Polynucleotides encoding linked antigens and uses thereof

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of U.S. Provisional Application No. 63 / 369,726, filed July 28, 2022, which is incorporated by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing The contents of the Sequence Listing have been submitted electronically (Name: 4821_086PC01_SequenceListing_ST26.XML, Size: 36,597 bytes, and Creation Date: July 28, 2023) and are filed with the application, which is incorporated by reference in its entirety herein.

[0003] The present disclosure generally relates to polynucleotides (e.g., isolated polynucleotides) that include multiple nucleotide sequences that encode antigens, where the multiple nucleotide sequences are linked (e.g., by a linker). [Background technology]

[0004] Cancer remains one of the leading causes of death in the modern world. Standard treatments currently implemented in clinics, including surgery, radiation, chemotherapy, and immunotherapy, have shown limited success. These therapies are typically only effective against early-stage, localized tumors and rarely effective against later-stage metastatic malignancies, leading to frequent recurrence or eventual therapy resistance (Sharma, P., et al., Cell 168(4):707-723(2017)). Furthermore, various drugs used in radiation and chemotherapy can damage normal tissues and lead to unwanted side effects. Therefore, there remains a need for new treatment options with acceptable safety profiles and high efficacy in cancer patients. Summary of the Invention

[0005] Provided herein is an isolated polynucleotide comprising a single ORF having a first nucleotide sequence ("first coding region") encoding a first antigen and a second nucleotide sequence ("second coding region") encoding a second antigen, wherein the first coding region and the second coding region are linked. In some aspects, the first coding region and the second coding region are linked by a linker.

[0006] In some embodiments, 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 embodiments, the order of the first coding region and the second coding region is different compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

[0007] In some embodiments, the single ORF of the polynucleotide further comprises one or more additional nucleotide sequences ("additional coding regions") encoding additional antigens. In some embodiments, the additional antigen is (i) not the same as the first antigen, (ii) not the same as the second antigen, or (iii) not the same as both the first antigen and the second antigen. In some embodiments, 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 embodiments, the additional coding region is linked to the first coding region or the second coding region. In some embodiments, the additional coding region is linked to the first coding region or the second coding region by a linker.

[0008] In some embodiments, the first coding region, second coding region, and 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), where L1 is a first linker and L2 is a second linker. In some embodiments, the first linker and the second linker are the same. In some embodiments, the first linker and the second linker are not the same. In some embodiments, the order of the first coding region, second coding region, and third coding region is different compared to the corresponding order present in a reference polynucleotide.

[0009] In some embodiments, the first antigen is about 25 amino acids in length. In some embodiments, the second antigen is about 25 amino acids in length. In some embodiments, the additional antigen is about 25 amino acids in length. In some embodiments, the first antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the second antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the additional antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof.

[0010] In some embodiments, the cancer antigen comprises a KRAS antigen. In some embodiments, the non-self antigen is derived from a pathogen selected from a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis B virus (HBV) antigen, or a combination thereof.

[0011] In some embodiments, the KRAS antigen comprises an amino acid sequence that differs in sequence from the amino acid sequence of a corresponding wild-type KRAS antigen. In some embodiments, 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 with the amino acid sequence of the corresponding wild-type KRAS antigen. In some embodiments, the amino acid sequence of the KRAS antigen is 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, G60 In some embodiments, the KRAS antigen comprises an amino acid substitution selected from the following: D, 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. 1-16 , G12D 2-19 , G12D 2-22 , G12D 2-29 , G12V 1-16 , G12V 2-19 , G12V 3-17 , or G12V 3-42 The antigens include one or more of:

[0012] In any of the above polynucleotides, in some embodiments, the linker comprises a peptide linker. In some embodiments, the peptide linker comprises a G4S linker or an EAAAK linker.

[0013] Some aspects of the present disclosure relate to an isolated polynucleotide comprising a single ORF having a first nucleotide sequence encoding a first antigen (the "first coding region"), a second nucleotide sequence encoding a second antigen (the "second coding region"), and a third nucleotide sequence encoding a third antigen (the "third coding region"), wherein the first coding region is linked to the second coding region by a first linker, and the second coding region is linked to the third coding region by a second linker. In some aspects, the first coding region, second coding region, and third coding region are arranged in an order that is different compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding naturally occurring polynucleotide.

[0014] 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"), and a fourth nucleotide sequence encoding a fourth antigen ("fourth coding region"), wherein the first coding region is linked to the second coding region by a first linker, the second coding region is linked to the third region by a second linker, and the third coding region is linked to the fourth coding region by a third linker. In some aspects, the first coding region, second coding region, third coding region, and fourth coding region are arranged in an order that is different compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding naturally occurring polynucleotide.

[0015] 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"), and a fifth nucleotide sequence encoding a fifth antigen ("fifth coding region"), wherein the first coding region is linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, and the fourth coding region is linked to the fifth coding region by a fourth linker. In some embodiments, the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are arranged in an order that differs compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

[0016] 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"). Also provided herein is an isolated polynucleotide, wherein a first coding region is linked to a second coding region by a first linker, the second coding region is linked to a third coding region by a second linker, the third coding region is linked to a fourth coding region by a third linker, the fourth coding region is linked to a fifth coding region by a fourth linker, and the fifth coding region is linked to a sixth coding region by a fifth linker. In some embodiments, 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 certain order, which is different from the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0017] 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"). Also provided herein is an isolated polynucleotide, wherein a first coding region is linked to a second coding region by a first linker, the second coding region is linked to a third coding region by a second linker, the third coding region is linked to a fourth coding region by a third linker, the fourth coding region is linked to a fifth coding region by a fourth linker, the fifth coding region is linked to a sixth coding region by a fifth linker, and the sixth coding region is linked to a seventh coding region by a sixth linker. In some embodiments, the first coding region, second coding region, third coding region, fourth coding region, and fifth coding region are arranged in an order that is different from the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding naturally occurring polynucleotide.

[0018] 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"). Also provided herein is an isolated polynucleotide, wherein a first coding region is linked to a second coding region by a first linker, the second coding region is linked to a third coding region by a second linker, the third coding region is linked to a fourth coding region by a third linker, the fourth coding region is linked to a fifth coding region by a fourth linker, and the fifth coding region is linked to a sixth coding region by a fifth linker. In some embodiments, 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 certain order, which is different from the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a naturally occurring corresponding polynucleotide.

[0019] 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"). Also provided herein is an isolated polynucleotide, wherein a first coding region is linked to a second coding region by a first linker, the second coding region is linked to a third coding region by a second linker, the third coding region is linked to a fourth coding region by a third linker, the fourth coding region is linked to a fifth coding region by a fourth linker, the fifth coding region is linked to a sixth coding region by a fifth linker, and the sixth coding region is linked to a seventh coding region by a sixth linker. In some embodiments, the first coding region, second coding region, third coding region, fourth coding region, and fifth coding region are arranged in an order that is different from the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding naturally occurring polynucleotide.

[0020] 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"). Further provided herein is an isolated polynucleotide comprising an oxidase, wherein a first coding region is linked to a second coding region by a first linker, the second coding region is linked to a third coding region by a second linker, the third coding region is linked to a fourth coding region by a third linker, the fourth coding region is linked to a fifth coding region by a fourth linker, the fifth coding region is linked to a sixth coding region by a fifth linker, the sixth coding region is linked to a seventh coding region by a sixth linker, and the seventh coding region is linked to an eighth coding region by a seventh linker. In some embodiments, 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 that differs compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

[0021] 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"). Provided herein is an isolated polynucleotide comprising a first coding region linked to a second coding region by a first linker, the second coding region linked to a third coding region by a second linker, the third coding region linked to a fourth coding region by a third linker, the fourth coding region linked to a fifth coding region by a fourth linker, the fifth coding region linked to a sixth coding region by a fifth linker, the sixth coding region linked to a seventh coding region by a sixth linker, the seventh coding region linked to an eighth coding region by a seventh linker, and the eighth coding region linked to a ninth coding region by an eighth linker. In some embodiments, the first coding region, second coding region, third coding region, fourth coding region, fifth coding region, sixth coding region, seventh coding region, eighth coding region, and ninth coding region are arranged in an order that differs compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

[0022] 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 ("tenth coding region"); Provided herein is an isolated polynucleotide comprising a first coding region linked to a second coding region by a first linker, the second coding region linked to a third coding region by a second linker, the third coding region linked to a fourth coding region by a third linker, the fourth coding region linked to a fifth coding region by a fourth linker, the fifth coding region linked to a sixth coding region by a fifth linker, the sixth coding region linked to a seventh coding region by a sixth linker, the seventh coding region linked to an eighth coding region by a seventh linker, the eighth coding region linked to a ninth coding region by an eighth linker, and the ninth coding region linked to a tenth coding region by a ninth linker. In some embodiments, the first coding region, second coding region, third coding region, fourth coding region, fifth coding region, sixth coding region, seventh coding region, eighth coding region, ninth coding region, and tenth coding region are arranged in an order that differs compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

[0023] In any of the above polynucleotides, in some embodiments, the first antigen is about 25 amino acids in length. In some embodiments, the second antigen is about 25 amino acids in length. In some embodiments, the third antigen is about 25 amino acids in length. In some embodiments, the fourth antigen is about 25 amino acids in length. In some embodiments, the fifth antigen is about 25 amino acids in length. In some embodiments, the sixth antigen is about 25 amino acids in length. In some embodiments, the seventh antigen is about 25 amino acids in length. In some embodiments, the eighth antigen is about 25 amino acids in length. In some embodiments, the ninth antigen is about 25 amino acids in length. In some embodiments, the tenth antigen is about 25 amino acids in length.

[0024] In some embodiments, the first antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the second antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the third antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the fourth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the fifth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the sixth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the seventh antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the eighth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the ninth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof. In some embodiments, the tenth antigen comprises a cancer antigen, a non-self antigen, a tumor-associated autoantigen, a disease-associated antigen, or a combination thereof.

[0025] In some embodiments, the cancer antigen comprises a KRAS antigen. In some embodiments, the non-self antigen is derived from a pathogen selected from a human papillomavirus (HPV) antigen, a human immunodeficiency virus (HIV) antigen, a hepatitis B virus (HBV) antigen, or a combination thereof. In some embodiments, the KRAS antigen comprises an amino acid sequence that differs from the amino acid sequence of a corresponding wild-type KRAS antigen. In some embodiments, 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 with the amino acid sequence of the corresponding wild-type KRAS antigen. In some embodiments, the amino acid sequence of the first KRAS antigen is 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, G In some embodiments, the KRAS antigen comprises an amino acid substitution selected from the following: 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. 1-16 , G12D 2-19 , G12D 2-22 , G12D 2-29 , G12V 1-16 , G12V 2-19 , G12V 3-17 , or G12V 3-42 The antigens include one or more of:

[0026] In some embodiments, any one of the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth linkers comprises a peptide linker. In some embodiments, the peptide linker comprises a G4S linker or an EAAAK linker.

[0027] For any of the polynucleotides described herein, in some embodiments, 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' tail region of linked nucleosides, (10) a 5' cap, (11) a sequence encoding a 2A ribosomal skipping peptide, or (12) any combination of (1)-(11). In some embodiments, the polynucleotide further comprises at least one modified nucleoside. In some embodiments, the at least one modified nucleoside is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 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, pseudo-uridine, inosine, α-thio-guanosine, 8-amino- and 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.

[0028] In some aspects, the polynucleotides described herein are mRNA.

[0029] Also provided herein are vectors comprising any of the polynucleotides described herein. Some aspects of the present disclosure relate to cells comprising any of the polynucleotides described herein. In some aspects, the cells comprise stem cells, somatic cells, or both. In some aspects, the stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells, tissue-specific stem cells, mesenchymal stem cells, or a combination thereof. In some aspects, the somatic cells comprise blood cells. In some aspects, the blood cells comprise PBMCs. In some aspects, the PBMCs comprise immune cells. In some aspects, the immune cells comprise T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof. In some aspects, the cells pass through a constriction under a set of parameters, thereby causing a perturbation in the cell such that the polynucleotide enters the cell through the perturbation upon contact with the cell.

[0030] Also provided herein are pharmaceutical compositions comprising any of the polynucleotides, vectors, or cells described herein and a pharmaceutically acceptable carrier. Also provided herein are kits comprising any of the polynucleotides, vectors, or cells described herein.

[0031] Provided herein are methods of making polynucleotides, comprising enzymatically or chemically synthesizing any of the polynucleotides described herein.

[0032] Also provided herein is a method for inducing expression of multiple antigens in a cell, the method comprising intracellularly delivering a polynucleotide described herein into the cell. In some aspects, the multiple antigens are simultaneously expressed in the cell after intracellular delivery. In some aspects, intracellularly delivering the polynucleotide into the cell comprises passing a cell suspension containing the cells through a constriction under a set of parameters, thereby causing a perturbation in the cell such that the polynucleotide enters the cell through the perturbation when it contacts the cell.

[0033] In some aspects, the method further comprises contacting the cells with the polynucleotide. In some aspects, contacting the cells with the polynucleotide comprises incubating a cell suspension with the polynucleotide such that the cells and polynucleotide are in contact. In some aspects, the contacting occurs before passing the cell suspension through the constriction. In some aspects, the contacting occurs during passage of the cell suspension through the constriction. In some aspects, the contacting occurs after passage of the cell suspension through the constriction.

[0034] In some embodiments, the set of parameters used to pass 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 cell; temperature; entrance angle of the constriction; exit angle of the constriction; length, width and / or width of the approach region; surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); actuation flow rate; payload concentration; viscosity, osmolality, salt concentration, serum content and / or pH of the cell suspension; time of constriction; shear rate of the constriction; type of payload; or combinations thereof.

[0035] In some embodiments, the cell density is at least about 6 x 10 7 cells / mL, at least approximately 7 x 10 7 cells / mL, at least approximately 8 x 10 7 cells / mL, at least approximately 9 x 10 7 cells / mL, at least approximately 1 x 10 8 cells / mL, at least approximately 1.1 x 10 8cells / mL, at least approximately 1.2 x 10 8 cells / mL, at least approximately 1.3 x 10 8 cells / mL, at least approximately 1.4 x 10 8 cells / mL, at least approximately 1.5 x 10 8 cells / mL, at least approximately 2.0 x 10 8 cells / mL, at least approximately 3.0 x 10 8 cells / mL, at least approximately 4.0 x 10 8 cells / mL, at least approximately 5.0 × 10 8 cells / mL, at least approximately 6.0 x 10 8 cells / mL, at least approximately 7.0 × 10 8 cells / mL, at least approximately 8.0 × 10 8 cells / mL, at least approximately 9.0 × 10 8 cells / mL or at least about 1.0 x 10 9 In some embodiments, 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.

[0036] In some embodiments, the constriction is contained within a microfluidic chip. In some embodiments, 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 embodiments, the width of the constriction is about 0 μm to about 10 μm. In some embodiments, 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 embodiments, the length of the constriction is about 0 μm to about 100 μm. In some embodiments, 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 embodiments, the depth of the constriction is at least about 1 μm to at least about 120 μm. In some embodiments, 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.

[0037] In some embodiments, the cell suspension containing the cells is passed through a plurality of constrictions. In some embodiments, 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 distinct constrictions. In some embodiments, each constriction in the plurality of constrictions is the same. In some embodiments, one or more of the constrictions in the plurality of constrictions are different. In some embodiments, one or more of the stenoses vary in their length, depth, width, or a combination thereof.

[0038] 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 of the polynucleotides, vectors, cells, or pharmaceutical compositions described herein. In some embodiments, the multispecific immune response comprises a CD8+ T cell response.

[0039] Also provided herein are methods of inducing an enhanced immune response in a subject in need thereof, comprising administering to the subject any of the polynucleotides, vectors, cells, or pharmaceutical compositions described herein. In some aspects, the enhanced immune response comprises (i) an increase in the magnitude of the induced immune response compared to a reference immune response, (ii) an increase in the breadth of the induced immune response compared to a reference immune response, (iii) an increase in the duration of the induced immune response compared to a reference immune response, or (iv) any combination of (i)-(iii), wherein the reference immune response comprises an immune response observed in a corresponding subject that did not receive the polynucleotide or modified cells.

[0040] Provided herein is a method for treating a disease or condition in a subject in need thereof, comprising administering to the subject any of the polynucleotides, vectors, cells or pharmaceutical compositions described herein. In some embodiments, the disease or condition comprises cancer. In some embodiments, the cancer is associated with abnormal KRAS expression. In some embodiments, the disease or condition is associated with a non-self antigen. In some embodiments, the non-self antigen is derived from a virus. In some embodiments, the virus comprises HPV, HIV or HBV. [Brief explanation of the drawings]

[0041] [Figure 1A] Three linked antigen mRNA constructs are shown, each encoding an approximately 25 amino acid long fragment of each of the five antigens (HPV16 E6, HPV16 E7, CMV pp65, KRASG12V, and KRASG12D) arranged in a different order. [Figure 1B]Figure 1A shows the activation of E6 TCR Jurkat cells (left graph), E7 TCR Jurkat cells (middle graph), and pp65-specific Cellero responder T cells (right graph) after coculture with PBMCs expressed with each of the three different linked antigen mRNA constructs shown in Figure 1A. Controls shown include: (i) unmodified PBMCs (i.e., no expression and no polynucleotide) ("NC"), (ii) PBMCs expressed without any mRNA ("Empty"), and (iii) responder cells treated with one of the following NC PBMCs spiked with the respective minimal epitope (second bar from the right for each of the different cell lines). Untreated responder cells were also used as a control (last bar from the right). [Figure 2A] Three concatenated antigen mRNA constructs are shown, two of which encode approximately 25 amino acid fragments of each of 10 antigens (HPV16 E6, HPV16 E7, CMV pp65, KRASG12V, KRASG12D, Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S, and MAGE-A10) arranged in different orders, and the third mRNA construct encodes approximately 25 amino acid fragments of each of five antigens (HPV16 E6, HPV16 E7, CMV pp65, KRASG12V, and KRASG12D). [Figure 2B] Figure 2 shows activation of E6 TCR Jurkat cells (left graph) and E7 TCR Jurkat cells (right graph) after co-culture with PMBCs expressed with each of the three different linked antigen mRNA constructs shown in Figure 2A. Controls are the same as those described in Figure 1B. [Figure 2C] Figure 2 shows activation of pp65-specific Cellero responder T cells (left graph), M1-specific Flu Cellero responder T cells (middle graph), and NY-ESO-1-specific Cellero responder T cells (right graph) after co-culture with PMBCs expressed with each of the three different linked antigen mRNA constructs shown in Figure 2A. Controls are the same as those described in Figure 1B. [Figure 3A]Five concatenated antigen mRNA constructs are shown, three of which encode approximately 25 amino acid long fragments of each of five antigens (HPV16 E6, HPV16 E7, CMV pp65, KRASG12V, and KRASG12D) arranged in different orders, and two of which encode approximately 25 amino acid long fragments of each of ten antigens (HPV16 E6, HPV16 E7, CMV pp65, KRASG12V, KRASG12D, Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S, and MAGE-A10) arranged in different orders. [Figure 3B] Western blotting of two SDS-PAGE gels run with samples of wheat germ extracts translating each of the five concatenated antigen mRNA constructs shown in Figure 3 A. The gels were blotted with a primary antibody against HPV E629-38 SLP (clone 5G10) and a goat anti-rabbit secondary antibody. [Figure 4] Figure 1 shows activation of KRASG12V TCR Jurkat cells after co-culture with PMBCs expressed with each of the three different linked antigen mRNA constructs shown in Figure 1A. Controls are the same as those described in Figure 1B. [Figure 5A] Schematic diagram of transduction of CD8 T cells with lentivirus expressing E629-38- or E711-19-specific TCRs and activation by co-culture with PMBCs expressed with either linked antigen mRNA, linked antigen mRNA and signal 2 / 3 mRNA, or signal 2 / 3 mRNA alone. Cells were cultured for 6 days before restimulation with the corresponding E629-38 or E711-19 peptide and measurement of cytokine production via intracellular cytokine staining (ICS). [Figure 5B] Figure 1 shows activation of E6 TCR-transduced and E7 TCR-transduced CD8+ T cells after co-culture with PMBCs expressed with linked antigen mRNA and signal 2 / 3 mRNA, compared to either linked antigen mRNA or signal 2 / 3 mRNA alone. [Figure 6A]The following mRNA constructs were prepared: (1) a concatenated mRNA construct ("Concatenated Antigens+2 / 3") encoding (a) an approximately 25 aa fragment (overlapping a sequence of 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) of the KRASG12D and KRASG12V antigens, (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); (2) an approximately 25 aa fragment (overlapping a sequence of 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) of the KRASG12D and KRASG12V antigens; (3) a concatenated mRNA construct encoding only the ASG12D and KRASG12V antigens ("G12D-G12V concatenated antigen"); (4) 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"); (5) a single approximately 25 aa fragment containing (a) 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). (5) a concatenated mRNA construct (“G12V+2 / 3”) encoding (a) a single approximately 25 aa fragment containing 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); (6) a approximately 25 aa fragment overlapping the first 1 to 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). (d) a concatenated mRNA construct encoding membrane-bound IL-12 (i.e., signal 3) (“WT+2 / 3”), (7) an unconcatenated mRNA construct encoding only a single approximately 25 aa fragment containing a G12V mutation (i.e., without signals 2 and 3) (“G12V”), (8) an unconcatenated mRNA construct encoding only a single approximately 25 aa fragment containing a G12D mutation (i.e., without signals 2 and 3) (“G12D”), and (9) an unconcatenated mRNA construct encoding only an approximately 25 aa fragment overlapping the first 1-25 aa of native wild-type KRAS (i.e.,Signals 2 and 3 (without) show G12V- and G12D-specific responses, respectively, in A*11-restricted G12V and G12D TCR Jurkat responder cells cultured with PBMCs squeezed with one of the unlinked mRNA constructs ("wild-type"). G12V- and G12D-specific responses are shown as evidenced by luminescence expression. Specific values ​​shown above some bars represent the fold change relative to luminescence expression observed in responder cells cultured with PBMCs squeezed without mRNA ("empty"). [Figure 6B]The following mRNA constructs were prepared: (1) a concatenated mRNA construct ("Concatenated Antigens+2 / 3") encoding (a) an approximately 25 aa fragment (overlapping a sequence of 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) of the KRASG12D and KRASG12V antigens, (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); (2) an approximately 25 aa fragment (overlapping a sequence of 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) of the KRASG12D and KRASG12V antigens; (3) a concatenated mRNA construct encoding only the ASG12D and KRASG12V antigens ("G12D-G12V concatenated antigen"); (4) 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"); (5) a single approximately 25 aa fragment containing (a) 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). (5) a concatenated mRNA construct (“G12V+2 / 3”) encoding (a) a single approximately 25 aa fragment containing 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); (6) a approximately 25 aa fragment overlapping the first 1 to 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). (d) a concatenated mRNA construct encoding membrane-bound IL-12 (i.e., signal 3) (“WT+2 / 3”), (7) an unconcatenated mRNA construct encoding only a single approximately 25 aa fragment containing a G12V mutation (i.e., without signals 2 and 3) (“G12V”), (8) an unconcatenated mRNA construct encoding only a single approximately 25 aa fragment containing a G12D mutation (i.e., without signals 2 and 3) (“G12D”), and (9) an unconcatenated mRNA construct encoding only an approximately 25 aa fragment overlapping the first 1-25 aa of native wild-type KRAS (i.e.,Signals 2 and 3 (without) show G12V- and G12D-specific responses, respectively, in A*11-restricted G12V and G12D TCR Jurkat responder cells cultured with PBMCs squeezed with one of the unlinked mRNA constructs ("wild-type"). G12V- and G12D-specific responses are shown as evidenced by luminescence expression. Specific values ​​shown above some bars represent the fold change relative to luminescence expression observed in responder cells cultured with PBMCs squeezed without mRNA ("empty"). [Figure 7A] Shown are G12V and G12D-specific responses in A*11-restricted G12V and G12D TCR Jurkat responder cells, respectively, cultured with PBMCs squeezed with an mRNA construct 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: (1) a concatenated mRNA construct ("7mut_v1") encoding only the approximately 25 aa fragment (overlapping a sequence from 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) KRASG12D, KRASG12V, KRASG12C, KRASG13D, KRASG12A, KRASG12R, and KRASG12S antigens; and (2) a concatenated mRNA construct ("7mut_v2") encoding only the approximately 25 aa fragment (overlapping a sequence from 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) KRASG12S, KRASG12R, KRASG12A, KRASG13D, KRASG12C, KRASG12V, and KRASG12D antigens. G12V- and G12D-specific responses are shown as evidenced by luminescence expression. The specific values ​​shown above some bars represent the fold change relative to luminescence expression observed in responder cells cultured with PBMCs squeezed without mRNA ("empty"). [Figure 7B]Shown are G12V and G12D-specific responses in A*11-restricted G12V and G12D TCR Jurkat responder cells, respectively, cultured with PBMCs squeezed with an mRNA construct 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: (1) a concatenated mRNA construct ("7mut_v1") encoding only the approximately 25 aa fragment (overlapping a sequence from 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) KRASG12D, KRASG12V, KRASG12C, KRASG13D, KRASG12A, KRASG12R, and KRASG12S antigens; and (2) a concatenated mRNA construct ("7mut_v2") encoding only the approximately 25 aa fragment (overlapping a sequence from 1 to 25 aa in the entire KRAS protein and a mutation on codon 12) KRASG12S, KRASG12R, KRASG12A, KRASG13D, KRASG12C, KRASG12V, and KRASG12D antigens. G12V- and G12D-specific responses are shown as evidenced by luminescence expression. The specific values ​​shown above some bars represent the fold change relative to luminescence expression observed in responder cells cultured with PBMCs squeezed without mRNA ("empty"). [Figure 8A]Figure 1 shows G12V- and G12D-specific responses, respectively, in A*11-restricted G12V and G12D TCR Jurkat responder cells cultured with PBMCs squeezed with mRNA constructs encoding seven linked KRAS mutant antigens and further encoding signals 2 (i.e., CD86) and 3 (i.e., membrane-bound IL-2 and IL-12).Specific mRNA constructs were as follows: (a) approximately 25 aa fragments (overlapping aa sequences 1-25 in the entire KRAS protein and a mutation on codon 12) KRASG12D, KRASG12V, KRASG12C, KRASG13D, KRASG12A, KRASG12R, and KRASG12S antigens (referred to as "KRAS 7mut_v1"); (b) CD86 (i.e., signal 2); (c) membrane-bound IL-2 (i.e., signal 3); and (d) a concatenated mRNA construct ("7mut_v1+signal 2 / 3") encoding membrane-bound IL-12 (i.e., signal 3); (2) approximately 25 aa fragments (overlapping aa sequences 1-25 in the entire KRAS protein and a mutation on codon 12) KRASG12D, KRASG12V, KRASG12C (3) (a) a concatenated mRNA construct ("7mut_v1") encoding only the KRASG13D, KRASG12A, KRASG12R, and KRASG12S antigens; (b) an approximately 25 aa fragment (overlapping the 1-25 aa sequence in the entire KRAS protein and a mutation on the 12th codon) encoding the KRASG12S, KRASG12R, KRASG12A, KRASG13D, KRASG12C, KRASG12V, and KRASG12D antigens (herein referred to as "KRAS (b) a concatenated mRNA construct encoding (referred to as "7mut_v2"), (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) a concatenated mRNA construct encoding only the approximately 25 aa fragment (overlap with the 1-25 aa sequence in the entire KRAS protein and a mutation on the 12th codon) KRASG12S, KRASG12R, KRASG12A, KRASG13D, KRASG12C, KRASG12V, and KRASG12D antigens ("mut_v2"); and (5) 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 evidenced by luminescence expression.Specific values ​​shown above some bars represent the fold change relative to luminescence expression observed in responder cells cultured with PBMCs squeezed without mRNA ("empty"). [Figure 8B]Figure 1 shows G12V- and G12D-specific responses, respectively, in A*11-restricted G12V and G12D TCR Jurkat responder cells cultured with PBMCs squeezed with mRNA constructs encoding seven linked KRAS mutant antigens and further encoding signals 2 (i.e., CD86) and 3 (i.e., membrane-bound IL-2 and IL-12).Specific mRNA constructs were as follows: (a) approximately 25 aa fragments (overlapping aa sequences 1-25 in the entire KRAS protein and a mutation on codon 12) KRASG12D, KRASG12V, KRASG12C, KRASG13D, KRASG12A, KRASG12R, and KRASG12S antigens (referred to as "KRAS 7mut_v1"); (b) CD86 (i.e., signal 2); (c) membrane-bound IL-2 (i.e., signal 3); and (d) a concatenated mRNA construct ("7mut_v1+signal 2 / 3") encoding membrane-bound IL-12 (i.e., signal 3); (2) approximately 25 aa fragments (overlapping aa sequences 1-25 in the entire KRAS protein and a mutation on codon 12) KRASG12D, KRASG12V, KRASG12C (3) (a) a concatenated mRNA construct ("7mut_v1") encoding only the KRASG13D, KRASG12A, KRASG12R, and KRASG12S antigens; (b) an approximately 25 aa fragment (overlapping the 1-25 aa sequence in the entire KRAS protein and a mutation on the 12th codon) encoding the KRASG12S, KRASG12R, KRASG12A, KRASG13D, KRASG12C, KRASG12V, and KRASG12D antigens (herein referred to as "KRAS (b) a concatenated mRNA construct encoding (referred to as "7mut_v2"), (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) a concatenated mRNA construct encoding only the approximately 25 aa fragment (overlap with the 1-25 aa sequence in the entire KRAS protein and a mutation on the 12th codon) KRASG12S, KRASG12R, KRASG12A, KRASG13D, KRASG12C, KRASG12V, and KRASG12D antigens ("mut_v2"); and (5) 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 evidenced by luminescence expression.Specific values ​​shown above some bars represent the fold change relative to luminescence expression observed in responder cells cultured with PBMCs squeezed without mRNA ("empty"). DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure is generally directed to isolated polynucleotides that can be used to induce the expression of multiple antigens in cells. More specifically, provided herein are polynucleotides comprising at least a first coding region encoding a first antigen and a second coding region encoding a second antigen, wherein the first and second antigens are not the same, and the first and second coding regions are linked (e.g., by a linker). As described and demonstrated herein, such polynucleotides can be delivered intracellularly to cells, which then express both the first and second antigens.

[0043] As further described herein, the exemplary delivery methods provided herein (i.e., aperture delivery) possess certain distinguishing characteristics not shared by other non-constriction-mediated delivery methods known in the art. For example, in addition to an improved ability to deliver various types of payloads to cells, the aperture processing methods described herein have minimal, persistent effects on cells. Compared to conventional delivery methods, such as electroporation, the aperture processing methods of the present disclosure maintain both the structural and functional integrity of the apertured cells. In contrast to the delivery methods provided herein, electroporation can induce widespread and persistent changes in gene expression, which can result in nonspecific activation of cells (e.g., human T cells) and delayed proliferation upon antigen stimulation. In this method, any changes to the cells (e.g., perturbations in the cell membrane) are transient, and the perturbations reseal when the cells are removed from the constriction. Non-limiting examples of various embodiments are presented in the present disclosure.

[0044] I. General techniques Some of the techniques and procedures described or referenced herein are generally well understood and may be found, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., 2004). th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012), Current Protocols in Molecular Biology (FMAusubel, et al. eds., 2003), the series Methods in Enzymology (Academic Press, Inc.), PCR 2:A Practical Approach (MJMacPherson, BD Hames and GRTaylor eds., 1995), Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988), Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RIFreshney, 6 thed.,J.Wiley and Sons,2010)、Oligonucleotide Synthesis(M.J.Gait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,Academic Press,1998)、Introduction to Cell and Tissue Culture(J.P.Mather and P.E.Roberts,Plenum Press,1998)、Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,J.Wiley and Sons,1993-8)、Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.,1996)、Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987)、PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994)、Current Protocols in Immunology(J.E.Coligan et al., eds.,1991)、Short Protocols in Molecular Biology(Ausubel et al.,eds.,J.Wiley and Sons,2002)、Immunobiology(C.A.Janeway et al.,2004)、Antibodies(P.Finch,1997)、Antibodies:A Practical Approach(D.Catty.,ed.,IRL Press,1988-1989)、Monoclonal Antibodies:A Practical Approach(P.Shepherd and C.Dean,eds., 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., J.B. Lippincott Company, 2011), are commonly used by those skilled in the art using conventional methodologies.

[0045] II. Definition For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth herein shall control. Additional definitions are set forth throughout the detailed description.

[0046] As used herein, the singular terms "a," "an," and "the" refer to one or more of that entity, unless otherwise indicated. Thus, the terms "a" (or "an" or "the"), "one or more," and "at least one" can be used interchangeably herein.

[0047] It is understood that the aspects and embodiments of the present disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. It is also understood that whenever aspects and embodiments are described herein with the term "comprising," similar aspects or embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0048] Furthermore, "and / or," when used herein, should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass 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).

[0049] For all compositions described herein and all methods of using the compositions described herein, the composition can include the recited components or steps or can "consist essentially of" the recited components or steps. When a composition is described as "consisting essentially of" recited components, the composition contains the recited components and can further contain other components that do not substantially affect the disclosed method, but does not contain any other components other than those explicitly recited that substantially affect the disclosed method, or if the composition contains additional components other than those recited that substantially affect the disclosed method, the composition does not contain the additional components in a concentration or amount sufficient to substantially affect the disclosed method. When a method is described as "consisting essentially of" recited steps, the method contains the recited steps and can further contain other steps that do not substantially affect the disclosed method, but the method does not contain any other steps other than those explicitly recited. As a non-limiting example, when a composition is described as "consisting essentially of" a component, the composition can further contain any amount of pharmaceutically acceptable carriers, vehicles or diluents, and other such components that do not substantially affect the disclosed methods.

[0050] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) recognized form. Numerical ranges are intended to be inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of this disclosure, which may be had by reference to the specification in its entirety. Accordingly, the terms defined immediately below are more fully defined in their entirety by reference to the specification.

[0051] The term "about" is used herein to mean approximately, in the region of, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. In general, the term "about" can modify a numerical value above and below the recited value, for example, by a variance of 10 percent above or below (higher or lower).

[0052] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance (i.e., 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.

[0053] As used herein, the term "associated with" refers to a close relationship between two or more entities or properties. For example, when used to describe a disease or condition, the term "associated with" refers to an increased likelihood that a subject will suffer from the disease or condition if the subject exhibits abnormal expression of a protein and / or gene (e.g., a KRAS mutant). In some embodiments, the abnormal expression of the protein and / or gene causes the disease or condition. In some embodiments, the abnormal expression does not necessarily cause the disease or condition, but is correlated with the disease or condition.

[0054] As used herein, the term "epitope" refers to a portion of a protein that can be recognized by the immune system (e.g., antibodies, B cells and / or T cells) and thereby induce an immune response.

[0055] As used herein, the term "linked" refers to a covalent or non-covalent bond formed between a first moiety and a second moiety, e.g., a first coding region and a second coding region. As further described herein, in some embodiments, the first and second moieties may be linked with a linker.

[0056] The term "constriction," as used herein, refers to a narrow passage. In some embodiments, the constriction is a microfluidic channel, such as one contained within a microfluidic device. In some embodiments, the constriction is a pore or is contained within a pore. When the constriction is a pore, in some embodiments, the pore is contained in a surface. Unless otherwise indicated, the term constriction refers to both microfluidic channels and pores, as well as other suitable constrictions available in the art. Thus, where applicable, disclosure regarding microfluidic channels can also be applied to pores and / or other suitable constrictions available in the art. Similarly, where applicable, disclosure regarding pores can equally be applied to microfluidic channels and / or other suitable constrictions available in the art.

[0057] The term "pore," as used herein, refers to an opening, including, but not limited to, a hole, crevice, cavity, opening, break, gap, or perforation in a material. In some aspects, (where indicated) the term refers to a pore in the surface of a microfluidic device, such as those described in this disclosure. In some aspects, (where indicated) the pore can refer to a pore in a cell wall and / or cell membrane.

[0058] The term "membrane," as used herein, refers to a selective barrier or sheet containing pores. The term includes, but is not limited to, flexible sheet-like structures that act as boundaries or backings. In some embodiments, the term refers to a surface or filter containing pores. This term can be distinguished from the term "cell membrane," which refers to a semipermeable membrane that surrounds the cytoplasm of a cell.

[0059] The term "filter," as used herein, refers to a porous article that allows selective passage through the pores. In some aspects, the term refers to a surface or membrane that contains pores.

[0060] As used herein, the terms "deform" and "deformation" (including derivatives thereof) refer to a physical change in a cell. As described herein, when a cell passes through a constriction (such as that disclosed herein), it experiences various forces due to the restrictive physical environment, including, but not limited to, mechanical deformation and / or shear forces, which cause perturbations in the cell membrane. As used herein, a "perturbation" in a cell membrane refers to any opening in the cell membrane that is not present under normal steady-state conditions (e.g., no deforming forces are applied to the cell). A perturbation can include a hole, a crevice, a cavity, an opening, a pore, a break, a gap, a perforation, or a combination thereof.

[0061] The term "polynucleotide" or "nucleic acid," as used herein, refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the 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 containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of a polynucleotide can contain sugar and phosphate groups (typically found in RNA or DNA), or modified or substituted sugar or phosphate groups. Alternatively, the backbone of a polynucleotide can comprise a polymer of synthetic subunits, such as phosphoramidates, and thus can be an oligodeoxynucleoside phosphoramidate (P-NH2) or a mixed phosphoramidate-phosphodiester oligomer. Additionally, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by either synthesizing the complementary strand and annealing the strands under appropriate conditions, or by de novo synthesizing the complementary strand using DNA polymerase with appropriate primers. As described herein, nucleic acids that can be delivered to cells using the filtration methods provided herein include RNA (e.g., mRNA). As used herein, "RNA" includes both 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, resulting in increased amounts of RNA and the protein (e.g., antigen) encoded by the RNA. As used herein, the term "mRNA" refers to any polynucleotide (either self-amplifying or non-self-amplifying) that encodes at least one polypeptide.

[0062] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues, without any minimum length limitation. Such polymers of amino acid residues can contain natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of this disclosure, "polypeptide" refers to a protein containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts producing the protein or errors due to PCR amplification.

[0063] "Immune response," as used herein, refers to a biological response in a vertebrate to foreign agents or abnormal, e.g., cancerous, cells, which protects the organism from these agents and the diseases they cause. 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 either these cells or the liver, resulting in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An immune response can include, for example, the activation or inhibition of T cells, e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or inhibition of any other cells of the immune system, e.g., NK cells. Thus, an immune response can include a humoral immune response (e.g., mediated by B cells), a cellular immune response (e.g., mediated by T cells), or both a humoral and a 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+ T cells and CD4+ T cells.

[0064] III. Polynucleotides 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), each of which encodes an antigen. A nucleotide sequence encoding an antigen is also referred to herein as a "coding region." As set forth and further described herein, the polynucleotides of the present disclosure differ (in structure and / or function) from corresponding polynucleotides naturally occurring in nature.

[0065] Code Region: In some embodiments, the polynucleotides described herein comprise coding regions from multiple proteins in a single open reading frame (ORF), where the coding regions do not naturally occur together in nature (e.g., within a single polynucleotide). For example, in some embodiments, the polynucleotides of the present disclosure comprise a single ORF having a first coding region and a second coding region, where the first coding region can encode a cancer antigen and the second coding region can encode a different cancer antigen. For example, in some embodiments, the polynucleotides of the present disclosure comprise a first coding region and a second coding region, where the first coding region can encode a viral antigen and the second coding region can encode a different viral antigen. For example, in some embodiments, the polynucleotides of the present disclosure comprise a single ORF having a first coding region and a second coding region, where 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 useful in the present disclosure are provided elsewhere in this disclosure. As will be apparent to one of skill in the art, such polynucleotides may be particularly useful in simultaneously combating many different pathogens and / or diseases.

[0066] In some embodiments, the polynucleotides described herein comprise coding regions, which can naturally occur together within a single polynucleotide (e.g., within the same ORF). For example, in some embodiments, the coding regions comprise a first coding region and a second coding region, where 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, and the proteins are naturally encoded by a single ORF of the polynucleotide. When a polynucleotide useful in the present disclosure comprises such a coding region, in some embodiments, the coding regions are arranged in a specific order within the ORF of the polynucleotide, and the specific order does not naturally occur in nature.

[0067] 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, where the first, second, and third coding regions are arranged (5' to 3') as follows: (first coding region), (second coding region), and (third coding region). When a polynucleotide described herein comprises such coding regions, in some embodiments, the first coding region, second coding region, and third coding region are arranged (5' to 3') within the single ORF of the polynucleotide in the following order: (a) (first coding region), (third coding region) and (second coding region), (b) (second coding region), (first coding region) and (third coding region), (c) (second coding region), (third coding region) and (first coding region), (d) (third coding region), (first coding region) and (second coding region), or (e) It is arranged in any one of (third coding region), (second coding region), and (first coding region).

[0068] Additional structural features of polynucleotides are provided throughout this disclosure. For example, in some embodiments, the polynucleotides described comprise a single ORF with multiple coding regions (e.g., a first coding region and a second coding region), where one or more of the coding regions encode a full-length protein. To illustrate, in some embodiments, a polynucleotide comprises a single ORF with a first coding region and a second coding region, where the first coding region encodes a full-length HPV E6 protein (e.g., HPV-16 or HPV-18), and the second coding region encodes a full-length HPV E7 protein (e.g., HPV-16 or HPV-18). However, as demonstrated herein, a coding region need not encode a full-length protein (or a large fragment thereof) to exert a therapeutic effect (e.g., induce an immune response against the protein). Thus, the polynucleotides described herein comprise a single ORF with multiple coding regions, where one or more of the antigens encoded by the multiple coding regions comprise a peptide fragment of a larger protein. In some embodiments, 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 length compared to the corresponding full-length fragment. In some embodiments, the antigen is encoded by one or more of a plurality of coding regions less than about 200 amino acids in length. In some embodiments, the antigen is less than about 150 amino acids in length. In some embodiments, the antigen is less than about 100 amino acids in length. In some embodiments, the antigen is less than about 50 amino acids in length. In some embodiments, the antigen encoded by one or more of the coding regions present within a single ORF of the polynucleotides described herein is from about 5 amino acids to about 200 amino acids in length, hi some embodiments, the antigen is from about 5 amino acids to about 150 amino acids in length.In some embodiments, the antigen is from about 5 amino acids to about 100 amino acids in length. In some embodiments, the antigen is from about 5 amino acids to about 50 amino acids in length. In some embodiments, 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, or about 50 amino acids in length. In some embodiments, the antigen is about 10 to about 50 amino acids in length. In some embodiments, the antigen is about 10 to about 40 amino acids in length. In some embodiments, the antigen is about 10 to about 30 amino acids in length. In some embodiments, the antigen is about 10 to about 20 amino acids in length. In some embodiments, the antigen is about 20 to about 50 amino acids in length. In some embodiments, the antigen is about 20 to about 40 amino acids in length. In some embodiments, the antigen is about 20 to about 30 amino acids in length. In some embodiments, the antigen is about 30 to about 50 amino acids in length. In some embodiments, the antigen is about 30 to about 40 amino acids in length. In some embodiments, the antigen is about 40 to about 50 amino acids in length. In some embodiments, the antigen is about 8 to about 11 amino acids in length. In some embodiments, the antigen is about 10 to about 17 amino acids in length. In some embodiments, the antigen is about 10 amino acids in length. In some embodiments, the antigen is about 15 amino acids in length. In some embodiments, the antigen is about 20 amino acids in length. In some embodiments, the antigen is about 25 amino acids in length. In some embodiments, the antigen is about 30 amino acids in length. In some embodiments, the antigen is about 35 amino acids in length. In some embodiments, the antigen is about 40 amino acids in length.In some embodiments, the antigen is about 45 amino acids in length. In some embodiments, the antigen is about 50 amino acids in length. In some embodiments, the antigen is about 8 amino acids in length.

[0069] Without being bound by any one theory, depending on the size of the antigen, the polynucleotides described herein can be designed to contain multiple coding regions. In some embodiments, the polynucleotides described contain a single ORF having about 2 coding regions, about 3 coding regions, about 4 coding regions, about 5 coding regions, about 6 coding regions, about 7 coding regions, about 8 coding regions, about 9 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 embodiments, two or more of the coding regions are linked. In some embodiments, each of the coding regions is linked. In some embodiments, the polynucleotides described herein comprise a single ORF having two linked coding regions. In some embodiments, the polynucleotides comprise a single ORF having three linked coding regions. In some embodiments, the polynucleotides comprise a single ORF having four linked coding regions. In some embodiments, the polynucleotides described herein comprise a single ORF having five linked coding regions. In some embodiments, the polynucleotides described herein comprise a single ORF having six linked coding regions. In some embodiments, the polynucleotides comprise a single ORF having seven linked coding regions. In some embodiments, the polynucleotides comprise a single ORF having eight linked coding regions. In some embodiments, the polynucleotides comprise a single ORF having nine linked coding regions. In some embodiments, the polynucleotides comprise a single ORF having ten linked coding regions.In some embodiments, the polynucleotide comprises a single ORF having 11 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 12 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 13 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 14 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 15 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 16 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 17 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 18 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 19 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 20 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 21 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 22 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 23 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 24 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 25 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 26 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 27 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 28 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 29 linked coding regions. In some embodiments, the polynucleotide comprises a single ORF having 30 linked coding regions.In some embodiments, each of the coding regions present within a single ORF of the polynucleotides described herein encodes a different antigen, hi some embodiments, at least two of the coding regions encode different antigens.

[0070] In some embodiments, any of the polynucleotides described herein may be used in combination. For example, in some embodiments, a composition may include a first polynucleotide and a second polynucleotide, where the first polynucleotide includes a single ORF having multiple (e.g., about 10) linked coding regions, and the second polynucleotide includes a single ORF having multiple (e.g., about 10) linked coding regions. In some embodiments, the multiple linked coding regions of the first polynucleotide and the multiple linked coding regions of the second polynucleotide encode different antigens. To illustrate, in some embodiments, the multiple coding regions of the first polynucleotide may encode tumor antigens (e.g., neoantigens), and the multiple coding regions of the second polynucleotide may encode non-self antigens (e.g., derived from a virus). Such first and second polynucleotides may be combined and administered to a subject as a single composition. In some embodiments, the first and second polynucleotides may be combined but used as separate compositions.

[0071] Accordingly, some aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) comprising a single 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, wherein the first coding region, the second coding region, and the third coding region are linked. In some aspects, at least two of the encoded linked antigens are not the same. In some aspects, each of the encoded linked antigens is different. In some aspects, at least two of the encoded linked antigens are the same. In some aspects, the first coding region, the second coding region, and the third coding region are arranged in an order that is different compared to the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some aspects, the particular order of the coding regions within the single ORF of the polynucleotide can help improve one or more properties of the polynucleotide (e.g., improved translation when delivered to a cell). For example, to help illustrate, in some embodiments, a first polynucleotide comprises a single ORF including 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 (5' to 3') within the ORF as follows: (first coding region)-(second coding region)-(third coding region). In some embodiments, a second polynucleotide comprises a single ORF with the same first, second, and third coding regions of the first polynucleotide, wherein the first, second, and third coding regions are arranged (5' to 3') within the ORF as follows: (first coding region)-(third coding region)-(second coding region). In some embodiments, the third polynucleotide comprises a single ORF having identical first, second, and third coding regions, the coding regions being arranged (5' to 3') as follows: (second coding region)-(third coding region)-(first coding region).For each of the first, second, and third polynucleotides, the three coding regions can be linked with and / or without a linker (e.g., directly conjugated via a natural peptide bond). Without being bound by any one theory, in some embodiments, each of the first, second, and third polynucleotides can be associated with a different therapeutic effect. For example, in some embodiments, when the first, second, and / or third polynucleotides are delivered to cells (e.g., using the dialysis process described herein), they can result in different translation efficiencies (e.g., cells modified to include the second polynucleotide can have higher encoded protein expression compared to cells modified to include the first or second polynucleotide). Thus, in some embodiments, the polynucleotides described herein can have improved effects (e.g., better translation) compared to corresponding polynucleotides having the same coding regions but arranged in a different order.

[0072] In some aspects, the polynucleotides described herein comprise a single ORF having 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, wherein the first coding region, the second coding region, the third coding region, and the fourth coding region are linked. In some aspects, the first coding region, the second coding region, the third coding region, and the fourth coding region are arranged in an order that is different from the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some aspects, at least two of the encoded linked antigens are not the same. In some aspects, each of the encoded linked antigens is different. In some aspects, at least two of the encoded linked antigens are the same.

[0073] In some aspects, the polynucleotides described herein comprise a single ORF having 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, wherein the first coding region, the second coding region, the third coding region, the fourth coding region, and the fifth coding region are linked. 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 an order that is different from the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some aspects, at least two of the encoded linked antigens are not the same. In some aspects, each of the encoded linked antigens is different. In some aspects, at least two of the encoded linked antigens are the same.

[0074] In some aspects, the polynucleotides described herein comprise a single ORF having 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, 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 linked. 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 an order that is different from the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some aspects, at least two of the encoded linked antigens are not the same. In some aspects, each of the encoded linked antigens is different. In some aspects, at least two of the encoded linked antigens are the same.

[0075] In some embodiments, the polynucleotides described herein comprise a single ORF having 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, 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 linked. In some embodiments, 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 an order that is different from the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some embodiments, at least two of the encoded linked antigens are not the same. In some embodiments, each of the encoded linked antigens is different. In some embodiments, at least two of the encoded linked antigens are the same.

[0076] In some aspects, the polynucleotides described herein comprise a single ORF having 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, 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 linked. 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 that is different compared to the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some embodiments, at least two of the encoded linked antigens are not the same. In some embodiments, each of the encoded linked antigens is different. In some embodiments, at least two of the encoded linked antigens are the same.

[0077] In some aspects, the polynucleotides described herein comprise a single ORF having 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, wherein the first coding region, second coding region, third coding region, fourth coding region, fifth coding region, sixth coding region, seventh coding region, eighth coding region, and ninth coding region are linked. In some embodiments, 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 that is different compared to the corresponding order present in a reference polynucleotide, wherein the reference polynucleotide comprises a corresponding naturally occurring polynucleotide. In some embodiments, at least two of the encoded linked antigens are not the same. In some embodiments, each of the encoded linked antigens is different. In some embodiments, at least two of the encoded linked antigens are the same.

[0078] In some aspects, the polynucleotides described herein comprise a single ORF having 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, 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 linked. In some embodiments, 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 an order that is different compared to the corresponding order present in a reference polynucleotide, which comprises a corresponding naturally occurring polynucleotide. In some embodiments, at least two of the encoded linked antigens are not the same. In some embodiments, each of the encoded linked antigens is different. In some embodiments, at least two of the encoded linked antigens are the same.

[0079] antigen: As described herein, the polynucleotides described herein comprise a single ORF having coding regions from multiple proteins, where the coding regions encode antigens. In some embodiments, the antigen can comprise an epitope. In some embodiments, the antigen can comprise an immunogenic epitope. Thus, in some embodiments, one or more of the multiple coding regions can encode distinct (i.e., different) epitopes of a single, larger protein, where the single, larger protein comprises multiple distinct epitopes. For example, in some embodiments, the polynucleotides described herein comprise a single ORF having at least a first coding region and a second coding region, where the first coding region and the second coding region encode distinct epitopes of the same protein. In some embodiments, one or more of the multiple coding regions can encode epitopes from different proteins. For example, in some aspects, a polynucleotide of the present disclosure comprises 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 and the second coding region encodes an epitope of a second protein, and the first protein and the second protein are not the same (e.g., a KRAS protein and an HPV protein, respectively). In some aspects, the antigen comprises a wild-type protein. In some aspects, the antigen comprises a peptide fragment derived from a wild-type protein. In some aspects, the antigen comprises a protein (also referred to herein as a "mutant protein") having at least one or more amino acid modifications (e.g., substitutions, deletions, additions, or indels) compared to the corresponding wild-type protein (e.g., a KRAS mutant). In some aspects, the antigen comprises a peptide fragment derived from such a mutant protein.

[0080] Non-limiting examples of antigens that may be used with the present disclosure include cancer antigens, non-self antigens, tumor-associated self antigens, disease-associated antigens, or combinations thereof.

[0081] In some embodiments, the antigen comprises a cancer antigen. As used herein, the terms "cancer antigen" and "tumor antigen" (and their derivatives) can be used interchangeably and refer to any antigen common to a particular hyperproliferative disorder, such as 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, Big3, 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 Cancer antigens include 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, neoantigen, KRAS, or a combination thereof. In some embodiments, the cancer antigen comprises KRAS.

[0082] Kirsten rat sarcoma viral oncogene homology (KRAS) is a member of the guanosine-5-triphosphatase (GTPase) protein superfamily, which also includes NRAS and HRAS. The primary role of members of this superfamily is to transduce signals from upstream cell surface receptors (e.g., EGFR, FGFR, and ERBB2-4) to downstream growth and survival pathways, such as RAF-MEK-ERK, PI3K-AKT-mTOR, and RALGDS-RA. (Adderley, H., et al., EBioMedicine 41:711-716 (2019)). KRAS mutations are implicated in many types of cancer, including over 90% of pancreatic cancers, 35-45% of colorectal cancers, and approximately 25% of lung cancers. 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 a very poor prognosis (e.g., a 5-year survival rate of approximately 9% in pancreatic cancer), and many patients with KRAS mutations are resistant to various cancer therapies. Del Re, M., et al., Oncotarget 9(5):6630-6643(2017). Therefore, there is a need for new and improved treatment options for cancers associated with KRAS mutations.

[0083] KRAS is known in the art by various names, including 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 CK-Ras oncogene, C-Kirsten-Ras protein, K-Ras P21 protein, and oncogene KRAS2.

[0084] There are two isoforms of the human wild-type KRAS protein (P01116) that result from 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.

[0085] Natural variants of human KRAS gene products are known. For example, natural variants of human KRAS proteins can contain one or more amino acid substitutions selected from the following: 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 combinations thereof. Natural variants that are specific for KRAS protein isoform 2B contain one or more amino acid substitutions selected from V152G, D153V, F156I, F156L, or a combination thereof.

[0086] As is apparent from the above disclosure, in some embodiments, one or more of the coding regions of the polynucleotides described herein encode a KRAS antigen, and the KRAS antigen comprises an amino acid sequence that differs from 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 embodiments, 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 with the amino acid sequence of the corresponding wild-type KRAS antigen. In some embodiments, the amino acid sequence of the KRAS mutant comprises one or more amino acid substitutions, and the one or more amino acid substitutions are associated with cancer. In some embodiments, the amino acid sequence of the KRAS mutant comprises any of the amino acid substitutions described herein. For example, in some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising a G12V amino acid substitution. In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising a G12D amino acid substitution. In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, and at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising a G12C amino acid substitution. In some embodiments, the polynucleotides described herein comprise a single ORF having multiple coding regions, wherein the multiple coding regions are linked, and at least one of the multiple coding regions encodes a KRAS antigen, and the amino acid sequence of the KRAS antigen comprises a G13D amino acid substitution.In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising a G12A amino acid substitution. In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising a G12R amino acid substitution. In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising a G12S amino acid substitution. In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, the multiple coding regions being linked, at least one of the multiple coding regions encoding a KRAS antigen, and the amino acid sequence of the KRAS antigen comprising the following: 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 The antigens include one or more of:

[0087] In some embodiments, at least one of the multiple coding regions encodes a KRAS antigen, and 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 compared to the amino acid sequence set forth in any one of SEQ ID NOS: 1-15. In some embodiments, the polynucleotides described herein comprise a single ORF having multiple coding regions, the multiple coding regions being linked and encoding at least one KRAS antigen, the KRAS antigen comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the polynucleotides described herein comprise a single ORF having multiple coding regions, the multiple coding regions being linked and encoding at least one KRAS antigen, the KRAS antigen comprising, consisting of, or consisting essentially of the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF having multiple coding regions that 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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 embodiments, the polynucleotides described herein comprise a single ORF with multiple coding regions, which 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: 15.

[0088] 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 linked, and 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 with multiple coding regions, wherein the multiple coding regions are linked, and wherein one or more of the multiple coding regions encode a mutant KRAS antigen (e.g., comprising a G12D and / or G12V substitution), and one or more of the multiple coding regions encode an additional tumor antigen (e.g., NY-ESO-1 and / or MAGEA10). In some aspects, the polynucleotides described herein comprise a single ORF with multiple coding regions, wherein the multiple coding regions are linked and encode multiple KRAS antigens. In some embodiments, the multiple KRAS antigens comprise one or more of the following amino acid substitutions: G12A, G12C, G12D, G12R, G12S G12V, and G13D. As described herein, in some embodiments, the multiple coding regions are arranged in a specific order within the polynucleotides described herein.

[0089] Thus, in some embodiments, the polynucleotides described herein comprise the following coding regions linked and arranged in the following order (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 embodiments, 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 multiple coding regions encoding (1) multiple KRAS mutant antigens, (2) costimulatory 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 are arranged (5' to 3') with the following mutations: G12D, G12V, G12C, G13D, G12A, G12R, and G12S. In some embodiments, the polynucleotides described herein comprise the following coding regions linked and arranged in the following order (5' to 3'): (1) a first coding region encoding a KRAS G12S mutant, (2) a second coding region encoding a KRAS G12R 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 embodiments, such polynucleotides comprise additional linked coding regions (e.g., encoding costimulatory molecules and / or cytokines).For example, in some embodiments, a polynucleotide of the present disclosure comprises multiple coding regions encoding (1) multiple KRAS mutant antigens, (2) a costimulatory molecule (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 comprise the following mutations arranged (5' to 3'): G12S, G12R, G21A, G13D, G12C, G12V, and G12D. When cells (e.g., PBMCs) are expressing such polynucleotides as described herein (see, e.g., Examples 6-8), each of the encoded antigens can be expressed in PBMCs such that the PBMCs are capable of inducing activation of T cells that recognize the encoded antigen.

[0090] In some embodiments, the antigen comprises a self-antigen. For example, in some embodiments, the antigen comprises a self-antigen that is overexpressed in certain tumors. In some embodiments, the antigen comprises a non-self-antigen. As used herein, the term "non-self-antigen" refers to any antigenic substance (i.e., capable of inducing an immune response when present in vivo) derived from a pathogen, including, but not limited to, a virus, a fungus, a protozoan, or a combination thereof.Non-limiting examples of non-self antigens include: human gammaherpesvirus 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., Plasmodium vivax and Plasmodium falciparum), Chikungunya virus, Human papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human T-lymphotropic virus (HTLV-1), Human herpesvirus 8 (HHV-8), Merkel cell polyomavirus (MCV), Bunyaviridae (e.g., Hantavirus), Arenaviruses (e.g., LCMV and Lassa virus), Flaviviruses (e.g., Dengue, Zika, Japanese encephalitis, West Nile, and Yellow fever), Enteroviruses (e.g., Polio), Astroviruses (e.g., Gastroenteritis), Rhabdoviridae (e.g., Rabies), Borrelia burgdorferi and Burrelia mayonii (e.g., Lyme disease), Herpes simplex virus 2 (HSV-2), Klebsiella species, Pseudomonas aeruginosa, Enterococcus species, Proteus species, Enterobacter species, Actinobacter species, coagulase-negative staphylococci (CoNS), Mycoplasma species, adenovirus, adeno-associated virus (AAV), or combinations thereof.

[0091] Thus, in some aspects, one or more of the coding regions of the polynucleotides described herein encode a non-self antigen. For example, in some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode a non-self antigen. In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode an HPV antigen (e.g., E7 and / or E6 proteins). In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode an HIV antigen. In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode an HBV antigen. In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode an HSV antigen (e.g., HSV gD). In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode an influenza antigen (e.g., an M1 protein). In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode a coronavirus antigen (e.g., an S protein). In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and the multiple coding regions encode multiple non-self antigens (e.g., an HPV antigen, an EBV antigen, an HSV antigen, an influenza antigen, a coronavirus antigen, and combinations thereof).In some aspects, the polynucleotides described herein comprise multiple coding regions, wherein the multiple coding regions are linked, and one or more of the multiple coding regions encode at least one cancer antigen (e.g., KRAS mutant, NY-ESO-1, MAGE A10, or a combination thereof), and one or more of the multiple coding regions encode at least one non-self antigen (e.g., HPV antigen, EBV antigen, HSV antigen, influenza antigen, coronavirus antigen, and combinations thereof).

[0092] Linker As described herein, the polynucleotides described herein comprise multiple coding regions, and the multiple coding regions are linked. Any useful means of linking two moieties (e.g., a first coding region and a second coding region) can be used with the present disclosure. In some embodiments, the multiple coding regions are linked with a linker. In some embodiments, the multiple coding regions can be linked without a linker. For example, in some embodiments, the first coding region and the second coding region can be positioned within an ORF such that they are directly adjacent to each other such that, when translated, the protein encoded by the first coding region and the protein encoded by the second coding region are linked by a conventional peptide amide bond. As used herein, the term "linker" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more linkers can be linked in tandem. When multiple linkers are present, each linker can be the same or different. Generally, the linker provides flexibility or prevents / ameliorates steric hindrance. Linkers are typically not cleavable, although in certain embodiments, such cleavage may be desirable. Thus, in some embodiments, linkers can contain one or more protease-cleavable sites, which can be located within the linker sequence or can be adjacent to the linker at either end of the linker sequence. Thus, in some embodiments, linkers useful in the present disclosure include cleavable linkers.

[0093] As used herein, the term "cleavable linker" refers to a linker that contains a cleavage site that, when expressed, can be selectively cleaved to produce two or more products. In some embodiments, the linker is selected from a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof (see Table 1 below). In some embodiments, the linker comprises a GSG linker sequence. In some embodiments, linkers useful in the present disclosure contain an internal ribosome entry site (IRES) such that distinct polypeptides encoded by the first and second genes are produced during translation. Further description of linkers that can be used with the present disclosure is provided, for example, in WO2020 / 223625A1 and US2019 / 0276801A1, each of which is incorporated by reference in its entirety. [Table 1]

[0094] In some embodiments, the linker moiety is a peptide linker. In some embodiments, the peptide linker comprises a glycine / serine linker. In some embodiments, the peptide linker is a glycine / serine linker according to the formula [(Gly)n-Ser]m (SEQ ID NO:23), where n is any integer between 1 and 100, and m is any integer between 1 and 100. In some embodiments, the glycine / serine linker is according to the formula [(Gly)x-(Ser)y]z (SEQ ID NO:24), where x is an integer between 1 and 4, y is 0 or 1, and z is an integer between 1 and 50. In some embodiments, the peptide linker comprises the sequence Gn (SEQ ID NO:25), where n can be an integer between 1 and 100. In some embodiments, the peptide linker can comprise the sequence (GlyAla)n (SEQ ID NO:26), where n is an integer between 1 and 100. In some embodiments, the peptide linker can comprise the sequence (GlyGlySer)n (SEQ ID NO:27), where n is an integer between 1 and 100. In some embodiments, the peptide linker comprises the sequence GGGG (SEQ ID NO:28).

[0095] In some embodiments, the peptide linker comprises the sequence (GGGS)n (SEQ ID NO:29). In certain embodiments, the peptide linker comprises the sequence (GGS)n(GGGGS)n (SEQ ID NO:30). In such embodiments, n can be an integer between 1 and 100. In some embodiments, n can be an integer between 1 and 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 embodiments, n is an integer between 1 and 100. In some embodiments, the peptide linker is (GS)3 (SEQ ID NO:31).

[0096] In some embodiments, the peptide linker comprises an EAAAK linker. In some embodiments, the EAAAK linker comprises the sequence (EAAAK)n, where n is an integer between 1 and 10 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker is (EAAAK)3 (SEQ ID NO: 32).

[0097] In some embodiments, the peptide linker is synthetic, i.e., not naturally occurring. In one embodiment, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally occurring peptide or a non-naturally occurring peptide) that comprises an amino acid sequence that links or genetically fuses a first linear sequence of amino acids to a second linear sequence of amino acids to which it is not naturally linked or genetically fused in nature. For example, in one embodiment, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., containing mutations such as additions, substitutions, or deletions).

[0098] In some aspects, the peptide linker can comprise non-naturally occurring amino acids. In yet other aspects, the peptide linker can comprise naturally occurring amino acids that occur in a linear sequence that does not occur in nature. In yet other aspects, the peptide linker can comprise a naturally occurring polypeptide sequence.

[0099] In some embodiments, the linker comprises a non-peptide linker. In other embodiments, the linker consists of a non-peptide linker. In some embodiments, the non-peptide linker can be, for example, maleimidocaproyl (MC), maleimidopropanoyl (MP), methoxyl polyethylene glycol (MPEG), succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 6-[3-(2-pyridyldithio)-propionamido]hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-alpha-α-(2-pyridyldithio)toluene (SMPT), or the like (see, e.g., U.S. Pat. No. 7,375,078, which is incorporated herein by reference in its entirety).

[0100] Other components: In addition to any of the above characteristics, polynucleotides useful in the present disclosure can include one or more additional components. In some embodiments, these additional components can assist the polynucleotide in exerting its therapeutic effect (e.g., allowing for improved translation and / or increased stability of the polynucleotide). Non-limiting examples of additional components that can be included in the polynucleotides described herein include an internal ribosome entry site (IRES), an intron sequence, a homology arma promoter, an enhancer, a UTR, a sequence encoding a signal peptide, a translation initiation sequence, a 3' tail region of linked nucleosides, a 5' cap, a sequence encoding a 2A ribosomal skip peptide, or any combination thereof.

[0101] Untranslated Regions (UTRs) In some embodiments, the polynucleotides described herein contain 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 of a gene that is transcribed but not translated. A "5'-UTR" begins at the transcription initiation site and follows, but does not include, the start codon, while a "3'-UTR" begins immediately after the stop codon and continues until the transcription termination signal. There is increasing evidence regarding the regulatory role that UTRs play in terms of the stability and translation of nucleic acid molecules. Regulatory features of UTRs can be incorporated into the polynucleotides described herein to enhance the stability of the molecule. Certain features can also be incorporated to ensure controlled downregulation of transcripts if they are misdirected to an undesirable organ site.

[0102] In some embodiments, UTRs useful in the present disclosure include those present in genes that are abundantly expressed in specific cells, tissues, and / or organs. By including such additional UTRs, in some embodiments, 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, additionally introducing a UTR (e.g., a 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 hepatocyte and / or liver 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.

[0103] Additional examples of UTRs that can be used with the present disclosure include those derived from globins such as α- or β-globin (e.g., Xenopus, mouse, rabbit, or human globin); strong Kozak translation initiation signals; CYBA (e.g., human cytochrome b-245α 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 viruses (e.g., hepatitis B virus), Sindbis virus, or PA virus). V Barley yellow dwarf virus); heat shock proteins (e.g., hsp70); translation initiation factors (e.g., eIF4G); glucose transporters (e.g., hGLUT1 (human glucose transporter 1)); actin (e.g., human α- or β-actin); GAPDH; tubulin; histones; citric acid cycle enzymes; topoisomerases (e.g., 5'-UTR of TOP genes lacking a 5' TOP motif (oligopyrimidine tract)); ribosomal protein large 32 (L32); ribosomal proteins (e.g., human or mouse ribosomal proteins such as rps9); ATP synthases (e.g., ATP5A1 or mitochondrial H +- ATP synthase beta subunit; growth hormone (e.g., bovine (bGH) or human (hGH)); elongation factor (e.g., elongation factor 1 alpha 1 (EEF1A1)); manganese superoxide dismutase (MnSOD); myocyte enhancer factor 2A (MEF2A); beta-F1-ATPase, creatine kinase, myoglobin, granulocyte-colony stimulating factor (G-CSF); collagen (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); ribophorin (e.g., ribophorin I (RPNI)); low-density lipoprotein receptor-related protein (e.g., LRP1); cardiotrophin-like cytokine factor (e.g., Nnt1); calreticulin (Calr); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (Plod1); nucleobindin (e.g., Nucb1); and combinations thereof.

[0104] Cap Structure In some embodiments, a polynucleotide described herein (eg, comprising multiple linked coding regions) comprises a 5' cap.

[0105] 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 nuclear export of the polynucleotide (e.g., into the cytoplasm where translation can occur) and / or can promote the stability of the polynucleotide. In some embodiments, a 5' cap can be linked to the 5' end of a polynucleotide described herein via a 5'-5'-triphosphate linkage. In certain embodiments, 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 with the present disclosure. Non-limiting examples of 5' caps that can be used with 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 may equivalently be 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’-OG(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 combinations thereof.

[0106] In some embodiments, 5' caps that can be used with the polynucleotides of the present disclosure include cap analogs (also known as "synthetic cap analogs," "chemical caps," "chemical cap analogs," or "structural or functional cap analogs"). A "cap analog" differs in its chemical structure from the 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.

[0107] In some embodiments, the 5' cap is modified. Modifications to the 5' cap can further increase the stability, half-life, and / or translation efficiency of the polynucleotide. In some embodiments, the modified 5' cap comprises one or more of the following modifications: a modification at the 2' and / or 3' position of the capped guanosine triphosphate (GTP), a replacement of the sugar ring oxygen (which created a carbocyclic ring) with a methylene moiety (CH), a modification in the triphosphate bridge portion of the cap structure, or a modification in the nucleobase (G) portion. See, e.g., US2014 / 0147454 and WO2018 / 160540, each of which is incorporated herein by reference in its entirety.

[0108] Poly(A) tail In some embodiments, the polynucleotides described herein (e.g., comprising multiple linked coding regions) comprise a long stretch of adenine nucleotides at the 3' end of the polynucleotide (referred to herein as a "poly(A) tail"). In some embodiments, the poly(A) tail is present alone or in combination with other components described herein (e.g., a 5' cap).

[0109] In some embodiments, the length of the poly(A) tail is greater than about 30 nucleotides in length. In certain embodiments, 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.

[0110] Any suitable poly(A) tail known in the art can be used with the present disclosure. Non-limiting examples of poly(A) tails that can be used with the present disclosure include SV40 poly(A), bGH poly(A), actin poly(A), hemoglobin poly(A), poly(A)-G quartet, or a combination thereof.

[0111] Enhancer In some embodiments, expression of an antigen encoded by one or more coding regions of a polynucleotide described herein can be further increased using one or more enhancer sequences (also referred to herein as "translation enhancer elements" or "TEEs"). Thus, in some embodiments, a polynucleotide described herein (e.g., comprising multiple linked coding regions) comprises one or more enhancer sequences. In some embodiments, a polynucleotide described herein comprises 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 a polynucleotide comprises multiple enhancers, in some embodiments, each enhancer is the same. In some embodiments, one or more of the enhancers are different. In some embodiments, one or more of the enhancers are separated by a spacer.

[0112] Any enhancer known in the art can be used with the polynucleotides of the present disclosure.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, and U.S. Patent No. 7,183,395, each of which is incorporated herein by reference in its entirety.In some embodiments, the enhancer useful in the present disclosure is a tissue-specific enhancer. In certain aspects, enhancers that may be used with the present disclosure are selected from a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor MEF (e.g., MEF2), a MyoD enhancer element, a cardiac enhancer factor (CEF) site, a mouse creatine kinase enhancer element, a fast skeletal troponin C gene element, a slow cardiac troponin C gene element, a slow troponin I gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, a glucorticoid response element (GRE), or any combination thereof.

[0113] IRES sequence In some embodiments, a polynucleotide described herein (e.g., comprising multiple linked coding regions) can further comprise a nucleotide sequence encoding an internal ribosome entry site (IRES). The IRES plays an important role in initiating protein synthesis in the absence of a 5' cap structure. The IRES can act as the only ribosome binding site or can function as one of multiple ribosome binding sites in a polynucleotide. A polynucleotide containing two or more functional ribosome binding sites can encode several peptides or polypeptides that are independently translated by the ribosome (a "polycistronic polynucleotide"). Thus, when a polynucleotide described herein comprises a sequence encoding an IRES, in certain embodiments, the polynucleotide can comprise multiple (e.g., at least two) translatable regions, e.g., a nucleotide sequence encoding a coronavirus spike protein and a nucleotide sequence encoding a different coronavirus protein (e.g., a nucleocapsid protein).

[0114] Any IRES sequence known in the art can be used with the present disclosure. Non-limiting examples of IRES sequences that can be used with the present disclosure include those derived from picornaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), hand, foot, and mouth disease viruses (FMDV), hepatitis C viruses (HCV), swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV), cricket paralysis viruses (CrPV), or combinations thereof.

[0115] Post-transcriptional regulatory elements In some embodiments, additional components that can be used with the polynucleotides described herein (e.g., comprising multiple linked coding regions) include post-transcriptional regulatory elements. In some embodiments, post-transcriptional regulatory elements can be present in the polynucleotides described herein in combination with one or more other components described herein (e.g., a 5' cap, a 3' poly(A) tail, an enhancer sequence, an IRES sequence, or a combination thereof). In some embodiments, post-translational regulatory elements are positioned 3' to the multiple coding regions of the polynucleotides described herein. Non-limiting examples of post-transcriptional regulatory elements useful in the present disclosure include a mutant woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a microRNA binding site, a DNA nuclear targeting sequence, or a combination thereof.

[0116] In some embodiments, one or more additional components that may be present in a polynucleotide described herein (e.g., comprising multiple linked coding regions) include a promoter. In certain embodiments, a polynucleotide may comprise a single promoter. In some embodiments, a polynucleotide may comprise multiple promoters (e.g., two, three, four, five, or more) operably linked to multiple coding regions of a polynucleotide described herein. When a polynucleotide comprises multiple promoters, in some embodiments, each of the multiple promoters is the same. In certain embodiments, one or more of the multiple promoters are different.

[0117] In some aspects, promoters useful in the present disclosure include mammalian promoters, viral promoters, or both. In certain aspects, promoters that may be used with the polynucleotides described herein include constitutive promoters, inducible promoters, or both.

[0118] Constitutive mammalian promoters include, but are not limited to, promoters of the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters that function constitutively in eukaryotic cells include, for example, the thymidine kinase promoters of cytomegalovirus (CMV), simian viruses (e.g., SV40), papillomavirus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus long terminal repeat (LTR), and other retroviruses, as well as herpes simplex virus. As described herein, in some aspects, promoters that can be used with the present disclosure are inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. In some aspects, promoters that can be used include the T7 promoter.

[0119] Modified Nucleosides / Nucleotides In some aspects, the polynucleotides described herein (eg, comprising multiple linked coding regions) comprise at least one chemically modified nucleoside and / or nucleotide.

[0120] "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). "Nucleotide" refers to a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.

[0121] A polynucleotide can comprise a region or regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide will comprise a region of nucleotides.

[0122] Polynucleotides of the present disclosure can include a variety of distinct modifications. In some embodiments, polynucleotides can contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, polynucleotides can exhibit one or more desirable properties, such as improved thermal or chemical stability, reduced immunogenicity, reduced degradation, increased binding to target microRNAs, or reduced non-specific binding to other microRNAs or other molecules, compared to unmodified polynucleotides.

[0123] In some aspects, the polynucleotides of the present disclosure are chemically modified. As used herein, with respect to a polynucleotide, the term "chemically modified" or, where appropriate, "chemically modified" refers to modification in one or more of its nucleobases, sugars, backbones, or any combination thereof, including but not limited to, with adenosine (A), guanosine (G), uridine (U), thymidine (T), or cytidine (C) ribonucleosides or deoxyribonucleosides, in their position, pattern, percentage, or population.

[0124] In some embodiments, polynucleotides of the disclosure can have uniform chemical modifications of all or any of the same nucleoside type, or a population of modifications generated by downward titration of the same starting modification of all or any of the same nucleoside type, or a measured percentage of chemical modifications of any of the same nucleoside type, but with random incorporation. In further embodiments, polynucleotides of the disclosure can have two, three, or four uniform chemical modifications of the same nucleoside type throughout the polynucleotide (e.g., all uridines and / or all cytidines, etc., modified in the same way).

[0125] Modified nucleotide base pairing encompasses not only standard adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides containing and / or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard base and a standard base, or between two complementary non-standard base structures. One 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 may be incorporated into the polynucleotides of the present disclosure.

[0126] Those skilled in the art will understand that unless otherwise specified, the polynucleotide sequences described in this application recite a "T" in a representative DNA sequence, but if the sequence represents RNA, the "T" will be replaced with a "U." For example, the TDs of the present disclosure can be administered as RNA, DNA, or as hybrid molecules containing both RNA and DNA units.

[0127] In some embodiments, the polynucleotides described herein comprise 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 nucleobase combinations.

[0128] In some embodiments, the nucleobases, sugars, backbone linkages, or any combination thereof in a polynucleotide are 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% modified.

[0129] Base Modification In some embodiments, the chemical modification is in a nucleobase in a polynucleotide of the present disclosure (e.g., comprising multiple linked coding regions). In some embodiments, 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 (m1A), 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.

[0130] In some embodiments, the polynucleotides described herein are uniformly modified for a particular modification (e.g., completely modified, modified throughout the entire sequence). For example, polynucleotides can be uniformly modified with the same type of base modification, such as 5-methyl-cytidine (m5C), which means that all cytosine residues in the polynucleotide sequence are replaced with 5-methyl-cytidine (m5C). Similarly, polynucleotides can be uniformly modified with any type of nucleoside residue present in the sequence by replacing them with any of the modified nucleosides described above.

[0131] In some embodiments, a polynucleotide comprises a combination of at least two (e.g., 2, 3, 4, or more) modified nucleobases. In some embodiments, 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 the nucleobases of a type in a polynucleotide of the disclosure are modified nucleobases.

[0132] skeletal modifications In some aspects, the polynucleotides described herein can include any useful internucleoside linkage. Such linkages, including backbone modifications, that are useful in the compositions of the present disclosure include the following: 3'-alkylene phosphonates, 3'-amino phosphoramidates, alkene containing backbones, aminoalkyl phosphoramidates, aminoalkyl phosphotriesters, boranophosphates, -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, -CH2-NH-CH2-, chiral phosphonates, chiral phosphorothioates, formacetyl and thioformacetyl backbones, methylene(methylimino), methyleneformacetyl and thioformacetyl backbones, methyleneimino. These include, but are not limited to, thiono and methylenehydrazino backbones, morpholino linkages, -N(CH3)-CH2-CH2-, oligonucleosides with heteroatom internucleoside linkages, phosphinates, phosphoramidates, phosphorodithioates, phosphorothioate internucleoside linkages, phosphorothioates, phosphorotriesters, PNAs, siloxane backbones, sulfamate backbones, sulfide sulfoxide and sulfone backbones, sulfonate and sulfonamide backbones, thionoalkylphosphonates, thionoalkylphosphotriesters, and thionophosphoramidates.

[0133] In some embodiments, the presence of the backbone linkages disclosed above increases the stability and resistance to degradation of the polynucleotides of the present disclosure. In some embodiments, 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 of them are phosphorothioate).

[0134] In some aspects, backbone modifications that may be included in polynucleotides of the present disclosure include phosphorodiamidate morpholino oligomer (PMO) and / or phosphorothioate (PS) modifications.

[0135] sugar modification 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, such as LNA or 2'-substituted sugars, can allow the length and / or size of the polynucleotide to be modified (e.g., reduced).

[0136] In some embodiments, 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 a polynucleotide of the disclosure contain a sugar modification (e.g., LNA).

[0137] As described herein, in some embodiments, the polynucleotides described herein can be RNA (e.g., mRNA). Generally, RNA comprises the sugar group ribose, which is a five-membered ring with oxygen. Exemplary, non-limiting modified nucleotides include replacement of oxygen in ribose (e.g., S, Se, or alkylene such as methylene or ethylene), addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a four-membered ring of cyclobutane or oxetane), ring expansion of ribose (e.g., to form six- or seven-membered rings with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have phosphoramidate backbones), polycyclic forms (e.g., tricyclo and "unlocked" rings), and the like. These include "unclear" forms such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, in which the ribose is replaced by a glycol unit attached to a phosphodiester bond), threose nucleic acids (TNAs, in which the ribose is replaced by α-L-threofuranosyl-(3'→2')), and peptide nucleic acids (PNAs, in which a 2-amino-ethyl-glycine linkage replaces the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that have the opposite stereochemical configuration as the corresponding carbon in ribose. Thus, a polynucleotide molecule can include nucleotides that contain, for example, arabinose as the sugar.

[0138] The 2' hydroxyl group (OH) of ribose can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2' position include H, halo, optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 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 C6-10 Aryl-C 1-6 Alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy, sugar (e.g., ribose, pentose, or any described herein), polyethylene glycol (PEG), —O(CH2CHO) n CH2CH2OR (wherein R is H or optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20)), "locked" nucleic acids (LNAs) (wherein the 2'-hydroxyl is 1-6 Alkylene or C 1-6 They are connected to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge, exemplary bridges include, but are not limited to, methylene, propylene, ether, amino bridge, amino alkyl, amino alkoxy, amino, and amino acid.

[0139] In some embodiments, 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, arabinonucleic acid (ANA) units, 2'-fluoro-ANA units, HNA units, INA (intercalating nucleic acid) units, 2'MOE units, or any combination thereof. In some embodiments, the LNA is, for example, oxy-LNA (such as beta-D-oxy-LNA or alpha-L-oxy-LNA), amino-LNA (such as beta-D-amino-LNA or alpha-L-amino-LNA), thio-LNA (such as beta-D-thio-LNA or alpha-L-thio-LNA), ENA (such as beta-D-ENA or alpha-L-ENA), or any combination thereof. In further aspects, nucleotide analogs that may be included in the polynucleotides of the present disclosure include locked nucleic acids (LNA), unlocked nucleic acids (UNA), arabinonucleic acids (ABA), bridged nucleic acids (BNA), and / or peptide nucleic acids (PNA).

[0140] In some embodiments, the polynucleotides of the present disclosure can comprise both modified RNA nucleotide analogs (e.g., LNA) and DNA units. In some embodiments, the polynucleotides described herein comprise gapmers. See, e.g., U.S. Patent Nos. 8,404,649, 8,580,756, 8,163,708, and 9,034,837, all of which are incorporated herein by reference in their entirety.

[0141] In some embodiments, polynucleotides of the present disclosure can include modifications to prevent rapid degradation by endonucleases and exonucleases, including, but not limited to, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, dephosphorylation, conjugation, reverse ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.), (b) base modifications, such as replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications or sugar replacements (e.g., at the 2' or 4' position), and (d) internucleoside linkage modifications, including modification or replacement of phosphodiester linkages.

[0142] IV. Vector In some aspects, provided herein are vectors (e.g., expression vectors) comprising the polynucleotides described herein (e.g., comprising linked coding regions). Suitable vectors for this disclosure include, but are not limited to, expression vectors, viral vectors, and plasmid vectors.

[0143] As used herein, "expression vector" refers to any nucleic acid construct that contains the necessary elements for the transcription and translation of an inserted coding sequence, or, in the case of RNA viral vectors, the necessary elements for replication and translation when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and their derivatives.

[0144] As used herein, "viral vector" includes, but is not limited to, the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; lentivirus; adenovirus; adeno-associated virus; SV40-type virus; polyoma virus; Epstein-Barr virus; papilloma virus; herpes virus; vaccinia virus; poliovirus; and nucleic acid sequences derived from RNA viruses such as retroviruses. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with a gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA followed by integration of the provirus into host cell DNA.

[0145] In some embodiments, the vector is derived from an adeno-associated virus. In some embodiments, the vector is derived from a lentivirus. Examples of lentiviral vectors are disclosed in WO9931251, WO9712622, WO9817815, WO9817816, and WO9818934, each of which is incorporated herein by reference in its entirety.

[0146] Other vectors include plasmid vectors. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the past few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because they cannot replicate or integrate into the host genome. However, these plasmids with a promoter compatible with the host cell can express peptides from genes operably encoded within 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, Calif. Additionally, plasmids can be custom designed using standard molecular biology techniques to remove and / or add specific segments of DNA.

[0147] V. Cell In some aspects, provided herein are cells comprising any of the polynucleotides described herein (e.g., comprising multiple linked coding regions). In some aspects, the cells described herein have been modified to comprise a polynucleotide described herein, such that the cell expresses antigens encoded by the multiple coding regions. As set forth herein, such antigens are simultaneously expressed in the cell. For example, in some aspects, the cells described herein have been modified with a polynucleotide comprising at least a first coding region and a second coding region, wherein the first coding region and the second coding region are linked. Such modified cells express both the antigen of the first coding region and the antigen of the second region. In some aspects, the cells do not naturally express the multiple antigens, such that the cells express the multiple antigens only after a polynucleotide described herein is introduced into the cells. In some aspects, the cells naturally express one or more of the multiple antigens, but after the cells are modified to comprise a polynucleotide described herein, expression of one or more of the multiple antigens is increased.

[0148] Unless otherwise indicated, any of the polynucleotides 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 into cells include transfection (also known as transformation and transduction), electroporation, non-viral delivery, viral transduction, lipid nanoparticle delivery, and combinations thereof. As stated herein, in some embodiments, 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 a cell passes through a constriction, the cell undergoes a transient deformation such that the cell's plasma membrane is perturbed. The perturbation in the plasma membrane can allow various payloads (e.g., polynucleotides comprising multiple linked coding regions) to enter the cell through the perturbation (e.g., through diffusion). The specific process by which a cell passes through a constriction and is transiently deformed is referred to herein as "aperture processing," "aperture delivery," or "aperture."

[0149] Thus, in some aspects, the present disclosure provides cells modified to simultaneously express multiple antigens not naturally expressed by the cell (e.g., cancer antigens and / or non-self antigens, e.g., as described herein), wherein the cell has passed through a constriction under a set of parameters, thereby causing a perturbation within the cell such that a polynucleotide enters the cell through the perturbation upon contact with the cell, and the polynucleotide comprises multiple linked coding regions encoding the multiple antigens. For example, in some aspects, the cells provided herein simultaneously express at least a first antigen, a second antigen, and a third antigen, wherein the first antigen, the second antigen, and the third antigen are not naturally expressed by the cell, and the cell has passed through a constriction under a set of parameters, thereby causing a perturbation within the cell such that a polynucleotide enters the cell through the perturbation upon contact with the cell, and the polynucleotide comprises a first coding region encoding the first antigen, a second coding region encoding the second antigen, and a third coding region encoding the third antigen, wherein the first coding region, the second coding region, and the third coding region are linked.

[0150] Any suitable cell known in the art can be modified as described herein.

[0151] In some embodiments, the cell is a stem cell. As used herein, the term "stem cell" refers to a cell that has not only the ability to self-renew but also the ability to differentiate into other types of cells (e.g., neurons). In some embodiments, stem cells useful in the present disclosure include induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), tissue-specific stem cells (e.g., liver stem cells, cardiac stem cells, or neural stem cells), mesenchymal stem cells, hematopoietic stem cells (HSCs), or combinations thereof. In some embodiments, the stem cell is an iPSC.

[0152] In some embodiments, 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 becomes 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 embodiments, somatic cells useful in the present disclosure include blood cells. In some embodiments, blood cells are peripheral blood mononuclear cells (PBMCs). As used herein, "PBMCs" refers to any peripheral blood cell with a round nucleus. In some embodiments, PBMCs include immune cells. As used herein, the term "immune cell" refers to any cell that plays a role in immune function. In some embodiments, immune cells include T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof. In some embodiments, blood cells are red blood cells. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is a cancer cell line cell, such as a HeLa cell. In some embodiments, the cancer cell is a tumor cell. In some embodiments, the cancer cell is a circulating tumor cell (CTC). In some embodiments, the cell is a fibroblast, such as a primary fibroblast or a neonatal human foreskin fibroblast (Nuff cell). In some embodiments, the cell is an immortalized cell line cell, such as a HEK293 cell or a CHO cell. In some embodiments, the cell is a skin cell. In some embodiments, the cell is a germ cell, such as an oocyte, egg, or zygote. In some embodiments, the cell is a cluster of cells, such as an embryo, given that the cluster of cells is not disrupted when passing through the pore.

[0153] VI. Composition In some aspects, the present disclosure further includes a composition comprising any of the polynucleotides, vectors, or cells described herein. In some aspects, the composition is a pharmaceutical composition. Accordingly, disclosed herein is a pharmaceutical composition comprising (i) a polynucleotide described herein (e.g., comprising at least a first coding region and a second coding region, wherein the first coding region and the second coding region are not identical and the first coding region and the second coding region are linked), and (ii) a pharmaceutically acceptable carrier. In some aspects, provided herein is a pharmaceutical composition comprising (i) a cell modified to comprise any of the polynucleotides described herein, and (ii) a pharmaceutically acceptable carrier. In some aspects, provided herein is a pharmaceutical composition comprising (i) a vector comprising any of the polynucleotides described herein, and (ii) a pharmaceutically acceptable carrier.

[0154] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound, e.g., any of the polynucleotides, vectors, or cells described herein. The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," and grammatical variations thereof, encompass any of the agents approved by a U.S. federal regulatory agency for use in animals, including humans, or listed in the U.S. Pharmacopeia, as well as any carrier or diluent that does not cause the production of undesirable physiological effects to an extent that would prohibit administration of the composition to a subject and does not abrogate the biological activity and properties of the administered compound. Included are generally safe, non-toxic, and desirable excipients and carriers that are useful in preparing pharmaceutical compositions.

[0155] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as 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) polypeptides; serum albumin, gelatin or proteins such as 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 non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0156] The pharmaceutical composition can be formulated for any route of administration to a subject. Specific examples of administration routes include intramuscular, subcutaneous, ocular, intravenous, intraperitoneal, intradermal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, or intratumoral administration. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, in solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, or glycerol. In addition, if desired, the administered pharmaceutical composition may also contain minor amounts of non-toxic auxiliary substances, pH buffering agents, stabilizers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0157] VII. Kit Also disclosed herein are kits comprising any of the polynucleotides, vectors, compositions, or cells described herein. In some embodiments, the kits are for use in immunotherapy for a disease or disorder (e.g., cancer) and / or in treating or reducing the risk of a disease or disorder (e.g., cancer). In some embodiments, the kits comprise one or more containers comprising any of the polynucleotides, vectors, compositions, or cells described herein.

[0158] In some embodiments, the kit includes instructions for use according to any of the methods described herein. For example, the included instructions can include instructions for administering a pharmaceutical composition described herein to treat, delay the onset of, or alleviate a target disease. In some embodiments, the instructions include instructions for administering a composition described herein to a subject at risk for a target disease / disorder (e.g., cancer).

[0159] In some embodiments, the instructions include dosage information, administration schedules, and routes of administration. In some embodiments, the containers are unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. In some embodiments, the instructions are written instructions on a label or package insert (e.g., a paper sheet included in the kit). In some embodiments, the instructions are machine-readable instructions (e.g., instructions transmitted on a magnetic or optical storage disk).

[0160] In some embodiments, the label or package insert indicates that the compositions disclosed herein are used for treating, delaying the onset of, and / or alleviating a cancer-related disease or disorder, such as those described herein. Instructions for practicing any of the methods described herein can be provided.

[0161] In some embodiments, the kits described herein are in suitable packaging. In some embodiments, suitable packaging includes vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), or combinations thereof. In some embodiments, the packaging includes packaging for use in combination with a specific device, such as an inhaler, a nasal administration device (e.g., an atomizer), or an infusion device such as a minipump. In some embodiments, the kit includes a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). In some embodiments, the container can also have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle).

[0162] In some aspects, the kit further comprises additional components such as buffers and interpretive information. In some aspects, the kit comprises a container and a label or package insert(s) on or associated with the container. In some aspects, the present disclosure provides an article of manufacture comprising the contents of the kit described herein.

[0163] VIII. Uses and Methods Expression of multiple antigens As is apparent from the present disclosure, the polynucleotides, vectors, cells and / or pharmaceutical compositions described herein have numerous in vitro and in vivo utilities. For example, the polynucleotides described herein can be administered to cells in culture, in vitro or ex vivo, or to a human subject, for example, in vivo, to induce expression of multiple antigens in the cells, and in some embodiments can be useful in treating a wide range of diseases or disorders.

[0164] Accordingly, some aspects of the present disclosure relate to a method for inducing expression of multiple antigens in a cell, the method comprising intracellularly delivering a polynucleotide described herein to the cell. As described and demonstrated herein, the multiple antigens are simultaneously expressed in the cell. For example, in some aspects, provided herein is a method for inducing expression of a first antigen and a second antigen in a cell, the method comprising intracellularly delivering a polynucleotide to the cell, the polynucleotide comprising a first coding region encoding the first antigen and a second coding region encoding the second antigen, wherein the first coding region and the second coding region are not identical, and the first coding region and the second coding region are linked. In some aspects, the first antigen and the second antigen are simultaneously expressed in the cell.

[0165] As described herein, in some aspects, the cells do not express the multiple antigens (e.g., the first antigen and the second antigen) before introducing the polynucleotide into the cells. In some aspects, the cells express one or more of the multiple antigens before introduction of the polynucleotide, but expression is further increased after introduction of the polynucleotide. In either situation, after introduction of the polynucleotide described herein, expression of the multiple antigens is increased compared to the reference expression. In some aspects, the reference expression includes expression in the cells before introduction of the polynucleotide. In some aspects, the reference expression includes expression in corresponding cells that have not been modified to include a polynucleotide described herein. In some aspects, the reference expression includes expression in corresponding cells that have been modified to include a plurality of polynucleotides, each of the plurality of polynucleotides encoding a distinct antigen. In some embodiments, after introduction of a polynucleotide described herein, expression of the plurality of antigens 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% compared to reference expression. In some embodiments, after introduction of a polynucleotide 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 compared to reference expression.

[0166] In some aspects, intracellular delivery of a polynucleotide to a cell comprises passing a cell suspension comprising the cells through a constriction under a set of parameters, thereby causing a perturbation within the cell such that the polynucleotide enters the cell through the perturbation upon contact with the cell. In some aspects, the method further comprises contacting the cell with the polynucleotide. As used herein, "contact" between a cell and a polynucleotide described herein does not require that the cell and the polynucleotide be in physical contact. As is apparent from the present disclosure, contact between the cell and the polynucleotide occurs as long as a perturbation in the cell membrane allows the polynucleotide to enter the cell. To aid in illustration, in some aspects, the cell and the polynucleotide are in contact when both are present in the same cell suspension, regardless of whether they are in physical contact. Thus, in some aspects, contacting the cell with the polynucleotide comprises incubating a cell suspension comprising the cells with the polynucleotide.

[0167] In some embodiments, the polynucleotides described herein (e.g., comprising multiple linked coding regions) can be delivered intracellularly alone or in combination with one or more additional cargos (also referred to herein as "payloads"). For example, in some embodiments, the additional cargo can comprise a separate polynucleotide. In some embodiments, the separate polynucleotide can encode a compound that improves and / or enhances the therapeutic effect of the polynucleotides described herein (e.g., comprising multiple 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 of the TCR to an antigenic peptide complexed with MHC; (2) "signal 2": mediated by engagement of costimulatory molecules such as 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 embodiments, a polynucleotide described herein (e.g., comprising multiple linked coding regions) is delivered into a cell in combination with one or more separate polynucleotides, where the one or more separate polynucleotides encode a costimulatory molecule (i.e., signal 2) and / or a cytokine (i.e., signal 3).

[0168] In some embodiments where multiple polynucleotides are being delivered, they may be delivered to cells using a single squeeze process, e.g., a cell suspension containing multiple polynucleotides delivered to cells in combination ("co-delivery"). In some embodiments, multiple polynucleotides (e.g., a polynucleotide described herein and a separate polynucleotide encoding a costimulatory molecule and / or cytokine) may be delivered to cells sequentially. As used herein, the term "sequential delivery" refers to the delivery of multiple polynucleotides to cells, where a first polynucleotide (e.g., comprising multiple linked coding regions) is delivered to a cell, and then a second (or subsequent) polynucleotide (e.g., a separate polynucleotide encoding a costimulatory molecule and / or cytokine) is delivered to a cell. In some embodiments, the first polynucleotide, the second polynucleotide, or both the first and second polynucleotides may be delivered to a cell using a squeeze process. For example, in some embodiments, the first polynucleotide may be delivered to a cell using a squeeze process, and the second polynucleotide may be delivered to a cell using a non-squeeze process (e.g., transfection). In some embodiments, a first polynucleotide can be delivered to a cell using a non-filtering process (e.g., transfection), and a second polynucleotide can be delivered to a cell using a filter process. In some embodiments, a first polynucleotide can be delivered to a cell using a first filter, and then a second polynucleotide can be delivered to a cell using a second filter (also referred to herein as "sequential filter" or "sequential filter process"). Thus, sequential delivery useful in the present disclosure can include multiple filter processes. In some embodiments, each of the multiple filter processes delivers a separate polynucleotide to a cell. In some embodiments, one or more of the multiple filter processes does not involve the delivery of a polynucleotide.For example, in some embodiments, the sequential delivery methods described herein include a first aperture, a second aperture, and a third aperture, where the first aperture includes passing cells through the first constriction without any payload, the second aperture includes passing cells from the first aperture to the second constriction to deliver a first polynucleotide (e.g., comprising multiple linked coding regions) to the cells, and the third aperture includes passing cells from the second aperture to the third constriction to deliver a second polynucleotide (e.g., encoding a costimulatory molecule and / or a cytokine) to the cells. Without being bound by any one theory, in some embodiments, passing cells through the first constriction without any payload (i.e., the first aperture) can serve to prepare the cells for subsequent delivery, e.g., improve the delivery efficiency of the first polynucleotide and / or the second polynucleotide.

[0169] In some embodiments, a combination of payloads (e.g., polynucleotides) can be repeatedly delivered to cells (e.g., stem cells or PBMCs). For example, in some embodiments, a combination of polynucleotides (e.g., a first polynucleotide comprising multiple linked coding regions and a second polynucleotide encoding a costimulatory molecule and / or cytokine) is delivered to cells using a first squeezing process, and then the combination of polynucleotides is again delivered to cells using a second squeezing process. In some embodiments, the first squeezing process comprises a microfluidic device (e.g., a chip) having multiple rows of constrictions, such that squeezing occurs on a single microfluidic device (e.g., a chip). As further described herein, in some embodiments, the second squeezing process can occur immediately after the cells have passed through the first squeezing process (e.g., immediately after the cells have passed through the constrictions of the first squeezing process). In some embodiments, the second squeezing process can occur some time after the first squeezing 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 cells have passed through the constriction of the first squeezing process).

[0170] constriction Microfluidic Channels As described herein, a constriction is used to cause a physical deformation of a cell such that a perturbation occurs in the cell's plasma membrane, allowing delivery of a payload (e.g., a polynucleotide described herein comprising multiple linked coding regions) into the cell. In some embodiments, 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 embodiments, the multiple channels may be arranged in parallel and / or series within the microfluidic device. In some embodiments, 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 distinct constrictions. In some embodiments, cells described herein pass through more than about 1,000 distinct constrictions. In some embodiments, multiple constrictions can be part of a single microfluidic device (e.g., a multi-row constriction chip). In some embodiments, one or more of the plurality of constrictions can be part of different microfluidic devices. For example, in some embodiments, cells (e.g., stem cells or PBMCs) described herein undergo a first constriction process, where the cells pass through a first constriction in a first microfluidic device (e.g., a chip). Then, after the cells have undergone the first constriction process (e.g., passed through the first constriction), the cells undergo a second constriction process, where the cells pass through a second constriction in a second microfluidic device (e.g., a chip). In some embodiments, each of the constrictions is the same (e.g., has the same length, width, and / or depth).In some embodiments, 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 multiple linked coding regions) and a second constriction associated with a second polynucleotide (e.g., encoding a costimulatory molecule and / or cytokine), where the cell suspension is passed through the first constriction to deliver the first polynucleotide to one or more cells of the plurality of cells, and then the cell suspension is passed through the second constriction to deliver the second polynucleotide to one or more cells of the plurality of cells. In some embodiments, the cell suspension is passed 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 has passed through the first constriction.

[0171] In some embodiments, when a cell suspension is passed through multiple constrictions (e.g., a multi-squeezing process), the cells remain viable after passing through each constriction. As will be apparent from the present disclosure, in some embodiments, the multiple constrictions can include two or more constrictions present within a single microfluidic device (e.g., a multi-squeezing chip) such that cells pass through the multiple constrictions sequentially. In some embodiments, the multiple constrictions are part of separate microfluidic devices, such that a first constriction is associated with the first microfluidic device and a second constriction is associated with the second microfluidic device. For example, in some embodiments, cells pass through a first constriction (i.e., a first constriction) associated with a first microfluidic device (e.g., a chip). After the cells pass through the first constriction, the cells pass through a second constriction (i.e., a second constriction) associated with a second microfluidic device (e.g., a chip). In some embodiments, after passing through the first constriction, the cells are cultured in a medium before passing the cells through the second constriction. In some embodiments, 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 before passing through the second constriction. As will be apparent from the present disclosure, in some embodiments, the first and second constrictions have the same length, depth, and / or width. In some embodiments, the first and second constrictions can have different lengths, depths, and / or widths.

[0172] In some embodiments, after passing through a constriction, at least about 50%, at least about 55%, at least 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 at least about 100% of the cells remain viable. When cells pass through multiple constrictions (e.g., in a single microfluidic device or as part of separate microfluidic devices), 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 remain viable after passing through each of the multiple constrictions. Cell viability can be measured using any suitable method known in the art. In some embodiments, cell viability can be measured using a Nucleocounter NC-200, an Orflo Moxi Go II Cell Counter, or both.

[0173] Exemplary microfluidic channels containing cell-deforming constrictions for use in the methods disclosed herein are described in U.S. Publication No. 2020 / 0277566A1, U.S. Publication No. 2020 / 0332243A1, U.S. Publication No. 2020 / 0316604A1, U.S. Provisional Application No. 63 / 131,423, and U.S. Provisional Application No. 63 / 131,430, each of which is incorporated by reference herein in its entirety.

[0174] In some embodiments, the microfluidic channels (i.e., including constrictions) described herein include a lumen and are configured to allow cells suspended in a buffer solution (e.g., a cell suspension) to pass through the channel. Microfluidic channels useful in the present disclosure can be fabricated 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 embodiments, the material is silicon. Fabrication of the microfluidic channels can be performed 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 masking, or a combination thereof. In some embodiments, fabrication is performed using dry etching.

[0175] In some embodiments, microfluidic channels useful in the present disclosure include an inlet portion, a center point, and an outlet portion. In some embodiments, the cross-section of one or more of the inlet portion, center point, and / or outlet portion can vary. For example, the cross-section can be circular, oval, elongated slit, square, hexagonal, or triangular in shape.

[0176] The inlet portion defines a constriction angle. In some embodiments, by adjusting (e.g., increasing or decreasing) the constriction angle, any clogging of the constriction can be reduced or prevented. In some embodiments, the angle of the outlet portion can also be adjusted. For example, in some embodiments, the angle of the outlet portion can be configured to reduce the possibility of turbulence, which can result in non-laminar flow. In some embodiments, the walls of the inlet portion and / or outlet portion are straight. In some embodiments, the walls of the inlet portion and / or outlet portion are curved.

[0177] In some embodiments, the length, depth, and / or width of the constriction can be varied. In some embodiments, by adjusting (e.g., increasing or decreasing) the length, depth, and / or width of the constriction, the efficiency of payload delivery can be adjusted. As used herein, the term "delivery efficiency" refers to the amount of payload delivered to a cell. For example, increased delivery efficiency can occur when the total amount of payload delivered is increased.

[0178] In some embodiments, the constriction has a length of less than about 1 μm. In some embodiments, the constriction has a length of about 0 μm to about 100 μm. In some embodiments, 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 embodiments, 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 embodiments, the length of the constriction is about 10 μm. In some embodiments, the constriction has a length of about 0 μm. For example, in some embodiments, a microfluidic device (eg, a chip) useful in the present disclosure comprises a constriction resembling two points of a diamond that come together such that the length of the constriction is about 0 μm.

[0179] In some embodiments, the width of the constriction is between about 0 μm and about 10 μm, or 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 embodiments, 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 embodiments, the width of the constriction is about 3 μm to about 10 μm. In some embodiments, the width of the constriction is about 6 μm.

[0180] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, 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 embodiments, the depth of the constriction is about 5 μm to about 90 μm. In some embodiments, 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 embodiments, the depth of the constriction is about 70 μm.

[0181] In some embodiments, 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 embodiments, 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.

[0182] In some embodiments, 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 embodiments, the diameter of the constriction is smaller than the diameter of the cell, such that a deforming force is applied to the cell as it passes through the constriction, resulting in a temporary physical deformation of the cell.

[0183] Thus, in some embodiments, the diameter of the constriction (also referred to herein as "constriction size") is about 20% to about 99% of the diameter of the cell. In some embodiments, 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 cell diameter. As is apparent from the present disclosure, by adjusting (e.g., increasing or decreasing) the diameter of the constriction, the efficiency of delivery of a payload to a cell can also be adjusted.

[0184] Porous surface In some embodiments, the constrictions described herein comprise surface-contained pores. Non-limiting examples of surface-contained pores that can be used with the present disclosure are described, for example, in U.S. Publication No. 2019 / 0382796A1, which is incorporated herein by reference in its entirety.

[0185] In some embodiments, surfaces useful in the present disclosure (i.e., comprising one or more pores capable of causing physical deformation of cells when passing through the pores) can be made using any suitable material available in the art and / or can take any one of a number of forms. Non-limiting examples of such materials include synthetic or natural polymers, polycarbonate, silicon, glass, metals, alloys, cellulose nitrate, silver, cellulose acetate, nylon, polyester, polyethersulfone, polyacrylonitrile (PAN), polypropylene, PVDF, polytetrafluoroethylene, mixed cellulose esters, porcelain, ceramic, or combinations thereof.

[0186] In some embodiments, the surface comprises a filter. In some embodiments, the filter is a tangential flow filter. In some embodiments, the surface comprises a membrane. In some embodiments, the surface comprises a sponge or sponge-like matrix. In some embodiments, the surface comprises a matrix. In some embodiments, the surface comprises a tortuous path surface. In some embodiments, the tortuous path surface comprises cellulose acetate.

[0187] The surface (i.e., comprising one or more pores) disclosed herein can have any suitable shape known in the art. When the surface has a two-dimensional shape, the surface can be, but is not limited to, a circle, an ellipse, a round, a square, a star, a triangle, a polygon, a pentagon, a hexagon, a heptagon, or an octagon. In some embodiments, the surface is round. When the surface has a three-dimensional shape, in some embodiments, the surface can be, but is not limited to, a cylinder, a cone, or a cube.

[0188] As will be apparent from the present disclosure, surfaces (e.g., comprising one or more pores) useful in the present disclosure can have a variety of cross-sectional widths and thicknesses. In some embodiments, the cross-sectional width of the surface is from about 1 mm to about 1 mm. In some embodiments, the surface has a defined thickness. In some embodiments, the surface thickness is uniform. In some embodiments, the surface thickness is variable. For example, in some embodiments, certain portions of the surface are thicker or thinner than other portions of the surface. In such embodiments, the thickness of different portions of the surface can vary by from about 1% to about 90%. In some embodiments, the surface is from about 0.01 μm to about 5 mm in thickness.

[0189] The cross-sectional width of the pore can depend on the type of cell being targeted with the payload. In some embodiments, the pore size is a function of the cell diameter of the targeted cell cluster. In some embodiments, the pore size is such that the cells are perturbed (i.e., physically deformed) as they pass through the pore. In some embodiments, the pore size is smaller than the cell diameter. In some embodiments, the pore size is about 20% to about 99% of the cell diameter. In some embodiments, the pore size 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 embodiments, 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 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.

[0190] The inlet and outlet of the pores can have various angles. In some embodiments, adjusting (e.g., increasing or decreasing) the pore angle can reduce or prevent any clogging of the pores. In some embodiments, the flow rate (i.e., the rate at which cells or cell-containing suspensions pass through the pores) is about 0.001 mL / cm / sec to about 100 L / cm / sec. For example, the angle of the inlet or outlet portion can be about 0 degrees to about 90 degrees. In some embodiments, the pores have the same inlet and outlet angles. In some embodiments, the pores have different inlet and outlet angles. In some embodiments, the pore edges are smooth, e.g., rounded or curved. As used herein, a "smooth" pore edge has a continuous, flat, and uniform surface without protrusions, ridges, or irregularities. In some embodiments, the pore edges are sharp. As used herein, a "sharp" pore edge has a thin edge that is pointed or has an acute angle. In some embodiments, the pore passage is straight. As used herein, a "straight" pore passage does not contain curves, bends, angles, or other irregularities. In some embodiments, the pore passage is curved. As used herein, a "curved" pore passage is bent or deviates from a straight line. In some embodiments, the pore passage has multiple curves, for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more curves.

[0191] The pores can have any shape known in the art, including two-dimensional or three-dimensional shapes. The pore shape (e.g., cross-sectional shape) can be, but is not limited to, circular, elliptical, round, square, star-shaped, triangular, polygonal, pentagonal, hexagonal, heptagonal, and octagonal. In some embodiments, the cross-section of the pore is round. In some embodiments, the three-dimensional shape of the pore is cylindrical or conical. In some embodiments, the pores have grooved inlet and outlet shapes. In some embodiments, the pore shape is uniform (i.e., consistent or regular) among the pores within a given surface. In some embodiments, the pore shape is non-uniform (i.e., mixed or varying) among the pores within a given surface.

[0192] Surfaces useful in the present disclosure can have a single pore. In some embodiments, surfaces useful in the present disclosure include multiple pores. In some embodiments, the pores comprise from about 10% to about 80% of the total surface area of ​​the surface. In some embodiments, the surface has a pore size of about 1.0 x 10 5 ~Approx. 1.0×10 30 In some embodiments, the surface contains mm 2 per surface area of ​​approximately 10 to 1.0 × 10 15 It contains pores.

[0193] Pores can be distributed in a number of ways within a given surface. In some embodiments, the pores are distributed parallel within a given surface. In some embodiments, the pores are distributed side by side in the same direction and spaced the same distance apart within a given surface. In some embodiments, the distribution of pores is ordered or uniform. In such embodiments, the pores can be distributed in a regular, systematic pattern or spaced the same distance apart within a given surface. In some embodiments, the distribution of pores is random or non-uniform. For example, in some embodiments, the pores are distributed in an irregular, unordered pattern or spaced different distances apart within a given surface.

[0194] In some embodiments, multiple surfaces are used such that cells pass through multiple pores, with the pores being on different surfaces. In some embodiments, the multiple surfaces are distributed in series. The multiple surfaces can be uniform or non-uniform in surface size, shape, and / or roughness. The multiple surfaces can further contain pores with uniform or non-uniform pore size, shape, and / or number, thereby enabling simultaneous delivery of a wide range of payloads to different cell types.

[0195] In some embodiments, for example, individual pores of a surface that may be used with the present disclosure have a uniform width dimension (i.e., a constant width along the length of the pore passage). In some embodiments, individual pores have a variable width (i.e., a width that increases or decreases along the length of the pore passage). In some embodiments, pores within a given surface have the same individual pore depth. In some embodiments, pores within a given surface have different individual pore depths. In some embodiments, pores are directly adjacent to one another. In some embodiments, pores are separated from one another by a distance. In some embodiments, pores are separated from one another by a distance of about 0.001 μm to about 30 mm.

[0196] In some embodiments, the surface is coated with a material. The material can be selected from any material known in the art, including, but not limited to, Teflon, adhesive coatings, surfactants, proteins, adhesion molecules, antibodies, anticoagulants, factors that regulate cell function, nucleic acids, lipids, carbohydrates, transmembrane proteins, or combinations thereof. In some embodiments, the surface is coated with polyvinylpyrrolidone. In some embodiments, the material is covalently bound to the surface. In some embodiments, the material is non-covalently bound to the surface. In some embodiments, the surface molecules are released when cells pass through the pores.

[0197] In some embodiments, the surface has modified chemical properties. In some embodiments, the surface is hydrophilic. In some embodiments, the surface is hydrophobic. In some embodiments, the surface is charged. In some embodiments, the surface is positively and / or negatively charged. In some embodiments, the surface may be positively charged in some areas and negatively charged in other areas. In some embodiments, the surface has an overall positive or overall negative charge. In some embodiments, the surface may be any one of a smooth surface, an electropolished surface, a roughened surface, or a plasma-treated surface. In some embodiments, the surface comprises a zwitterion or dipolar compound. In some embodiments, the surface is plasma-treated.

[0198] In some embodiments, the surface is contained within a larger module. In some embodiments, the surface is contained within a syringe, such as a plastic or glass syringe. In some embodiments, the surface is contained within a plastic filter holder. In some embodiments, the surface is contained within a pipette tip.

[0199] Cellular perturbation As described herein, when a cell passes through a constriction, it is physically deformed, resulting in the presence of a perturbation (e.g., a hole, a crevice, a cavity, an opening, a pore, a break, a gap, a perforation) in the cell's plasma membrane. Such a perturbation in the plasma membrane is temporary and sufficient to deliver any of the payloads described herein (e.g., a polynucleotide comprising multiple linked coding regions) to the cell. Cells have self-repair mechanisms that allow them to repair disruptions to their plasma membrane. See Blazek et al., Physiology (Bethesda) 30(6):438-48 (Nov. 2015), which is incorporated herein by reference in its entirety. Thus, in some embodiments, when a cell passes through a constriction (e.g., a microfluidic channel or a pore), the perturbation in the plasma membrane can be reduced or eliminated so that the payload delivered to the cell does not exit the cell.

[0200] In some embodiments, the perturbation in the cell membrane is about 1.0×10 after the pressure is removed (e.g., the cell passes through a constriction). -9 In some embodiments, the cellular perturbation lasts from about 1.0 x 10 seconds to about 2 hours. -9 In some embodiments, the cellular perturbation lasts from about 1.0 x 10 seconds to about 1 second, from about 1 second to about 1 minute, or from about 1 minute to about 1 hour. -9 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -2 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -3 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -4seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -5 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -6 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -7 seconds, or approximately 1.0 x 10 -9 seconds ~ approx. 1.0×10 -8 In some embodiments, the cellular perturbation lasts for about 1.0 x 10 seconds. -8 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -7 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -6 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -5 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -4 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -3 seconds ~ approx. 1.0×10 -1 seconds, or approximately 1.0 x 10 -2 seconds ~ approx. 1.0×10 -1 The cellular perturbations (e.g., pores or holes) produced by the methods described herein are not formed as a result of assembly of polypeptide subunits to form multimeric pore structures such as those produced by complement or bacterial hemolysin.

[0201] In some embodiments, as cells pass through the constriction, the pressure applied to the cells temporarily damages the cell membrane, which causes passive diffusion of material through the perturbation. In some embodiments, cells are deformed or perturbed for a short period of time, e.g., about 100 μs or less, to minimize the possibility of activating apoptotic pathways through cell signaling mechanisms, although other durations (e.g., ranging from nanoseconds to several hours) are possible. In some embodiments, cells are deformed or perturbed for a short period of time, e.g., about 1.0 × 10 -9 In some embodiments, the cells are transformed in a volume of about 1.0 x 10 -9 In some embodiments, the cells are deformed for less than about 1.0 x 10 seconds to less than about 1 second, less than about 1 second to less than about 1 minute, or less than about 1 minute to less than about 1 hour.-9 In some embodiments, the cells are transformed at a concentration of about 1.0 x 10 -9 In some embodiments, the cells are deformed for about 1.0 x 10 seconds to about 1 second, about 1 second to about 1 minute, or about 1 minute to about 1 hour. -9 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -2 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -3 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -4 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -5 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -6 seconds, approximately 1.0×10 -9 seconds ~ approx. 1.0×10 -7 seconds, or approximately 1.0 x 10 -9 seconds ~ approx. 1.0×10 -8 In some embodiments, the cells are deformed for any one of about 1.0 x 10 seconds. -8 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -7 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -6 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -5 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -4 seconds ~ approx. 1.0×10 -1 seconds, approximately 1.0×10 -3 seconds ~ approx. 1.0×10 -1 seconds, or approximately 1.0 x 10 -2 seconds ~ approx. 1.0×10 -1 In some embodiments, deforming the cells includes deforming the cells for a time ranging from about 1 μs to at least about 750 μs, e.g., but not limited to, 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.

[0202] In some embodiments, delivery of a polynucleotide described herein (e.g., comprising multiple linked coding regions) to a cell occurs simultaneously with the cell passing through the constriction. In some embodiments, delivery of the polynucleotide to a cell can occur after the cell has passed through the constriction (i.e., while the cell membrane perturbation is still present and before the cell's cell membrane is restored). In some embodiments, delivery of the polynucleotide to a cell occurs about several minutes after the cell has passed through the constriction. In some embodiments, the perturbation in the cell after the cell has passed through the constriction is corrected within about 5 minutes after the cell has passed through the constriction.

[0203] In some embodiments, the viability of cells (e.g., stem cells or PBMCs) after passing through the stenosis is between about 5% and about 100%. In some embodiments, the viability of cells after passing through the stenosis is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least 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 embodiments, the viability of cells is between about 1.0 x 10 cells passing through the stenosis. -2 The viability of cells is measured after 2 seconds to at least about 10 days. For example, the viability of cells is measured after approximately 1.0 x 10 cells have passed through the constriction. -2 The cell viability can be measured after about 1.0 x 10 cells have passed through the constriction. -2 seconds to approximately 2 hours, approximately 1.0 x 10 -2 seconds to approximately 1 hour, approximately 1.0 x 10 -2 seconds to approximately 30 minutes, approximately 11.0×10 -2 seconds to approximately 1 minute, approximately 1.0×10 -2 seconds ~ approx. 30 seconds, approx. 1.0 x 10 -2 seconds to approximately 1 second, or approximately 1.0 x 10 -2In some embodiments, cell viability is measured 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 cells have passed through the constriction. In some embodiments, cell viability is measured 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 cells have passed through the constriction.

[0204] Delivery Parameters As is apparent from the present disclosure, numerous parameters can affect the efficiency of delivery of a polynucleotide described herein (e.g., comprising multiple linked coding regions) to cells using the squeezing methods provided herein. Thus, adjusting (e.g., increasing or decreasing) one or more of the delivery parameters can improve delivery of a payload to cells. Thus, in some aspects, the present disclosure relates to a method of increasing delivery of a payload (e.g., a polynucleotide described herein) to cells, the method comprising adjusting one or more parameters through which a cell suspension passes through a constriction, the cell suspension comprising a population of cells, wherein the one or more parameters increase delivery of the payload to one or more cells of the population of cells compared to reference parameters. As described elsewhere in this disclosure, the payload can be contacted with the population of cells before, during, or after the squeezing step.

[0205] In some embodiments, by adjusting one or more of the 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 the reference parameters.

[0206] In some embodiments, one or more delivery parameters that can be adjusted to increase the efficiency of delivery of the parameters include cell density (i.e., the concentration of cells present in, for example, a cell suspension), pressure, or both. Additional examples of delivery parameters that can be adjusted are provided elsewhere in this disclosure.

[0207] In some embodiments, the cell density is about 1 x 10 7 cells / mL, approximately 2×10 7 cells / mL, approximately 3×10 7 cells / mL, approximately 4×10 7 cells / mL, approximately 5×10 7 cells / mL, approximately 6×10 7 cells / mL, approximately 7×10 7 cells / mL, approximately 8×10 7 cells / mL, approximately 9×10 7 cells / mL, approximately 1×10 8 cells / mL, approximately 1.1×10 8 cells / mL, approximately 1.2×10 8 cells / mL, approximately 1.3×10 8 cells / mL, approximately 1.4×10 8 cells / mL, approximately 1.5×10 8 cells / mL, approximately 2.0×10 8 cells / mL, approximately 3.0×10 8 cells / mL, approximately 4.0×10 8 cells / mL, approximately 5.0×10 8 cells / mL, approximately 6.0×10 8 cells / mL, approximately 7.0×10 8 cells / mL, approximately 8.0×10 8 cells / mL, approximately 9.0×10 8 cells / mL or approximately 1.0 x 10 9 In some embodiments, the cell density is about 6 x 10 cells / mL or more. 7 cells / mL ~ approx. 1.2×10 8 cells / mL.

[0208] In some embodiments, the pressure is about 20 psi, about 25 psi, about 30 psi, about 35 psi, about 40 psi, about 50 psi, about 55 psi, about 60 psi, about 65 psi, about 70 psi, about 75 psi, about 80 psi, about 85 psi, about 90 psi, about 95 psi, about 100 psi, about 110 psi, about 120 psi, about 130 psi, about 140 psi, about 150 psi, about 160 psi, about 170 psi, about 180 psi, about 190 psi, or about 200 psi, or more. In some embodiments, the pressure is from about 30 psi to about 90 psi.

[0209] In some embodiments, the specific type of device (e.g., a microfluidic chip) can also affect the efficiency of delivery of a payload (e.g., a polynucleotide) described herein. In the case of a microfluidic chip, different chips can have different constriction parameters, such as the length, depth, and width of the constriction; the entrance angle, exit angle, length, depth, and width of the approach region, etc. As described herein, such variables can affect the delivery of a payload to a cell using the constriction processing methods of the present disclosure. In some embodiments, the length of the constriction is up to 100 μm. For example, in some embodiments, 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 embodiments, the length of the constriction is less than 1 μm. In some embodiments, the length of the stenosis 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 embodiments, the stenosis has a length of about 10 μm.

[0210] In some embodiments, the width of the constriction is up to about 10 μm. In some embodiments, 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 embodiments, the width is between about 3 μm and about 10 μm. In some embodiments, 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 embodiments, the width of the constriction is about 6 μm.

[0211] In some embodiments, the depth of the constriction is at least about 1 μm. In some embodiments, 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 embodiments, the depth is about 5 μm to about 90 μm. In some embodiments, 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 embodiments, the depth of the constriction is about 70 μm.

[0212] In some embodiments, the length is about 10 μm, the width is about 6 μm, and the depth is about 70 μm.

[0213] Additional examples of parameters that can affect the delivery of a payload to cells include, but are not limited to, the dimensions of the constriction (e.g., length, width, and / or depth), the entrance angle of the constriction, the surface characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity), the actuation flow rate, the payload concentration, the amount of time the cells are allowed to recover, or a combination thereof. Further parameters that can affect the efficiency of delivery of a payload (e.g., a polynucleotide described herein) can include the velocity of the cells in the constriction, the shear rate in the constriction, the viscosity of the cell suspension, the velocity component perpendicular to the flow rate, and the time in the constriction. Such parameters can be designed to control the delivery of the payload.

[0214] In some embodiments, the temperature used in the disclosed methods can affect the efficiency of delivery of the payload to the cells, as well as cell viability. In some embodiments, the squeeze process is performed at about -5°C to about 45°C. For example, the method can be performed at room temperature (e.g., about 20°C), physiological temperature (e.g., about 37°C), higher than physiological temperature (e.g., about 37°C to 45°C or higher), or lower temperature (e.g., about -5°C to about 4°C), or temperatures between these exemplary temperatures.

[0215] Various methods can be used to force cells through the constriction. For example, pressure can be applied by a pump (e.g., a gas cylinder or compressor) on the inlet side, vacuum can be applied by a vacuum pump on the outlet side, capillary action can be applied through tubing, and / or the system can be gravity-fed. Displacement-based flow systems (e.g., syringe pumps, peristaltic pumps, manual syringes or pipettes, pistons, etc.) can also be used. In some embodiments, cells are forced through the constriction by positive pressure. In some embodiments, cells are forced through the constriction by constant or variable pressure. In some embodiments, pressure is applied using a syringe. In some embodiments, pressure is applied using a pump. In some embodiments, the pump is a peristaltic pump or a diaphragm pump. In some embodiments, pressure is applied using a vacuum. In some embodiments, cells are forced through the constriction by g-force. In some embodiments, cells are forced through the constriction by capillary pressure.

[0216] In some aspects, the fluid flow guides the cells through the constriction. In some aspects, the fluid flow is turbulent before the cells pass through the constriction. Turbulent flow is 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. Laminar flow involves uninterrupted flow in a fluid close to a solid boundary, where the direction of flow remains constant at all points. In some aspects, the fluid flow is turbulent after the cells pass through the constriction. The velocity at which the cells pass through the constriction can vary. In some aspects, the cells pass through the constriction at a uniform cell velocity. In some aspects, the cells pass through the constriction at fluctuating cell velocities.

[0217] In some embodiments, a combination treatment, such as a method described herein, followed by exposure to an electric field downstream of the constriction is used to deliver a payload. In some embodiments, after passing through the constriction, the cells pass through an electric field generated by at least one electrode. In some embodiments, the electric field assists in delivering the payload to a second location within the cell, such as the cell nucleus. In some embodiments, one or more electrodes are in close proximity to the cell-deforming constriction to generate the electric field. In some embodiments, the electric field is about 0.1 kV / m to about 100 MV / m. In some embodiments, an integrated circuit is used to provide the electrical signal to drive the electrodes. In some embodiments, the cells are exposed to the electric field with a pulse width of about 1 ns to about 1 s and a duration of about 100 ns to about 10 s.

[0218] Induction of immune responses As is apparent from the present disclosure, the polynucleotides described herein (e.g., comprising multiple linked coding regions) can be useful for inducing an immune response in a subject in need thereof. For example, as set forth herein, because the polynucleotides described herein comprise multiple linked coding regions, administering a polynucleotide of the present disclosure to a subject can result in the induction of an immune response against one or more of the antigens encoded by the multiple coding regions. Accordingly, in some aspects, provided herein are methods for inducing a multi-antigen-specific immune response in a subject in need thereof, comprising administering to the subject a polynucleotide comprising multiple linked coding regions, wherein the multiple coding regions encode multiple antigens. In some aspects, the method comprises administering any of the polynucleotides of the present disclosure. In some aspects, the method comprises administering to the subject a 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 linked, and following administration, an immune response against both the first antigen and the second antigen is induced in the subject. In some aspects, the methods can include administering to a subject cells that have been modified to contain a polynucleotide such that the cells express multiple antigens.

[0219] In some embodiments, after administration, the immune response to the first antigen is increased compared to the immune response in a reference subject. In some embodiments, the reference subject includes the subject before administration. In some embodiments, the reference subject includes a corresponding subject who did not receive the polynucleotide (i.e., comprising at least a first coding region encoding the first antigen and a second coding region encoding the second antigen, wherein the first coding region and the second coding region are linked). In some embodiments, the reference subject includes a corresponding subject who received at least two separate polynucleotides, wherein the first antigen and the second antigen are encoded on separate polynucleotides. In some embodiments, the immune response to the first antigen in the subject after administration 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% compared to the reference subject. In some embodiments, the immune response to the first antigen in the subject 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 ultimately by about 50-fold or more compared to the reference subject.

[0220] In some embodiments, after administration, the immune response to the second antigen is increased compared to the immune response in the reference subject. In some embodiments, compared to the reference subject, the immune response to the second antigen in the subject after administration 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%. In some embodiments, compared to the reference subject, the immune response to the second antigen in the subject 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 ultimately by more than about 50-fold.

[0221] In some embodiments, after administration, the immune response to both the first antigen and the second antigen is increased compared to the immune response in a reference subject. In some embodiments, the immune response to both the first antigen and the second antigen in the subject after administration 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% compared to the reference subject. In some embodiments, the immune response to the first antigen and the second antigen in the subject 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 ultimately by more than about 50-fold compared to the reference subject.

[0222] Thus, in some aspects, provided herein are methods for inducing an enhanced immune response in a subject in need thereof. In some aspects, such methods include administering to the subject a polynucleotide comprising multiple coding regions, wherein the multiple coding regions are linked and the multiple coding regions encode multiple antigens. In some aspects, the methods can include administering to the subject a cell that has been modified to include the polynucleotide such that the cell expresses the multiple antigens.

[0223] In some embodiments, an enhanced immune response includes (i) an increase in the magnitude of the induced immune response compared to a reference immune response, (ii) an increase in the breadth of the induced immune response compared to a reference immune response, (iii) an increase in the duration of the induced immune response compared to a reference immune response, or (iv) any combination of (i)-(iii). As described herein, in some embodiments, the reference subject includes a subject prior to administration. In some embodiments, the reference subject includes a corresponding subject who did not receive administration of a polynucleotide (i.e., comprising multiple coding regions linked together and encoding multiple antigens). In some embodiments, the reference subject includes a corresponding subject who received administration of multiple polynucleotides, each of the multiple antigens being encoded on a separate polynucleotide.

[0224] In some embodiments, 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% compared to the reference subject. In some embodiments, the magnitude of the induced immune response 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 ultimately by about 50-fold or more compared to the reference subject.

[0225] In some embodiments, 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% compared to the reference subject. In some embodiments, the breadth of the induced immune response 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 ultimately by more than about 50-fold compared to the reference subject.

[0226] In some embodiments, 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% compared to the reference subject. In some embodiments, the duration of the induced immune response 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 ultimately by about 50-fold or more compared to the reference subject.

[0227] In some aspects, both the magnitude and breadth of the induced immune response are increased in the subject compared to the reference subject. In some aspects, both the magnitude and duration of the induced immune response are increased in the subject compared to the reference subject. In some aspects, both the breadth and duration of the induced immune response are increased in the subject compared to the reference subject. In some aspects, the magnitude, breadth, and duration of the induced immune response are increased in the subject compared to the reference subject.

[0228] As will become apparent from the present disclosure, the enhanced immune response induced by the polynucleotides described herein, or by cells modified to contain the polynucleotides, can be useful in treating a wide range of diseases or conditions. In some aspects, provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject any of the polynucleotides disclosed herein. Some aspects of the present disclosure relate to methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a 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 linked, and the first antigen and the second antigen are associated with the disease or condition. Thus, it will be apparent 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 condition associated with a particular antigen. Without being bound by any one theory, an immune response against multiple antigens can be induced by encoding multiple antigens associated with a disease or condition within multiple coding regions of the polynucleotides described herein. In some embodiments, such immune responses can be useful in treating diseases or conditions. For example, in some embodiments, 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.

[0229] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0230] Example 1: Analysis of antigen expression using five linked antigens To determine whether immune cells expressing a single mRNA encoding five antigen fragments linked together could simultaneously induce multiple antigen-specific immune responses, human PBMCs were transfected with mRNA encoding HPV16 E6, HPV16 E7, CMV pp65, and KRAS. G12V and KRAS G12D Three different linked antigen mRNA constructs were developed, each encoding approximately 25 amino acid fragments of the antigen (see Figure 1A). The five antigen fragments in each mRNA construct were linked in a different order than in the other two mRNA constructs. Presentation of immunogenic epitopes from these encoded antigens on MHC class I cells was evaluated using antigen-specific responder cells.

[0231] KRAS G12V Specific TCR, E6 29-38 Specific TCR or E7 11-19 We transduced Jurkat-Lucia NFAT cells (InvivoGen) in which the endogenous TCRα / β was knocked out with either the E6 T cell receptor (TCR), E7 TCR, or KRAS specific TCR-expressing lentivirus. G12V TCR Jurkat-Lucia NFAT reporter cells were generated. Engagement of these three TCRs with the corresponding peptide-MHC (pMHC) complexes leads to activation of the NFAT signaling pathway and secretion of Lucia luciferase.

[0232] PBMCs from a human HLA-A*02+HLA-A*11+ donor were cultured at 5 × 10 7 Cells were prepared at a density of 1000 cells / mL and squeezed at 60 psi through a 3.5 μm wide, 10 μm long, and 70 μm deep constriction in RPMI 1640 medium at room temperature with 250 μg / mL of mRNA constructs encoding five linked antigen fragments (5xL1, 5xL2, or 5xL3) or without cargo (empty squeeze). The squeezed PBMCs were transferred to RPMI + 10% fetal bovine serum + 1x Pen / Strep (R10) and quenched at room temperature. The squeezed PBMCs were washed twice in co-culture medium (R10) and then resuspended in fresh co-culture medium.

[0233] Next, 2.5 x 10 5 1.25 x 10 squeeze-filled PBMCs in a 96-well plate 5 E6 TCR Jurkat cells or 5 x 10 4 These were then placed in co-culture with 5 x 10 E7 TCR Jurkat cells. 5 2.5 x 10 squeeze-filled PBMCs in a 96-well plate 5 KRAS G12V As a positive control, 1 μM E6 was added to the co-culture with TCR Jurkat cells. 29-38 , E7 11-19 or KRAS G12V The (7-16) minimal epitope was directly added to co-cultures of naive PBMCs and relevant Jurkat responder cells. The co-cultures were incubated at 37°C for 16–18 h, after which the co-culture supernatants were harvested. The level of secreted lucia luciferase in the culture supernatants was measured via luminescence after the addition of coelenterazine substrate.

[0234] To measure the response to the pp65 fragment in the concatenated antigen mRNA, 2.5 x 10 5 6.25 x 10 squeeze-filled PBMCs in a 96-well plate 4 As a positive control, 1 μM pp65 was placed in co-culture with CMV pp65-specific T cells (Cellero). 495-503 The minimal epitope was directly added to cocultures of naive PBMCs and CMV pp65-specific T cells. The cocultures were incubated at 37°C for 16–18 hours, after which the coculture supernatants were collected. To assess pp65-specific T cell activation, IFNγ concentrations in the coculture supernatants were measured using an Ella immunoassay instrument (Bio-Techne) according to the manufacturer's protocol.

[0235] As shown in Figure 1B, CMV pp65-specific T cells were stimulated with the ligated antigen mRNA and expressed KRAS G12VCo-culture of human PBMCs with either TCR Jurkat cells, E6 TCR Jurkat cells, or E7 TCR Jurkat cells resulted in increased activation of relevant responder cells compared with empty-pressure controls. These results demonstrate that human PBMCs pressurized with linked antigen mRNA can simultaneously elicit immune responses against multiple antigen fragments.

[0236] Example 2: Analysis of antigen expression using 10 linked antigens To determine whether immune cells expressing a single mRNA encoding up to 10 antigen fragments linked together could simultaneously elicit multiple antigen-specific immune responses, human PBMCs were cultured with mRNA encoding HPV16 E6, HPV16 E7, CMV pp65, and KRAS. G12V , KRAS G12D Two different concatenated antigen mRNA constructs were developed, each encoding approximately 25 amino acid fragments of the following antigens: Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S, and MAGE-A10. The 10 antigen fragments in each mRNA construct were concatenated in a different order than the others (see Figure 2A). Presentation of immunogenic epitopes from these encoded antigens on MHC class I cells was assessed using antigen-specific responder cells.

[0237] E6 29-38 Specific TCR or E7 11-19 E6 and E7 TCR Jurkat-Lucia NFAT reporter cells were generated by transducing either specific TCR-expressing lentiviruses into Jurkat-Lucia NFAT cells (InvivoGen) in which the endogenous TCRα / β was knocked out. Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes leads to activation of the NFAT signaling pathway and secretion of lucia luciferase.

[0238] Human HLA-A*02 + HLA-A*11 + 5 x 10 PBMCs from donors 7PBMCs were prepared at a density of 1000 cells / mL and squeezed at 60 psi through a 3.5 μm wide, 10 μm long, and 70 μm deep constriction in RPMI 1640 medium at room temperature with 250 μg / mL of mRNA constructs encoding 10 antigen fragments linked together (10×L1 or 10×L2), 250 μg / mL of mRNA constructs encoding 5 antigen fragments linked together (5×L3), or no cargo (empty squeeze). The squeezed PBMCs were transferred to RPMI + 10% fetal bovine serum + 1× Pen / Strep (R10) and quenched at room temperature. The squeezed PBMCs were washed twice in co-culture medium (R10) and then resuspended in fresh co-culture medium.

[0239] Next, 2.5 x 10 5 1.25 x 10 squeeze-filled PBMCs in a 96-well plate 5 E6 TCR Jurkat cells or 5 x 10 4 As a positive control, 1 μM E6 29-38 or E7 11-19 The minimal epitope was directly added to co-cultures of naive PBMCs and relevant Jurkat responder cells. The co-cultures were incubated at 37°C for 16–18 hours, after which the co-culture supernatants were harvested. The level of secreted lucia luciferase in the culture supernatants was measured via luminescence after the addition of coelenterazine substrate.

[0240] Next, 2.5 x 10 5 6.25 x 10 squeeze-filled PBMCs in a 96-well plate 4 As a positive control, 1 μM of M1 was placed in co-culture with CMV pp65, M1 Flu, NY-ESO-1, or MAGE-A10-specific T cells (Cellero). 58-66 or pp65 495-503The minimal epitope was added directly to co-cultures of naive PBMCs and the relevant antigen-specific T cells. The co-cultures were incubated at 37°C for 16–18 h, after which the co-culture supernatants were collected. To assess antigen-specific T cell activation, IFNγ concentrations in the co-culture supernatants were measured using an Ella immunoassay instrument (Bio-Techne) according to the manufacturer's protocol.

[0241] As shown in Figures 2B and 2C, human PBMCs were squeezed with mRNA constructs encoding 10 linked antigen fragments and cocultured with either 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, resulting in increased activation of relevant responder cells compared with empty squeeze controls. These results demonstrate that human PBMCs squeezed with linked antigen mRNAs can simultaneously elicit immune responses against multiple antigen fragments.

[0242] Example 3: In vitro translation efficiency of mRNA containing linked antigens Five antigen fragments linked together (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V and KRAS G12D ) or 10 antigen fragments linked together (HPV16 E6, HPV16 E7, CMV pp65, KRAS G12V , KRAS G12D To evaluate the in vitro translation efficiency of mRNA constructs encoding the antigens (Flu M1, NY-ESO-1, HSV gD, SARS-CoV2 S, and MAGE-A10), each linked mRNA construct was individually mixed with wheat germ extract, a cell-free expression system (see Figure 3A).

[0243] TnT® SP6 High-Yield Wheat Germ Protein Master Mix (Promega) was thawed and immediately placed on ice. Each ligated antigen mRNA construct was diluted with diH2O to a final concentration of 1 μg / μL. For each ligated antigen mRNA construct, 2 μg of mRNA was added to 30 μL of wheat germ extract, brought to a total volume of 50 μL with diH2O, and the reaction was incubated at 25°C for 2 hours. After the 2-hour incubation, the sample was placed on ice to terminate the reaction, and 50 μL of the translation sample was mixed with 25 μL of sample buffer.

[0244] For SDS-PAGE gels, samples were heated at 95°C for 2 minutes and then loaded onto a 4-12% protein gel (Invitrogen). The samples were run on the gel at 100-130V for approximately 90 minutes until the dye reached the bottom of the gel. The gel was transferred onto a nitrocellulose membrane, and HPV E6 29-38 The linked antigens were blotted 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 for the mRNA construct encoding five linked antigen fragments and approximately 35 kDa for the mRNA construct encoding ten linked antigen fragments.

[0245] As shown in Figure 3B, translation of each linked antigen mRNA construct was detected at various levels. Of the three mRNA constructs, each encoding five linked model antigen fragments, construct 5xL3 was the most highly translated, followed by 5xL2 and finally 5xL1. Of the two mRNA constructs, each encoding ten linked model antigen fragments, 10xL2 was more highly translated than 10xL1.

[0246] Example 4: Induction of KRAS mutant-specific immune responses Immune cells conditioned with a single mRNA encoding five antigen fragments linked together express KRAS G12V and KRAS G12DTo determine whether antigen-specific immune responses can be simultaneously induced, human PBMCs were transfected with HPV16 E6, HPV16 E7, CMV pp65, and KRAS. G12V and KRAS G12D Three different linked antigen mRNA constructs were developed, each encoding approximately 25 amino acid fragments of the antigen. The five antigen fragments in each mRNA construct were linked in a different order than in the other two mRNA constructs. Presentation of immunogenic epitopes from these encoded antigens on MHC class I cells was assessed using antigen-specific responder cells.

[0247] KRAS G12V Specific TCR or KRAS G12D KRAS was expressed by transducing either a specific TCR-expressing lentivirus into Jurkat-Lucia NFAT cells (InvivoGen) in which endogenous TCRα / β was knocked out. G12D TCR and KRAS G12V TCR Jurkat-Lucia NFAT reporter cells were generated, and engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes leads to activation of the NFAT signaling pathway and secretion of Lucia luciferase.

[0248] PBMCs from a human HLA-A*02+HLA-A*11+ donor were cultured at 5 × 10 7 Cells were prepared at a density of 1000 cells / mL and squeezed at 60 psi through a 3.5 μm wide, 10 μm long, and 70 μm deep constriction in RPMI 1640 medium at room temperature with 250 μg / mL of mRNA constructs encoding five linked antigen fragments (5xL1, 5xL2, or 5xL3) or without cargo (empty squeeze). The squeezed PBMCs were transferred to RPMI + 10% fetal bovine serum + 1x Pen / Strep (R10) and quenched at room temperature. The squeezed PBMCs were washed twice in co-culture medium (R10) and then resuspended in fresh co-culture medium.

[0249] Next, 5 x 10 5 2.5 x 10 squeeze-filled PBMCs in a 96-well plate5 KRAS G12V TCR Jurkat cells or KRAS G12D TCR Jurkat cells were co-cultured with 1 μM KRAS as a positive control. G12V (7-16) or KRAS G12D (7-16) The minimal epitope was directly added to co-cultures of naive PBMCs and relevant Jurkat responder cells. The co-cultures were incubated at 37°C for 16–18 h, after which the co-culture supernatants were harvested. The level of secreted lucia luciferase in the co-culture supernatants was measured via luminescence after the addition of coelenterazine substrate.

[0250] As shown in Figure 4, the aperture was filled with the linked antigen mRNA and the KRAS G12V Co-culture of human PBMCs with TCR Jurkat cells resulted in increased activation of relevant responder cells compared to empty-pressure controls. These results demonstrate that human PBMCs pressurized with linked antigen mRNA can elicit immune responses against KRAS mutant antigens.

[0251] Example 5: Effect of signal 2 / 3 on immune response induction by mRNA containing linked antigen To measure functional CD8+ T cell responses of immune cells expressed with a single mRNA encoding five antigen fragments linked together, with or without additional signal 2 / 3 mRNA (encoding CD86, mbIL-2, and mbIL-12), human PBMCs were cultured with or without signal 2 / 3 mRNA encoding HPV16 E6, HPV16 E7, CMV pp65, KRAS, and other antigen fragments. G12V and KRAS G12D We targeted the antigens with mRNA constructs encoding approximately 25-aa fragments or with signal 2 / 3 mRNA alone. Functional CD8+ T cell responses from these antigens, with or without signal 2 / 3, were assessed using antigen-specific primary cells.

[0252] As illustrated in Figure 5A, CD8+ T cells were isolated from an HLA-A*02+ donor and activated with CD3 / CD28 Dynabeads and rhIL-2 for 2 days. 29-38 Specific TCR or E7 11-19 Cells were transduced on two consecutive days with either a specific TCR-expressing lentivirus or a specific TCR-expressing lentivirus. 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 results in the production of IFNγ.

[0253] PBMCs from the same HLA-A*02+ donor were cultured at 5 × 10 7 Cells were prepared at a density of 1000 cells / mL and squeezed at 60 psi through a 3.5 μm wide, 10 μm long, and 70 μm deep constriction in room temperature RPMI 1640 medium with 250 μg / mL of the linked antigen (L2), the linked antigen and signal 2 / 3 mRNA, signal 2 / 3 mRNA alone, or no cargo (empty squeeze). The squeezed PBMCs were transferred to RPMI + 10% fetal bovine serum + 1x Pen / Strep (R10) and quenched at room temperature. The squeezed PBMCs were washed twice in co-culture medium (R10) and then resuspended in fresh co-culture medium.

[0254] Next, 5 x 10 4 The squeeze-filled PBMCs were cultured in a 96-well plate with 0.2% E6 TCR or E7 TCR tetramer CD8 T cells and 1.28 x 10 5 After incubating the co-cultures at 37°C for 6 days, the co-cultured cells were then transfected with the corresponding E6 29-38 or E7 11-19 The cells were restimulated with peptide and Golgi block, or with Golgi block alone as a negative control, and incubated at 37°C. Six hours after restimulation, the cells were intracellularly stained and evaluated for IFNγ and TNFα expression via flow cytometry. E7 cells after 6 days of co-culture 11-29 Pentamer+ or E6 29-38 The frequency of tetramer+CD8+ T cells was also assessed via flow cytometry.

[0255] As shown in Figure 5B, human PBMCs pressure-loaded with linked antigen mRNA and signal 2 / 3 mRNA and then cocultured with E6 TCR-transduced or E7 TCR-transduced CD8+ T cells resulted in increased activation of antigen-specific CD8+ T cells compared with human PBMCs pressure-loaded with signal 2 / 3 mRNA alone. These results demonstrate that human PBMCs pressure-loaded with linked antigen mRNA can induce potent immune responses when combined with signal 2 / 3 mRNA.

[0256] Example 6: Analysis of KRAS G12V and G12D-specific responses after squeezing using mRNA encoding two KRAS mutant-linked antigens To further evaluate the functional capabilities of the polynucleotides described herein (e.g., containing multiple linked coding regions), human PBMCs were stimulated with one of the linked mRNAs described below. (1) (a) Approximately 25 aa fragment (overlapping with the 1-25 aa sequence in the entire KRAS protein and a mutation on codon 12) KRAS G12D and KRAS G12V a concatenated mRNA construct encoding the antigen, (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) ("Concatenated Antigen+2 / 3"); (2) Approximately 25 aa fragment (overlapping the 1-25 aa sequence of the entire KRAS protein and a mutation on the 12th codon) KRAS G12D and KRAS G12V a linked mRNA construct encoding only the antigen ("G12D-G12V linked antigen"); (3) 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"); (4) a concatenated mRNA construct (“G12V+2 / 3”) encoding (a) a single approximately 25 aa fragment containing 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); (5) a concatenated mRNA construct (“G12D+2 / 3”) encoding (a) a single approximately 25 aa fragment containing 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); (6) a concatenated mRNA construct (“WT+2 / 3”) 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); (7) An mRNA construct (“G12V”) encoding only a single approximately 25 aa fragment containing the G12V mutation (i.e., without signals 2 and 3); (8) an mRNA construct (“G12D”) encoding only a single approximately 25 aa fragment containing the G12D mutation (i.e., without signals 2 and 3); and (9) An mRNA construct (“wild-type”) encoding only an approximately 25-aa fragment overlapping the first 1–25 aa of native wild-type KRAS (i.e., without signals 2 and 3).

[0257] method Generation of responder cells: To generate A*11-restricted G12V and G12D TCR Jurkat responder cells, Jurkat-Lucia NFAT TCR α / β KO cells (custom-manufactured at Creative Biolabs) were transfected with A*11-restricted G12V 7-16 and G12D 7-16 After transduction with a specific TCR-expressing lentivirus, residual virus was removed, and the cells were then subjected to puromycin selection and culture for several days before undergoing mouse TCR (mTCR) enrichment. +The frequency of mTCR-enriched cells was increased. After mTCR enrichment, cells were cultured for several days, characterized by flow cytometry, and frozen for use in co-culture assays. The resulting A*11-restricted G12V TCR Jurkat and G12D TCR Jurkat responder cells expressed HLA-A*11-restricted G12V TCR Jurkat on the cell surface, respectively. 7-16 Specific TCR or HLA-A*11 restricted G12D 7-16 Specific TCRs were expressed in PBMCs either filtered with mutant KRAS mRNA or incubated with the G12V7-16 or G12D7-16 A*11-restricted peptide. Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes resulted in activation of the NFAT signaling pathway and secretion of lucia luciferase. The level of secreted lucia luciferase in the culture supernatant was measured using a coelenterazine substrate. The resulting luminescence signal correlated with the amount of secreted lucia luciferase, confirming that the cells were properly generated.

[0258] Squeeze process: HLA-A*11 + 200 x 10 PBMCs from donors 6 PBMCs were prepared at a density of 1000 cells / mL and allowed to rest for approximately 15 minutes at 4-8°C. PBMCs were then squeezed in room temperature RPMI 1640 medium with 250 μg / mL of one of the above mRNA constructs at 60 psi using a 3.5 μm wide, 10 μm long, and 70 μm deep constriction (the mRNA solution was cooled at 4-8°C for approximately 15 minutes before mixing with the cells and squeezing). PBMCs squeezed without mRNA (i.e., empty squeeze) served as one of the controls. Following squeezing, the squeezed PBMCs were transferred to R10 medium (RPMI + 10% fetal bovine serum (FBS) + 1× Pen / Strep) and quenched at room temperature. Subsequently, they were washed with R10 and diluted to 5×10 6 cells / mL and stored in liquid nitrogen for future use.

[0259] analysis: On the day of co-culture setup, 2.5 x 10 HLA-A*11 restricted G12V and G12D TCR Jurkat cells were cultured in IMDM + 10% FBS. 6 Cryopreserved, squeezed, or untreated PBMCs were thawed and cultured at 5 × 10 cells / mL. 6 The cells were then replaced with IMDM + 10% FBS at a density of 100,000 cells / mL. G12V or G12D TCR Jurkat cells were co-cultured with PBMCs at a 1:2 ratio (250,000 Jurkat cells to 500,000 PBMCs) in tissue culture-treated (TCT) 96-well U-bottom plates for 18–24 hours. HLA-A*11-restricted G12V 7-16 and G12D 7-16 or W.T. 7-16 A minimal epitope was added to Jurkat cells and co-cultures with naive PBMCs to control for antigen variant-specific Jurkat reactivity. Cell culture supernatants were collected and assayed for levels of secreted lucia luciferase via Quanti LUC.

[0260] result As shown in Figures 6A and 6B, human PBMCs squeezed with KRAS G12D-G12V-linked antigen mRNA, with or without signal 2 / 3 mRNA, and cocultured with G12V or G12D TCR-transformed Jurkat cells resulted in KRAS mutant-specific increases in activation of G12V and G12D TCR-transformed Jurkat cells compared with controls (empty squeeze, signal 2 / 3 mRNA alone, KRAS WT mRNA with or without signal 2 / 3 mRNA). PBMCs squeezed with KRAS G12D-G12V-linked antigen mRNA elicited mutant-specific responses from G12V or G12D TCR-transformed Jurkat cells that were similar to or stronger than those induced by the relevant KRAS mutant mRNA alone, respectively.

[0261] These results demonstrate that human PBMCs squeezed with KRAS mutant-linked antigen mRNA can elicit potent mutant-specific responses from G12V and G12D TCR-transduced Jurkat cells that are similar to or stronger than their respective KRAS mutant single antigen mRNAs.

[0262] Example 7: Analysis of KRAS G12V and G12D-specific responses after swabbing using mRNA encoding seven KRAS mutant-linked antigens To evaluate the functional activity of mRNA encoding more than two linked antigens, immune cells (PBMCs) were treated with an mRNA construct 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. (1) Approximately 25 aa fragment (overlapping the 1-25 aa sequence of the entire KRAS protein and a mutation on codon 12) KRAS G12D , KRAS G12V , KRAS G12C , KRAS G13D , KRAS G12A , KRAS G12R and KRAS G12S A concatenated mRNA construct encoding only the antigen ("7mut_v1"), and (2) Approximately 25 aa fragment (overlapping the 1-25 aa sequence of the entire KRAS protein and a mutation on the 12th codon) KRAS G12S , KRAS G12R , KRAS G12A , KRAS G13D , KRAS G12C , KRAS G12V and KRAS G12D A concatenated mRNA construct encoding the antigen alone (“7mut_v2”).

[0263] method Generation of responder cells: To generate A*11-restricted G12V and G12D TCR Jurkat responder cells, Jurkat-Lucia NFAT TCR α / β KO cells (custom-manufactured at Creative Biolabs) were transfected with A*11-restricted G12V 7-16 and G12D 7-16 The cells were transduced once with a specific TCR-expressing lentivirus. Residual virus was removed one day after TCR transduction, and the cells were then subjected to puromycin selection and cultured for several days before undergoing murine TCR (mTCR) enrichment. + The frequency of mTCR-enriched cells was increased. After mTCR enrichment, cells were cultured for several days, characterized by flow cytometry, and frozen for use in co-culture assays. The resulting A*11-restricted G12V TCR Jurkat and G12D TCR Jurkat responder cells expressed HLA-A*11-restricted G12V TCR Jurkat on the cell surface, respectively. 7-16 Specific TCR or HLA-A*11 restricted G12D 7-16 These express specific TCRs and are provided in the form of PBMCs squeezed with KRAS mutant mRNA or G12V 7-16 Or G12D 7-16 Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes incubated with the A*11 restricted peptide results in activation of the NFAT signaling pathway and secretion of lucia luciferase. The level of secreted lucia luciferase in the culture supernatant can be measured using a coelenterazine substrate. The resulting luminescent signal correlates with the amount of secreted lucia luciferase.

[0264] Squeeze process: HLA-A*11 + 200 x 10 PBMCs from donors 6After incubation at 4-8°C for approximately 10-15 minutes, the cells were squeezed through a 3.5 μm wide, 10 μm long, and 70 μm deep constriction at 60 psi in room temperature RPMI 1640 medium with 250 or 500 μg / mL KRAS 7mut_v1 mRNA, 250 or 500 μg / mL KRAS 7mut_v2 mRNA, or no cargo (empty squeeze). (The mRNA solution was cooled at 4-8°C for approximately 10-15 minutes before mixing with the cells and squeezing.) Following squeezing, the squeezed PBMCs were transferred to R10 medium (RPMI + 10% fetal bovine serum (FBS) + 1x Pen / Strep) and quenched at room temperature. Subsequently, the cells were washed with R10 and diluted to 5-10x10 6 cells / mL and stored in liquid nitrogen for future use.

[0265] analysis: On the day of co-culture setup, 2.5 x 10 HLA-A*11 restricted G12V and G12D TCR Jurkat cells were cultured in IMDM + 10% FBS. 6 Cryopreserved, squeezed, or untreated PBMCs were thawed and cultured in medium at 5 x 10 cells / mL. 6 The cells were then replaced with IMDM + 10% FBS at a density of 100,000 cells / mL. G12V or G12D TCR Jurkat cells were co-cultured with PBMCs at a 1:2 ratio (250,000 Jurkat cells to 500,000 PBMCs) in tissue culture-treated (TCT) 96-well U-bottom plates for 18–24 hours. HLA-A*11-restricted G12V 7-16 and G12D 7-16 The minimal epitope was added to Jurkat cells and co-cultures with naive PBMCs to control for antigen variant-specific Jurkat reactivity. Cell culture supernatants were collected and assayed for secreted lucia luciferase levels via Quanti LUC. The resulting luminescent signal correlates with the amount of secreted lucia luciferase.

[0266] result: As shown in Figures 7A and 7B, human PBMCs squeezed with KRAS 7 mutant-linked antigen mRNA and cocultured with G12V TCR-transduced or G12D TCR-transduced Jurkat cells elicited KRAS mutant-specific responses from TCR-transduced Jurkat cells compared to controls (empty squeeze, untreated PBMCs, and irrelevant mutant minimal epitope stimulation). G12V-specific responses: PBMCs squeezed with KRAS 7mut_v1 mRNA induced G12V-specific responses from G12V TCR-transduced Jurkat cells in a dose-dependent manner. PBMCs squeezed with KRAS 7mut_v2 mRNA induced similar responses at both squeeze concentrations (250 and 500 μg / mL) and responses comparable to those elicited by PBMCs squeezed with KRAS 7mut_v1 mRNA at 250 μg / mL. G12D-specific responses: PBMCs filtered with KRAS 7mut_v1 mRNA induced G12D-specific responses from G12D TCR-transduced Jurkat cells in a dose-dependent manner. The level of G12D responses induced by PBMCs filtered with KRAS 7mut_v2 mRNA was similar to that observed in the control groups (untreated PBMCs and empty filtered).

[0267] These results demonstrate that human PBMCs squeezed with KRAS 7 mutation-associated antigen mRNA (KRAS 7mut_v1 construct) can elicit potent mutation-specific responses from both G12V and G12D TCR-transduced Jurkat cells.

[0268] Example 8: Further analysis of KRAS G12V and G12D-specific responses after squeezing using mRNAs encoding seven KRAS mutant-linked antigens To assess the functional activity of mRNAs encoding both 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 transfected with the following mRNA constructs: (1) (a) Approximately 25 aa fragment (overlapping with the 1-25 aa sequence in the entire KRAS protein and a mutation on codon 12) KRAS G12D , KRASG12V , KRAS G12C , KRAS G13D , KRAS G12A , KRAS G12R and KRAS G12S antigen (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), and a concatenated mRNA construct ("7mut_v1+signal 2 / 3") encoding the 7mut_v1 antigen (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); (2) Approximately 25 aa fragment (overlapping the 1-25 aa sequence of the entire KRAS protein and a mutation on the 12th codon) KRAS G12D , KRAS G12V , KRAS G12C , KRAS G13D , KRAS G12A , KRAS G12R and KRAS G12S a concatenated mRNA construct encoding only the antigen ("7mut_v1"); (3) (a) Approximately 25 aa fragment (overlapping with the 1-25 aa sequence in the entire KRAS protein and a mutation on codon 12) KRAS G12S , KRAS G12R , KRAS G12A , KRAS G13D , KRAS G12C , KRAS G12V and KRAS G12D a concatenated mRNA construct ("7mut_v2+signal 2 / 3") encoding an antigen (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); (4) Approximately 25 aa fragment (overlapping the 1-25 aa sequence of the entire KRAS protein and a mutation on codon 12) KRAS G12S , KRAS G12R , KRAS G12A , KRAS G13D , KRAS G12C , KRAS G12V and KRAS G12Da concatenated mRNA construct encoding only the antigen ("mut_v2"), and (5) were treated with one of the concatenated mRNA constructs 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").

[0269] method Generation of responder cells: To generate A*11-restricted G12V and G12D TCR Jurkat responder cells, Jurkat-Lucia NFAT TCR α / β KO cells (custom-manufactured at Creative Biolabs) were transfected with A*11-restricted G12V 7-16 and G12D 7-16 The cells were transduced once with a specific TCR-expressing lentivirus. Residual virus was removed one day after TCR transduction, and the cells were then subjected to puromycin selection and cultured for several days before undergoing murine TCR (mTCR) enrichment. + The frequency of mTCR-enriched cells was increased. After mTCR enrichment, cells were cultured for several days, characterized by flow cytometry, and frozen for use in co-culture assays. The resulting A*11-restricted G12V TCR Jurkat and G12D TCR Jurkat responder cells expressed HLA-A*11-restricted G12V TCR Jurkat on the cell surface, respectively. 7-16 Specific TCR or HLA-A*11 restricted G12D 7-16 These express specific TCRs and are provided in the form of PBMCs squeezed with KRAS mutant mRNA or G12V 7-16 Or G12D 7-16 Engagement of these TCRs with the corresponding peptide-MHC (pMHC) complexes incubated with the A*11 restricted peptide results in activation of the NFAT signaling pathway and secretion of lucia luciferase. The level of secreted lucia luciferase in the culture supernatant can be measured using a coelenterazine substrate. The resulting luminescent signal correlates with the amount of secreted lucia luciferase.

[0270] Squeeze process: HLA-A*11 + 200 x 10 PBMCs from donors 6 Density of cells / mL (cell squeezing concentration 100 x 10 6 The mRNA was prepared at a concentration of 1000 μg / mL (cells / mL) and allowed to stand at 4-8°C for approximately 10-15 minutes. PBMCs were then squeezed in room temperature RPMI 1640 medium with 500 μg / mL of one of the above mRNA constructs at 60 psi using a 3.5 μm wide, 10 μm long, and 70 μm deep constriction (the mRNA solution was cooled at 4-8°C for approximately 15 minutes before mixing with the cells and squeezing). Following squeezing, the squeezed PBMCs were transferred to R10 medium (RPMI + 10% fetal bovine serum (FBS) + 1× Pen / Strep) and quenched at room temperature. Subsequently, the PBMCs were washed with R10 and diluted with 5×10 6 cells / mL and stored in liquid nitrogen for future use.

[0271] analysis: On the day of co-culture setup, 2.5 x 10 HLA-A*11 restricted G12V and G12D TCR Jurkat cells were cultured in IMDM + 10% FBS. 6 Cryopreserved, squeezed, or untreated PBMCs were thawed and cultured in medium at 5 x 10 cells / mL. 6 The cells were then replaced with IMDM + 10% FBS at a density of 100,000 cells / mL. G12V or G12D TCR Jurkat cells were co-cultured with PBMCs at a 1:2 ratio (250,000 Jurkat cells to 500,000 PBMCs) in tissue culture-treated (TCT) 96-well U-bottom plates for 18–24 hours. HLA-A*11-restricted G12V 7-16 and G12D 7-16 The minimal epitope was added to Jurkat cells and co-cultures with naive PBMCs to control for antigen variant-specific Jurkat reactivity. Cell culture supernatants were collected and assayed for secreted lucia luciferase levels via Quanti LUC. The resulting luminescent signal correlates with the amount of secreted lucia luciferase.

[0272] result As shown in Figures 8A and 8B, human PBMCs expressed with KRAS 7mutant-linked antigen mRNA, with or without signal 2 / 3 mRNA, and cocultured with G12V TCR-transduced or G12D TCR-transduced Jurkat cells elicited KRAS mutant-specific responses from TCR-transduced Jurkat cells compared to controls (empty expression, untreated PBMCs, signal 2 / 3 mRNA alone, and irrelevant mutant minimal epitope stimulation). G12V-specific responses: PBMCs expressed with 500 μg / mL of KRAS 7mut_v1 mRNA or KRAS 7mut_v2 mRNA (with or without signal 2 / 3 mRNA) elicited G12V-specific responses from G12V TCR-transduced Jurkat cells. G12D-specific responses: PBMCs expressed with 500 μg / mL of KRAS 7mut_v1 mRNA (with or without signal 2 / 3 mRNA) induced G12D-specific responses from G12D TCR-transduced Jurkat cells.

[0273] These results further demonstrate that human PBMCs squeezed with KRAS 7 mutation-associated antigen mRNA (KRAS 7mut_v1 construct) can elicit potent mutation-specific responses from both G12V and G12D TCR-transduced Jurkat cells. [Table 2] TIFF2025528742000004.tif36159

[0274] Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are incorporated herein 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.

[0275] equivalent While various specific embodiments have been illustrated and described, the above description is not intended to be limiting. It will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). Many variations will be apparent to those skilled in the art upon consideration of this specification.

Claims

1. 1. An isolated polynucleotide comprising a single ORF having a first nucleotide sequence ("first coding region") that encodes a first antigen and a second nucleotide sequence ("second coding region") that encodes a second antigen, 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. 3. The polynucleotide of claim 1, 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. 4. The polynucleotide of claim 3, wherein the order of the first coding region and the second coding region is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

5. 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. 6. The polynucleotide of claim 5, wherein the additional antigen is (i) not the same as the first antigen, (ii) not the same as the second antigen, or (iii) not the same as both the first antigen and the second antigen.

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

10. The first coding region, the second coding region and the additional coding region are 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); The polynucleotide of any one of claims 5 to 9, wherein L1 is a first linker and L2 is a second linker.

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

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

13. 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 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. 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. 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 antigen comprises a cancer antigen, a non-self antigen, a tumor-associated self antigen, a disease-associated antigen, or a combination thereof.

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

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

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

23. 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 compared to the amino acid sequence of the corresponding wild-type KRAS antigen.

24. The amino acid sequence of the KRAS antigen is 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, G6 24. The polynucleotide of claim 22 or 23, comprising an amino acid substitution selected from OV, 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 KRAS antigen is selected from the group consisting 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 The polynucleotide of any one of claims 22 to 24, comprising one or more of the antigens.

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

27. The peptide linker is G ４ 27. The polynucleotide of claim 26, comprising an S linker or an EAAAK linker.

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

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

30. 1. 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 linked to the second coding region by a first linker, the second coding region is linked to the third region by a second linker, and the third coding region is linked to the fourth coding region by a third linker.

31. 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 an order that is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

32. 1. 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 linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, and the fourth coding region is linked to the fifth coding region by a fourth linker.

33. 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 an order that is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs 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"). the first coding region is linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, the fourth coding region is linked to the fifth coding region by a fourth linker, and the fifth coding region is linked to the sixth coding region by a fifth linker.

35. 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 an order that is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

36. 1. 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 linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, the fourth coding region is linked to the fifth coding region by a fourth linker, the fifth coding region is linked to the sixth coding region by a fifth linker, and the sixth coding region is linked to the seventh coding region by a sixth linker.

37. 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 an order that is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

38. 1. 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"), the first coding region is linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, the fourth coding region is linked to the fifth coding region by a fourth linker, the fifth coding region is linked to the sixth coding region by a fifth linker, the sixth coding region is linked to the seventh coding region by a sixth linker, and the seventh coding region is linked to the eighth coding region by a seventh linker.

39. 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 an order that is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

40. 1. 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 polynucleotide comprises: the isolated polynucleotide, wherein a first coding region is linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, the fourth coding region is linked to the fifth coding region by a fourth linker, the fifth coding region is linked to the sixth coding region by a fifth linker, the sixth coding region is linked to the seventh coding region by a sixth linker, the seventh coding region is linked to the eighth coding region by a seventh linker, and the eighth coding region is linked to the ninth coding region by an eighth linker.

41. 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 an order that is different compared to the corresponding order present in a reference polynucleotide, and the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

42. 1. 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 ("tenth coding region"), wherein said first the coding region is linked to the second coding region by a first linker, the second coding region is linked to the third coding region by a second linker, the third coding region is linked to the fourth coding region by a third linker, the fourth coding region is linked to the fifth coding region by a fourth linker, the fifth coding region is linked to the sixth coding region by a fifth linker, the sixth coding region is linked to the seventh coding region by a sixth linker, the seventh coding region is linked to the eighth coding region by a seventh linker, the eighth coding region is linked to the ninth coding region by an eighth linker, and the ninth coding region is linked to the tenth coding region by a ninth linker.

43. 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 an order that differs compared to the corresponding order present in a reference polynucleotide, and wherein the reference polynucleotide comprises a corresponding polynucleotide that occurs in nature.

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

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

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

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

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

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

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

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

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

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

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

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

68. the amino acid sequence of the first KRAS antigen is 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, G6 68. The polynucleotide of claim 66 or 67, comprising an amino acid substitution selected from OV, 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 KRAS antigen is selected from the group consisting 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 69. The polynucleotide of any one of claims 66 to 68, comprising one or more of the following antigens:

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

71. The peptide linker is G ４ 71. The polynucleotide of claim 70, comprising an S linker or an EAAAK linker.

72. 72. The polynucleotide of 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' tail region of linked nucleosides, (10) a 5' cap, (11) a sequence encoding a 2A ribosomal skipping peptide, or (12) any combination of (1) to (11).

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

74. The at least one modified nucleoside is 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 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, pseudo-uridine, inosine, α-thio-guanosine, 8-oxo-guanosine, O6-methyl- 74. The polynucleotide of claim 73, comprising thio-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. 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. 78. The cell of claim 77, wherein the cell comprises a stem cell, a somatic cell, or both.

79. 78. The cell of claim 77, wherein the stem cells comprise induced pluripotent stem cells (iPSCs), embryonic stem cells, tissue-specific stem cells, mesenchymal stem cells, or a combination thereof.

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

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

82. 82. The cell of claim 81, wherein the PBMC comprises an immune cell.

83. 83. The cell of claim 82, wherein the immune cells comprise T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), NKT cells, mast cells, monocytes, macrophages, basophils, eosinophils, neutrophils, DC2.4 dendritic cells, or a combination thereof.

84. 84. The cell of any one of claims 77-83, wherein the cell passes through a constriction under a set of parameters, thereby causing a perturbation in the cell such that the polynucleotide, upon contact with the cell, enters the cell through the perturbation.

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

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

87. A method for producing a polynucleotide, comprising enzymatically or chemically synthesizing a polynucleotide according to any one of claims 1 to 75.

88. 76. A method for inducing expression of multiple antigens in a cell, the method comprising intracellular delivery of a polynucleotide according to any one of claims 1 to 75 into said cell.

89. 89. The method of claim 88, wherein the multiple antigens are simultaneously expressed within the cell after the intracellular delivery.

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

91. 91. The method of Claim 90, further comprising contacting said cell with said polynucleotide.

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

93. 93. The method of claim 91 or 92, wherein said contacting occurs prior to passing said cell suspension through said constriction.

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

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

96. 96. The method of any one of claims 84 to 95, wherein the set of parameters 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 characteristics of the constriction (e.g., roughness, chemical modification, hydrophilicity, hydrophobicity); operating flow rate; payload concentration; viscosity, osmolality, salt concentration, serum content and / or pH of the cell suspension; duration of the constriction; shear rate of the constriction; type of payload; or a combination thereof.

97. The cell density is at least about 6 x 10 ７ cells / mL, at least about 7 x 10 ７ cells / mL, at least about 8 x 10 ７ cells / mL, at least about 9 x 10 ７ cells / mL, at least about 1 x 10 ８ cells / mL, at least about 1.1 x 10 ８ cells / mL, at least about 1.2 x 10 ８ cells / mL, at least about 1.3 x 10 ８ cells / mL, at least about 1.4 x 10 ８ cells / mL, at least about 1.5 x 10 ８ cells / mL, at least about 2.0 x 10 ８ cells / mL, at least about 3.0 x 10 ８ cells / mL, at least about 4.0 x 10 ８ cells / mL, at least about 5.0 x 10 ８ cells / mL, at least about 6.0 x 10 ８ cells / mL, at least about 7.0 x 10 ８ cells / mL, at least about 8.0 x 10 ８ cells / mL, at least about 9.0 x 10 ８ cells / mL or at least about 1.0 x 10 ９ 97. The method of claim 96, wherein the concentration is 1000 or more cells / mL.

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

100. 100. The method of any one of claims 96-99, 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. 101. The method of claim 100, wherein the width of the constriction is from about 0 μm to about 10 μm.

102. 102. The method of 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. 100. The method of any one of claims 92 to 99, wherein the length of the constriction is from about 0 μm to about 100 μm.

104. 100. The method of 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. 101. The method of any one of claims 92 to 100, wherein the depth of the constriction is from at least about 1 μm to at least about 120 μm.

106. 102. The method of 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. 103. The method of any one of claims 92 to 102, wherein the cell suspension containing the cells is passed through a plurality of constrictions.

108. 104. The method of claim 103, wherein the plurality of stenoses 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 distinct stenoses.

109. 105. The method of claim 103 or 104, wherein each stenosis of the plurality of stenoses is the same.

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

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

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

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

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

115. The method of claim 110, wherein the enhanced immune response comprises: (i) an increase in the magnitude of the induced immune response compared to a reference immune response; (ii) an increase in the breadth of the induced immune response compared to a reference immune response; (iii) an increase in the duration of the induced immune response compared to a reference immune response; or (iv) any combination of (i)-(iii), wherein the reference immune response comprises an immune response observed in a corresponding subject that did not receive administration of the polynucleotide or modified cells.

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

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

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

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

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

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

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