesized with cytidine and either uridine or pseudouridine Samples of mRNA prepared for XFP1 (both codon-optimized and non-codon-optimized) See Figure 4 for sample Bioanalyzer traces.

[0405] Transfection of mRNA into cultured mammalian cells and expression of the encoded protein Western blot to confirm Thermo Fisher Scientific's Lipofectamine ( In vitro transfer of mRNA into cultured mammalian cells using 2000 Reagent The translational potential was evaluated by transfection. After 4-48 hours, the cells were lysed and the proteins in the lysates were analyzed by polyacrylamide gel electrophoresis. followed by immunofluorescence with antibodies specific for the protein encoded by the mRNA. Immunoblotting was performed (Western blot of the samples confirming the expression of human RXFP1 from mRNA). See Figure 5 for blots).

[0406] Step 4. Packaging the modified synthetic mRNA into a delivery vehicle The materials used in this example were as follows:

[0407] [Table 47]

[0408] [ka] 1. Cationic lipid amine groups to mRNA phosphate groups (N:P) molar ratio of 4:1 for target protein Modified synthetic mRNA encoding a protein (see, for example, Tables 1-7) is inserted into lipid nanoparticles. Packaged, dialyzed, and concentrated. As an example, mRNP with a concentration of more than 0.4 mg / mL For the protocol resulting in approximately 2 mg of packaged modified synthetic mRNA, show. 2. Use RNAse-free reagents, tubes, tips and procedures described in Table 13 The lipid nanoparticle mixture reagents were weighed out and mixed in a vial as described above.

[0409] [Table 48]

[0410] 3. To facilitate processing, ethanol is added to the lipids to give a 1.1x ratio of the required amount. The mixture was briefly sonicated and gently stirred for 5 min at 37°C. The mixture was incubated at 37°C without stirring until ready. 4. Fill the Amicon Ultra-15 centrifuge device with the mRNA solution and place it at 4°C. The modified synthetic mRNA was purified from the water by centrifugation at 4,000 rpm for 15 min. The concentrated mRNA was resuspended in pH 6.0 citrate buffer and then exchanged into H6.0 buffer. The mRNA concentration was measured. 5. A final concentration of 0.5 mg / mL of modified synthetic mRNA in citrate buffer at pH 6.0 Concentrations were prepared in rinsed scintillation vials (4 mg of mRNP in 8 mL). A) The final concentration of the mRNA solution was measured. The mRNA dilution solution was stored until it was ready for use. was incubated at 37°C. 6. Mix 8 mL each of (a) lipid mixture; (b) mRNA solution; and (c) citrate buffer. Three 10 ml syringes were prepared. Syringes (a) and (b) were inserted into the lumen of the T-junction. In summary, a P727 T-mixer with a 0.5mm inner diameter was fitted to the P6 52 adapter (IDEX, Oak Harbor WA USA). Syringes (a) and (b) were attached to P658 Luer fittings (IDEX). Diagrams (a) and (b) were attached to the P938x nuts and ferrules (IDEX). TFE 0.8mm ID tubing (#3200068, Dolomite, Royston The syringe (c) was connected to a T-mixer by a P938x nut and The tube was attached to a luer fitting that was connected to a final single tube by a ferrule. The end of the tube is then clamped over a pre-rinsed beaker containing a stirring bar and gently stirred. did. 7. Set the syringe pump settings to the appropriate syringe manufacturer and size and volume (8 The flow rate of 1.0 mL / min was entered. The pump was started and the resulting material was stirred. The mRNA was collected in a 50 mL RNase-free plastic beaker with a rod. Transfer the lipid nanoparticle suspension containing 2-3 mL per bag into a dialysis tube and The mixture was dialyzed into phosphate buffered saline (PBS) at 4° C. overnight. 8. The aliquots were pooled into one 15 mL conical tube. A lipid nanoparticle (LNP) suspension was concentrated using trickle flow filtration (TFF). Using the new 500K molecular weight cutoff capsules with the Minimate system and rinse with 500 mL of RNA-free water at a flow rate of 150 rpm. The TFF system was prepared as follows. 9. Load the lipid nanoparticle / modified synthetic mRNA suspension into the TFF unit reservoir and allow to sit for 75 min. The mixture was concentrated to a final volume of 2-3 ml at a flow rate of mL / min. 10. Percent encapsulation of modified synthetic mRNA was performed using Life Technologies (G Quant-iT Ribogreen R from Ranch Island NY USA The NA was determined using an assay kit. The lipid nanoparticle / modified synthetic mRNA suspension was placed in buffer. For fluorescence measurement in liquid (mRNA outside the particles) and in buffer containing surfactant (total mRNA) The analysis was performed by preparing a 1000ng / mL stock solution from the provided ribosomal RNA. This was used to generate a standard curve for the Ribogreen assay. Prepare samples in TE buffer or TE with Triton added, and add fluorescent reagent to each. The difference calculated is the mRNA inside the particle.

[0411] [Table 49]

[0412] 11. Dilute the sample in TE buffer and PBS so that the measured value is within the standard curve (400-600 times). and appropriate dilutions were prepared in TE buffer + 0.75% Triton X-100. 100 μL of standard / sample was added to each well of the well plate. Drugs were diluted 1:200 in TE buffer and 100 μL was added to each well. 12. Sample fluorescence was measured using a fluorescence microplate reader at excitation of 480 nm and fluorescence intensity of 520 nm. The fluorescence value of the reagent blank was subtracted from that of each RNA sample to obtain the fluorescence value. A standard curve of fluorescence versus RNA concentration was generated. The fluorescence value of each sample was subtracted from the fluorescence value of the reagent blank. The RNA concentration of the samples was determined from the standard curve. The difference in concentration between the sample with Triton and the sample alone was calculated by the concentration of the sample with Triton. The lipid nanoparticles / modified synthetic suspension was diluted 6-fold to obtain a 1:1 solution. The data were collected using a Zetasizer Nano ZS instrument (Malvern Instruments, The diameter and polydispersity index were measured using a 100% NMR spectrometer (H.T.S., Ltd., Worcestershire, UK). It has been decided.

[0413] [Table 50]

[0414] Example 2 - Immunization strategy against RXFP1 An overview of immunization strategies against GPCR target proteins such as human RXFP1 is shown in Table 16. vinegar.

[0415] [Table 51]

[0416] Female BALB / c mice were injected with 100 mg of 10 ... 100 μg of packaged human RXFP1 mRNA (see, for example, Table 1) ), 10 6 cells / ml of Ba / F3 cells overexpressing hRXFP1, or 300.1 50μL overexpressing human RXFP1 derived from either HEK293 or HEK293 cells The subjects were immunized with either priming immunization or immunization with 100 μg of virus-like particle (VLP). The boosting immunization was performed 10 days after vaccination (as seen in Figure 1A). The test was performed 21 days after immunization with .

[0417] A total of 207 hybridomas producing antibodies specific to RXFP1 were obtained, and further We screened 10 anti-RXFs with high affinities in the pM and nM range. The P1 monoclonal antibody was identified.

[0418] FIG. 1B shows that 10 days after immunization, virus-like particles or RXFP1 (an exemplary target) Immunization with cells overexpressing a target protein immunogen failed to induce significant antibody titers. In contrast, immunization with mRNA-LNPs resulted in a significantly higher than background These data suggest that immunization with mRNA-LNPs results in 2- to 12-fold higher titers of the This is more efficient than conventional methods such as using virus-like particles or cells that overexpress the immunogen of the target gene. More robust immune responses (e.g., higher serum levels) to the target protein immunogen than These results suggest that the vaccine may confer higher antibody titers.

[0419] FIG. 1C shows animals after a boosting immunization step (e.g., a second administration of immunogen). (Figure 1B) shows antibody titers in mice immunized with mRNA for both the first and second immunizations. Mice immunized (sc) were immunized twice (priming and boosting immunizations). Mice immunized with mRNA (sc) for only one of the two immunizations and for one of the remaining immunizations, immunization with VLPs or cells overexpressing RXFP1 was performed. Mice immunized via i.v. administration showed higher antibody titers than mice immunized via sc. For immunization, mRNA was used for both priming and boosting immunizations. Immunization strategies using mRNA for only one of the two immunizations were compared. The difference in antibody titers was smaller for all iv immunizations. The mean titers for all iv immunizations were was higher than that of immunization.

[0420] Figure 1D shows three monoclonal antibodies specific for RXFP1 obtained from mRNA-based immunization. FACS plots of single hybridoma culture samples are shown. It showed minimal cross-reactivity to expressing cells (parental 300.19) and overexpressed human RXFP1. Significantly higher levels of 300.19hRXFP1 and Ba / F3hRXFP1 were observed in cells expressing Shows bond.

[0421] Figure 2 shows the immunization strategy against the multi-pass transmembrane protein SLC52A2 and the resulting The FACS-based serum responses are shown. Only two rounds of immunization with NA can induce target-specific antibody titers The only antigen; overexpressing cells, VLPs and peptides encoding the extracellular loop (EC2) Two to four rounds of immunization with conventional antigens such as peptides in various combinations have been Target-specific titers were not elicited from mice immunized with these immunogen forms. Hybridoma fusion was initiated because target-specific IgG was not detected in serum. No target-specific antibodies were detected in plasma from animals immunized with these conventional antigens. The inability to detect IgG indicates that SLC52A2 is a poorly immunogenic protein However, in mice immunized with mRNA-LNP, We initiated doma fusions and isolated a total of 228 clones capable of producing SLC52A2-specific antibodies. Hybridomas were identified from a pool of 12,880 hybridoma wells. Generally, about one-third (1 / 3) of these wells contain hybridomas. (>4,290 hybridomas). Therefore, the data shown in Figure 2 The mRNA immunization methods described herein can be used to, for example, immunize multiple transmembrane proteins, e.g. , which is surprisingly superior to conventional antigen formats for transmembrane proteins such as SLC52A2. This suggests that the only method that could produce target-specific serum titers Because it was a means to an end.

[0422] Example 3 - Immunization strategies against ANGPTL8 We present an overview of immunization strategies for difficult-to-express target proteins such as human ANGPTL8. 17. Related to recombinant expression of ANGPTL8 to generate specific antibodies Examples of problems include low yield, insufficient secretion, and aggregation. Using the expression protocol, the resulting protein appears to be over 90% aggregated. I got it.

[0423] [Table 52]

[0424] Female BALB / c mice were cultured with 50 μg of packaged human ANGPTL8 mRNA. A (see, e.g., Table 3) or 50 μg of HSA-ANGPTL8 fusion protein Titers were measured 10 days after the priming immunization and at the first booster immunization. The first boosting immunization was given 21 days after the priming immunization. The final boost was given 25 days after the first boost. Mice administered either mRNA or purified recombinant human ANGPTL8 protein , which elicited roughly equivalently robust immune responses to human ANGPTL8 in mice Hybridomas producing ANGPTL8-specific antibodies were generated and further screened. These results are consistent with the results of the mR The immunization method with NA-LNPs is a promising approach for targeting difficult-to-express proteins such as human ANGPTL8. These results demonstrate that this is an effective strategy for generating antibodies against antigens.

[0425] Example 4 - Immunization strategies against Galectin-3 An overview of immunization strategies against lectin-binding proteins such as galectin-3 is shown in Table 18. vinegar.

[0426] BALB / c mice were treated with 2 mg / kg The mice were immunized with either 20 μg of recombinant galectin-1 or 20 μg of mRNA. 3 IgG titers were assessed 7 days after the final boost given on day 55.

[0427] FIG. 3 shows that the use of galectin-3 mRNA as a final boosting agent was Significantly higher target-specific IgA than recombinant proteins (conventional immunogens) This effect was observed whether the antigen was given subcutaneously or intravenously. Regardless, it was observed.

[0428] Hybridomas producing galectin-3-specific antibodies were generated and further selected. A high affinity monoclonal anti-galectin-3 antibody was obtained.

[0429] [Table 53]

[0430] Overview of compatibility rates achievable by mRNA-mediated immunization Table 19 shows the total hybridoma wells selected (generally about 3 of these wells). Summary of target proteins from 1 / 3 of all hybridomas (1 / 3 contain hybridomas) and immunoassays The hybridoma wells were obtained after using lipid-encapsulated mRNA as an antigen. The number of confirmed target-specific antibodies that can be identified is provided.

[0431] Table 20 shows the target-specificity of the mRNA-LNP immunization method compared to other conventional immunization methods. The data provide a comparison of the number of hybridomas producing the desired antibodies. Immunization with mRNA-LNPs induces an immune response against the target protein antigen, It is an effective method to obtain a higher number / rate of antibodies specific to a target protein. In particular, these results suggest that immunization with mRNA-LNPs may be able to induce antibody production. Transmembrane proteins that are difficult to identify, such as multi-transmembrane proteins such as GPCRs quality, and poorly immunogenic proteins (e.g., plasma from animals immunized with conventional antigens). Proteins that give low or no detectable target-specific IgG in It is surprisingly more effective than conventional immunization methods at obtaining specific antibodies against the This proves that...

[0432] [Table 54]

[0433] [Table 55]

[0434] Generally, the successful generation of antigen-specific antibody-producing hybridomas is described in detail herein. To achieve this, we have developed at least 15 different target genes using the mRNA-LNP immunization method. These results were achieved by the mRNA immunization method described herein. In the host animal, various antigens (e.g., transmembrane proteins, e.g., GPCRs, etc.) It is possible to induce an immune response against the chimeric mutant, human High affinity models that can serve as parents for the generation of affinity-matured and affinity-immobilized variants. This shows that this is an effective method for generating monoclonal antibodies.

[0435] Incorporation by Reference All references and supporting documents cited herein, including patents, patent applications, articles, textbooks, etc. and the references cited therein are incorporated herein by reference unless they have already been cited. is incorporated in its entirety by

[0436] Equivalent The foregoing specification is considered to be sufficient to enable one skilled in the art to practice the invention. The following detailed description and examples detail certain embodiments of the present invention. No matter how specifically described herein, the invention can be practiced in various ways. that the invention should be construed according to the appended claims and any equivalents thereof. Let it be understood.

Claims

1. A method for producing a monoclonal antibody against a target protein, comprising: (a) mixing at least one cationic lipid with messenger RNA (mRNA) encoding said target protein or a fragment thereof, thereby forming a cationic lipid-mRNA complex; (b) administering the lipid-mRNA complex to a non-human animal; (c1) obtaining an antibody that specifically binds to the target protein by removing antibody-producing cells from the non-human animal, fusing the antibody-producing cells with myeloma cells to produce hybridomas, and isolating the antibody from a culture medium of the hybridoma; or (c2) obtaining an antibody that specifically binds to the target protein by removing antibody-producing cells from the non-human animal, culturing the antibody-producing cells to produce an antibody, and isolating the antibody from the culture of antibody-producing cells; and the mRNA of the complex contains pseudouridine, and The mRNA is (i) the consensus Kozak sequence; (ii) a 7-methylguanosine cap on the 5' end of the mRNA; (iii) a polyadenosine (poly A) tail found at the 3' end of the mRNA transcript; and (iv) 5'- and 3'-untranslated regions (UTRs) and one or more features selected from the group consisting of the target protein is a transmembrane protein; method.

2. The method of claim 1, wherein the target protein is a transmembrane protein and the monoclonal antibody is a therapeutic monoclonal antibody.

3. The transmembrane protein comprising: (i) G protein-coupled receptors (GPCRs); (ii) a single-pass transmembrane protein receptor; (iii) tumor necrosis factor receptor superfamily (TNFRSF) members; (iv) interleukin (IL) receptor; (v) ion channels; (vi) solute transporter; (vii) an immune receptor; and (viii) multi-transmembrane proteins The method of claim 2, wherein the compound is selected from the group consisting of

4. The target protein is one of the following: ACKR1, ACKR2, ACKR3, ACKR4, ADCYAP1R1, ADGRA1, ADGRA2, ADGRA3, ADGRB1, ADGRB2, ADGRB3, ADGRD1, ADGRD2, ADGRE1, ADGRE2, ADGRE3, ADGRE4P, ADGRE5, ADGRF1, ADGRF2, ADGRF3, ADGRF4, ADGRF5, ADGRG1, ADGRG2, ADGRG3, ADGRG4, ADGRG5, ADGRG6, ADGRG7, ADGRL1, ADGRL2, ADGRL3, AD GRL4, ADGRV1, ADORA1, ADORA2A, ADORA2B, ADORA3, ADRA1A, ADRA1B, ADRA1D , ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3, AGTR1, AGTR2, APLNR / APJ, ASGR1, ASGR2, AVPR1A, AVPR1B, AVPR2, BDKRB1, BDKRB2, BRS3, BRS3, C3AR1, C5AR1, C5AR2, CALCR, CALCRL, CASR, CCKAR, CCKBR, CCR1, CCR10, CCR2, CCR3 , CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCRL2, CELSR1, CELSR2, CELSR3, CHRM 1, CHRM2, CHRM3, CHRM4, CHRM5, CMKLR1, CNR1, CNR2, CRHR1, CRHR2, CX3CR1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CYSLTR1, CYSLTR2, DRD1, DRD2, DRD3, DRD4, DRD5, EDNRA, EDNRB, F2R, F2RL1, F2RL2, F2RL3, FFAR1, FFAR2, F FAR3, FFAR4, FPR1, FPR2, FPR2, FPR3, FSHR, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, GABBR1, GABBR2, GALR1, GALR2, GALR3, GCGR, GHRHR, GHSR, GIPR, GLP1R, GLP2R, GNRHR, GNRHR2, GPBAR1, GPER1, GPR1, GPR 4, GPR12, GPR15, GPR17, GPR18, GPR19, GPR20, GPR21, GPR22, GPR25, GPR26,GPR27、GPR3、GPR31、GPR32、GPR33、GPR34、GPR35、GPR37、GPR37L1、GPR39、GPR40、GPR42、GPR42、GPR45、GPR50、GPR52、GPR55、GPR6、GPR61、GPR62、GPR63、GPR65、GPR68、GPR75、GPR78、GPR79、GPR82、GPR83、GPR84、GPR85、GPR87、GPR88、GPR101、GPR107、GPR132、GPR135、GPR137、GPR139、GPR141、GPR142、GPR143、GPR146、GPR148、GPR149、GPR15、GPR150、GPR151、GPR152、GPR153、GPR156、GPR157、GPR158、GPR160、GPR161、GPR162、GPR171、GPR173、GPR174、GPR176、GPR179、GPR182、GPR183、GPRC5A、GPRC5B、GPRC5C、GPRC5D、GPRC6A、GRM1、GRM2、GRM3、GRM4、GRM5、GRM6、GRM7、GRM8、GRPR、HCAR1、HCAR2、HCAR3、HCRTR1、HCRTR2、HRH1、HRH2、HRH3、HRH4、HTR1A、HTR1B、HTR1D、HTR1E、HTR1F、HTR2A、HTR2B、HTR2C、HTR4、HTR5A、HTR5BP、HTR6、HTR7、KISS1R、LGR3、LGR4、LGR5、LGR6、LHCGR、LPAR1、LPAR2、LPAR3、LPAR4、LPAR5、LPAR6、LTB4R、LTB4R2、MAS1、MAS1L、MC1R、MC2R、MC3R、MC4R、MC5R、MCHR1、MCHR2、MLNR、MRGPRD、MRGPRE、MRGPRF、MRGPRG、MRGPRX1、MRGPRX2、MRGPRX3、MRGPRX4、MTNR1A、MTNR1B、NMBR、NMUR1、NMUR2、NPBWR1、NPBWR2、NPFFR1、NPFFR2、NPSR1、NPY1R、NPY2R、NPY4R、NPY5R、NPY6R、NTSR1、NTSR2、OPN3、OPN4、OPN5、OPRD1、OPRK1、OPRL1、OPRM1、OR51E1、OXER1、OXGR1、OXTR、P2RY1、P2RY10、P2RY11、P2RY12、P2RY13, P2RY14, P2RY2, P2RY4, P2RY6, P2RY8, PRLHR, PROKR1, PROKR2, PTA FR, PTGDR, PTGDR2, PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGIR, PTH1R, PTH2R, QRFPR, RXFP1, RXFP2, RXFP3, RXFP4, S1PR1, S1PR2, S1PR3, S1PR4, S1 PR5, SCTR, SMO, SSTR1, SSTR2, SSTR3, SSTR4, SSTR5, SUCNR1, TAAR1, TAAR2, TAAR3, TAAR4P, TAAR5, TAAR6, TAAR8, TAAR9, TACR1, TACR2, TACR3, TAS1R1 , TAS1R2, TAS1R3, TAS2R1, TAS2R10, TAS2R13, TAS2R14, TAS2R16, TAS2R19, TAS2R20, TAS2R3, TAS2R30, TAS2R31, TAS2R38, TAS2R39, TAS2R4, TAS2R40, TAS2R41, TAS2R42, TAS2R43, TAS2R45, TAS2R46, TAS2R5, TAS2R50, TAS2R60 , TAS2R7, TAS2R8, TAS2R9, TBXA2R, TPRA1, TRHR, TSHR, UTS2R, VIPR1, VIPR2, XCR1, TCR-α, TCR-β, CD3, ζ-chain accessory, CD4, CD8, SIGIRR, mannose receptor (MR), asialoglycoprotein receptor family (e.g., asialoglycoprotein receptor macrophage galactose-type lectin (MGL)), DC-SIGN (CLEC4L), Langerin (CLEC4K), myeloid DAP12-associated lectin (MDL)-1 (CLEC5A), Dectin 1 / CLEC7A, DNGR1 / CLEC9A, myeloid C-type lectin-like receptor (MICL) (CLEC12A), CLEC2 (also called CLEC1B), CLEC12B, DCIR / CLEC4A, Dectin2 / CLEC6A, blood DC antigen 2 (BDCA2) (CLEC4C), macrophage-inducible C-type lectin (CLEC4E), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, FcγRI (CD64), FcγRIIA (CD32),FcγRIIB1 (CD32), FcγRIIB2 (CD32), FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI, FcεRII (CD23), FcαR1 (CD89), Fcα / μR, FcRn, CD27, CD40, OX40, GITR, CD137, PD-1, CTLA-4, PD-L1, TIGIT, T-cell immunoglobulin domain and mucin domain 3 (TIM3), V-domain Ig inhibitor of T-cell activation (VISTA), CD28, CD122, ICOS, A2AR, B7-H3, B7-H4, B and and T-lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), FAM159B, HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, gp130, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4 receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor, IL-9 receptor, IL-1 0 receptor, IL-11 receptor, IL-12 receptor, IL-13 receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17 receptor, IL-18 receptor, IL-19 receptor, IL-20 receptor, IL-21 receptor, IL-22 receptor, IL-23 receptor, IL-24 receptor, IL-25 receptor, IL-26 receptor, IL-27 receptor, IL-28 receptor, IL-29 receptor, IL-30 receptor, IL-31 receptor, IL-32 receptor, IL-33 receptor, IL-35 receptor, IL-36 receptor, FGFR1, FGFR2, FGFR3, F GFR4, TNFRSF1A, TNFRSF1B, TNFRSF3, TNFRSF4, TNFRSF5, TNFRSF6, TNFRSF6 B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF1 0D, TNFRSF11A, TNFRSF11B, TNFRSF12A, TNFRSF13B, TNFRSF13C, TNFRSF14, TNFRSF16, TNFRSF17, TNFRSF18, TNFRSF19, TNFRSF21, TNFRSF25, TNFRSF27,SCN1A、SCN1B、SCN2A、SCN2B、SCN3A、SCN3B、SCN4A、SCN5A、SCN7A、SCN8A、SCN9A、SCN10A、SCN11A、CACNA1A、CACNA1B、CACNA1C、CACNA1D、CACNA1E、CACNA1F、CACNA1G、CACNA1H、CACNA1I、CACNA1S、TRPA1、TRPC1、TRPC2、TRPC3、TRPC4、TRPC5、TRPC6、TRPC7、TRPM1、TRPM2、TRPM3、TRPM4、TRPM5、TRPM6、TRPM7、TRPM8、MCOLN1、MCOLN2、MCOLN3、PKD1、PKD2、PKD2L1、PKD2L2、TRPV1、TRPV2、TRPV3、TRPV4、TRPV5、TRPV6、CATSPER1、CATSPER2、CATSPER3、CATSPER4、TPCN1、TPCN2、CNGA1、CNGA2、CNGA3、CNGA4、CNGB1、CNGB3、HCN1、HCN2、HCN3、HCN4、KCNMA1、KCNN1、KCNN2、KCNN3、KCNN4、KCNT1、KCNT2、KCNU1、KCNA1、KCNA2、KCNA3、KCNA4、KCNA5、KCNA6、KCNA7、KCNA10、KCNB1、KCNB2、KCNC1、KCNC2、KCNC3、KCNC4、KCND1、KCND2、KCND3、KCNF1、KCNG1、KCNG2、KCNG3、KCNG4、KCNH1、KCNH2、KCNH3、KCNH4、KCNH5、KCNH6、KCNH7、KCNH8、KCNQ1、KCNQ2、KCNQ3、KCNQ4、KCNQ5、KCNS1、KCNS2、KCNS3、KCNV1、KCNV2、KCNJ1、KCNJ2、KCNJ3、KCNJ4、KCNJ5、KCNJ6、KCNJ8、KCNJ9、KCNJ10、KCNJ11、KCNJ12、KCNJ13、KCNJ14、KCNJ15、KCNJ16、KCNJ18、KCNK1、KCNK2、KCNK3、KCNK4、KCNK5、KCNK6、KCNK7、KCNK9、KCNK10、KCNK12、KCNK13、KCNK15、KCNK16、KCNK17、KCNK18、HVCN1、HTR3A、HTR3B、HTR3C、HTR3D、HTR3E、CHRNA1、CHRNA2、CHRNA3、CHRNA4、CHRNA5、CHRNA6、CHRNA7、CHRNA9、CHRNA10、CHRNB1、CHRNB2、CHRNB3、CHRNB4、CHRND、CHRNE、CHRNG、GABRA1、GABRA2、GABRA3、GABRA4、GABRA5、GABRA6、GABRB1、GABRB2、GABRB3、 GABRD, GABRE, GABRG1, GABRG2, GABRG3, GABRP, GABRQ, GABRR1, GABRR2, GA BRR3, GRIA1, GRIA2, GRIA3, GRIA4, GRID1, GRID2, GRIK1, GRIK2, GRIK3, GRI K4, GRIK5, GRIN1, GRIN2A, GRIN2B, GRIN2C, GRIN2D, GRIN3A, GRIN3B, GLRA1 , GLRA2, GLRA3, GLRA4, P2RX1, P2RX2, P2RX3, P2RX4, P2RX5, P2RX6, P2RX7, Z ACN, ASIC1, ASIC2, ASIC3, ASIC4, AQP1, AQP2, AQP3, AQP4, AQP5, AQP6, AQP7, AQP8, AQP9, AQP10, AQP11, AQP12A, AQP12B, MIP, CLCN1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, CLCNKB, cystic fibrosis transmembrane conductance regulator (CFTR), ANO1, ANO2, ANO3, ANO4, ANO5, ANO6, ANO7, ANO8, ANO9, ANO10, BEST1, BEST2, B EST3, BEST4, CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, CLIC6, GJA1, GJA3, GJA4, GJA5, GJA6P, GJA8, GJA9, GJA10, GJB1, GJB2, GJB3, GJB4, GJB5, GJB6, GJB7, GJC1, GJC2, GJC3, GJD2, GJD3, GJD4, GJE1, ITPR1, ITPR2, ITPR3, PANX1, PAN X2, PANX3, RYR1, RYR2, RYR3, NALCN, SCNN1A, SCNN1B, SCNN1D, SCNN1G, TEM1 6A, ADAMTS7, ANGPTL3, ANGPTL4, ANGPTL8, LPL, GDF15, galectin-1, galectin-2, galectin-3, galectin-4, galectin-7, galectin-8, galectin-9, galectin-10, galectin-12, galectin-13, matrix gla protein (MGP), PRNP, DGAT1, GPAT3, DMC1, BLM, BRCA2, members of the human endogenous retrovirus type K (HERV-K) family, ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1),Ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2), SLC1A1, SLC1A2, SLC1A3, SLC1A4, SLC1A5, SLC1A6, SLC1A7, SLC2A1, SLC2A2, SLC2A3, SLC2A4, SLC2A5, SLC2A6, SLC2A7, SLC2A8, SLC2A9, SLC2A10, SLC2A11, SLC2A12, SLC2A13, SLC2A14, SLC3A1, SLC3A2, SLC4A1, SLC4A2, SLC4A3, SLC4A4, SLC4A5, SLC4A6, SLC4A7, SLC4A8, SLC4A9, SLC4A10, SLC4A11, SLC5A1, SLC5A2, SLC5A3, SLC5A4, SLC5A5, SLC5A6, SLC5A7, SLC5A8, SLC5A9, SLC5A10, SLC5A11, SLC5A12, SLC6A1, SLC6A2, SLC6A3, SLC6A4, SLC6A5, SLC6A6, SLC6A7, SLC6A8, SLC6A9, SLC6A10, SLC6A11, SLC6A12, SLC6A13, SLC6A14, SLC6A15, SLC6A16, SLC6A17, SLC6A18, SLC6A19, SLC6A20, SLC7A5, SLC7A6, SLC7A7, SLC7A8, SLC7A9, SLC7A10, SLC7A11, SLC7A13, SLC7A14, SLC8A1, SLC8A2, SLC8A3, SLC9A1, SLC9A2, SLC9A3, SLC9A4, SLC9A5, SLC9A6, SLC9A7, SLC9A8, SLC9A9, SLC9A10, SLC9A11, SLC9B1, SLC9B2, SLC10A1, SLC10A2, SLC10A3, SLC10A4, SLC10A5, SLC10A6, SLC10A7, SLC11A1, SLC11A2, SLC12A1, SLC12A2, SLC12A3, SLC12A4, SLC12A5, SLC12A6, SLC12A7, SLC12A8, SLC12A9, SLC13A1, SLC13A2, SLC13A3, SLC13A4, SLC13A5, SLC14A1, SLC14A2, SLC15A1, SLC15A2, SLC15A3, SLC15A4, SLC16A1, SLC16A2, SLC16A3, SLC16A4, SLC16A5, SLC16A6, SLC16A7, SLC16A8, SLC16A9,<h2 style=";text-align:left;direction:ltr">SLC16A10、SLC16A11、SLC16A12、SLC1 6A13、SLC16A14、SLC17A1、SLC17A2、S LC17A3、SLC17A4、SLC17A5、SLC17A6、SLC17A7、SLC17A8、SLC18A1 、SLC18A2、SLC19A1、SLC19A2、SLC19A3、SLC20A1、SLC20A2、SLCO1 A2、SLCO1B1、SLCO1B3、SLCO1C1、SLCO2A1、SLCO2B1、SLCO3A1、SLCO4A1、SLCO 4C1、SLCO5A1、SLC22A1、SLC22A2、SLC22A3、SLC22A4、SLC22A5、SL C222A6、SLC22A7、SLC22A8、SLC22A9、SLC22A10、SLC22A11、SLC22A12、SLC22 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 67, 68, 69, 69, 69, 64, 69, 65, 69, 66, 67, 69, 69, 65 C222A19、SLC22A20、SLC22A23、SLC22A24、SLC22A25、SLC22A31、SLC23A1、SLC 233A2、SLC23A3、SLC23A4、SLC24A1、SLC24A2、SLC24A3、SLC24A4、SLC24A5、S LC24A6、SLC25A1、SLC25A2、SLC25A3、SLC25A4、SLC25A5、SLC25A6、SLC25A7 、SLC25A8、SLC25A9、SLC25A10、SLC25A11、SLC25A13、SLC25A14、 5LC25A15、SLC25A16、SLC25A17、SLC25A18、SLC25A19,SLC25A20、SLC25A21、 5LC25A22、SLC25A23、SLC25A24、SLC25A25、SLC25A26、SLC25A27、SLC25A28 、SLC25A29、SLC25A30、SLC25A31、SLC25A32、SLC25A34、SLC25A3 5、SLC25A36、SLC25A37,SLC25A38、SLC25A39、SLC25A40、SLC25A41、SLC25A 42、5SLC25A43、5SLC25A44、5SLC25A45、5SLC25A46、5SLC26A1、5SLC26A2、5SLC26A3、LC26A4、LC26A5、LC26A6、LC26A7、LL26A8、LL26A9、LL26A10、LL26 111、SL"27A1、"""27A2、"""27A3、"""27A4、"""27A5、"""27A6、"""28A1、SL CLCA2、LLLLLL、LLL29A1、LLL29A2、LLL29A3、LLL29A4、LLL30A1、LL30A2、 BLC30A3、BLC30A4、BLC30A5、BLC30A6、BLC30A7、BLC30A8、BLC30A9、BLC30A1 0. - "35B4、SL"35"1、""35"2、"""35#1、""35"、"""35"、"""35"1、""35"2、 LC35E3、LC35E4、LC35K1、LL35K2、LL35K3、LL35K4、LL35K5、LL35G1 、SL"35G3、"""35G4、"""35G5、""35G6、""36A1、""36A2、""36A3、""36 A4、SLC37A1、BLC37A2、BLC37A3、BLC37A4、BLC38A1、BLC38A2、BLC38A3、LC 38A4、AF00050000000000000000000000000000 、SLC39A1、BLC39A2、BLC39A3、BLC39A4、BLC39A5、BLC39A6、BLC39A7、BLC39A 8、SLC39A9、BLC39A10、BLC39A11、BLC39A12、BLC39A13、BLC39A14、BLC40A1 、SLC41A1、LC41A2、LC41A3、GGGGGG、GGGGGGGGGGGGGGGGGGGGGGGGG 、SLC44A1、LC44A2、LC44A3、LC44A4、LC44A5、LC45A1、LC45A2、LC45 А3、SLC45A4、BLC46A1、BLC46A2、BLC46A3、BLC47A1、BLC47A2、HC--、5I-1、2. The method of claim 1, wherein the target polypeptide is selected from MFSD10, SLC50A1, OSTA, OSTβ, SLC52A1, SLC52A2, and SLC52A3.

5. The method of claim 1, wherein the administration is parenteral, intravenous, intramuscular, intranasal, or subcutaneous.

6. The method of claim 1, wherein the complex comprises mRNA comprising a nucleic acid sequence of SEQ ID NO: 7, or any one of SEQ ID NOs: 5, 7 and 40.

7. The method of claim 1, wherein the complex has a diameter of about 30 to 150 nm.

8. The method of claim 1, wherein the complex comprises a helper lipid.

9. The complex comprising a combination of (i) a cationic lipid, (ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), (iii) cholesterol, and (iv) lipidated polyethylene glycol (PEG lipid); The mRNA is (i) the consensus Kozak sequence; (ii) a 7-methylguanosine cap on the 5' end of the mRNA; (iii) a polyadenosine (poly A) tail found at the 3' end of the mRNA transcript, the poly A tail being between 60 bps and 120 bps; and (iv) comprising a 5' untranslated region (UTR) derived from Tobacco etch virus (TEV) and a tandem repeat of the 3' UTR found in human β-globin; 2. The method of claim 1, wherein the target protein is selected from the group consisting of RXFP1, SLC52A2, ANGPTL8, Galectin-3, TSHR, APJ and GP130.

10. The method of claim 1, wherein the animal is administered 10 μg, 12.5 μg, 20 μg, 25 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg of mRNA.

11. The cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), dilauryl (C 12:0 ) trimethylammonium propane (DLTAP), dioctadecylamidoglycylspermine (DOGS), DC-Chol, dioleoyloxy-N-[2-sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy) 10. The method of claim 9, wherein the hydroxyl group is selected from the group consisting of N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 2-[5'-(cholest-5-en-3β-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA) and N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP).