Polynucleotides encoding ligation antigens and uses thereof

Abstract

The present disclosure provides an isolated polynucleotide comprising a plurality of linked coding regions. In some aspects, a polynucleotide comprises at least a first coding region encoding a first antigen and a second coding region encoding a second antigen wherein the first antigen and the second antigen are not identical, and wherein the first coding region and the second coding region are linked. The disclosure also relates to the use of such polynucleotides to express multiple antigens in a cell and induce a multi-antigen-specific immune response in vivo.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to U.S. Provisional Application No. 63 / 369,726, filed on July 28, 2022, which is incorporated herein by reference in its entirety.

[0003] References to sequence listings submitted electronically

[0004] The contents of the sequence listing are submitted electronically (name: 4821_086PC01_SequenceListing_ST26.XML; size: 36,597 bytes; and creation date: July 28, 2023) and submitted with this application, and are incorporated herein by reference in their entirety. Field of the Invention

[0005] The present disclosure generally relates to polynucleotides (eg, isolated polynucleotides) comprising multiple nucleotide sequences encoding antigens, wherein the multiple nucleotide sequences are linked (eg, via a linker). Background of the Invention

[0007] Cancer remains one of the leading causes of death in the modern world. Standard treatments currently implemented in the clinic include surgery, radiation, chemotherapy, and immunotherapy, which show limited success. These therapies are generally only effective for early localized tumors and rarely for advanced metastatic malignancies, leading to frequent recurrences or eventual resistance to therapy. Sharma, P. et al., Cell 168(4): 707-723 (2017). In addition, various agents used in radiation and chemotherapy can damage normal tissues, which can lead to adverse side effects. Therefore, cancer patients still need new treatment options with an acceptable safety profile and high efficacy. Summary of the invention

[0008] A kind of isolated polynucleotide is provided herein, the isolated polynucleotide comprises a single ORF, the ORF has a first nucleotide sequence encoding a first antigen ("first coding region") and a second nucleotide sequence encoding a second antigen ("second coding region"), wherein the first coding region and the second coding region are connected. In some aspects, the first coding region and the second coding region are connected by a joint.

[0009] In some aspects, the first coding region and the second coding region are arranged in the following order: the first coding region is upstream of the second coding region. In some aspects, the order of the first coding region and the second coding region is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0010] In some aspects, the single ORF of the polynucleotide also comprises one or more additional nucleotide sequences ("additional coding regions") encoding additional antigens. In some aspects, the additional antigens: (i) are different from the first antigen, (ii) are different from the second antigen, or (iii) are different from the first antigen and the second antigen. In some aspects, the single ORF of the polynucleotide comprises at least two additional coding regions, at least three additional coding regions, at least four additional coding regions, at least five additional coding regions, at least six additional coding regions, at least seven additional coding regions, at least eight additional coding regions, at least nine additional coding regions, or at least ten additional coding regions. In some aspects, the additional coding region is connected to the first coding region or the second coding region. In some aspects, the additional coding region is connected to the first coding region or the second coding region by a joint.

[0011] In some aspects, the first coding region, the second coding region, and the additional coding region are arranged in the following order: (a) (first coding region)-L1-(second coding region)-L2-(additional coding region); (b) (first coding region)-L1-(additional coding region)-L2-(second coding region); or (c) (additional coding region)-L1-(first coding region)-L2-(second coding region); wherein L1 is the first joint and L2 is the second joint. In some aspects, the first joint and the second joint are the same. In some aspects, the first joint and the second joint are not the same. In some aspects, the order of the first coding region, the second coding region, and the third coding region is different as compared to the corresponding order present in the reference polynucleotide.

[0012] In some aspects, the first antigen is about 25 amino acids long. In some aspects, the second antigen is about 25 amino acids long. In some aspects, the additional antigen is about 25 amino acids long. In some aspects, the first antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the second antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the additional antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof.

[0013] In some aspects, the cancer antigen comprises a KRAS antigen. In some aspects, the non-self antigen is derived from a pathogen selected from the group consisting of a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis B virus (HBV) antigen, or a combination thereof.

[0014] In some aspects, the KRAS antigen comprises an amino acid sequence that differs in sequence as compared to the amino acid sequence of the corresponding wild-type KRAS antigen. In some aspects, the amino acid sequence of the KRAS antigen has less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, or less than about 90% sequence identity as compared to the amino acid sequence of the corresponding wild-type KRAS antigen. In some aspects, the amino acid sequence of the KRAS antigen comprises an amino acid substitution selected from the group consisting of K5E, K5N, G10GG, G10V, G12A, G12C, G12D, G12F, G12I, G12L, G12R, G12S, G12V, G13C, G13D, G13E, G13R, G13V, V14I, L19F, T20M, Q22E, Q22H, Q22K, Q22R, Q25H, N26Y, F28L, E31K, D33E, P34L, P34Q, P34R, I36M, R41K, D57 N, T58I, A59T, G60D, G60R, G60S, G60V, Q61A, Q61H, Q61K, Q61L, Q61P, Q61R, E63K, S65N, R68S, Y71H, T74A, L79I, R97I, Q99E, M111L, K117N, K117R, D119G, S122F, T144P, A146P, A146T, A146V, K147E, K147T, R149K, L159S, I163S, R164Q, I183N, I84M, or a combination thereof. In some aspects, the KRAS antigen comprises one or more of: G12D 1-16 、G12D 2-19 、G12D 2-22 、G12D 2-29 、G12V 1-16 、G12V 2-19 、G12V 3-17 or G12V 3-42 antigen.

[0015] In any of the above polynucleotides, in some aspects, the linker comprises a peptide linker. In some aspects, the peptide linker comprises a G4S linker or an EAAAK linker.

[0016] Some aspects of the present disclosure relate to a kind of isolated polynucleotide, the isolated polynucleotide comprises a single ORF, the ORF has a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), and a third nucleotide sequence encoding a third antigen ("third coding region"), wherein the first coding region is connected to the second coding region by a first joint, and wherein the second coding region is connected to the third coding region by a second joint. In some aspects, the first coding region, the second coding region, and the third coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0017] Also provided herein is a separated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), and a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), wherein the first coding region is connected to the second coding region by a first joint, wherein the second coding region is connected to the third region by a second joint, and wherein the third coding region is connected to the fourth coding region by a third joint. In some aspects, the first coding region, the second coding region, the third coding region, and the fourth coding region are arranged in order, wherein the order is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0018] Also provided herein is a separated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), and a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), wherein the first coding region is connected to the second coding region by a first joint, wherein the second coding region is connected to the third coding region by a second joint, wherein the third coding region is connected to the fourth coding region by a third joint, and wherein the fourth coding region is connected to the fifth coding region by a fourth joint. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are arranged in order, wherein the order is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0019] Also provided herein is a separated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), and a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), wherein the first coding region is connected to the second coding region by a first joint, wherein the second coding region is connected to the third coding region by a second joint, wherein the third coding region is connected to the fourth coding region by a third joint, wherein the fourth coding region is connected to the fifth coding region by a fourth joint, and wherein the fifth coding region is connected to the sixth coding region by a fifth joint. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, and the sixth coding region are arranged in order, wherein the order is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0020] Also provided herein is a separated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), and a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), wherein the first coding region is connected to the second coding region by a first joint, wherein the second coding region is connected to the third coding region by a second joint, wherein the third coding region is connected to the fourth coding region by a third joint, wherein the fourth coding region is connected to the fifth coding region by a fourth joint, wherein the fifth coding region is connected to the sixth coding region by a fifth joint, and wherein the sixth coding region is connected to the seventh coding region by a sixth joint. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are arranged in order, wherein the order is different as compared to the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0021] Also provided herein is a separated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), and a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), wherein the first coding region is connected to the second coding region by a first joint, wherein the second coding region is connected to the third coding region by a second joint, wherein the third coding region is connected to the fourth coding region by a third joint, wherein the fourth coding region is connected to the fifth coding region by a fourth joint, and wherein the fifth coding region is connected to the sixth coding region by a fifth joint. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, and the sixth coding region are arranged in order, wherein the order is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0022] Also provided herein is a separated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), and a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), wherein the first coding region is connected to the second coding region by a first joint, wherein the second coding region is connected to the third coding region by a second joint, wherein the third coding region is connected to the fourth coding region by a third joint, wherein the fourth coding region is connected to the fifth coding region by a fourth joint, wherein the fifth coding region is connected to the sixth coding region by a fifth joint, and wherein the sixth coding region is connected to the seventh coding region by a sixth joint. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are arranged in order, wherein the order is different as compared to the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0023] Also provided herein is an isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), and an eighth nucleotide sequence encoding an eighth antigen ("eighth coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third coding region via a second linker, wherein the third coding region is connected to the fourth coding region via a third linker, wherein the fourth coding region is connected to the fifth coding region via a fourth linker, wherein the fifth coding region is connected to the sixth coding region via a fifth linker, wherein the sixth coding region is connected to the seventh coding region via a sixth linker, and wherein the seventh coding region is connected to the eighth coding region via a seventh linker. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, and the eighth coding region are arranged in an order, wherein the order is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises the corresponding polynucleotide occurring in nature.

[0024] Provided herein is an isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), an eighth nucleotide sequence encoding an eighth antigen ("eighth coding region"), and a ninth nucleotide sequence encoding a ninth antigen ("ninth coding region"), wherein the first coding region is linked to the second coding region via a first linker, wherein the second coding region is linked to the third coding region via a second linker, wherein the third coding region is linked to the fourth coding region via a third linker, wherein the fourth coding region is linked to the fifth coding region via a fourth linker, wherein the fifth coding region is linked to the sixth coding region via a fifth linker, wherein the sixth coding region is linked to the seventh coding region via a sixth linker, wherein the seventh coding region is linked to the eighth coding region via a seventh linker, and wherein the eighth coding region is linked to the ninth coding region via an eighth linker. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, and the ninth coding region are arranged in an order, wherein the order is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises the corresponding polynucleotide occurring in nature.

[0025] Provided herein is an isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), an eighth nucleotide sequence encoding an eighth antigen ("eighth coding region"), a ninth nucleotide sequence encoding a ninth antigen ("ninth coding region"), and a ninth nucleotide sequence encoding a ninth antigen. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, the ninth coding region, and the tenth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0026] In any of the above polynucleotides, in some aspects, the first antigen is about 25 amino acids long. In some aspects, the second antigen is about 25 amino acids long. In some aspects, the third antigen is about 25 amino acids long. In some aspects, the fourth antigen is about 25 amino acids long. In some aspects, the fifth antigen is about 25 amino acids long. In some aspects, the sixth antigen is about 25 amino acids long. In some aspects, the seventh antigen is about 25 amino acids long. In some aspects, the eighth antigen is about 25 amino acids long. In some aspects, the ninth antigen is about 25 amino acids long. In some aspects, the tenth antigen is about 25 amino acids long.

[0027] In some aspects, the first antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the second antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the third antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the fourth antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the fifth antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the sixth antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the seventh antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the eighth antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the ninth antigen comprises a cancer antigen, a non-self antigen, an autoantigen associated with a tumor, a disease-associated antigen, or a combination thereof. In some aspects, the tenth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

[0028] In some aspects, the cancer antigen comprises a KRAS antigen. In some aspects, the non-self antigen is derived from a pathogen selected from the following: a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis B virus (HBV) antigen, or a combination thereof. In some aspects, the KRAS antigen comprises an amino acid sequence that is different from the amino acid sequence of the corresponding wild-type KRAS antigen. In some aspects, the amino acid sequence of the KRAS antigen has less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85% or less than about 90% sequence identity compared to the amino acid sequence of the corresponding wild-type KRAS antigen. In some aspects, the amino acid sequence of the first KRAS antigen comprises an amino acid substitution selected from the group consisting of K5E, K5N, G10GG, G10V, G12A, G12C, G12D, G12F, G12I, G12L, G12R, G12S, G12V, G13C, G13D, G13E, G13R, G13V, V14I, L19F, T20M, Q22E, Q22H, Q22K, Q22R, Q25H, N26Y, F28L, E31K, D33E, P34L, P34Q, P34R, 136M, R41K, D5 7N, T58I, A59T, G60D, G60R, G60S, G60V, Q61A, Q61H, Q61K, Q61L, Q61P, Q61R, E63K, S65N, R68S, Y71H, T74A, L79I, R97I, Q99E, M111L, K117N, K117R, D119G, S122F, T144P, A146P, A146T, A146V, K147E, K147T, R149K, L159S, I163S, R164Q, I183N, I84M, or a combination thereof. In some aspects, the KRAS antigen comprises one or more of: G12D 1-16 、G12D 2-19 、G12D 2-22 、G12D 2-29 、G12V 1-16 、G12V 2-19 、G12V 3-17 or G12V 3-42 antigen.

[0029] In some aspects, any one of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth linker comprises a peptide linker. In some aspects, the peptide linker comprises a G4S linker or an EAAAK linker.

[0030] For any polynucleotide described herein, in some aspects, the polynucleotide further comprises one or more of the following components: (1) an internal ribosome entry site (IRES), (2) an intron sequence, (3) a homology arm, (4) a promoter, (5) an enhancer, (6) a UTR, (7) a sequence encoding a signal peptide, (8) a translation initiation sequence, (9) a 3′ tailing region for linking nucleosides, (10) a 5′ cap, (11) a sequence encoding a 2A ribosomal skipping peptide, or (12) any combination of (1) to (11). In some aspects, the polynucleotide further comprises at least one modified nucleoside. In some aspects, the at least one modified nucleoside comprises 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudouridine, inosine, α-thio-guanosine, 8-oxo- Guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine or a combination thereof.

[0031] In some aspects, the polynucleotides described herein are mRNA.

[0032] A carrier is also provided herein, and the carrier comprises any polynucleotide described herein. Some aspects of the present disclosure relate to cells, and the cells comprise any polynucleotide described herein. In some aspects, the cells comprise stem cells, somatic cells, or both. In some aspects, stem cells comprise induced pluripotent stem cells (iPSC), embryonic stem cells, tissue-specific stem cells, mesenchymal stem cells, or a combination thereof. In some aspects, somatic cells comprise blood cells. In some aspects, blood cells comprise PBMC. In some aspects, PBMC comprise immune cells. In some aspects, immune cells comprise T cells, B cells, natural killer (NK) cells, dendritic cells (DC), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof. In some aspects, the cells have passed through the contraction under a set of parameters, thereby causing disturbances in the cells, so that the polynucleotides enter the cells by disturbances when in contact with the cells.

[0033] Also provided herein is a pharmaceutical composition comprising any polynucleotide, vector or cell described herein and a pharmaceutically acceptable carrier. Also provided herein is a kit comprising any polynucleotide, vector or cell described herein.

[0034] Provided herein is a method of preparing a polynucleotide, the method comprising enzymatically or chemically synthesizing any of the polynucleotides described herein.

[0035] Also provided herein is a method for inducing multiple antigens to be expressed in cells, the method comprising delivering the polynucleotides described herein to cells in cells. In some aspects, the multiple antigens are expressed in cells simultaneously after intracellular delivery. In some aspects, the polynucleotides are delivered to cells in cells including passing a cell suspension comprising cells through a contraction under a set of parameters, thereby causing disturbances in the cells, so that the polynucleotides enter the cells by disturbances when in contact with the cells.

[0036] In some aspects, the method further comprises contacting the cell with the polynucleotide. In some aspects, contacting the cell with the polynucleotide comprises incubating a cell suspension with the polynucleotide so that the cell and the polynucleotide are in contact. In some aspects, contact occurs before the cell suspension passes through the constriction. In some aspects, contact occurs during the cell suspension passes through the constriction. In some aspects, contact occurs after the cell suspension passes through the constriction.

[0037] In some aspects, the set of parameters for passing cells through the constriction is selected from cell density; pressure; length, width and / or depth of the constriction; diameter of the constriction; diameter of the cells; temperature; entrance angle of the constriction; exit angle of the constriction; length, width and / or width of the approach zone; surface properties of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration; viscosity, osmolarity, salt concentration, serum content and / or pH of the cell suspension; time in the constriction; shear rate in the constriction; type of payload, or a combination thereof.

[0038] In some aspects, the cell density is at least about 6 x 10 7 cells / mL, at least about 7x10 7 cells / mL, at least about 8x10 7 cells / mL, at least about 9x10 7 cells / mL, at least about 1x10 8 cells / mL, at least about 1.1x10 8 cells / mL, at least about 1.2x10 8 cells / mL, at least about 1.3x10 8cells / mL, at least about 1.4x10 8 cells / mL, at least about 1.5x10 8 cells / mL, at least about 2.0x10 8 cells / mL, at least about 3.0x10 8 cells / mL, at least about 4.0x10 8 cells / mL, at least about 5.0x10 8 cells / mL, at least about 6.0x10 8 cells / mL, at least about 7.0x10 8 cells / mL, at least about 8.0x10 8 cells / mL, at least about 9.0x10 8 cells / mL or at least about 1.0x10 9 In some aspects, the pressure is at least about 30 psi, at least about 35 psi, at least about 40 psi, at least about 45 psi, at least about 50 psi, at least about 55 psi, at least about 60 psi, at least about 65 psi, at least about 70 psi, at least about 75 psi, at least about 80 psi, at least about 85 psi, at least about 90 psi, at least about 95 psi, at least about 100 psi, at least about 110 psi, at least about 120 psi, at least about 130 psi, at least about 140 psi, or at least about 150 psi.

[0039] In some aspects, the constriction is contained in the microfluidic chip. In some aspects, the diameter of the constriction is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the diameter of the cell. In some aspects, the width of the constriction is between about 0 μm and about 10 μm. In some aspects, the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm or less than about 10 μm. In some aspects, the length of the constriction is between about 0 μm and about 100 μm. In some aspects, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some aspects, the depth of the constriction is between at least about 1 μm and at least about 120 μm. In some aspects, the depth of the constriction is at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm, or at least about 120 μm.

[0040] In some aspects, a cell suspension comprising cells is passed through a plurality of constrictions. In some aspects, the plurality of constrictions comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more individual constrictions. In some aspects, each of the plurality of constrictions is identical. In some aspects, one or more of the plurality of constrictions are different. In some aspects, the one or more constrictions are different in length, depth, width, or a combination thereof.

[0041] The present disclosure also provides a method of inducing a multispecific immune response in a subject in need thereof, the method comprising administering to the subject any polynucleotide, vector, cell or pharmaceutical composition described herein. In some aspects, the multispecific immune response comprises a CD8+ T cell response.

[0042] Also provided herein is a method of inducing an enhanced immune response in a subject in need thereof, the method comprising administering to the subject any polynucleotide, vector, cell or pharmaceutical composition described herein. In some aspects, the enhanced immune response comprises: (i) an increase in the magnitude of the induced immune response as compared to a reference immune response, (ii) an increase in the breadth of the induced immune response as compared to a reference immune response, (iii) an increase in the duration of the induced immune response as compared to a reference immune response, or (iv) any combination of (i) to (iii); wherein the reference immune comprises an immune response observed in a corresponding subject who has not received administration of the polynucleotide or modified cell.

[0043] Provided herein is a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject any polynucleotide, vector, cell, or pharmaceutical composition described herein. In some aspects, the disease or disorder comprises cancer. In some aspects, the cancer is associated with abnormal KRAS expression. In some aspects, the disease or disorder is associated with a non-self antigen. In some aspects, the non-self antigen is derived from a virus. In some aspects, the virus comprises HPV, HIV, or HBV. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A Three junction antigen mRNA constructs are shown, each encoding five antigens (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D ) is a fragment of about 25 amino acids in length of each of the .

[0045] Figure 1B Shown in the use Figure 1A After each of the three different connection antigen mRNA constructs shown in the co-culture of PMBCs squeezed, E6 TCR Jurkat cells (left figure), E7 TCR Jurkat cells (middle figure) and pp65 specific Cellero response T cells (right figure) activation. The control shown includes the response cells treated with one of the following: (i) unmodified PBMC (i.e., no squeezing and no polynucleotide) ("NC"), (ii) PBMCs not squeezed with any mRNA ("empty"), and (iii) NC PBMCs (for each different cell line, the second bar on the right) mixed with the respective minimal epitopes. Untreated response cells are also used as controls (last bar on the right).

[0046] Figure 2A Three conjugate antigen mRNA constructs are shown, two of which encode 10 antigens (HPV16 E6, HPV16 E7, CMV pp65, KRAS Gl2V 、KRAS G12D , Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S, and MAGE-A10), and a third mRNA construct encoded 5 antigens (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D ) is a fragment of about 25 amino acids in length of each of the .

[0047] Figure 2B Shown in the use Figure 2A Activation of E6 TCR Jurkat cells (left) and E7 TCR Jurkat cells (right) after co-culture with PMBCs extruded with each of the three different linked antigen mRNA constructs shown in . Figure 1B Same as those described in .

[0048] Figure 2C Shown in the use Figure 2AActivation of pp65-specific Cellero-responsive T cells (left), M1-specific Flu Cellero-responsive T cells (middle), and NY-ESO-1-specific Cellero-responsive T cells (right) after co-culture with PMBCs extruded with each of the three different linked antigen mRNA constructs shown in . Figure 1B Same as those described in .

[0049] Figure 3A Five junction antigen mRNA constructs are shown, three of which encode five antigens (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D ), and two of them encode 10 antigens arranged in different orders (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V 、KRAS G12D , Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S and MAGE-A10).

[0050] Figure 3B Shown are Western blots of two SDS-PAGE gels on which samples of wheat germ extract were run, which translated Figure 3A Each of the five linked antigen mRNA constructs shown in . 29-38 The gel was blotted with primary antibody against SLP (clone 5G10) and goat anti-rabbit secondary antibody.

[0051] Figure 4 Shown in the use Figure 1A After co-culture of extruded PMBCs with each of the three different linked antigen mRNA constructs shown in G12V Activation of TCR Jurkat cells. Control vs. Figure 1B Same as those described in .

[0052] Figure 5A CD8 T cells expressing E6 29-38 Specific TCR or E7 11-19 Schematic diagram of lentiviral transduction of specific TCR and activation by co-culture with PMBCs extruded with junctional antigen mRNA, junctional antigen mRNA and signal 2 / 3 mRNA, or signal 2 / 3 mRNA alone. Cells were cultured for 6 days and then treated with the corresponding E6 29-38 or E7 11-19Peptide restimulation was performed and cytokine production was measured by intracellular cytokine staining (ICS).

[0053] Figure 5B Shown is the activation of E6 TCR-transduced and E7 TCR-transduced CD8+ T cells after co-culture with PMBCs extruded with both junctive antigen mRNA and signal 2 / 3 mRNA, compared to junctive antigen mRNA or signal 2 / 3 mRNA alone.

[0054] Fig. 6A and Figure 6B Figure 3 shows G12V and G12D specific responses in A*11 restricted G12V and G12D TCR Jurkat responder cells, respectively, cultured with PBMCs extruded with one of the following mRNA constructs: (1) ligated mRNA constructs encoding: (a) KRAS G12D and KRAS G12V The antigen is a fragment of about 25aa (overlapping with the 1-25aa sequence in the entire KRAS protein and with a mutation at codon 12), (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("junction antigen +2 / 3"); (2) only encodes KRAS G12D and KRAS G12V(i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3); (4) a concatenated mRNA construct encoding the following: NA constructs: (a) a single approximately 25 aa fragment comprising the G12V mutation, (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("G12V+2 / 3"); (5) a concatenated mRNA construct encoding: (a) a single approximately 25 aa fragment comprising the G12D mutation, (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3). (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("G12D+2 / 3"); (6) a concatenated mRNA construct encoding: (a) an approximately 25 aa fragment overlapping the first 1-25 aa of native wild-type KRAS; (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("WT+2 / 3"); (7) a concatenated mRNA construct encoding only the G12V (8) a non-linked mRNA construct encoding a single approximately 25aa fragment containing a G12D mutation (i.e., no signals 2 and 3) ("G12V"); (8) a non-linked mRNA construct encoding only a single approximately 25aa fragment containing a G12D mutation (i.e., no signals 2 and 3) ("G12D"); and (9) a non-linked mRNA construct encoding only a fragment of approximately 25aa overlapping the first 1-25aa of native wild-type KRAS (i.e., no signals 2 and 3) ("wild type"). G12V and G12D specific responses are shown, as confirmed by luminescent expression. The specific values ​​shown above some bars represent the fold change of luminescent expression observed in responding cells cultured with PBMCs that were not squeezed with mRNA ("empty").

[0055] Fig. 7A and Figure 7B Figure 3 shows G12V and G12D specific responses in A*11 restricted G12V and G12D TCR Jurkat responder cells, respectively, cultured with PBMCs extruded with mRNA constructs encoding seven linked KRAS mutant antigens. The mRNA constructs were used at one of two doses (250 μg / mL and 500 μg / mL) and included the following: (1) encoding only KRAS G12D 、KRAS G12V 、KRAS G12C 、KRASG13D 、KRAS G12A 、KRAS G12R and KRAS G12S The mRNA constructs (“7mut_v1”) of a fragment of approximately 25 aa of the antigen (overlapping with the 1-25 aa sequence in the entire KRAS protein and with a mutation at the 12th codon); and (2) mRNA constructs encoding only KRAS G12S 、KRAS G12R 、KRAS G12A 、KRAS G13D 、KRAS G12C 、KRAS G12V and KRAS G12D A conjugated mRNA construct ("7mut_v2") of an approximately 25aa fragment of the antigen (overlapping the 1-25aa sequence in the entire KRAS protein and with a mutation at the 12th codon). G12V and G12D specific responses are shown, as confirmed by luminescence expression. The specific values ​​shown above some bars represent the fold change of luminescence expression observed in responder cells cultured with PBMCs that were not squeezed with mRNA ("empty").

[0056] Fig. 8A and Figure 8B G12V and G12D specific responses are shown in A*11 restricted G12V and G12D TCR Jurkat responder cells, respectively, which were cultured with PBMCs extruded with mRNA constructs encoding seven linked KRAS mutant antigens and other mRNA constructs encoding signals 2 (i.e., CD86) and 3 (i.e., membrane-bound IL-2 and IL-12). The specific mRNA constructs are as follows: (1) Linked mRNA constructs encoding: (a) KRAS G12D 、KRAS G12V 、KRAS G12C 、KRAS G13D 、KRAS G12A 、KRAS G12R and KRAS G12S The antigen is a fragment of about 25aa (overlapping with the 1-25aa sequence in the entire KRAS protein and with a mutation at the 12th codon) (called "KRAS 7mut_v1"), (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("7mut_v1+signal 2 / 3"); (2) only encodes KRAS G12D 、KRAS Gl2V 、KRAS G12C 、KRAS G13D 、KRASG12A 、KRAS G12R and KRAS G12S A conjugated mRNA construct ("7mut_v1") encoding a fragment of about 25 aa of the antigen (overlapping with the 1-25 aa sequence in the entire KRAS protein and having a mutation at the 12th codon); (3) a conjugated mRNA construct encoding: (a) KRAS G12S 、KRAS G12R 、KRAS G12A 、KRAS G13D 、KRAS G12C 、KRAS G12V and KRAS G12D The antigen is a fragment of about 25aa (overlapping with the 1-25aa sequence in the entire KRAS protein and with a mutation at the 12th codon) (herein referred to as KRAS 7mut_v2), (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("7mut_v2+signal 2 / 3"); (4) encoding only KRAS G12S 、KRAS G12R 、KRAS G12A 、KRAS G13D 、KRAS G12C 、KRAS G12V and KRAS G12D (a) a fragment of about 25aa of the antigen (overlapping with the 1-25aa sequence in the entire KRAS protein and with a mutation at the 12th codon) ("mut_v2"); and (b) a concatenated mRNA construct encoding only CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("signal 2 / 3 alone"). G12V and G12D specific responses are shown, as confirmed by luminescent expression. The specific values ​​shown above some bars represent the fold change of luminescent expression observed in responder cells cultured with PBMCs that were not squeezed with mRNA ("empty"). DETAILED DESCRIPTION

[0057] The present disclosure is generally directed to isolated polynucleotides that can be used to induce expression of multiple antigens in cells. More particularly, provided herein are polynucleotides that at least comprise a first coding region encoding a first antigen and a second coding region encoding a second antigen, wherein the first antigen and the second antigen are not identical, and wherein the first coding region and the second coding region are connected (e.g., through a joint). As described herein and demonstrated, such polynucleotides can be delivered to cells intracellularly, wherein the cells subsequently express both the first antigen and the second antigen.

[0058] As further described herein, the exemplary delivery methods provided herein (i.e., extrusion delivery) have certain unique characteristics that other non-contraction-mediated delivery methods known in the art do not have. For example, in addition to improving the ability to deliver various types of payloads to cells, the extrusion treatment methods described herein also have minimal lasting effects on cell production. Compared with traditional delivery methods such as electroporation, the extrusion treatment methods disclosed herein retain both the structural and functional integrity of extruded cells. In contrast to the delivery methods provided herein, electroporation can induce extensive and lasting changes in gene expression, which may lead to non-specific activation of cells (e.g., human T cells) and delayed proliferation after antigen stimulation. Using the inventive method, any changes to cells (e.g., disturbances in the cell membrane) are temporary, and once the cells are removed from the contraction, the disturbances will be resealed. The present disclosure shows non-limiting examples of various aspects.

[0059] I. General Technology

[0060] Some of the techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly used using routine methods, such as the widely used methods described in the following: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 2003); the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, J.Wiley and Sons, 1993-8); Handbook of Experimental Immunology (DMWeir and CC Blackwell, 1996); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (J. E. Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., J. Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011).

[0061] II. Definitions

[0062] For purposes of interpreting this specification, the following definitions shall apply and, where appropriate, terms used in the singular shall include the plural and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall prevail. Additional definitions are set forth throughout the Detailed Description.

[0063] As used herein, the singular terms "a", "an", and "the" entity refer to one or more of the entity unless otherwise specified. Thus, the terms "a" (or "the"), "one or more", and "at least one" are used interchangeably herein.

[0064] It should be understood that the disclosed aspects and aspects described herein include "comprising" aspects and aspects, "consisting of" aspects and aspects, and "consisting essentially of aspects and aspects." It should also be understood that whenever aspects and aspects are described herein with the language "comprising," other similar aspects or aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0065] In addition, when used in this article, "and / or" should be considered as specifically disclosing each of the two specified features or components, with or without other features or components. Therefore, as used in phrases such as "A and / or B" herein, the term "and / or" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, as used in phrases such as "A, B, and / or C", the term "and / or" is intended to cover each of the following aspects: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0066] For all compositions described herein, and all methods using the compositions described herein, the compositions may include the listed components or steps, or may "consist essentially of the listed components or steps." When a composition is described as "consisting essentially of the listed components," the composition contains the listed components, and may also contain other components that do not substantially affect the disclosed methods, but does not contain any other components other than those explicitly listed that do substantially affect the disclosed methods; or, if the composition does contain additional components other than those listed that do substantially affect the disclosed methods, the composition does not contain the additional components in sufficient concentrations or amounts to substantially affect the disclosed methods. When a method is described as "consisting essentially of the listed steps," the method contains the listed steps, and may also contain other steps that do not substantially affect the disclosed methods, but the method does not contain any other steps other than those explicitly listed that do substantially affect the disclosed methods. As a non-limiting specific example, when a composition is described as "consisting essentially of one component," the composition may additionally contain any amount of pharmaceutically acceptable carriers, excipients or diluents, and other such components that do not substantially affect the disclosed methods.

[0067] Units, prefixes and symbols are expressed in their form accepted by the International System of Units (SI). Numerical ranges include numbers that define ranges. The headings provided herein are not limitations on the various aspects of the disclosure, which can be obtained by reference to this specification throughout. Therefore, by reference to this specification throughout, the terms defined immediately below are defined in more detail.

[0068] The term "about" is used herein to mean approximately, roughly, approximately, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the numerical values ​​set forth. Typically, the term "about" can modify the numerical value above and below the stated value by, for example, a variance of 10%, up or down (higher or lower).

[0069] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance (ie, capable of inducing an immune response in vitro and / or in vivo), such as a protein, peptide, or hapten. Non-limiting examples of antigens are provided elsewhere in this disclosure.

[0070] As used herein, the term "associated with" refers to a close relationship between two or more entities or characteristics. For example, when used to describe a disease or illness, the term "associated with" means that when a subject exhibits abnormal expression of a protein and / or gene (e.g., a KRAS mutant), the subject has an increased likelihood of developing the disease or illness. In some aspects, abnormal expression of a protein and / or gene causes a disease or illness. In some aspects, abnormal expression does not necessarily cause a disease or illness, but is associated with a disease or illness.

[0071] As used herein, the term "epitope" refers to a portion of a protein that can be recognized by the immune system (eg, antibodies, B cells and / or T cells) and thereby induce an immune response.

[0072] As used herein, the term "linked" refers to a covalent or non-covalent bond formed between a first portion and a second portion (e.g., a first coding region and a second coding region). As further described herein, in some aspects, the first portion and the second portion can be connected with a linker.

[0073] As used herein, the term "constriction" refers to a narrow channel. In some aspects, the constriction is a microfluidic channel, such as a channel contained in a microfluidic device. In some aspects, the constriction is a hole or contained in a hole. When the constriction is a hole, in some aspects, the hole is contained in the surface. Unless otherwise stated, the term constriction refers to both microfluidic channels and holes, as well as other suitable constrictions available in the art. Therefore, where applicable, the disclosures related to microfluidic channels may also apply to holes and / or other suitable constrictions available in the art. Similarly, where applicable, the disclosures related to holes may also apply to microfluidic channels and / or other suitable constrictions available in the art.

[0074] As used herein, the term "pore" refers to an opening, including but not limited to a hole, a crack, a cavity, a pore, a break, a gap, or a perforation in a material. In some aspects, if specified, the term refers to a pore in the surface of a microfluidic device (such as those described in the present disclosure). In some aspects, if specified, a pore can refer to a pore in a cell wall and / or a cell membrane.

[0075] As used herein, the term "membrane" refers to a selective barrier or sheet containing pores. The term includes, but is not limited to, a flexible sheet-like structure that acts as a border or lining. In some aspects, the term refers to a surface or filter containing pores. This term is different from "cell membrane," which refers to a semipermeable membrane surrounding the cytoplasm of a cell.

[0076] As used herein, the term "filter" refers to a porous article that allows selective passage through the pores. In some aspects, the term refers to a surface or membrane containing pores.

[0077] As used herein, the terms "deformation" and "deformation" (including derivatives thereof) refer to physical changes in cells. As described herein, when a cell passes through a constriction (such as those disclosed herein), it is subjected to various forces, including but not limited to mechanical deformation forces and / or shear forces that cause disturbances in the cell membrane due to the constraining physical environment. As used herein, a "disturbance" in a cell membrane refers to any opening in the cell membrane that does not exist under normal steady-state conditions (e.g., no deforming forces are applied to the cell). A disturbance can comprise a hole, a cleft, a cavity, a pore, a hole, a fracture, a gap, a perforation, or a combination thereof.

[0078] As used herein, the term "polynucleotide" or "nucleic acid" refers to the polymer form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes but is not limited to single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. The backbone of a polynucleotide can include sugar and phosphate groups (usually found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can include a polymer (such as phosphoramidate) of a synthetic subunit, and therefore can be oligodeoxynucleoside phosphoramidate (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. In addition, a double-stranded polynucleotide can be obtained from a chemically synthesized single-stranded polynucleotide product by synthesizing a complementary chain and annealing the chain under appropriate conditions, or by using a DNA polymerase to synthesize a complementary chain from the beginning with a suitable primer. As described herein, the nucleic acid that can be delivered to a cell using the extrusion treatment method provided herein includes RNA (for example, mRNA). As used herein, "RNA" includes self-amplifying RNA (e.g., self-amplifying mRNA) and non-self-amplifying RNA (e.g., non-self-amplifying mRNA). As used herein, the term "self-amplifying RNA" refers to an RNA molecule that can replicate in a host, thereby resulting in an increase in the amount of RNA and the protein (e.g., antigen) encoded by the RNA. As used herein, the term "mRNA" refers to any polynucleotide (self-amplifying or non-self-amplifying) encoding at least one polypeptide.

[0079] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), as long as the protein maintains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host that produces the protein or errors due to PCR amplification.

[0080] As used herein, "immune response" refers to a biological response in a vertebrate body to foreign agents or abnormalities (e.g., cancer cells), which protects the organism from these agents and diseases caused by them. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in selective targeting, binding to, damage, destruction, and / or elimination of invasive pathogens, cells or tissues infected by pathogens, cancer cells, or other abnormal cells, or (in the case of autoimmunity or pathological inflammation) normal human cells or tissues. An immune response includes, for example, activation or suppression of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+T cells, or Treg cells, or activation or suppression of any other cells of the immune system, such as NK cells. Therefore, the immune response can include a humoral immune response (e.g., mediated by a B cell), a cellular immune response (e.g., mediated by a T cell), or both a humoral and cellular immune response. In some aspects, the immune response induced by the polynucleotides described herein includes a T cell response. In some aspects, the T cell response is mediated by CD8+T cells. In some aspects, the T cell response is mediated by CD4+T cells. In some aspects, the T cell response is mediated by both CD8+ and CD4+T cells.

[0081] III. Polynucleotides

[0082] The present disclosure is directed to polynucleotides (e.g., isolated polynucleotides) comprising a plurality of nucleotide sequences (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten), wherein each of the plurality of nucleotide sequences encodes an antigen. Nucleotide sequences encoding an antigen are also referred to herein as "coding regions." As demonstrated and further described herein, the polynucleotides of the present disclosure differ (in structure and / or function) as compared to corresponding polynucleotides naturally occurring in nature.

[0083] Coding region:

[0084] In some aspects, the polynucleotides described herein include coding regions from multiple proteins in a single open reading frame (ORF), wherein the coding regions do not naturally exist together in nature (e.g., within a single polynucleotide). For example, in some aspects, the polynucleotides of the present disclosure include a single ORF having a first coding region and a second coding region, wherein the first coding region can encode a cancer antigen and the second coding region can encode different cancer antigens. For example, in some aspects, the polynucleotides of the present disclosure include a first coding region and a second coding region, wherein the first coding region can encode a viral antigen and the second coding region can encode different viral antigens. For example, in some aspects, the polynucleotides of the present disclosure include a single ORF having a first coding region and a second coding region, wherein the first coding region can encode a cancer antigen (e.g., a KRAS antigen) and the second coding region can encode a viral antigen (e.g., HPV, HIV, or HBV). Non-limiting examples of antigens that can be used for the present disclosure are provided elsewhere in the present disclosure. It is obvious to those skilled in the art that such polynucleotides can be particularly used to simultaneously combat a variety of different pathogens and / or diseases.

[0085] In some aspects, the polynucleotides described herein include coding regions, wherein the coding regions can naturally exist together in a single polynucleotide (e.g., within the same ORF). For example, in some aspects, the coding region includes a first coding region and a second coding region, wherein the first coding region can encode a first epitope of a protein (e.g., a viral protein) and the second coding region can encode a second epitope of the same protein, wherein the protein is encoded by a single ORF of the polynucleotide in nature. When the polynucleotides useful for the present disclosure include such coding regions, in some aspects, the coding regions are arranged in a specific order within the ORF of the polynucleotide, wherein the specific order does not naturally exist in nature.

[0086] To aid in illustration, an exemplary polynucleotide found in nature comprises a single ORF having a first coding region, a second coding region, and a third coding region, wherein the first, second, and third coding regions are arranged as follows (from 5' to 3'): (first coding region), (second coding region), and (third coding region). When the polynucleotides described herein comprise such coding regions, in some aspects, the first coding region, the second coding region, and the third coding region are arranged in any of the following orders (from 5' to 3') within the single ORF of the polynucleotide:

[0087] (a) (first coding region), (third coding region) and (second coding region);

[0088] (b) (second coding region), (first coding region) and (third coding region);

[0089] (c) (second coding region), (third coding region) and (first coding region);

[0090] (d) (third coding region), (first coding region) and (second coding region); or

[0091] (e) (third coding region), (second coding region) and (first coding region).

[0092] Additional structural features of the polynucleotides are provided throughout the disclosure. For example, in some aspects, the polynucleotides described comprise a single ORF having multiple coding regions (e.g., a first coding region and a second coding region), wherein one or more coding regions encode full-length proteins. For illustration, in some aspects, the polynucleotides comprise a single ORF having a first coding region and a second coding region, wherein the first coding region encodes a full-length protein of an HPV E6 protein (e.g., HPV-16 or HPV-18), and the second coding region encodes a full-length protein of an HPV E7 protein (e.g., HPV-16 or HPV-18). However, as demonstrated herein, it is not necessary for the coding region to encode a full-length protein (or a large fragment thereof) to exert a therapeutic effect (e.g., to induce an immune response to the protein). Therefore, the polynucleotides described herein comprise a single ORF containing multiple coding regions, wherein one or more antigens encoded by the multiple coding regions comprise peptide fragments of larger proteins. In some aspects, as compared with the corresponding full-length fragment, the length of the peptide fragment is less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10% or less than about 5%. In some aspects, the antigen encoded by one or more of the multiple coding regions is less than about 200 amino acids long. In some aspects, antigen is less than about 150 amino acids long. In some aspects, antigen is less than about 100 amino acids long. In some aspects, antigen is less than about 50 amino acids long. In some aspects, the antigen encoded by one or more of the multiple coding regions present in the single ORF of the polynucleotides described herein is about 5 amino acids to about 200 amino acids long. In some aspects, the antigen is about 5 amino acids to about 150 amino acids long. In some aspects, the antigen is about 5 amino acids to about 100 amino acids long. In some aspects, the antigen is about 5 amino acids to about 50 amino acids long.In some aspects, the antigen is about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 26 amino acids, about 27 amino acids, about 28 amino acids, about 29 amino acids, about 30 amino acids, about 31 amino acids, about 32 amino acids, about 33 amino acids, about 34 amino acids, about 35 amino acids, about 36 amino acids, about 37 amino acids, about 38 amino acids, about 39 amino acids, about 40 amino acids, about 41 amino acids, about 42 amino acids, about 43 amino acids, about 44 amino acids, about 45 amino acids, about 46 amino acids, about 47 amino acids, about 48 amino acids, about 49 amino acids, about 50 amino acids, about 51 amino acids, about 52 amino acids, about 53 amino acids, about 54 amino acids, about 55 In some aspects, the antigen is about 10 to about 50 amino acids long. In some aspects, the antigen is about 10 to about 40 amino acids long. In some aspects, the antigen is about 10 to about 30 amino acids long. In some aspects, the antigen is about 10 to about 20 amino acids long. In some aspects, the antigen is about 20 to about 50 amino acids long. In some aspects, the antigen is about 20 to about 40 amino acids long. In some aspects, the antigen is about 20 to about 30 amino acids long. In some aspects, the antigen is about 30 to about 50 amino acids long. In some aspects, the antigen is about 30 to 40 amino acids long. In some aspects, the antigen is about 40 to about 50 amino acids long. In some aspects, the antigen is about 8 to about 11 amino acids long. In some aspects, the antigen is about 10 to about 17 amino acids long. In some aspects, the antigen is about 10 amino acids long. In some aspects, the antigen is about 15 amino acids long. In some aspects, the antigen is about 20 amino acids long. In some aspects, the antigen is about 25 amino acids long. In some aspects, the antigen is about 30 amino acids long. In some aspects, the antigen is about 35 amino acids long. In some aspects, the antigen is about 40 amino acids long. In some aspects, the antigen is about 45 amino acids long. In some aspects, the antigen is about 50 amino acids long. In some aspects, the antigen is about 8 amino acids long.

[0093] Not bound by any one theory, due to the size of antigen, polynucleotide as herein described can be designed to comprise many coding regions.In some aspects, described polynucleotide comprises single ORF, and described single ORF has about two coding regions, about three coding regions, about four coding regions, about five coding regions, about six coding regions, about seven coding regions, about eight coding regions, about nine coding regions, about 10 coding regions, about 11 coding regions, about 12 coding regions, about 13 coding regions, about 14 coding regions, about 15 coding regions, about 16 coding regions, about 17 coding regions, about 18 coding regions, about 19 coding regions, about 20 coding regions, about 21 coding regions, about 22 coding regions, about 23 coding regions, about 24 coding regions, about 25 coding regions, about 26 coding regions, about 27 coding regions, about 28 coding regions, about 29 coding regions or about 30 coding regions.As described herein, in some aspects, two or more coding regions are connected. In some aspects, each coding region is connected. In some aspects, the polynucleotides described herein include a single ORF with two connected coding regions. In some aspects, the polynucleotides include a single ORF with three connected coding regions. In some aspects, the polynucleotides include a single ORF with four connected coding regions. In some aspects, the polynucleotides described herein include a single ORF with five connected coding regions. In some aspects, the polynucleotides described herein include a single ORF with six connected coding regions. In some aspects, the polynucleotides include a single ORF with seven connected coding regions. In some aspects, the polynucleotides include a single ORF with eight connected coding regions. In some aspects, the polynucleotides include a single ORF with nine connected coding regions. In some aspects, the polynucleotides include a single ORF with ten connected coding regions. In some aspects, the polynucleotides include a single ORF with 11 connected coding regions. In some aspects, the polynucleotides include a single ORF with 12 connected coding regions. In some aspects, the polynucleotides include a single ORF with 13 connected coding regions. In some aspects, the polynucleotides include a single ORF with 14 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 15 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 16 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 17 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 18 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 19 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 20 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 21 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 22 connected coding regions.In some aspects, the polynucleotide comprises a single ORF with 23 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 24 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 25 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 26 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 27 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 28 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 29 connected coding regions. In some aspects, the polynucleotide comprises a single ORF with 30 connected coding regions. In some aspects, each coding region present in the single ORF of the polynucleotide described herein encodes different antigens. In some aspects, at least two coding regions encode different antigens.

[0094] In some aspects, any polynucleotide described herein can be used in combination.For example, in some aspects, the composition may include a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a single ORF with a coding region having multiple (e.g., about 10) connections, and the second polynucleotide comprises a single ORF with a coding region having multiple (e.g., about 10) connections. In some aspects, the coding regions of multiple connections of the first polynucleotide and the coding regions of multiple connections of the second polynucleotide encode different antigens. To illustrate, in some aspects, multiple coding regions of the first polynucleotide can encode tumor antigens (e.g., new antigens), and multiple coding regions of the second polynucleotide can encode non-self antigens (e.g., derived from viruses). Such first and second polynucleotides can be applied to a subject as a single composition combination. In some aspects, the first and second polynucleotides can be combined but used as a separate composition.

[0095] Therefore, some aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) comprising a single ORF, the ORF having a first coding region encoding a first antigen, a second coding region encoding a second antigen, and a third coding region encoding a third antigen, and wherein the first coding region, the second coding region, and the third coding region are connected. In some respects, at least two of the connection antigens of the coding are not identical. In some respects, each of the connection antigens of the coding is different. In some respects, at least two of the connection antigens of the coding are identical. In some respects, the first coding region, the second coding region, and the third coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide comprises naturally occurring corresponding polynucleotides. In some respects, the specific order of the coding region in the single ORF of a polynucleotide may help improve one or more characteristics of the polynucleotide (e.g., improving translation when delivered to a cell). For example, to help illustrate, in some aspects, the first polynucleotide comprises a single ORF, the ORF comprising a first coding region encoding an antigen, a second coding region encoding an antigen, and a third coding region encoding an antigen, wherein the first, second, and third coding regions are arranged as follows within the ORF (from 5' to 3'): (first coding region)-(second coding region)-(third coding region). In some aspects, the second polynucleotide comprises a single ORF, the ORF having the same first, second, and third coding regions as the first polynucleotide, but wherein the first, second, and third coding regions are arranged as follows within the ORF (from 5' to 3'): (first coding region)-(third coding region)-(second coding region). In some aspects, the third polynucleotide comprises a single ORF, the ORF having the same first, second, and third coding regions, but wherein the coding regions are arranged as follows (from 5' to 3'): (second coding region)-(third coding region)-(first coding region). For each of the first, second, and third polynucleotides, the three coding regions can be connected with a linker and / or without a linker (e.g., directly conjugated by a natural peptide bond). Without being bound by any one theory, in some aspects, each of the first, second and third polynucleotides may produce different therapeutic effects. For example, in some aspects, when the first, second and / or polynucleotides are delivered to cells (e.g., using extrusion treatment as described herein), they can result in different translation efficiencies (e.g., cells modified to include the second polynucleotide may have higher expression of encoded proteins compared to cells modified to include the first or second polynucleotides). Therefore, in some aspects, the polynucleotides described herein may have improved effects (e.g., better translation) as compared to corresponding polynucleotides having the same coding region but arranged in a different order.

[0096] In some respects, polynucleotides as described herein comprise a single ORF, and the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, and a fourth coding region encoding a fourth antigen, and wherein the first coding region, the second coding region, the third coding region, and the fourth coding region are connected. In some respects, the first coding region, the second coding region, the third coding region, and the fourth coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotides, wherein the reference polynucleotides comprise naturally occurring corresponding polynucleotides. In some respects, at least two of the connection antigens of the coding are not identical. In some respects, each of the connection antigens of the coding is different. In some respects, at least two of the connection antigens of the coding are identical.

[0097] In some respects, polynucleotides as described herein comprise a single ORF, and the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, a fourth coding region encoding a fourth antigen, and a fifth coding region encoding a fifth antigen, and wherein the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are connected. In some respects, the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotides, wherein the reference polynucleotides comprise naturally occurring corresponding polynucleotides. In some respects, at least two of the connection antigens of the coding are not identical. In some respects, each of the connection antigens of the coding is different. In some respects, at least two of the connection antigens of the coding are identical.

[0098] In some respects, polynucleotides as described herein comprise a single ORF, and the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, a fourth coding region encoding a fourth antigen, a fifth coding region encoding a fifth antigen, and a sixth coding region encoding a sixth antigen, and wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, and the sixth coding region are connected. In some respects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, and the sixth coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide comprises naturally occurring corresponding polynucleotides. In some respects, at least two of the connection antigens of the coding are not identical. In some respects, each of the connection antigens of the coding is different. In some respects, at least two of the connection antigens of the coding are identical.

[0099] In some aspects, polynucleotides as described herein include a single ORF, the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, a fourth coding region encoding a fourth antigen, a fifth coding region encoding a fifth antigen, a sixth coding region encoding a sixth antigen, and a seventh coding region encoding a seventh antigen, and wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, and the seventh coding region are connected. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, and the seventh coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide includes naturally occurring corresponding polynucleotides. In some aspects, at least two of the connection antigens of the coding are not the same. In some aspects, each of the connection antigens of the coding is different. In some aspects, at least two of the connection antigens of the coding are the same.

[0100] In some aspects, polynucleotides as described herein include a single ORF, the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, a fourth coding region encoding a fourth antigen, a fifth coding region encoding a fifth antigen, a sixth coding region encoding a sixth antigen, a seventh coding region encoding a seventh antigen, and an eighth coding region encoding an eighth antigen, and wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, and the eighth coding region are connected. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, and the eighth coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide includes naturally occurring corresponding polynucleotides. In some aspects, at least two of the connection antigens of the encoding are not the same. In some aspects, each of the connection antigens of the encoding is different. In some aspects, at least two of the connection antigens of the encoding are the same.

[0101] In some aspects, polynucleotides as described herein include a single ORF, the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, a fourth coding region encoding a fourth antigen, a fifth coding region encoding a fifth antigen, a sixth coding region encoding a sixth antigen, a seventh coding region encoding a seventh antigen, an eighth coding region encoding an eighth antigen, and a ninth coding region encoding a ninth antigen, and wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, and the ninth coding region are connected. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, and the ninth coding region are arranged in order, wherein the order is different as compared with the corresponding order present in the reference polynucleotide, wherein the reference polynucleotide includes the corresponding polynucleotides of natural occurrence. In some aspects, at least two of the connection antigens of the encoding are not the same. In some aspects, each of the connection antigens of the encoding is different. In some aspects, at least two of the connection antigens of the encoding are the same.

[0102] In some aspects, polynucleotides as described herein include a single ORF, the single ORF has a first coding region encoding a first antigen, a second coding region encoding a second antigen, a third coding region encoding a third antigen, a fourth coding region encoding a fourth antigen, a fifth coding region encoding a fifth antigen, a sixth coding region encoding a sixth antigen, a seventh coding region encoding a seventh antigen, an eighth coding region encoding an eighth antigen, a ninth coding region encoding a ninth antigen, and a tenth coding region encoding a tenth antigen, and wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, the ninth coding region, and the tenth coding region are connected. In some aspects, the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, the ninth coding region, and the tenth coding region are arranged in order, wherein as compared with the corresponding order present in the reference polynucleotide, the order is different, wherein the reference polynucleotide includes the corresponding polynucleotides of natural occurrence. In some aspects, at least two of the connection antigens of the coding are not the same. In some aspects, each of the connection antigens of the coding is different. In some aspects, at least two of the encoded junctional antigens are identical.

[0103] antigen:

[0104] As described herein, the polynucleotides described herein include a single ORF with a coding region from a variety of proteins, wherein the coding region encodes an antigen. In some aspects, the antigen may include an epitope. In some aspects, the antigen may include an immunogenic epitope. Therefore, in some aspects, one or more of the multiple coding regions may encode a unique (i.e., different) epitope of a single larger protein, wherein the single larger protein includes multiple unique epitopes. For example, in some aspects, the polynucleotides described herein include a single ORF having at least a first coding region and a second coding region, and wherein the first coding region and the second coding region encode a unique epitope of the same protein. In some aspects, one or more of the multiple coding regions may encode epitopes from different proteins. For example, in some aspects, the polynucleotides disclosed herein include a single ORF having at least a first coding region and a second coding region, wherein the first coding region encodes an epitope of a first protein, wherein the second coding region encodes an epitope of a second protein, and wherein the first protein and the second protein are not identical (e.g., KRAS protein and HPV protein, respectively). In some aspects, the antigen includes a wild-type protein. In some aspects, the antigen includes a peptide fragment derived from a wild-type protein. In some aspects, the antigen comprises a protein having at least one or more amino acid modifications (e.g., substitutions, deletions, additions, or indels) compared to a corresponding wild-type protein (also referred to herein as a "mutant protein") (e.g., a KRAS mutant). In some aspects, the antigen comprises a peptide fragment derived from such a mutant protein.

[0105] Non-limiting examples of antigens useful in the present disclosure include cancer antigens, non-self antigens, tumor-associated self antigens, disease-associated antigens, or combinations thereof.

[0106] In some aspects, the antigen comprises a cancer antigen. As used herein, the terms "cancer antigen" and "tumor antigen" (and derivatives thereof) are used interchangeably and refer to any antigen common to a specific hyperproliferative disorder (e.g., cancer). Non-limiting examples of cancer antigens include: guanylate cyclase C (GC-C), epidermal growth factor receptor (EGFR or erbB-1), human epidermal growth factor receptor 2 (HER2 or erbB2), erbB-3, erbB-4, MUC-1, melanoma-associated chondroitin sulfate proteoglycan (MCSP), mesothelin (MSLN), folate receptor 1 (FOLR1), CD4, CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8 、CD70、CD123、CD138、CD171、CEA、CSPG4、CXCR5、c-Met、HERV envelope protein、eriostin、Bigh3、SPARC、BCR、CD79、CD37、EGFRvIII、EGP2、EGP40、IGFr、L1CAM、AXL、tissue factor (TF)、CD74、EpCAM、EphA2、MRP3 cadherin 19 (CDH19), epidermal growth factor 2 (HER2), 5T4、8H9、α v β6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, FAP, FBP, fetal AchR, FRcc, GD2, GD3, Glypican-1 (GPC1), Glypican-2 (GPC2), Glypican-3 (GPC3), MAGE1, MAGEA10, NY-ESO-1, IL-13Rcc2, Lewis-Y, KDR, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, PRAME, PSC1, PSCA, PSMA, ROR1, ROR2, SP17, survivin, TAG72, TEM, carcinoembryonic antigen, HMW-MAA, VEGF, CLDN18.2, neoantigens, KRAS, or a combination thereof. In some aspects, the cancer antigen comprises KRAS.

[0107] Kirsten rat sarcoma viral oncogene homolog (KRAS) is a member of the guanosine-5-triphosphatase (GTPase) protein superfamily, which also includes NRAS and HRAS. The main role of members of this superfamily is to transmit signals from upstream cell surface receptors (e.g., EGFR, FGFR, and ERBB2-4) to downstream proliferation and survival pathways (e.g., RAF-MEK-ERK, PI3K-AKT-mTOR, and RALGDS-RA). Adderley, H. et al., EBioMedicine 41:711-716 (2019). KRAS mutations are associated with multiple types of cancer, including more than 90% of pancreatic cancer, 35-45% of colorectal cancer, and approximately 25% of lung cancer. Zeitouni, D. et al., Cancers 8(4):45 (2016); Tan, C. et al., World J Gastroenterol 18(37):5171-5180 (2012); and Roman, M. et al., Molecular Cancer 17:33 (2018). KRAS mutations are also associated with very poor prognosis (e.g., 5-year survival rate for pancreatic cancer is about 9%), and many patients with KRAS mutations are resistant to various cancer therapies. Del Re, M. et al., Oncotarget 9(5):6630-6643 (2017). Therefore, new and improved treatment options are needed for cancers associated with KRAS mutations.

[0108] KRAS is referred to in the art by a variety of names. Such names include: KRAS proto-oncogene GTPase; V-Ki-Ras2 Kirsten rat sarcoma 2 viral oncogene homolog; GTPase KRas; C-Ki-Ras; K-Ras 2; KRAS2; RASK2; V-Ki-Ras2 Kirsten rat sarcoma viral oncogene homolog; Kirsten rat sarcoma viral proto-oncogene; cellular transforming proto-oncogene; cellular C-Ki-Ras2 proto-oncogene; transforming protein P21; PR310 C-K-Ras oncogene; C-Kirsten-Ras protein; K-Ras P21 protein; and oncogene KRAS2.

[0109] There are two isoforms of human wild-type KRAS protein (P01116) that are produced by alternative splicing. Isoform 2A (Accession No.: P01116-1; SEQ ID NO: 16) is the canonical sequence. It is also known as K-Ras4A. Isoform 2B (Accession No.: P01116-2; also known as K-Ras4B; SEQ ID NO: 17) differs from the canonical sequence as follows: (i) 151-153: RVE→GVD; and (ii) 165-189: QYRLKKISKEEKTPGCVKIKKCIIM→KHKEKMSKDGKKKKKKSKTKCVIM.

[0110] Natural variants of human KRAS gene products are known. For example, natural variants of human KRAS protein can contain one or more amino acid substitutions selected from the group consisting of K5E, K5N, G10GG, G10V, G12A, G12C, G12D, G12F, G12I, G12L, G12R, G12S, G12V, G13C, G13D, G13E, G13R, G13V, V14I, L19F, T20M, Q22E, Q22H, Q22K, Q22R, Q25H, N26Y, F28L, E31K, D33E, P34L, P34Q, P34R, I36M, R41K, D 98N, T57N, T58I, A59T, G60D, G60R, G60S, G60V, Q61A, Q61H, Q61K, Q61L, Q61P, Q61R, E63K, S65N, R68S, Y71H, T74A, L79I, R97I, Q99E, M111L, K117N, K117R, D119G, S122F, T144P, A146P, A146T, A146V, K147E, K147T, R149K, L159S, I163S, R164Q, I183N, I84M, or a combination thereof. The natural variants characteristic of KRAS protein isoform 2B contain one or more amino acid substitutions selected from the group consisting of V152G, D153V, F156I, F156L, or a combination thereof.

[0111] As is apparent from the above disclosure, in some aspects, one or more coding regions of the polynucleotides described herein encode a KRAS antigen, wherein the KRAS antigen comprises an amino acid sequence that is different in sequence as compared to the amino acid sequence of the corresponding wild-type KRAS antigen. Such KRAS antigens are also referred to herein as "KRAS mutants" or "KRAS variants". In some aspects, the amino acid sequence of the KRAS mutant has less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, or less than about 90% sequence identity as compared to the amino acid sequence of the corresponding wild-type KRAS antigen. In some aspects, the amino acid sequence of the KRAS mutant comprises one or more amino acid substitutions, wherein the one or more amino acid substitutions are associated with cancer. In some aspects, the amino acid sequence of the KRAS mutant comprises any amino acid substitution described herein. For example, in some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are connected, wherein at least one of the multiple coding regions encodes a KRAS antigen, wherein the amino acid sequence of the KRAS antigen comprises a G12V amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are connected, wherein at least one of the multiple coding regions encodes a KRAS antigen, wherein the amino acid sequence of the KRAS antigen comprises a G12D amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are connected, wherein at least one of the multiple coding regions encodes a KRAS antigen, and wherein the amino acid sequence of the KRAS antigen comprises a G12C amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are connected, wherein at least one of the multiple coding regions encodes a KRAS antigen, and wherein the amino acid sequence of the KRAS antigen comprises a G13D amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked, wherein at least one of the multiple coding regions encodes a KRAS antigen, and wherein the amino acid sequence of the KRAS antigen comprises a G12A amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked, wherein at least one of the multiple coding regions encodes a KRAS antigen, and wherein the amino acid sequence of the KRAS antigen comprises a G12R amino acid substitution.In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked, wherein at least one of the multiple coding regions encodes a KRAS antigen, and wherein the amino acid sequence of the KRAS antigen comprises a G12S amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked, wherein at least one of the multiple coding regions encodes a KRAS antigen, and wherein the amino acid sequence of the KRAS antigen comprises one or more of: G12D. 1-16 、G12D 2-19 、G12D 2-22 、G12D 2 -29 、G12V 1-16 、G12V 2-19 、G12V 3-17 or G12V 3-42 antigen.

[0112] In some aspects, at least one of the plurality of coding regions encodes a KRAS antigen, wherein the amino acid sequence of the KRAS antigen has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or about 99% sequence identity as compared to the amino acid sequence set forth in any one of SEQ ID NOs: 1-15. In some aspects, the polynucleotides described herein comprise a single ORF having a plurality of coding regions, wherein the plurality of coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the polynucleotides described herein comprise a single ORF having a plurality of coding regions, wherein the plurality of coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 2. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:3. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:4. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:5. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:6. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:7.In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 8. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 9. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 10. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 11. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 12. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 14. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked and encode at least one KRAS antigen, wherein the KRAS antigen comprises, consists of, or consists essentially of the amino acid sequence shown in SEQ ID NO: 15.

[0113] In some aspects, the polynucleotides described herein comprise a single ORF having at least a first coding region encoding a first KRAS antigen and a second coding region encoding a second KRAS antigen, wherein the first coding region and the second coding region are connected, wherein the amino acid sequence of the first KRAS antigen comprises a G12V amino acid substitution, and the amino acid sequence of the second KRAS antigen comprises a G12D amino acid substitution. In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are connected, wherein one or more of the multiple coding regions encode KRAS mutant antigens (e.g., comprising G12D and / or G12V substitutions), and wherein one or more of the multiple coding regions encode additional tumor antigens (e.g., NY-ESO-1 and / or MAGEA10). In some aspects, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are connected and encode multiple KRAS antigens. In some aspects, the plurality of KRAS antigens comprises one or more of the following amino acid substitutions: G12A, G12C, G12D, G12R, G12SG12V, and G13D. As described herein, in some aspects, the plurality of coding regions are arranged in a specific order within the polynucleotides described herein.

[0114] Thus, in some aspects, the polynucleotides described herein comprise the following coding regions, which are linked and arranged in the following order (from 5' to 3'): (1) a first coding region encoding a KRAS G12D mutant (i.e., having a G12D amino acid substitution), (2) a second coding region encoding a KRAS G12V mutant, (3) a third coding region encoding a KRAS G12C mutant, (4) a fourth coding region encoding a KRAS G13D mutant, (5) a fifth coding region encoding a KRAS G12A mutant, (6) a sixth coding region encoding a KRAS G12R mutant, and (7) a seventh coding region encoding a KRAS G12S mutant. In some aspects, such polynucleotides comprise additional linked coding regions (e.g., encoding a co-stimulatory molecule and / or a cytokine). For example, in some aspects, the polynucleotides of the present disclosure comprise multiple coding regions encoding: (1) multiple KRAS mutant antigens, (2) co-stimulatory molecules (e.g., CD86), and (3) one or more cytokines (e.g., membrane-bound IL-2 and membrane-bound IL-12), wherein the multiple KRAS mutant antigens comprising the following mutations are arranged as follows (from 5′ to 3′): G12D, G12V, G12C, G13D, G12A, G12R, and G12S. In some aspects, the polynucleotides described herein comprise the following coding regions, which are linked and arranged in the following order (from 5' to 3'): (1) a first coding region encoding a KRAS G12S mutant, (2) a second coding region encoding a KRASG12R mutant, (3) a third coding region encoding a KRAS G12A mutant, (4) a fourth coding region encoding a KRAS G13D mutant, (5) a fifth coding region encoding a KRAS G12C mutant, (6) a sixth coding region encoding a KRAS G12V mutant, and (7) a seventh coding region encoding a KRAS G12D mutant. In some aspects, such polynucleotides comprise additional linked coding regions (e.g., encoding costimulatory molecules and / or cytokines). For example, in some aspects, the polynucleotides of the present disclosure comprise a plurality of coding regions encoding: (1) a plurality of KRAS mutant antigens, (2) a co-stimulatory molecule (e.g., CD86), and (3) one or more cytokines (e.g., membrane-bound IL-2 and membrane-bound IL-12), wherein the plurality of KRAS mutant antigens comprising the following mutations are arranged as follows (from 5' to 3'): G12S, G12R, G21A, G13D, G12C, G12V, and G12D. As demonstrated herein (see, e.g., Examples 6-8), when such polynucleotides are used to squeeze cells (e.g., PBMCs), each encoded antigen can be expressed in the PBMCs, enabling the PBMCs to induce activation of T cells that recognize the encoded antigens.

[0115] In some aspects, the antigen comprises an autoantigen. For example, in some aspects, the antigen comprises an autoantigen that is overexpressed in certain tumors. In some aspects, the antigen comprises a non-self antigen. As used herein, the term "non-self antigen" refers to any antigenic substance derived from a pathogen (i.e., capable of inducing an immune response when present in the body), including but not limited to viruses, fungi, protozoa, or combinations thereof. Non-limiting examples of non-self antigens include antigens derived from one or more of the following: human gamma herpes virus 4 (i.e., Epstein Barr virus (EBV)), influenza A virus, influenza B virus, cytomegalovirus, Staphylococcus aureus, Mycobacterium tuberculosis, Chlamydia trachomatis, HIV (e.g., HIV-2), coronaviruses (e.g., COVID-19, MERS-CoV, and SARS CoV), filoviruses (e.g., Marburg and Ebola), Streptococcus pyogenes, Streptococcus pneumoniae, Plasmodium species (e.g., P. vivax and P. falciparum), chikungunya virus, human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-lymphotropic virus (HTLV1), human herpesvirus 8 (HHV8), Merkel cell polyomavirus (MCV), bunyaviruses (e.g., hantavirus), arenaviruses (e.g., LCMV and Lassa virus), yellow fever virus Viruses (e.g., dengue, Zika, Japanese encephalitis, West Nile, and yellow fever), enteroviruses (e.g., poliomyelitis), astroviruses (e.g., gastroenteritis), Rhabdoviridae (e.g., rabies), Borrelia burgdorferi and Borrelia mayoyi (e.g., Lyme disease), herpes simplex virus 2 (HSV-2), Klebsiella, Pseudomonas aeruginosa, Enterococcus, Proteus, Enterobacter, Actinomyces, coagulase-negative Staphylococcus (CoNS), Mycoplasma, adenovirus, adeno-associated virus (AAV), or a combination thereof.

[0116] Therefore, in some aspects, one or more coding regions of the polynucleotides described herein encode non-self antigens. For example, in some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode non-self antigens. In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode HPV antigens (e.g., E7 and / or E6 proteins). In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode HIV antigens. In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode HBV antigens. In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode HSV antigens (e.g., HSV gD). In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode influenza antigens (e.g., M1 proteins). In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode coronavirus antigens (e.g., S protein). In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein the multiple coding regions encode multiple non-self antigens (e.g., HPV antigens, EBV antigens, HSV antigens, influenza antigens, coronavirus antigens, and combinations thereof). In some aspects, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected, and wherein one or more of the multiple coding regions encode at least one cancer antigen (e.g., KRAS mutants, NY-ESO-1, MAGE A10, or combinations thereof) and one or more of the multiple coding regions encode at least one non-self antigen (e.g., HPV antigens, EBV antigens, HSV antigens, influenza antigens, coronavirus antigens, and combinations thereof).

[0117] Connectors

[0118] As described herein, the polynucleotides described herein include multiple coding regions, wherein the multiple coding regions are connected. Any useful method for connecting two parts (e.g., the first coding region and the second coding region) can be used in the present disclosure. In some aspects, the multiple coding regions are connected with a joint. In some aspects, the multiple coding regions can be connected without a joint. For example, in some aspects, the first coding region and the second coding region can be arranged in the ORF so that they are adjacent to each other, so that when translated, the protein encoded by the first coding region and the protein encoded by the second coding region will be connected by traditional peptide amide bonds. As used herein, the term "joint" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide (e.g., an alkyl chain). In some aspects, two or more joints can be connected in series. When there are multiple joints, each of the joints can be the same or different. In general, joints provide flexibility or prevent / improve steric hindrance. Joints are usually not cleaved; however, in some aspects, such cleavage can be desired. Therefore, in some aspects, the joint can include one or more protease cleavable sites, which can be located in the joint sequence or at the joint flank at either end of the joint sequence. Thus, in some aspects, linkers useful in the present disclosure comprise cleavable linkers.

[0119] As used herein, the term "cleavable linker" refers to a linker comprising a cleavage site so that it can be selectively cleaved when expressed to produce two or more products. In some aspects, the linker is selected from a P2A linker, a T2A linker, a F2A linker, an E2A linker, a furin cleavage site, or any combination thereof (see Table 1 below). In some aspects, the linker also includes a GSG linker sequence. In some aspects, the linker that can be used for the present disclosure includes an internal ribosome entry site (IRES) so that a separate polypeptide encoded by the first and second genes is produced during translation. For example, WO 2020 / 223625A1 and US2019 / 0276801A1 provide additional descriptions of linkers that can be used for the present disclosure, and each of the documents is incorporated herein by reference in its entirety.

[0120] Table 1: Linker sequences

[0121] P2A ATNFSLLKQAGDVEENPGP (SEQ ID NO: 18) T2A EGRGSLLTCGDVEENPGP (SEQ ID NO: 19) F2A VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 20) E2A QCTNYALLKLAGDVESNPGP(SEQ ID NO: 21) Furin cleavage site RAKR (SEQ ID NO: 22)

[0122] In some aspects, the linker is a peptide linker. In some aspects, the peptide linker comprises a glycine / serine linker. In some aspects, the peptide linker is a glycine / serine linker according to the formula [(Gly)n-Ser]m (SEQ ID NO: 23), wherein n is any integer from 1 to 100 and m is any integer from 1 to 100. In some aspects, the glycine / serine linker is according to the formula [(Gly)x-(Ser)y]z (SEQ ID NO: 24), wherein x is an integer from 1 to 4, y is 0 or 1, and z is an integer from 1 to 50. In some aspects, the peptide linker comprises the sequence Gn (SEQ ID NO: 25), wherein n can be an integer from 1 to 100. In some aspects, the peptide linker may comprise the sequence (GlyAla)n (SEQ ID NO: 26), wherein n is an integer between 1 and 100. In some aspects, the peptide linker may comprise the sequence (GlyGlySer)n (SEQ ID NO: 27), wherein n is an integer between 1 and 100. In some aspects, the peptide linker comprises the sequence GGGG (SEQ ID NO: 28).

[0123] In some aspects, the peptide linker comprises the sequence (GGGS)n (SEQ ID NO: 29). In some aspects, the peptide linker comprises the sequence (GGS)n(GGGGS)n (SEQ ID NO: 30). In such aspects, n can be an integer from 1 to 100. In some aspects, n can be an integer from 1 to 20, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some aspects, n is an integer from 1 to 100. In some aspects, the peptide linker is (G4S)3 (SEQ ID NO: 31).

[0124] In some aspects, the peptide linker comprises an EAAAK linker. In some aspects, the EAAAK linker comprises the sequence (EAAAK)n, wherein n is an integer between 1 and 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some aspects, the peptide linker is (EAAAK)3 (SEQ ID NO: 32).

[0125] In some aspects, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker includes a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising an amino acid sequence that connects or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence to which it is not naturally connected or genetically fused in nature. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation, such as an addition, substitution, or deletion).

[0126] In some aspects, the peptide linker can comprise non-naturally occurring amino acids. In other aspects, the peptide linker can comprise naturally occurring amino acids present in a linear sequence that does not exist in nature. In other aspects, the peptide linker can comprise a naturally occurring polypeptide sequence.

[0127] In some aspects, the linker comprises a non-peptide linker. In other aspects, the linker consists of a non-peptide linker. In some aspects, the non-peptide linker can be, for example, maleimidocaproyl (MC), maleimidopropionyl (MP), methoxypolyethylene glycol (MPEG), 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(p-maleimidophenyl)butyric acid succinimidyl ester (SMPB), (4-iodoacetyl)aminobenzoic acid N-succinimidyl ester (SIAB), 6-[3-(2-pyridyldithio)-propionamido]hexanoic acid succinimidyl ester (LC-SPDP), 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), etc. (see, for example, U.S. Pat. No. 7,375,078, which is incorporated herein by reference in its entirety).

[0128] Other components:

[0129] In addition to any of the above features, the polynucleotides that can be used for the present disclosure may also include one or more additional components. In some aspects, these additional components can help the polynucleotides to exert their therapeutic effects (e.g., allowing improved translation and / or increasing the stability of the polynucleotides). Non-limiting examples of additional components that may be included in the polynucleotides described herein include: internal ribosome entry site (IRES), intron sequences, homology arm promoters, enhancers, UTRs, sequences encoding signal peptides, translation initiation sequences, 3' tailing regions of linked nucleosides, 5' caps, sequences encoding 2A ribosomal skipping peptides, or any combination thereof.

[0130] Untranslated region (UTR)

[0131] In some aspects, the polynucleotides described herein comprise one or more UTR sequences (e.g., 5′-UTR and / or 3′-UTR). As used herein, the term “untranslated region” or “UTR” refers to a region in a gene that is transcribed but not translated. “5′-UTR” starts from the transcription start site and continues to the start codon, but does not include the start codon; whereas “3′-UTR” starts immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence that UTR plays a regulatory role in the stability and translation of nucleic acid molecules. The regulatory features of UTR can be incorporated into the polynucleotides described herein to enhance the stability of the molecule. Specific features can also be incorporated to ensure that transcripts are controlled down-regulated when they are misdirected to unwanted organ sites.

[0132] In some aspects, the UTRs useful for the present disclosure include those present in genes that are expressed in large quantities in specific cells, tissues, and / or organs. By including such additional UTRs, in some aspects, the polynucleotides of the present disclosure can be preferentially expressed in specific cells, tissues, and / or organs, for example, when administered to a subject. For example, additional introduction of a UTR (e.g., 5-UTR) of an mRNA expressed in the liver (e.g., albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII) can enhance the expression of the polynucleotides described herein in the liver and / or hepatocyte cell lines. Non-limiting examples of such tissue-specific UTRs include those from: (a) muscle: myoD, myosin, myoglobin, myogenin, and herculin; (b) endothelial cells: Tie-1 and CD36; (c) myeloid cells: C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, and i-NOS; (d) leukocytes: CD45 and CD18; (e) adipose tissue: CD36, GLUT4, ACRP30, and adiponectin; and (f) lung epithelial cells: SP-A / B / C / D.

[0133] Additional examples of UTRs useful in the present disclosure include one or more 5′-UTRs and / or 3′-UTRs derived from the following nucleic acid sequences: globulins, such as α-globulins or β-globulins (e.g., Xenopus, mouse, rabbit, or human globulins); strong Kozak translation initiation signals; CYBAs (e.g., human cytochrome b-245 alpha polypeptide); albumins (e.g., human albumin 7); HSD17B4 (hydroxysteroid (17-β) dehydrogenase); viruses (e.g., tobacco etch virus (TEV), Venezuelan equine encephalitis virus (VEEV), dengue virus, cytomegalovirus ( CMV) (e.g., CMV immediate early 1 (IE1)), hepatitis virus (e.g., hepatitis B virus), Sindbis virus or PAV barley yellow dwarf virus); heat shock protein (e.g., hsp70); translation initiation factor (e.g., elF4G); glucose transporter (e.g., hGLUT1 (human glucose transporter 1)); actin (e.g., human α or β actin); GAPDH; tubulin; histone; citric acid cycle enzyme; topoisomerase (e.g., 5′-UTR of TOP gene lacking 5′ TOP motif (oligopyrimidine tract)); ribosomal protein Large 32 (L32); ribosomal protein (e.g., human or mouse ribosomal protein, such as rps9); ATP synthase (e.g., ATP5A1 or mitochondrial H + -ATP synthase beta subunit); growth hormone e (e.g., bovine (bGH) or human (hGH)); elongation factors (e.g., elongation factor 1 alpha 1 (EEF1A1)); manganese superoxide dismutase (MnSOD); myocyte enhancer factor 2A (MEF2A); β-F1-ATPase, creatine kinase, myoglobin, granulocyte colony stimulating factor (G-CSF); collagens (e.g., type I collagen alpha 2 (Col1A2), type I collagen alpha 1 (Col1A1 ), type VI collagen alpha 2 (Col6A2), type VI collagen alpha 1 (Col6A1)); ribosome-binding glycoproteins (e.g., ribosome-binding glycoprotein I (RPNI)); low-density lipoprotein receptor-related proteins (e.g., LRP1); cardiotrophin-like cytokines (e.g., Nnt1); calreticulin (Calr); procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 (Plod1); nuclear ligins (e.g., Nucb1); and combinations thereof.

[0134] Cap structure

[0135] In some aspects, a polynucleotide described herein (eg, comprising multiple linked coding regions) comprises a 5'-cap.

[0136] As used herein, the term "5'-cap" refers to a modified nucleotide (e.g., guanine) that can be added to the 5' end of a polynucleotide (e.g., mRNA). The 5'-cap structure can play a role in the nuclear export of the polynucleotide (e.g., export to the cytoplasm where translation can occur) and / or promote the stability of the polynucleotide. In some aspects, the 5'-cap can be connected to the 5' end of the polynucleotide described herein via a 5'-5'-triphosphate linkage. In certain aspects, the 5'-cap can be methylated (e.g., m7GpppN, where N is the terminal 5' nucleotide of the polynucleotide). Any suitable 5'-cap known in the art can be used in the present disclosure. Non-limiting examples of 5'-caps that can be used in the present disclosure include: m2 7，2′-O Gpp s pGRNA, m 7 GpppG、m 7 Gppppm 7 G、m2 (7，3′-O) GpppG, m2 (7，2′-O) GppspG(D1),m2 (7，2′-O) GppspG(D2),m2 7，3’-O Gppp(m1 2’-O )ApG, (m 7 G-3'mppp-G; which can be equivalently designated as 3'O-Me-m7G(5')ppp(5')G), N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G, N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G, N7-(4-chlorophenoxyethyl)-m 3′-O G(5′)ppp(5′)G, 7mG(5′)ppp(5′)N, pN2p, 7mG(5′)ppp(5′)NlmpNp, 7mG(5′)-ppp(5′)NlmpN2 mp, m(7)Gpppm(3)(6,6,2′)Apm(2′)Apm(2′)Cpm(2)(3,2′)Up, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N1-methylpseudouridine, m7G(5′)ppp(5′)(2'OMeA)pG or a combination thereof.

[0137] In some aspects, the 5′-caps useful for the polynucleotides of the present disclosure comprise cap analogs (also referred to as “synthetic cap analogs,” “chemical caps,” “chemical cap analogs,” or “structural or functional cap analogs”). A “cap analog” differs in chemical structure from a natural (i.e., endogenous, wild-type, or physiological) 5′-cap while retaining cap function. Non-limiting examples of cap analogs are described in US 8,519,110 and Kore et al., Bioorganic & Medicinal Chemistry 21: 4570-4574 (2013), each of which is incorporated herein by reference in its entirety.

[0138] In some aspects, the 5'-cap is modified. The modification on the 5'-cap can further increase the stability, half-life and / or translation efficiency of the polynucleotide. In some aspects, the modified 5'cap comprises one or more of the following modifications: modification at the 2' and / or 3' position of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (producing a carbocycle) with a methylene moiety (CH2), modification at the triphosphate bridge portion of the cap structure, or modification at the core base (G) portion. See, for example, US2014 / 0147454 and WO 2018 / 160540, each of which is incorporated herein by reference in its entirety.

[0139] Poly(A) tail

[0140] In some aspects, the polynucleotides described herein (e.g., comprising a plurality of linked coding regions) comprise a long chain of adenine nucleotides (referred to herein as a "poly(A) tail") at the 3' end of the polynucleotide. In some aspects, the poly(A) tail is present alone or in combination with other components described herein (e.g., a 5'-cap).

[0141] In some aspects, the length of the poly (A) tail is greater than about 30 nucleotides long. In certain aspects, the length of the poly (A) tail is at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 350 nucleotides, at least about 400 nucleotides, at least about 450 nucleotides or at least about 500 nucleotides or more.

[0142] Any suitable poly(A) tail known in the art can be used in the present disclosure. Non-limiting examples of poly(A) tails that can be used in the present disclosure include: SV40 poly(A), bGH poly(A), actin poly(A), hemoglobin poly(A), poly(A)-G tetrad, or a combination thereof.

[0143] Enhancer

[0144] In some aspects, one or more enhancer sequences (also referred to herein as "translation enhancer element" or "TEE") can be used to further increase the expression of antigens encoded by one or more coding regions of polynucleotides as described herein. Therefore, in some aspects, polynucleotides as described herein (for example, comprising a plurality of connected coding regions) include one or more enhancer sequences. In some aspects, polynucleotides as described herein include at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45 or at least about 50 or more enhancer sequences. When polynucleotides include multiple enhancers, in some aspects, each of the enhancers is the same. In some aspects, one or more of the enhancers are different. In some aspects, one or more of the enhancers are separated by spacers.

[0145] Any enhancer known in the art can be used for the polynucleotides disclosed herein. See, for example, WO1999024595, WO2012009644, WO2009075886 and WO2007025008, European Patent Publication Nos. EP2610341A1 and EP2610340A1, U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, U.S. Patent No. 7,183,395, each of which is incorporated herein by reference in its entirety. In some aspects, the enhancer that can be used for the present disclosure is a tissue-specific enhancer. In certain aspects, enhancers useful in the present disclosure are selected from human skeletal actin gene elements, cardiac actin gene elements, myocyte-specific enhancer binding factor MEF (e.g., MEF2), MyoD enhancer elements, cardiac enhancer factor (CEF) sites, murine creatine kinase enhancer elements, skeletal fast-twitch troponin C gene elements, slow-twitch cardiac troponin C gene elements, slow-twitch troponin I gene elements, hypoxia-inducible nuclear factor, steroid-inducible elements, glucocorticoid response elements (GREs), or any combination thereof.

[0146] IRES sequence

[0147] In some aspects, the polynucleotides described herein (e.g., comprising a plurality of linked coding regions) may further comprise a nucleotide sequence encoding an internal ribosome entry site (IRES). In the absence of a 5′-cap structure, IRES plays an important role in initiating protein synthesis. IRES can be used as the only ribosome binding site, or can be used as one of the multiple ribosome binding sites of a polynucleotide. A polynucleotide containing more than one functional ribosome binding site can encode several peptides or polypeptides independently translated by a ribosome (“polycistronic polynucleotide”). Therefore, when the polynucleotides described herein comprise a sequence encoding an IRES, in some aspects, the polynucleotide may comprise a plurality of (e.g., at least two) translatable regions—e.g., a nucleotide sequence encoding a coronavirus spike protein; and a nucleotide sequence encoding different coronavirus proteins (e.g., nucleocapsid proteins).

[0148] Any IRES sequence known in the art can be used in the present disclosure. Non-limiting examples of IRES sequences that can be used in the present disclosure include those from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), cricket paralysis virus (CrPV), or a combination thereof.

[0149] Post-transcriptional regulatory elements

[0150] In some aspects, additional components that can be used for polynucleotides described herein (e.g., coding regions comprising multiple connections) include post-transcriptional regulatory elements. In some aspects, post-transcriptional regulatory elements can be present in polynucleotides described herein in combination with one or more other components described herein (e.g., 5′-cap, 3′-poly (A) tail, enhancer sequence, IRES sequence, or a combination thereof). In some aspects, post-translational regulatory elements are located at 3′-of multiple coding regions of polynucleotides described herein. Non-limiting examples of post-transcriptional regulatory elements that can be used for the present disclosure include mutant woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE), microRNA binding sites, DNA nuclear targeting sequences, or a combination thereof.

[0151] In some aspects, one or more additional components that may be present in a polynucleotide as described herein (e.g., comprising a plurality of connected coding regions) include a promoter. In some aspects, a polynucleotide may include a single promoter. In some aspects, a polynucleotide may include a plurality of promoters (e.g., two, three, four or five or more), which are operably connected to a plurality of coding regions of a polynucleotide as described herein. When a polynucleotide includes a plurality of promoters, in some aspects, each of the plurality of promoters is identical. In some aspects, one or more of the plurality of promoters are different.

[0152] In some aspects, promoters useful in the present disclosure comprise mammalian promoters, viral promoters, or both. In certain aspects, promoters useful in the polynucleotides described herein comprise constitutive promoters, inducible promoters, or both.

[0153] Constitutive mammalian promoters include, but are not limited to, promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin promoter and other constitutive promoters. Exemplary viral promoters that play a constitutive role in eukaryotic cells include, for example, promoters from cytomegalovirus (CMV), simian virus (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeats (LTR) and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. As described herein, in some aspects, promoters that can be used in the present disclosure are inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, in the presence of certain metal ions, the metallothionein promoter is induced, thereby promoting transcription and translation. In some aspects, the promoter that can be used comprises the T7 promoter.

[0154] Modified Nucleosides / Nucleotides

[0155] In some aspects, a polynucleotide described herein (eg, comprising multiple linked coding regions) comprises at least one chemically modified nucleoside and / or nucleotide.

[0156] "Nucleoside" refers to a compound containing a combination of a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). "Nucleotide" refers to a nucleoside that includes a phosphate group. Modified nucleotides can be synthesized by any useful method, such as chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.

[0157] A polynucleotide may comprise one or more regions linked by nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotide will comprise a nucleotide region.

[0158] Polynucleotides disclosed herein can comprise various unique modifications.In some respects, polynucleotides can contain one, two or more (optionally different) nucleosides or nucleotide modifications.In some respects, as compared with unmodified polynucleotides, polynucleotides can show one or more desired characteristics, for example, improved heat or chemical stability, reduced immunogenicity, reduced degradation, increased in combination with target microRNA, reduced with non-specific binding of other microRNAs or other molecules.

[0159] In some aspects, the polynucleotides of the present disclosure are chemically modified. As used herein, with respect to polynucleotides, the term "chemical modification" or, where appropriate, "chemically modified" refers to the modification of one or more of the position, pattern, percentage, or population of adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides (including, but not limited to, their core bases, sugars, backbones, or any combination thereof).

[0160] In some aspects, the polynucleotides of the present disclosure can have uniform chemical modifications of all or any of the same nucleoside types, or a population of modifications generated by titrating down the same starting modification in all or any of the same nucleoside types, or a measured percentage of chemical modifications of all or any of the same nucleoside types but randomly incorporated. In other aspects, the polynucleotides of the present disclosure can have uniform chemical modifications of two, three, or four of the same nucleoside types throughout the polynucleotide (e.g., all uridines and / or all cytidines, etc., are modified in the same manner).

[0161] The modified nucleotide base pairing includes not only standard adenine-thymine, adenine-uracil or guanine-cytosine base pairs, but also includes base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between non-standard bases and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleobase inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker can be incorporated into the polynucleotides of the present disclosure.

[0162] The skilled artisan will understand that unless otherwise indicated, the polynucleotide sequences set forth in this application will list "T" in representative DNA sequences, but when the sequence represents RNA, "T" will be replaced with "U." For example, the TD of the present disclosure can be administered as RNA, DNA, or a hybrid molecule comprising both RNA and DNA units.

[0163] In some aspects, the polynucleotides described herein include a combination of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 20, or more) modified nucleobases.

[0164] In some aspects, the nucleobases, sugars, backbone linkages, or any combination thereof in a polynucleotide are modified by at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0165] Base modification

[0166] In some aspects, the chemical modification is at a nucleobase in a polynucleotide of the present disclosure (e.g., a coding region comprising multiple connections). In some aspects, at least one chemically modified nucleoside is a modified uridine (e.g., pseudouridine (ψ), 2-thiouridine (s2U), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ) or 5-methoxy-uridine (mo5U)), a modified cytosine (e.g., 5-methyl-cytidine (m5C)), a modified adenosine (e.g., 1-methyl-adenosine (miA), N6-methyl-adenosine (m6A) or 2-methyl-adenine (m2A)), a modified guanosine (e.g., 7-methyl-guanosine (m7G) or 1-methyl-guanosine (m1G)) or a combination thereof.

[0167] In some aspects, the polynucleotides described herein are uniformly modified (e.g., completely modified, modified throughout the sequence) for a particular modification. For example, polynucleotides can be uniformly modified with base modifications of the same type, such as 5-methyl-cytidine (m5C), which means that all cytosine residues in the polynucleotide sequence are replaced by 5-methyl-cytidine (m5C). Similarly, polynucleotides can be uniformly modified by replacing any type of nucleoside residue present in the sequence with modified nucleosides (e.g., any of those described above).

[0168] In some aspects, the polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) modified nucleobases. In some aspects, at least about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% of a type of nucleobase in the polynucleotides of the present disclosure is a modified nucleobase.

[0169] Skeleton modification

[0170] In some aspects, polynucleotides described herein may include any useful linkages between nucleosides. Such linkages (including backbone modifications) that may be used for compositions of the present disclosure include but are not limited to the following: 3′-alkylene phosphonates, 3′-amino phosphoramidates, olefin-containing backbones, aminoalkyl phosphoramidates, aminoalkyl phosphotriesters, borane phosphates, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral thiophosphates, formyl acetyl and thioformyl acetyl backbones, methylene (methyl imino) ), methyleneformylacetyl and thioformylacetyl backbones, methyleneimino and methylenehydrazine backbones, morpholino linkages, -N(CH3)-CH2-CH2-, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphotriesters, PNAs, siloxane backbones, sulfamic acid ester backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thiocarbonyl alkylphosphonates, thiocarbonyl alkylphosphotriesters and thiocarbonyl phosphoramidates.

[0171] In some aspects, the presence of the backbone linkages disclosed above increases the stability and degradation resistance of the polynucleotides of the present disclosure. In some aspects, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% of the backbone linkages in the polynucleotides of the present disclosure are modified (e.g., all phosphorothioates).

[0172] In some aspects, backbone modifications that may be included in the polynucleotides of the present disclosure comprise phosphorodiamidate morpholino oligomers (PMOs) and / or phosphorothioate (PS) modifications.

[0173] Sugar modification

[0174] Modified nucleosides and nucleotides that can be incorporated into the polynucleotides of the present disclosure can be modified on the sugar of the nucleic acid. Incorporation of affinity-enhancing nucleotide analogs (eg, LNA or 2'-substituted sugars) can modify (eg, reduce) the length and / or size of a polynucleotide.

[0175] In some aspects, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the nucleotides in the polynucleotides of the disclosure contain a sugar modification (e.g., LNA).

[0176] As described herein, in some aspects, the polynucleotides described herein can be RNA (e.g., mRNA). In general, RNA includes the sugar group ribose, which is a five-membered ring containing oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in the ribose (e.g., with S, Se, or an alkylene group such as methylene or ethylene); addition of double bonds (e.g., replacement of ribose with a cyclopentenyl or cyclohexenyl group); ring contraction of the ribose (e.g., to form a 4-membered ring of a cyclobutane or oxetane); ring expansion of the ribose (e.g., to form a 6- or 7-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexyl, cyclohexenyl, and morpholino, which also have an aminophosphoroester backbone); polycyclic forms (e.g., tricyclic; and "unlocked" forms, such as Such as glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, in which ribose is replaced by an ethylene glycol unit linked to a phosphodiester bond), threose nucleic acids (TNA, in which ribose is replaced by α-L-threofuranosyl-(3′→2′)), and peptide nucleic acids (PNA, in which 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbones). The sugar group may also contain one or more carbon atoms that have the opposite stereochemical configuration to the corresponding carbon atom in ribose. Thus, a polynucleotide molecule may include nucleotides containing, for example, arabinose as a sugar.

[0177] The 2' hydroxyl (OH) of the ribose can be modified or replaced by a variety of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C 1-6 Alkyl; optionally substituted C1-6 Alkoxy; optionally substituted C 6-10 Aryloxy; optionally substituted C 3-8 Cycloalkyl; optionally substituted C 3-8 Cycloalkoxy; optionally substituted C 6-10 Aryloxy; optionally substituted C 6-10 Aryl-C 1-6 Alkoxy, optionally substituted C 1-12 (Heterocyclyl)oxy; sugar (e.g., ribose, pentose, or any sugar described herein); polyethylene glycol (PEG), i.e., -O(CH2CH2O) n CH2CH2OR, wherein R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); "locked" nucleic acids (LNAs) in which the 2'-hydroxyl group is connected by a C 1-6 Alkylene or C 1-6 The heteroalkylene bridge is attached to the 4'-carbon of the same ribose, where exemplary bridges include methylene, propylene, ether, amino bridges, aminoalkyl, aminoalkoxy, amino, and amino acid.

[0178] In some aspects, the nucleotide analogs present in the polynucleotides of the present disclosure include, for example, 2'-O-alkyl-RNA units, 2'-OMe-RNA units, 2'-O-alkyl-SNA, 2'-amino-DNA units, 2'-fluoro-DNA units, LNA units, arabinose nucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2'MOE units, or any combination thereof. In some aspects, LNA is, for example, oxy-LNA (e.g., β-D-oxy-LNA or α-L-oxy-LNA), amino-LNA (e.g., β-D-amino-LNA or α-L-amino-LNA), thio-LNA (e.g., β-D-thio-LNA or α-L-thio-LNA), ENA (e.g., β-D-ENA or α-L-ENA), or any combination thereof. In other aspects, the nucleotide analogs that can be included in the polynucleotides of the present disclosure include locked nucleic acids (LNA), unlocked nucleic acids (UNA), arabinoic acids (ABA), bridged nucleic acids (BNA) and / or peptide nucleic acids (PNA).

[0179] In some aspects, the polynucleotides of the present disclosure may comprise both modified RNA nucleotide analogs (e.g., LNA) and DNA units. In some aspects, the polynucleotides described herein are gapmers. See, e.g., U.S. Patent Nos. 8,404,649; 8,580,756; 8,163,708; 9,034,837; all of which are incorporated herein by reference in their entirety.

[0180] In some aspects, the polynucleotides of the present disclosure may include modifications to prevent rapid degradation by endonucleases and exonucleases. Modifications include, but are not limited to, for example (a) terminal modifications, such as 5' terminal modifications (phosphorylation, dephosphorylation, conjugation, reverse linkage, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as replacement of bases or conjugated bases with modified bases, stabilized bases, destabilized bases, or bases that are base-paired with an expanded library of paired objects, (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars, and (d) internucleotide linkage modifications, including modification or replacement of phosphodiester linkages.

[0181] IV. Vector

[0182] In some aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides described herein (e.g., comprising multiple linked coding regions). Suitable vectors for use in the present disclosure include, but are not limited to, expression vectors, viral vectors, and plasmid vectors.

[0183] As used herein, "expression vector" refers to any nucleic acid construct that contains the necessary elements for transcription and translation of the inserted coding sequence or the necessary elements for replication and translation in the case of RNA viral vectors when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses and derivatives thereof.

[0184] As used herein, "viral vectors" include, but are not limited to, nucleic acid sequences from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; lentiviruses; adenoviruses; adeno-associated viruses; SV40-type viruses; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia virus; polioviruses; and RNA viruses, such as retroviruses. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced by genes of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by proviral integration into host cell DNA.

[0185] In some aspects, the vector is derived from an adeno-associated virus. In some aspects, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, W09712622, W09817815, W09817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0186] Other vectors include plasmid vectors. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989. In the past few years, it has been found that plasmid vectors are particularly useful for delivering genes to cells in vivo because they cannot replicate and integrate into the host genome in the host genome. However, these plasmids with promoters compatible with host cells can express peptides from genes operably encoded in the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC / CMV, various pCMV plasmids, pSV40 and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1 / hygro, catalog number V87020; pcDNA4 / myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, CA.). In addition, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0187] V. Cells

[0188] In some aspects, provided herein is a cell comprising any polynucleotide described herein (e.g., comprising a plurality of connected coding regions). In some aspects, cells as described herein have been modified to comprise polynucleotides as described herein so that cells express antigens encoded by the plurality of coding regions. As demonstrated herein, such antigens are expressed simultaneously in cells. For example, in some aspects, cells as described herein have been modified with polynucleotides at least comprising the first coding region and the second coding region, wherein the first coding region and the second coding region are connected. Such modified cells express both antigens in the first coding region and the second coding region. In some aspects, the cell does not express multiple antigens naturally, so that only after the polynucleotides as described herein are introduced into the cell, the cell expresses multiple antigens. In some aspects, the cell expresses one or more of the multiple antigens naturally, but after the cell is modified to comprise polynucleotides as described herein, the expression of one or more of the multiple antigens increases.

[0189] Unless otherwise stated, any polynucleotide described herein can be introduced into cells using any suitable method known in the art. Non-limiting examples of suitable methods for delivering one or more exogenous nucleotide sequences to cells include: transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof. As demonstrated herein, in some aspects, the polynucleotides described herein (e.g., comprising multiple linked coding regions) can be introduced into cells using constriction-mediated delivery as described herein. As further described elsewhere in this disclosure, when cells pass through a constriction, they are temporarily deformed, causing the cell membrane of the cell to be disturbed. Disturbances within the cell membrane can allow various payloads (e.g., polynucleotides comprising multiple linked coding regions) to enter the cell through disturbances (e.g., by diffusion). The specific process in which cells pass through a constriction and are temporarily deformed is referred to herein as "extrusion processing." 、 "Extrusion delivery" or "extrusion".

[0190] Therefore, in some aspects, the disclosure provides cells that have been modified to express multiple antigens (e.g., described herein, such as cancer antigens and / or non-self antigens) that are not naturally expressed by cells simultaneously, wherein the cells have passed through a constriction under a set of parameters, thereby causing a disturbance in the cell, so that the polynucleotide enters the cell by the disturbance when contacting the cell, and wherein the polynucleotide comprises a plurality of coding regions, which are connected and encode multiple antigens. For example, in some aspects, the cells provided herein at least simultaneously express a first antigen, a second antigen, and a third antigen, wherein the first, second, and third antigens are not naturally expressed by the cell, wherein the cells have passed through a constriction under a set of parameters, thereby causing a disturbance in the cell, so that the polynucleotide enters the cell by the disturbance when contacting the cell, and wherein the polynucleotide comprises a first coding region encoding a first antigen, a second coding region encoding a second antigen, and a third coding region encoding a third antigen, and wherein the first coding region, the second coding region, and the third coding region are connected.

[0191] Any suitable cell known in the art can be modified as described herein.

[0192] In some aspects, the cell is a stem cell. As used herein, the term "stem cell" refers to a cell that not only has self-replication ability, but also has the ability to differentiate into other types of cells (e.g., neurons). In some aspects, stem cells that can be used for the present disclosure include induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), tissue-specific stem cells (e.g., liver stem cells, cardiac stem cells or neural stem cells), mesenchymal stem cells, hematopoietic stem cells (HSC) or a combination thereof. In some aspects, the stem cell is an iPSC.

[0193] In some aspects, the cell is a somatic cell. As used herein, the term "somatic cell" refers to any cell in the body that is not a gamete (sperm or egg), a germ cell (a cell that continues to become a gamete) or a stem cell. Non-limiting examples of somatic cells include blood cells, bone cells, muscle cells, nerve cells, or a combination thereof. In some aspects, the somatic cells that can be used for the present disclosure include blood cells. In some aspects, blood cells are peripheral blood mononuclear cells (PBMCs). As used herein, "PBMC" refers to any peripheral blood cells with a round nucleus. In some aspects, PBMCs include immune cells. As used herein, the term "immune cell" refers to any cell that plays a role in immune function. In some aspects, immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells (DC), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof. In some aspects, blood cells are red blood cells. In some aspects, the cell is a cancer cell. In some aspects, the cancer cell is a cancer cell line cell, such as a HeLa cell. In some aspects, the cancer cell is a tumor cell. In some aspects, the cancer cell is a circulating tumor cell (CTC). In some aspects, the cell is a fibroblast, such as a primary fibroblast or a neonatal foreskin fibroblast (Nuff cell). In some aspects, the cell is an immortalized cell line cell, such as a HEK293 cell or a CHO cell. In some aspects, the cell is a skin cell. In some aspects, the cell is a reproductive cell, such as an oocyte, an ovum or a fertilized egg. In some aspects, the cell is a cell cluster, such as an embryo, provided that the cell cluster is not destroyed when passing through a hole.

[0194] VL Composition

[0195] In some aspects, the disclosure also includes a composition comprising any polynucleotide, vector or cell described herein. In some aspects, the composition is a pharmaceutical composition. Therefore, a pharmaceutical composition is disclosed herein, comprising (i) a polynucleotide as described herein (e.g., at least comprising a first coding region and a second coding region, wherein the first coding region and the second coding region are not identical, and wherein the first coding region and the second coding region are connected), and (ii) a pharmaceutically acceptable carrier. In some aspects, a pharmaceutical composition is provided herein, comprising (i) a cell modified to comprise any polynucleotide as described herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, a pharmaceutical composition is provided herein, comprising (i) a vector comprising any polynucleotide as described herein, and (ii) a pharmaceutically acceptable carrier.

[0196] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound (e.g., any polynucleotide, vector, or cell described herein). The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," and grammatical variations thereof encompass any agent approved by a U.S. federal government regulatory agency or listed in the U.S. Pharmacopeia for use in animals (including humans), and any carrier or diluent that does not cause adverse physiological effects to the extent that administration of the composition to a subject is prohibited and does not eliminate the biological activity and properties of the administered compound. Excipients and carriers that are generally safe, nontoxic, and desirable are included for use in the preparation of pharmaceutical compositions.

[0197] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as or polyethylene glycol (PEG).

[0198] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of route of administration include intramuscular, subcutaneous, ocular, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular or intratumoral. Parenteral administration is also contemplated herein, characterized in that it is injected subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquids before injection, or emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose or glycerol. In addition, if necessary, the pharmaceutical composition to be administered can also contain a small amount of non-toxic auxiliary substances, such as pH buffers, stabilizers and other such agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrin.

[0199] VII. Kit

[0200] Also disclosed herein is a kit comprising any polynucleotide, carrier, composition or cell described herein. In some aspects, the kit is used for immunotherapy and / or treatment of a disease or condition (e.g., cancer) or reducing its risk. In some aspects, the kit includes one or more containers including any polynucleotide, carrier, composition or cell described herein.

[0201] In some aspects, the kit includes instructions for use according to any method described herein. For example, the included instructions may include a description of administering a pharmaceutical composition as described herein to treat, delay the onset of, or alleviate a target disease. In some aspects, the instructions include a description of administering a composition as described herein to a subject at risk of a target disease / disorder (e.g., cancer).

[0202] In some aspects, the instructions include dosage information, dosing schedule, and route of administration. In some aspects, the container is a unit dose, bulk packaging (e.g., multi-dose packaging), or a subunit dose. In some aspects, the instructions are written instructions on a label or package insert (e.g., paper included in a test kit). In some aspects, the instructions are machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk).

[0203] In some aspects, the label or package insert indicates that the compositions disclosed herein are used to treat, delay the onset of, and / or ameliorate a disease or condition associated with cancer, such as those described herein.Instructions for practicing any of the methods described herein can be provided.

[0204] In some respects, the kit described herein adopts suitable packaging. In some respects, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags) or combinations thereof. In some respects, packaging includes packaging used in combination with a specific device (e.g., inhaler, nasal application device (e.g., nebulizer) or infusion device (e.g., miniature pump)). In some respects, the kit includes a sterile access port (e.g., the container can be an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle). In some respects, the container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle).

[0205] In some aspects, the kit further comprises additional components, such as buffer and interpretative information. In some aspects, the kit comprises a container and a label or package insert on or associated with the container. In some aspects, the disclosure provides an article comprising the contents of the kit described herein.

[0206] VIII. Use and Methods

[0207] Expression of multiple antigens

[0208] It is apparent from this disclosure that the polynucleotides, vectors, cells and / or pharmaceutical compositions described herein have a variety of in vitro and in vivo utilities. For example, the polynucleotides described herein can be administered to cultured cells in vitro or ex vivo, or, for example, administered to human subjects in vivo, to induce expression of a variety of antigens in cells, which in some aspects can help treat a variety of diseases or conditions.

[0209] Therefore, some aspects of the present disclosure relate to a method for inducing multiple antigens to be expressed in cells, the method comprising delivering polynucleotides as described herein to cells in cells. As described herein and confirmed, the multiple antigens are expressed in cells simultaneously. For example, in some aspects, a method for inducing a first antigen and a second antigen to be expressed in cells is provided herein, the method comprising delivering polynucleotides to cells in cells, wherein the polynucleotides comprise a first coding region encoding a first antigen and a second coding region encoding a second antigen, wherein the first coding region and the second coding region are not identical, and wherein the first coding region and the second coding region are connected. In some aspects, the first antigen and the second antigen are expressed in cells simultaneously.

[0210] As described herein, in some aspects, before the polynucleotides are introduced into the cell, the cell does not express the multiple antigens (e.g., the first antigen and the second antigen). In some aspects, the cell expresses one or more of the multiple antigens before the polynucleotides are introduced, but after the polynucleotides are introduced, the expression further increases. In either case, after the polynucleotides as described herein are introduced, as compared with reference expression, the expression of the multiple antigens increases. In some aspects, the reference expression is included in the expression in the cell before the polynucleotides are introduced. In some aspects, the reference expression includes the expression in the corresponding cells that are not modified to include the polynucleotides as described herein. In some aspects, the reference expression includes the expression in the corresponding cells that are modified to include the multiple polynucleotides, wherein each of the multiple polynucleotides encodes a separate antigen. In some aspects, after the polynucleotides as described herein are introduced, as compared with reference expression, the expression of the multiple antigens increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. In some aspects, following introduction of the polynucleotides described herein, expression of the plurality of antigens is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold or more as compared to a reference expression.

[0211] In some aspects, the polynucleotide is delivered to the cell in the cell, including passing a cell suspension comprising the cell through a contraction under a set of parameters, thereby causing a disturbance in the cell, so that the polynucleotide enters the cell by the disturbance when in contact with the cell. In some aspects, the method also includes contacting the cell with the polynucleotide. As used herein, "contact" between a cell and a polynucleotide as described herein does not require that the cell and the polynucleotide are in physical contact. It is obvious from the disclosure that as long as the polynucleotide can enter the cell when a disturbance occurs in the cell membrane of the cell, contact between the cell and the polynucleotide will occur. To help illustrate, in some aspects, if the cell and the polynucleotide are both present in the same cell suspension, then whether the cell and the polynucleotide are in physical contact, the cell and the polynucleotide are in contact. Therefore, in some aspects, contacting the cell with the polynucleotide includes incubating the cell suspension comprising the cell with the polynucleotide.

[0212] In some aspects, the polynucleotides described herein (e.g., comprising a plurality of linked coding regions) can be delivered to a cell intracellularly, either alone or in combination with one or more additional cargoes (also referred to herein as "payloads"). For example, in some aspects, the additional cargoes can comprise a single polynucleotide. In some aspects, the single polynucleotide can encode a compound that improves and / or enhances the therapeutic effect of the polynucleotides described herein (e.g., comprising a plurality of linked coding regions). As is generally understood in the art, multiple signals are required for optimal T cell activation: (1) "Signal 1": an antigen-specific signal provided by binding the TCR to an antigenic peptide complexed with an MHC; (2) "Signal 2": mediated by the engagement of co-stimulatory molecules (e.g., CD80 and CD86) on antigen presenting cells (APCs); and (3) "Signal 3": mediated by cytokines (e.g., IL-2 and / or IL-12). Thus, in some aspects, a polynucleotide described herein (e.g., comprising multiple linked coding regions) is delivered intracellularly to a cell in combination with one or more separate polynucleotides, wherein the one or more separate polynucleotides encode a co-stimulatory molecule (i.e., signal 2) and / or a cytokine (i.e., signal 3).

[0213] In some aspects, when delivering a variety of polynucleotides, they can be delivered to cells using a single extrusion process (for example, a cell suspension includes the multiple polynucleotides, which are delivered to cells in combination; "simultaneous delivery"). In some aspects, the multiple polynucleotides (for example, polynucleotides described herein and separate polynucleotides encoding costimulatory molecules and / or cytokines) can be delivered to cells in sequence. As used herein, the term "sequential delivery" refers to the delivery of a variety of polynucleotides to cells, wherein the first polynucleotide (for example, comprising a plurality of connected coding regions) is delivered to cells and then the second (or subsequent) polynucleotide (for example, separate polynucleotides encoding costimulatory molecules and / or cytokines) is delivered to cells. In some aspects, the first polynucleotide, the second polynucleotide, or both the first and second polynucleotides can be delivered to cells using an extrusion process. For example, in some aspects, the first polynucleotide can be delivered to cells using an extrusion process, and the second polynucleotide can be delivered to cells using a non-extrusion process (for example, transfection). In some aspects, the first polynucleotide can be delivered to cells using a non-extrusion process (for example, transfection), and the second polynucleotide can be delivered to cells using an extrusion process. In some aspects, the first extrusion can be used to deliver the first polynucleotide to the cell, and then the second extrusion can be used to deliver the second polynucleotide to the cell (also referred to herein as "sequential extrusion" or "sequential extrusion treatment"). Therefore, the sequential delivery that can be used for the present disclosure can include multiple extrusion treatments. In some aspects, each of the multiple extrusion treatments will deliver a separate polynucleotide to the cell. In some aspects, one or more of the multiple extrusion treatments do not involve the delivery of polynucleotides. For example, in some aspects, the sequential delivery method described herein includes a first extrusion, a second extrusion, and a third extrusion, wherein the first extrusion includes passing a cell without any payload through a first constriction, the second extrusion includes passing the cell after the first extrusion through a second constriction to deliver the first polynucleotide (e.g., comprising a plurality of connected coding regions) to the cell, and the third extrusion includes passing the cell after the second extrusion through a third constriction to deliver the second polynucleotide (e.g., encoding costimulatory molecules and / or cytokines) to the cell. Without being bound by any one theory, in some aspects, passing the cells through a first constriction (i.e., a first extrusion) without any payload can help prepare the cells for subsequent delivery, for example, can improve the delivery efficiency of the first polynucleotide and / or the second polynucleotide.

[0214] In some aspects, the combination of payload (e.g., polynucleotide) can be repeatedly delivered to cells (e.g., stem cells or PBMC). For example, in some aspects, a combination of polynucleotides (e.g., a first polynucleotide comprising a plurality of connected coding regions and a second polynucleotide encoding a co-stimulatory molecule and / or a cytokine) is delivered to cells by a first extrusion process; then, the combination of the polynucleotides is delivered to cells again by a second extrusion process. In some aspects, the first extrusion process includes a microfluidic device (e.g., a chip) having a multi-row constriction, so that the extrusion process occurs on a single microfluidic device (e.g., a chip). As further described herein, in some aspects, the second extrusion process can occur immediately after the cell passes through the first extrusion process (e.g., immediately after the cell passes through the constriction of the first extrusion process). In some aspects, the second extrusion process can occur at a certain time after the first extrusion process (e.g., at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day after the cell passes through the constriction of the first extrusion process).

[0215] Contraction

[0216] Microfluidic channels

[0217] As described herein, a constriction is used to cause physical deformation of a cell, so that a disturbance is generated in the cell membrane of the cell, thereby allowing a payload (e.g., a polynucleotide comprising a plurality of connected coding regions as described herein) to be delivered to the cell. In some aspects, the constriction is within a channel (referred to herein as a "microfluidic channel" or "channel") contained within a microfluidic device. When multiple channels are involved, in some aspects, the multiple channels can be placed in parallel and / or in series within a microfluidic device. In some aspects, the cells described herein can pass through at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more individual constrictions. In some aspects, the cells described herein pass through more than about 1,000 individual constrictions. In some aspects, the multiple constrictions can be part of a single microfluidic device (e.g., a chip with multiple rows of constrictions). In some aspects, one or more of the multiple constrictions can be part of different microfluidic devices. For example, in some aspects, the cells described herein (e.g., stem cells or PBMCs) undergo a first extrusion process, in which the cells pass through a first constriction in a first microfluidic device (e.g., a chip). Then, after the cells have undergone a first extrusion process (e.g., passing through a first constriction), the cells undergo a second extrusion process, in which the cells pass through a second constriction in a second microfluidic device (e.g., a chip). In some aspects, each of the constrictions is the same (e.g., having the same length, width, and / or depth). In some aspects, one or more of the constrictions are different. When multiple constrictions are used, the multiple constrictions can include a first constriction associated with a first polynucleotide (e.g., comprising a plurality of linked coding regions), and a second constriction associated with a second polynucleotide (e.g., encoding a co-stimulatory molecule and / or a cytokine), wherein the cell suspension passes through the first constriction such that the first polynucleotide is delivered to one or more cells in the plurality of cells, and then, the cell suspension passes through the second constriction such that the second polynucleotide is delivered to one or more cells in the plurality of cells. In some aspects, the cell suspension passes through the second constriction at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day after the cell suspension passes through the first constriction.

[0218] In some aspects, when a cell suspension passes through a plurality of constrictions (e.g., multiple extrusion processes), the cells are still alive after passing through each constriction. It is apparent from the present disclosure that in some aspects, the plurality of constrictions may include two or more constrictions present in a single microfluidic device (e.g., a chip with multiple rows of constrictions), such that the cells pass through the plurality of constrictions in sequence. In some aspects, the plurality of constrictions are part of a separate microfluidic device, such that a first constriction is associated with a first microfluidic device, and a second constriction is associated with a second microfluidic device. For example, in some aspects, the cell passes through a first constriction (i.e., a first extrusion process), the first constriction being associated with a first microfluidic device (e.g., a chip). After the cell has passed through the first constriction, the cell is passed through a second constriction (i.e., a second extrusion process), the second constriction being associated with a second microfluidic device (e.g., a chip). In some aspects, after passing through the first constriction, the cell is cultured in a culture medium, and then the cell is passed through the second constriction. In some aspects, the cells are cultured for at least about 1 minute, at least about 30 minutes, at least about 1 hour, at least about 6 hours, at least about 12 hours, or at least about 1 day prior to passing the cells through the second constriction. It will be apparent from this disclosure that in some aspects, the first and second constrictions have the same length, depth, and / or width. In some aspects, the first and second constrictions can have different lengths, depths, and / or widths.

[0219] In some aspects, after passing through the constriction, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the cells are still alive. When the cell passes through a plurality of constrictions (e.g., a part of a single microfluidic device or a separate microfluidic device), at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100% of the cells are still alive after passing through each of the plurality of constrictions. Any suitable method known in the art can be used to measure the viability of the cell. In some aspects, the viability of the cell can be measured using Nucleocounter NC-200, Orflo Moxi Go II cell counter or both.

[0220] Exemplary microfluidic channels containing cell-deforming constrictions for use with the methods disclosed herein are described in U.S. Publication No. 2020 / 0277566 A1, U.S. Publication No. 2020 / 0332243 A1, U.S. Publication No. 2020 / 0316604 A1, U.S. Provisional Application No. 63 / 131,423, and U.S. Provisional Application No. 63 / 131,430, each of which is incorporated herein by reference in its entirety.

[0221] In some aspects, microfluidic channel as described herein (that is, including constriction) includes lumen and is configured so that cells suspended in buffer (e.g., cell suspension) can pass through the channel. Microfluidic channel that can be used for the present disclosure can be made using any suitable material available in the art, including but not limited to silicon, metal (e.g., stainless steel), plastic (e.g., polystyrene), ceramic, glass, crystalline substrate, amorphous substrate, polymer (e.g., polymethyl methacrylate (PMMA), PDMS, cyclic olefin copolymer (COC)) or a combination thereof. In some aspects, the material is silicon. The manufacture of microfluidic channel can be carried out by any method known in the art, including but not limited to dry etching (e.g., deep reactive ion etching), wet etching, photolithography, injection molding, laser ablation, SU-8 mask or a combination thereof. In some aspects, the manufacture is carried out using dry etching.

[0222] In some aspects, the microfluidic channel that can be used for the present disclosure includes an inlet portion, a center point, and an outlet portion. In some aspects, the cross-section of one or more of the inlet portion, the center point, and / or the outlet portion can vary. For example, the shape of the cross-section can be circular, elliptical, elongated slits, square, hexagonal, or triangular.

[0223] The inlet portion defines a contraction angle. In some respects, by adjusting (e.g., increasing or decreasing) the contraction angle, any blockage of the contraction portion can be reduced or prevented. In some respects, the angle of the outlet portion can also be adjusted. For example, in some respects, the angle of the outlet portion can be configured to reduce the possibility of turbulence that can cause non-laminar flow. In some respects, the wall of the inlet portion and / or the outlet portion is linear. In some respects, the wall of the inlet portion and / or the outlet portion is curved.

[0224] In some aspects, the length, depth and / or width of the contraction can vary. In some aspects, the delivery efficiency of the payload can be adjusted by adjusting (e.g., increasing or decreasing) the length, depth and / or width of the contraction. As used herein, the term "delivery efficiency" refers to the amount of the payload delivered to the cell. For example, when the total amount of the payload delivered increases, an increase in delivery efficiency may occur.

[0225] In some aspects, the constriction has a length of less than about 1 μm. In some aspects, the constriction has a length of about 0 μm to about 100 μm. In some aspects, the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some aspects, the length of the constriction is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2.5 μm, about 5 μm, about 7.5 μm, about 10 μm, about 12.5 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm or about 100 μm. In some aspects, the length of the constriction is about 10 μm. In some aspects, the constriction has a length of about 0 μm. For example, in some aspects, a microfluidic device (e.g., a chip) that can be used for the present disclosure includes a constriction that is similar to two points of a diamond that converge together so that the length of the constriction is about 0 μm.

[0226] In some aspects, the width of the constriction is between about 0 μm and about 10 μm. In some aspects, the width of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some aspects, the width of the constriction is about 0.1 μm, about 0.2 μm, about 0.3 μm, about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm or about 10 μm. In some aspects, the width of the constriction is between about 3 μm and about 10 μm. In some aspects, the width of the constriction is about 6 μm.

[0227] In some aspects, the depth of the constriction is at least about 1 μm. In some aspects, the depth of the constriction is at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm or at least about 120 μm. In some aspects, the depth of the constriction is about 5 μm to about 90 μm. In some aspects, the depth of the constriction is about 5 μm, about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm or about 90 μm. In some aspects, the depth of the constriction is about 70 μm.

[0228] In some aspects, the length of the constriction is about 10 μm, the width of the constriction is about 6 μm, and the depth of the constriction is about 70 μm. In some aspects, the length of the constriction is 10 μm, the width of the constriction is 6 μm, and the depth of the constriction is 70 μm.

[0229] In some aspects, the diameter of the constriction (e.g., contained within a microfluidic channel) is a function of the diameter of one or more cells passing through the constriction. Without being bound by any one theory, in some aspects, the diameter of the constriction is smaller than the diameter of the cell, such that a deforming force is applied to the cell as the cell passes through the constriction, resulting in a temporary physical deformation of the cell.

[0230] Thus, in some aspects, the diameter of the constriction (also referred to herein as the "constriction size") is about 20% to about 99% of the diameter of the cell. In some aspects, the constriction size is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99% of the diameter of the cell. It will be apparent from this disclosure that by adjusting (e.g., increasing or decreasing) the diameter of the constriction, the efficiency of delivering the payload to the cell can also be adjusted.

[0231] Surface with holes

[0232] In some aspects, the constriction described herein includes pores contained in the surface. Non-limiting examples of pores contained in the surface that can be used for the present disclosure are described in, for example, U.S. Publication No. 2019 / 0382796 A1, which is incorporated herein by reference in its entirety.

[0233] In some aspects, surfaces useful in the present disclosure (i.e., comprising one or more pores that can cause physical deformation of cells when cells pass through the pores) can be made using any suitable material available in the art and / or in any of a variety of forms. Non-limiting examples of such materials include synthetic or natural polymers, polycarbonates, silicon, glass, metals, alloys, nitrocellulose, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, ceramics, or combinations thereof.

[0234] In some aspects, the surface comprises a filter. In some aspects, the filter is a tangential flow filter. In some aspects, the surface comprises a membrane. In some aspects, the surface comprises a sponge or sponge-like matrix. In some aspects, the surface comprises a matrix. In some aspects, the surface comprises a tortuous path surface. In some aspects, the tortuous path surface comprises cellulose acetate.

[0235] The surface disclosed herein (i.e., including one or more holes) can have any suitable shape known in the art. When the surface has a 2-dimensional shape, the surface can be, but is not limited to, circular, elliptical, round, square, star, triangle, polygon, pentagon, hexagon, heptagon or octagon. In some aspects, the shape of the surface is circular. When the surface has a 3-dimensional shape, in some aspects, the surface can be, but is not limited to, cylindrical, conical or cubic.

[0236] It is obvious from the present disclosure that the surface (for example, including one or more holes) that can be used for the present disclosure can have various cross-sectional widths and thicknesses. In some aspects, the cross-sectional width on the surface is between about 1mm and about 1m. In some aspects, the surface has a determined thickness. In some aspects, the surface thickness is uniform. In some aspects, the surface thickness is variable. For example, in some aspects, some parts of the surface are thicker or thinner than other parts of the surface. In such aspects, the thickness of different parts of the surface can vary from about 1% to about 90%. In some aspects, the thickness on the surface is between about 0.01 μm to about 5mm.

[0237] The cross-sectional width of the hole may depend on the type of the cell targeted by the payload. In some aspects, the aperture is a function of the diameter of the cell in the cell cluster to be targeted. In some aspects, the aperture causes the cell to be disturbed (i.e., physical deformation) when passing through the hole. In some aspects, the aperture is less than the cell diameter. In some aspects, the aperture is about 20% to about 99% of the cell diameter. In some aspects, the aperture is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the cell diameter. In some aspects, the pore size is about 0.4 μm, about 0.5 μm, about 0.6 μm, about 0.7 μm, about 0.8 μm, about 0.9 μm, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, or about 15 μm or more.

[0238] The entrance and exit of a hole can have a variety of angles. In some respects, by adjusting (for example, increasing or decreasing) hole angle, any obstruction in a hole can be reduced or prevented. In some respects, the flow velocity (that is, the rate at which a cell or a suspension comprising cells passes through a hole) is between about 0.001 mL / cm / sec and about 100 L / cm / sec. For example, the angle of an entrance or exit portion can be between about 0 and about 90 degrees. In some respects, the hole has the same entrance angle and exit angle. In some respects, the hole has different entrance angles and exit angles. In some respects, the hole edge is smooth, such as round or curved. As used herein, "smooth" hole edge has a continuous, flat and uniform surface without protrusions, ridges or uneven parts. In some respects, the hole edge is sharp. As used herein, "sharp" hole edge has a thin edge that is sharp or acute. In some respects, the hole channel is straight. As used herein, "straight" hole channel does not contain curves, bends, angles or other irregular shapes. In some respects, the hole channel is curved. As used herein, a "curved" pore channel is curved or deviates from a straight line. In some aspects, the pore channel has a plurality of curves, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more curves.

[0239] The hole can have any shape as known in the art, including 2-dimensional or 3-dimensional shapes. The hole shape (e.g., cross-sectional shape) can be but is not limited to circle, ellipse, circle, square, star, triangle, polygon, pentagon, hexagon, heptagon and octagon. In some respects, the cross-sectional shape of the hole is circular. In some respects, the 3-dimensional shape of the hole is cylindrical or conical. In some respects, the hole has a groove inlet and outlet shape. In some respects, among the holes in a given surface, the hole shape is homogeneous (i.e., consistent or regular). In some respects, among the holes in a given surface, the hole shape is heterogeneous (i.e., mixed or varied).

[0240] The surface useful for the present disclosure may have a single pore. In some aspects, the surface useful for the present disclosure includes a plurality of pores. In some aspects, the pores account for about 10% to about 80% of the total surface area of ​​the surface. In some aspects, the surface contains a total of about 1.0x10 5 to about 1.0x10 30 In some aspects, the surface includes about 10 holes with about 1.0×10 15 Holes between each / mm 2 Surface area.

[0241] The holes can be distributed in a given surface in a variety of ways. In some aspects, the holes are distributed in parallel in a given surface. In some aspects, the holes are distributed side by side in the same direction and are spaced the same distance apart in a given surface. In some aspects, the distribution of the holes is ordered or homogeneous. In such aspects, the holes can be distributed in a regular, systematic pattern, or can be spaced the same distance apart in a given surface. In some aspects, the distribution of the holes is random or heterogeneous. For example, in some aspects, the holes are distributed in an irregular, disordered pattern, or are spaced different distances apart in a given surface.

[0242] In some aspects, multiple surfaces are used so that cells pass through multiple holes, wherein the holes are on different surfaces. In some aspects, multiple surfaces are distributed in series. The multiple surfaces can be homogeneous or heterogeneous in surface size, shape and / or roughness. The multiple surfaces can also contain holes with homogeneous or heterogeneous pore size, shape and / or quantity, thereby enabling multiple payloads to be delivered to different cell types simultaneously.

[0243] In some aspects, a single hole (e.g., a hole that can be used for a surface of the present disclosure) has a uniform width dimension (i.e., a constant width along the length of the hole channel). In some aspects, a single hole has a variable width (i.e., an increased or decreased width along the length of the hole channel). In some aspects, the holes in a given surface have the same single hole depth. In some aspects, the holes in a given surface have different single hole depths. In some aspects, the holes are adjacent to each other. In some aspects, the holes are spaced a certain distance apart from each other. In some aspects, the holes are spaced a distance of about 0.001 μm to about 30 mm apart from each other.

[0244] In some aspects, the surface is coated with material. The material can be selected from any material known in the art, including but not limited to Teflon (Teflon), adhesive coating, surfactant, protein, adhesion molecule, antibody, anticoagulant, factor regulating cell function, nucleic acid, lipid, carbohydrate, transmembrane protein or their combination. In some aspects, the surface is coated with polyvinyl pyrrolidone. In some aspects, the material is covalently attached to the surface. In some aspects, the material is non-covalently attached to the surface. In some aspects, the surface molecule is released when the cell passes through the hole.

[0245] In some aspects, the surface has a changed chemical property. In some aspects, the surface is hydrophilic. In some aspects, the surface is hydrophobic. In some aspects, the surface is charged. In some aspects, the surface is positively and / or negatively charged. In some aspects, the surface can be positively charged in some areas and negatively charged in other areas. In some aspects, the surface has an overall positive charge or an overall negative charge. In some aspects, the surface can be any of the following: smooth, electropolished, rough, or plasma treated. In some aspects, the surface comprises zwitterions or dipole compounds. In some aspects, the surface is plasma treated.

[0246] In some aspects, the surface is contained within a larger module. In some aspects, the surface is contained within a syringe (e.g., a plastic or glass syringe). In some aspects, the surface is contained within a plastic filter holder. In some aspects, the surface is contained within a pipette tip.

[0247] Cell perturbations

[0248] As described herein, when a cell passes through a constriction, it is physically deformed such that a perturbation (e.g., a hole, a crack, a cavity, a pore, a hole, a break, a gap, a perforation) occurs in the cell's cell membrane. Such perturbations in the cell membrane are temporary and sufficient to deliver any payload described herein (e.g., a polynucleotide comprising a plurality of linked coding regions) into the cell. Cells have self-repair mechanisms that allow cells to repair any damage to their cell membranes. See Blazek et al., Physiology (Bethesda) 30(6):438-48 (November 2015), which is incorporated herein by reference in its entirety. Thus, in some aspects, once a cell has passed through a constriction (e.g., a microfluidic channel or pore), perturbations in the cell membrane can be reduced or eliminated so that a payload delivered to the cell does not leave the cell.

[0249] In some aspects, after the pressure is removed (e.g., the cell has passed through the constriction), the perturbation in the cell membrane persists for about 1.0 x 10 -9 In some aspects, the cell perturbation lasts for about 1.0 x 10 -9 In some aspects, the cell perturbation lasts for about 1.0 x 10 -9 seconds to about 1.0x10 -1 seconds, about 1.0x10 -9 seconds to about 1.0x10 -2 seconds, about 1.0x10 -9 seconds to about 1.0x10 -3 seconds, about 1.0x10 -9 seconds to about 1.0x10 -4 seconds, about 1.0x10 -9 seconds to about 1.0x10 -5 seconds, about 1.0x10 -9 seconds to about 1.0x10 -6 seconds, about 1.0x10 -9 seconds to about 1.0x10 -7 seconds or about 1.0x10 -9 seconds to about 1.0x10 -8 In some aspects, the cell perturbation lasts about 1.0x10 -8 seconds to about 1.0x10 -1 seconds, about 1.0x10 -7 seconds to about 1.0x10 -1 seconds, about 1.0x10 -6 seconds to about 1.0x10 -1 seconds, about 1.0x10 -5 seconds to about 1.0x10-1 seconds, about 1.0x10 -4 seconds to about 1.0x10 -1 seconds, about 1.0x10 -3 seconds to about 1.0x10 -1 seconds or about 1.0x10 -2 seconds to about 1.0x10 -1 The cellular perturbations (eg, pores or holes) generated by the methods described herein are not formed due to the assembly of polypeptide subunits to form multimeric pore structures (eg, multimeric pore structures generated by complement or bacterial hemolysin).

[0250] In some aspects, the pressure applied to the cell as it passes through the constriction temporarily damages the cell membrane, resulting in passive diffusion of the material through the perturbation. In some aspects, the cell is deformed or perturbed for only a short period of time, such as about 100 μs or less, to minimize the possibility of activating apoptotic pathways through cell signaling mechanisms, although other durations are possible (e.g., in the nanosecond to hour range). In some aspects, the cell deformation lasts less than about 1.0 x 10 -9 In some aspects, the cell deformation lasts less than about 1.0 x 10 -9 In some aspects, the cell deformation lasts for about 1.0 x 10 -9 In some aspects, the cell deformation lasts for about 1.0 x 10 -9 10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. In some aspects, the cell deformation lasts between any of: about 1.0 x 10 -9 seconds to about 1.0x10 -1 seconds, about 1.0x10 -9 seconds to about 1.0x10 -2 seconds, about 1.0x10 -9 seconds to about 1.0x10 -3 seconds, about 1.0x10 -9 seconds to about 1.0x10 -4 seconds, about 1.0x10 -9 seconds to about 1.0x10 -5 seconds, about 1.0x10 -9 seconds to about 1.0x10 -6 seconds, about 1.0x10 -9 seconds to about 1.0x10 -7 seconds or about 1.0x10 -9 seconds to about 1.0x10 -8 In some aspects, the cell is deformed or perturbed for about 1.0 x 10 -8seconds to about 1.0x10 -1 seconds, about 1.0x10 -7 seconds to about 1.0x10 -1 seconds, about 1.0x10 -6 seconds to about 1.0x10 -1 seconds, about 1.0x10 -5 seconds to about 1.0x10 -1 seconds, about 1.0x10 -4 seconds to about 1.0x10 -1 seconds, about 1.0x10 -3 seconds to about 1.0x10 -1 seconds or about 1.0x10 -2 seconds to about 1.0x10 -1 In some aspects, deforming the cell comprises deforming the cell for a period of time ranging from, but not limited to, about 1 μs to at least about 750 μs, such as at least about 1 μs, at least about 10 μs, at least about 50 μs, at least about 100 μs, at least about 500 μs, or at least about 750 μs.

[0251] In some aspects, polynucleotides as described herein (e.g., coding regions comprising multiple connections) are delivered to cells and cells pass through constrictions simultaneously. In some aspects, polynucleotides are delivered to cells and can occur after cells pass through constrictions (i.e., when the disturbance of cell membrane still exists and before the cell membrane of cells recovers). In some aspects, polynucleotides are delivered to cells and occur in about a few minutes after cells pass through constrictions. In some aspects, the disturbance after cells pass through constrictions is corrected in about five minutes after cells pass through constrictions.

[0252] In some aspects, the viability of cells (e.g., stem cells or PBMCs) after passing through the constriction is about 5% to about 100%. In some aspects, the viability of cells after passing through the constriction is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some aspects, about 1.0x10 -2 Cell viability can be measured within about 10 seconds to at least about 10 days. For example, the viability of cells can be measured about 1.0×10 -2 Cell viability is measured from about 1.0 x 10 cells per second to about 1 second, from about 1 second to about 1 minute, from about 1 minute to about 30 minutes, or from about 30 minutes to about 2 hours. In some aspects, about 1.0 x 10 -2 seconds to about 2 hours, about 1.0x10 -2 seconds to about 1 hour, about 1.0x10-2 seconds to about 30 minutes, about 11.0x10 -2 seconds to about 1 minute, about 1.0x10 -2 seconds to about 30 seconds, about 1.0x10 -2 seconds to about 1 second or about 1.0x10 -2 In some aspects, cell viability is measured in about 1.5 hours to about 2 hours, about 1 hour to about 2 hours, about 30 minutes to about 2 hours, about 15 minutes to about 2 hours, about 1 minute to about 2 hours, about 30 seconds to about 2 hours, or about 1 second to about 2 hours after the cell passes through the constriction. In some aspects, cell viability is measured in about 2 hours to about 5 hours, about 5 hours to about 12 hours, about 12 hours to about 24 hours, or about 24 hours to about 10 days after the cell passes through the constriction.

[0253] Delivery parameters

[0254] It is obvious from the present disclosure that many parameters can affect the efficiency of delivering polynucleotides described herein (e.g., coding regions comprising multiple connections) to cells using the extrusion treatment method provided herein. Therefore, by adjusting (e.g., increasing or decreasing) one or more delivery parameters, the delivery of payload to cells can be improved. Therefore, in some aspects, the present disclosure relates to a method for increasing the delivery of payload (e.g., polynucleotides described herein) to cells, wherein the method includes one or more parameters when adjusting a cell suspension through a constriction, wherein the cell suspension comprises a cell colony, and wherein the one or more parameters increase the delivery of payload to one or more cells in the cell colony compared to a reference parameter. As described elsewhere in the present disclosure, the payload can be contacted with the cell colony before, during, or after the extrusion step.

[0255] In some aspects, by adjusting one or more delivery parameters, delivery of a payload (e.g., a polynucleotide described herein) to one or more cells is increased by at least about 1 fold, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 40 fold, or at least about 50 fold compared to delivery of the payload agent to the corresponding cell using reference parameters.

[0256] In some aspects, the one or more delivery parameters that can be adjusted to increase the delivery efficiency of the parameter include cell density (ie, the concentration of cells present in the cell suspension, for example), pressure, or both. Additional examples of delivery parameters that can be adjusted are provided elsewhere in this disclosure.

[0257] In some aspects, the cell density is about 1 x 107 cells / mL, about 2x10 7 cells / mL, about 3x10 7 cells / mL, about 4x10 7 cells / mL, about 5x10 7 cells / mL, about 6x10 7 cells / mL, about 7x10 7 cells / mL, about 8x10 7 cells / mL, about 9x10 7 cells / mL, about 1x10 8 cells / mL, about 1.1x10 8 cells / mL, about 1.2x10 8 cells / mL, about 1.3x10 8 cells / mL, about 1.4x10 8 cells / mL, about 1.5x10 8 cells / mL, about 2.0x10 8 cells / mL, about 3.0x10 8 cells / mL, about 4.0x10 8 cells / mL, about 5.0x10 8 cells / mL, about 6.0x10 8 cells / mL, about 7.0x10 8 cells / mL, about 8.0x10 8 cells / mL, about 9.0x10 8 cells / mL or about 1.0x10 9 In some aspects, the cell density is about 6 x 10 7 cells / mL and about 1.2x10 8 cells / mL.

[0258] In some aspects, the pressure is about 20psi, about 25psi, about 30psi, about 35psi, about 40psi, about 50psi, about 55psi, about 60psi, about 65psi, about 70psi, about 75psi, about 80psi, about 85psi, about 90psi, about 95psi, about 100psi, about 110psi, about 120psi, about 130psi, about 140psi, about 150psi, about 160psi, about 170psi, about 180psi, about 190psi or about 200psi or more. In some aspects, the pressure is between about 30psi and about 90psi.

[0259] In some aspects, a particular type of device (e.g., a microfluidic chip) may also have an impact on the delivery efficiency of the payload described herein (e.g., a polynucleotide described herein). For microfluidic chips, different chips may have different contraction parameters, such as the length, depth, and width of the contraction; the inlet angle, outlet angle, length, depth, and width of the approaching area, etc. As described herein, such variables may affect the delivery of the payload to the cell using the extrusion processing method disclosed herein. In some aspects, the length of the contraction is up to 100 μm. For example, in some aspects, the length is about 1 μm, about 5 μm, 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm. In some aspects, the length of the contraction is less than 1 μm. In some aspects, the length of the constriction is less than about 1 μm, less than about 5 μm, less than about 10 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, or less than about 100 μm. In some aspects, the constriction has a length of about 10 μm.

[0260] In some aspects, the width of the constriction is up to about 10 μm. In some aspects, the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm, or less than about 10 μm. In some aspects, the width is between about 3 μm and about 10 μm. In some aspects, the width is about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, or about 10 μm. In some aspects, the width of the constriction is about 6 μm.

[0261] In some aspects, the depth of the constriction is at least about 1 μm. In some aspects, the depth of the constriction is at least about 1 μm, at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm or at least about 120 μm. In some aspects, the depth is between about 5 μm and about 90 μm. In some aspects, the depth is about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm or about 90 μm. In some aspects, the depth of the constriction is about 70 μm.

[0262] In some aspects, the length is about 10 μm, the width is about 6 μm, and the depth is about 70 μm.

[0263] Additional examples of parameters that may affect the delivery of a payload to a cell include, but are not limited to, the size of the contraction (e.g., length, width, and / or depth), the inlet angle of the contraction, the surface characteristics of the contraction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity), the operating flow rate, the payload concentration, the amount of time for cell recovery, or a combination thereof. Other parameters that may affect the delivery efficiency of a payload (e.g., a polynucleotide as described herein) may include the velocity of the cell in the contraction, the shear rate in the contraction, the viscosity of the cell suspension, the velocity component perpendicular to the flow velocity, and the time in the contraction. Such parameters may be designed to control the delivery of a payload.

[0264] In some aspects, the temperature used in the method of the present disclosure may also have an impact on the efficiency of the payload being delivered to the cell and the viability of the cell. In some aspects, the extrusion treatment method is carried out between about -5 ° C and about 45 ° C. For example, the method can be carried out at room temperature (e.g., about 20 ° C), physiological temperature (e.g., about 37 ° C), above physiological temperature (e.g., greater than about 37 ° C to 45 ° C or higher) or low temperature (e.g., about -5 ° C to about 4 ° C) or at a temperature between these exemplary temperatures.

[0265] Various methods can be used to drive cells through the contraction. For example, pressure can be applied by a pump (e.g., a gas cylinder or a compressor) on the inlet side, a vacuum can be applied by a vacuum pump on the outlet side, capillary action can be applied by a tube, and / or the system can be gravity-fed. A displacement-based flow system (e.g., a syringe pump, a peristaltic pump, a manual syringe or a pipette, a piston, etc.) can also be used. In some aspects, cells pass through the contraction by positive pressure. In some aspects, cells pass through the contraction by constant pressure or variable pressure. In some aspects, a syringe is used to apply pressure. In some aspects, a pump is used to apply pressure. In some aspects, the pump is a peristaltic pump or a diaphragm pump. In some aspects, a vacuum is used to apply pressure. In some aspects, cells pass through the contraction by gravity. In some aspects, cells pass through the contraction by capillary pressure.

[0266] In some aspects, the liquid flow guides the cells through the constriction. In some aspects, before the cells pass through the constriction, the fluid flow is turbulent. Turbulent flow is a fluid flow in which the velocity at a given point varies irregularly in magnitude and direction. In some aspects, the fluid flow through the constriction is laminar flow. Laminar flow involves the uninterrupted flow of a fluid near a solid boundary in which the direction of flow at each point remains constant. In some aspects, after the cells pass through the constriction, the fluid flow is turbulent. The velocity of the cells when passing through the constriction can be variable. In some aspects, the cells pass through the constriction at a uniform cell velocity. In some aspects, the cells pass through the constriction at a fluctuating cell velocity.

[0267] In some aspects, a combination therapy is used to deliver a payload, for example, using the methods described herein, followed by exposure to an electric field downstream of the constriction. In some aspects, the cell passes through an electric field generated by at least one electrode after passing through the constriction. In some aspects, the electric field helps deliver the payload to a second location within the cell, such as a cell nucleus. In some aspects, one or more electrodes are positioned near the cell deformation constriction to generate an electric field. In some aspects, the electric field is between about 0.1 kV / m and about 100 MV / m. In some aspects, an integrated circuit is used to provide an electrical signal to drive the electrode. In some aspects, the cell is exposed to an electric field with a pulse width between about 1 ns and about 1 s and a period between about 100 ns and about 10 s.

[0268] Inducing immune response

[0269] It is obvious from the present disclosure that the polynucleotides described herein (e.g., comprising a plurality of connected coding regions) can be used to induce an immune response in a subject in need. For example, as demonstrated herein, because the polynucleotides described herein comprise a plurality of connected coding regions, administering the polynucleotides disclosed herein to a subject can result in inducing an immune response to one or more antigens encoded by the plurality of coding regions. Therefore, in some aspects, a method for inducing a multi-antigen-specific immune response in a subject in need is provided herein, the method comprising administering a polynucleotide to the subject, the polynucleotide comprising a plurality of connected coding regions, and wherein the plurality of coding regions encodes a plurality of antigens. In some aspects, the method comprises administering any polynucleotide of the present disclosure. In some aspects, the method comprises administering a polynucleotide to the subject, the polynucleotide comprising at least a first coding region encoding a first antigen and a second coding region encoding a second antigen, wherein the first coding region and the second coding region are connected, and wherein after administration, an immune response to both the first antigen and the second antigen is induced in the subject. In some aspects, the method may include administering to the subject a cell modified to comprise the polynucleotide so that the cell expresses the plurality of antigens.

[0270] In some respects, after administration, as compared with the immune response of a reference subject, the immune response for the first antigen increases. In some respects, the reference subject includes the subject before administration. In some respects, the reference subject includes the corresponding subject of the administration that does not accept the polynucleotide (that is, at least including the first coding region encoding the first antigen and the second coding region encoding the second antigen, wherein the first coding region and the second coding region are connected). In some respects, the reference subject includes the corresponding subject of the administration that has accepted at least two independent polynucleotides, wherein the first antigen and the second antigen are encoded on independent polynucleotides. In some respects, as compared with the reference subject, the subject increases at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100% for the immune response for the first antigen after administration. In some aspects, the subject's immune response to the first antigen after administration is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold or more, as compared to a reference subject.

[0271] In some respects, after administration, as compared with the immune response of a reference subject, the immune response for the second antigen increases. In some respects, as compared with the reference subject, the immune response for the second antigen after administration of the subject increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. In some respects, as compared with the reference subject, the immune response for the second antigen after administration of the subject increases by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times or at least about 50 times or more.

[0272] In some respects, after administration, as compared with the immune response of a reference subject, the immune response for both the first antigen and the second antigen increases. In some respects, as compared with the reference subject, the immune response for both the first antigen and the second antigen after administration of the subject increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. In some respects, as compared with the reference subject, the immune response for both the first antigen and the second antigen after administration of the subject increases by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times or at least about 50 times or more.

[0273] Therefore, in some aspects, a method for inducing an enhanced immune response of a subject in need is provided herein. In some aspects, such methods include administering to the subject a polynucleotide comprising a plurality of coding regions, wherein the plurality of coding regions are connected, and wherein the plurality of coding regions encode a plurality of antigens. In some aspects, the method may include administering to the subject a cell modified to comprise the polynucleotide so that the cell expresses the plurality of antigens.

[0274] In some aspects, the enhanced immune response comprises: (i) as compared with a reference immune response, the amplitude of the induced immune response increases, (ii) as compared with a reference immune response, the breadth of the induced immune response increases, (iii) as compared with a reference immune response, the duration of the induced immune response increases, or (iv) any combination of (i) to (iii). As described herein, in some aspects, the reference subject comprises a subject before administration. In some aspects, the reference subject comprises a corresponding subject that has not received the administration of the polynucleotide (i.e., comprising a plurality of coding regions, the coding regions are connected and encode a plurality of antigens). In some aspects, the reference subject comprises a corresponding subject that has received the administration of a plurality of polynucleotides, wherein each of the plurality of antigens is encoded on a separate polynucleotide.

[0275] In some aspects, the magnitude of the induced immune response is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject. In some aspects, the magnitude of the induced immune response is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or more, as compared to a reference subject.

[0276] In some aspects, the breadth of the induced immune response is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject. In some aspects, the breadth of the induced immune response is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or more, as compared to a reference subject.

[0277] In some aspects, the duration of the induced immune response is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, as compared to a reference subject. In some aspects, the duration of the induced immune response is increased by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times or more, as compared to a reference subject.

[0278] In some aspects, the magnitude and breadth of the immune response induced in the subject are increased, such as compared to a reference subject. In some aspects, the magnitude and duration of the immune response induced in the subject are increased, such as compared to a reference subject. In some aspects, the breadth and duration of the immune response induced in the subject are increased, such as compared to a reference subject. In some aspects, the magnitude, breadth, and duration of the immune response induced in the subject are increased, such as compared to a reference subject.

[0279] It is obvious from the present disclosure that the enhanced immune response induced by the polynucleotides described herein or cells modified to contain the polynucleotides can be used to treat a variety of diseases or disorders. In some aspects, a method for treating a disease or disorder of a subject in need is provided herein, the method comprising administering any polynucleotide of the present disclosure to the subject. Some aspects of the present disclosure relate to a method for treating a disease or disorder of a subject in need, the method comprising administering a polynucleotide to the subject, the polynucleotide comprising at least a first coding region encoding a first antigen and a second coding region encoding a second antigen, wherein the first coding region and the second coding region are connected, wherein the first antigen and the second antigen are associated with the disease or disorder. Therefore, it is obvious to those skilled in the art that the polynucleotides described herein (or cells modified to contain the polynucleotides) can be used to treat any disease or disorder associated with a specific antigen. Without being bound by any one theory, an immune response to the multiple antigens can be induced by encoding multiple antigens associated with a disease or disorder in multiple coding regions of the polynucleotides described herein. In some aspects, such immune responses can be used to treat the disease or disorder. For example, in some aspects, the polynucleotides described herein comprise multiple linked coding regions, wherein the multiple coding regions encode multiple KRAS mutant antigens (e.g., comprising G12D and / or G12V amino acid substitutions). Such polynucleotides can be used to treat cancers associated with KRAS mutant expression.

[0280] The following examples are offered by way of illustration and not by way of limitation.

[0281] Example

[0282] Example 1: Analysis of antigen expression using 5 linked antigens

[0283] To determine whether immune cells extruded with a single mRNA encoding five linked antigen fragments could elicit multiple antigen-specific immune responses simultaneously, human PBMCs were extruded with three different linked antigen mRNA constructs, each encoding HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D A fragment of an antigen that is approximately 25 amino acids long. Figure 1A The five antigen fragments in each mRNA construct were linked in a different order than in the other two mRNA constructs. Antigen-specific responder cells were used to assess the presentation of immunogenic epitopes of these encoded antigens on MHC class I cells.

[0284] By expressing KRAS G12V Specific TCR, E6 29-38 Specific TCR or E7 11-19The E6 T cell receptor (TCR), E7 TCR, and KRAS were generated by lentiviral transduction of specific TCRs into Jurkat-Lucia NFAT cells (InvivoGen) in which endogenous TCRα / β had been knocked out. G12V TCR Jurkat-Lucia NFAT reporter cells Engagement of these three TCRs with the corresponding peptide-MHC (pMHC) complexes results in activation of the NFAT signaling pathway and secretion of Lucia luciferase.

[0285] 5×10 7 The density of cells / mL is prepared from PBMC of human HLA-A*02+HLA-A*11+ donors, and at room temperature in RPMI 1640 culture medium, the mRNA A construct of encoding 5 linked antigen fragments (5xL1, 5xL2 or 5xL3) of 250 μg / mL or without cargo (empty extrusion) is squeezed through the contraction of 3.5 μm width, 10 μm length and 70 μm depth at 60 psi. The PMBC of extrusion load is transferred to RPMI+10% fetal bovine serum+1x Pen / Strep (R10), quenched at room temperature. The PBMC of extrusion load is washed twice in co-culture medium (R10), then resuspended in fresh co-culture medium.

[0286] Then 2.5×10 5 PBMCs with extrusion load of 1.25×10 5 E6 TCR Jurkat cells or 5 × 10 4 E7 TCR Jurkat cells were placed in a 96-well plate for co-culture. 5 PBMCs were extruded with 2.5 × 10 5 KRAS G12V TCR Jurkat cells were placed in 96-well plates for co-culture. As a positive control, 1 μM E6 29-38 、E7 11-19 or KRAS G12V (7-16) The minimal epitope was added directly to the co-culture of untreated PBMC and relevant Jurkat responder cells. After incubation of the co-culture at 37°C for 16 to 18 hours, the co-culture supernatant was harvested. After addition of coelenterazine substrate, the level of secreted Lucia luciferase in the culture supernatant was measured by luminescence.

[0287] To measure the response to the pp65 fragment in the ligated antigen mRNA, 2.5 × 10 5 PBMCs with 6.25×10 4CMV pp65-specific T cells (Cellero) were placed in 96-well plates for co-culture. As a positive control, 1 μM pp65 495-503 The minimal epitope was added directly to the co-culture of untreated PBMC and CMV pp65-specific T cells. After incubation of the co-culture at 37°C for 16 to 18 hours, the co-culture supernatant was harvested. To assess the activation of pp65-specific T cells, the IFNγ concentration in the co-culture supernatant was measured using an Ella immunoassay instrument (Bio-Techne) according to the manufacturer's protocol.

[0288] like Figure 1B As shown, compared with empty extrusion controls, extrusions loaded with ligation antigen mRNA and associated with CMV pp65-specific T cells, KRAS G12V Co-culture of human PBMCs with TCR Jurkat cells, E6 TCR Jurkat cells, or E7 TCR Jurkat cells resulted in increased activation of relevant responder cells. These results suggest that extrusion of human PBMCs loaded with linked antigen mRNA can elicit immune responses to multiple antigen fragments simultaneously.

[0289] Example 2: Analysis of antigen expression using 10 linked antigens

[0290] To determine whether immune cells extruded with a single mRNA encoding up to 10 linked antigen fragments could elicit multiple antigen-specific immune responses simultaneously, human PBMCs were extruded with two different linked antigen mRNA constructs, each encoding HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V 、KRAS G12D , Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S, and MAGE-A10 antigens. The 10 antigen fragments in each mRNA construct were linked in a different order from the other constructs. Figure 2A Antigen-specific responder cells are used to assess the presentation of immunogenic epitopes of these encoded antigens on MHC class I cells.

[0291] By expressing E6 29-38 Specific TCR or E7 11-19E6 TCR and E7 TCR Jurkat-LuciaNFAT reporter cells were generated by lentiviral transduction of specific TCRs into Jurkat-Lucia NFAT cells (InvivoGen) in which endogenous TCRα / β had been knocked out. Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes results in activation of the NFAT signaling pathway and secretion of Lucia luciferase.

[0292] 5×10 7 The density of cells / mL was used to prepare the HLA-A*02 + HLA-A*11 + Donor's PBMC, and at room temperature in RPMI 1640 culture medium, with 250 μ g / mL of encoding 10 antigen fragments (10xL1 or 10xL2) mRNA constructs, with 250 μ g / mL of encoding 5 antigen fragments (5xL3) mRNA constructs or without goods (empty extrusion) under 60psi, extruded through the contraction of 3.5 μ m width, 10 μ m length and 70 μ m depth. The PMBC of extruding load is transferred among RPMI+10% fetal calf serum+1x Pen / Strep (R10) and at room temperature quenched. The PBMC of extruding load is washed twice in co-culture medium (R10), then resuspended in fresh co-culture medium.

[0293] Then 2.5×10 5 PBMCs with extrusion load of 1.25×10 5 E6 TCR Jurkat cells or 5 × 10 4 E7 TCR Jurkat cells were placed in 96-well plates for co-culture. As a positive control, 1 μM E6 29-38 or E7 11-19 The minimal epitope was added directly to the co-culture of untreated PBMC and relevant Jurkat responder cells. After incubation of the co-culture at 37°C for 16 to 18 hours, the co-culture supernatant was harvested. After addition of coelenterazine substrate, the level of secreted Lucia luciferase in the culture supernatant was measured by luminescence.

[0294] 2.5×10 5 PBMCs with 6.25×10 4 CMV pp65, M1 Flu, NY-ESO-1, or MAGE-A10 specific T cells (Cellero) were placed in 96-well plates for co-culture. As a positive control, 1 μM M1 58-66 or pp65 495-503The minimal epitope was added directly to the co-culture of untreated PBMC and relevant antigen-specific T cells. After incubation of the co-culture at 37°C for 16 to 18 hours, the co-culture supernatant was harvested. To assess the activation of antigen-specific T cells, the IFNγ concentration in the co-culture supernatant was measured using an Ella immunoassay instrument (Bio-Techne) according to the manufacturer's protocol.

[0295] like Figure 2B and Figure 2C As shown, extrusion of human PBMCs loaded with mRNA constructs encoding 10 linked antigen fragments and co-cultured with CMV pp65-specific T cells, Flu M1-specific T cells, NY-ESO-1-specific T cells, E6 TCR Jurkat cells, or E7 TCR Jurkat cells resulted in increased activation of the relevant responder cells compared to empty extrusion controls. These results suggest that extrusion of human PBMCs loaded with linked antigen mRNAs can simultaneously elicit immune responses to multiple antigen fragments.

[0296] Example 3: In vitro translation efficiency of mRNA containing linked antigens

[0297] To evaluate the expression of proteins encoding five linked antigenic fragments (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D ) or encodes 10 linked antigen fragments (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V 、KRAS G12D In vitro translation efficiency of mRNA constructs of 1, 2, 3, 4, 5, 7, 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67 Figure 3A .

[0298] Will SP6 high-yield wheat germ protein premix (Promega) was thawed and immediately placed on ice. Each connection antigen mRNA construct was diluted to a final concentration of 1 μg / μL with diH2O. For each connection antigen mRNA construct, 2 μg mRNA was added to 30 μL wheat germ extract, and diH2O was used to make it reach a total volume of 50 μL, and the reactants were incubated at 25°C for 2 hours. After 2 hours of incubation, the sample was placed on ice to terminate the reaction, and 50 μL of the translated sample was mixed with 25 μL of sample buffer.

[0299] For SDS-PAGE gels, samples were heated at 95°C for 2 minutes and then loaded onto 4 to 12% protein gels (Invitrogen). The samples were run on the gel at 100-130V for about 90 minutes until the dye reached the bottom of the gel. The gel was transferred to a nitrocellulose membrane and stained with a 50% ELISA kit for HPV E6. 29-38 The junction antigens were analyzed by blot analysis using a primary antibody raised against SLP (clone 5G10) and a goat anti-rabbit secondary antibody. Translation was assessed by the presence of a protein band of approximately 26 kDa in mRNA constructs encoding 5 junction antigen fragments and a protein band of approximately 35 kDa in mRNA constructs encoding 10 junction antigen fragments.

[0300] like Figure 3B As shown, the translation of every kind of connection antigen mRNA construct has been detected under various amplitudes.In three kinds of mRNA constructs encoding 5 model antigen fragments that link together separately, the translation degree of construct 5xL3 is the highest, followed by 5xL2 and 5xL1 at last.In two kinds of mRNA constructs encoding 10 model antigen fragments that link together separately, the translation degree of 10xL2 is higher than 10xL1.

[0301] Example 4: Inducing KRAS mutant-specific immune responses

[0302] To determine whether immune cells stimulated with a single mRNA encoding five linked antigen fragments could simultaneously elicit KRAS G12V and KRAS G12D Antigen-specific immune responses were obtained by extruding human PBMCs with three different linked antigen mRNA constructs, each encoding HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D The antigen is a fragment of about 25 amino acids long. The five antigen fragments in each mRNA construct are linked in a different order than in the other two mRNA constructs. Antigen-specific responder cells are used to evaluate the presentation of these immunogenic epitopes of the encoded antigen on MHC class I cells.

[0303] By expressing KRAS G12V Specific TCR or KRAS G12D The KRAS-specific TCR was transduced into Jurkat-Lucia NFAT cells (InvivoGen) in which endogenous TCRα / β was knocked out. G12D TCR and KRAS G12VTCRJurkat-Lucia NFAT reporter cells. Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes results in activation of the NFAT signaling pathway and secretion of Lucia luciferase.

[0304] 5×10 7 The density of individual cells / mL is prepared from the PBMC of human HLA-A*02+HLA-A*11+ donor, and at room temperature in RPMI 1640 culture medium, with 250 μ g / mL of mRNA constructs encoding 5 linked antigen fragments (5xL1, 5xL2 or 5xL3) or without cargo (empty extrusion) under 60psi, extruded through the contraction of 3.5 μm width, 10 μm length and 70 μm depth. The PMBC of extrusion load is transferred to RPMI+10% fetal bovine serum+1x Pen / Strep (R10), quenched at room temperature. The PBMC of extrusion load is washed twice in co-culture medium (R10), then resuspended in fresh co-culture medium.

[0305] Then 5×10 5 PBMCs were extruded with 2.5 × 10 5 KRAS G12V TCR Jurkat cells or KRAS G12D TCR Jurkat cells were placed in 96-well plates for co-culture. As a positive control, 1 μM KRAS G12V (7-16) or KRAS G12D (7-16) The minimal epitope was added directly to the co-culture of untreated PBMC and relevant Jurkat responder cells. After incubation of the co-culture at 37°C for 16 to 18 hours, the co-culture supernatant was harvested. After addition of coelenterazine substrate, the level of secreted Lucia luciferase in the co-culture supernatant was measured by luminescence.

[0306] like Figure 4 As shown, compared with empty extrusion controls, extrusions loaded with junction antigen mRNA and KRAS G12V Co-culture of human PBMCs with TCRJurkat cells resulted in increased activation of relevant responder cells. These results suggest that extrusion of human PBMCs loaded with linked antigen mRNA can elicit an immune response to KRAS mutant antigens.

[0307] Example 5: Effects of Signal 2 / 3 on Immune Responses Induced by mRNA Containing Linked Antigens

[0308] To measure the functional CD8+ T cell responses of immune cells primed with a single mRNA encoding 5 linked antigen fragments plus or without the additional signal 2 / 3 mRNAs (encoding CD86, mbIL-2, and mbIL-12), the mRNAs encoding HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D Human PBMCs were extruded with mRNA constructs of approximately 25 amino acid long aa fragments of the antigen with or without signal 2 / 3 mRNA, or with signal 2 / 3 mRNA alone. Antigen-specific primary cells were used to assess functional CD8+ T cell responses to these antigens with or without signal 2 / 3.

[0309] like Figure 5A As shown, CD8+ T cells were isolated from HLA-A*02+ donors and activated for 2 days with CD3 / CD28 Dynabeads and rhIL-2. 29-38 Specific TCR or E7 11-19 CD8+ T cells were transduced with lentiviruses expressing specific TCRs. Residual lentivirus was removed and cells were expanded for 5 days before co-culture. Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes resulted in IFNγ production.

[0310] 5×10 7 The PBMC from the same HLA-A*02+ donor was prepared at a density of 1 cell / mL, and at room temperature in RPMI 1640 culture medium, with 250 μg / mL of connection antigen mRNA (L2), connection antigen mRNA and signal 2 / 3mRNA, signal 2 / 3mRNA alone or without cargo (empty extrusion) at 60psi through a contraction of 3.5 μm width, 10 μm length and 70 μm depth. The PMBC of the extrusion load is transferred to RPMI+10% fetal bovine serum+1x Pen / Strep (R10) and quenched at room temperature. The PBMC of the extrusion load is washed twice in co-culture medium (R10), then resuspended in fresh co-culture medium.

[0311] Then 5×10 4 PBMCs were loaded with 0.2% E6 TCR or E7 TCR tetramer + CD8 + T cells and 1.28 × 10 5 Untreated PBMCs were placed in 96-well plates for co-culture. After incubation at 37°C for 6 days, the co-culture was treated with the corresponding E6 29-38 or E7 11-19Co-cultured cells were restimulated with peptide and golgiblock or with golgiblock alone as a negative control and incubated at 37°C. After 6 h of restimulation, cells were intracellularly stained and the expression of IFNγ and TNFα was assessed by flow cytometry. E7 was also assessed by flow cytometry after 6 days of co-culture. 11-29 Pentamer+ or E6 29-38 Frequency of tetramer+CD8+ T cells.

[0312] like Figure 5B As shown, extrusion of human PBMCs loaded with junction antigen mRNA and signal 2 / 3 mRNA and then co-cultured with E6 TCR-transduced CD8+ T cells or E7 TCR-transduced CD8+ T cells resulted in increased activation of antigen-specific CD8+ T cells compared to human PBMCs extruded with signal 2 / 3 mRNA alone. These results suggest that extrusion of human PBMCs loaded with junction antigen mRNA can elicit a strong immune response when combined with signal 2 / 3 mRNA.

[0313] Example 6: Analysis of KRAS G12V and G12D-specific responses after extrusion with mRNA encoding two KRAS mutant linked antigens

[0314] To further assess the functional capacity of the polynucleotides described herein (eg, comprising multiple linked coding regions), human PBMCs were extruded with one of the linked mRNAs described below.

[0315] (1) Concatenated mRNA constructs encoding: (a) KRAS G12D and KRAS G12V an approximately 25aa fragment of the antigen (overlapping with the 1-25aa sequence in the entire KRAS protein and with a mutation at codon 12), (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) (“junction antigen +2 / 3”);

[0316] (2) Encoding only KRAS G12D and KRAS G12V A conjugated mRNA construct of an approximately 25aa fragment of the antigen (overlapping the 1-25aa sequence in the entire KRAS protein and having a mutation at the 12th codon) ("G12D-G12V conjugated antigen");

[0317] (3) a linked mRNA construct encoding only CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) (“signal 2 / 3”);

[0318] (4) a concatenated mRNA construct encoding: (a) a single approximately 25 aa fragment comprising the G12V mutation, (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("G12V+2 / 3");

[0319] (5) a concatenated mRNA construct encoding: (a) a single approximately 25 aa fragment comprising the G12D mutation, (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("G12D+2 / 3");

[0320] (6) a concatenated mRNA construct encoding: (a) an approximately 25 aa fragment overlapping the first 1-25 aa of native wild-type KRAS; (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("WT+2 / 3");

[0321] (7) an mRNA construct encoding only a single approximately 25 aa fragment containing the G12V mutation (i.e., without signals 2 and 3) ("G12V");

[0322] (8) an mRNA construct encoding only a single approximately 25 aa fragment containing the G12D mutation (i.e., without signals 2 and 3) ("G12D"); and

[0323] (9) An mRNA construct encoding only a fragment of approximately 25 aa that overlaps with the first 1-25 aa of native wild-type KRAS (ie, without signals 2 and 3) ("wild-type").

[0324] method

[0325] Generation of responder cells:

[0326] To generate A*11-restricted G12V and G12D TCR Jurkat responder cells, cells expressing A*11-restricted G12V 7-16 and G12D 7-16 Jurkat-LuciaNFAT TCRα / βKO cells were custom-made by Creative Biolabs for lentiviral transduction of specific TCR. The residual virus was removed, and the cells were then selected with puromycin and cultured for several days, followed by mouse TCR (mTCR) enrichment to increase mTCR +After mTCR enrichment, cells were cultured for several days, characterized by flow cytometry and frozen for co-culture analysis. The resulting A*11 restricted G12V TCR Jurkat and G12D TCR Jurkat responder cells expressed HLA-A*11 restricted G12V on the cell surface, respectively. 7-16 Specific TCR or HLA-A*11 restricted G12D 7-16 Specific TCR. The engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes (provided in the form of PBMCs squeezed with KRAS mutant mRNA, or incubated with G12V7-16 or G12D7-16A*11 restricted peptides) leads to the activation of the NFAT signaling pathway and the secretion of Lucia luciferase. The level of Lucia luciferase secreted in the culture supernatant is measured using a coelenterazine substrate. The luminescent signal generated is related to the amount of Lucia luciferase secreted, confirming that the cells have been produced correctly.

[0327] Extrusion treatment:

[0328] 200×10 6 The density of cells / mL was prepared from HLA-A*11 + PBMCs of donors were added and allowed to stand at 4-8°C for about 15 minutes. Next, the PBMCs were extruded with 250 μg / mL of one of the above mRNA constructs in RPMI 1640 medium at 60 psi using a constriction of 3.5 μm width, 10 μm length, and 70 μm depth (the mRNA solution was cooled at 4-8°C for about 15 minutes, then mixed with the cells and extruded). PBMCs that were not extruded with mRNA (i.e., empty extrusion) were used as a control. After extrusion, the extruded PBMCs were transferred to R10 medium (RPMI+10% fetal bovine serum (FBS)+1x Pen / Strep), quenched at room temperature, then washed in R10 and incubated at 5x10 6 Cells / mL were cryopreserved and stored in liquid nitrogen for future use.

[0329] analyze:

[0330] On the day of co-culture setup, culture the cells at 2.5x10 μl in IMDM + 10% FBS. 6 Prepare HLA-A*11 restricted G12V and G12D TCR Jurkat at 10 cells / mL. Thaw cryopreserved squeezed or untreated PBMCs and replace the medium with IMDM + 10% FBS at a density of 5x10 6G12V or G12D TCR Jurkat was co-cultured with PBMC at a 1:2 ratio (250,000 Jurkat cells to 500,000 PBMC) in a tissue culture treated (TCT) 96-well U-bottom plate overnight (18-24 hours). 7-16 and G12D 7-16 or WT 7-16 The minimal epitope was added to co-cultures with Jurkat cells and untreated PBMCs to control for antigen mutation-specific Jurkat reactivity. Cell culture supernatants were collected and analyzed for levels of secreted Lucia luciferase by Quanti LUC.

[0331] result

[0332] like Fig. 6A and Figure 6B As shown, extrusion of human PBMCs loaded with KRAS G12D-G12V junction antigen mRNA with or without signal 2 / 3 mRNA and co-cultured with G12V TCR-transduced Jurkat cells or G12D TCR-transduced Jurkat cells resulted in KRAS mutant-specific increases in activation of G12V and G12D TCR-transduced Jurkat cells relative to controls (empty extrusion, signal 2 / 3 mRNA alone, KRAS WT mRNA with or without signal 2 / 3 mRNA). PBMCs extruded with KRAS G12D-G12V junction antigen mRNA elicited similar or stronger mutation-specific responses than their associated KRAS mutant single mRNAs in G12V TCR-transduced or G12D TCR-transduced Jurkat cells, respectively.

[0333] These results indicate that extrusion of human PBMCs loaded with KRAS mutant-linked antigen mRNAs can elicit strong mutation-specific responses in G12V- and G12DTCR-transduced Jurkat cells that are similar to or stronger than their respective KRAS mutant single antigen mRNAs.

[0334] Example 7: Analysis of KRAS G12V and G12D-specific responses after extrusion with mRNA encoding seven KRAS mutant-linked antigens

[0335] To evaluate the functional activity of mRNA encoding more than two linked antigens, immune cells (PBMCs) were extruded with mRNA constructs containing seven linked KRAS mutant antigens. The specific mRNA constructs used are provided below. Each construct was used at one of two doses: 250 μg / mL and 500 μg / mL.

[0336] (1) Encoding only KRAS G12D 、KRAS G12V 、KRAS G12C 、KRAS G13D 、KRAS G12A 、KRAS G12R and KRAS G12S A conjugated mRNA construct of an approximately 25aa fragment of the antigen (overlapping the 1-25aa sequence in the entire KRAS protein and having a mutation at the 12th codon) ("7mut_v1"); and

[0337] (2) Encoding only KRAS G12S 、KRAS G12R 、KRAS G12A 、KRAS G13D 、KRAS G12C 、KRAS G12V and KRAS G12D A ligated mRNA construct ("7mut_v2") of a fragment of approximately 25 aa of the antigen that overlaps the 1-25 aa sequence in the entire KRAS protein and has a mutation at the 12th codon.

[0338] method

[0339] Generation of responder cells:

[0340] To generate A*11-restricted G12V and G12D TCR Jurkat responder cells, cells expressing A*11-restricted G12V 7-16 and G12D 7-16 Lentiviral transduction of specific TCR was custom-made by Creative Biolabs for Jurkat-LuciaNFAT TCRα / βKO cells (once). The residual virus was removed one day after TCR transduction, and the cells were then selected with puromycin and cultured for several days, followed by mouse TCR (mTCR) enrichment to increase mTCR + After mTCR enrichment, cells were cultured for several days, characterized by flow cytometry and frozen for co-culture analysis. The resulting A*11 restricted G12V TCR Jurkat and G12D TCR Jurkat responder cells expressed HLA-A*11 restricted G12V on the cell surface, respectively. 7-16 Specific TCR or HLA-A*11 restricted G12D 7-16 Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes (provided in the form of PBMCs extruded with KRAS mutant mRNA or with G12V 7-16 or G12D7-16 A*11 restriction peptide incubation together) can cause the activation of NFAT signal transduction pathway and the secretion of Lucia luciferase. The level of Lucia luciferase secreted in the culture supernatant can be measured using coelenterazine substrate. The luminescent signal generated is related to the amount of Lucia luciferase secreted.

[0341] Extrusion treatment:

[0342] 200×10 6 The density of cells / mL was prepared from HLA-A*11 + PBMCs from donors were allowed to rest at 4-8°C for about 10-15 minutes, then extruded at 60 psi through a 3.5 μm wide, 10 μm long, and 70 μm deep constriction in RPMI 1640 medium with 250 or 500 μg / mL of KRAS 7mut_v1 mRNA or 250 or 500 μg / mL of KRAS 7mut_v2 mRNA or no cargo (empty extrusion) at room temperature (the mRNA solution was cooled at 4-8°C for about 10-15 minutes, then mixed with cells and extruded). After extrusion, the extruded PBMCs were transferred to R10 medium (RPMI+10% fetal bovine serum (FBS)+1x Pen / Strep), quenched at room temperature, then washed in R10 and incubated at 5-10x10 6 Cells / mL were cryopreserved and stored in liquid nitrogen for future use.

[0343] analyze:

[0344] On the day of co-culture setup, culture the cells at 2.5x10 μl in IMDM + 10% FBS. 6 Prepare HLA-A*11 restricted G12V and G12D TCR Jurkat at 10 cells / mL. Thaw cryopreserved squeezed or untreated PBMCs and replace the medium with IMDM + 10% FBS at a density of 5x10 6 G12V or G12D TCR Jurkat was co-cultured with PBMC at a 1:2 ratio (250,000 Jurkat cells to 500,000 PBMC) in a tissue culture treated (TCT) 96-well U-bottom plate overnight (18-24 hours). 7-16 and G12D 7-16The minimal epitope is added to the co-culture with Jurkat cells and untreated PBMCs to control the antigen mutation specific Jurkat reactivity. Cell culture supernatants are collected and the level of secreted Lucia luciferase is analyzed by Quanti LUC. The luminescent signal produced is related to the amount of secreted Lucia luciferase.

[0345] result:

[0346] As shown in 7A and 7B, human PBMCs extruded with KRAS seven mutant linked antigen mRNA and co-cultured with G12V TCR-transduced or G12DTCR-transduced Jurkat cells resulted in KRAS mutant-specific responses in TCR-transduced Jurkat cells relative to controls (empty extrusion, untreated PBMCs, and irrelevant mutant minimal epitope stimulation). G12V-specific responses: PBMCs extruded with KRAS 7mut_v1 mRNA elicited G12V-specific responses in G12V TCR-transduced Jurkat cells in a dose-dependent manner. PBMCs extruded with KRAS 7mut_v2 mRNA elicited similar responses at two extrusion concentrations (250, 500 μg / mL) and were comparable to responses elicited by PBMCs extruded with 250 μg / mL of KRAS 7mut_v1 mRNA. G12D-specific responses: PBMCs extruded with KRAS7mut_v1 mRNA elicited G12D-specific responses in Jurkat cells transduced with G12D TCR in a dose-dependent manner. PBMCs extruded with KRAS7mut_v2 mRNA elicited G12D responses at levels similar to those observed in controls (untreated PBMCs and empty extrusions).

[0347] These results indicate that extrusion of human PBMCs loaded with KRAS 7 mutant-linked antigen mRNA (KRAS 7mut_v1 construct) can elicit strong mutation-specific responses in both G12V and G12D TCR-transduced Jurkat cells.

[0348] Example 8: Further analysis of KRAS G12V and G12D-specific responses after extrusion with mRNA encoding seven KRAS mutant-linked antigens

[0349] To evaluate the functional activity of mRNA encoding seven KRAS mutant antigens and further encoding CD86 (signal 2), membrane-bound IL-2 (signal 3), and membrane-bound IL-12 (signal 3), immune cells (PBMCs) were extruded with one of the following mRNA constructs:

[0350] (1) Concatenated mRNA constructs encoding: (a) KRAS G12D 、KRAS G12V 、KRAS G12C 、KRAS G13D 、KRAS G12A 、KRAS G12R and KRAS G12S A fragment of about 25 aa of the antigen (overlapping with the 1-25 aa sequence in the entire KRAS protein and with a mutation at the 12th codon) (referred to as "KRAS 7mut_v1"), (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("7mut_v1+signal 2 / 3");

[0351] (2) Encoding only KRAS G12D 、KRAS G12V 、KRAS G12C 、KRAS G13D 、KRAS G12A 、KRAS G12R and KRAS G12S A conjugated mRNA construct of a fragment of about 25 aa of the antigen (overlapping the 1-25 aa sequence in the entire KRAS protein and having a mutation at the 12th codon) ("7mut_v1");

[0352] (3) Concatenated mRNA constructs encoding: (a) KRAS G12s 、KRAS G12R 、KRAS G12A 、KRAS G13D 、KRAS G12C 、KRAS G12V and KRAS G12D A fragment of about 25 aa of the antigen (overlapping with the 1-25 aa sequence in the entire KRAS protein and having a mutation at the 12th codon) (referred to herein as KRAS 7mut_v2), (b) CD86 (i.e., signal 2), (c) membrane-bound IL-2 (i.e., signal 3), and (d) membrane-bound IL-12 (i.e., signal 3) ("7mut_v2+signal 2 / 3");

[0353] (4) Encoding only KRAS G12S 、KRAS G12R 、KRAS G12A 、KRAS G13D 、KRAS G12C 、KRAS G12V and KRAS G12DA conjugated mRNA construct of an approximately 25aa fragment of the antigen (overlapping the 1-25aa sequence in the entire KRAS protein and having a mutation at the 12th codon) ("mut_v2"); and

[0354] (5) A linked mRNA construct encoding only CD86 (ie, signal 2), (c) membrane-bound IL-2 (ie, signal 3), and (d) membrane-bound IL-12 (ie, signal 3) ("signal 2 / 3 alone").

[0355] method

[0356] Generation of responder cells:

[0357] To generate A*11-restricted G12V and G12D TCR Jurkat responder cells, cells expressing A*11-restricted G12V 7-16 and G12D 7-16 Lentiviral transduction of specific TCR was custom-made by Creative Biolabs for Jurkat-LuciaNFAT TCRα / βKO cells (once). The residual virus was removed one day after TCR transduction, and the cells were then selected with puromycin and cultured for several days, followed by mouse TCR (mTCR) enrichment to increase mTCR + After mTCR enrichment, cells were cultured for several days, characterized by flow cytometry and frozen for co-culture analysis. The resulting A*11 restricted G12V TCR Jurkat and G12D TCR Jurkat responder cells expressed HLA-A*11 restricted G12V on the cell surface, respectively. 7-16 Specific TCR or HLA-A*11 restricted G12D 7-16 Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes (provided in the form of PBMCs extruded with KRAS mutant mRNA or with G12V 7-16 or G12D 7-16 A*11 restriction peptide incubation together) can cause the activation of NFAT signal transduction pathway and the secretion of Lucia luciferase. The level of Lucia luciferase secreted in the culture supernatant can be measured using coelenterazine substrate. The luminescent signal generated is related to the amount of Lucia luciferase secreted.

[0358] Extrusion treatment:

[0359] 200×10 6 The density of cells / mL (cell squeezing concentration 100×10 6 cells / mL) from HLA-A*11 +PBMCs of donors were placed in a 3.5 μm wide, 10 μm long, and 70 μm deep constriction at 60 psi and room temperature in RPMI 1640 medium with 500 μg / mL of one of the above mRNA constructs. (The mRNA solution was cooled at 4-8°C for about 15 minutes, then mixed with cells and squeezed.) After squeezing, the squeezing PBMCs were transferred to R10 medium (RPMI+10% fetal bovine serum (FBS)+1xPen / Strep), quenched at room temperature, then washed in R10 and washed at 5x10 6 Cells / mL were cryopreserved and stored in liquid nitrogen for future use.

[0360] analyze:

[0361] On the day of co-culture setup, culture the cells at 2.5x10 μl in IMDM + 10% FBS. 6 Prepare HLA-A*11 restricted G12V and G12D TCR Jurkat at 10 cells / mL. Thaw cryopreserved squeezed or untreated PBMCs and replace the medium with IMDM + 10% FBS at a density of 5x10 6 G12V or G12D TCR Jurkat was co-cultured with PBMC at a 1:2 ratio (250,000 Jurkat cells to 500,000 PBMC) in a tissue culture treated (TCT) 96-well U-bottom plate overnight (18-24 hours). 7-16 and G12D 7-16 The minimal epitope is added to the co-culture with Jurkat cells and untreated PBMCs to control the antigen mutation specific Jurkat reactivity. Cell culture supernatants are collected and the level of secreted Lucia luciferase is analyzed by Quanti LUC. The luminescent signal produced is related to the amount of secreted Lucia luciferase.

[0362] result

[0363] like Fig. 8A and Figure 8BAs shown, extrusion of human PBMCs loaded with KRAS seven mutant junction antigen mRNA plus or minus signal 2 / 3 mRNA and co-cultured with G12V TCR-transduced or G12D TCR-transduced Jurkat cells resulted in KRAS mutant-specific responses in TCR-transduced Jurkat cells relative to controls (empty extrusion, untreated PBMCs, signal 2 / 3 mRNA alone and irrelevant mutant minimal epitope stimulation). G12V-specific responses: PBMCs extruded with 500 μg / mL of KRAS 7mut_v1 mRNA or KRAS 7mut_v2 mRNA (plus or minus signal 2 / 3 mRNA) elicited G12V-specific responses in G12V TCR-transduced Jurkat cells. G12D-specific responses: PBMCs extruded with 500 μg / mL of KRAS 7mut_v1 mRNA (plus or minus signal 2 / 3 mRNA) elicited G12D-specific responses in G12D TCR-transduced Jurkat cells.

[0364] These results also demonstrate that extrusion of human PBMCs loaded with KRAS 7 mutant-linked antigen mRNA (KRAS 7mut_v1 construct) can elicit strong mutation-specific responses in both G12V and G12D TCR-transduced Jurkat cells.

[0365] Table 2. Exemplary sequences

[0366]

[0367] Incorporated by Reference

[0368] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0369] Equivalent solutions

[0370] Although various specific aspects have been illustrated and described, the above description is not restrictive. It should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. After reading this specification, many changes will become apparent to those skilled in the art.

Claims

1. An isolated polynucleotide comprising a single ORF having a first nucleotide sequence encoding a first antigen ("first coding region") and a second nucleotide sequence encoding a second antigen ("second coding region"), wherein the first coding region and the second coding region are linked.

2. The polynucleotide of claim 1, wherein the first coding region and the second coding region are connected by a linker.

3. The polynucleotide of claim 1 or 2, wherein the first coding region and the second coding region are arranged in the following order: the first coding region is upstream of the second coding region.

4. The polynucleotide of claim 3, wherein the order of the first coding region and the second coding region is different as compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

5. The polynucleotide of any one of claims 1 to 4, wherein the single ORF further comprises one or more additional nucleotide sequences encoding additional antigens ("additional coding regions").

6. The polynucleotide of claim 5, wherein the additional antigen is: (i) different from the first antigen, (ii) different from the second antigen, or (iii) different from both the first antigen and the second antigen.

7. The polynucleotide of claim 5 or 6, wherein the single ORF comprises at least two additional coding regions, at least three additional coding regions, at least four additional coding regions, at least five additional coding regions, at least six additional coding regions, at least seven additional coding regions, at least eight additional coding regions, at least nine additional coding regions, or at least ten additional coding regions.

8. The polynucleotide of any one of claims 5 to 7, wherein the additional coding region is linked to the first coding region or the second coding region.

9. The polynucleotide of claim 8, wherein the additional coding region is connected to the first coding region or the second coding region via a linker.

10. The polynucleotide of any one of claims 5 to 9, wherein the first coding region, the second coding region and the additional coding region are arranged in the following order: (a) (first coding region)-L1-(second coding region)-L2-(additional coding region); (b) (first coding region)-L1-(additional coding region)-L2-(second coding region); or (c) (additional coding region)-L1-(first coding region)-L2-(second coding region); Wherein L1 is the first linker and L2 is the second linker.

11. The polynucleotide of claim 10, wherein the first linker and the second linker are identical.

12. The polynucleotide of claim 10, wherein the first linker and the second linker are not identical.

13. The polynucleotide of any one of claims 10 to 12, wherein the order of the first coding region, the second coding region, and the third coding region is different as compared to the corresponding order present in the reference polynucleotide.

14. The polynucleotide of any one of claims 1 to 13, wherein the first antigen is about 25 amino acids in length.

15. The polynucleotide of any one of claims 1 to 14, wherein the second antigen is about 25 amino acids in length.

16. The polynucleotide of any one of claims 4 to 15, wherein the additional antigen is about 25 amino acids in length.

17. The polynucleotide of any one of claims 1 to 16, wherein the first antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

18. The polynucleotide of any one of claims 1 to 17, wherein the second antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

19. The polynucleotide of any one of claims 5 to 18, wherein the additional antigens comprise cancer antigens, non-self antigens, tumor-associated self antigens, disease-associated antigens, or a combination thereof.

20. The polynucleotide of any one of claims 17 to 19, wherein the cancer antigen comprises a KRAS antigen.

21. The polynucleotide of any one of claims 17 to 20, wherein the non-self antigen is derived from a pathogen selected from the group consisting of a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis B virus (HBV) antigen, or a combination thereof.

22. The polynucleotide of claim 20 or 21, wherein the KRAS antigen comprises an amino acid sequence that differs in sequence as compared to the amino acid sequence of the corresponding wild-type KRAS antigen.

23. The polynucleotide of claim 22, wherein the amino acid sequence of the KRAS antigen has less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, or less than about 90% sequence identity as compared to the amino acid sequence of the corresponding wild-type KRAS antigen.

24. The polynucleotide of claim 22 or 23, wherein the amino acid sequence of the KRAS antigen comprises an amino acid substitution selected from the group consisting of K5E, K5N, G10GG, G10V, G12A, G12C, G12D, G12F, G12I, G12L, G12R, G12S, G12V, G13C, G13D, G13E, G13R, G13V, V14I, L19F, T20M, Q22E, Q22H, Q22K, Q22R, Q25H, N26Y, F28L, E31K, D33E, P34L, P34Q, P34R, I36M , R41K, D57N, T58I, A59T, G60D, G60R, G60S, G60V, Q61A, Q61H, Q61K, Q61L, Q61P, Q61R, E63K, S65N, R68S, Y71H, T74A, L79I, R97I, Q99E, M111L, K117N, K117R, D119G, S122F, T144P, A146P, A146T, A146V, K147E, K147T, R149K, L159S, I163S, R164Q, I183N, I84M, or a combination thereof.

25. The polynucleotide of any one of claims 22 to 24, wherein the KRAS antigen comprises one or more of: G12D 1-16 、G12D 2-19 、G12D 2-22 、G12D 2-29 、G12V 1-16 、G12V 2-19 、G12V 3-17 or G12V 3-42 antigen.

26. The polynucleotide of any one of claims 2 to 25, wherein the linker comprises a peptide linker.

27. The polynucleotide of claim 26, wherein the peptide linker comprises a G4S linker or an EAAAK linker.

28. An isolated polynucleotide comprising a single ORF having a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), and a third nucleotide sequence encoding a third antigen ("third coding region"), wherein the first coding region is connected to the second coding region via a first linker, and wherein the second coding region is connected to the third coding region via a second linker.

29. The polynucleotide of claim 28, wherein the first coding region, the second coding region, and the third coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

30. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), and a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third region via a second linker, and wherein the third coding region is connected to the fourth coding region via a third linker.

31. The polynucleotide of claim 30, wherein the first coding region, the second coding region, the third coding region, and the fourth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

32. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), and a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third coding region via a second linker, wherein the third coding region is connected to the fourth coding region via a third linker, and wherein the fourth coding region is connected to the fifth coding region via a fourth linker.

33. The polynucleotide of claim 32, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

34. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), and a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third coding region via a second linker, wherein the third coding region is connected to the fourth coding region via a third linker, wherein the fourth coding region is connected to the fifth coding region via a fourth linker, and wherein the fifth coding region is connected to the sixth coding region via a fifth linker.

35. The polynucleotide of claim 34, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, and the sixth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

36. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), and a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third coding region via a second linker, wherein the third coding region is connected to the fourth coding region via a third linker, wherein the fourth coding region is connected to the fifth coding region via a fourth linker, wherein the fifth coding region is connected to the sixth coding region via a fifth linker, and wherein the sixth coding region is connected to the seventh coding region via a sixth linker.

37. The polynucleotide of claim 36, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, and the seventh coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

38. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), and an eighth nucleotide sequence encoding an eighth antigen ("eighth coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third coding region via a second linker, wherein the third coding region is connected to the fourth coding region via a third linker, wherein the fourth coding region is connected to the fifth coding region via a fourth linker, wherein the fifth coding region is connected to the sixth coding region via a fifth linker, wherein the sixth coding region is connected to the seventh coding region via a sixth linker, and wherein the seventh coding region is connected to the eighth coding region via a seventh linker.

39. The polynucleotide of claim 38, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, and the eighth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

40. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), an eighth nucleotide sequence encoding an eighth antigen ("eighth coding region"), and a fourth nucleotide sequence encoding a ninth antigen ("eighth coding region") A nine-nucleotide sequence ("ninth coding region"), wherein the first coding region is connected to the second coding region via a first linker, wherein the second coding region is connected to the third coding region via a second linker, wherein the third coding region is connected to the fourth coding region via a third linker, wherein the fourth coding region is connected to the fifth coding region via a fourth linker, wherein the fifth coding region is connected to the sixth coding region via a fifth linker, wherein the sixth coding region is connected to the seventh coding region via a sixth linker, wherein the seventh coding region is connected to the eighth coding region via a seventh linker, and wherein the eighth coding region is connected to the ninth coding region via an eighth linker.

41. The polynucleotide of claim 40, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, and the ninth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

42. An isolated polynucleotide comprising a first nucleotide sequence encoding a first antigen ("first coding region"), a second nucleotide sequence encoding a second antigen ("second coding region"), a third nucleotide sequence encoding a third antigen ("third coding region"), a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), a sixth nucleotide sequence encoding a sixth antigen ("sixth coding region"), a seventh nucleotide sequence encoding a seventh antigen ("seventh coding region"), an eighth nucleotide sequence encoding an eighth antigen ("eighth coding region"), a ninth nucleotide sequence encoding a ninth antigen ("ninth coding region"), and a tenth nucleotide sequence encoding a tenth antigen. The nucleotide sequence ("the tenth coding region") is as follows: wherein the first coding region is connected to the second coding region through a first linker, wherein the second coding region is connected to the third coding region through a second linker, wherein the third coding region is connected to the fourth coding region through a third linker, wherein the fourth coding region is connected to the fifth coding region through a fourth linker, wherein the fifth coding region is connected to the sixth coding region through a fifth linker, wherein the sixth coding region is connected to the seventh coding region through a sixth linker, wherein the seventh coding region is connected to the eighth coding region through a seventh linker, wherein the eighth coding region is connected to the ninth coding region through an eighth linker, and wherein the ninth coding region is connected to the tenth coding region through a ninth linker.

43. The polynucleotide of claim 42, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, the fifth coding region, the sixth coding region, the seventh coding region, the eighth coding region, the ninth coding region, and the tenth coding region are arranged in a sequence, wherein the sequence is different as compared to the corresponding sequence present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide occurring in nature.

44. The polynucleotide of any one of claims 28 to 43, wherein the first antigen is about 25 amino acids in length.

45. The polynucleotide of any one of claims 28 to 44, wherein the second antigen is about 25 amino acids in length.

46. ​​The polynucleotide of any one of claims 28 to 45, wherein the third antigen is about 25 amino acids in length.

47. The polynucleotide of any one of claims 30 to 46, wherein the fourth antigen is about 25 amino acids in length.

48. The polynucleotide of any one of claims 32 to 47, wherein the fifth antigen is about 25 amino acids in length.

49. The polynucleotide of any one of claims 34 to 48, wherein the sixth antigen is about 25 amino acids long.

50. The polynucleotide of any one of claims 36 to 49, wherein the seventh antigen is about 25 amino acids in length.

51. The polynucleotide of any one of claims 38 to 50, wherein the eighth antigen is about 25 amino acids long.

52. The polynucleotide of any one of claims 40 to 51, wherein the ninth antigen is about 25 amino acids long.

53. The polynucleotide of any one of claims 42 to 52, wherein the tenth antigen is about 25 amino acids long.

54. The polynucleotide of any one of claims 28 to 53, wherein the first antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

55. The polynucleotide of any one of claims 28 to 54, wherein the second antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

56. The polynucleotide of any one of claims 28 to 55, wherein the third antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

57. The polynucleotide of any one of claims 30 to 56, wherein the fourth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

58. The polynucleotide of any one of claims 32 to 57, wherein the fifth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

59. The polynucleotide of any one of claims 34 to 58, wherein the sixth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

60. The polynucleotide of any one of claims 36 to 59, wherein the seventh antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

61. The polynucleotide of any one of claims 38 to 60, wherein the eighth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

62. The polynucleotide of any one of claims 40 to 61, wherein the ninth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

63. The polynucleotide of any one of claims 42 to 62, wherein the tenth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

64. The polynucleotide of any one of claims 54 to 63, wherein the cancer antigen comprises a KRAS antigen.

65. The polynucleotide of any one of claims 54 to 64, wherein the non-self antigen is derived from a pathogen selected from the group consisting of a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis B virus (HBV) antigen, or a combination thereof.

66. The polynucleotide of claim 64 or 65, wherein the KRAS antigen comprises an amino acid sequence that differs in sequence as compared to the amino acid sequence of the corresponding wild-type KRAS antigen.

67. The polynucleotide of claim 66, wherein the amino acid sequence of the KRAS antigen has less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, or less than about 90% sequence identity as compared to the amino acid sequence of the corresponding wild-type KRAS antigen.

68. The polynucleotide of claim 66 or 67, wherein the amino acid sequence of the first KRAS antigen comprises an amino acid substitution selected from the group consisting of K5E, K5N, G10GG, G10V, G12A, G12C, G12D, G12F, G12I, G12L, G12R, G12S, G12V, G13C, G13D, G13E, G13R, G13V, V14I, L19F, T20M, Q22E, Q22H, Q22K, Q22R, Q25H, N26Y, F28L, E31K, D33E, P34L, P34Q, P34R, I36 M, R41K, D57N, T58I, A59T, G60D, G60R, G60S, G60V, Q61A, Q61H, Q61K, Q61L, Q61P, Q61R, E63K, S65N, R68S, Y71H, T74A, L79I, R97I, Q99E, M111L, K117N, K117R, D119G, S122F, T144P, A146P, A146T, A146V, K147E, K147T, R149K, L159S, I163S, R164Q, I183N, I84M, or a combination thereof.

69. The polynucleotide of any one of claims 66 to 68, wherein the KRAS antigen comprises one or more of: G12D 1-16 、G12D 2-19 、G12D 2-22 、G12D 2-29 、G12V 1-16 、G12V 2-19 、G12V 3-17 or G12V 3-42 antigen.

70. The polynucleotide of any one of claims 28 to 69, wherein any of the first, the second, the third, the fourth, the fifth, the sixth, the seventh, the eighth or the ninth linker comprises a peptide linker.

71. The polynucleotide of claim 70, wherein the peptide linker comprises a G4S linker or an EAAAK linker.

72. A polynucleotide as described in any one of claims 1 to 71, further comprising one or more of the following components: (1) an internal ribosome entry site (IRES), (2) an intron sequence, (3) a homology arm, (4) a promoter, (5) an enhancer, (6) a UTR, (7) a sequence encoding a signal peptide, (8) a translation initiation sequence, (9) a 3′ tailing region for linking nucleosides, (10) a 5′ cap, (11) a sequence encoding a 2A ribosomal skipping peptide, or (12) any combination of (1) to (11).

73. The polynucleotide of any one of claims 1 to 72, comprising at least one modified nucleoside.

74. The polynucleotide of claim 73, wherein the at least one modified nucleoside comprises 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, pseudouridine, inosine, α-thio-guanosine, guanosine, 8-oxo-guanosine, O6-methyl-guanosine, 7-deaza-guanosine, N1-methyladenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine, pyrrolo-cytidine, 5-methyl-cytidine, N4-acetyl-cytidine, 5-methyl-uridine, 5-iodo-cytidine, or a combination thereof.

75. The polynucleotide of any one of claims 1 to 74, which is mRNA.

76. A vector comprising the polynucleotide of any one of claims 1 to 75.

77. A cell comprising the polynucleotide of any one of claims 1 to 76.

78. The cell of claim 77, wherein the cell comprises a stem cell, a somatic cell, or both.

79. The cell of claim 77, wherein the stem cell comprises an induced pluripotent stem cell (iPSC), an embryonic stem cell, a tissue-specific stem cell, a mesenchymal stem cell, or a combination thereof.

80. The cell of claim 78 or 79, wherein the somatic cell comprises a blood cell.

81. The cell of claim 80, wherein the blood cells comprise PBMCs.

82. The cell of claim 81, wherein the PBMCs comprise immune cells.

83. The cell of claim 82, wherein the immune cell comprises a T cell, a B cell, a natural killer (NK) cell, a dendritic cell (DC), a NKT cell, a mast cell, a monocyte, a macrophage, a basophil, an eosinophil, a neutrophil, a DC2.4 dendritic cell, or a combination thereof.

84. The cell of any one of claims 77 to 83, which has been passed through a constriction under a set of parameters causing a perturbation within the cell such that the polynucleotide enters the cell through the perturbation upon contact with the cell.

85. A pharmaceutical composition comprising a polynucleotide as described in any one of claims 1 to 75, a vector as described in claim 76 or a cell as described in any one of claims 77 to 84, and a pharmaceutically acceptable carrier.

86. A kit comprising a polynucleotide as described in any one of claims 1 to 75, a vector as described in claim 76 or a cell as described in any one of claims 77 to 84, and instructions for use.

87. A method of preparing a polynucleotide, the method comprising enzymatically or chemically synthesizing the polynucleotide of any one of claims 1 to 75.

88. A method of inducing expression of multiple antigens in a cell, the method comprising delivering a polynucleotide as described in any one of claims 1 to 75 to the cell intracellularly.

89. The method of claim 88, wherein the plurality of antigens are simultaneously expressed in the cell following intracellular delivery.

90. The method of claim 88 or 89, wherein delivering the polynucleotide to the cell intracellularly comprises passing a cell suspension comprising the cells through a constriction under a set of parameters, thereby causing a disturbance within the cell such that the polynucleotide enters the cell through the disturbance when in contact with the cell.

91. The method of claim 90, further comprising contacting the cell with the polynucleotide.

92. The method of claim 91, wherein contacting the cells with the polynucleotide comprises incubating the cell suspension with the polynucleotide such that the cells and the polynucleotide are in contact.

93. The method of claim 91 or 92, wherein the contacting occurs before the cell suspension passes through the constriction.

94. The method of any one of claims 87 to 89, wherein the contacting occurs during passage of the cell suspension through the constriction.

95. The method of any one of claims 91 to 94, wherein the contacting occurs after the cell suspension passes through the constriction.

96. A method as described in any one of claims 84 to 95, wherein the parameter group is selected from cell density; pressure; length, width and / or depth of the constriction; diameter of the constriction; diameter of the cells; temperature; entrance angle of the constriction; exit angle of the constriction; length, width and / or width of the approach zone; surface properties of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration; viscosity, osmolarity, salt concentration, serum content and / or pH of the cell suspension; time in the constriction; shear rate in the constriction; type of payload, or a combination thereof.

97. The method of claim 96, wherein the cell density is at least about 6 x 10 7 cells / mL, at least about 7x10 7 cells / mL, at least about 8x10 7 cells / mL, at least about 9x10 7 cells / mL, at least about 1x10 8 cells / mL, at least about 1.1x10 8 cells / mL, at least about 1.2x10 8 cells / mL, at least about 1.3x10 8 cells / mL, at least about 1.4x10 8 cells / mL, at least about 1.5x10 8 cells / mL, at least about 2.0x10 8 cells / mL, at least about 3.0x10 8 cells / mL, at least about 4.0x10 8 cells / mL, at least about 5.0x10 8 cells / mL, at least about 6.0x10 8 cells / mL, at least about 7.0x10 8 cells / mL, at least about 8.0x10 8 cells / mL, at least about 9.0x10 8 cells / mL or at least about 1.0x10 9 cells / mL or higher.

98. The method of claim 96 or 97, wherein the pressure is at least about 30 psi, at least about 35 psi, at least about 40 psi, at least about 45 psi, at least about 50 psi, at least about 55 psi, at least about 60 psi, at least about 65 psi, at least about 70 psi, at least about 75 psi, at least about 80 psi, at least about 85 psi, at least about 90 psi, at least about 95 psi, at least about 100 psi, at least about 110 psi, at least about 120 psi, at least about 130 psi, at least about 140 psi, or at least about 150 psi.

99. The method of any one of claims 90 to 98, wherein the constriction is contained within a microfluidic chip.

100. The method of any one of claims 96 to 99, wherein the diameter of the constriction is about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 99% of the diameter of the cell.

101. The method of claim 100, wherein the constriction has a width between about 0 μm and about 10 μm.

102. A method as claimed in claim 101, wherein the width of the constriction is less than about 1 μm, less than about 2 μm, less than about 3 μm, less than about 4 μm, less than about 5 μm, less than about 6 μm, less than about 7 μm, less than about 8 μm, less than about 9 μm or less than about 10 μm.

103. The method of any one of claims 92 to 99, wherein the constriction has a length between about 0 μm and about 100 μm.

104. A method as claimed in claim 99, wherein the length of the constriction is less than about 0.1 μm, less than about 0.2 μm, less than about 0.3 μm, less than about 0.4 μm, less than about 0.5 μm, less than about 0.6 μm, less than about 0.7 μm, less than about 0.8 μm, less than about 0.9 μm, less than about 1 μm, less than about 2.5 μm, less than about 5 μm, less than about 7.5 μm, less than about 10 μm, less than about 12.5 μm, less than about 15 μm, less than about 20 μm, less than about 30 μm, less than about 40 μm, less than about 50 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm or less than about 100 μm.

105. The method of any one of claims 92 to 100, wherein the constriction has a depth of between at least about 1 μm and at least about 120 μm.

106. A method as claimed in claim 101, wherein the depth of the constriction is at least about 2 μm, at least about 3 μm, at least about 4 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 60 μm, at least about 70 μm, at least about 80 μm, at least about 90 μm, at least about 100 μm, at least about 110 μm or at least about 120 μm.

107. The method of any one of claims 92 to 102, wherein the cell suspension comprising the cells is passed through a plurality of constrictions.

108. The method of claim 103, wherein the plurality of constrictions comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 75, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600, at least about 650, at least about 700, at least about 750, at least about 800, at least about 850, at least about 900, at least about 950, at least about 1,000 or more individual constrictions.

109. The method of claim 103 or 104, wherein each of the plurality of constrictions is identical.

110. The method of claim 103 or 104, wherein one or more of the plurality of constrictions are different.

111. The method of claim 106, wherein the one or more constrictions vary in length, depth, width, or a combination thereof.

112. A method of inducing a multispecific immune response in a subject in need thereof, the method comprising administering to the subject a polynucleotide as described in any one of claims 1 to 71, a vector as described in claim 72, a cell as described in any one of claims 73 to 80, or a pharmaceutical composition as described in claim 81.

113. The method of claim 108, wherein the multispecific immune response comprises a CD8+ T cell response.

114. A method of inducing an enhanced immune response in a subject in need thereof, the method comprising administering to the subject a polynucleotide as described in any one of claims 1 to 71, a vector as described in claim 72, a cell as described in any one of claims 73 to 80, or a pharmaceutical composition as described in claim 81.

115. A method as claimed in claim 110, wherein the enhanced immune response comprises: (i) an increase in the amplitude of the induced immune response as compared to a reference immune response, (ii) an increase in the breadth of the induced immune response as compared to a reference immune response, (iii) an increase in the duration of the induced immune response as compared to a reference immune response, or (iv) any combination of (i) to (iii); wherein the reference immune comprises an immune response observed in a corresponding subject who has not received administration of the polynucleotide or the modified cell.

116. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a polynucleotide as described in any one of claims 1 to 71, a vector as described in claim 72, a cell as described in any one of claims 73 to 80, or a pharmaceutical composition as described in claim 81.

117. The method of claim 112, wherein the disease or disorder comprises cancer.

118. The method of claim 113, wherein the cancer is associated with aberrant KRAS expression.

119. The method of claim 112 or 113, wherein the disease or disorder is associated with a non-self antigen.

120. The method of claim 115, wherein the non-self antigen is derived from a virus.

121. The method of claim 116, wherein the virus comprises HPV, HIV, or HBV.

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