WO2025153053A1

WO2025153053A1 - Composition and methods for inducing antigen specific immunity against kras mutant cells

Info

Publication number: WO2025153053A1
Application number: PCT/CN2025/072964
Authority: WO
Inventors: Jingshu MA; Liang Du; Bo YING; Jijun Yuan
Original assignee: Abogen Biosciences Shanghai Co Ltd
Current assignee: Abogen Biosciences Shanghai Co Ltd
Priority date: 2024-01-17
Filing date: 2025-01-17
Publication date: 2025-07-24
Anticipated expiration: 2026-07-17

Abstract

Provided herein are therapeutic nucleic acid molecules for managing, preventing and/or treating neoplastic diseases or disorders caused by or associated with oncogenic mutations in KRAS gene. Also provided herein are therapeutic compositions, including vaccines and lipid nanoparticles, comprising the therapeutic nucleic acids and related therapeutic methods and uses.

Description

COMPOSITION AND METHODS FOR INDUCING ANTIGEN SPECIFIC IMMUNITY AGAINST KRAS MUTANT CELLS

1. FIELD

The present disclosure generally relates to nucleic acid molecules that can be used for the management, prevention, and treatment of KRAS mutant cancers. The present disclosure also relates to lipid-containing compositions, including vaccines, of the nucleic acid molecules, and related methods of delivery.

2. BACKGROUND

KRAS mutations account for approximately 30%of human cancers including pancreatic ductal adenocarcinoma (PDAC) , non-small cell lung cancer (NSCLC) , and colorectal cancer (CRC) . KRAS is the first oncogene identified in human cancer with highest mutation rate of the RAS family. Vasan et al. Clinical Cancer Research, 2014, 20 (15) : 3921-3930. doi: 10.1158/1078-0432. CCR-13-1762; Huang et al., Signal Transduction and Targeted Therapy volume 6, Article number: 386 (2021) . doi: 10.1038/s41392-021-00780-4.

Due to the mechanisms of KRAS activation, there are several point mutations could lead to same outcome, thus resulting a high degree of heterogeneity in KRAS mutants in tumors. In the past nearly 40 years since the discovery of KRAS gene in 1982, the attempts of targeting KRAS mutant has been intensively focused, however, there is only a limited progress in the field, due to the poor druggability of KRAS. Firstly, the protein molecule is small, and the surface of the protein is relatively smooth and nearly spherical, thus difficult to find a binding pocket of small molecules. Secondly, KRAS has high affinity to GTP, so the competitive binding molecules could rarely compete out GTP from binding to KRAS. Thirdly, different mutation sites of KRAS have different GTP binding structures, which also increased the difficulties of targeting.

There is no sign of succeed yet for identifying small inhibitory molecules for various KRAS oncogenic mutations. Additionally, although inhibitors like Sotorasib and Adagrasib may be identified for other KRAS mutants, acquired drug resistance always rapidly desensitize clinical response. Therefore, there exist an urgent need for effective therapeutics, including vaccines for curbing cancer driven by KRAS mutations. The present disclosure meets this need.

3. SUMMARY

KRAS is one of the front-line sensors that activate a group of signal molecules, controlling multiple signal cascades by switching between active and inactivated conformations. KRAS protein has high affinity with guanosine diphosphate (GDP) and guanosine triphosphate (GTP) , and it is inactivated when binding to GDP while activated when binding to GTP ^[2, 3, 4] . KRAS signaling pathway could be activated by growth factors, chemokines, Ca²⁺, or receptor tyrosine kinases (RTK) . Activated KRAS protein subsequently triggers a variety of signaling pathways, including rapidly accelerating fibrosarcoma (RAF) , mitogen-activated protein kinase (MEK) , extracellular regulatory protein kinase (ERK) signaling pathway, phosphoinositide 3-kinase (PI3K) , protein kinase B (AKT) -mammalian target of rapamycin (mTOR) signaling pathway, etc. It also modulates fundamental cellular processes, such as cell differentiation, growth, and apoptosis. Liu et al., Acta Pharm Sin B. 2019 Sep; 9 (5) : 871-879. doi: 10.1016/j. apsb. 2019.03.002. Mugarza Strobl, Edurne; (2021) The role of oncogenic KRAS in the immune evasion of non-small cell lung cancer. Doctoral thesis (Ph. D) , UCL (University College London) .

KRAS gene mutations occurs mainly concentrated in the positions of codon 12, 13 and 61, of which, 97%are in amino acid residues 12 or 13, including G12A, G12C, G12D, G12V and G13D. The mutation of KRAS gene leads to the continuous binding of KRAS protein to GTP, so as to maintain the active state, and then initiate a variety of downstream signaling pathways, resulting in uncontrollable cell growth and proliferation, thus promote the occurrence and development of cancer. Cox A D, Fesik S W, Kimmelman A C, et al. Nature Reviews Drug Discovery, 2014, 13 (11) : 828. doi: 10.1038/nrd4389. Kessler et al., (2019) . PNAS; doi: 10.1073/pnas. 1904529116.

Accordingly, provided herein are therapeutic nucleic acid molecules encoding KRAS neoepitopes that can be used for the management, prevention, and treatment of KRAS mutant cancers. The present disclosure also relates to lipid nanoparticle compositions, including vaccines, of the nucleic acid molecules, and related methods of delivery and use.

In one aspect, provided herein are polypeptides, including therapeutic polypeptides comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of human KRAS. In some embodiments, the at least one mutated fragment of human KRAS comprises one or more KRAS mutations selected from G12A, G12C, G12D, G12V, G13D and Q61R.

In some embodiments, the polypeptide further comprises a trafficking peptide configured for trafficking the KRAS domain to endosomes and lysosomes. In some embodiments, the polypeptide further comprises a degradation domain configured for marking the KRAS domain for proteasomal degradation.

In some embodiments, the polypeptide further comprises one or more additional elements configured to enhance immunogenicity of the polypeptide. In some embodiments, such additional elements are selected from a helper T cell epitope, a signaling peptide, and an immunomodulator.

In a related aspect, provided herein are also nucleic acid molecules encoding the polypeptides described herein. In a related aspect, provided herein are also vectors and cells comprising the nucleic acid molecules described herein.

In some embodiments, provided herein is an immunomodulating nucleic acid system encoding the KRAS domain according to the present invention, wherein the system comprises a first coding sequence encoding a helper T cell epitope, wherein the helper T cell epitope is configured to modulate immunogenicity of the KRAS domain upon co-expression with the KRAS domain.

In some embodiments, the system further comprises a second coding sequence encoding a polypeptide comprising an immunomodulator, wherein the immunomodulator is configured to enhance immunogenicity of the KRAS domain upon co-expression with the KRAS domain.

In some embodiments, the helper T cell epitope and the immunomodulator are configured to enhance immunogenicity of the KRAS domain upon co-expression with the KRAS domain.

In some embodiments, the system further comprises a third coding sequence encoding a polypeptide comprising a trafficking peptide configured for intracellularly trafficking the KRAS domain towards proteosome upon expression.

In some embodiments, the system further comprises a fourth coding sequence encoding a polypeptide comprising a signal peptide.

In some embodiments, the system further comprises a fifth coding sequence encoding a polypeptide comprising a ubiquitin peptide.

In some embodiments, the first coding sequence encodes a polypeptide comprising the helper T cell epitope and the KRAS domain according to the present invention.

In some embodiments, the second coding sequence encodes a polypeptide comprising the immunomodulator and the KRAS domain according to the present invention; optionally wherein the immunomodulator is positioned N-terminal to the KRAS domain or is positioned C-terminal to the KRAS domain in the polypeptide.

In some embodiments, the third coding sequence encodes a polypeptide comprising the trafficking peptide and the KRAS domain; optionally wherein the trafficking peptide is positioned C-terminal to the KRAS domain in the polypeptide.

In some embodiments, the fourth coding sequence encodes a polypeptide comprising the signal peptide and the KRAS domain; optionally wherein the signal peptide is positioned N-terminal to the KRAS domain in the polypeptide.

In some embodiments, the fifth coding sequence encodes a polypeptide comprising the ubiquitin peptide and the KRAS domain; optionally wherein the ubiquitin peptide is positioned N-terminal to the KRAS domain in the polypeptide; optionally wherein polypeptide further comprises a spacer peptide positioned in between the ubiquitin peptide and the KRAS domain in the polypeptide; optionally the spacer peptide is at least about 25 amino acids in length.

In some embodiments, the first coding sequenc is in a first nucleic acid molecule.

In some embodiments, at least one of the first coding sequence and the second coding sequence is in a first nucleic acid molecule.

In some embodiments, at least one of the first coding sequence, the second coding sequence and the third coding sequence is in a first nucleic acid molecule.

In some embodiments, at least one of the first coding sequence, the second coding sequence, the third coding sequence and the fourth coding sequence is in a first nucleic acid molecule.

In some embodiments, at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences are in a first nucleic acid molecule.

In some embodiments of the immunomodulating nucleic acid system, (a) the second coding sequence is in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulatory and the KRAS domain; (b) the first coding sequence and the second coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator and the KRAS domain; (c) the first coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the trafficking peptide and the KRAS domain; (d) the first coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the signal peptide and the KRAS domain; (e) the first coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the ubiquitin peptide and the KRAS domain; (f) the second coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide and the KRAS domain; (g) the second coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the signal peptide, and the KRAS domain; (h) the second coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the ubiquitin peptide and the KRAS domain; (i) the third coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the signal peptide and the KRAS domain; (j) the third coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the ubiquitin peptide and the KRAS domain; (k) the fourth coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the signal peptide, the ubiquitin peptide and the KRAS domain; (l) the first coding sequence, the second coding sequence, and the third coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, and the KRAS domain; (m) the first coding sequence, the second coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the signal peptide, and the KRAS domain; (n) the first coding sequence, the second coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the ubiquitin peptide, and the KRAS domain; (o) the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide, the signal peptide, and the KRAS domain; (p) the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the KRAS domain; (q) the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the signal peptide, the ubiquitin peptide, and the KRAS domain; (r) the first coding sequence, the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the KRAS domain; (s) the first coding sequence, the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the KRAS domain; or (t) the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the ubiquitin peptide, and the KRAS domain.

In some embodiments, at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences is in a second nucleic acid molecule, and wherein the first nucleic acid molecule and the second nucleic acid molecule are different molecules.

In some embodiments, the second nucleic acid molecule does not encode the KRAS domain.

In some embodiments, the first nucleic acid molecule encodes the polypeptide comprising the KRAS domain; wherein the polypeptide does not comprise the immunomodulator; and wherein the second nucleic acid molecule comprises the second coding sequence encoding the immunomodulator.

In some embodiments, at least the second coding sequence is in the first nucleic acid molecule encoding the polypeptide comprising at least the immunomodulator and the KRAS domain; and wherein the first nucleic acid molecule further comprises a means for producing the immunomodulator and the KRAS domain as separate proteins or peptides.

In some embodiments, the means for producing the immunomodulator and the KRAS domain as separate proteins or peptides comprises a sixth coding sequence encoding a cleavable linker in between the second coding sequence and a coding sequence for the KRAS domain in the first nucleic acid.

In some embodiments, the cleavable linker is a 2A peptide, selected from the group consisting of P2A, F2A, T2A, and E2A. In some embodiments, the P2A peptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 105 or 258.

In some embodiments, the helper T cell epitope is a universal CD4 epitope. In some embodiments, the helper T cell epitope is an epitope of tetanus and diphtheria toxoids. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE. In some embodiments, the first coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 95-98 and 186. In some embodiments, the first coding sequence encodes the helper T cell epitope that is a pan DR-binding epitope (PADRE) , and wherein the first coding sequence comprises the nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence of SEQ ID NO: 98 or 186. In some embodiments, the PADRE comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 94. In some embodiments, the helper T cell epitope comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 187, 190, or 192-197.

In some embodiments, the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof. In some embodiments, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.

In some embodiments, the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) . In some embodiments, the GM-CSF is a full-length GM-CSF polypeptide. In some embodiments, the full-length GM-CSF polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268. In some embodiments, the GM-CSF comprises a truncated GM-CSF or a mutant GM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268, and wherein the truncated GM-CSF or mutant GM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) . In some embodiments, the STING is human STING (hSTING) (V155M) . In some embodiments, the STING comprises a polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence of SEQ ID NO: 220 or 253. In some embodiments, the second coding sequence encoding the immunomodulator comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 112-114, 215-217, 116-118, 218-219, 221, 254, 265, and 269-271.

In some embodiments, the immunomodulating nucleic acid system comprises (a) the first coding sequence encodes a polypeptide comprising the KRAS domain and a helper T cell epitope, and the second coding sequence encodes an immunomodulator, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.

In some embodiments, the trafficking peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.

In some embodiments, the trafficking peptide is MHC class I trafficking peptide. In some embodiments, the trafficking peptide is MHC class I trafficking peptide or LAMP3 transmembrane domain (LAMP3 TM) . In some embodiments, the third coding sequence encoding the trafficking peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 100-104 and 198-202. In some embodiments, the MHC class I trafficking peptide comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence set forth in SEQ ID NO: 99.

In some embodiments, the fourth coding sequence encoding the signal peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 90-93 and 171-175. In some embodiments, the signal peptide comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequences set forth in SEQ ID NO: 89 or 170.

In some embodiments, the ubiquitin peptide comprises a naturally-existing ubiquitin peptide or a functional derivative thereof. In some embodiments, the fifth coding sequence encoding the ubiquitin peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence set forth in SEQ ID NO: 2, 3 or 169. In some embodiments, the ubiquitin peptide comprises the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequences set forth in SEQ ID NO: 1.

In some embodiments, the first nucleic acid molecule is DNA or RNA. In some embodiments, the second nucleic acid molecule is DNA or RNA. In some embodiments, the RNA is mRNA, self-amplifying RNA, or circular RNA. In some embodiments, the one or more coding sequences are codon optimized. In some embodiments, the one or more coding sequences are in one or more mRNA molecule.

In some embodiments, the mRNA molecule further comprises a 5’ untranslated region (UTR) , a 3’ UTR, or both a 5’ UTR and a 3’ UTR. In some embodiments, the 5’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 136, 251 or 275. In some embodiments, the 3’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 137 or 252. In some embodiments, the mRNA molecule further comprises a 5’ Cap. In some embodiments, the mRNA molecule further comprises a poly (A) sequence. In some embodiments, the poly (A) sequence has a length of about 50 nucleotides or longer.

In some embodiments, the first nucleic acid molecule comprises a chemical modification. In some embodiments, the second nucleic acid molecule comprises a chemical modification. In some embodiments, the chemical modification comprises pseudouridine. In some embodiments, the pseudouridine is 1-methylpseudouridine.

In some embodiments, the first nucleic acid molecule is in a first vector. In some embodiments, the second nucleic acid molecule is a second vector.

In a related aspect, provided herein is also a vector system comprising the nucleic acid molecules described herein. In some embodiments, the vector system comprises a vector comprising the nucleic acid molecule according to the present disclosure. In some embodiments, the vector system comprises two or more vectors comprising two or more nucleic acid molecules of the present disclosure.

In another related aspect, provided herein is also a polypeptide encoded by the nucleic acid molecule of the immunomodulating nucleic acid system according to the present disclosure.

In a related aspect, compositions including pharmaceutical compositions such as a vaccine, encompassing the polypeptides or nucleic acid molecules described herein are provided. In a particular embodiment, the composition comprises a lipid nanoparticle composition. In a related aspect, methods and uses of the polypeptides, nucleic acids, and compositions comprising the polypeptides or nucleic acids are provided, including therapeutic methods for the prevention, management and treatment of cancer (e.g., cancer associated with KRAS oncogenic mutations) .
4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1E show schematic illustrations of several mRNA constructs encoding a mutant KRAS polypeptide. The mRNA molecules can have one or more chemically modified nucleotide analogs, such as pseudouridine, 1-methyl-pseudouridine (ψ) or 5-methylcytosine.

As shown in FIG. 1A, the KRAS-001 construct contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a mutant KRAS polypeptide, and a 3’ poly-adenylation (poly-A) region.

As shown in FIG. 1B, the KRAS-002 and KRAS-003 constructs each contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein of an ubiquitin peptide fused to a mutant KRAS polypeptide, and a 3’ poly-adenylation (poly-A) region. The KRAS-002 and KRAS-003 constructs have different codon optimization in their respective coding regions.

As shown in FIG. 1C, the KRAS-004, KRAS-005, and KRAS-006 constructs each contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein, and a 3’ poly-adenylation (poly-A) region. The fusion protein contains a signal peptide (SP) , a mutant KRAS polypeptide, a helper T cell epitope (PADRE) , a MHC Class I trafficking peptide, a cleavable linker (p2A) and a mouse GM-CSF polypeptide. The KRAS-004, KRAS-005, and KRAS-006 constructs have different codon optimization in their respective coding regions.

As shown in FIG. 1D, the KRAS-007, KRAS-008, and KRAS-009 constructs each contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein, and a 3’ poly-adenylation (poly-A) region. The fusion protein contains a signal peptide (SP) , a mutant KRAS polypeptide, a helper T cell epitope (PADRE) , a MHC Class I trafficking peptide, a cleavable linker (p2A) and a human GM-CSF polypeptide. The KRAS-007, KRAS-008, and KRAS-009 constructs have different codon optimization in their respective coding regions.

As shown in FIG. 1E, the BMK construct contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein, and a 3’ poly-adenylation (poly-A) region. The fusion protein contains a mutant KRAS polypeptide, a cleavable linker (p2A) and a Sting polypeptide.

FIG. 2. shows results of an ELISPOT assay measuring the level of IFN-γ production by splenocytes of mice immunized with mRNA molecules KRAS-002, KRAS-003, and KRAS-001, in the presence of mutant KRAS G12V fragment, G12D fragment, G13D fragment, G12A fragment, G12C fragment, Q61R fragment or corresponding wild-type KRAS fragments.

FIG. 3A shows results of an ELISPOT assay measuring the level of IFN-γ production by splenocytes of HLA-A*1101 transgene mice immunized with 5μg or 20μg mRNA molecules KRAS-002 in the presence of mutant KRAS G12V fragment, G12D fragment, G12A fragment, G12C fragment, G13D fragment, or corresponding wild-type KRAS fragment. Splenocytes isolated from mice treated with PBS were included as negative controls. FIG. 3B shows the quantitation of the various panels shown in FIG. 3A.

FIG. 4 shows progression of tumor volumes in a colorectal cancer model established with xenografting SW480-KRAS^G12V cells in B-NDG immunodeficient mice with or without treatment. Mice in the treatment group (Group 3) received adoptive transfer of splenocytes isolated from mice immunized with mRNA molecule KRAS-002. Mice in a control group (Group 2) received adoptive transfer of splenocytes isolated from mice received only PBS. Mice in another control group (Group 1) received no treatment.

FIG. 5 shows progression of tumor volumes in a pancreatic cancer model established with xenografting Panc-1-KRAS^G12D cells in B-NDG immunodeficient mice with or without treatment. Mice in the treatment group (Group 3) received adoptive transfer of splenocytes isolated from mice immunized with mRNA molecule KRAS-002. Mice in a control group (Group 2) received adoptive transfer of splenocytes isolated from mice received only PBS. Mice in another control group (Group 1) received no treatment.

FIG. 6A shows GM-CSF expression levels in cells transfected with 0.2μg, 1μg, or 5μg mRNA molecule KRAS-004, KRAS-005, or KRAS-006, or negative control groups of cells that either received no treatment (blank) , or treated with transfection reagents containing no mRNA (Lipo) , or transfected with a functionally unrelated mock mRNA molecule (NST) . FIG. 6B shows GM-CSF expression levels in cells transfected with 0.2μg, 1μg, or 5μg mRNA molecule KRAS-007, KRAS-008, or KRAS-009, or negative control groups of cells that either received no treatment (blank) , or treated with transfection reagents containing no mRNA (Lipo) , or transfected with a functionally unrelated mock mRNA molecule (NST) .

FIG. 7 shows results of an ELISPOT assay measuring the level of IFN-γ production by activated antigen specific T cells co-cultured with dendritic cells transfected with mRNA molecule KRAS-003 (middle panels) in the presence of a mutant KRAS fragment selected from the G12A fragment, the G12C fragment, the G12D fragment, and the G12V fragment, or pulsed with directly with 10μg/ml of such mutant KRAS fragment (upper panels) . Untreated effector cells were included as negative controls (bottom panels) .

FIG. 8 shows results of an ELISPOT assay measuring the level of IFN-γ production by splenocytes of mice immunized with mRNA molecule KRAS-002, KRAS-004, or BMK, in the presence of mutant KRAS G12V fragment, G12D fragment, G12C fragment, or G12A fragment.

FIG. 9 shows quantitation of mGM-CSF expression level by cells transfected with mRNA molecule KRAS-004, KRAS-005, KRAS-006 or BMK.

FIG. 10 shows images (upper panel) and quantitation (lower panel) of an ELISPOT assay measuring the level of IFN-γ production by splenocytes of mice immunized with PBS or mRNA molecules KRAS-004, KRAS-004-ψ, or KRAS-004-M1-ψ, in the presence of mutant KRAS G12V fragment, G12D fragment, G12C fragment, or G12A fragment.

FIG. 11A show images (upper panels) and quantitation (lower panels) of an ELISPOT assay measuring the level of IFN-γ production by splenocytes of mice immunized with mRNA molecules KRAS-004 or BMK, in the presence of mutant KRAS G12V fragment, G12D fragment, G12C fragment, or G12A fragment.

FIG. 11B shows progression of tumor volumes in colon cancer model established with SW480-KRAS^G12V cells in HLA-A*1101 mice with or without treatment. Mice in a treatment group (down triangle; group 1) received adoptive transfer of 2E7 splenocytes isolated from mice immunized with 20μg mRNA molecule KRAS-004 and mice in another treatment group (cross; group 2) received adoptive transfer of 2E7 splenocytes isolated from mice immunized with 20μg mRNA molecule BMK. Mice in a control group (open circle) received adoptive transfer of 2E7 splenocytes isolated from mice received only PBS. Mice in another control group (closed circle) received no treatment. At the end of the 18-day observation window, the tumor size differences between the two treatment groups were statistically significant (P<0.01) . In the treatment group 1, 4 out of 5 animals achieved complete remission. In the treatment group 2, 0 out of 5 animals achieved complete remission.

FIG. 12 shows progression of tumor volumes in colon cancer model established with SW480-KRAS^G12V cells in HLA-A*1101 mice with or without treatment. Mice in a treatment group (down triangle; group 1) received adoptive transfer of 1E7 splenocytes isolated from mice immunized with 20μg mRNA molecule KRAS-004 on day 0. Mice in another treatment group (up triangle; group 2) received adoptive transfer of 1E7 splenocytes isolated from mice received PBS on day 0, and 10 mpk mPD-1 antibody on days 0, 4, 7, 11, 14 and 18. Mice in another treatment group (diamond; group 3) received adoptive transfer of 1E7 splenocytes isolated from mice immunized with 20μg mRNA molecule KRAS-004 on day 0, and 10 mpk mPD-1 antibody on days 0, 4, 7, 11, 14 and 18. Mice in a control group (square) received adoptive transfer of 1E7 splenocytes isolated from mice received only PBS on Day 0. Mice in another control group (circle) received no treatment. At the end of the 18-day observation window, the tumor size differences between treatment groups 1 and 3, as well as between groups 2 and 3 were statistically significant (p<0.001) .

FIG. 13 shows results of an ELISPOT assay measuring the level of IFN-γ production in PBMCs reduced by mRNA molecule KRAS-007, in the presence of mutant KRAS G12V fragment, G12D fragment, G12C fragment, G12A fragment, G13D fragment or PADRE.
[0100]FIG. 14 shows specific killing of KRAS-G13D positive tumor cells by mRNA molecule KRAS-007
between non-specific T cells and specific T cells. The target cell viabilities between specific T cells (KRAS-007) and non-specific control groups (only culture medium) were statistically significant (p<0.001) .
[0101]FIG. 15 shows GM-CSF levels in serum of mice immunized with KRAS-004 vaccine, or PBS.

FIG. 16 shows levels of various cytokines, including IFN-α, IFN-β, IFN-γ and IL-6 in mouse serum after vaccination with the KRAS-004 vaccine.

FIG. 17 shows specific killing of KRAS-G12A, G12C, G12V and G12D positive tumor cells by mRNA molecule KRAS-004. The target cell viabilities in specific T cells (KRAS-004) were significantly lower than non-specific PBS control groups, for all of tumor antigen positive tumor cell groups.

5. DETAILED DESCRIPTION

RAS is one of the most frequently mutated oncogenes in human cancer. The frequency and distribution of RAS gene mutations are not uniform. Kirsten rat sarcoma viral oncogene homologue (KRAS) is the isoform most frequently mutated, which constitutes 86%of RAS mutations. KRAS is associated with a series of highly fatal cancers, including pancreatic ductal adenocarcinoma (PDAC) , non-small-cell lung cancer (NSCLC) , and colorectal cancer (CRC) . KRAS mutations are also present in biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, and breast cancer.

Provided herein are therapeutic nucleic acid molecules useful for the prevention, management and treatment of neoplastic diseases or conditions, including cancer, that are driven by oncogenic mutations of KRAS. Also provided herein are pharmaceutical compositions comprising the therapeutic nucleic acid molecules, including pharmaceutical composition formulated as lipid nanoparticles and related therapeutic methods and uses for preventing, managing and treating of such neoplastic diseases. Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of particular embodiments.
5.1 General Techniques

Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001) ; Current Protocols in Molecular Biology (Ausubel et al. eds., 2003) .
5.2 Terminology

Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

The term “polynucleotide” or “nucleic acid, ” as used interchangeably herein, refers to polymers of nucleotides of any length and includes, e.g., DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acid can be in either single-or double-stranded forms. As used herein and unless otherwise specified, “nucleic acid” also includes nucleic acid mimics such as locked nucleic acids (LNAs) , peptide nucleic acids (PNAs) , and morpholinos.

The term “corresponding DNA sequence” when used in connection with an RNA sequence refers to the sequence where each “U” in the RNA sequence is replaced by a “T. ” Reversely, the term “corresponding RNA sequence” when used in connection with a DNA sequence refers to the sequence where each “T” in the DNA sequence is replaced by a “U. ”

“Oligonucleotide, ” as used herein, refers to short synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences” ; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences. ”

As used herein, the term “non-naturally occurring” when used in reference to a nucleic acid molecule as described herein is intended to mean that the nucleic acid molecule is not found in nature. A non-naturally occurring nucleic acid encoding a protein (e.g., KRAS) contains at least one genetic alternation or chemical modification not normally found in a naturally occurring nucleic acid, including a wild-type nucleic acid. Genetic alterations include, for example, modifications to an expressible nucleic acid sequence encoding heterologous peptides or polypeptides, other nucleic acid additions, nucleic acid deletions, nucleic acid substitution, and/or other functional disruption of a coding sequence. Such modifications include, for example, modifications in the coding regions and functional fragments thereof, for heterologous, homologous or both heterologous and homologous polypeptides. Additional modifications include, for example, modifications in non-coding regulatory regions in which the modifications alter expression of a gene or operon. Additional modifications also include, for example, incorporation of a nucleic acid sequence into a vector, such as a plasmid or an artificial chromosome. Chemical modifications include, for example, one or more functional nucleotide analog as described herein.

An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as an mRNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antigen as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

The term “encoding nucleic acid” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule encompasses (a) a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA which is then translated into a peptide and/or polypeptide, and (b) the mRNA molecule itself. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom. The term “coding region” refers to a portion in an encoding nucleic acid sequence that is translated into a peptide or polypeptide. The term “untranslated region” or “UTR” refers to the portion of an encoding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of a UTR with respect to the coding region of a nucleic acid molecule, a UTR is referred to as the 5’-UTR if located to the 5’-end of a coding region, and a UTR is referred to as the 3’-UTR if located to the 3’-end of a coding region.

An encoding nucleic acid can be mono-cistronic or multi-cistronic. A “mono-cistronic sequence” refers to a polynucleotide that comprises coding sequence for a single peptide or polypeptide chain. A “multi-cistronic sequence” refers to a polynucleotide that comprises coding sequences for two or more peptide and/or polypeptide chains.

The term “mRNA” as used herein refers to a message RNA molecule comprising one or more coding regions, such as open reading frames (ORF) , that can be translated by a cell or an organism provided with the mRNA to produce one or more peptide or protein product. The region containing the one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs) .

In certain embodiments, the mRNA is a monocistronic mRNA that comprises only one ORF. In certain embodiments, the monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor associated antigen) . In other embodiments, the mRNA is a multicistronic mRNA that comprises two or more ORFs. In certain embodiments, the multiecistronic mRNA encodes two or more peptides or proteins that can be the same or different from each other. In certain embodiments, each peptide or protein encoded by a multicistronic mRNA comprises at least one epitope of a selected antigen. In certain embodiments, different peptide or protein encoded by a multicistronic mRNA each comprises at least one epitope of different antigens. In any of the embodiments described herein, the at least one epitope can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 epitopes of an antigen.

The terms “circRNA” or “circular RNA” are used interchangeably and refer to a polyribonucleotide that forms a circular structure through covalent bonds. Circular RNA (circRNA) is a type of single-stranded RNA which forms a 3’-5’ covalently closed loop. CircRNA, or circular RNA, can be produced through various mechanisms. One primary method is backsplicing, a non-canonical splicing process mediated by the spliceosome. In this process, a downstream splice donor site is joined to an upstream splice acceptor site, forming a covalently closed loop. Additionally, circRNAs can also be generated through chemical ligation, enzymatic ligation, and ribozyme methods. These alternative approaches enable the formation of circular RNA structures, expanding the variety of circRNAs that can be synthesized for research and therapeutic applications. Unlike linear mRNAs, circRNAs do not require a 5’-cap or 3’-poly (A) tail for their stability. The closed ring structure of circRNAs protects them from exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and having a longer half-life compared to their linear mRNA counterparts.

Self-replicating RNA (saRNA) , such as that derived from viral replicons, is useful for expression of proteins. It results in the production of numerous copies of the original RNA, leading to high and sustained expression levels of the protein of interest.

As used herein, the term “ribosomal skipping element” refers to a nucleotide sequence capable of causing generation of two polypeptide chains from translation of one RNA molecule. In some embodiments, the ribosomal skipping element can terminate translation of the first polypeptide chain and re-initiating translation of the second polypeptide chain from the RNA molecule. In alternative embodiments, the ribosomal skipping element encodes a protease cleavage site in the polypeptide encoded by the RNA molecule, so that the polypeptide can be cleaved by an intrinsic protease activity of its own, or by another protease in its environment to produce two polypeptide chains. In specific embodiments, the ribosomal skipping element encodes a cleavable linker.

The term “cleavable linker” as used herein refers to a type of linker, typically a peptide linker (usually composed of about 5 to 30 amino acids, typically around 10 to 20 amino acids long) . This linker can be integrated into multicistronic mRNA constructs, allowing for the simultaneous production of multiple genes in equimolar amounts from the same mRNA. Examples of such cleavable linkers include peptides from the 2A family, such as thosea-asigna virus 2A peptide (T2A) , porcine teschovirus-12 A peptide (P2A) , foot-and-mouth disease virus 2 A peptide (F2A) , equine rhinitis A vims 2A peptide (E2A) , cytoplasmic polyhedrosis vims 2A peptide (BmCPV 2A) , or flacherie vims of B. mori2A peptide (BmIFV 2A) . When these linkers are inserted into an mRNA construct, typically between two polypeptide domains, they cause the ribosome to skip the synthesis of a peptide bond at the C-terminus of the 2A element. This, in turn, leads to the separation between the end of the 2A sequence and the next downstream peptide.

Codon substitution or codon replacement in the context of codon optimization refer to replacing a codon present in a candidate nucleotide sequence (e.g., an mRNA encoding a therapeutic agent) with another codon. Thus, a codon can be substituted in a candidate nucleic acid sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a “substitution” or “replacement” at a certain location in a nucleic acid sequence (e.g., an mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon. As used herein, the term “codon optimized variant” refers to a synonymous nucleotide sequence that encodes the same polypeptide sequence encoded by a candidate nucleotide sequence (e.g., a nucleotide sequence encoding a KRAS polypeptide or mutated fragment thereof) . Thus, there are no amino acid substitutions in the polypeptide encoded by the codon optimized nucleotide sequence with respect to the polypeptide encoded by the candidate nucleotide sequence. A candidate nucleic acid sequence can be codon-optimized by replacing all or part of its codons according to a substitution table map. According to the present disclosure, a candidate nucleotide sequence can be codon-optimized, for example, to improve its translation efficacy of the encoded polypeptide.

The term “nucleobases” encompasses purines and pyrimidines, including natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

The term “functional nucleotide analog” as used herein refers to a modified version of a canonical nucleotide A, G, C, U or T that (a) retains the base-pairing properties of the corresponding canonical nucleotide, and (b) contains at least one chemical modification to (i) the nucleobase, (ii) the sugar group, (iii) the phosphate group, or (iv) any combinations of (i) to (iii) , of the corresponding natural nucleotide. As used herein, base pairing encompasses not only the canonical Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between canonical nucleotides and functional nucleotide analogs or between a pair of functional nucleotide analogs, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a modified nucleobase and a canonical nucleobase or between two complementary modified nucleobase structures. For example, a functional analog of guanosine (G) retains the ability to base-pair with cytosine (C) or a functional analog of cytosine. One example of such non-canonical base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, a functional nucleotide analog can be either naturally occurring or non-naturally occurring. Accordingly, a nucleic acid molecule containing a functional nucleotide analog can have at least one modified nucleobase, sugar group and/or internucleoside linkage. Exemplary chemical modifications to the nucleobases, sugar groups, or internucleoside linkages of a nucleic acid molecule are provided herein.

The terms “translational enhancer element, ” “TEE” and “translational enhancers” as used herein refers to a region in a nucleic acid molecule that functions to promotes translation of a coding sequence of the nucleic acid into a protein or peptide product, such as via cap-dependent or cap-independent translation. A TEE typically locates in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the translational level of a coding sequence located either upstream or downstream. For example, a TEE in a 5’-UTR of a nucleic acid molecule can locate between the promoter and the starting codon of the nucleic acid molecule. Various TEE sequences are known in the art (Wellensiek et al. Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10 (8) : 747–750; Chappell et al. PNAS June 29, 2004101 (26) 9590-9594) . Some TEEs are known to be conserved across multiple species (et al. Nucleic Acids Research, Volume 41, Issue 16, 1 September 2013, Pages 7625–7634) .

As used herein, the term “stem-loop sequence” refers to a single-stranded polynucleotide sequence having at least two regions that are complementary or substantially complementary to each other when read in opposite directions, and thus capable of base-pairing with each other to form at least one double helix and an unpaired loop. The resulting structure is known as a stem-loop structure, a hairpin, or a hairpin loop, which is a secondary structure found in many RNA molecules.

The term “Kirsten rat sarcoma viral oncogene homologue” or “KRAS” as used herein, refers to any native KRAS from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed KRAS as well as any form of KRAS that results from processing in the cell. The term also encompasses naturally occurring variants of KRAS, e.g. splice variants or allelic variants. The amino acid sequence of an exemplary human KRAS is MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMR DQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARS YGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKKKKKKSKTKCVIM (SEQ ID NO: 168; GenBank accession: NP_004976.2) ; G12, G13, and Q61 are underlined and shown in boldface. A “full-length” KRAS as used herein refers to the mature, natural length KRAS molecule. For example, full-length human KRAS refers to a molecule that has 188 amino acids (see e.g. SEQ ID NO: 168) .

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse KRAS and human KRAS can be considered orthologs for the biological function of regulation of cell division as a result of its ability to relay external signals to the cell nucleus. See e.g., et al. J Biomed Biotechnol. 2010; 2010: 150960. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs ifthey share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25%to 100%amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less than 25%can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. Those skilled in the art will understand how to identify orthologous genes harboring a biological function of interest. For example, a list of orthologous KRAS genes and encoded KRAS protein sequences can be found on GenBank website: https: //www. ncbi. nlm. nih. gov/gene/3845/ortholog/? scope=7776&term=KRAS. In some embodiments, A group orthologs genes encode protein products that can be considered functional derivatives of one another.

The term “KRAS mutant” or “mutant KRAS” as used herein refers to an KRAS molecule that contains one or more modifications of the wild-type amino acid residue normally located at that position. As used herein, the term “wild-type” refers to organisms, cells, genes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (in the wild) .

The term “peptide, ” “polypeptide, ” and “protein” are used interchangeably herein to refer to a polymer containing between two or more amino acid residues linked by one or more covalent peptide bond (s) . The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog or non-natural amino acid) . Although not strictly applied, the term “peptide” is used sometimes to refer to a polymer of two (2) to fifty (50) amino acid residues linked by covalent peptide bonds, and the term “protein” is used sometimes herein to refer to a polymer of greater than fifty (50) amino acid residues linked by covalent peptide bonds. As used herein, the term “polypeptide” encompasses amino acid chains of any length, including full-length proteins (e.g., antigens) or fragments thereof that is shorter than the full-length proteins. In some embodiments, the term “polypeptide” also encompasses a fusion protein as recited in the present disclosure. In some embodiments, the term “peptide, ” “polypeptide, ” and “protein” also encompasses a domain as recited in the present disclosure.

In the context of a peptide or polypeptide, the term “derivative” as used herein refers to a peptide or polypeptide that comprises an amino acid sequence of the peptide or polypeptide, or a fragment of a peptide or polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions, or additions. The term “derivative” as used herein also refers to a peptide or polypeptide, or a fragment of a peptide or polypeptide, which has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a peptide or polypeptide or a fragment of the peptide or polypeptide may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or protein. Further, a derivative of a peptide or protein or a fragment of a peptide or protein may contain one or more non-classical amino acids. In specific embodiments, a derivative is a functional derivative of the native or unmodified peptide or polypeptide from which it was derived.

The term “functional derivative” refers to a derivative that retains one or more functions or activities of the naturally occurring or starting peptide or polypeptide from which it was derived. For example, a functional derivative of a proteosome substrate may retain the ability to recruit a proteosome complex for degradation of a polypeptide sequence connected therewith.

The term “ubiquitin” is a term of art. Ubiquitin is an evolutionarily conserved, ubiquitously expressed eukaryotic protein. When comparing the amino acid sequence of ubiquitin from plants, humans, and yeast. plant and yeast ubiquitin differ by only 2 amino acids and only 3-amino-acid differences are observed when plant ubiquitin is compared to human ubiquitin (Sharp and Li, 1987; Callis and Vierstra, 1989) . Among plant species the amino acid sequence of ubiquitin is identical (Callis and Vierstra, 1989; Callis et al., 1995) . The ubiquitin gene translation product is a polyubiquitin precursor protein containing ubiquitin molecules fused in tandem. Ubiquitin is also produced as a fusion with unrelated ribosomal proteins. The fusion proteins are processed by deubiquitinating enzymes (DUBs) to produce the mature free ubiquitin monomer. (Amerik and Hochstrasser, 2004; Reyes-Turcu et al., 2009) . In certain species, the ubiquitin monomer is small, consisting of 76 amino acids having that fold into a compact, globular, tightly hydrogen-bonded structure with a terminal diglycine sequence (Vijay-Kumar et al., 1987) . In certain embodiments, the “ubiquitin monomer” has the amino acid of SEQ ID NO: 1 as disclosed herein. An “ubiquitin multimer” as used herein refers to a polypeptide comprising multiple copies of ubiquitin monomers fused directly or via linkers in a tandem. In certain embodiments, a ubiquitin multiple comprises 2, 3, 4, or more than 4 copies of ubiquitin monomers.

The terms “ubiquitin-like domain” or “UbL” are also terms of art and refer to the domain of certain proteins that are homologous to ubiquitin. Various Ubiquitin-like domain proteins (UDPs) and their UbL domains are known in the art (Yu et al., The EMBO Journal (2016) 35: 1522-1536https: //doi. org/10.15252/embj. 201593147) . For example, Rad23, Dsk2, and Ddi1 contain N-terminal UbLs, which are recognized by the proteasome subunits Rpn1, Rpn10, and Rpn13 (Elsasser et al, 2002; Funakoshi et al, 2002; Kaplun et al, 2005; Husnjak et al, 2008; Zhang et al, 2009; Gomez et al, 2011) .

The terms “proteosome” or “proteasome system” are used interchangeably herein to refer to the protease complex that are naturally occurring in vivo or artificially designed to carry out selective degradation of a polypeptide having a substrate recognized by the proteosome. Components, structures and functions of proteosome systems are well known in the art (for a review, see Tanaka Proc Jpn Acad Ser B Phys Biol Sci. 2009 Jan; 85 (1) : 12–36. ) Those of ordinary skill in the art would understand how to purify proteasome system from eukaryotic cellular preparations.

A “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/position. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or substantial substitution) , insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position. For example, a human KRAS mutant contains one or more modifications in its amino acid sequence with respect to the sequence shown in SEQ ID NO: 168, such modifications are sometime referred to as the “KRAS mutations” in this application. Various designations may be used herein to indicate the same modification. For example, amodification from a glycine at position 12 of human KRAS to an Alanine can be indicated as G12A or Gly12Ala. A modification from a glycine at position 12 of human KRAS to a cysteine can be indicated as G12C or Gly12Cys. A modification from a glycine at position 12 of human KRAS to an aspartic acid can be indicated as G12D or Gly12Asp. A modification from a glycine at position 12 of human KRAS to a valine can be indicated as G12V or Gly12Val. A modification from a glycine at position 13 of human KRAS to an aspartic acid can be indicated as G13D or Gly13Asp. A modification from a glutamine at position 61 of human KRAS to an arginine can be indicated as Q61R or Gln61Arg. In some embodiments, a mutant EGFR shares at least 75%, 80%, 85%, 90%, 95%or 97%sequence identity to the wild-type EGFR protein.

As used herein, a residue in a first KRAS sequence that “corresponds” to a reference residue in a second KRAS sequence (e.g., wild-type human KRAS or SEQ ID NO: 168) means after aligning the amino acid sequences of the first and second KRAS sequences, the residue of the first KRAS molecule that aligns at the same position as the reference residue of the second KRAS molecule. In some embodiments, the first sequence and the second KRAS sequence share at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97%sequence identity as determined by the alignment. In some embodiments, the first sequence aligns with a fragment of the second sequence, and the sequence identity between the first sequence and the aligned portion of the second sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97%. In some embodiments, the first KRAS sequence is human KRAS, or a fragment or variant thereof, and the second KRAS sequence is human KRAS (e.g., SEQ ID NO: 168) . In alternative embodiments, the first KRAS sequence is a non-human vertebrate KRAS or a fragment or variant thereof, and the second KRAS sequence is human KRAS (e.g., SEQ ID NO: 168) .

The term “sequence identity” refers to a relationship between the sequences of two or more biological molecules (e.g., a pair of polynucleotides or multiple polypeptides) , as determined by aligning and comparing the respective sequences. “Percent (%) amino acid sequence identity” with respect to a reference amino acid sequence (e.g., a reference polypeptide) is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference amino acid sequence, after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNAStar, Inc. ) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm, for example, can be as set forth below. Briefly, amino acid sequence alignments can be performed using BLASTP version 2.0.8 (Jan-05-1999) and the following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on. Nucleic acid sequence alignments can be performed using BLASTN version 2.0.6 (Sept-16-1998) and the following parameters: Match: 1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50; expect: 10.0; wordsize: 11; filter: off. Those skilled in the art will know what modifications can be made to the above parameters to either increase or decrease the stringency of the comparison, for example, and determine the relatedness of two or more sequences.

A “fragment” when used in connection of a protein or polypeptide refers to a continuous sequence of amino acids of such protein or polypeptide that is less than the full length amino acid sequence of such polypeptide. Such a fragment may arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue (s) from the amino acid sequence. Fragments may, for example, result from alternative RNA splicing or from in vivo protease activity. In certain embodiments, fragments refers to polypeptides comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, or at least 950 contiguous amino acid residues of the amino acid sequence of a polypeptide. In a specific embodiment, a fragment of a polypeptide retains at least 1, at least 2, at least 3, or more functions of the polypeptide.

The term “immunogenic fragment” as used herein in the context of a peptide or polypeptide (e.g., aprotein) , refers to a fragment of a peptide or polypeptide that retains the ability of the peptide or polypeptide in eliciting an immune response upon contacting the immune system of a mammal, including innate immune responses and/or adaptive immune responses, and/or including B cell and/or T cell mediated immunity. In some embodiments, an immunogenic fragment of a peptide or polypeptide can be an epitope. In some embodiments, an immunogenic fragment of a peptide or polypeptide can contain one or more epitopes of a disease associated protein.

The term “antigen” refers to a substance that can be recognized by the immune system of a subject (including by the adaptive immune system) , and is capable of triggering an immune response after the subject is contacted with the antigen (including an antigen-specific immune response) . In certain embodiments, the antigen is a protein associated with a diseased cell, such as a neoplastic cell. In certain embodiments, the antigen is the product of an oncogenic gene. In certain embodiments, the antigen is a mutated version of an antigen (e.g., amutated KRAS protein or a fragment thereof containing one or more mutations as compared to wild-type KRAS protein) .

A “T-cell epitope” is the site on the surface of an antigen molecule that can be recognized by the T cell receptors after the antigen has been intracellularly processed, bound to at least one MHC molecule and expressed on the surface of the antigen presenting cell (such as a dendritic cell) as an MHC-peptide complex.

A “B-cell epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human) , that is capable of eliciting an immune response. An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure. Induced epitopes are formed when the three dimensional structure of the protein is in an altered conformation, such as following activation or binding of another protein or ligand. In certain embodiments, an epitope is a three-dimensional surface feature of a polypeptide. In other embodiments, an epitope is linear feature of a polypeptide. Generally an antigen has several or many different epitopes and may react with many different antibodies.

The term “genetic vaccine” as used herein refers to a therapeutic or prophylactic composition comprising at least one nucleic acid molecule encoding an antigen associated with a target disease (e.g., an infectious disease or a neoplastic disease) . Administration of the vaccine to a subject ( “vaccination” ) allows for the production of the encoded peptide or polypeptide, thereby eliciting an immune response against the target disease in the subject. In certain embodiments, the immune response comprises adaptive immune response, such as the production of antibodies against the encoded antigen, and/or activation and proliferations of immune cells capable of specifically eliminating diseased cells expressing the antigen. In certain embodiments, the immune response further comprises innate immune response. According to the present disclosure, a vaccine can be administered to a subject either before or after the onset of clinical symptoms of the target disease. In some embodiments, vaccination of a healthy or asymptomatic subject renders the vaccinated subject immune or less susceptible to the development of the target disease. In some embodiments, vaccination of a subject showing symptoms of the disease improves the condition of, or treats, the disease in the vaccinated subject.

The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a KRAS polypeptide as described herein, in order to introduce a nucleic acid sequence into a host cell, or serve as a transcription template to carry out in vitro transcription reaction in a cell-free system to produce mRNA. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate transcription or translation control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Transcription or translation control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-transcribed or co-translated (e.g., nucleic acid molecules encoding two or more different peptides or proteins) , both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector transcription and/or translation, the encoding nucleic acids can be operationally linked to one common transcription or translation control sequence or linked to different transcription or translation control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., a mRNA transcript of the nucleic acid as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

The terms “innate immune response” and “innate immunity” are recognized in the art, and refer to non-specific defense mechanism a body’s immune system initiates upon recognition of pathogen-associated molecular patterns, which involves different forms of cellular activities, including cytokine production and cell death through various pathways. As used herein, innate immune responses include, without limitation, increased production of inflammation cytokines (e.g., type I interferon or IL-10 production) , activation of the NFκB pathway, increased proliferation, maturation, differentiation and/or survival of immune cells, and in some cases, induction of cell apoptosis. Activation of the innate immunity can be detected using methods known in the art, such as measuring the (NF) -κB activation.

The terms “adaptive immune response” and “adaptive immunity” are recognized in the art, and refer to antigen-specific defense mechanism a body’s immune system initiates upon recognition of a specific antigen, which include both humoral response and cell-mediated responses. As used herein, adaptive immune responses include cellular responses that is triggered and/or augmented by a vaccine composition, such as a genetic composition described herein. In some embodiments, the vaccine composition comprises an antigen that is the target of the antigen-specific adaptive immune response. In other embodiments, the vaccine composition, upon administration, allows the production in an immunized subject of an antigen that is the target of the antigen-specific adaptive immune response. Activation of an adaptive immune response can be detected using methods known in the art, such as measuring the antigen-specific antibody production, or the level of antigen-specific cell-mediated cytotoxicity.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted immunoglobulin bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9: 457-92) . To assess ADCC activity of a molecule of interest, an in vitro ADCC assay (see, e.g., US Pat. Nos. 5,500,362 and 5,821,337) can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model (see, e.g., Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95: 652-56) . Antibodies with little or no ADCC activity may be selected for use.

“Antibody-dependent cellular phagocytosis” or “ADCP” refers to the destruction of target cells via monocyte or macrophage-mediated phagocytosis when immunoglobulin bound onto Fc receptors (FcRs) present on certain phagocytotic cells (e.g., neutrophils, monocytes, and macrophages) enable these phagocytotic cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell. To assess ADCP activity of a molecule of interest, an in vitro ADCP assay (see, e.g., Bracher et al., 2007, J. Immunol. Methods 323: 160-71) can be performed. Useful phagocytotic cells for such assays include peripheral blood mononuclear cells (PBMC) , purified monocytes from PBMC, or U937 cells differentiated to the mononuclear type. Alternatively or additionally, ADCP activity of the molecule of interest may be assessed in vivo, for example, in an animal model (see, e.g., Wallace et al., 2001, J. Immunol. Methods 248: 167-82) . Antibodies with little or no ADCP activity may be selected for use.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. Moreover, an exemplary FcR is one that binds an IgG antibody (e.g., agamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor” ) and FcγRIIB (an “inhibiting receptor” ) , which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof (see, e.g., 1997, Annu. Rev. Immunol. 15: 203-34) . Various FcRs are known (see, e.g., Ravetch and Kinet, 1991, Annu. Rev. Immunol. 9: 457-92; Capel et al., 1994, Immunomethods 4: 25-34; and de Haas et al., 1995, J. Lab. Clin. Med. 126: 330-41) . Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (see, e.g., Guyer et al., 1976, J. Immunol. 117: 587-93; and Kim et al., 1994, Eu. J. Immunol. 24: 2429-34) . Antibody variants with improved or diminished binding to FcRs have been described (see, e.g., WO 2000/42072; U.S. Pat. Nos. 7,183,387; 7,332,581; and 7.335, 742; Shields et al. 2001, J. Biol. Chem. 9 (2) : 6591-604) .

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay (see, e.g., Gazzano-Santoro et al., 1996, J. Immunol. Methods 202: 163) may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased C1q binding capability have been described (see, e.g., US Pat. No. 6,194,551; WO 1999/51642; Idusogie et al., 2000, J. Immunol. 164: 4178-84) . Antibodies with little or no CDC activity may be selected for use.

The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) , each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995) ; and Kuby, Immunology (3d ed. 1997) . In specific embodiments, the specific molecular antigen can be bound by an antibody provided herein, including a polypeptide, a fragment or an epitope thereof. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc. ) , Fab fragments, F (ab’) fragments, F (ab) ₂ fragments, F (ab’) ₂ fragments, disulfide-linked Fvs (dsFv) , Fd fragments, Fv fragments, diabody, triabody, tetrabody, and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen-binding domains or molecules that contain an antigen-binding site (e.g., one or more CDRs of an antibody) . Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989) ; Mol. Biology and Biotechnology: A Comprehensive DeskReference (Myers ed., 1995) ; Huston et al., 1993, Cell Biophysics 22: 189-224; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2d ed. 1990) . The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule.

The term “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a lipid nanoparticle composition as described herein) into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art. When a disease, disorder, condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptoms thereof. When a disease, disorder, condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptoms thereof.

“Chronic” administration refers to administration of the agent (s) in a continuous mode (e.g., for a period of time such as days, weeks, months, or years) as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

The term “targeted delivery” or the verb form “target” as used herein refers to the process that promotes the arrival of a delivered agent (such as a therapeutic payload molecule in a lipid nanoparticle composition as described herein) at a specific organ, tissue, cell and/or intracellular compartment (referred to as the targeted location) more than any other organ, tissue, cell or intracellular compartment (referred to as the non-target location) . Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in a targeted cell population with the concentration of the delivered agent at a non-target cell population after systemic administration. In certain embodiments, targeted delivery results in at least 2 folds higher concentration at a targeted location as compared to a non-target location.

An “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a disease, disorder, or condition, including, for example, infection and neoplasia. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., avaccine composition) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder, or condition, and/or a symptom related thereto (e.g., an infectious disease such as caused by viral infection, or a neoplastic disease such as cancer) . A “therapeutically effective amount” of a substance/molecule/agent of the present disclosure (e.g., the lipid nanoparticle composition as described herein) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term “therapeutically effective amount” refers to an amount of a lipid nanoparticle composition as described herein or a therapeutic or prophylactic agent contained therein (e.g., a therapeutic mRNA) effective to “treat” a disease, disorder, or condition, in a subject or mammal.

A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing, delaying, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, condition, or associated symptom (s) (e.g., an infectious disease such as caused by viral infection, or a neoplastic disease such as cancer) . Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, a prophylactically effective amount may be less than a therapeutically effective amount. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

The terms “treat, ” “treating, ” and “treatment” refer to an alleviation, in whole or in part, of a disorder, disease or condition, or one or more of the symptoms associated with a disorder, disease, or condition, or slowing or halting of further progression or worsening of those symptoms, or alleviating or eradicating the cause (s) of the disorder, disease, or condition itself.

The terms “prevent, ” “preventing, ” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom (s) (e.g., an infectious disease such as caused by viral infection, or a neoplastic disease such as cancer) .

The terms “manage, ” “managing, ” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent) , which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as a lipid nanoparticle composition as described herein) to “manage” an infectious or neoplastic disease, one or more symptoms thereof, so as to prevent the progression or worsening of the disease.

The term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset, or spread of disease and/or symptom related thereto in a subject.

The term “therapeutic agent” refers to any agent that can be used in treating, preventing, or alleviating a disease, disorder, or condition, including in the treatment, prevention, or alleviation of one or more symptoms of a disease, disorder, or condition and/or a symptom related thereto.

The term “therapy” refers to any protocol, method, and/or agent that can be used in the prevention, management, treatment, and/or amelioration of a disease, disorder, or condition. In certain embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment, and/or amelioration of a disease, disorder, or condition, known to one of skill in the art such as medical personnel.

As used herein, a “prophylactically effective serum titer” is the serum titer of an antibody in a subject (e.g., a human) , that totally or partially inhibits the development, recurrence, onset, or spread of a disease, disorder, or condition, and/or symptom related thereto in the subject.

In certain embodiments, a “therapeutically effective serum titer” is the serum titer of an antibody in a subject (e.g., a human) , that reduces the severity, the duration, and/or the symptoms associated with a disease, disorder, or condition, in the subject.

The term “serum titer” refers to an average serum titer in a subject from multiple samples (e.g., at multiple time points) or in a population of at least 10, at least 20, at least 40 subjects, up to about 100, 1000, or more.

The term “side effects” encompasses unwanted and/or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent) . Unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., a prophylactic or therapeutic agent) might be harmful, uncomfortable, or risky. Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills, and fatigue, digestive tract problems, and allergic reactions. Additional undesired effects experienced by patients are numerous and known in the art. Many are described in Physician’s Desk Reference (68th ed. 2014) .

The terms “subject” and “patient” may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc. ) or a primate (e.g., monkey and human) . In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having an infectious disease or neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or neoplastic disease.

The term “detectable probe” refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

The term “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an antigen encoded by an mRNA molecule as described herein, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation) .

As used herein and unless otherwise specified, the term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many nonpolar organic solvents. While lipids generally have poor solubility in water, there are certain categories of lipids (e.g., lipids modified by polar groups, e.g., DMG-PEG2000) that have limited aqueous solubility and can dissolve in water under certain conditions. Known types of lipids include biological molecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three classes: (1) “simple lipids, ” which include fats and oils as well as waxes; (2) “compound lipids, ” which include phospholipids and glycolipids (e.g., DMPE-PEG2000) ; and (3) “derived lipids” such as steroids. Further, as used herein, lipids also encompass lipidoid compounds. The term “lipidoid compound, ” also simply “lipidoid” , refers to a lipid-like compound (e.g., an amphiphilic compound with lipid-like physical properties) .

The term “lipid nanoparticle” or “LNP” refers to a particle having at least one dimension on the order of nanometers (nm) (e.g., 1 to 1,000 nm) , which contains one or more types of lipid molecules. The LNP provided herein can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules) . In some embodiments, the LNP comprises a non-lipid payload molecule either partially or completely encapsulated inside a lipid shell. Particularly, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA encoding a KRAS polypeptide or fragment thereof) , and the lipid components of the LNP comprise at least one cationic lipid. Without being bound by the theory, it is contemplated that the cationic lipids can interact with the negatively charged payload molecules and facilitates incorporation and/or encapsulation of the payload into the LNP during LNP formation. Other lipids that can form part of a LNP as provided herein include but are not limited to neutral lipids and charged lipids, such as steroids, polymer conjugated lipids, and various zwitterionic lipids.

The term “cationic lipid” refers to a lipid that is either positively charged at any pH value or hydrogen ion activity of its environment, or capable of being positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use) . Thus, the term “cationic” encompasses both “permanently cationic” and “cationisable. ” In certain embodiments, the positive charge in a cationic lipid results from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge in the environment of its intended use (e.g., at physiological pH) . In certain embodiments, the cationic lipid is one or more lipids of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) as described herein.

The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid (PEG-lipid) , in which the polymer portion comprises a polyethylene glycol.

The term “neutral lipid” encompasses any lipid molecules existing in uncharged forms or neutral zwitterionic forms at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH condition in an environment of the intended uses of the lipids, such as the physiological pH. As non-limiting examples, neutral lipids that can be used in connection with the present disclosure include, but are not limited to, phosphotidylcholines such as 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , phophatidylethanolamines such as 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 2- ( (2, 3-bis (oleoyloxy) propyl) dimethylammonio) ethyl hydrogen phosphate (DOCP) , sphingomyelins (SM) , ceramides, steroids such as sterols and their derivatives. Neutral lipids as provided herein may be synthetic or derived (isolated or modified) from a natural source or compound.

The term “charged lipid” encompasses any lipid molecules that exist in either positively charged or negatively charged forms at a selected pH or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH condition in an environment of the intended uses of the lipids, such as the physiological pH. As non-limiting examples, neutral lipids that can be used in connection with the present disclosure include, but are not limited to, phosphatidylserines, phosphatidic acids, phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates, dialkyl trimethylarnmonium-propanes, (e.g., DOTAP, DOTMA) , dialkyl dimethylaminopropanes, ethyl phosphocholines, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol) , 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na) , 1, 2-dioleoyl-sn-glycero-3-phospho- (1'-rac-glycerol) sodium salt (DOPG-Na) , and 1, 2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA-Na) . Charged lipids as provided herein may be synthetic or derived (isolated or modified) from a natural source or compound.

As used herein, and unless otherwise specified, the term “alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated. In one embodiment, the alkyl group has, for example, from one to twenty-four carbon atoms (C₁-C₂₄ alkyl) , four to twenty carbon atoms (C₄-C₂₀ alkyl) , six to sixteen carbon atoms (C₆-C₁₆ alkyl) , six to nine carbon atoms (C₆-C₉ alkyl) , one to fifteen carbon atoms (C₁-C₁₅ alkyl) , one to twelve carbon atoms (C₁-C₁₂ alkyl) , one to eight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl) , n-butyl, n-pentyl, 1, 1-dimethylethyl (t-butyl) , 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, an alkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkenyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds. The term “alkenyl” also embraces radicals having “cis” and “trans” configurations, or alternatively, “E” and “Z” configurations, as appreciated by those of ordinary skill in the art. In one embodiment, the alkenyl group has, , for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkenyl) , four to twenty carbon atoms (C₄-C₂₀ alkenyl) , six to sixteen carbon atoms (C₆-C₁₆ alkenyl) , six to nine carbon atoms (C₆-C₉ alkenyl) , two to fifteen carbon atoms (C₂-C₁₅ alkenyl) , two to twelve carbon atoms (C₂-C₁₂ alkenyl) , two to eight carbon atoms (C₂-C₈ alkenyl) or two to six carbon atoms (C₂-C₆ alkenyl) and which is attached to the rest of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1, 4-dienyl, and the like. Unless otherwise specified, an alkenyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkynyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon triple bonds. In one embodiment, the alkynyl group has, for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkynyl) , four to twenty carbon atoms (C₄-C₂₀ alkynyl) , six to sixteen carbon atoms (C₆-C₁₆ alkynyl) , six to nine carbon atoms (C₆-C₉ alkynyl) , two to fifteen carbon atoms (C₂-C₁₅ alkynyl) , two to twelve carbon atoms (C₂-C₁₂ alkynyl) , two to eight carbon atoms (C₂-C₈ alkynyl) or two to six carbon atoms (C₂-C₆ alkynyl) and which is attached to the rest of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise specified, an alkynyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “alkylene” or “alkylene chain” refers to a straight or branched multivalent (e.g., divalent or trivalent) hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated. In one embodiment, the alkylene has, for example, from one to twenty-four carbon atoms (C₁-C₂₄ alkylene) , one to fifteen carbon atoms (C₁-C₁₅ alkylene) , one to twelve carbon atoms (C₁-C₁₂ alkylene) , one to eight carbon atoms (C₁-C₈ alkylene) , one to six carbon atoms (C₁-C₆ alkylene) , two to four carbon atoms (C₂-C₄ alkylene) , one to two carbon atoms (C₁-C₂ alkylene) . Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group canbe through one carbon or any two carbons within the chain. Unless otherwise specified, an alkylene chain is optionally substituted.

As used herein, and unless otherwise specified, the term “alkenylene” refers to a straight or branched multivalent (e.g., divalent or trivalent) hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which contains one or more carbon-carbon double bonds. In one embodiment, the alkenylene has, for example, from two to twenty-four carbon atoms (C₂-C₂₄ alkenylene) , two to fifteen carbon atoms (C₂-C₁₅ alkenylene) , two to twelve carbon atoms (C₂-C₁₂ alkenylene) , two to eight carbon atoms (C₂-C₈ alkenylene) , two to six carbon atoms (C₂-C₆ alkenylene) or two to four carbon atoms (C₂-C₄ alkenylene) . Examples of alkenylene include, but are not limited to, ethenylene, propenylene, n-butenylene, and the like. The alkenylene is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkenylene to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise specified, an alkenylene is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which is saturated. Cycloalkyl group may include fused or bridged ring systems. In one embodiment, the cycloalkyl has, for example, from 3 to 15 ring carbon atoms (C₃-C₁₅ cycloalkyl) , from 3 to 10 ring carbon atoms (C₃-C₁₀ cycloalkyl) , or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkyl) . The cycloalkyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl radicals include, but are not limited to, adamantyl, norbornyl, decalinyl, 7, 7-dimethyl-bicyclo [2.2.1] heptanyl, and the like. Unless otherwise specified, a cycloalkyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkylene” is a multivalent (e.g., divalent or trivalent) cycloalkyl group. Unless otherwise specified, a cycloalkylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkenyl” refers to a non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, and which includes one or more carbon-carbon double bonds. Cycloalkenyl may include fused or bridged ring systems. In one embodiment, the cycloalkenyl has, for example, from 3 to 15 ring carbon atoms (C₃-C₁₅ cycloalkenyl) , from 3 to 10 ring carbon atoms (C₃-C₁₀ cycloalkenyl) , or from 3 to 8 ring carbon atoms (C₃-C₈ cycloalkenyl) . The cycloalkenyl is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl radicals include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise specified, a cycloalkenyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “cycloalkenylene” is a multivalent (e.g., divalent or trivalent) cycloalkenyl group. Unless otherwise specified, a cycloalkenylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “heterocyclyl” refers to a non-aromatic radical monocyclic or polycyclic moiety that contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorous, and sulfur. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom. A heterocyclyl group can be a monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic ring system, wherein the polycyclic ring systems can be a fused, bridged or spiro ring system. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or more rings. A heterocyclyl group can be saturated or partially unsaturated. Saturated heterocycloalkyl groups can be termed “heterocycloalkyl” . Partially unsaturated heterocycloalkyl groups can be termed “heterocycloalkenyl” if the heterocyclyl contains at least one double bond, or “heterocycloalkynyl” if the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3-to 18-membered heterocyclyl) , 4 to 18 ring atoms (4-to 18-membered heterocyclyl) , 5 to 18 ring atoms (3-to 18-membered heterocyclyl) , 4 to 8 ring atoms (4-to 8-membered heterocyclyl) , or 5 to 8 ring atoms (5-to 8-membered heterocyclyl) . Whenever it appears herein, a numerical range such as “3 to 18” refers to each integer in the given range; e.g., “3 to 18 ring atoms” means that the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc., up to and including 18 ring atoms. Examples of heterocyclyl groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl, and isoquinolyl. Unless otherwise specified, a heterocyclyl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heterocyclylene” is a multivalent (e.g., divalent or trivalent) heterocyclyl group. Unless otherwise specified, a heterocyclylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “aryl” refers to a monocyclic aromatic group and/or multicyclic monovalent aromatic group that contain at least one aromatic hydrocarbon ring. In certain embodiments, the aryl has from 6 to 18 ring carbon atoms (C₆-C₁₈ aryl) , from 6 to 14 ring carbon atoms (C₆-C₁₄ aryl) , or from 6 to 10 ring carbon atoms (C₆-C₁₀ aryl) . Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl. The term “aryl” also refers to bicyclic, tricyclic, or other multicyclic hydrocarbon rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, for example, dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetralinyl) . Unless otherwise specified, an aryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “arylene” is a multivalent (e.g., divalent or trivalent) aryl group. Unless otherwise specified, an arylene group is optionally substituted.

As used herein, and unless otherwise specified, the term “heteroaryl” refers to a monocyclic aromatic group and/or multicyclic aromatic group that contains at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. The heteroaryl may be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10 ring atoms. The term “heteroaryl” also refers to bicyclic, tricyclic, or other multicyclic rings, where at least one of the rings is aromatic and the others of which may be saturated, partially unsaturated, or aromatic, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrollinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise specified, a heteroaryl group is optionally substituted.

As used herein, and unless otherwise specified, the term “heteroarylene” is a multivalent (e.g., divalent or trivalent) heteroaryl group. Unless otherwise specified, a heteroarylene group is optionally substituted.

When the groups described herein are said to be “substituted, ” they may be substituted with any appropriate substituent or substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: a halogen atom such as F, CI, Br, or I; cyano; oxo (=O) ; hydroxyl (-OH) ; alkyl; alkenyl; alkynyl; cycloalkyl; aryl; - (C=O) OR’; -O (C=O) R’; -C (=O) R’; -OR’; -S (O) _xR’; -S-SR’; -C (=O) SR’; -SC (=O) R’; -NR’R’; -NR’ C (=O) R’; -C (=O) NR’R’; -NR’C (=O) NR’R’; -OC (=O) NR’R’; -NR’ C (=O) OR’; -NR’S (O) _xNR’R’; -NR’S (O) _xR’; and-S (O) _xNR’R’, wherein: R’ is, at each occurrence, independently H, C₁-C₁₅ alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments the substituent is a C₁-C₁₂ alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR’) . In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR’R’) .

As used herein, and unless otherwise specified, the term “optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

As used herein, and unless otherwise specified, the term “prodrug” of a biologically active compound refers to a compound that may be converted under physiological conditions or by solvolysis to the biologically active compound. In one embodiment, the term “prodrug” refers to a metabolic precursor of the biologically active compound that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to the biologically active compound. Prodrugs are typically rapidly transformed in vivo to yield the parent biologically active compound, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985) , pp. 7-9, 21-24 (Elsevier, Amsterdam) ) . A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

In one embodiment, the term “prodrug” is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds wherein a hydroxyl, amino or mercapto group is bonded to any group that, when the prodrug of the compound is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino or free mercapto group, respectively.

Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol or amide derivatives of amine functional groups in the compounds provided herein.

As used herein, and unless otherwise specified, the term “pharmaceutically acceptable salt” includes both acid and base addition salts.

Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

Examples of pharmaceutically acceptable base addition salt include, but are not limited to, salts prepared from addition of an inorganic base or an organic base to a free acid compound. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. In one embodiment, the inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. In one embodiment, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

A compound provided herein may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R) -or (S) -or, as (D) -or (L) -for amino acids. Unless otherwise specified, a compound provided herein is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-) , (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC) . When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

As used herein, and unless otherwise specified, the term “isomer” refers to different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space. “Atropisomers” are stereoisomers from hindered rotation about single bonds. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any proportion can be known as a “racemic” mixture. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.

“Stereoisomers” can also include E and Z isomers, or a mixture thereof, and cis and trans isomers or a mixture thereof. In certain embodiments, a compound described herein is isolated as either the E or Z isomer. In other embodiments, a compound described herein is a mixture of the E and Z isomers.

“Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution.

It should also be noted a compound described herein can contain unnatural proportions of atomic isotopes at one or more of the atoms. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H) , iodine-125 (¹²⁵I) , sulfur-35 (³⁵S) , or carbon-14 (¹⁴C) , or may be isotopically enriched, such as with deuterium (²H) , carbon-13 (¹³C) , or nitrogen-15 (¹⁵N) . As used herein, an “isotopolog” is an isotopically enriched compound. The term “isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. “Isotopically enriched” may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. The term “isotopic composition” refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of a compound described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, there are provided isotopologs of a compound described herein, for example, the isotopologs are deuterium, carbon-13, and/or nitrogen-15 enriched. As used herein, “deuterated” , means a compound wherein at least one hydrogen (H) has been replaced by deuterium (indicated by D or ²H) , that is, the compound is enriched in deuterium in at least one position.

It should be noted that if there is a discrepancy between a depicted structure and a name for that structure, the depicted structure is to be accorded more weight.

As used herein, and unless otherwise specified, the term “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

The term “composition” is intended to encompass a product containing the specified ingredients (e.g., a mRNA molecule provided herein) in, optionally, the specified amounts.

“Substantially all” refers to 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 98%, at least about 99%, or about 100%.

As used herein, and unless otherwise indicated, the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.05%, or less of a given value or range. As used herein, when “about” is used in connection with a numerical range, the term “about” is meant to apply to both ends of such modified range (e.g., “about 5 to 10” means “about 5 to about 10” ) .

The singular terms “a, ” “an, ” and “the” as used herein include the plural reference unless the context clearly indicates otherwise.

All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference in their entirety as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the Experimental section and examples are intended to illustrate but not limit the scope of invention described in the claims.
5.3 Therapeutic Nucleic Acids

In one aspect, provided herein is an immunomodulating nucleic acid system (e.g., DNA or RNA based system) comprising one or more nucleic acid molecules encoding for a target antigen, such as the KRAS domain according to the present invention and one or more peptide-based elements configured for modulating, such as enhancing, immunogenicity of the target antigen upon co-expression with the target antigen.

In some embodiments, the immunomodulating nucleic acid system is configured for enhancing a T cell mediated immune response against the target antigen. In some embodiments, the immunomodulating nucleic acid system comprises a first coding sequence encoding a helper T cell epitope. In some embodiments, the immunomodulating nucleic acid system is configured for enhancing a B cell mediated immune response against the target antigen.

In some embodiments, the immunomodulating nucleic acid system comprises a second coding sequence encoding a protein that modulates an immune response against the target antigen, and such protein, functional fragment or functional derivative thereof is herein referred to as an “immunomodulator. ” In some embodiments, the immunomodulator stimulates an immune response against the target antigen. In some embodiments, the immunomodulator inhibits an immune response against the target antigen. In some embodiments, the immunomodulator enhance T cell mediated immune response against the target antigen. In some embodiments, the immunomodulator enhance B cell mediated immune response against the target antigen.

In some embodiments, the immunomodulating nucleic acid system comprises a third coding sequence encoding a trafficking peptide. In some embodiments, the trafficking peptide is configured for intracellularly transporting the target antigen towards proteosome upon expression. In some embodiments, the trafficking peptide is configured for intracellularly transporting the target antigen towards endosomes or lysosomes upon expression.

In some embodiments, the immunomodulating nucleic acid system comprises a fourth coding sequence encoding a signal peptide.

In some embodiments, the immunomodulating nucleic acid system comprises a fifth coding sequence encoding a ubiquitin domain. In some embodiments, the ubiquitin peptide is configured for marking the target antigen for intracellular degradation through the ubiquitin pathway.

In some embodiments, the immunomodulating nucleic acid system comprises a sixth and/or additional coding sequences encoding one or more peptide linkers connecting one or more immunomodulating peptides provided herein with the target antigen as a fusion protein. In some embodiments, the peptide linker is a spacer peptide. In some embodiments, the spacer peptide is configured to determine a suitable distance between the ubiquitin peptide and the target antigen, thereby marking the target antigen for efficient degradation.

In some embodiments, it is desirable to provide the target antigen and one or more of the immunomodulating peptides as separate protein or peptide-based entities. For example, in some embodiments, the target antigen is configured to be transported to one intracellular compartment for post-translational processing and the immunomodulating peptide is configured for transportation to a different population of cells to stimulate immune response of such cell population. Hence, in some embodiments, the peptide linker is a cleavable linker configured to be cleaved to release multiple domains or portions of a fusion protein as separate peptides upon expression of the fusion protein.

In some embodiments, the peptide linker is not configured for intracellular cleavage. For example, in some embodiments, the peptide linker is configured to provide a connection of suitable flexibility and length between two functional domains of a fusion protein. Hence, in some embodiments, the peptide linker is not cleavable.

In some embodiments, the immunomodulator and target antigen (or target antigen linked to functional elements, such as MITD) expressed by one nucleic acid molecule are not included in the same fusion protein.

According to the present disclosure, the one or more coding sequences encoding the target antigen and the additional functional peptides can be arranged in a single expression cassette in the same nucleic acid molecule, or as multiple expression cassettes in at least two different nucleic acid molecules, as long as the functional peptides as provided herein can be co-expressed with the target antigen and are capable of enhancing immunogenicity of the target antigen as intended, upon co-expression with the target antigen. In some embodiments, the immunomodulating nucleic acid system comprises one isolated nucleic acid molecule (e.g., DNA or RNA) encoding a fusion protein comprising a target antigen and one or more of the functional peptides as provided herein. In some embodiments, the immunomodulating nucleic acid system comprises a vector containing the nucleic acid molecule. In some embodiments, the immunomodulating nucleic acid system comprises at least two isolated nucleic acid molecules (e.g., DNA or RNA) collectively encoding a target antigen and one or more functional peptides as provided herein, respectively. In some embodiments, the immunomodulating nucleic acid system comprises at least two vectors each containing one of the at least two nucleic acid molecules.

In some embodiments, the present invention provides an isolated nucleic acid (e.g., DNA or RNA) comprising a coding sequence encoding a target antigen. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a helper T cell epitope. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising the target antigen and the helper T cell epitope. In some embodiment, the T cell epitope is configured for enhancing a T cell mediated immune response towards the target antigen. In some embodiments, the helper T cell epitope described herein is a universal CD4 epitope. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE. In some embodiments, the helper T cell epitope is PADRE.

In alternative embodiments, the isolated nucleic acid molecule comprises two coding sequences encoding the target antigen and the helper T cell epitope as separate polypeptides, respectively. In specific embodiments one polypeptide comprises (a) the target antigen, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen.

In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a trafficking peptide. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising the target antigen and the trafficking peptide. In alternative embodiments, the isolated nucleic acid molecule comprises at least two coding sequences encoding the target antigen and the trafficking peptide as separate polypeptides, respectively.

In one aspect, the present invention provides an isolated nucleic acid (e.g., DNA or RNA) comprising a coding sequence encoding a fusion protein comprising a target antigen and a trafficking peptide, wherein the trafficking peptide is derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor. In some embodiments, the trafficking peptide is positioned C-terminal to the target antigen in the fusion protein. In some embodiments, the trafficking peptide is positioned N-terminal to the target antigen in the fusion protein. In some embodiments, the fusion protein further comprises a helper T cell epitope (such as an epitope of tetanus and diphtheria toxoids, for example PADRE) , optionally positioned between the target antigen and the trafficking peptide.

In specific embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide. In alternative specific embodiments, the isolated nucleic acid molecule comprises multiple coding sequences collectively encoding (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide in at least two separate polypeptides. In specific embodiments one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In alternative specific embodiments, both of the at least two polypeptides comprise the target antigen.

In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the trafficking peptide, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In specific embodiments, one polypeptide comprises (a) the target antigen and (c) the trafficking peptide, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the trafficking peptide.

In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding an immunomodulator peptide. In some embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising the target antigen and the immunomodulator. In alternative embodiments, the isolated nucleic acid molecule comprises at least two coding sequences encoding the target antigen and the immunomodulator as separate polypeptides, respectively. In some embodiments, the immunomodulator is configured for enhancing immune response against the target antigen. In some embodiments, the immunomodulator is configured for maintaining an immune response against the target antigen. In some embodiments, the immunomodulator is configured for reducing an immune response against the target antigen.

In some embodiments, the isolated nucleic acid molecule further comprises a coding sequence encoding an immunomodulator, such as GM-CSF and/or STING, optionally connected with the fusion protein with a cleavable linker (e.g., a P2A linker) . In some embodiments, the fusion protein further comprises a signal peptide (such as a signal peptide derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor) .

In specific embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator. In alternative specific embodiments, the isolated nucleic acid molecule comprises multiple coding sequences collectively encoding (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator in at least two separate polypeptides. In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In alternative specific embodiments, both of the at least two polypeptides comprise the target antigen.

In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope, and (c) the immunomodulator, and does not contain the target antigen.

In alternative specific embodiments, both of the at least two polypeptides comprise the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the otherpolypeptide comprises (a) the target antigen and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen and (c) the immunomodulator.

In specific embodiments, the isolated nucleic acid molecule comprises a coding sequence encoding a fusion protein comprising (a) the target antigen, (b) the helper T cell epitope, (c) the immunomodulator, and (d) the trafficking peptide. In alternative specific embodiments, the isolated nucleic acid molecule comprises multiple coding sequences collectively encoding (a) the target antigen, (b) the helper T cell epitope, (c) the immunomodulator, and (d) the trafficking peptide in at least two separate polypeptides. In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In alternative embodiments, both of the at least two polypeptides comprise the target antigen.

In specific embodiments, one polypeptide comprises the target antigen, while the other polypeptide does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (d) the trafficking peptide, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (c) the immunomodulator and does not contain the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (b) the helper T cell epitope and (c) the immunomodulator, and does not contain the target antigen.

In alternative embodiments, both of the at least two polypeptides comprise the target antigen. In yet specific embodiments, one polypeptide comprises (a) the target antigen and (d) the trafficking peptide, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope and (c) the immunomodulator. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (b) the helper T cell epitope. In yet specific embodiments, one polypeptide comprises (a) the target antigen, (d) the trafficking peptide, (b) the helper T cell epitope, and (c) the immunomodulator, while the other polypeptide comprises (a) the target antigen, (d) the trafficking peptide, and (c) the immunomodulator.

In some embodiments, the isolated nucleic acid further comprises an enhancement component, e.g., a ubiquitin peptide that optionally further comprises a C-terminal extension peptide. In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA, e.g., an mRNA, a self-amplifying RNA, or a circular RNA.

In some embodiments, the one or more polypeptides encoded by the present immunomodulating nucleic acid system further comprises a signal peptide. Signal peptides that can be used in connection with the present disclosure can be selected from any signal peptides of the present disclosure.

In specific embodiments, the immunomodulating nucleic acid system comprises an isolated nucleic acid (e.g., DNA or RNA) comprising: 1) a coding sequence encoding a fusion protein comprising a target antigen and a helper T cell epitope (such as an epitope of tetanus and diphtheria toxoids, for example PADRE) , and 2) a coding sequence encoding an immunomodulator, wherein the immunomodulator comprises GM-CSF and/or STING. In some embodiments, the isolated nucleic acid is DNA or RNA (e.g., mRNA) . In some embodiments, the helper T cell epitope is positioned C-terminal to the target antigen in the fusion protein. In some embodiments, the immunomodulator is connected with the fusion protein with a cleavable linker (e.g., a P2A linker) . In some embodiments, the fusion protein further comprises a trafficking peptide (such as a trafficking peptide derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor) . In some embodiments, the fusion protein further comprises a signal peptide (such as a signal peptide derived from a protein selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor) . In some embodiments, the isolated nucleic acid is DNA. In some embodiments, the isolated nucleic acid is RNA, e.g., an mRNA, a self-amplifying RNA, a circular RNA or a cyclization precursor RNA. In some embodiments, the isolated nucleic acid further comprises an enhancement component, e.g., a ubiquitin peptide that optionally further comprises a C-terminal extension peptide.

In specific embodiments, the immunomodulating nucleic acid system comprises a nucleic acid (e.g., DNR or RNA) encoding a fusion protein. In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide (e.g., the KRAS domain according to the present invention) , a target antigen, a helper T cell epitope (e.g., PADRE) , a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) , a cleavable linker (e.g., p2A) and a immunomodulator (e.g., STING or GM-CSF) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) , a helper T cell epitope (e.g., PADRE) , a cleavable linker (e.g., p2A) and a immunomodulator (e.g., STING or GM-CSF) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a ubiquitin peptide fused to a target antigen. In specific embodiments, the ubiquitin peptide is connected to the target antigen by a spacer having at least 25 amino acids.

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., P2, P16) , a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) , and a helper T cell epitope (e.g., P2, P16) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a ubiquitin peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) . In specific embodiments, the ubiquitin peptide is connected with the target antigen by a spacer peptide having at least 25 amino acids.

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) , a cleavable linker (e.g., p2A) and a immunomodulator (e.g., STING or GM-CSF) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., pp65) , and a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) , and a helper T cell epitope (e.g., pp65) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a helper T cell epitope (e.g., PADRE) , and a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, a trafficking peptide (e.g., MHC class I trafficking peptide; MITD) , and a helper T cell epitope (e.g., PADRE) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide (e.g., LAMP3 signal peptide) , a target antigen, a helper T cell epitope (e.g., p2p16) , and a trafficking peptide (e.g., LAMP3 transmembrane domain) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide (e.g., LAMP3 signal peptide) , a target antigen, a trafficking peptide (e.g., LAMP3 transmembrane domain) , and a helper T cell epitope (e.g., p2p16) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., MHC class I trafficking peptide) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., HLA-E trafficking peptide or trafficking signal) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., LAMP1 trafficking peptide or trafficking signal) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., LAMP3 trafficking peptide or trafficking signal) . In specific embodiments, the fusion protein comprises, from the N-to-C direction, a signal peptide, a target antigen, and a trafficking peptide (e.g., HLA-DMB trafficking peptide or trafficking signal) .

In specific embodiments, the fusion protein comprises, from the N-to-C direction, a target antigen, a helper T cell epitope (e.g., PADRE) , a cleavable linker (e.g., P2A) and an immunomodulator (e.g., GM-CSF) .

In one aspect, provided herein are therapeutic nucleic acid molecules for the management, prevention and treatment of a neoplastic disease driven by one or more activating oncogenic mutations. In some embodiments, the therapeutic nucleic acid encodes a polypeptide comprising a tumor neoepitope, such as a mutant fragment from a tumor antigen. In some embodiments, upon administration of the therapeutic nucleic acid into a subject in need thereof, it is expressed by the cells in the subject to produce the encoded polypeptide. In some embodiments, the therapeutic nucleic acid molecules are DNA molecules. In other embodiments, the therapeutic nucleic acid molecules are RNA molecules. In particular embodiments, the therapeutic nucleic acid molecules are mRNA molecules.

In some embodiments, the therapeutic nucleic acid molecule is formulated in a vaccine composition. In some embodiments, the vaccine composition is a genetic vaccine as described herein. In some embodiments, the vaccine composition comprises an mRNA molecule as described herein.

In some embodiments, the mRNA molecule of the present disclosure encodes a polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A peptide or polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by an mRNA payload can have a therapeutic effect when expressed in a cell.

In some embodiment, the mRNA molecule of the present disclosure comprises at least one coding region encoding the polypeptide of interest (e.g., an open reading frame (ORF) ) . In some embodiments, the nucleic acid molecule further comprises at least one untranslated region (UTR) . In particular embodiments, the untranslated region (UTR) is located upstream (to the 5’-end) of the coding region, and is referred to herein as the 5’-UTR. In particular embodiments, the untranslated region (UTR) is located downstream (to the 3’-end) of the coding region, and is referred to herein as the 3’-UTR. In particular embodiments, the nucleic acid molecule comprises both a 5’-UTR and a 3’-UTR. In some embodiments, the 5’-UTR comprises a 5’-Cap structure. In some embodiments, the nucleic acid molecule comprises a Kozak sequence (e.g., in the 5’-UTR) . In some embodiments, the nucleic acid molecule comprises a poly-A region (e.g., in the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises a polyadenylation signal (e.g., in the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises stabilizing region (e.g., in the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in the 5’-UTR and/or the 3’-UTR) . In some embodiments, the nucleic acid molecule comprises one or more intronic regions capable of being excised during splicing. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a 5’-UTR, and a coding region. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a coding region and a 3’-UTR. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a 5’-UTR, a coding region, and a 3’-UTR.
5.3.1 Coding Region

In some embodiments, the nucleic acid molecule of the present disclosure comprises at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) that encodes for a single polypeptide. In some embodiments, the coding region comprises at least two ORFs, each encoding a polypeptide. In those embodiments where the coding region comprises more than one ORFs, the encoded peptides and/or proteins can be the same as or different from each other. In some embodiments, the multiple ORFs in a coding region are separated by non-coding sequences. In specific embodiments, a non-coding sequence separating two ORFs comprises an internal ribosome entry sites (IRES) .

Without being bound by the theory, it is contemplated that an internal ribosome entry sites (IRES) can act as the sole ribosome binding site, or serve as one of multiple ribosome binding sites of an mRNA. An mRNA molecule containing more than one functional ribosome binding site can encode several peptides or proteins that are translated independently by the ribosomes (e.g., multicistronic mRNA) . Accordingly, in some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises one or more internal ribosome entry sites (IRES) . Examples of IRES sequences that can be used in connection with the present disclosure include, without limitation, those from picomaviruses (e.g., FMDV) , pest viruses (CFFV) , polio viruses (PV) , encephalomyocarditis viruses (ECMV) , foot-and-mouth disease viruses (FMDV) , hepatitis C viruses (HCV) , classical swine fever viruses (CSFV) , murine leukemia virus (MLV) , simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV) .

In various embodiments, the nucleic acid molecule of the present disclosure encodes for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 polypeptides. Polypeptides encoded by a nucleic acid molecule can be the same or different. In some embodiments, the nucleic acid molecule of the present disclosure encodes a dipeptide. In some embodiments, the nucleic acid molecule encodes a tripeptide. In some embodiments, the nucleic acid molecule encodes a tetrapeptide. In some embodiments, the nucleic acid molecule encodes a pentapeptide. In some embodiments, the nucleic acid molecule encodes a hexapeptide. In some embodiments, the nucleic acid molecule encodes a heptapeptide. In some embodiments, the nucleic acid molecule encodes an octapeptide. In some embodiments, the nucleic acid molecule encodes a nonapeptide. In some embodiments, the nucleic acid molecule encodes a decapeptide. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 15 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 50 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 100 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 150 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 300 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 500 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide that has at least about 1000 amino acids.

In some embodiments, the nucleic acid molecule of the present disclosure is at least about 30 nucleotides (nt) in length. In some embodiments, the nucleic acid molecule is at least about 35 nt in length. In some embodiments, the nucleic acid molecule is at least about 40 nt in length. In some embodiments, the nucleic acid molecule is at least about 45 nt in length. In some embodiments the nucleic acid molecule is at least about 50 nt in length. In some embodiments, the nucleic acid molecule is at least about 55 nt in length. In some embodiments, the nucleic acid molecule is at least about 60 nt in length. In some embodiments, the nucleic acid molecule is at least about 65 nt in length. In some embodiments, the nucleic acid molecule is at least about 70 nt in length. In some embodiments, the nucleic acid molecule is at least about 75 nt in length. In some embodiments, the nucleic acid molecule is at least about 80 nt in length. In some embodiments the nucleic acid molecule is at least about 85 nt in length. In some embodiments, the nucleic acid molecule is at least about 90 nt in length. In some embodiments, the nucleic acid molecule is at least about 95 nt in length. In some embodiments, the nucleic acid molecule is at least about 100 nt in length. In some embodiments, the nucleic acid molecule is at least about 120 nt in length. In some embodiments, the nucleic acid molecule is at least about 140 nt in length. In some embodiments, the nucleic acid molecule is at least about 160 nt in length. In some embodiments, the nucleic acid molecule is at least about 180 nt in length. In some embodiments, the nucleic acid molecule is at least about 200 nt in length. In some embodiments, the nucleic acid molecule is at least about 250 nt in length. In some embodiments, the nucleic acid molecule is at least about 300 nt in length. In some embodiments, the nucleic acid molecule is at least about 400 nt in length. In some embodiments, the nucleic acid molecule is at least about 500 nt in length. In some embodiments, the nucleic acid molecule is at least about 600 nt in length. In some embodiments, the nucleic acid molecule is at least about 700 nt in length. In some embodiments, the nucleic acid molecule is at least about 800 nt in length. In some embodiments, the nucleic acid molecule is at least about 900 nt in length. In some embodiments, the nucleic acid molecule is at least about 1000 nt in length. In some embodiments, the nucleic acid molecule is at least about 1100 nt in length. In some embodiments, the nucleic acid molecule is at least about 1200 nt in length. In some embodiments, the nucleic acid molecule is at least about 1300 nt in length. In some embodiments, the nucleic acid molecule is at least about 1400 nt in length. In some embodiments, the nucleic acid molecule is at least about 1500 nt in length. In some embodiments, the nucleic acid molecule is at least about 1600 nt in length. In some embodiments, the nucleic acid molecule is at least about 1700 nt in length. In some embodiments, the nucleic acid molecule is at least about 1800 nt in length. In some embodiments, the nucleic acid molecule is at least about 1900 nt in length. In some embodiments, the nucleic acid molecule is at least about 2000 nt in length. In some embodiments, the nucleic acid molecule is at least about 2500 nt in length. In some embodiments, the nucleic acid molecule is at least about 3000 nt in length. In some embodiments, the nucleic acid molecule is at least about 3500 nt in length. In some embodiments, the nucleic acid molecule is at least about 4000 nt in length. In some embodiments, the nucleic acid molecule is at least about 4500 nt in length. In some embodiments, the nucleic acid molecule is at least about 5000 nt in length.

In specific embodiments, the therapeutic nucleic acid of the present disclosure is formulated as a vaccine composition (e.g., a genetic vaccine) as described herein. In some embodiments, the therapeutic nucleic acid encodes a polypeptide capable of eliciting immunity against one or more target conditions or disease. In some embodiments, the target disease or condition is a neoplastic disease or condition driven by one or more activating oncogenic mutations.

In some embodiments, the therapeutic nucleic acid sequence (e.g., mRNA) encodes a diseased-associated protein expressed by a diseased cell, or an immunogenic fragment (e.g., epitope) or derivative thereof. The vaccine, upon administration to a vaccinated subject, allows for expression of the encoded disease-associated protein (or the immunogenic fragment or derivative thereof) , thereby eliciting immunity in the subject against the pathogen.

In specific embodiments, provided herein are therapeutic compositions (e.g., vaccine compositions) for the management, prevention and treatment of neoplastic diseases or conditions caused by oncogenic mutations of KRAS. In some embodiments, the neoplastic disease is cancer having a KRAS mutation at the position corresponding to G12 in human KRAS (SEQ ID NO: 168) . In specific embodiments, the neoplastic disease is cancer having a KRAS mutation corresponding to G12A, G12C, G12D, or G12V in human KRAS. In specific embodiments, the neoplastic disease is cancer having a mutation selected from G12A, G12C, G12D, and G12V in human KRAS. In some embodiments, the neoplastic disease is cancer having a KRAS mutation at the position corresponding to G13 in human KRAS. In specific embodiments, the neoplastic disease is cancer having a KRAS mutation corresponding to G13D in human KRAS. In specific embodiments, the neoplastic disease is cancer having the G13D mutation in human KRAS. In some embodiments, the neoplastic disease is cancer having a KRAS mutation at the position corresponding to Q61 in human KRAS. In specific embodiments, the neoplastic disease is cancer having a KRAS mutation corresponding to Q61R in human KRAS. In specific embodiments, the neoplastic disease is cancer having the Q61R mutation in human KRAS.
5.3.1.1 KRAS Domain

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising at least one mutated fragment originating from a tumor antigen. In some embodiments, the therapeutic nucleic acid encodes multiple mutated fragments of a tumor antigen linked in a tandem directly or through a linker, which is sometimes referred herein to as “a tandem string of neoepitopes. ” In some embodiments, the multiple mutated fragments of the tumor antigen contain the same oncogenic mutation of the tumor antigen. In some embodiments, the multiple mutated fragments of the tumor antigen contain different oncogenic mutations of the tumor antigen. In some embodiments, the tumor antigen is a Ras protein. In some embodiments, the tumor antigen is KRAS. In some embodiments, the tumor antigen is human KRAS (SEQ ID NO: 168) . In some embodiments, the tumor antigen is a KRAS polypeptide originated from a non-human species.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises one or more mutations at residues corresponding to G12, G13 and Q61 of human KRAS (SEQ ID NO: 168) . In particular embodiments, the mutation at the residue corresponding to G12 of human KRAS (SEQ ID NO: 1) is selected from G12A, G12C, G12D, and G12V. In particular embodiments, the mutation at the residue corresponding to G13 of human KRAS (SEQ ID NO: 168) is G13D. In particular embodiments, the mutation at the residue corresponding to Q61 of human KRAS (SEQ ID NO: 168) is Q61R. In some embodiments, the at least one mutated fragment of KRAS is at least about 5, about 10, about 15, about 20, about 25, about 30 or about 35 amino acids in length. In some embodiments, the at least one mutated fragment of KRAS is more than 35 amino acids in length.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein each mutated fragment comprises one or more amino acid substitutions corresponding to G12A, G12C, G12D, G12V, G13D, and Q61R in human KRAS (SEQ ID NO: 168) . In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitution corresponding to G12A in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitution corresponding to G12C in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitution corresponding to G12D in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitution corresponding to G12V in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitution corresponding to G13D in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitution corresponding to Q61R in human KRAS. In some embodiments, the mutated fragment of KRAS is a mutated fragment of human KRAS (SEQ ID NO: 168) .

In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12A and G13D in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12C and G13D in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12D and G13D in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12V and G13D in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12A and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12C and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12D and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12V and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G13D and Q61R in human KRAS.

In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12A, G13D and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12C, G13D and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12D, G13D and Q61R in human KRAS. In some embodiments, the KRAS domain comprises at least one mutated fragment of KRAS, wherein the mutated fragment of KRAS comprises the amino acid substitutions corresponding to G12V, G13D and Q61R in human KRAS. In some embodiments, the mutated fragment of KRAS is a mutated fragment of human KRAS (SEQ ID NO: 168) .

In some embodiments, the at least one mutated fragment of human KRAS comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different. In some embodiments, the KRAS domain comprises two or more mutated fragments of human KRAS, and wherein at least two of the two or more mutated fragments are fused directly. In some embodiments, the KRAS domain comprises two or more mutated fragments of human KRAS, and wherein at least two of the two or more mutated fragments are fused through a linker peptide.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutated fragments of KRAS, wherein each mutated fragment comprises one amino acid substitution independently selected from those corresponding to G12A, G12C, G12D, G12V, G13D, and Q61R in human KRAS (SEQ ID NO: 168) . In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more mutated fragments of KRAS, wherein each mutated fragment comprises one amino acid substitution independently selected from those corresponding to G12A, G12C, G12D, G12V, and G13D in human KRAS (SEQ ID NO: 168) .

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein the at least one mutated fragment of KRAS comprises a G12A fragment. As used herein, a “G12A fragment” refers to a fragment of about 15 to 30 continuous amino acids of KRAS that comprises the amino acid substitution corresponding to G12A in human KRAS (SEQ ID NO: 168) . In some embodiments, a G12A fragment is a mutated human KRAS fragment containing the G12A mutation. In some embodiments, the KRAS domain comprises two or more identical copies of the G12A fragment. In some embodiments, the G12A fragment consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the G12A fragment consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 7. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12A fragment, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 5 to 19, or a codon optimized variant of any sequence selected from SEQ ID NOS: 5 to 19. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12A fragment, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 5 to 19, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein the at least one mutated fragment of KRAS comprises a G12C fragment. As used herein, a “G12C fragment” refers to a fragment of about 15 to 30 continuous amino acids of KRAS that comprises the amino acid substitution corresponding to G12C in human KRAS. In some embodiments, a G12C fragment is a mutated human KRAS fragment containing the G12C mutation. In some embodiments, the KRAS domain comprises two or more identical copies of the G12C fragment. In some embodiments, the G12C fragment consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the G12C fragment consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 20. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12C fragment, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 21 to 35, or a codon optimized variant of any sequence selected from SEQ ID NOS: 21 to 35. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12C fragment, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 21 to 35, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein the at least one mutated fragment of KRAS comprises a G12D fragment. As used herein, a “G12D fragment” refers to a fragment of about 15 to 30 continuous amino acids of KRAS that comprises the amino acid substitution corresponding to G12D in human KRAS. In some embodiments, a G12D fragment is a mutated human KRAS fragment containing the G12D mutation. In some embodiments, the KRAS domain comprises two or more identical copies of the G12D fragment. In some embodiments, the G12D fragment consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the G12D fragment consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 36. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12D fragment, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 37 to 51, or a codon optimized variant of any sequence selected from SEQ ID NOS: 37 to 51. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12D fragment, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 37 to 51, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein the at least one mutated fragment of KRAS comprises a G12V fragment. As used herein, a “G12V fragment” refers to a fragment of about 15 to 30 continuous amino acids of KRAS that comprises the amino acid substitution corresponding to G12V in human KRAS. In some embodiments, a G12V fragment is a mutated human KRAS fragment containing the G12V mutation. In some embodiments, the KRAS domain comprises two or more identical copies of the G12V fragment. In some embodiments, the G12V fragment consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the G12V fragment consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 52. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12V fragment, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 53 to 67, or a codon optimized variant of any sequence selected from SEQ ID NOS: 53 to 67. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G12V fragment, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 53 to 67, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein the at least one mutated fragment of KRAS comprises a G13D fragment. As used herein, a “G13D fragment” refers to a fragment of about 15 to 30 continuous amino acids of KRAS that comprises the amino acid substitution corresponding to G13D in human KRAS. In some embodiments, a G13D fragment is a mutated human KRAS fragment containing the G13D mutation. In some embodiments, the KRAS domain comprises two or more identical copies of the G13D fragment. In some embodiments, the G13D fragment consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 68. I In some embodiments, the G13D fragment consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 68. n some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G13D fragment, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 69 to 82, or a codon optimized variant of any sequence selected from SEQ ID NOS: 69 to 82. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the G13D fragment, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 69 to 82, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of KRAS, wherein the at least one mutated fragment of KRAS comprises a Q61R fragment. As used herein, a “Q61R fragment” refers to a fragment of about 15 to 30 continuous amino acids of KRAS that comprises the amino acid substitution corresponding to Q61R in human KRAS. In some embodiments, a Q61R fragment is a mutated human KRAS fragment containing the Q61R mutation. In some embodiments, the KRAS domain comprises two or more identical copies of the Q61R fragment. In some embodiments, the Q61R fragment consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the Q61R fragment consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 83. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the Q61R fragment, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 84 to 86, or a codon optimized variant of any sequence selected from SEQ ID NOS: 84 to 86. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the Q61R fragment, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 84 to 86, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises two G12A fragments, two G12C fragments, two G12D fragments, two G12V fragments, two G13D fragments, and two Q61R fragments. In some embodiments, the KRAS domain comprises two repeating units each comprising in the N-to-C direction: the G12C fragment, the G12V fragment, the G12D fragment, the G13D fragment, the G12A fragment, and the Q61R fragment. In some embodiments, each of the G12A fragments has the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20. In some embodiments, each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36. In some embodiments, each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52. In some embodiments, each of the G13D fragments has the amino acid sequence of SEQ ID NO: 68. In some embodiments, each of the Q61R fragments has the amino acid sequence of SEQ ID NO: 83. In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the KRAS domain consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 138. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 138. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding a polypeptide comprising a KRAS domain, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 87, 88, and 160, or a codon optimized variant of any sequence selected from SEQ ID NOS: 87, 88, and 160. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding a polypeptide comprising a KRAS domain, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 87, 88, and 160, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain comprises three G12A fragments, three G12C fragments, three G12D fragments, three G12V fragments, and three G13D fragments. In some embodiments, wherein the KRAS domain comprises three repeating units each comprising in the N-to-C direction: a copy of the G12C fragment, a copy of the G12V fragment, a copy of the G12D fragment, a copy of the G13D fragment and a copy of the G12A fragment. In some embodiments, each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7. In some embodiments, each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20. In some embodiments, each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36. In some embodiments, each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52. In some embodiments, the therapeutic nucleic acid of the present disclosure encodes a polypeptide comprising a KRAS domain, wherein the KRAS domain consists of, essentially consists of, or comprises the amino acid sequence set forth in SEQ ID NO: 161. In some embodiments, the KRAS domain consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 161. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 161. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding a polypeptide comprising a KRAS domain, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 162 to 167, or a codon optimized variant of any sequence selected from SEQ ID NOS: 162 to 167. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding a polypeptide comprising a KRAS domain, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 162 to 167, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.
5.3.1.2 Protease Degradation Domain

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain and degradation domain that is configured to mark the KRAS domain for proteasomal degradation. Without being bound by any theory, the degraded KRAS domain peptides are then processed and presented by antigen presenting cells (e.g., dendritic cells) in complex with major histocompatibility complex I or II (MHC) proteins to train lymphocytes (e.g., B cells and T cells) to recognize and eliminate any cells that express the target antigen (e.g., a cancer cell expressing the mutated KRAS protein) . Antigen processing can proceed through two distinct pathways: proteasomal degradation and phagolysosomal degradation. Antigens found in the dendritic cell cytoplasm can be targeted by chaperone proteins to the proteasome for degradation. Antigens that are taken up via phagocytosis into the phagosome can be targeted for processing by the fusion of phagosome and lysosome for partial degradation.

The ubiquitin pathway is well described for targeting proteins to the proteasome. Ubiquitin molecules can be found in multiple peptide forms, such as polyubiquitin chains that are branched or linear and that are heterotypic or homotypic chains. For example, homotypic K48-linked ubiquitin drives proteasomal degradation, whereas branched ubiquitin can act to promote a diverse range of cellular functions (see, e.g., Kolla et al., (2022) , Trends Biochem Sci; 47 (9) : 759-771, hereby incorporated by reference in its entirety) . Polyubiquitin chains attached to a target protein function differently based on the Lysine residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in proteotoxic stress response and cell cycle; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses, and signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Methionine lead to cell signaling. Ubiquitin typically is conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed.

Ubiquitin peptides are encoded by one of four genes. UBA52 and RPS27A genes code for a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively. UBB and UBC genes code for a polyubiquitin precursor with exact head to tail repeats, the number of which are species dependent.

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain and degradation domain that is configured to mark the KRAS domain for proteasomal degradation. In some embodiments, the degradation domain is fused to the KRAS domain. In some embodiments, the polypeptide comprises a N-terminal portion comprising the degradation domain and a C-terminal portion comprising the KRAS domain. In some embodiments, the polypeptide comprises a N-terminal portion comprising the KRAS domain and a C-terminal portion comprising the degradation domain.

In some embodiments, the degradation domain comprises a ubiquitin peptide. In some embodiments, the degradation domain comprises a ubiquitin monomer, or a functional derivative thereof. In some embodiments, the degradation domain comprises a ubiquitin multimer, or a functional derivative thereof. In some embodiments, the degradation domain comprises a ubiquitin like domain (ULD) , or a functional derivative thereof.

In some embodiments, the ubiquitin peptide comprises an amino acid sequence that is encoded by any one of the UBA52, RPS27A, UBB, or UBC genes. In some embodiments, the ubiquitin peptide comprises an amino acid sequence of about 80% (such as any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence that is encoded by any one of the UBA52, RPS27A, UBB, or UBC genes.

In some embodiments, the degradation domain consists of, essentially consists of, or comprises the sequence set forth in SEQ ID NO: 1. In some embodiments, the degradation domain consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 1. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 1. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the degradation domain, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 2 and 3, or a codon optimized variant of any sequence selected from SEQ ID NOS: 2 and 3. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the degradation domain, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 2 and 3, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some of the embodiments described in this paragraph, the degradation domain is located in the N-terminal portion of the polypeptide and the KRAS domain is located in the C-terminal portion of the polypeptide. In some embodiments, the KRAS domain is located in the C-terminal portion of the polypeptide and fused to the C terminal of the degradation domain. In some embodiments, the C-terminal portion of the polypeptide has a length of at least about 5, at least about 10 amino acids, at least about 15, at least about 20, at least about 25, at least about 30 amino acids. In some embodiments, the KRAS domain is fused directly to the degradation domain, and wherein the KRAS domain is at least about 5, at least about 10 amino acids, at least about 15, at least about 20, at least about 25, at least about 30 amino acids in length.

An alternative transcript of a ubiquitin peptide encoded by the UBB gene has been identified wherein the peptide can further include a 19 amino acid C-terminal extension that inhibits proteasomal degradation activity, thereby stabilizing the protein to which this ubiquitin peptide is attached. However, when extended to 25 amino acids, the alternative ubiquitin peptide attached at the N-terminus of a larger protein can target efficiently the larger protein for proteasomal degradation (see Vergoef, et al. (2009) , FASEB J; 23 (1) : 123-133, hereby incorporated by reference in its entirety) . Within the greater context of eliciting an immune response to a vaccine, efficient proteasomal processing and degradation of target antigens increases the likelihood of vaccine efficacy. Therefore, enhancements to the vaccine construct such as the fusion of the alternative ubiquitin peptide to the vaccine construct can improve overall efficacy and likelihood of successful vaccination and/or treatment.

Accordingly, in some embodiments, a spacer peptide is located between the degradation domain and the KRAS domain. In some embodiments, the spacer peptide is at least about 15 amino acids in length. In some embodiments, the spacer peptide is about 15-30 amino acids in length. In some embodiments, the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids in length. In some embodiments, the spacer peptide is a 25 amino acids peptide of any sequence.

In some embodiments, the spacer peptide consists of, essentially consists of, or comprises the sequence set forth in SEQ ID NO: 4. In some embodiments, the spacer peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 4. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 4. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the spacer peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 5 and 6, or a codon optimized variant of any sequence selected from SEQ ID NOS: 5 and 6. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the spacer peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 5 and 6, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, degradation domain that is configured to mark the KRAS domain for proteasomal degradation, and a spacer peptide located between the KRAS domain and the degradation domain, wherein the polypeptide consists of, essentially consists of, or comprises the sequence set forth in SEQ ID NO: 141. In some embodiments, the polypeptide consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 141. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 141. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the polypeptide comprising a KRAS domain, a degradation domain, and a spacer peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 142 and 144, or a codon optimized variant of any sequence selected from SEQ ID NOS: 142 and 144. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the polypeptide comprising a KRAS domain, a degradation domain and a spacer peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 142 and 144, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.
5.3.1.3 Traffickingpeptide

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, and a trafficking peptide that is configured for trafficking the KRAS domain to endosomes and lysosomes. In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain and a trafficking peptide, wherein the trafficking peptide is located to the N terminus of the KRAS domain. In some embodiments, the trafficking peptide is located to the C terminus of the KRAS domain.

Without being bound by theory, it is contemplated that sorting proteins to endosomes and lysosomes is mediated by sorting signals presented within the proteins. Many endosomal-lysosomal sorting signals have been characterized, and most of these signals are contained within the cytosolic domains of transmembrane proteins. See Bonifacino and Traub; Annu. Rev. Biochem. 2003. 72: 395–447 for a review, the content of which is incorporated herein by reference in its entirety. In general, the signals consist of short, linear arrays of amino acid residues. These arrays are not exactly conserved sequences but degenerate motifs of four to seven residues of which two or three are often critical for function. The critical residues are generally bulky and hydrophobic, although charged residues are also important determinants of specificity for some signals. Accordingly, in some embodiments, the trafficking peptide of the polypeptide encoded by the present therapeutic nucleic acid can be derived from a protein sequence that contains the sorting signal.

Without being bound by any theory, it is contemplated that two major classes of endosomal-lysosomal sorting signals are referred to as “tyrosine-based” and “dileucine based” signals, respectively owing to the identity of their most critical residues. Particularly, the tyrosine-based sorting signals conform to the NPXY or consensus motifs, and the dileucine-based signals conforms to [DE] XXXL [LI] or DXXLL consensus motifs, where X stands for any amino acid, stands for an amino acid residue with a bulky hydrophobic side chain, [DE] stands for D or E, and [LI] stands for L or I. Other sorting signals include acidic clusters, lysosomal avoidance signals, NPFX (1, 2) D-type signals and ubiquitin-based signals. See Table 1 below. Bonifacino and Traub; Annu. Rev. Biochem. 2003.72: 395–447.
Table 1 Endosomal/lysosomal sorting signals

Motifs in this table are denoted using the PROSITE syntax (www. expasy. ch/prosite/) . Amino acid residues are
designated according to the single letter code as follows: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan, and Y, tyrosine. X stands for any amino acid andstands for an amino acid residue with a bulky hydrophobic side chain. Abbreviations: PTB, phosphotyrosine-binding; Dab2, disabled-2; AP, adaptorprotein; VHS domainpresent in Vps27p, Hrs, Stam; GGAs, Golgi-localized, γ-ear-containing, ARF-binding proteins; PACS-1, phosphofurin acidic cluster sorting protein 1; TIP47, tail-interacting protein of 47kDa; SHD1, Sla1p homology domain 1; UBA, ubiquitin associated; UBC, ubiquitin conjugating; UIM, ubiquitin interaction motif.
Tables B to G list trafficking signals for various proteins of human or non-human origins. According to the
present disclosure, the trafficking peptide described herein can be derived from a protein selected from (but is not limited to) those proteins listed in Tables B to G. In some embodiments, the trafficking peptide comprises a portion of the one or more proteins listed in Tables B to G. In some embodiments, the trafficking peptide comprises the trafficking signal sequence listed for the corresponding protein as shown in Tables B to G.
Table B Endocytic machinery motifs and their recognition domains

Motifs are denoted as indicated in the legend to Table 1. EH, eps15 homology.
Table C NPXY-type signals

Numbers in parentheses indicate motifs that are present in more than one copy within the same protein. The
signals in this and other tables should be considered examples. Not all of these sequences have been shown to be active in sorting; some are included because of their conservation in members of the same protein family. Key residues are indicated in bold type. Numbers of amino acids before (i.e., amino-terminal) and after (i.e., carboxy-terminal) the signals are indicated. Abbreviations: Tm, transmembrane; LDL, low density lipoprotein; LRP1, LDL receptor related protein 1; APP, _-amyloid precursor protein; APLP1, APP-like protein 1.
Table Dsignals

See legends to Tables 1, B-Cfor explanation of signal format.
Table E [DE] XXX [LI] -type signals

See legends to Tables 1, B-C for explanation of signal format.
Table F DXXLL-type dileucine-based signals

See legends to Tables 1, B-C for explanation of signal format. Serine and threonine residues are underlined.
Table G Acidic cluster signals

See legends to Tables 1, B-C for explanation of signal format. Serine and threonine residues are underlined.
*The number in parentheses is the motif number.

In specific embodiments, the trafficking peptides described herein can be derived from a protein selected from (but is not limited to) , MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.

In some embodiments, the trafficking peptide comprises a portion of the one or more polypeptides. In some embodiments, the trafficking peptide comprises the trafficking signal of the one or more polypeptides as described in Tables B to G herein. In some embodiments, the trafficking peptide comprises one or more variations (e.g., substitutions, deletions, and/or additions) in the wild-type sequence of the one or more polypeptides. In some embodiments, the trafficking peptide comprises one or more variations (e.g., substitutions, deletions, and/or additions) in or to the wild-type sequence of the trafficking signal of the one or more polypeptides as described in Tables B to G herein. In some embodiments, the trafficking peptide comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of the one or more polypeptides. In some embodiments, the trafficking peptide comprises at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of the trafficking signal of the one or more polypeptides as described in Tables B to G herein.

Sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present within the cytosolic domains of the proteins. Most signals consist of short, linear sequences of amino acid residues. Signals are referred to as either tyrosine-based sorting signals or dileucine-based signals, each of which can be identified based on specific consensus motifs. All of these signals are recognized by components of protein coats peripherally associated with the cytosolic face of membranes. Phosphorylation events regulate signal recognition. In addition to peptide motifs, ubiquitination of cytosolic lysine residues also serves as a signal for sorting at various stages of the endosomal-lysosomal system. Non-limiting examples of trafficking peptides can be found in, e.g., J.S. Bonifacino&L.M. Traub (2003) , Annu Rev Biochem 72: 395-447, hereby incorporated by reference in its entirety. These peptides can be utilized as described herein by identifying the trafficking peptides based on the presence of the relevant consensus motifs and then isolating the nucleotides that encode said peptides and inserting these nucleotides into any of the nucleic acid constructs described herein by any method known in the art, such as by standard cloning techniques such as restriction enzyme digestion, PCR amplification, homologous recombination, or by nucleic acid synthesis. Nucleotide sequences encoding said peptides may be inserted 5′ or 3′ of the target antigen to promote the trafficking of the target antigen through the endosomal-lysosomal pathway of the antigen presenting cells.

CD1d: The CD1 family is an antigen presenting protein related to MHC class I but technically distinct from both MHC class I and II. The CD1 family mainly presents bacterial and pathogenic lipid and glycolipid antigens to T cells, specifically the natural killer T cell (NKT) subtype that produces IL-4 and IFNγ. CD1d plays a critical role in facilitating late endosomal trafficking of the processed antigen to the plasma membrane for presentation. See, e.g., Lawton et al., Immunol. Cell Biol., 2004; de Araujo et al., J Immunol Res., 2021; Miura et al., J. Virol., 2010; Jawawardena-Wolf et al., Immunity, 2001.

HLA-E: HLA-E is a non-canonical MHC class I molecule that binds to CD94/NKG2A/B/C receptor on NK cells. Binding to A/B receptors prevents target cell lysis by NK cells, while C receptor binding triggers expansion of NK cells to aid in fighting infection. HLA-E is also capable of presenting antigens to CD8+T cells. HLA-E can simultaneously stimulate NK-and CD8+T cell-mediated immune response, prevent rejection of transplanted cells by NK self-recognition mechanism, and resist the pathogen-mediated downregulation of HLA-I molecules that allows infection to evade immune response. Furthermore, HLA-E molecules are preferentially recycled and reused for antigen presentation rather than degraded, and HLA-E molecules have a longer residence in late and recycled endosomes. See, e.g., Braud et al., Nature, 1998; Rolle et al., J Clin Invest., 2014.; and He et al., JEM, 2023

LAMP1: LAMP1 is a glycoprotein found on various membranes within a cell, for example lysosome membranes. See, e.g., Watts, FEBS Open Bio., 2022; Cheng et al., J Cell Biol., 2018.

LAMP3: LAMP3 (DC-LAMP) is a glycoprotein found on the surface of lysosomes similar to LAMP1 and LAMP2. LAMP3 is specific only to mature dendritic cells and plays a key role in the maturation of DCs. LAMP3 specifically localizes to the MHC class II compartment immediately before translocation of MHC-II molecules to the cell surface, and helps with antigen processing and loading onto the MHC-II molecule. See, e.g., Barois et al., Traffic, 2002; de Saint-Vis et al., Immunity, 1998.

HLA-DMB: New MHC-II molecules leave the ER and are directed to the endocytic pathway by the Invariant chain (Ii) chaperone, which is subsequently degraded to CLIP (class II-associated Ii-chain peptide) while still bound to MHC II. HLA-DMB is a non-classical MHC-II molecule that displaces CLIP and helps load a processed antigen onto the target MHC-II molecule for antigen presentation. See, e.g., Barois et al., Traffic, 2002; Santambrogio, Front. Immunol., 2022.

MHC CLASS I: MHC class I is involved in antigen presentation. Antigen presentation in APCs is accomplished by loading antigen peptides onto MHC-II molecules, however it is known that MHC class I molecules also reside in and travel through the cellular compartments involved in MHC class II antigen processing and presentation. MHC CLASS I has been shown to improve antigen presentation and immune response in a manner dependent on MHC-I function. See, e.g., Kreiter et al., J. Immunol., 2008.

LAMP-2a: LAMP2a is associated with lysosomal function, specifically with chaperone-mediated autophagy. See, e.g., Kaushik and Cuervo, Nat. Rev. Mol. Cell Biol., 2018.

CD63: CD63 is a surface glycoprotein also found on membranes of intracellular vesicles, for example of immature dendritic cells, alongside MHC class II molecules. See, e.g., Piper and Katzmann, Annu. Rev. Cell Dev. Biol., 2010.

GMP-17: Granule membrane protein 17 (i.e., NKG7 or GIG-1) is a membrane protein typically localized to cytotoxic granules and highly expressed in NK cells and CD8+T cells. GMP-17 expression was recently found to be induced in activated CD4+T cells stimulated by IL-27. See, e.g., Ng et al., Nat. Immunol., 2020; Malarkannan, Nat. Immunol., 2020.

CD1b: CD1b is a glycoprotein expressed on APCs such as dendritic cells that binds to self-and non-self-lipid antigens for presentation. CD1b is specifically capable of binding and presenting microbial lipid antigens of various alkyl chain lengths. See, e.g., Gras et al., Nat. Comm., 2016.

CD1c: CD1c is a glycoprotein expressed on APCs such as dendritic cells that binds self-and non-self-lipid antigens for presentation. CD1c labels a distinct subpopulation of DCs that secrete high levels of IL-12 and potently prime cytotoxic T-cell responses. See, e.g., Nizzoli et al., Blood, 2013; Heger et al., Front. Immunol., 2020.

Cystinosin: Cystinosin is a lysosomal membrane protein that primarily transports cystine from lysosomes in a H+-dependent manner. Cystine is a disulfide form of the amino acid cysteine that forms from lysosomal protein hydrolysis, and mutations in the Cystinosin gene cause the lysosomal storage disease cystinosis. See, e.g., Kalatzis et al., EMBO J., 2001.

CTLA-4: Cytotoxic T cell antigen 4 is a surface membrane protein expressed by activated T cells and binds to the B7 ligand to negatively regulate T cell function and proliferation. See, e.g., Bashyam, J. Exp. Med., 2007; Salvatori et al., npj Vaccines, 2022.

CD4: CD4 is a receptor for helper T cells that assists the T cell receptor (TCR) in binding to antigen-presenting MHC class II molecules on APCs. See, e.g., Miceli and Parnes, Seminars in Immunology, 1991.

NPC1: Niemann-Pick disease type C1 is a membrane protein that mediates intracellular cholesterol trafficking. Mutations in NPC1 cause Niemann-Pick disease, where lipids accumulate in late endosomes and lysosomes.

CIMPR: Cation-independent mannose 6-phosphate receptor (CIMPR, or insulin-like growth factor 2 receptor (IGF2R) ) binds to IGF2 at the cell surface for transport to early endosomes for the release of IGF2 and attenuation of IGF2 signaling. CIMPR also shuttles mannose 6-phosphate containing lysosomal enzymes through the endosomal system. See, e.g., Bohnsack et al., J. Biol. Chem., 2009; and Ghosh et al., Nat. Rev. Mol. Cell Biol., 2003.

LRP3: low density lipoprotein (LDL) receptor-related protein 3 is a probable surface receptor for internalization of lipophilic molecules and signal transduction, however its precise role in this process remains unclear. See, e.g., Ishii et al., Genomics, 1998.

Furin: Furin is a protease in the trans-Golgi network that cleaves a number of target proteins for activation. In T cells, Furin helps maintain peripheral tolerance by cleaving the anti-inflammatory cytokine TGF-β1 and generally preventing overproduction of cytokines. Furin is involved in proteolytic maturation of substrate proteins in the secretory pathway. See, e.g., Pesu et al., Nature, 2008.

VAMP4: Vesicle-associated membrane protein 4 is involved in membrane fusion during vesicle transport, specifically by mediating the transport of vesicles from the trans-Golgi network to endosomes. See, e.g., Steegmaier et al., Mol. Biol. Cell, 1999.

VMAT1: Vesicular monoamine transporter 1 (SLC181) is an integral membrane protein in neuronal and endocrine cells that allows for monoamine neurotransmitter transport into secretory vesicles to then be discharged into extracellular space by exocytosis. See, e.g., Eiden et al., Pflugers Archiv., 2004.

VMAT2: Vesicular monoamine transporter 2 (SLC182) is an integral membrane protein in neuronal and endocrine cells that allows for monoamine neurotransmitter transport into secretory vesicles to then be discharged into extracellular space by exocytosis. See, e.g., Eiden et al., Pflugers Archiv., 2004.

PAM: Peptidyl-glycine alpha-amidating monooxygenase helps to form active neuroendocrine peptides by catalyzing their alpha-amidation. See, e.g., Driscoll et al., Mol. Pharmacol., 1999.

CPD: Carboxypeptidase D is a trans-Golgi resident enzyme that cleaves C-terminal arginine or lysine residues to aid in biosynthesis of neuropeptides and peptide hormones. See, e.g., Song and Fricker, Enzymology, 1995.

PC7: Proprotein convertase 7 is a secretory pathway enzyme that helps process secreted proteins to active state. PC7 can reach the cell surface through canonical ER/Golgi-dependent secretory pathway, i.e., predominantly through its pro-segment. The PC7 transmembrane domain also regulates trafficking to the surface through an unconventional pathway that is not dependent on COPII vesicles. See, e.g., Rousselet et al., J. Biol. Chem., 2011.

Beta-secretase (BACE) : BACE is an aspartic-acid protease important in the formation of myelin sheaths in peripheral nerve cells. BACE is transported through the secretory pathway to the cell surface. See, e.g., Capell et al., J. Biol. Chem., 2000.

Sortilin: Sortilin is a membrane glycoprotein responsible for mediating protein transport between the Golgi, endosomes, lysosomes, and plasma membrane. See, e.g., Nielsen et al., EMBO J., 2001.

GLUT4: Glucose transporter type 4 is a plasma membrane protein that facilitates passive diffusion of circulating glucose down a concentration gradient into muscle and fat cells in an insulin-dependent manner. See, e.g., James et al., Nature, 1989.

TRP-1 (TYRP1) : Tyrosinase-related protein 1 is a transmembrane protein involved in the processing and maturation of melanin. TRP-1 is trafficked through the melanosomes of melanocytes. See, e.g., Rzepka et al., Postepy Hig. Med. Dosw., 2016; and Kameyama et al., Pigment Cell Res., 1995.

LDL receptor: Low-density lipoprotein receptor is a cell surface receptor that mediates endocytosis of low-density lipoprotein (LDLs) by recognition of apolipoprotein B100. See, e.g., Sudhof et al., Science, 1985.

LRP1: low density lipoprotein (LDL) receptor-related protein 1 is involved in receptor mediated endocytosis and plays a role in many related biological processes. See, e.g., Etique et al., Biomed Research International, 2013.

Megalin (LRP2) : Megalin is a mediator of ligand endocytosis, leading to the degradation of these target molecules within lysosomes. See, e.g., Eshbach et al., Annu. Rev. Physiol., 2017.

Integrin beta-1: Integrin beta-1 is a cell surface protein that functions as a collagen receptor with Integrin alpha 1 and Integrin alpha 2. See, e.g., Hynes, Cell, 1992.

APLP1: The amyloid-like protein 1 is a membrane-associated glycoprotein that modulates glucose and insulin homeostasis. APLP1 is cleaved by secretases within the secretory pathway. See, e.g., Bayer et al., Mol. Psychiatry, 2000.

APP: The amyloid-beta precursor protein is an integral membrane protein that functions as a cell surface receptor involved in synapse formation and neural plasticity, among other functions. APP is trafficked to the cell surface through the secretory system. See, e.g., Turner et al., Progress in Neurobiology, 2003.

Insulin receptor: The insulin receptor (IR) is a transmembrane protein that binds circulating insulin in order to promote glucose homeostasis and initiate cellular uptake of glucose molecules. See, e.g., McKern et al., Nature, 2006.

EGF receptor: Epidermal growth factor receptor (EGFR) is a cell surface receptor for epidermal growth factor (EGF) . Activating mutations in EGFR have been associated with a number of cancers.

In specific embodiments, the trafficking peptide is derived from MHC Class I (MHC CLASS I) . In some embodiments, the trafficking peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 99. In some embodiments, the trafficking peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 99. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 99. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 100 to 104 and 198 to 202, or a codon optimized variant of any sequence selected from SEQ ID NOS: 100 to 104 and 198 to 202. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 100 to 104 and 198 to 202, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the trafficking peptide is derived from LAMP3 transmembrane domain (LAMP3 TM) . In some embodiments, the trafficking peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 203 or 205. In some embodiments, the trafficking peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 203 or 205. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 203 or 205. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NO: 204 or 206, or a codon optimized variant of SEQ ID NO: 204 or 206. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 204 or 206, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the trafficking peptide is derived from HLA-E domain. In some embodiments, the trafficking peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 207. In some embodiments, the trafficking peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 207. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 207. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NO: 208, or a codon optimized variant of SEQ ID NO: 208. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 208, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the trafficking peptide is derived from LAMP1 domain. In some embodiments, the trafficking peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 209. In some embodiments, the trafficking peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 209. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 209. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NO: 210, or a codon optimized variant of SEQ ID NO: 210. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 210, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the trafficking peptide is derived from HLA-DMB domain. In some embodiments, the trafficking peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 211. In some embodiments, the trafficking peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 211. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 211. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NO: 212, or a codon optimized variant of SEQ ID NO: 212. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the trafficking peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 212, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.
5.3.1.4 Helper T Cell Epitope

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, and a helper T cell epitope configured to augment T cell mediated immunity against the KRAS domain. In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain and a helper T cell epitope, wherein the helper T cell epitope is located to the N terminus of the KRAS domain. In some embodiments, the helper T cell epitope is located to the C terminus of the KRAS domain.

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, and further encodes a helper T cell epitope configured to augment T cell mediated immunity against the KRAS domain. In some embodiments, the therapeutic nucleic acid molecule comprises a multicistronic coding region that encodes the KRAS domain and the helper T cell epitope as separate polypeptides. In some embodiments, the therapeutic nucleic acid molecule comprises at least two coding regions that encode the KRAS domain and the helper T cell epitope, respectively. In some embodiments, the therapeutic nucleic acid molecule comprises at least two separate nucleic acid molecules that encode the KRAS domain and the helper T cell epitope, respectively.

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, a helper T cell epitope and a trafficking peptide. In some embodiments, the polypeptide comprises, in its N-to-C direction, the KRAS domain, the helper T cell epitope, and the trafficking peptide. In some embodiments, the polypeptide further comprises a linker peptide between the KRAS domain and the helper T cell epitope. In some embodiments, the polypeptide further comprises a linker peptide between the helper T cell epitope and the trafficking peptide.

In some embodiments, the helper T cell epitope is a universal CD4 epitope. In some embodiments, the helper T cell epitope is an epitope of tetanus and diphtheria toxoids. In some embodiments, the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE.

The P2 helper T cell epitope is a signaling peptide from the tetanus toxoid amino acid residues 830-844 that functions as a universal CD4+T-cell activator. The P16 helper T cell epitope is a signaling peptide from tetanus toxoid that also functions as a universal CD4+T-cell activator. These two epitopes can be connected into the P2P16 epitope, for example via a linker peptide such as a flexible linker or a cleavable linker as described below.

The TT_827-841 helper T cell epitope is a signaling peptide from the tetanus toxoid amino acid residues 827-841 that have been shown to stimulate T-helper cell activity. The pDT_331-350 helper T cell epitope is a region of diphtheria toxin (DTX) of amino acid residues 331-350 that is recognized by human CD4+T cells and may help to activate the helper T cell response to a target antigen. The TT_632-651 helper T cell epitope is a signaling peptide from the tetanus toxoid amino acid residues 632-651 that has been shown to stimulate helper T cell activity.

The pan-DR epitope (PADRE) is a 13 amino acid synthetic peptide that stimulates CD4+T cell activation by binding to fifteen of the sixteen most common HLA-DR types. In proliferation assays, PADRE has been shown to induce up to a 100-fold more potent helper T cell response than other universal helper T cell epitopes such as tetanus toxin-derived epitopes.

In specific embodiments, the helper T cell epitope is the pan-DR epitope PADRE. In some embodiments, the helper T cell epitope consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 94. In some embodiments, the helper T cell epitope consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 94. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 94. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the helper T cell epitope, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 95 to 98 and 186, or a codon optimized variant of any sequence selected from SEQ ID NOS: 95 to 98 and 186. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the helper T cell epitope, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 95 to 98 and 186, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the helper T cell epitope is the P2P16. In some embodiments, the helper T cell epitope consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 187. In some embodiments, the helper T cell epitope consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 187. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 187. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the helper T cell epitope, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 188 and 189, or a codon optimized variant of any sequence selected from SEQ ID NOS: 188 and 189. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the helper T cell epitope, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 188 and 189, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the helper T cell epitope is the PP65. In some embodiments, the helper T cell epitope consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 190. In some embodiments, the helper T cell epitope consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 190. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 190. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the helper T cell epitope, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 191, or a codon optimized variant of SEQ ID NO: 191. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the helper T cell epitope, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 191, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In specific embodiments, the helper T cell epitope consists, essentially consists, or comprises the sequence selected from those set forth SEQ ID NOS: 192 to 197. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence selected from those set forth SEQ ID NOS: 192 to 197.
5.3.1.5 Immunomodulator

In some embodiments, the therapeutic nucleic acid encodes a polypeptide comprising a KRAS domain, and an immunomodulator configured to augment T cell mediated immunity against the KRAS domain. In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain and an immunomodulator, wherein the immunomodulator is located to the N terminus of the KRAS domain. In some embodiments, the immunomodulator is located to the C terminus of the KRAS domain. In some embodiments, the KRAS domain and the immunomodulator are connected through a cleavable linker, such as a 2A peptide (e.g., P2A, F2A, T2A, and E2A) , as described herein.

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, and further encodes an immunomodulator configured to augment T cell mediated immunity against the KRAS domain. In some embodiments, the therapeutic nucleic acid molecule comprises a multicistronic coding region that encodes the KRAS domain and the immunomodulator as separate polypeptides. In some embodiments, the therapeutic nucleic acid molecule comprises at least two coding regions that encode the KRAS domain and the immunomodulator, respectively. In some embodiments, the therapeutic nucleic acid molecule comprises at least two separate nucleic acid molecules that encode the KRAS domain and the immunomodulator, respectively.

In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising a KRAS domain, a helper T cell epitope, a trafficking peptide, and an immunomodulator. In some embodiments, the polypeptide comprises, in the N-to-C direction, the KRAS domain, the helper T cell epitope, the trafficking peptide and the immunomodulator. In some embodiments, the polypeptide further comprises a linker peptide between the KRAS domain and the helper T cell epitope. In some embodiments, the polypeptide further comprises a linker peptide between the helper T cell epitope and the trafficking peptide. In some embodiments, the polypeptide further comprises a linker peptide between the trafficking peptide and the immunomodulator. In some embodiments, the linker peptide between the trafficking peptide and the immunomodulator is a cleavable linker.

In some embodiments, the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, a portion thereof, and any combination thereof.

GM-CSF: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the differentiation of granulocytes, macrophages, eosinophils, and erythrocytes from hematopoietic precursors, specifically myeloid-biased common myeloid progenitors (CMPs) . GM-CSF is also critical for the maturation of hematopoietic progenitors, specifically circulating monocytes, to dendritic cells (DCs) . GM-CSF therefore promotes DC maturation. See, e.g., Banchereau and Palucka, Nature Reviews Immunology, 2005; Greter et al., Immunity, 2012. In some embodiments, the GM-CSF is mouse GM-CSF (mGM-CSF) . In some embodiments, the GM-CSF is human GM-CSF (hGM-CSF) . In some embodiments, the GM-CSF is a wild-type polypeptide. In some embodiments, the GM-CSF polypeptide is full-length. In some embodiments, the GM-CSF polypeptide is a naturally occurring isoform. In some embodiments, the GM-CSF is a naturally occurring variant polypeptide. In some embodiments, the GM-CSF is a mutated polypeptide comprising one or more variations (e.g., substitutions, deletions, and/or additions) with respect to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268. In some embodiments, the GM-CSF polypeptide is truncated.

STING: STING (stimulator of interferon genes) is an Endoplasmic Reticulum (ER) -localized membrane protein that upregulates the expression of interferon-related genes and production of type I interferons in response to pathogenic nucleic acids released from viruses, bacteria, and/or parasites. DCs upregulate antigen presentation in response to interferon signaling and inflammation generally, indicating that constitutive STING activity may help stimulate DC function. Therefore, expression of STING stimulates DCs to produce interferons, thereby upregulating their own maturation and antigen-presenting functions. See, e.g., Nakhaei et al., J. Mol. Cell Biol., 2009; and Jneid et al., Science Immunology, 2023.

FLT3L: Fms-related tyrosine kinase 3 ligand is a cytokine involved in regulation of hematopoietic progenitor function in mouse and hematopoietic stem cell homeostasis in human. FLT3L is also critical to development of both classic and plasmacytoid dendritic cells, and intratumoral FLT3L has been shown to induce accumulation and antigen presentation of DCs along with an in situ vaccine and a TLR3 agonist. See, e.g., Shortman and Naik, Nature Review Immunology, 2007; and Hammerich et al., Nature Medicine, 2019.

c-FLIP: Cellular FLICE-inhibitory protein is a master anti-apoptotic regulator that functions by suppressing TNF-α, Fas-L, and TNF-related (TRAIL) -induced apoptosis. c-FLIP can also activate other pro-survival signaling proteins such as Akt, ERK, and NF-κB. It is found to be upregulated in many tumor types to prevent cancer cell death. See, e.g., Safa, Exp. Oncol., 2012.

IKKβ: Inhibitor of NF-κB kinase subunit beta phosphorylates inhibitor of NF-κB (IkB) , targeting it for degradation and thereby driving nuclear translocation of NF-κB to act as a transcription factor. Among other roles, active NF-κB mediates maturation of DCs to become fully functional. See, e.g., Voigt et al., Nature Comm., 2020; and Creusot, Nature Medicine, 2011.

RIPK1: Receptor interacting protein 1 plays a critical role in TNF receptor-mediated activation of NF-κB, which would benefit DC maturation and function. Furthermore, RIPK1 has also been shown to play a role in activating TNF-induced apoptosis and necrosis under certain conditions. See, e.g., Lin, Necrotic CellDeath, 2014.

Btk: Bruton’s tyrosine kinase activates NF-κB. B cell receptor (BCR) -dependent NF-κB signaling requires functional Btk. See, e.g., Mohamed et al., Immunol. Rev., 2009.

TAK1: Mitogen-activated protein 3 kinase 7 has a powerful pro-survival role in activating the IKK-NF-κB pathway to block apoptosis, promote cell proliferation, and stimulate inflammatory responses. TAK1 may also play a role in mediating necroptosis with RIPK1 and RIPK3. Ultimately, TAK1 may therefore help to activate NF-κB in DCs to improve DC survival. See, e.g., Paul, Oncogene, 1999; Mihaly et al., Cell Death and Differentiation, 2014.

TAK-TAB1: Binding partner of TAK1 is needed for TAK1 functional activation of NF-κB. TAK-TAB1 therefore may be similar to TAK1 by helping to activate NF-κB in DCs to improve DC survival. See, e.g., Xu and Lei, Front. Immunol., 2021.

TBK1: TANK-binding kinase 1 is a non-canonical IKK kinase that phosphorylates IkB to activate NF-κB, similar to IKKB.

MyD88: Myeloid differentiation primary response 88 is a universal adapter protein that activates NF-κB in response to toll-like receptor (TLR) activity. See, e.g., Lord et al., Oncogene, 1990.

IRAK1: Interleukin receptor-associated kinase 1 is activated downstream of toll-like receptor (TLR) or interleukin-1 receptor (IL-1R) activation by inflammatory signaling to upregulate and activate NF-κB. See, e.g., Cao et al., Science, 1996; and Muzio et al., Science, 1997.

IRAK2: Interleukin receptor-associated kinase 2 is activated downstream of TLR or IL-1R activation by inflammatory signaling to upregulate and activate NF-κB.

TAB2: TGF-beta activated kinase 1 (MAP3K7) binding protein 2 is required for the IL-1 induced activation of NF-κB and MAPK by forming a kinase complex with TRAF6, MAP3K7, and TAB1.

TAB3: TGF-beta activated kinase 1 (MAP3K7) binding protein 3 forms a complex with MAP3K7 and either TRAF2 or TRAF6 in response to TNF or IL-1 signaling to activate NF-κB.

TRAF6: TNF receptor associated factor 6 mediates signal transduction from TNF and IL-1 to activate IKK in response to inflammation for downstream NF-κB activation.

TRAM: Translocation associated membrane protein 2 is a component of the translocon gated channel that controls the posttranslational processing of nascent secretory and membrane proteins at the ER.

MKK3: Mitogen-activatedprotein 2 kinase 3 activates MAPK14/p-38MAPK in response to mitogenic and environmental stress. MAPK activation drives cell differentiation and proliferation.

MKK4: Mitogen-activated protein 2 kinase 4 activates the MAPK pathway in response to mitogenic and environmental stress. MAPK activation drives cell differentiation and proliferation.

MKK6: Mitogen-activated protein 2 kinase 6 activates the MAPK pathway in response to mitogenic and environmental stress. MAPK activation drives cell differentiation and proliferation.

Type I IFN: Type I interferons are cytokines that drive inflammation and can induce T cell responses. Type I IFNs can stimulate DC maturation and function, and preliminary studies have shown that cancer vaccines employing IFNα/βimprove DC-mediated neoantigen-specific T cell activation and NK cell activation. See, e.g., Sprooten et al., International Review of Cell and Molecular Biology, 2019.

In some embodiments, the immunomodulator is GM-CSF. In some embodiments, the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) . In some embodiments, the GM-CSF is a full-length GM-CSF polypeptide. In some embodiments, the immunomodulator consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 111 or 264. In some embodiments, the immunomodulator consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 111 or 264. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 111 or 264. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the immunomodulator, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 112 to 114, 215 to 217, and 265, or a codon optimized variant of any sequence selected from SEQ ID NOS: 112 to 114, 215 to 217, and 265. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the immunomodulator, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 112 to 114, 215 to 217, and 265, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the immunomodulator consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 115 or 268. In some embodiments, the immunomodulator consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 115 or 268. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 115 or 268. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the immunomodulator, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 116 to 118, 218 to 219 and 269-271, or a codon optimized variant of any sequence selected from SEQ ID NOS: 116 to 118, 218 to 219 and 269-271. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the immunomodulator, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 116 to 118, 218 to 219 and 269-271, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the immunomodulator consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 220 or 253. In some embodiments, the immunomodulator consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 220 or 253. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 220 or 253. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the immunomodulator, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 221 or 254, or a codon optimized variant of SEQ ID NO: 221 or 254. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the immunomodulator, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 221 or 254, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.
5.3.1.6 Signaling peptide

In some embodiments, the therapeutic nucleic acid encodes a polypeptide comprising a KRAS domain fused to a signal peptide configured for translocate the polypeptide in a cell. In some embodiments, the signal peptide is located to the N terminus of the KRAS domain. In some embodiments, the signal peptide is located to the C terminus of the KRAS domain.

In some embodiments, the therapeutic nucleic acid encodes a polypeptide comprising a signal peptide, a KRAS domain, a helper T cell epitope, a trafficking peptide and an immunomodulator. In some embodiments, the polypeptide comprises, in the N-to-C direction, a signal peptide, a KRAS domain, a helper T cell epitope, a trafficking peptide and an immunomodulator. In some embodiments, the polypeptide further comprises a linker peptide between the signal peptide and the KRAS domain. In some embodiments, the polypeptide further comprises a linker peptide between the KRAS domain and the helper T cell epitope. In some embodiments, the polypeptide further comprises a linker peptide between the helper T cell epitope and the trafficking peptide. In some embodiments, the polypeptide further comprises a linker peptide between the trafficking peptide and the immunomodulator. In some embodiments, the linker peptide between the trafficking peptide and the immunomodulator is a cleavable linker.

In some embodiments, the signaling peptide assists with translocating the polypeptide encoded by the therapeutic nucleic acid of the present disclosure to the Golgi apparatus. In some embodiments, the signaling peptide assists with translocating the polypeptide encoded by the therapeutic nucleic acid of the present disclosure to the Endoplasmic Reticulum (ER) . In some embodiments, the signal peptide assists with translocating the polypeptide to the endosome, lysosome, or proteosome for processing of the polypeptide for antigen presentation in antigen presenting cells (APCs) . Many signal peptides of prokaryotic and eukaryotic proteins are known in the art. For review, see e.g., Owji et al. Eur J Cell Biol. 2018 Aug; 97 (6) : 422-441.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 89. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 89. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 89. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 90 to 93, or a codon optimized variant of any sequence selected from SEQ ID NOS: 90 to 93. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 90 to 93, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 170. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 170. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 170. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 171 to 175, or a codon optimized variant of any sequence selected from SEQ ID NOS: 171 to 175. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 171 to 175, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 176. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 176. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 176. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 177, or a codon optimized variant of SEQ ID NO: 177. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 177, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 178. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 178. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 178. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 179, or a codon optimized variant of SEQ ID NO: 179. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 179, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 180. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 180. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 180. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 181, or a codon optimized variant of SEQ ID NO: 181. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 181, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 182. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 182. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 182. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 183, or a codon optimized variant of SEQ ID NO: 183. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 183, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the signal peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 184. In some embodiments, the signal peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 184. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 184. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the signal peptide, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 185, or a codon optimized variant of SEQ ID NO: 185. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the signal peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 185, or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.
5.3.1.7 Linker Peptides and Fusion Proteins

In some embodiments, the therapeutic nucleic acid described herein encodes a polypeptide comprising (i) a KRAS domain as described in Section 5.3.1.1 and one or more selected from (ii) a protease degradation domain as described in Section 5.3.1.2; (iii) a trafficking peptide as described in Section 5.3.1.3, (iv) a helper T cell epitope as described in Section 5.3.1.4, (v) an immunomodulators as described in Section 5.3.1.5, and (vi) a signal peptide as described in Section 5.3.1.6.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a linker peptide. In some embodiments, the linker peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 119. In some embodiments, the linker peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 119. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 119. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the linker peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 120 to 124, 222, and 223, or a codon optimized variant of any sequence selected from SEQ ID NOS: 120 to 124, 222, and 223. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the linker peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 120 to 124, 222, and 223 or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a linker peptide. In some embodiments, the linker peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 125. In some embodiments, the linker peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 125. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 125. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the linker peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 126 to 131 and 224, or a codon optimized variant of any sequence selected from SEQ ID NOS: 126 to 131 and 224. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the linker peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 126 to 131 and 224 or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a linker peptide. In some embodiments, the linker peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 132. In some embodiments, the linker peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 132. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 132. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the linker peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 133 to 135, or a codon optimized variant of any sequence selected from SEQ ID NOS: 133 to 135. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the linker peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 133 to 135 or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a linker peptide. In some embodiments, the linker peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 225. In some embodiments, the linker peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 225. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 225. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the linker peptide, wherein the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 226, or a codon optimized variant of SEQ ID NO: 226. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the linker peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence of SEQ ID NO: 226 or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a linker peptide. In some embodiments, the linker peptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 227. In some embodiments, the linker peptide consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 227. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 227. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the linker peptide, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 228 to 232, or a codon optimized variant of any sequence selected from SEQ ID NOS: 228 to 232. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the linker peptide, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 228 to 232 or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a linker peptide. In some embodiments, the linker peptide consists, essentially consists, or comprises the sequence selected from those set forth in SEQ ID NOS: 233 to 248. In some embodiments, the linker peptide consists of, essentially consists of, or comprises a functional variant of the sequence selected from those set forth in SEQ ID NOS: 233 to 248. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the sequence selected from those set forth in SEQ ID NOS: 233 to 248.

In some embodiments, connection between the two or more domains of the encoded polypeptide is through a cleavable linker peptide, such as a 2A peptide (e.g., P2A, F2A, T2A, and E2A) . In some embodiments, a P2A linker consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 105. In some embodiments, the P2A linker consists of, essentially consists of, or comprises a functional variant of the sequence set forth in SEQ ID NO: 105 or 258. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 105 or 258. In some embodiments, the therapeutic nucleic acid comprising a coding sequence encoding the P2A linker, wherein the coding sequence comprises the nucleic acid sequence selected from those set forth in SEQ ID NOS: 106 to 110, 213, 214 and 259, or a codon optimized variant of any sequence selected from SEQ ID NOS: 106 to 110, 213, 214 and 259. In some embodiments, the therapeutic nucleic acid comprises a coding sequence encoding the P2A linker, wherein the coding sequence comprises the corresponding DNA sequence of a sequence selected from SEQ ID NOS: 106 to 110, 213, 214 and 259 or a codon optimized variant of such DNA sequence. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid molecule of the present disclosure encodes a polypeptide comprising, from the N-to-C direction: a signal peptide, a first linker peptide, a KRAS domain, a second linker peptide, a helper T cell epitope, a third linker peptide, a trafficking peptide, a cleavable linker, and an immunomodulator. In some embodiments, the polypeptide consists, essentially consists, or comprises the sequence set forth in SEQ ID NO: 146. In some embodiments, the polypeptide consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 146. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 146. In some embodiments, the therapeutic nucleic acid molecule encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 153. In some embodiments, the polypeptide consists of, essentially consists of, or comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 153. In some embodiments, the functional variant has at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 153.
5.3.2 Untranslated Regions (UTRs)

In some embodiments, the therapeutic nucleic acid molecules of the present disclosure comprise one or more untranslated regions (UTRs) . In some embodiments, an UTR is positioned upstream to a coding region in the nucleic acid molecule, and is termed 5’-UTR. The 5’ UTRs provided herein may be recognized by the ribosome, thereby allowing the ribosome to bind and initiate translation of the mRNA (e.g., translation of the coding sequence and/or nucleic acid encoding the mRNA) . In some embodiments, an UTR is positioned downstream to a coding region in the nucleic acid molecule, and is termed 3’-UTR. The 3’ UTRs provided herein may be involved in translation termination (e.g., translation of the coding sequence and/or nucleic acid encoding the mRNA) and can also be important for post-transcriptional modifications. The sequence of an UTR can be homologous or heterologous to the sequence of the coding region found in a nucleic acid molecule. Multiple UTRs can be included in a nucleic acid molecule and can be of the same or different sequences, and/or genetic origin. According to the present disclosure, any portion of UTRs in a nucleic acid molecule (including none) can be codon optimized and any may independently contain one or more different structural or chemical modification, before and/or after codon optimization.

The UTR of the isolated nucleic acid (e.g., mRNA) may be involved in various regulatory aspects of gene expression. It should be understood that the UTRs (e.g., the 5’ UTRs and/or the 3’ UTRs) provided herein are examples, and that the isolated nucleic acid (e.g., mRNA) may comprise any UTR from any gene. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide synthetic (e.g., artificial UTRs) which are not variants of wild-type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5’ or 3’ UTR may be inverted, shortened, lengthened, or made chimeric with one or more other 5’ UTRs or 3’ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3’ or 5’ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, or swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’) comprise a variant UTR.

In some embodiments, a therapeutic nucleic acid molecule of the present disclosure (e.g., mRNA) comprises UTRs and coding regions that are homologous with respect to each other. In other embodiments, a nucleic acid molecule of the present disclosure (e.g., mRNA) comprises UTRs and coding regions that are heterologous with respect to each other. In some embodiments, to monitor the activity of a UTR sequence, anucleic acid molecule comprising the UTR and a coding sequence of a detectable probe can be administered in vitro (e.g., cell or tissue culture) or in vivo (e.g., to a subject) , and an effect of the UTR sequence (e.g., modulation on the expression level, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

In some embodiments, a double, triple, or quadruple UTR, such as a 5’ or 3’ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. It is also within the scope of the present invention to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. The UTRs provided herein may also include translation enhancer elements (TEE) .

In some embodiments, the UTR of a nucleic acid molecule of the present disclosure (e.g., mRNA) comprises at least one translation enhancer element (TEE) that functions to increase the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the TEE is located in the 5’-UTR of the nucleic acid molecule. In other embodiments, the TEE is located at the 3’-UTR of the nucleic acid molecule. In yet other embodiments, at least two TEE are located at the 5’-UTR and 3’-UTR of the nucleic acid molecule respectively. In some embodiments, a nucleic acid molecule of the present disclosure (e.g., mRNA) can comprise one or more copies of a TEE sequence or comprise more than one different TEE sequences. In some embodiments, different TEE sequences that are present in a nucleic acid molecule of the present disclosure can be homologues or heterologous with respect to one another.

Various TEE sequences that are known in the art and can be used in connection with the present disclosure. For example, in some embodiments, the TEE can be an internal ribosome entry site (IRES) , HCV-IRES or an IRES element. Chappell et al. Proc. Natl. Acad. Sci. USA 101: 9590-9594, 2004; Zhou et al. Proc. Natl. Acad. Sci. 102: 6273-6278, 2005. Additional internal ribosome entry site (IRES) that can be used in connection with the present disclosure include but are not limited to those described in U.S. Patent No. 7,468,275, U.S. Patent Publication No. 2007/0048776 and U.S. Patent Publication No. 2011/0124100 and International Patent Publication No. WO2007/025008 and International Patent Publication No. WO2001/055369, the content of each of which is enclosed herein by reference in its entirety. In some embodiments, the TEE can be those described in Supplemental Table 1 and in Supplemental Table 2 of Wellensiek et al Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10 (8) : 747–750; the content of which is incorporated by reference in its entirety.

Additional exemplary TEEs that can be used in connection with the present disclosure include but are not limited to the TEE sequences disclosed in U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, U.S. Patent No. 7,183,395, U.S. Patent Publication No. 2009/0226470, U.S. Patent Publication No. 2013/0177581, U.S. Patent Publication No. 2007/0048776, U.S. Patent Publication No. 2011/0124100, U.S. Patent Publication No. 2009/0093049, International Patent Publication No. WO2009/075886, International Patent Publication No. WO2012/009644, and International Patent Publication No. WO1999/024595, International Patent Publication No. WO2007/025008, International Patent Publication No. WO2001/055371, European Patent No. 2610341, European Patent No. 2610340, the content of each of which is enclosed herein by reference in its entirety.

In various embodiments, a nucleic acid molecule of the present disclosure (e.g., mRNA) comprises at least one UTR that comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In some embodiments, the TEE sequences in the UTR of a nucleic acid molecule are copies of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of a nucleic acid molecule are of different TEE sequences. In some embodiments, multiple different TEE sequences are arranged in one or more repeating patterns in the UTR region of a nucleic acid molecule. For illustrating purpose only, a repeating pattern can be, for example, ABABAB, AABBAABBAABB, ABCABCABC, or the like, where in these exemplary patterns, each capitalized letter (A, B, or C) represents a different TEE sequence. In some embodiments, at least two TEE sequences are consecutive with one another (i.e., no spacer sequence in between) in a UTR of a nucleic acid molecule. In other embodiments, at least two TEE sequences are separated by a spacer sequence. In some embodiments, a UTR can comprise a TEE sequence-spacer sequence module that is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or more than 9 times in the UTR. In any of the embodiments described in this paragraph, the UTR can be a 5’-UTR, a 3’-UTR or both 5’-UTR and 3’-UTR of a nucleic acid molecule.

In some embodiments, the UTR of a nucleic acid molecule of the present disclosure (e.g., mRNA) comprises at least one translation suppressing element that functions to decrease the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the UTR of the nucleic acid molecule comprises one or more miR sequences or fragment thereof (e.g., miR seed sequences) that are recognized by one or more microRNA. In some embodiments, the UTR of the nucleic acid molecule comprises one or more stem-loop structure that downregulates translational activity of the nucleic acid molecule. Other mechanisms for suppressing translational activities associated with nucleic acid molecules are known in the art. In any of the embodiments described in this paragraph, the UTR can be a 5’-UTR, a 3’-UTR or both 5’-UTR and 3’-UTR of a nucleic acid molecule.

In some embodiments, the 5’ UTR is from an organism or is synthetic. In some embodiments, the 5’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 136, 251 or 275. In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 5’-UTR comprising the sequence set forth in SEQ ID NO: 136. In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 5’-UTR comprising the sequence set forth in SEQ ID NO: 251. In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 5’-UTR comprising the sequence set forth in SEQ ID NO: 275.

In some embodiments, the 3’ UTR is from an organism or is synthetic. In some embodiments, the 3’ UTR comprises a nucleic acid sequence comprising at least about 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 137 or 252. In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 3’-UTR comprising the sequence set forth in SEQ ID NO: 137. In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 3’-UTR comprising the sequence set forth in SEQ ID NO: 252.

In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 5’-UTR comprising the sequence set forth in SEQ ID NO: 136 and a 3’-UTR comprising the sequence set forth in SEQ ID NO: 137. In specific embodiments, the therapeutic nucleic acid molecule of the present disclose comprises a 5’-UTR comprising the sequence set forth in SEQ ID NO: 251 and a 3’-UTR comprising the sequence set forth in SEQ ID NO: 252.

In any of the embodiments described in this paragraph, the nucleic acid molecule may further comprise a coding region having a sequence as described in Section 5.3.1 (Coding Region) , such as any of the RNA coding sequences in the Section 7 (Sequence Chart) or corresponding DNA sequences thereof. In particular embodiments, the nucleic acid molecules described in this paragraph can be RNA molecules in vitro transcribed.
5.3.3 mRNA Molecules

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising a coding sequence encoding a KRAS domain as described in Section 5.3.1.1. In some embodiments, the mRNA molecule further comprises a 5’-UTR and a 3’-UTR as described in Section 5.3.2.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof. In some embodiments, the mRNA molecule further comprises a 5’-UTR comprising the RNA sequence set forth in SEQ ID NO: 136. In some embodiments, the mRNA molecule further comprises a 3’-UTR comprising the RNA sequence set forth in SEQ ID NO: 137.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 139, or a codon optimized variant thereof SEQ ID NO: 139. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising, acoding sequence encoding a protease degradation domain as described in Section 5.3.1.2, and a coding sequence encoding a KRAS domain as described in Section 5.3.1.1. In some embodiments, the mRNA molecule further comprises a 5’-UTR and a 3’-UTR as described in Section 5.3.2.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising a coding sequence encoding a ubiquitin peptide comprising the RNA sequence selected from SEQ ID NOS: 2 and 3 or a codon optimized variant thereof, a coding sequence encoding a spacer comprising the RNA sequence selected from SEQ ID NOS: 5 and 6 or a codon optimized variant thereof, and a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising, in the 5’-to-3’ direction, a coding sequence encoding a ubiquitin peptide comprising the RNA sequence selected from SEQ ID NOS: 2 and 3 or a codon optimized variant thereof, a coding sequence encoding a spacer comprising the RNA sequence selected from SEQ ID NOS: 5 and 6 or a codon optimized variant thereof, and a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof.

In some embodiments, the mRNA molecule further comprises a 5’-UTR comprising the RNA sequence set forth in SEQ ID NO: 136. In some embodiments, the mRNA molecule further comprises a 3’-UTR comprising the RNA sequence set forth in SEQ ID NO: 137.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 143, or a codon optimized variant thereof SEQ ID NO: 143. In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 145, or a codon optimized variant thereof SEQ ID NO: 145. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising, acoding sequence encoding a signal peptide as described in Section 5.3.1.6, a coding sequence encoding a KRAS domain as described in Section 5.3.1.1, a coding sequence encoding a helper T cell epitope as described in Section 5.3.1.4, a coding sequence encoding a trafficking peptide as described in Section 5.3.1.3, a coding sequence encoding a cleavable linker as described in Section 5.3.1.7, and a coding sequence encoding an immunomodulator as described in Section 5.3.1.5. In some embodiments, the mRNA molecule further comprises at least one coding sequence encoding a linker peptide between adjacent coding sequences encoding the functional domains selected from the signal peptide, the KRAS domain, the helper T cell epitope, and the trafficking peptide, where the coding sequences encoding the linker peptide is as described in Section 5.3.1.7. In some embodiments, the mRNA molecule further comprises a 5’-UTR and a 3’-UTR as described in Section 5.3.2.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising, a coding sequence encoding a signal peptide comprising the RNA sequence selected from SEQ ID NOS: 90 to 93 or a codon optimized variant thereof, a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof, a coding sequence encoding a helper T cell epitope comprising the RNA sequence selected from SEQ ID NOS: 95 to 98 or a codon optimized variant thereof, a coding sequence encoding a trafficking peptide comprising the RNA sequence selected from SEQ ID NOS: 100 to 104 or a codon optimized variant thereof, a coding sequence encoding a cleavable linker comprising the RNA sequence selected from SEQ ID NOS: 106 to 110 or a codon optimized variant thereof, a coding sequence encoding an immunomodulator comprising the RNA sequence selected from SEQ ID NOS: 112 to 114, and 116 to 118 or a codon optimized variant thereof.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising, in the 5’-to-3’ direction, a coding sequence encoding a signal peptide comprising the RNA sequence selected from SEQ ID NOS: 90 to 93 or a codon optimized variant thereof, a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof, a coding sequence encoding a helper T cell epitope comprising the RNA sequence selected from SEQ ID NOS: 95 to 98 or a codon optimized variant thereof, a coding sequence encoding a trafficking peptide comprising the RNA sequence selected from SEQ ID NOS: 100 to 104 or a codon optimized variant thereof, a coding sequence encoding a cleavable linker comprising the RNA sequence selected from SEQ ID NOS: 106 to 110 or a codon optimized variant thereof, a coding sequence encoding an immunomodulator comprising the RNA sequence selected from SEQ ID NOS: 112 to 114, and 116 to 118 or a codon optimized variant thereof. In some embodiments, the mRNA molecule further comprises at least one coding sequence encoding a linker peptide between adjacent coding sequences for the signal peptide, the KRAS domain, the helper T cell epitope, and the trafficking peptide. In some embodiments, the at least one coding sequence encoding the linker peptide comprises the RNA sequence selected from SEQ ID NOS: 120 to 124, 126 to 131, and 133 to 135, or a codon optimized variant thereof.

In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 148, or a codon optimized variant thereof SEQ ID NO: 148. In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 150, or a codon optimized variant thereof SEQ ID NO: 150. In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 152, or a codon optimized variant thereof SEQ ID NO: 152. In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 155, or a codon optimized variant thereof SEQ ID NO: 155. In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 157, or a codon optimized variant thereof SEQ ID NO: 157. In some embodiments, the therapeutic nucleic acid molecule is a mRNA molecule comprising the sequence set forth in SEQ ID NO: 159, or a codon optimized variant thereof SEQ ID NO: 159. In some embodiments, the codon optimized variant of a coding RNA or DNA sequence has at least 80% (such as about any of 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to such coding RNA or DNA sequence.

In some embodiments, the therapeutic nucleic acid molecule is a multi-cistronic mRNA molecule comprising a first coding region and a second coding region, wherein the first coding region comprises a coding sequence encoding a signal peptide as described in Section 5.3.1.6, a coding sequence encoding a KRAS domain as described in Section 5.3.1.1, a coding sequence encoding a helper T cell epitope as described in Section 5.3.1.4, and a coding sequence encoding a trafficking peptide as described in Section 5.3.1.3; and where the second coding region comprises a coding sequence encoding an immunomodulator as described in Section 5.3.1.5. In some embodiments, in the first coding region, the mRNA molecule further comprises at least one coding sequence encoding a linker peptide between adjacent coding sequences encoding the functional domains selected from the signal peptide, the KRAS domain, the helper T cell epitope, and the trafficking peptide, where the coding sequences encoding the linker peptide is as described in Section 5.3.1.7. In some embodiments, the mRNA molecule further comprises a 5’-UTR and a 3’-UTR as described in Section 5.3.2.

In some embodiments, the therapeutic nucleic acid molecule is a multi-cistronic mRNA molecule comprising a first coding region and a second coding region, wherein the first coding region comprises a coding sequence encoding a signal peptide comprising the RNA sequence selected from SEQ ID NOS: 90 to 93 or a codon optimized variant thereof, a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof, a coding sequence encoding a helper T cell epitope comprising the RNA sequence selected from SEQ ID NOS: 95 to 98 or a codon optimized variant thereof, and a coding sequence encoding a trafficking peptide comprising the RNA sequence selected from SEQ ID NOS: 100 to 104 or a codon optimized variant thereof; and wherein the second coding region comprises a coding sequence encoding a coding sequence encoding an immunomodulator comprising the RNA sequence selected from SEQ ID NOS: 112 to 114, and 116 to 118 or a codon optimized variant thereof. In some embodiments, in the first coding region, the mRNA molecule further comprises at least one coding sequence encoding a linker peptide between adjacent coding sequences for the signal peptide, the KRAS domain, the helper T cell epitope, and the trafficking peptide. In some embodiments, the at least one coding sequence encoding the linker peptide comprises the RNA sequence selected from SEQ ID NOS: 120 to 124, 126 to 131, and 133 to 135, or a codon optimized variant thereof. In some embodiments, the mRNA molecule further comprises a 5’-UTR comprising the RNA sequence set forth in SEQ ID NO: 136. In some embodiments, the mRNA molecule further comprises a 3’-UTR comprising the RNA sequence set forth in SEQ ID NO: 137.

In some embodiments, the therapeutic nucleic acid molecule comprises a first mRNA molecule and a second mRNA molecule, wherein the first mRNA molecule comprises a coding sequence encoding a signal peptide as described in Section 5.3.1.6, a coding sequence encoding a KRAS domain as described in Section 5.3.1.1, a coding sequence encoding a helper T cell epitope as described in Section 5.3.1.4, and a coding sequence encoding a trafficking peptide as described in Section 5.3.1.3; and wherein the second mRNA molecule comprises a coding sequence encoding an immunomodulator as described in Section 5.3.1.5. In some embodiments, the first mRNA molecule further comprises at least one coding sequence encoding a linker peptide between adjacent coding sequences encoding the functional domains selected from the signal peptide, the KRAS domain, the helper T cell epitope, and the trafficking peptide, where the coding sequences encoding the linker peptide is as described in Section 5.3.1.7. In some embodiments, the first and/or second mRNA molecules further comprises a 5’-UTR and a 3’-UTR as described in Section 5.3.2.

In some embodiments, the therapeutic nucleic acid molecule comprises a first mRNA molecule and a second mRNA molecule, wherein the first mRNA molecule comprises a coding sequence encoding a signal peptide comprising the RNA sequence selected from SEQ ID NOS: 90 to 93 or a codon optimized variant thereof, a coding sequence encoding a KRAS domain comprising the RNA sequence selected from SEQ ID NOS: 87, 88, 160, 162 to 167 or a codon optimized variant thereof, a coding sequence encoding a helper T cell epitope comprising the RNA sequence selected from SEQ ID NOS: 95 to 98 or a codon optimized variant thereof, and a coding sequence encoding a trafficking peptide comprising the RNA sequence selected from SEQ ID NOS: 100 to 104 or a codon optimized variant thereof; and wherein the second mRNA molecule comprises a coding sequence encoding an immunomodulator comprising the RNA sequence selected from SEQ ID NOS: 112 to 114, and 116 to 118 or a codon optimized variant thereof. In some embodiments, the first mRNA molecule further comprises at least one coding sequence encoding a linker peptide between adjacent coding sequences for the signal peptide, the KRAS domain, the helper T cell epitope, and the trafficking peptide. In some embodiments, the at least one coding sequence encoding the linker peptide comprises the RNA sequence selected from SEQ ID NOS: 120 to 124, 126 to 131, and 133 to 135, or a codon optimized variant thereof. In some embodiments, the first and/or second mRNA molecule further comprises a 5’-UTR comprising the RNA sequence set forth in SEQ ID NO: 136. In some embodiments, the first and/or second mRNA molecule further comprises a 3’-UTR comprising the RNA sequence set forth in SEQ ID NO: 137.

In some embodiments, the mRNA molecule described herein further comprises a 5’ cap structure as described in Section 5.3.4. In some embodiments, the mRNA molecule described herein further comprises a poly-A region as described in Section 5.3.5.
5.3.4 5’-Cap Structure

Without being bound by the theory, it is contemplated that, a 5’-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP) , which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. The 5’-cap structure further assists the removal of 5’-proximal introns removal during mRNA splicing. Accordingly, in some embodiments, the nucleic acid molecules of the present disclosure comprise a 5’-cap structure.

Nucleic acid molecules may be 5’-end capped by the endogenous transcription machinery of a cell to generate a 5’-ppp-5’-triphosphate linkage between a terminal guanosine cap residue and the 5’-terminal transcribed sense nucleotide of the polynucleotide. This 5’-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5’ end of the polynucleotide may optionally also be 2’-O-methylated. 5’-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations to the natural 5’-cap structure generated by the endogenous process. Without being bound by the theory, a modification on the 5’-cap may increase the stability of polynucleotide, increase the half-life of the polynucleotide, and could increase the polynucleotide translational efficiency.

Exemplary alterations to the natural 5’-Cap structure include generation of a non-hydrolyzable cap structure preventing decapping and thus increasing polynucleotide half-life. In some embodiments, because cap structure hydrolysis requires cleavage of 5’-ppp-5’ phosphorodiester linkages, in some embodiments, modified nucleotides may be used during the capping reaction. For example, in some embodiments, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass. ) may be used withα-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5’-ppp-5’ cap. Additional modified guanosine nucleotides may be used, such asα-methyl-phosphonate and seleno-phosphate nucleotides.

Additional exemplary alterations to the natural 5’-Cap structure also include modification at the 2’-and/or 3’-position of a capped guanosine triphosphate (GTP) , a replacement of the sugar ring oxygen (that produced the carbocyclic ring) with a methylene moiety (CH₂) , a modification at the triphosphate bridge moiety of the cap structure, or a modification at the nucleobase (G) moiety.

Additional exemplary alterations to the natural 5’-cap structure include, but are not limited to, 2’-O-methylation of the ribose sugars of 5’-terminal and/or 5’-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2’-hydroxy group of the sugar. Multiple distinct 5’-cap structures can be used to generate the 5’-cap of a polynucleotide, such as an mRNA molecule. Additional exemplary 5’-Cap structures that can be used in connection with the present disclosure further include those described in International Patent Publication Nos. WO2008127688, WO 2008016473, and WO 2011015347, the entire contents of each of which are incorporated herein by reference.

In various embodiments, 5’-terminal caps can include cap analogs. Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wild-type, or physiological) 5’-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and/linked to a polynucleotide.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanosines linked by a 5’-5’-triphosphate group, wherein one guanosine contains an N7-methyl group as well as a 3’-O-methyl group (i.e., N7, 3’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine, m⁷G-3’mppp-G, which may equivalently be designated 3’ O-Me-m7G (5’) ppp (5’) G) . The 3’-O atom of the other, unaltered, guanosine becomes linked to the 5’-terminal nucleotide of the capped polynucleotide (e.g., an mRNA) . The N7-and 3’-O-methlyated guanosine provides the terminal moiety of the capped polynucleotide (e.g., mRNA) . Another exemplary cap structure is mCAP, which is similar to ARCA but has a 2’-O-methyl group on guanosine (i.e., N7, 2’-O-dimethyl-guanosine-5’-triphosphate-5’-guanosine, m⁷Gm-ppp-G) .

In some embodiments, a cap analog can be a dinucleotide cap analog. As a non-limiting example, the dinucleotide cap analog may be modified at different phosphate positions with a boranophosphate group or a phophoroselenoate group such as the dinucleotide cap analogs described in U.S. Patent No.: 8,519,110, the entire content of which is herein incorporated by reference in its entirety.

In some embodiments, a cap analog can be a N7- (4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and/or described herein. Non-limiting examples of N7- (4-chlorophenoxyethyl) substituted dinucleotide cap analogs include a N7- (4-chlorophenoxyethyl) -G (5’) ppp (5’) G and a N7- (4-chlorophenoxyethyl) -m3’-OG (5’) ppp (5’) G cap analog (see, e.g., the various cap analogs and the methods of synthesizing cap analogs described in Kore et al. Bioorganic&Medicinal Chemistry 2013 21: 4570-4574; the entire content of which is herein incorporated by reference) . In other embodiments, a cap analog useful in connection with the nucleic acid molecules of the present disclosure is a 4-chloro/bromophenoxyethyl analog.

In various embodiments, a cap analog can include a guanosine analog. Useful guanosine analogs include but are not limited to inosine, N1-methyl-guanosine, 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Without being bound by the theory, it is contemplated that while cap analogs allow for the concomitant capping of a polynucleotide in an in vitro transcription reaction, up to 20%of transcripts remain uncapped. This, as well as the structural differences of a cap analog from the natural 5’-cap structures of polynucleotides produced by the endogenous transcription machinery of a cell, may lead to reduced translational competency and reduced cellular stability.

Accordingly, in some embodiments, a nucleic acid molecule of the present disclosure can also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5’-cap structures. As used herein, the phrase “more authentic” refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a “more authentic” feature is better representative of an endogenous, wild-type, natural or physiological cellular function, and/or structure as compared to synthetic features or analogs of the prior art, or which outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more authentic 5’-cap structures useful in connection with the nucleic acid molecules of the present disclosure are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5’-endonucleases, and/or reduced 5’-decapping, as compared to synthetic 5’-cap structures known in the art (or to a wild-type, natural or physiological 5’-cap structure) . For example, in some embodiments, recombinant Vaccinia Virus Capping Enzyme and recombinant 2’-O-methyltransferase enzyme can create a canonical 5’-5’-triphosphate linkage between the 5’-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide wherein the cap guanosine contains an N7-methylation and the 5’-terminal nucleotide of the polynucleotide contains a 2’-O-methyl. Such a structure is termed the Cap1 structure. This cap results in a higher translational-competency, cellular stability, and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5’cap analog structures known in the art. Other exemplary cap structures include 7mG (5’) ppp (5’) N, pN2p (Cap 0) , 7mG (5’) ppp (5’) NlmpNp (Cap 1) , ^7MeGpppG_2’ OMe (Cap 1) , 7mG (5’) -ppp (5’) NlmpN2mp (Cap 2) , and m (7) Gpppm (3) (6, 6, 2’) Apm (2’) Apm (2’) Cpm (2) (3, 2’) Up (Cap 4) .

Without being bound by the theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped post-transcriptionally, and because this process is more efficient, nearly 100%of the nucleic acid molecules may be capped.
5.3.5 The Polyadenylation (Poly-A) Regions

During natural RNA processing, a long chain of adenosine nucleotides (poly-A region) is normally added to messenger RNA (mRNA) molecules to increase the stability of the molecule. Immediately after transcription, the 3’-end of the transcript is cleaved to free a 3’-hydroxy. Then poly-A polymerase adds a chain of adenosine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A region that is between 100 and 250 residues long. Without being bound by the theory, it is contemplated that a poly-A region can confer various advantages to the nucleic acid molecule of the present disclosure.

Accordingly, in some embodiments, a nucleic acid molecule of the present disclosure (e.g., an mRNA) comprises a polyadenylation signal. In some embodiments, a nucleic acid molecule of the present disclosure (e.g., an mRNA) comprises one or more polyadenylation (poly-A) regions. In some embodiments, a poly-A region is composed entirely of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3’-end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5’-end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5’-end and at least one poly-A region at its 3’-end.

According to the present disclosure, the poly-A region can have varied lengths in different embodiments. Particularly, in some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 30 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 35 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 40 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 45 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 50 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 55 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 60 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 65 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 70 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 75 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 80 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 85 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 90 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 95 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 100 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 110 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 120 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 130 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 140 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 150 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 160 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 170 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 180 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 190 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 200 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 225 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 250 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 275 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 300 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 350 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 400 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 450 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 600 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 700 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 800 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 900 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1000 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1100 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1200 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1300 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1400 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1600 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1700 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1800 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1900 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2000 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2250 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2750 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 3000 nucleotides in length.

In some embodiments, length of a poly-A region in a nucleic acid molecule can be selected based on the overall length of the nucleic acid molecule, or a portion thereof (such as the length of the coding region or the length of an open reading frame of the nucleic acid molecule, etc. ) . For example, in some embodiments, the poly-A region accounts for about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more of the total length of nucleic acid molecule containing the poly-A region.

Without being bound by the theory, it is contemplated that certain RNA-binding proteins can bind to the poly-A region located at the 3’-end of an mRNA molecule. These poly-A binding proteins (PABP) can modulate mRNA expression, such as interacting with translation initiation machinery in a cell and/or protecting the 3’-poly-A tails from degradation. Accordingly, in some embodiments, in some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises at least one binding site for poly-A binding protein (PABP) . In other embodiments, the nucleic acid molecule is conjugated or complex with a PABP before loaded into a delivery vehicle (e.g., lipid nanoparticles) .

In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises a poly-A-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanosine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A region. The resultant polynucleotides (e.g., mRNA) may be assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet structure results in protein production equivalent to at least 75%of that seen using a poly-A region of 120 nucleotides alone.

In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) may include a poly-A region and may be stabilized by the addition of a 3’-stabilizing region. In some embodiments, the 3’-stabilizing region which may be used to stabilize a nucleic acid molecule (e.g., mRNA) including the poly-A or poly-A-G Quartet structures as described in International Patent Publication No. WO2013/103659, the content of which is incorporated herein by reference in its entirety.

In other embodiments, the 3’-stabilizing region which may be used in connection with the nucleic acid molecules of the present disclosure include a chain termination nucleoside such as but is not limited to 3’-deoxyadenosine (cordycepin) , 3’-deoxyuridine, 3’-deoxycytosine, 3’-deoxyguanosine, 3’-deoxythymine, 2’, 3’-dideoxynucleosides, such as 2’, 3’-dideoxyadenosine, 2’, 3’-dideoxyuridine, 2’, 3’-dideoxycytosine, 2’, 3’-dideoxyguanosine, 2’, 3’-dideoxythymine, a 2’-deoxynucleoside, or an O-methylnucleoside, 3’-deoxynucleoside, 2’, 3’-dideoxynucleoside 3’-O-methylnucleosides, 3’-O-ethylnucleosides, 3’-arabinosides, and other alternative nucleosides known in the art and/or described herein.
5.3.6 Secondary Structure

Without being bound by the theory, it is contemplated that a stem-loop structure can direct RNA folding, protect structural stability of a nucleic acid molecule (e.g., mRNA) , provide recognition sites for RNA binding proteins, and serve as a substrate for enzymatic reactions. For example, the incorporation of a miR sequence and/or a TEE sequence changes the shape of the stem loop region which may increase and/or decrease translation (Kedde et al. A Pumilio-induced RNA structure switch in p27-3’ UTR controls miR-221 and miR-222 accessibility. Nat Cell Biol., 2010 Oct; 12 (10) : 1014-20, the content of which is herein incorporated by reference in its entirety) .

Accordingly, in some embodiments, the nucleic acid molecules as described herein (e.g., mRNA) or a portion thereof may assume a stem-loop structure, such as but is not limited to a histone stem loop. In some embodiments, the stem-loop structure is formed from a stem-loop sequence that is about 25 or about 26 nucleotides in length such as, but not limited to, those as described in International Patent Publication No. WO2013/103659, the content of which is incorporated herein by reference in its entirety. Additional examples of stem-loop sequences include those described in International Patent Publication No. WO2012/019780 and International Patent Publication No. WO201502667, the contents of which are incorporated herein by reference. In some embodiments, the step-loop sequence comprises a TEE as described herein. In some embodiments, the step-loop sequence comprises a miR sequence as described herein. In specific embodiments, the stem loop sequence may include a miR-122 seed sequence.

In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises a stem-loop sequence located upstream (to the 5’-end) of the coding region in a nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 5’-UTR of the nucleic acid molecule. In some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises a stem-loop sequence located downstream (to the 3’-end) of the coding region in a nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 3’-UTR of the nucleic acid molecule. In some cases, a nucleic acid molecule can contain more than one stem-loop sequences. In some embodiment, the nucleic acid molecule comprises at least one stem-loop sequence in the 5’-UTR, and at least one stem-loop sequence in the 3’-UTR.

In some embodiments, a nucleic acid molecule comprising a stem-loop structure further comprises a stabilization region. In some embodiment, the stabilization region comprises at least one chain terminating nucleoside that functions to slow down degradation and thus increases the half-life of the nucleic acid molecule. Exemplary chain terminating nucleoside that can be used in connection with the present disclosure include but are not limited to 3’-deoxyadenosine (cordycepin) , 3’-deoxyuridine, 3’-deoxycytosine, 3’-deoxyguanosine, 3’-deoxythymine, 2’, 3’-dideoxynucleosides, such as 2’, 3’-dideoxyadenosine, 2’, 3’-dideoxyuridine, 2’, 3’-dideoxycytosine, 2’, 3’-dideoxyguanosine, 2’, 3’-dideoxythymine, a 2’-deoxynucleoside, or an O-methylnucleoside, 3’-deoxynucleoside, 2’, 3’-dideoxynucleoside 3’-O-methylnucleosides, 3’-O-ethylnucleosides, 3’-arabinosides, and other alternative nucleosides known in the art and/or described herein. In other embodiments, a stem-loop structure may be stabilized by an alteration to the 3’-region of the polynucleotide that can prevent and/or inhibit the addition of oligio (U) (International Patent Publication No. WO2013/103659, incorporated herein by reference in its entirety) .

In some embodiments, a nucleic acid molecule of the present disclosure comprises at least one stem-loop sequence and a poly-A region or polyadenylation signal. Non-limiting examples of polynucleotide sequences comprising at least one stem-loop sequence and a poly-A region or a polyadenylation signal include those described in International Patent Publication No. WO2013/120497, International Patent Publication No. WO2013/120629, International Patent Publication No. WO2013/120500, International Patent Publication No. WO2013/120627, International Patent Publication No. WO2013/120498, International Patent Publication No. WO2013/120626, International Patent Publication No. WO2013/120499 and International Patent Publication No. WO2013/120628, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can encode for a pathogen antigen or fragment thereof such as the polynucleotide sequences described in International Patent Publication No. WO2013/120499 and International Patent Publication No. WO2013/120628, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can encode for a therapeutic protein such as the polynucleotide sequences described in International Patent Publication No. WO2013/120497 and International Patent Publication No. WO2013/120629, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can encode for a tumor antigen or fragment thereof such as the polynucleotide sequences described in International Patent Publication No. WO2013/120500 and International Patent Publication No. WO2013/120627, the content of each of which is incorporated herein by reference in its entirety.

In some embodiments, the nucleic acid molecule comprising a stem-loop sequence and a poly-A region or a polyadenylation signal can code for an allergenic antigen or an autoimmune self-antigen such as the polynucleotide sequences described in International Patent Publication No. WO2013/120498 and International Patent Publication No. WO2013/120626, the content of each of which is incorporated herein by reference in its entirety.
5.3.7 Functional nucleotide analogs

In some embodiments, a payload nucleic acid molecule described herein contains only canonical nucleotides selected from A (adenosine) , G (guanosine) , C (cytosine) , U (uridine) , and T (thymidine) . Without being boundby the theory, it is contemplated that certain functional nucleotide analogs can confer useful properties to a nucleic acid molecule. Examples of such as useful properties in the context of the present disclosure include but are not limited to increased stability of the nucleic acid molecule, reduced immunogenicity of the nucleic acid molecule in inducing innate immune responses, enhanced production of protein encoded by the nucleic acid molecule, increased intracellular delivery and/or retention of the nucleic acid molecule, and/or reduced cellular toxicity of the nucleic acid molecule, etc.

Accordingly, in some embodiments, a payload nucleic acid molecule comprises at least one functional nucleotide analog as described herein. In some embodiments, the functional nucleotide analog contains at least one chemical modification to the nucleobase, the sugar group and/or the phosphate group. Accordingly, a payload nucleic acid molecule comprising at least one functional nucleotide analog contains at least one chemical modification to the nucleobases, the sugar groups, and/or the internucleoside linkage. Exemplary chemical modifications to the nucleobases, sugar groups, or internucleoside linkages of a nucleic acid molecule are provided herein.

As described herein, ranging from 0%to 100%of all nucleotides in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1%to about 20%, from about 1%to about 25%, from about 1%to about 50%, from about 1%to about 60%, from about 1%to about 70%, from about 1%to about 80%, from about 1%to about 90%, from about 1%to about 95%, from about 10%to about 20%, from about 10%to about 25%, from about 10%to about 50%, from about 10%to about 60%, from about 10%to about 70%, from about 10%to about 80%, from about 10%to about 90%, from about 10%to about 95%, from about 10%to about 100%, from about 20%to about 25%, from about 20%to about 50%, from about 20%to about 60%, from about 20%to about 70%, from about 20%to about 80%, from about 20%to about 90%, from about 20%to about 95%, from about 20%to about 100%, from about 50%to about 60%, from about 50%to about 70%, from about 50%to about 80%, from about 50%to about 90%, from about 50%to about 95%, from about 50%to about 100%, from about 70%to about 80%, from about 70%to about 90%, from about 70%to about 95%, from about 70%to about 100%, from about 80%to about 90%, from about 80%to about 95%, from about 80%to about 100%, from about 90%to about 95%, from about 90%to about 100%, or from about 95%to about 100%of all nucleotides in a nucleic acid molecule are functional nucleotide analogs described herein. In any of these embodiments, a functional nucleotide analog can be present at any position (s) of a nucleic acid molecule, including the 5’-terminus, 3’-terminus, and/or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and/or different types internucleoside linkages (e.g., backbone structures) .

As described herein, ranging from 0%to 100%of all nucleotides of a kind (e.g., all purine-containing nucleotides as a kind, or all pyrimidine-containing nucleotides as a kind, or all A, G, C, T or U as a kind) in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1%to about 20%, from about 1%to about 25%, from about 1%to about 50%, from about 1%to about 60%, from about 1%to about 70%, from about 1%to about 80%, from about 1%to about 90%, from about 1%to about 95%, from about 10%to about 20%, from about 10%to about 25%, from about 10%to about 50%, from about 10%to about 60%, from about 10%to about 70%, from about 10%to about 80%, from about 10%to about 90%, from about 10%to about 95%, from about 10%to about 100%, from about 20%to about 25%, from about 20%to about 50%, from about 20%to about 60%, from about 20%to about 70%, from about 20%to about 80%, from about 20%to about 90%, from about 20%to about 95%, from about 20%to about 100%, from about 50%to about 60%, from about 50%to about 70%, from about 50%to about 80%, from about 50%to about 90%, from about 50%to about 95%, from about 50%to about 100%, from about 70%to about 80%, from about 70%to about 90%, from about 70%to about 95%, from about 70%to about 100%, from about 80%to about 90%, from about 80%to about 95%, from about 80%to about 100%, from about 90%to about 95%, from about 90%to about 100%, or from about 95%to about 100%of a kind of nucleotides in a nucleic acid molecule are functional nucleotide analogs described herein. In any of these embodiments, a functional nucleotide analog can be present at any position (s) of a nucleic acid molecule, including the 5’-terminus, 3’-terminus, and/or one or more internal positions. In some embodiments, a single nucleic acid molecule can contain different sugar modifications, different nucleobase modifications, and/or different types internucleoside linkages (e.g., backbone structures) .
5.3.8 Modification to Nucleobases

In some embodiments, a functional nucleotide analog contains a non-canonical nucleobase. In some embodiments, canonical nucleobases (e.g., adenine, guanine, uracil, thymine, and cytosine) in a nucleotide can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modification to nucleobases include but are not limited to one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and/or thio substitutions; one or more fused or open rings, oxidation, and/or reduction.

In some embodiments, the non-canonical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having an modified uracil include pseudouridine (ψ) , pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s²U) , 4-thio-uracil (s⁴U) , 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho⁵U) , 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil) , 3-methyl-uracil (m³U) , 5-methoxy-uracil (mo⁵U) , uracil 5-oxyacetic acid (cmo⁵U) , uracil 5-oxyacetic acid methyl ester (mcmo⁵U) , 5-carboxymethyl-uracil (cm⁵U) , 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm⁵U) , 5-carboxyhydroxymethyl-uracil methyl ester (mchm⁵U) , 5-methoxycarbonylmethyl-uracil (mcm⁵U) , 5-methoxycarbonylmethyl-2-thio-uracil (mcm⁵s²U) , 5-aminomethyl-2-thio-uracil (nm⁵s²U) , 5-methylaminomethyl-uracil (mnm⁵U) , 5-methylaminomethyl-2-thio-uracil (mnm⁵s²U) , 5-methylaminomethyl-2-seleno-uracil (mnm⁵se²U) , 5-carbamoylmethyl-uracil (ncm⁵U) , 5-carboxymethylaminomethyl-uracil (cmnm⁵U) , 5-carboxymethylaminomethyl-2-thio-uracil (cmnm⁵s²U) , 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm ⁵U) , 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm⁵5s²U) , 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m⁵U, i.e., having the nucleobase deoxythymine) , 1-methyl-pseudouridine (m¹ψ) , 1-ethyl-pseudouridine (Et¹ψ) , 5-methyl-2-thio-uracil (m⁵s²U) , 1-methyl-4-thio-pseudouridine (m¹s⁴ψ) , 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ) , 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D) , dihydropseudouridine, 5, 6-dihydrouracil, 5-methyl-dihydrouracil (m⁵D) , 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uracil (acp³U) , 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp³ψ) , 5- (isopentenylaminomethyl) uracil (m⁵U) , 5- (isopentenylaminomethyl) -2-thio-uracil (m⁵s²U) , 5, 2’-O-dimethyl-uridine (m⁵Um) , 2-thio-2’-O-methyl-uridine (s²Um) , 5-methoxycarbonylmethyl-2’-O-methyl-uridine (mcm⁵Um) , 5-carbamoylmethyl-2’-O-methyl-uridine (ncm⁵Um) , 5-carboxymethylaminomethyl-2’-O-methyl-uridine (cmnm⁵Um) , 3, 2’-O-dimethyl-uridine (m³Um) , and 5- (isopentenylaminomethyl) -2’-O-methyl-uridine (inm⁵Um) , 1-thio-uracil, deoxythymidine, 5- (2-carbomethoxyvinyl) -uracil, 5- (carbamoylhydroxymethyl) -uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil, and 5- [3- (1-E-propenylamino) ] uracil.

In some embodiments, the non-canonical nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C) , N4-acetyl-cytosine (ac4C) , 5-formyl-cytosine (f5C) , N4-methyl-cytosine (m4C) , 5-methyl-cytosine (m5C) , 5-halo-cytosine (e.g., 5-iodo-cytosine) , 5-hydroxymethyl-cytosine (hm5C) , 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C) , 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5-methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C) , 5, 2’-O-dimethyl-cytidine (m5Cm) , N4-acetyl-2’-O-methyl-cytidine (ac4Cm) , N4, 2’-O-dimethyl-cytidine (m4Cm) , 5-formyl-2’-O-methyl-cytidine (fSCm) , N4, N4, 2’-O-trimethyl-cytidine (m42Cm) , 1-thio-cytosine, 5-hydroxy-cytosine, 5- (3-azidopropyl) -cytosine, and 5- (2-azidoethyl) -cytosine.

In some embodiments, the non-canonical nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine) , 6-halo-purine (e.g., 6-chloro-purine) , 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenine (m1A) , 2-methyl-adenine (m2A) , N6-methyl-adenine (m6A) , 2-methylthio-N6-methyl-adenine (ms2m6A) , N6-isopentenyl-adenine (i6A) , 2-methylthio-N6-isopentenyl-adenine (ms2i6A) , N6- (cis-hydroxyisopentenyl) adenine (io6A) , 2-methylthio-N6- (cis-hydroxyisopentenyl) adenine (ms2io6A) , N6-glycinylcarbamoyl-adenine (g6A) , N6-threonylcarbamoyl-adenine (t6A) , N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A) , 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A) , N6, N6-dimethyl-adenine (m62A) , N6-hydroxynorvalylcarbamoyl-adenine (hn6A) , 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A) , N6-acetyl-adenine (ac6A) , 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6, 2’-O-dimethyl-adenosine (m6Am) , N6, N6, 2’-O-trimethyl-adenosine (m62Am) , 1, 2’-O-dimethyl-adenosine (m1Am) , 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6- (19-amino-pentaoxanonadecyl) -adenine, 2, 8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

In some embodiments, the non-canonical nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I) , 1-methyl-inosine (m1I) , wyosine (imG) , methylwyosine (mimG) , 4-demethyl-wyosine (imG-14) , isowyosine (imG2) , wybutosine (yW) , peroxywybutosine (o2yW) , hydroxywybutosine (OHyW) , undermodified hydroxywybutosine (OHyW*) , 7-deaza-guanine, queuosine (Q) , epoxyqueuosine (oQ) , galactosyl-queuosine (galQ) , mannosyl-queuosine (manQ) , 7-cyano-7-deaza-guanine (preQO) , 7-aminomethyl-7-deaza-guanine (preQ1) , archaeosine (G+) , 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G) , 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G) , N2-methyl-guanine (m2G) , N2, N2-dimethyl-guanine (m22G) , N2, 7-dimethyl-guanine (m2, 7G) , N2, N2, 7-dimethyl-guanine (m2, 2, 7G) , 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2, N2-dimethyl-6-thio-guanine, N2-methyl-2’-O-methyl-guanosine (m2Gm) , N2, N2-dimethyl-2’-O-methyl-guanosine (m22Gm) , 1-methyl-2’-O-methyl-guanosine (m1Gm) , N2, 7-dimethyl-2’-O-methyl-guanosine (m2, 7Gm) , 2’-O-methyl-inosine (Im) , 1, 2’-O-dimethyl-inosine (m1Im) , 1-thio-guanine, and O-6-methyl-guanine.

In some embodiments, the non-canonical nucleobase of a functional nucleotide analog can be independently a purine, a pyrimidine, a purine or pyrimidine analog. For example, in some embodiments, the non-canonical nucleobase can be modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, the non-canonical nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo [3, 4-d] pyrimidines, 5-methylcytosine (5-me-C) , 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil) , 4-thiouracil, 8-halo (e.g., 8-bromo) , 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo [3, 4-d] pyrimidine, imidazo [1, 5-a] 1, 3, 5 triazinones, 9-deazapurines, imidazo [4, 5-d] pyrazines, thiazolo [4, 5-d] pyrimidines, pyrazin-2-ones, 1, 2, 4-triazine, pyridazine; or 1, 3, 5 triazine.
In some embodiments, the isolated nucleic acid, e.g., mRNA comprises a chemical modification. In
some embodiments, at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or all) of the nucleotides on the isolated nucleic acid are chemically modified. In some embodiments, the chemical modification occurs in the coding sequence, an intron, the 3’ UTR, or the 5’ UTR of the mRNA. In some embodiments, the chemical modification includes a modification to an adenosine, cytidine, guanosine, and/or a uridine base. In some embodiments, at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%adenosine base of the isolated nucleic acid comprises a chemical modification. In some embodiments, at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%cytidines of the isolated nucleic acid comprise a chemical modification. In some embodiments, at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%guanosine base of the isolated nucleic acid comprises a chemical modification. In some embodiments, at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%uridine base of the isolated nucleic acid comprises a chemical modification. In some embodiments, the adenosine is converted to an inosine, or methylated to N¹-methyladenosine, N⁶-methyladenosine, or N⁶, N⁶-dimethyladenosine. In some embodiments, the cytidine is converted to uridine, acetylated to N⁴-acetylcytidine, or methylated to 3-methylcytidine or 5-methylcytidine. In some embodiments, the 5-methylcytidine is further converted to 5-hydroxymethylcytidine. In some embodiments, the guanosine is methylated to 7-methylguanosine or oxidized to 7, 8-dihydro-8-oxoguanosine. In some embodiments, the ribose sugars of all nucleotides can be 2'-O-methylated. In some embodiments, the uridine is be converted to pseudouridine (Ψ) . In some embodiments, each uridine of the (e.g. ) mRNA is converted to a pseudouridine. In some embodiments, the (e.g. ) mRNA comprises an N¹-methylpseudouridine chemical modification. In some embodiments, each uridine of the (e.g. ) mRNA is converted to an N¹-methylpseudouridine. In some embodiments, M1-ψ means one or more or all uridines are converted to an N¹-methylpseudouridine.
5.3.9 Modification to the Sugar

In some embodiments, a functional nucleotide analog contains a non-canonical sugar group. In various embodiments, the non-canonical sugar group can be a 5-carbon or 6-carbon sugar (such as pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof) with one or more substitutions, such as a halo group, a hydroxy group, a thiol group, an alkyl group, an alkoxy group, an alkenyloxy group, an alkynyloxy group, an cycloalkyl group, an aminoalkoxy group, an alkoxyalkoxy group, an hydroxyalkoxy group, an amino group, an azido group, an aryl group, an aminoalkyl group, an aminoalkenyl group, an aminoalkynyl group, etc.

Generally, RNA molecules contain the ribose sugar group, which is a 5-membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with 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 4-membered ring of cyclobutane or oxetane) ; ring expansion of ribose (e.g., to form a 6-or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (that also has a phosphoramidate backbone) ) ; multicyclic forms (e.g., tricyclo and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds) , threose nucleic acid (TNA, where ribose is replace withα-L-threofuranosyl- (3’ →2’) ) , and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone) .

In some embodiments, the sugar group contains one or more carbons that possess the opposite stereochemical configuration of the corresponding carbon in ribose. Thus, a nucleic acid molecule can include nucleotides containing, e.g., arabinose or L-ribose, as the sugar. In some embodiments, the nucleic acid molecule includes at least one nucleoside wherein the sugar is L-ribose, 2’-O-methyl-ribose, 2’-fluoro-ribose, arabinose, hexitol, an LNA, or a PNA.
5.3.10 Modifications to the Internucleoside Linkage

In some embodiments, the payload nucleic acid molecule of the present disclosure can contain one or more modified internucleoside linkage (e.g., phosphate backbone) . Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent.

In some embodiments, the functional nucleotide analogs can include the replacement of an unaltered phosphate moiety with another internucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates) , sulfur (bridged phosphorothioates) , and carbon (bridged methylene-phosphonates) .

The alternative nucleosides and nucleotides can include the replacement of one or more of the non-bridging oxygens with a borane moiety (BH₃) , sulfur (thio) , methyl, ethyl, and/or methoxy. As a non-limiting example, two non-bridging oxygens at the same position (e.g., the alpha (α) , beta (β) or gamma (γ) position) can be replaced with a sulfur (thio) and a methoxy. The replacement of one or more of the oxygen atoms at the position of the phosphate moiety (e.g., α-thio phosphate) is provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

Other internucleoside linkages that may be employed according to the present disclosure, including internucleoside linkages which do not contain a phosphorous atom, are described herein.

Additional examples of nucleic acid molecules (e.g., mRNA) , compositions, formulations and/or methods associated therewith that can be used in connection with the present disclosure further include those described in WO2002/098443, WO2003/051401, WO2008/052770, WO2009127230, WO2006122828, WO2008/083949, WO2010088927, WO2010/037539, WO2004/004743, WO2005/016376, WO2006/024518, WO2007/095976, WO2008/014979, WO2008/077592, WO2009/030481, WO2009/095226, WO2011069586, WO2011026641, WO2011/144358, WO2012019780, WO2012013326, WO2012089338, WO2012113513, WO2012116811, WO2012116810, WO2013113502, WO2013113501, WO2013113736, WO2013143698, WO2013143699, WO2013143700, WO2013/120626, WO2013120627, WO2013120628, WO2013120629, WO2013174409, WO2014127917, WO2015/024669, WO2015/024668, WO2015/024667, WO2015/024665, WO2015/024666, WO2015/024664, WO2015101415, WO2015101414, WO2015024667, WO2015062738, WO2015101416, the content of each of which is incorporated herein in its entirety.

Therapeutic nucleic acid molecules as described herein can by isolated or synthesized using methods known in the art. In some embodiments, DNA or RNA molecules to be used in connection with the present disclosure are chemically synthesized. In other embodiments, DNA or RNA molecules to be used in connection with the present disclosure are isolated from a natural source.

In some embodiments, mRNA molecules to be used in connection with the present disclosure are biosynthesized using a host cell. In particular embodiments, an mRNA is produced by transcribing a corresponding DNA sequencing using a host cell. In some embodiments, a DNA sequence encoding an mRNA sequence is incorporated into an expression vector, which vector is then introduced into a host cell (e.g., E. coli) using methods known in the art. The host cell is then cultured under a suitable condition to produce mRNA transcripts. Other methods for producing an mRNA molecule from an encoding DNA are known in the art. For example, in some embodiments, a cell-free (in vitro) transcription system comprising enzymes of the transcription machinery of a host cell can be used to produce mRNA transcripts.
5.4 Nanoparticle Compositions

In one aspect, therapeutic nucleic acid molecules described herein are formulated for in vitro and in vivo delivery. Particularly, in some embodiments the nucleic acid molecule is formulated into a lipid-containing composition. In some embodiments, the lipid-containing composition forms lipid nanoparticles enclosing the nucleic acid molecule within a lipid shell. In some embodiments, the lipid shells protect the nucleic acid molecules from degradation. In some embodiments, the lipid nanoparticles also facilitate transportation of the enclosed nucleic acid molecules into intracellular compartments and/or machinery to exert an intended therapeutic of prophylactic function. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, such as those disclosed in, e.g., U.S. Patent Publication No. 2004/0142025, U.S. Patent Publication No. 2007/0042031, PCT Publication No. WO 2017/004143, PCT Publication No. WO 2015/199952, PCT Publication No. WO 2013/016058, and PCT Publication No. WO 2013/086373, the full disclosures of each of which are herein incorporated by reference in their entirety for all purposes.

In some embodiments, the largest dimension of a nanoparticle composition provided herein is 1μm or shorter (e.g., ≤1μm, ≤900 nm, ≤800 nm, ≤700 nm, ≤600 nm, ≤500 nm, ≤400 nm, ≤300 nm, ≤200 nm, ≤175 nm, ≤150 nm, ≤125 nm, ≤100 nm, ≤75 nm, ≤50 nm, or shorter) , such as when measured by dynamic light scattering (DLS) , transmission electron microscopy, scanning electron microscopy, or another method. In one embodiment, the lipid nanoparticle provided herein has at least one dimension that is in the range of from about 40 to about 200 nm. In one embodiment, the at least one dimension is in the range of from about 40 to about 100 nm.

Nanoparticle compositions that can be used in connection with the present disclosure include, for example, lipid nanoparticles (LNPs) , nano lipoprotein particles, liposomes, lipid vesicles, and lipoplexes. In some embodiments, nanoparticle compositions are vesicles including one or more lipid bilayers. In some embodiments, a nanoparticle composition includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and/or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.
In some embodiments, nanoparticle compositions as described comprise a lipid component including at least one
lipid, such as a compound according to one of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) as described herein. For example, in some embodiments, a nanoparticle composition may include a lipid component including one of compounds provided herein. Nanoparticle compositions may also include one or more other lipid or non-lipid components as described below.
5.4.1 Cationic Lipids

In one embodiment, the cationic lipid contained in the lipid nanoparticle compositions described herein is a cationic lipid described in International Patent Publication No. WO2021204175, the entirety of which is incorporated herein by reference.

In one embodiment, the cationic lipid is a compound of Formula (01-I) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or C₂-C₁₂ alkenylene, wherein one or more-CH₂-in
the alkylene or alkenylene is optionally replaced by-O-;
L¹ is–OC (=O) R¹, -C (=O) OR¹, -OC (=O) OR¹, -C (=O) R¹, -OR¹, -S (O) _xR¹, -S-SR¹, -C (=O) SR¹, -SC (=O) R¹, -
NR^aC (=O) R¹, -C (=O) NR^bR^c, -NR^aC (=O) NR^bR^c, -OC (=O) NR^bR^c, -NR^aC (=O) OR¹, -SC (=S) R¹, -C (=S) SR¹, -C (=S) R¹, -CH (OH) R¹, -P (=O) (OR^b) (OR^c) , - (C₆-C₁₀ arylene) -R¹, - (6-to 10-membered heteroarylene) -R¹, or R¹;
L² is–OC (=O) R², -C (=O) OR², -OC (=O) OR², -C (=O) R², -OR², -S (O) _xR², -S-SR², -C (=O) SR², -SC (=O) R², -
NR^dC (=O) R², -C (=O) NR^eR^f, -NR^dC (=O) NR^eR^f, -OC (=O) NR^eR^f, -NR^dC (=O) OR², -SC (=S) R², -C (=S) SR², -C (=S) R², -CH (OH) R², -P (=O) (OR^e) (OR^f) , - (C₆-C₁₀ arylene) -R², - (6-to 10-membered heteroarylene) -R², or R²;
R¹ and R² are each independently C₆-C₃₂ alkyl or C₆-C₃₂ alkenyl;
R^a, R^b, R^d, and R^e are each independently H, C₁-C₂₄ alkyl, or C₂-C₂₄ alkenyl;
R^c and R^fare each independently C₁-C₃₂ alkyl or C₂-C₃₂ alkenyl;
G³ is C₂-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₃-C₈ cycloalkylene, or C₃-C₈ cycloalkenylene;
R³ is-N (R⁴) R⁵;
R⁴ is C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 4-to 8-membered heterocyclyl, or C₆-C₁₀ aryl; or R⁴, G³ or part of G³,
together with the nitrogen to which they are attached form a cyclic moiety;
R⁵ is C₁-C₁₂ alkyl or C₃-C₈ cycloalkyl; or R⁴, R⁵, together with the nitrogen to which they are attached form a
cyclic moiety;
x is 0, 1 or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene,
cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the cationic lipid is a compound of Formula (01-II) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
is a single bond or a double bond;
G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or C₂-C₁₂ alkenylene, wherein one or more-CH₂-in
the alkylene or alkenylene is optionally replaced by-O-;
L¹ is–OC (=O) R¹, -C (=O) OR¹, -OC (=O) OR¹, -C (=O) R¹, -OR¹, -S (O) _xR¹, -S-SR¹, -C (=O) SR¹, -SC (=O) R¹, -
NR^aC (=O) R¹, -C (=O) NR^bR^c, -NR^aC (=O) NR^bR^c, -OC (=O) NR^bR^c, -NR^aC (=O) OR¹, -SC (=S) R¹, -C (=S) SR¹, -C (=S) R¹, -CH (OH) R¹, -P (=O) (OR^b) (OR^c) , - (C₆-C₁₀ arylene) -R¹, - (6-to 10-membered heteroarylene) -R¹, or R¹;
L² is–OC (=O) R², -C (=O) OR², -OC (=O) OR², -C (=O) R², -OR², -S (O) _xR², -S-SR², -C (=O) SR², -SC (=O) R², -
NR^dC (=O) R², -C (=O) NR^eR^f, -NR^dC (=O) NR^eR^f, -OC (=O) NR^eR^f, -NR^dC (=O) OR², -SC (=S) R², -C (=S) SR², -C (=S) R², -CH (OH) R², -P (=O) (OR^e) (OR^f) , - (C₆-C₁₀ arylene) -R², - (6-to 10-membered heteroarylene) -R², or R²;
R¹ and R² are each independently C₆-C₃₂ alkyl or C₆-C₃₂ alkenyl;
R^a, R^b, R^d, and R^e are each independently H, C₁-C₂₄ alkyl, or C₂-C₂₄ alkenyl;
R^c and R^fare each independently C₁-C₃₂ alkyl or C₂-C₃₂ alkenyl;
G⁴ is a bond, C₁-C₂₃ alkylene, C₂-C₂₃ alkenylene, C₃-C₈ cycloalkylene, or C₃-C₈ cycloalkenylene;
R³ is-N (R⁴) R⁵;
R⁴ is C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, 4-to 8-membered heterocyclyl, or C₆-C₁₀ aryl; or R⁴, G³
or part of G³, together with the nitrogen to which they are attached form a cyclic moiety;
R⁵ is C₁-C₁₂ alkyl or C₃-C₈ cycloalkyl; or R⁴, R⁵, together with the nitrogen to which they are attached form a
cyclic moiety;
x is 0, 1 or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene,
cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the compound is a compound of Formula (01-I-B) , (01-I-B’) , (01-I-B” ) , (01-I-C) , (01-I-D) , or (01-I-E) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G¹ and G² are each independently C₃-C₇ alkylene. In one embodiment, G¹ and G² are each independently C₅ alkylene. In one embodiment, G³ is C₂-C₄ alkylene. In one embodiment, G³ is C₂ alkylene. In one embodiment, G³ is C₄ alkylene.

In one embodiment, R³ has one of the following structures:

In one embodiment, R¹, R², R^c and R^f are each independently branched C₆-C₃₂ alkyl or branched C₆-C₃₂ alkenyl. In one embodiment, R¹, R², R^c and R^fare each independently branched C₆-C₂₄ alkyl or branched C₆-C₂₄ alkenyl. In one embodiment, R¹, R², R^c and R^f are each independently-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl. In one embodiment, R¹, R², R^c and R^f are each independently-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ are independently C₄-C₈ alkyl.

In one embodiment, the compound is a compound in Table 2, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
Table 2.

In one embodiment, the cationic lipid contained in the lipid nanoparticle compositions provided herein is a cationic lipid described in International Patent Application No. WO/2023/138611, the entirety of which is incorporated herein by reference. In one embodiment, the cationic lipid is a compound of Formula (02-I) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene, wherein one or more-CH₂-in G¹ and
G² is optionally replaced by-O-, -C (=O) O-, or-OC (=O) -;
each L¹ is independently–OC (=O) R¹, -C (=O) OR¹, -OC (=O) OR¹, -C (=O) R¹, -OR¹, -S (O) _xR¹, -S-SR¹, -C (=O) SR¹,
-SC (=O) R¹, -NR^aC (=O) R¹, -C (=O) NR^bR^c, -NR^aC (=O) NR^bR^c, -OC (=O) NR^bR^c, -NR^aC (=O) OR¹, -SC (=S) R¹, -C (=S) SR¹, -C (=S) R¹, -CH (OH) R¹, -P (=O) (OR^b) (OR^c) , -NR^aP (=O) (OR^b) (OR^c) ;
each L² is independently–OC (=O) R², -C (=O) OR², -OC (=O) OR², -C (=O) R², -OR², -S (O) _xR², -S-SR², -C (=O) SR²,
-SC (=O) R², -NR^dC (=O) R², -C (=O) NR^eR^f, -NR^dC (=O) NR^eR^f, -OC (=O) NR^eR^f, -NR^dC (=O) OR², -SC (=S) R², -C (=S) SR², -C (=S) R², -CH (OH) R², -P (=O) (OR^e) (OR^f) , -NR^dP (=O) (OR^e) (OR^f) ;
R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;
R^a, R^b, R^d, and R^e are each independently H, C₁-C₂₄ alkyl, or C₂-C₂₄ alkenyl;
R^c and R^fare each independently C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl;
G³ is C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by
a C₃-C₈ cycloalkylene or C₃-C₈ cycloalkenylene;
R³ is-N (R⁴) R⁵, -OR⁶, or-SR⁶;
R⁴ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered
heterocycloalkyl;
R⁵ is H, C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered heterocycloalkyl;
R⁶is hydrogen, C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or C₆-C₁₀ aryl;
x is 0, 1, or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene,
and cycloalkenylene is independently optionally substituted.

In one embodiment, the cationic lipid is a compound of Formula (02-II) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene, wherein one or more-CH₂-in G¹ and
G² is optionally replaced by-O-, -C (=O) O-, or-OC (=O) -;
each L¹ is independently–OC (=O) R¹, -C (=O) OR¹, -OC (=O) OR¹, -C (=O) R¹, -OR¹, -S (O) _xR¹, -S-SR¹, -C (=O) SR¹,
-SC (=O) R¹, -NR^aC (=O) R¹, -C (=O) NR^bR^c, -NR^aC (=O) NR^bR^c, -OC (=O) NR^bR^c, -NR^aC (=O) OR¹, -SC (=S) R¹, -C (=S) SR¹, -C (=S) R¹, -CH (OH) R¹, -P (=O) (OR^b) (OR^c) , -NR^aP (=O) (OR^b) (OR^c) ;
each L² is independently–OC (=O) R², -C (=O) OR², -OC (=O) OR², -C (=O) R², -OR², -S (O) _xR², -S-SR², -C (=O) SR²,
-SC (=O) R², -NR^dC (=O) R², -C (=O) NR^eR^f, -NR^dC (=O) NR^eR^f, -OC (=O) NR^eR^f, -NR^dC (=O) OR², -SC (=S) R², -C (=S) SR², -C (=S) R², -CH (OH) R², -P (=O) (OR^e) (OR^f) , -NR^dP (=O) (OR^e) (OR^f) ;
R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;
R^a, R^b, R^d, and R^e are each independently H, C₁-C₂₄ alkyl, or C₂-C₂₄ alkenyl;
R^c and R^fare each independently C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl;
G³ is C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by
a C₃-C₈ cycloalkylene or C₃-C₈ cycloalkenylene;
R³ is-N (R⁴) R⁵, -OR⁶, or-SR⁶;
R⁴ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered
heterocycloalkyl;
R⁵ is H, C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered heterocycloalkyl;
R⁶is hydrogen, C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or C₆-C₁₀ aryl;
x is 0, 1, or 2; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene,
and cycloalkenylene is independently optionally substituted.

In one embodiment, the compound is a compound of Formula (02-V-A) , (02-V-B) , (02-V-C) , (02-V-D) , (02-V-E) , (02-V-F) :

wherein z is an integer from 2 to 12,
x0 is an integer from 1 to 11;
y0 is an integer from 1 to 11;
x1 is an integer from 0 to 9;
y1 is an integer from 0 to 9;
x2 is an integer from 2 to 5;
x3 is an integer from 1 to 5;
x4 is an integer from 0 to 3;
y2 is an integer from 2 to 5;
y3 is an integer from 1 to 5; and
y4 is an integer from 0 to 3;
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, z is an integer from 2 to 6. In one embodiment, z is 2, 4, or 5. In one embodiment, x0 and y0 are independently 2 to 6. In one embodiment, x0 and y0 are independently 4 or 5. In one embodiment, x1 and y1 are independently 2 to 6. In one embodiment, x1 and y1 are independently 4 or 5. In one embodiment, x2 and y2 are independently an integer from 2 to 5. In one embodiment, x2 and y2 are independently 3 or 5. In one embodiment, x3 and y3 are both 1. In one embodiment, x4 and y4 are independently 0 or 1.

In one embodiment, each L¹ is independently-OR¹, -OC (=O) R¹ or-C (=O) OR¹, and each L² is independently–OR², -OC (=O) R² or-C (=O) OR². In one embodiment, R¹ and R² are independently straight C₆-C₁₀ alkyl, or-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl or C₂-C₁₀ alkenyl.

In one embodiment, the compound is a compound of formula (02-VI-A) , (02-VI-B) , (02-VI-C) , (02-VI-D) , (02-VI-E) , or (02-VI-F) :

wherein z is an integer from 2 to 12;
y is an integer from 2 to 12;
x0 is an integer from 1 to 11;
x1 is an integer from 0 to 9;
x2 is an integer from 2 to 5;
x3 is an integer from 1 to 5; and
x4 is an integer from 0 to 3;
or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, z is an integer from 2 to 6. In one embodiment, z is 2, 4 or 5. In one embodiment, x0 is 4 or 5. In one embodiment, x1 is 4 or 5. In one embodiment, x2 is an integer from 2to 5. In one embodiment, x2 is 3 or 5. In one embodiment, x3 is 0 or 1. In one embodiment, y is an integer from 2 to 6. In one embodiment, y is 5.

In one embodiment, each L¹ is independently-OR¹, -OC (=O) R¹ or-C (=O) OR¹, and L² is-OC (=O) R²or -C (=O) OR², -NR^dC (=O) R², or-C (=O) NR^eR^f. In one embodiment, R¹ is straight C₆-C₁₀ alkyl or-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₅ alkylene, and R⁸and R⁹ are independently C₂-C₁₀ alkyl or C₂-C₁₀ alkenyl. In one embodiment, R² and R^f are each independently straight C₆-C₁₈ alkyl, C₆-C₁₈ alkenyl, or-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl or C₂-C₁₀ alkenyl. In one embodiment, R^d and R^e are each independently H.

In one embodiment, the compound is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
Table 3.

In one embodiment, the cationic lipid contained in the lipid nanoparticle compositions described herein is a cationic lipid described in International Patent Publication No. WO2022152109, the entirety of which is incorporated herein by reference.

In one embodiment, the cationic lipid is a compound of Formula (03-I) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or C₂-C₁₂ alkenylene, wherein one or more-CH₂-in
G¹ and G² is optionally replaced by-O-;
each L¹ is independently–OC (=O) R¹, -C (=O) OR¹, -OC (=O) OR¹, -C (=O) R¹, -OR¹, -S (O) _xR¹, -S-SR¹, -C (=O) SR¹,
-SC (=O) R¹, -NR^aC (=O) R¹, -C (=O) NR^bR^c, -NR^aC (=O) NR^bR^c, -OC (=O) NR^bR^c, -NR^aC (=O) OR¹, -SC (=S) R¹, -C (=S) SR¹, -C (=S) R¹, -CH (OH) R¹, -P (=O) (OR^b) (OR^c) , -NR^aP (=O) (OR^b) (OR^c) , - (C₆-C₁₀ arylene) -R¹, - (6-to 10-membered heteroarylene) -R¹, - (4-to 8-membered heterocyclylene) -R¹, or R¹;
each L² is independently–OC (=O) R², -C (=O) OR², -OC (=O) OR², -C (=O) R², -OR², -S (O) _xR², -S-SR², -C (=O) SR²,
-SC (=O) R², -NR^dC (=O) R², -C (=O) NR^eR^f, -NR^dC (=O) NR^eR^f, -OC (=O) NR^eR^f, -NR^dC (=O) OR², -SC (=S) R², -C (=S) SR², -C (=S) R², -CH (OH) R², -P (=O) (OR^e) (OR^f) , -NR^dP (=O) (OR^e) (OR^f) , - (C₆-C₁₀ arylene) -R², - (6-to 10-membered heteroarylene) -R², - (4-to 8-membered heterocyclylene) -R², or R²;
R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;
R^a, R^b, R^d, and R^e are each independently H, C₁-C₂₄ alkyl, or C₂-C₂₄ alkenyl;
R^c and R^fare each independently C₁-C₂₄ alkyl or C₂-C₂₄ alkenyl;
G³ is C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene, wherein part or all of alkylene or alkenylene is optionally replaced by
C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene, C₃-C₈ cycloalkynylene, 4-to 8-membered heterocyclylene, C₆-C₁₀ arylene, or 5-to 10-membered heteroarylene;
R³ is hydrogen, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₃-
C₈ cycloalkynyl, 4-to 8-membered heterocyclyl, C₆-C₁₀ aryl, or 5-to 10-membered heteroaryl; or R³, G¹ or part of G¹, together with the nitrogen to which they are attached form a cyclic moiety; or R³, G³ or part of G³, together with the nitrogen to which they are attached form a cyclic moiety;
R⁴ is C₁-C₁₂ alkyl or C₃-C₈ cycloalkyl;
x is 0, 1, or 2;
n is 1 or 2;
m is 1 or 2; and
wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl,
alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the compound is a compound of Formula (03-II-A) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (03-II-B) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (03-II-C) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, the compound is a compound of Formula (03-II-D) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G¹ and G² are each independently C₂-C₁₂ alkylene. In one embodiment, G¹ and G² are each independently C₅ alkylene. In one embodiment, G³ is C₂-C₆ alkylene.

In one embodiment, R³ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, or C₃-C₈ cycloalkyl. In one embodiment, R³ is C₃-C₈ cycloalkyl. In one embodiment, R³ is unsubstituted. In one embodiment, R⁴ is substituted C₁-C₁₂ alkyl. In one embodiment, R⁴ is–CH₂CH₂OH.

In one embodiment, L¹ is–OC (=O) R¹, -C (=O) OR¹, -NR^aC (=O) R¹, or-C (=O) NR^bR^c; and L² is–OC (=O) R², -C (=O) OR², -NR^dC (=O) R², or-C (=O) NR^eR^f. In one embodiment, R¹, R², R^c, and R^f are each independently straight C₆-C₁₈ alkyl, straight C₆-C₁₈ alkenyl, or-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl or C₂-C₁₀ alkenyl. In one embodiment, R¹, R², R^c, and R^f are each independently straight C₇-C₁₅ alkyl, straight C₇-C₁₅ alkenyl, or-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₀-C₁ alkylene, and R⁸ and R⁹ are independently C₄-C₈ alkyl or C₆-C₁₀ alkenyl. In one embodiment, R^a, R^b, R^d, and R^e are each independently H.

In one embodiment, the compound is a compound in Table 5, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
Table 5

In one embodiment, the cationic lipid contained in the particles or compositions provided herein is a cationic lipid described in International Patent Application No. WO/2022/247755, the entirety of which is incorporated herein by reference.

In one embodiment, the cationic lipid is a compound of Formula (04-I) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
G¹ and G² are each independently a bond, C₂-C₁₂ alkylene, or C₂-C₁₂ alkenylene;
L¹ is–OC (=O) R¹, -C (=O) OR¹, -OC (=O) OR¹, -C (=O) R¹, -OR¹, -S (O) _xR¹, -S-SR¹, -C (=O) SR¹, -SC (=O) R¹, -
NR^aC (=O) R¹, -C (=O) NR^bR^c, -NR^aC (=O) NR^bR^c, -OC (=O) NR^bR^c, -NR^aC (=O) OR¹, -SC (=S) R¹, -C (=S) SR¹, -C (=S) R¹, -CH (OH) R¹, -P (=O) (OR^b) (OR^c) , - (C₆-C₁₀arylene) -R¹, - (6-to 10-membered heteroarylene) -R¹, or R¹;
L² is–OC (=O) R², -C (=O) OR², -OC (=O) OR², -C (=O) R², -OR², -S (O) _xR², -S-SR², -C (=O) SR², -SC (=O) R², -
NR^dC (=O) R², -C (=O) NR^eR^f, -NR^dC (=O) NR^eR^f, -OC (=O) NR^eR^f, -NR^dC (=O) OR², -SC (=S) R², -C (=S) SR², -C (=S) R², -CH (OH) R², -P (=O) (OR^e) (OR^f) , - (C₆-C₁₀ arylene) -R², - (6-to 10-membered heteroarylene) -R², or R²;
R¹ and R² are each independently C₅-C₃₂alkyl or C₅-C₃₂alkenyl;
R^a, R^b, R^d, and R^e are each independently H, C₁-C₂₄alkyl, or C₂-C₂₄alkenyl;
R^c and R^fare each independently C₁-C₃₂ alkyl or C₂-C₃₂ alkenyl;
R⁰ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered
heterocycloalkyl;
G³ is C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene;
R⁴ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered
heterocycloalkyl;
R⁵ is C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered heterocycloalkyl;
x is 0, 1, or 2;
s is 0 or 1; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and
heteroarylene, is independently optionally substituted.

In one embodiment, the cationic lipid is a compound of Formula (04-III) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:
R¹ and R² are each independently C₅-C₃₂alkyl or C₅-C₃₂alkenyl;
R⁰ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered
heterocycloalkyl;
G³ is C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene;
G⁴ is C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene;
R³ is-N (R⁴) R⁵ or-OR⁶;
R⁴ is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered
heterocycloalkyl;
R⁵ is C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, C₆-C₁₀ aryl, or 4-to 8-membered heterocycloalkyl; or R⁴,
R⁵, together with the nitrogen to which they are attached form a cyclic moiety;
R⁶is hydrogen, C₁-C₁₂ alkyl, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or C₆-C₁₀ aryl; and
wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, and cyclic
moiety is independently optionally substituted.

In one embodiment, the compound is a compound of Formula (04-IV) :

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

In one embodiment, G³ is C₂-C₄ alkylene. In one embodiment, G⁴ is C₂-C₄ alkylene.

In one embodiment, R⁰ is C₁-C₆ alkyl. In one embodiment, R³ is-OH. In one embodiment, R³ is-N (R⁴) R⁵. In one embodiment, R⁴ is C₃-C₈ cycloalkyl. In one embodiment, R⁴ is unsubstituted. In one embodiment, R⁵ is–CH₂CH₂OH.

In one embodiment, L¹ is–OC (=O) R¹, -C (=O) OR¹, -C (=O) R¹, -C (=O) NR^bR^c, or R¹; and L² is–OC (=O) R², -C (=O) OR², -C (=O) R², -C (=O) NR^eR^f, or R². In one embodiment, R¹ and R² are each independently branched C₆-C₂₄ alkyl or branched C₆-C₂₄ alkenyl. In one embodiment, R¹ and R² are each independently-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₁-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl or C₂-C₁₀ alkenyl. In one embodiment, R¹ is straight C₆-C₂₄ alkyl and R² is branched C₆-C₂₄ alkyl. In one embodiment, R¹ is straight C₆-C₂₄ alkyl and R² is-R⁷-CH (R⁸) (R⁹) , wherein R⁷ is C₁-C₅ alkylene, and R⁸ and R⁹ are independently C₂-C₁₀ alkyl.

In one embodiment, the compound is a compound in Table 6, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.
Table 6.

In one embodiment, the cationic lipid contained in the particles or compositions provided herein is a cationic lipid described in U.S. Patent Nos. US10442756B2, US9868691B2, and US9868692B2, the entire teachings of which are incorporated herein by reference.

It is understood that any embodiment of the compounds provided herein, as set forth above, and any specific substituent and/or variable in the compound provided herein, as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of the compounds to form embodiments not specifically set forth above. In addition, in the event that a list of substituents and/or variables is listed for any particular group or variable, it is understood that each individual substituent and/or variable may be deleted from the particular embodiment and/or claim and that the remaining list of substituents and/or variables will be considered to be within the scope of embodiments provided herein.

It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compounds.
5.4.2 Other Ionizable Lipids

As described herein, in some embodiments, a nanoparticle composition provided herein comprises one or more charged or ionizable lipids in addition to a lipid according to Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) . Without being bound by the theory, it is contemplated that certain charged or zwitterionic lipid components of a nanoparticle composition resembles the lipid component in the cell membrane, thereby can improve cellular uptake of the nanoparticle. Exemplary charged or ionizable lipids that can form part of the present nanoparticle composition include but are not limited to 3- (didodecylamino) -N1, N1, 4-tridodecyl-1-piperazineethanamine (KL10) , N1- [2- (didodecylamino) ethyl] -N1, N4, N4-tridodecyl-1, 4-piperazinediethanamine (KL22) , 14, 25-ditridecyl-15, 18, 21, 24-tetraaza-octatriacontane (KL25) , 1, 2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA) , 2, 2-dilinoleyl-4-dimethylaminomethyl- [1, 3] -dioxolane (DLin-K-DMA) , heptatriaconta-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (DLin-MC3-DMA) , 2, 2-dilinoleyl-4- (2-dimethylaminoethyl) - [1, 3] -dioxolane (DLin-KC2-DMA) , 1, 2-dioleyloxy-N, N-dimethylaminopropane (DODMA) , 2- ( {8- [ (3β) -cholest-5-en-3-yloxy] octyl} oxy) -N, N-dimethyl-3 [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA) , (2R) -2- ( {8- [ (3β) -cholest-5-en-3-yloxy] octyl} oxy) -N, N-dimethyl-3- [ (9Z, 12Z) --octadeca-9, 12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA (2R) ) , (2S) -2- ( {8- [ (3β) -cholest-5-en-3-yloxy] octyl} oxy) -N, N-dimethyl-3- [ (9Z-, 12Z) -octadeca-9, 12-dien-1-yloxy] propan-1-amine (Octyl-CLinDMA (2S) ) , (12Z, 15Z) -N, N-dimethyl-2-nonylhenicosa-12, 15-den-1-amine, N, N-dimethyl-1- { (1S, 2R) -2-octylcyclopropyl} heptadecan-8-amine. Additional exemplary charged or ionizable lipids that can form part of the present nanoparticle composition include the lipids (e.g., lipid 5) described in Sabnis et al. “A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates” , Molecular Therapy Vol. 26 No 6, 2018, the entirety of which is incorporated herein by reference.

In some embodiments, suitable cationic lipids include N- [1- (2, 3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA) ; N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTAP) ; 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC) ; 1, 2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC) ; 1, 2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC) ; 1, 2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14: 1) ; N1- [2- ( (1S) -1- [ (3-aminopropyl) amino] -4- [di (3-amino-propyl) amino] butylcarboxamido) ethyl] -3, 4-di [oleyloxy] -benzamide (MVL5) ; dioctadecylamido-glycylspermine (DOGS) ; 3b- [N- (N', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) ; dioctadecyldimethylammonium bromide (DDAB) ; SAINT-2, N-methyl-4- (dioleyl) methylpyridinium; 1, 2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE) ; 1, 2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE) ; 1, 2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium chloride (DORI) ; di-alkylated amino acid (DILA²) (e.g., C18: 1-norArg-C16) ; dioleyldimethylammonium chloride (DODAC) ; 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) ; 1, 2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC) ; (R) -5- (dimethylamino) pentane-1, 2-diyl dioleate hydrochloride (DODAPen-Cl) ; (R) -5-guanidinopentane-1, 2-diyl dioleate hydrochloride (DOPen-G) ; and (R) -N, N, N-trimethyl-4, 5-bis (oleoyloxy) pentan-1-aminium chloride (DOTAPen) . Also suitable are cationic lipids with headgroups that are charged at physiological pH, such as primary amines (e.g., DODAG N', N'-dioctadecyl-N-4, 8-diaza-10-aminodecanoylglycine amide) and guanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol (BGSC) , bis-guanidiniumtren-cholesterol (BGTC) , PONA, and (R) -5-guanidinopentane-1, 2-diyl dioleate hydrochloride (DOPen-G) ) . Yet another suitable cationic lipid is (R) -5- (dimethylamino) pentane-1, 2-diyl dioleate hydrochloride (DODAPen-Cl) . In certain embodiments, the cationic lipid is a particular enantiomer or the racemic form, and includes the various salt forms of a cationic lipid as above (e.g., chloride or sulfate) . For example, in some embodiments, the cationic lipid is N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTAP-Cl) or N- [1- (2, 3-dioleoyloxy) propyl] -N, N, N-trimethylammonium sulfate (DOTAP-Sulfate) . In some embodiments, the cationic lipid is an ionizable cationic lipid such as, e.g., dioctadecyldimethylammonium bromide (DDAB) ; 1, 2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA) ; 2, 2-dilinoleyl-4- (2dimethylaminoethyl) - [1, 3] -dioxolane (DLin-KC2-DMA) ; heptatriaconta-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (DLin-MC3-DMA) ; 1, 2-dioleoyloxy-3-dimethylaminopropane (DODAP) ; 1, 2-dioleyloxy-3-dimethylaminopropane (DODMA) ; and morpholinocholesterol (Mo-CHOL) . In certain embodiments, a lipid nanoparticle includes a combination or two or more cationic lipids (e.g., two or more cationic lipids as above) .

Additionally, in some embodiments, the charged or ionizable lipid that can form part of the present nanoparticle composition is a lipid including a cyclic amine group. Additional cationic lipids that are suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each of which are hereby incorporated by reference in their entireties. Additionally, in some embodiments, the charged or ionizable lipid that can form part of the present nanoparticle composition is a lipid including a cyclic amine group. Additional cationic lipids that are suitable for the formulations and methods disclosed herein include those described in WO2015199952, WO2016176330, and WO2015011633, the entire contents of each of which are hereby incorporated by reference in their entireties.
5.4.3 Polymer ConjugatedLipids

In some embodiments, the lipid component of a nanoparticle composition can include one or more polymer conjugated lipids, such as PEGylated lipids (PEG lipids) . Without being bound by the theory, it is contemplated that a polymer conjugated lipid component in a nanoparticle composition can improve of colloidal stability and/or reduce protein absorption of the nanoparticles. Exemplary cationic lipids that can be used in connection with the present disclosure include but are not limited to PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, Ceramide-PEG2000, or Chol-PEG2000.

In one embodiment, the polymer conjugated lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol) -2, 3-dimyristoylglycerol (PEG-DMG) , a pegylated phosphatidylethanoloamine (PEG-PE) , a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O- (2’, 3’-di (tetradecanoyloxy) propyl-1-O- (ω-methoxy (polyethoxy) ethyl) butanedioate (PEG-S-DMG) , a pegylated ceramide (PEG-cer) , or a PEG dialkoxypropylcarbamate such as ω-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecanoxy) propyl) carbamate or 2, 3-di (tetradecanoxy) propyl-N- (ω-methoxy (polyethoxy) ethyl) carbamate.

In one embodiment, the polymer conjugated lipid is present in a concentration ranging from 1.0 to 2.5 molar percent. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.7 molar percent. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.5 molar percent.

In one embodiment, the molar ratio of cationic lipid to the polymer conjugated lipid ranges from about 35: 1 to about 25: 1. In one embodiment, the molar ratio of cationic lipid to polymer conjugated lipid ranges from about 100: 1 to about 20: 1.

In one embodiment, the pegylated lipid has the following Formula:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
R¹² and R¹³ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10
to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In one embodiment, R¹² and R¹³ are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In other embodiments, the average w ranges from 42 to 55, for example, the average w is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55. In some specific embodiments, the average w is about 49.

In one embodiment, the pegylated lipid has the following Formula:

wherein the average w is about 49.
5.4.4 Structural Lipids

In some embodiments, the lipid component of a nanoparticle composition can include one or more structural lipids. Without being bound by the theory, it is contemplated that structural lipids can stabilize the amphiphilic structure of a nanoparticle, such as but not limited to the lipid bilayer structure of a nanoparticle. Exemplary structural lipids that can be used in connection with the present disclosure include but are not limited to cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) , or a combination thereof.

In one embodiment, the lipid nanoparticles provided herein comprise a steroid or steroid analogue. In one embodiment, the steroid or steroid analogue is cholesterol. In one embodiment, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In one embodiment, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent.

In one embodiment, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol ranges from about 5: 1 to 1: 1. In one embodiment, the steroid is present in a concentration ranging from 32 to 40 mol percent of the steroid.

In one embodiment, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol ranges from about 5: 1 to 1: 1. In one embodiment, the steroid is present in a concentration ranging from 32 to 40 mol percent of the steroid.
5.4.5 Phospholipids

In some embodiments, the lipid component of a nanoparticle composition can include one or more phospholipids, such as one or more (poly) unsaturated lipids. Without being bound by the theory, it is contemplated that phospholipids may assemble into one or more lipid bilayers structures. Exemplary phospholipids that can form part of the present nanoparticle composition include but are not limited to 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC) , 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) , 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) , 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC) , 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) , 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) , 1, 2-diundecanoyl-sn-glycero-phosphocholine (DUPC) , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , 1, 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18: 0 Diether PC) , 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC) , 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) , 1, 2-dilinolenoyl-sn-glycero-3-phosphocholine, 1, 2-diarachidonoyl-sn-glycero-3-phosphocholine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE) , 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG) , and sphingomyelin. In certain embodiments, a nanoparticle composition includes DSPC. In certain embodiments, a nanoparticle composition includes DOPE. In some embodiments, a nanoparticle composition includes both DSPC and DOPE.

Additional exemplary neutral lipids include, for example, dipalmitoylphosphatidylglycerol (DPPG) , palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1carboxylate (DOPE-mal) , dipalmitoyl phosphatidyl ethanolamine (DPPE) , dimyristoylphosphoethanolamine (DMPE) , distearoyl-phosphatidylethanolamine (DSPE) , 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE) , and 1, 2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE) . In one embodiment, the neutral lipid is 1, 2-distearoyl-sn-glycero-3phosphocholine (DSPC) . In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

In one embodiment, the neutral lipid is phosphatidylcholine (PC) , phosphatidylethanolamine (PE) phosphatidylserine (PS) , phosphatidic acid (PA) , or phosphatidylglycerol (PG) .

Additionally phospholipids that can form part of the present nanoparticle composition also include those described in WO2017/112865, the entire content of which is hereby incorporated by reference in its entirety.
5.4.6 Formulation

According to the present disclosure, nanoparticle compositions described herein can include at least one lipid component and one or more additional components, such as a therapeutic and/or prophylactic agent (e.g., the therapeutic nucleic acid described herein) . A nanoparticle composition may be designed for one or more specific applications or targets. The elements of a nanoparticle composition may be selected based on a particular application or target, and/or based on the efficacy, toxicity, expense, ease of use, availability, or other feature of one or more elements. Similarly, the particular formulation of a nanoparticle composition may be selected for a particular application or target according to, for example, the efficacy and toxicity of particular combinations of elements.

The lipid component of a nanoparticle composition may include, for example, a lipid according to one of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) described herein, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC) , a PEG lipid, and a structural lipid. The elements of the lipid component may be provided in specific fractions.

In one embodiment, provided herein is a nanoparticle compositions comprising a cationic or ionizable lipid compound provided herein, a therapeutic agent, and one or more excipients. In one embodiment, cationic or ionizable lipid compound comprises a compound according to one of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) as described herein, and optionally one or more additional ionizable lipid compounds. In one embodiment, the one or more excipients are selected from neutral lipids, steroids, and polymer conjugated lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with the lipid nanoparticle.

In one embodiment, provided herein is a nanoparticle composition (lipid nanoparticle) comprising:
i) between 40 and 50 mol percent of a cationic lipid;
ii) a neutral lipid;
iii) a steroid;
iv)a polymer conjugated lipid; and
v)a therapeutic agent.

As used herein, “mol percent” refers to a component’s molar percentage relative to total mols of all lipid components in the LNP (i.e., total mols of cationic lipid (s) , the neutral lipid, the steroid and the polymer conjugated lipid) .

In one embodiment, the lipid nanoparticle comprises from 20 to 65 mol percent, from 25 to 60 mol percent, from 30 to 55 mol percent, from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In one embodiment, the lipid nanoparticle comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid.

In one embodiment, the neutral lipid is present in a concentration ranging from 5 to 40 mol percent, 5 to 30 mol percent, 5 to 25 mol percent, 5 to 20 mol percent, 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9, 9.5, 10, 10.5, 11 or 15 mol percent. In one embodiment, the molar ratio of the cationic lipid to the neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8: 1.0, or from about 4.7: 1.0 to 4.8: 1.0.

In one embodiment, the steroid is present in a concentration ranging from 20 to 50 molar percent, 25 to 45 molar percent, from 30 to 45 molar percent, from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In one embodiment, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In one embodiment, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0to 1.0: 1.2. In one embodiment, the steroid is cholesterol.

In one embodiment, the therapeutic agent to lipid ratio in the LNP (i.e., N/P, were N represents the moles of cationic lipid and P represents the moles of phosphate present as part of the nucleic acid backbone) range from 2: 1 to 30: 1, for example 3: 1 to 22: 1. In one embodiment, N/P ranges from 6: 1 to 20: 1 or 2: 1 to 12: 1. Exemplary N/P ranges include about 3: 1. About 6: 1, about 12: 1 and about 22: 1.
In one embodiment, provided herein is a lipid nanoparticle comprising:
i) between 20 and 65 mol percent of a cationic lipid;
ii) between 5 and 40 mol percent of a neutral lipid;
iii) between 20 and 50 mol percent of a steroid;
iv) between 0.5 and 5 mol percent of a polymer conjugated lipid; and
v) a therapeutic agent.
In another embodiment, provided herein is a lipid nanoparticle comprising:
i) between 30 and 55 mol percent of a cationic lipid;
ii) between 5 and 40 mol percent of a neutral lipid;
iii) between 20 and 50 mol percent of a steroid;
iv) between 0.5 and 5 mol percent of a polymer conjugated lipid; and
v) a therapeutic agent.

In one embodiment, provided herein is a lipid nanoparticle comprising:
i) a cationic lipid having an effective pKa greater than 6.0;
ii) from 5 to 15 mol percent of a neutral lipid;
iii) from 1 to 15 mol percent of an anionic lipid;
iv) from 30 to 45 mol percent of a steroid;
v) a polymer conjugated lipid; and
vi) a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof,
wherein the mol percent is determined based on total mol of lipid present in the lipid nanoparticle.

In one embodiment, the cationic lipid can be any of a number of lipid species which carry a net positive charge at a selected pH, such as physiological pH. Exemplary cationic lipids are described herein below. In one embodiment, the cationic lipid has a pKa greater than 6.25. In one embodiment, the cationic lipid has a pKa greater than 6.5. In one embodiment, the cationic lipid has a pKa greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greater than 6.7.

In one embodiment, the lipid nanoparticle comprises from 40 to 45 mol percent of the cationic lipid. In one embodiment, the lipid nanoparticle comprises from 45 to 50 mole percent of the cationic lipid.

In one embodiment, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1. In one embodiment, the lipid nanoparticle comprises from 5 to 10 mol percent of the neutral lipid.

Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoylphosphatidylglycerol (DOPG) , dipalmitoylphosphatidylglycerol (DPPG) or 1, 2-distearoyl-sn-glycero-3-phospho- (1'-rac-glycerol) (DSPG) .

In one embodiment, the lipid nanoparticle comprises from 1 to 10 mole percent of the anionic lipid. In one embodiment, the lipid nanoparticle comprises from 1 to 5 mole percent of the anionic lipid. In one embodiment, the lipid nanoparticle comprises from 1 to 9 mole percent, from 1 to 8 mole percent, from 1 to 7 mole percent, or from 1 to 6 mole percent of the anionic lipid. In one embodiment, the mol ratio of anionic lipid to neutral lipid ranges from 1: 1 to 1: 10.

In one embodiment, the steroid cholesterol. In one embodiment, the molar ratio of the cationic lipid to cholesterol ranges from about 5: 1 to 1: 1. In one embodiment, the lipid nanoparticle comprises from 32 to 40 mol percent of the steroid.

In one embodiment, the sum of the mol percent of neutral lipid and mol percent of anionic lipid ranges from 5 to 15 mol percent. In one embodiment, wherein the sum of the mol percent of neutral lipid and mol percent of anionic lipid ranges from 7 to 12 mol percent.

In one embodiment, the mol ratio of anionic lipid to neutral lipid ranges from 1: 1 to 1: 10. In one embodiment, the sum of the mol percent of neutral lipid and mol percent steroid ranges from 35 to 45 mol percent.

In one embodiment, the lipid nanoparticle comprises:
i) from 45 to 55 mol percent of the cationic lipid;
ii) from 5 to 10 mol percent of the neutral lipid;
iii) from 1 to 5 mol percent of the anionic lipid; and
iv) from 32 to 40 mol percent of the steroid.

In one embodiment, the polymer conjugated lipid is present in a concentration ranging from 0.5 to 5.0 molar percent. In one embodiment, the lipid nanoparticle comprises from 1.0 to 2.5 mol percent of the conjugated lipid. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.5 mol percent.

In one embodiment, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, 7 to 13 mol percent, or 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent. In one embodiment, the molar ratio of the cationic lipid to the neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8: 1.0, or from about 4.7: 1.0 to 4.8: 1.0.

In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present in a concentration ranging from 39 to 49 molar percent, 40 to 46 molar percent, from 40 to 44 molar percent, from 40 to 42 molar percent, from 42 to 44 molar percent, or from 44 to 46 molar percent. In one embodiment, the steroid is present in a concentration of 40, 41, 42, 43, 44, 45, or 46 molar percent. In certain embodiments, the molar ratio of cationic lipid to the steroid ranges from 1.0: 0.9 to 1.0: 1.2, or from 1.0: 1.0 to 1.0: 1.2.

In one embodiment, the molar ratio of cationic lipid to steroid ranges from 5: 1 to 1: 1.

In one embodiment, the lipid nanoparticle comprises from 1.0 to 2.5 mol percent of the conjugated lipid. In one embodiment, the polymer conjugated lipid is present in a concentration of about 1.5 mol percent.

In one embodiment, the molar ratio of cationic lipid to polymer conjugated lipid ranges from about 100: 1 to about 20: 1. In one embodiment, the molar ratio of cationic lipid to the polymer conjugated lipid ranges from about 35: 1 to about 25: 1.

In one embodiment, the lipid nanoparticle has a mean diameter ranging from 50 nm to 100 nm, or from 60 nm to 85 nm.

In one embodiment, the composition comprises a cationic lipid provided herein, DSPC, cholesterol, and PEG-lipid, and mRNA. In one embodiment, the cationic lipid provided herein, DSPC, cholesterol, and PEG-lipid are at a molar ratio of about 50: 10: 38.5: 1.5.

Nanoparticle compositions can be designed for one or more specific applications or targets. For example, a nanoparticle composition can be designed to deliver a therapeutic and/or prophylactic agent such as an RNA to a particular cell, tissue, organ, or system or group thereof in a mammal’s body. Physiochemical properties of nanoparticle compositions can be altered in order to increase selectivity for particular bodily targets. For instance, particle sizes can be adjusted based on the fenestration sizes of different organs. The therapeutic and/or prophylactic agent included in a nanoparticle composition can also be selected based on the desired delivery target or targets. For example, a therapeutic and/or prophylactic agent can be selected for a particular indication, condition, disease, or disorder and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., localized or specific delivery) . In certain embodiments, a nanoparticle composition can include an mRNA encoding a polypeptide of interest capable of being translated within a cell to produce the polypeptide of interest. Such a composition can be designed to be specifically delivered to a particular organ. In certain embodiments, acomposition can be designed to be specifically delivered to a mammalian liver.

The amount of a therapeutic and/or prophylactic agent in a nanoparticle composition can depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the therapeutic and/or prophylactic agent. For example, the amount of an RNA useful in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of a therapeutic and/or prophylactic agent and other elements (e.g., lipids) in a nanoparticle composition can also vary. In some embodiments, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic agent in a nanoparticle composition can be from about 5: 1 to about 60: 1, such as about 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 22: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt/wt ratio of the lipid component to a therapeutic and/or prophylactic agent can be from about 10: 1 to about 40: 1. In certain embodiments, the wt/wt ratio is about 20: 1. The amount of a therapeutic and/or prophylactic agent in a nanoparticle composition can, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy) .

In some embodiments, a nanoparticle composition includes one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a specific N: P ratio. The N: P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA. In some embodiments, a lower N: P ratio is selected. The one or more RNA, lipids, and amounts thereof can be selected to provide an N: P ratio from about 2: 1 to about 30: 1, such as 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 12: 1, 14: 1, 16: 1, 18: 1, 20: 1, 22: 1, 24: 1, 26: 1, 28: 1, or 30: 1. In certain embodiments, the N: P ratio can be from about 2: 1 to about 8: 1. In other embodiments, the N: P ratio is from about 5: 1 to about 8: 1. For example, the N: P ratio may be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N: P ratio may be about 5.67: 1.

The physical properties of a nanoparticle composition can depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid can have different characteristics compared to a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) may also be used to measure multiple characteristics of a nanoparticle composition, such as particle size, polydispersity index, and zeta potential.

In various embodiments, the mean size of a nanoparticle composition can be between 10s of nm and 100s of nm. For example, the mean size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition can be from about 30 nm to about 200 nm, from about 50 nm to about 150 nm, from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition can be from about 70 nm to about 100 nm. In some embodiments, the mean size can be about 80 nm. In other embodiments, the mean size can be about 100 nm.

A nanoparticle composition can be relatively homogenous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition can have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a nanoparticle composition can be from about 0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species can interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a nanoparticle composition can be from about-10 mV to about+20 mV, from about-10 mV to about+15 mV, from about-10 mV to about+10 mV, from about-10 mV to about+5 mV, from about-10 mV to about 0 mV, from about-10 mV to about-5 mV, from about-5 mV to about+20 mV, from about -5 mV to about+15 mV, from about-5 mV to about+10 mV, from about-5 mV to about+5 mV, from about-5 mV to about 0 mV, from about 0 mV to about+20 mV, from about 0 mV to about+15 mV, from about 0 mV to about+10 mV, from about 0 mV to about+5 mV, from about+5 mV to about+20 mV, from about+5 mV to about+15 mV, or from about+5 mV to about+10 mV.

The efficiency of encapsulation of a therapeutic and/or prophylactic agent describes the amount of therapeutic and/or prophylactic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%) . The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and/or prophylactic agent in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agent (e.g., RNA) in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and/or prophylactic agent can be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency can be at least 90%.

A nanoparticle composition can optionally comprise one or more coatings. For example, a nanoparticle composition can be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein can have any useful size, tensile strength, hardness, or density.
5.4.7 Pharmaceutical Compositions

According to the present disclosure, nanoparticle compositions can be formulated in whole or in part as pharmaceutical compositions. Pharmaceutical compositions can include one or more nanoparticle compositions. For example, a pharmaceutical composition can include one or more nanoparticle compositions including one or more different therapeutic and/or prophylactic agents. Pharmaceutical compositions can further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington’s The Science and Practice of Pharmacy, 21^stEdition, A.R. Gennaro; Lippincott, Williams &Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients can be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient can be incompatible with one or more components of a nanoparticle composition. An excipient or accessory ingredient can be incompatible with a component of a nanoparticle composition if its combination with the component can result in any undesirable biological effect or otherwise deleterious effect.

In some embodiments, one or more excipients or accessory ingredients can make up greater than 50%of the total mass or volume of a pharmaceutical composition including a nanoparticle composition. For example, the one or more excipients or accessory ingredients can make up 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP) , the European Pharmacopoeia (EP) , the British Pharmacopoeia, and/or the International Pharmacopoeia.

Relative amounts of the one or more nanoparticle compositions, the one or more pharmaceutically acceptable excipients, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition can comprise between 0.1%and 100% (wt/wt) of one or more nanoparticle compositions.

In certain embodiments, the nanoparticle compositions and/or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and/or shipment (e.g., being stored at a temperature of 4℃. or lower, such as a temperature between about-150℃ and about0℃ or between about-80℃ and about-20℃ (e.g., about-5℃, -10℃, -15℃, -20℃, -25℃, -30℃, -40℃, -50℃, -60℃, -70℃, -80℃, -90℃, -130℃ or-150 ℃) . For example, the pharmaceutical composition comprising a compound of any of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) is a solution that is refrigerated for storage and/or shipment at, for example, about-20℃, -30℃, -40℃, -50℃, -60℃, -70℃, or-80℃ In certain embodiments, the disclosure also relates to a method of increasing stability of the nanoparticle compositions and/or pharmaceutical compositions comprising a compound of any of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) by storing the nanoparticle compositions and/or pharmaceutical compositions at a temperature of 4℃ or lower, such as a temperature between about-150℃ and about 0℃ or between about-80℃ and about-20℃, e.g., about-5℃, -10℃, -15℃, -20℃, -25℃, -30℃, -40℃, -50℃, -60℃, -70℃, -80℃, -90℃, -130℃ or-150℃) . For example, the nanoparticle compositions and/or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months, e.g., at a temperature of 4℃ or lower (e.g., between about 4℃ and-20℃) . In one embodiment, the formulation is stabilized for at least 4 weeks at about 4℃ In certain embodiments, the pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, an acetate (e.g., sodium acetate) , an citrate (e.g., sodium citrate) , saline, PBS, and sucrose. In certain embodiments, the pharmaceutical composition of the disclosure has a pH value between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, or between 7.5 and 8 or between 7 and 7.8) . For example, a pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein, Tris, saline and sucrose, and has a pH of about 7.5-8, which is suitable for storage and/or shipment at, for example, about-20℃ For example, a pharmaceutical composition of the disclosure comprises a nanoparticle composition disclosed herein and PBS and has a pH of about 7-7.8, suitable for storage and/or shipment at, for example, about 4℃ or lower. “Stability, ” “stabilized, ” and “stable” in the context of the present disclosure refers to the resistance of nanoparticle compositions and/or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc. ) under given manufacturing, preparation, transportation, storage and/or in-use conditions, e.g., when stress is applied such as shear force, freeze/thaw stress, etc.

Nanoparticle compositions and/or pharmaceutical compositions including one or more nanoparticle compositions can be administered to any patient or subject, including those patients or subjects that can benefit from a therapeutic effect provided by the delivery of a therapeutic and/or prophylactic agent to one or more particular cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions provided herein of nanoparticle compositions and pharmaceutical compositions including nanoparticle compositions are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other mammal. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, hoses, sheep, cats, dogs, mice, and/or rats.

A pharmaceutical composition including one or more nanoparticle compositions can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, ifdesirable or necessary, dividing, shaping, and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosure can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., nanoparticle composition) . The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Pharmaceutical compositions can be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions can be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs) , injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules) , dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches) , suspensions, powders, and other forms.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms can comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils) , glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include additional therapeutic and/or prophylactic agents, additional agents such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as Cremophor^TM, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

The disclosure features methods of delivering a therapeutic and/or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and/or contacting a mammalian cell with a nanoparticle composition including a therapeutic and/or prophylactic agent.
5.5 Methods

In one aspect, provided herein are also methods for managing, preventing, and treating neoplastic diseases or conditions that are associated with one or more oncogenic mutations in the KRAS gene. In some embodiments, the neoplastic disease is cancer or tumor associated with one or more oncogenic mutations in the KRAS gene, such as epithelial cancers, lung cancers, particularly non-small cell lung cancers, colorectal cancers, and pancreatic cancers, cholangiocarcinoma, uterine endometrial carcinoma, testicular germ cell cancer, cervical squamous cell carcinoma, myelodysplastic carcinoma. Huang et al. “KRAS mutation: from undruggable to druggable in cancer” Signal Transduction and Targeted Therapy volume 6, Article number: 386 (2021) etc.

As used herein, “tumor” refers to any neoplastic cell growth or proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to: breast cancer, colon cancer, renal cancer, lung cancer, squamous cell myeloid leukemia, hemangiomas, melanomas, astrocytomas, and glioblastomas as well as other cellular-proliferative disease states, including but not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma) , myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma) , alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hanlartoma, inesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma) , stomach (carcinoma, lymphoma, leiomyosarcoma) , pancreas (ductal adenocarcinoma, insulinorna, glucagonoma, gastrinoma, carcinoid tumors, vipoma) , small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma) , large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) ; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma} , lymphoma, leukemia, renal cell carcinoma) , bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma) , prostate (adenocarcinoma, sarcoma, small cell carcinoma of the prostate) , testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma) ; Liver: hepatoma (hepatocellular carcinoma) , cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma) , fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma) , malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses) , benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis defornians) , meninges (meningioma, meningiosarcoma, gliomatosis) , brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma) , glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors) , spinal cord neurofibroma, meningioma, glioma, sarcoma) ; Gynecological: uterus (endometrial carcinoma) , cervix (cervical carcinoma, pre-tumor cervical dysplasia) , ovaries (ovarian carcinoma) serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma) , vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma) , vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma] , fallopian tubes (carcinoma) ; Hematologic: blood (myeloid leukemia (acute and chronic) , acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome) , Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma) ; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma; as well as cancers of the thyroid including medullary thyroid cancer.

In some embodiments, the tumor is a solid tumor associated with one or more oncogenic mutations in the KRAS gene. In some embodiments, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene is not a solid tumor. In further embodiments, the cancer associated with one or more oncogenic mutations in the KRAS gene is a leukemia cancer. In some embodiments, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene is a relapsed tumor or cancer. In some embodiments, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene is a metastatic tumor or cancer. In some embodiments, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene is a primary tumor or cancer. In some embodiments, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene reaches a remission but can relapse. In some embodiments, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene is unresectable. Additionally or alternatively, the tumor or cancer associated with one or more oncogenic mutations in the KRAS gene is resistant to a chemotherapy or other anti-cancer therapy.

In some embodiments, the one or more oncogenic mutations comprises a substitution at a position corresponding to G12V in human KRAS (SEQ ID NO: 168) . In specific embodiments, the neoplastic disease (e.g., cancer) is associated with the G12A mutation, G12C mutation, G12D mutation, or G12V mutation in KRAS. In some embodiments, the one or more oncogenic mutations comprises a substitution at a position corresponding to G13 in human KRAS (SEQ ID NO: 168) . In specific embodiments, the neoplastic disease (e.g., cancer) is associated with the G13D mutation in KRAS. In some embodiments, the one or more oncogenic mutations comprises a substitution at a position corresponding to Q61 in human KRAS (SEQ ID NO: 168) . In some embodiments, the neoplastic disease (e.g., cancer) is associated with the Q61R mutation in KRAS.

According to the present disclosure, the therapeutic nucleic acids encoding a polypeptide comprising a KRAS domain (e.g., a tandem string of KRAS neoepitopes) as described herein can be administered to a subject in need thereof for managing, preventing, and treating a neoplastic disease associated with one or more oncogenic mutations of KRAS.

In some embodiments, a pharmaceutical composition comprising multiple therapeutic nucleic acids each encoding a different polypeptide comprising a KRAS domain as described herein can be administered to a subject in need thereof for managing, preventing, and treating a neoplastic disease associated with one or more oncogenic mutations of KRAS.

In a particular embodiment, the therapeutic nucleic acid is formulated as a lipid nanoparticle composition as described herein and administered to the subject in need thereof for managing, preventing, and treating a neoplastic disease associated with one or more oncogenic mutations of KRAS. In a particular embodiment, the therapeutic nucleic acid formulated as a lipid nanoparticle composition is a mRNA molecule as described herein. In some embodiments, the present method for managing or treating a neoplastic disease associated with one or more oncogenic mutations of KRAS in a subject comprises administering to the subject a therapeutic effective amount of a lipid-containing composition comprising a therapeutic nucleic acid as described herein, wherein the lipid-containing composition is formulated as a lipid nanoparticle encapsulating the therapeutic nucleic acid in a lipid shell. In some embodiments, the present method for preventing a neoplastic disease associated with one or more oncogenic mutations of KRAS in a subject comprises administering to the subject a prophylactically effective amount of a lipid-containing composition comprising a therapeutic nucleic acid as described herein, wherein the lipid-containing composition is formulated as a lipid nanoparticle encapsulating the therapeutic nucleic acid in a lipid shell. In specific embodiments, the therapeutic nucleic acid comprises one or more mRNA molecule as described herein. In specific embodiments, the cells in the subject effectively intake the lipid-containing composition (e.g., lipid nanoparticles) described herein upon administration. In specific embodiments, lipid-containing composition (e.g., lipid nanoparticles) described herein are endocytosed by cells of the subject. In some embodiments, the composition is a genetic vaccine composition described herein.

In some embodiments, a polypeptide comprising a KRAS domain (e.g., a tandem string of KRAS neoepitopes) as described herein can be administered to a subject in need thereof for managing, preventing, and treating a neoplastic disease associated with one or more oncogenic mutations of KRAS. In some embodiments, a pharmaceutical composition comprising multiple different polypeptides each comprising a KRAS domain as described herein can be administered to a subject in need thereof for managing, preventing, and treating a neoplastic disease associated with one or more oncogenic mutations of KRAS.

In some embodiments, upon administration to a subject in need thereof of the therapeutic nucleic acid as described herein, a vaccine composition comprising the therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, the cells in the subject uptake and express the administered therapeutic nucleic acids to produce a polypeptide encoded by the nucleic acid. In some embodiments, the encoded polypeptide is processed (e.g., degradation by proteosome, lysosome or exosome) into neoepitopes, and presented by antigen presenting cells (e.g., dendritic cells) , and activate cell-mediated immunity (e.g., antigen-specific T cell mediated cytotoxicity) against cancer cell expressing such neoepitopes.

In some embodiments, upon administration to a subject in need thereof of the therapeutic nucleic acid encoding a polypeptide comprising the KRAS domain or the encoded polypeptide comprising the KRAS domain as described herein, a vaccine composition comprising the therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, one or more immune responses against the a neoplastic cell expressing a mutant KRAS is elicited in the subject. In some embodiments, the elicited immune response comprises one or more adaptive immune responses against the neoplastic cells. In some embodiments, the elicited immune response comprises one or more innate immune responses against the neoplastic cells. The one or more immune responses can be in the form of, e.g., an antibody response (humoral response) or a cellular immune response, e.g., cytokine secretion (e.g., interferon-gamma) , helper activity or cellular cytotoxicity. In some embodiments, expression of an activation marker on immune cells, expression of a co-stimulatory receptor on immune cells, expression of a ligand for a co-stimulatory receptor, cytokine secretion, infiltration of immune cells (e.g., T-lymphocytes, B lymphocytes and/or NK cells) to the neoplastic cell, production of antibody specifically recognizing one or more antigens associated with the neoplastic cell (e.g., the mutant KRAS epitope encoded by the therapeutic nucleic acid) , effector function, T cell activation, T cell differentiation, T cell proliferation, B cell differentiation, B cell proliferation, and/or NK cell proliferation is induced, activated and/or enhanced. In some embodiments, activation and proliferation of myeloid-derived suppressor cell (MDSC) and Treg cells are inhibited.

In some embodiments, upon administration to a subject in need thereof of the therapeutic nucleic acid as described herein, a vaccine composition comprising the therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, one or more antibody against the neoplastic cells expressing mutant KRAS is produced in the subject.

In specific embodiments, the antibody binds to one or more proteins present on the surface of diseased cells and mark the diseased cells for destruction by the subject’s immune system. In some embodiments, endocytosis of diseased cells by white blood cells (e.g., macrophage) is induced or enhanced. In some embodiments, antibody-dependent cell-mediated cytotoxicity (ADCC) against infected cells in the subject is induced or enhanced. In some embodiments, antibody-dependent cellular phagocytosis (ADCP) against infected cells in the subject is induced or enhanced. In some embodiments, complement dependent cytotoxicity (CDC) against infected cells in the subject is induced or enhanced.

In some embodiments, provided herein is a method of activating KRAS-mutant specific immune effector cells. In some embodiments, the method comprises providing a starting population of immune effector cells and contacting the starting population of immune effector cells with a composition comprising the therapeutic nucleic acid encoding a polypeptide comprising a KRAS domain as described herein under a suitable condition, wherein upon the contacting, KRAS-mutant specific immune effector cells are activated. In some embodiments, the method comprises providing a starting population of immune effector cells, and contacting the starting population of immune effector cells with a composition comprising the polypeptide comprising a KRAS domain as described herein under a suitable condition, wherein upon the contacting, KRAS-mutant specific immune effector cells are activated.

In some embodiments, the immune effector cell is an antigen presenting cell. In some embodiments, the immune effector cell is an immune cell. In some embodiments, the immune effector cell is B cells, natural killer cells, dendritic cells, macrophages, monocytes, granulocytes, eosinophils, neutrophils, or a combination thereof. In one embodiment, the immune effector cell is a dendritic cell. In one embodiment, the immune effector cell is a macrophage. In one embodiment, the immune effector cell is a natural killer cell. In one embodiment, the immune effector cell is a monocyte. In one embodiment, the immune effector cell is a granulocyte. In one embodiment, the immune effector cell is an eosinophil. In one embodiment, the immune effector cell is a granulocyte. In one embodiment, the immune effector cell is a neutrophil. In some embodiments, the immune effector cell is a Langerhans cell. In some embodiments, the immune effector cell is a prostatic glandular cell. In some embodiments, the immune effector cell is a B cell. In some embodiments, the immune effector cell is a B cell. In some embodiments, the immune effector cell is a memory B cell. In some embodiments, the immune effector cell is a basal respiratory cell. In some embodiments, the immune effector cell is a CD4+T cell. In some embodiments the immune effector cell is a CD8+T cell. In some embodiments the immune effector cell is a MAIT cell. In some embodiments, the immune effector cell is a natural killer cell.

In some embodiments, the activated immune effector cell activates another cell. In some embodiments, the activated immune effector cell activates another immune cell. In some embodiments, the activated immune effector cell activates a T cell. In some embodiments, the activated immune effector cell activates CD4+T cells, CD8+T cells, MAIT, natural killer cells, neutrophils, or a combination thereof. In one embodiment, the activated immune effector cell activates a CD4+T cell. In one embodiment, the activated immune effector cell activates a CD8+T cell. In one embodiment, the activated immune effector cell activates a MAIT. In one embodiment, the activated immune effector cell activates a natural killer cell. In one embodiment, the activated immune effector cell activates a neutrophil.

In some embodiments, activation of the immune effector cell is measured as increased proliferation or maturation of the immune effector cell. In one embodiment, activation of the immune effector cell is measured as increased proliferation of the immune effector cell. In one embodiment, activation of the immune effector cell is measured as increased maturation of the immune effector cell. In particular embodiments, proliferation or maturation of the immune effector cell is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%or 1000%. In one embodiment, proliferation of the immune effector cell is increased by about 10-100%. In another embodiment, proliferation of the immune effector cell is increased by about 100-200%. In another embodiment, proliferation of the immune effector cell is increased by about 200-300%. In another embodiment, proliferation of the immune effector cell is increased by about 300-400%. In another embodiment, proliferation of the immune effector cell is increased by about 400-500%. In another embodiment, proliferation of the immune effector cell is increased by about 500-600%. In another embodiment, proliferation of the immune effector cell is increased by about 600-700%. In another embodiment, proliferation of the immune effector cell is increased by about 700-800%. In another embodiment, proliferation of the immune effector cell is increased by about 800-900%. In another embodiment, proliferation of the immune effector cell is increased by about 900-1000%. In one embodiment, proliferation of the immune effector cell is increased by about 10%. In one embodiment, proliferation of the immune effector cell is increased by about 20%. In one embodiment, proliferation of the immune effector cell is increased by about 30%. In one embodiment, proliferation of the immune effector cell is increased by about 40%. In one embodiment, proliferation of the immune effector cell is increased by about 45%. In one embodiment, proliferation of the immune effector cell is increased by about 50%. In one embodiment, proliferation of the immune effector cell is increased by about 55%. In one embodiment, proliferation of the immune effector cell is increased by about 60%. In one embodiment, proliferation of the immune effector cell is increased by about 65%. In one embodiment, proliferation of the immune effector cell is increased by about 70%. In one embodiment, proliferation of the immune effector cell is increased by about 75%. In one embodiment, proliferation of the immune effector cell is increased by about 80%. In one embodiment, proliferation of the immune effector cell is increased by about 85%. In one embodiment, proliferation of the immune effector cell is increased by about 90%. In one embodiment, proliferation of the immune effector cell is increased by about 95%. In one embodiment, proliferation of the immune effector cell is increased by about 100%. In one embodiment, proliferation of the immune effector cell is increased by about 125%. In one embodiment, proliferation of the immune effector cell is increased by about 150%. In one embodiment, proliferation of the immune effector cell is increased by about 175%. In one embodiment, proliferation of the immune effector cell is increased by about 200%. In one embodiment, proliferation of the immune effector cell is increased by about 250%. In one embodiment, proliferation of the immune effector cell is increased by about 300%. In one embodiment, proliferation of the immune effector cell is increased by about 400%. In one embodiment, proliferation of the immune effector cell is increased by about 500%. In one embodiment, proliferation of the immune effector cell is increased by about 600%. In one embodiment, proliferation of the immune effector cell is increased by about 700%. In one embodiment, proliferation of the immune effector cell is increased by about 800%. In one embodiment, proliferation of the immune effector cell is increased by about 900%. In one embodiment, proliferation of the immune effector cell is increased by about 1000%. In one embodiment, maturation of the immune effector cell is increased by about 10-100%. In another embodiment, maturation of the immune effector cell is increased by about 100-200%. In another embodiment, maturation of the immune effector cell is increased by about 200-300%. In another embodiment, maturation of the immune effector cell is increased by about 300-400%. In another embodiment, maturation of the immune effector cell is increased by about 400-500%. In another embodiment, maturation of the immune effector cell is increased by about 500-600%. In another embodiment, maturation of the immune effector cell is increased by about 600-700%. In another embodiment, maturation of the immune effector cell is increased by about 700-800%. In another embodiment, maturation of the immune effector cell is increased by about 800-900%. In another embodiment, maturation of the immune effector cell is increased by about 900-1000%. In one embodiment, maturation of the immune effector cell is increased by about 10%. In one embodiment, maturation of the immune effector cell is increased by about 20%. In one embodiment, maturation of the immune effector cell is increased by about 30%. In one embodiment, maturation of the immune effector cell is increased by about 40%. In one embodiment, maturation of the immune effector cell is increased by about 45%. In one embodiment, maturation of the immune effector cell is increased by about 50%. In one embodiment, maturation of the immune effector cell is increased by about 55%. In one embodiment, maturation of the immune effector cell is increased by about 60%. In one embodiment, maturation of the immune effector cell is increased by about 65%. In one embodiment, maturation of the immune effector cell is increased by about 70%. In one embodiment, maturation of the immune effector cell is increased by about 75%. In one embodiment, maturation of the immune effector cell is increased by about 80%. In one embodiment, maturation of the immune effector cell is increased by about 85%. In one embodiment, maturation of the immune effector cell is increased by about 90%. In one embodiment, maturation of the immune effector cell is increased by about 95%. In one embodiment, maturation of the immune effector cell is increased by about 100%. In one embodiment, maturation of the immune effector cell is increased by about 125%. In one embodiment, maturation of the immune effector cell is increased by about 150%. In one embodiment, maturation of the immune effector cell is increased by about 175%. In one embodiment, maturation of the immune effector cell is increased by about 200%. In one embodiment, maturation of the immune effector cell is increased by about 250%. In one embodiment, maturation of the immune effector cell is increased by about 300%. In one embodiment, maturation of the immune effector cell is increased by about 400%. In one embodiment, maturation of the immune effector cell is increased by about 500%. In one embodiment, maturation of the immune effector cell is increased by about 600%. In one embodiment, maturation of the immune effector cell is increased by about 700%. In one embodiment, maturation of the immune effector cell is increased by about 800%. In one embodiment, maturation of the immune effector cell is increased by about 900%. In one embodiment, maturation of the immune effector cell is increased by about 1000%.

In some embodiments, activation of the immune effector cell is measured as prolonged survival time of the immune effector cell. In particular embodiments, survival time of the immune effector cell is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%or 1000%. In one embodiment, survival time of the immune effector cell is increased by about 10-100%. In another embodiment, survival time of the immune effector cell is increased by about 100-200%. In another embodiment, survival time of the immune effector cell is increased by about 200-300%. In another embodiment, survival time of the immune effector cell is increased by about 300-400%. In another embodiment, survival time of the immune effector cell is increased by about 400-500%. In another embodiment, survival time of the immune effector cell is increased by about 500-600%. In another embodiment, survival time of the immune effector cell is increased by about 600-700%. In another embodiment, survival time of the immune effector cell is increased by about 700-800%. In another embodiment, survival time of the immune effector cell is increased by about 800-900%. In another embodiment, survival time of the immune effector cell is increased by about 900-1000%. In one embodiment, survival time of the immune effector cell is increased by about 10%. In one embodiment, survival time of the immune effector cell is increased by about 20%. In one embodiment, survival time of the immune effector cell is increased by about 30%. In one embodiment, survival time of the immune effector cell is increased by about 40%. In one embodiment, survival time of the immune effector cell is increased by about 45%. In one embodiment, survival time of the immune effector cell is increased by about 50%. In one embodiment, survival time of the immune effector cell is increased by about 55%. In one embodiment, survival time of the immune effector cell is increased by about 60%. In one embodiment, survival time of the immune effector cell is increased by about 65%. In one embodiment, survival time of the immune effector cell is increased by about 70%. In one embodiment, survival time of the immune effector cell is increased by about 75%. In one embodiment, survival time of the immune effector cell is increased by about 80%. In one embodiment, survival time of the immune effector cell is increased by about 85%. In one embodiment, survival time of the immune effector cell is increased by about 90%. In one embodiment, survival time of the immune effector cell is increased by about 95%. In one embodiment, survival time of the immune effector cell is increased by about 100%. In one embodiment, survival time of the immune effector cell is increased by about 125%. In one embodiment, survival time of the immune effector cell is increased by about 150%. In one embodiment, survival time of the immune effector cell is increased by about 175%. In one embodiment, survival time of the immune effector cell is increased by about 200%. In one embodiment, survival time of the immune effector cell is increased by about 250%. In one embodiment, survival time of the immune effector cell is increased by about 300%. In one embodiment, survival time of the immune effector cell is increased by about 400%. In one embodiment, survival time of the immune effector cell is increased by about 500%. In one embodiment, survival time of the immune effector cell is increased by about 600%. In one embodiment, survival time of the immune effector cell is increased by about 700%. In one embodiment, survival time of the immune effector cell is increased by about 800%. In one embodiment, survival time of the immune effector cell is increased by about 900%. In one embodiment, survival time of the immune effector cell is increased by about 1000%.

Also provided are methods of activating an immune effector cell comprising contacting the immune effector cell with an antigen presenting cell (APC) in the presence of a nucleic acid encoding a polypeptide comprising a KRAS domain and a degradation system. Also provided are methods of activating an immune effector cell comprising contacting the immune effector cell with an antigen presenting cell in the presence of a polypeptide comprising a KRAS domain and a degradation system. In some embodiments, the degradation system degrades the KRAS domain into one or more fragments that can be presented by APCs. In some embodiments, the degradation system is a proteosome system, a phagosome system, a lysosome system, or an endosome system. In specific embodiments, the degraded KRAS fragment is of about 9 to 15 amino acids in length.

In some embodiments, the APCs present a degraded fragment of the KRAS domain on their surface to activate the immune effector cell. In some embodiments, the contacting is performed in the presence of antigen presenting cells (APCs) , wherein the APCs present the degraded fragment of the KRAS domain on their surface. In some embodiments, the APCs are selected from dendritic cells, macrophages, Langerhans cells, B cells, natural killer cells, monocytes, granulocytes, eosinophils, neutrophils, or a combination thereof. In some embodiments, the APCs express HLA class I and/or HLA class II molecules. In some embodiments, the HLA class I molecule is of the HLA-A*11: 01 subtype.

In some embodiments, the immune effector cells are selected from T cells, peripheral blood lymphocytes, and natural killer cells; optionally wherein the T cells comprises CD4+T cells, CD8+T cells, antigen-specific T cells, tumor infiltration T cells, γδT cells, cytotoxic T cells, natural killer T cells, and mucosal associated invariant T cells (MAIT) .

In some embodiments, the immune effector cells are T cells. In some embodiments, activation of a population of T cells is measured by increased secretion of pro-inflammatory cytokines by the T cells. In some embodiments, the pro-inflammatory cytokines are selected from IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1 or any combination thereof. In one embodiment, the cytokine is IL-1. In one embodiment, the cytokine is IL-2. In one embodiment, the cytokine is IL-6. In one embodiment, the cytokine is IL-12. In one embodiment, the cytokine is IL-17. In one embodiment, the cytokine is IL-22. In one embodiment, the cytokine is IL-23. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IFN-γ. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IP-10. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is CXCL1. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is MCP-1.

In some embodiments, the immune effector cells, once activated, mediate cytotoxicity specifically against a target cell expressing the fragment of the KRAS domain presented by the APCs. In some embodiments, the immune effector cells are antigen-specific T cells specifically recognize and mediate cytotoxicity against a cancer cell expressing a mutant KRAS. In some embodiments, the cancer cell expressing a mutant KRAS comprising one or more mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R. In some embodiments, the cancer cell is selected from epithelial cancers, lung cancers, particularly non-small cell lung cancers, colorectal cancers, and pancreatic cancers.

In some embodiments, provided herein is a method for promoting antibody production by a population of B cells, comprising contacting the B cells with a therapeutic nucleic acid encoding a polypeptide comprising a KRAS domain according to the present disclosure. In some embodiments, provided herein is a method for promoting antibody production by a population of B cells, comprising contacting the B cells with a polypeptide comprising a KRAS domain according to the present disclosure.

In some embodiments, upon the contacting, the B cells produce an antibody that specifically binds to a fragment in the KRAS domain. In some embodiments, the fragment of the KRAS domain is presented by an antigen-presenting cell. In some embodiments, the fragment of the KRAS domain is associated with an MHC class I complex. In some embodiments, the fragment of the KRAS domain is associated with an MHC class II complex. In some embodiments, the fragment of the KRAS domain is originated or derived from a neoplastic cell (e.g., cancer cell) . In some embodiments, the neoplastic cell is a cancer cell expressing a mutant KRAS. In some embodiments, the cancer cell expressing a mutant KRAS comprising one or more mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.

In some embodiments, antibody production by the population of B cells is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%or 1000%. In one embodiment, antibody production by a population of B cells is increased by about 10-100%. In another embodiment, antibody production by a population of B cells is increased by about 100-200%. In another embodiment, antibody production by a population of B cells is increased by about 200-300%. In another embodiment, antibody production by a population of B cells is increased by about 300-400%. In another embodiment, antibody production by a population of B cells is increased by about 400-500%. In another embodiment, antibody production by a population of B cells is increased by about 500-600%. In another embodiment, antibody production by a population of B cells is increased by about 600-700%. In another embodiment, antibody production by a population of B cells is increased by about 700-800%. In another embodiment, antibody production by a population of B cells is increasedby about 800-900%. In another embodiment, antibody production by a population of B cells is increased by about 900-1000%. In one embodiment, antibody production by a population of B cells is increased by about 10%. In one embodiment, antibody production by a population of B cells is increased by about 20%. In one embodiment, antibody production by a population of B cells is increased by about 30%. In one embodiment, antibody production by a population of B cells is increased by about 40%. In one embodiment, antibody production by a population of B cells is increased by about 45%. In one embodiment, antibody production by a population of B cells is increased by about 50%. In one embodiment, antibody production by a population of B cells is increased by about 55%. In one embodiment, antibody production by a population of B cells is increased by about 60%. In one embodiment, antibody production by a population of B cells is increased by about 65%. In one embodiment, antibody production by a population of B cells is increased by about 70%. In one embodiment, antibody production by a population of B cells is increased by about 75%. In one embodiment, antibody production by a population of B cells is increased by about 80%. In one embodiment, antibody production by a population of B cells is increased by about 85%. In one embodiment, antibody production by a population of B cells is increased by about 90%. In one embodiment, antibody production by a population of B cells is increased by about 95%. In one embodiment, antibody production by a population of B cells is increased by about 100%. In one embodiment, antibody production by a population of B cells is increased by about 125%. In one embodiment, antibody production by a population of B cells is increased by about 150%. In one embodiment, antibody production by a population of B cells is increased by about 175%. In one embodiment, antibody production by a population of B cells is increased by about 200%. In one embodiment, antibody production by a population of B cells is increased by about 250%. In one embodiment, antibody production by a population of B cells is increased by about 300%. In one embodiment, antibody production by a population of B cells is increased by about 400%. In one embodiment, antibody production by a population of B cells is increased by about 500%. In one embodiment, antibody production by a population of B cells is increased by about 600%. In one embodiment, antibody production by a population of B cells is increased by about 700%. In one embodiment, antibody production by a population of B cells is increased by about 800%. In one embodiment, antibody production by a population of B cells is increased by about 900%. In one embodiment, antibody production by a population of B cells is increased by about 1000%. In one embodiment, antibody production by a population of B cells is increased by about 10-100%. In another embodiment, antibody production by a population of B cells is increased by about 100-200%. In another embodiment, antibody production by a population of B cells is increased by about 200-300%. In another embodiment, antibody production by a population of B cells is increased by about 300-400%. In another embodiment, antibody production by a population of B cells is increasedby about 400-500%. In another embodiment, antibody production by a population of B cells is increased by about 500-600%. In another embodiment, antibody production by a population of B cells is increased by about 600-700%. In another embodiment, antibody production by a population of B cells is increased by about 700-800%. In another embodiment, antibody production by a population of B cells is increased by about 800-900%. In another embodiment, antibody production by a population of B cells is increased by about 900-1000%. In some embodiments, the method further promotes formation of memory B cells capable of producing the antibody in response to the fragment of the KRAS domain.

In some embodiments, provided herein is a method for increasing secretion of pro-inflammatory cytokines by a population of immune cells, comprising contacting the population of immune cells with a population of antigen presenting cells in the presence of a therapeutic nucleic acid encoding a polypeptide comprising a KRAS domain according to the present disclosure. In some embodiments, provided herein is a method for increasing secretion of pro-inflammatory cytokines by a population of immune cells, comprising contacting the population of immune cells with a population of antigen presenting cells in the presence of a polypeptide comprising a KRAS domain according to the present disclosure.

In some embodiments, the population of antigen presenting cells present a fragment of the KRAS domain to the population of immune cells. In some embodiments, the population of immune cells comprises T cells. In some embodiments, the population of immune cells is a population of T cells. In specific embodiments, upon activation of the antigen presenting cells, the presentation of the KRAS fragment by the population of antigen presenting cells to the population of immune cells is increased. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 10-20%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 20-30%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 30-40%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 40-50%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 50-60%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 60-70%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 70-80%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 80-90%. In another embodiment, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 90-99%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 10%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 20%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 30%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 40%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 45%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 50%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 55%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 60%. In some embodiments, aminimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 65%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 70%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 75%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 80%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 85%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 90%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about 95%. In some embodiments, a minimal percentage of antigen presenting cells presenting the KRAS fragment in the population of antigen presenting cells is increased by about or 99%. In some embodiments, the population of antigen presenting cells comprises B cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, neutrophils, or a combination thereof. In some embodiments, the population of antigen presenting cells comprise B cells. In some embodiments, the population of antigen presenting cells comprise macrophages. In some embodiments, the population of antigen presenting cells comprise dendritic cells. In some embodiments, the population of antigen presenting cells comprise natural killer cells. In some embodiments, the population of antigen presenting cells comprise monocytes. In some embodiments, the population of antigen presenting cells comprise granulocytes. In some embodiments, the population of antigen presenting cells comprise eosinophils. In some embodiments, the population of antigen presenting cells comprise neutrophils. In some embodiments, the cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1 or any combination thereof. In one embodiment, the cytokine is IL-1. In one embodiment, the cytokine is IL-2. In one embodiment, the cytokine is IL-6. In one embodiment, the cytokine is IL-12. In one embodiment, the cytokine is IL-17. In one embodiment, the cytokine is IL-22. In one embodiment, the cytokine is IL-23. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IFN-γ. In one embodiment, the cytokine is IP-10. In one embodiment, the cytokine is CXCL1. In one embodiment, the cytokine is MCP-1. In some embodiments, the cytokine production is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%or 1000%. In one embodiment, cytokine production is increased by about 10-100%. In another embodiment, cytokine production is increased by about 100-200%. In another embodiment, cytokine production is increased by about 200-300%. In another embodiment, cytokine production is increased by about 300-400%. In another embodiment, cytokine production is increased by about 400-500%. In another embodiment, cytokine production is increased by about 500-600%. In another embodiment, cytokine production is increased by about 600-700%. In another embodiment, cytokine production is increased by about 700-800%. In another embodiment, cytokine production is increased by about 800-900%. In another embodiment, cytokine production is increased by about 900-1000%. In another embodiment, cytokine production is increased by about 10-1000%. In one embodiment, cytokine production is increased by about 20%. In one embodiment, cytokine production is increased by about 30%. In one embodiment, cytokine production is increased by about 40%. In one embodiment, cytokine production is increased by about 45%. In one embodiment, cytokine production is increased by about 50%. In one embodiment, cytokine production is increased by about 55%. In one embodiment, cytokine production is increased by about 60%. In one embodiment, cytokine production is increased by about 65%. In one embodiment, cytokine production is increased by about 70%. In one embodiment, cytokine production is increased by about 75%. In one embodiment, cytokine production is increased by about 80%. In one embodiment, cytokine production is increased by about 85%. In one embodiment, cytokine production is increased by about 90%. In one embodiment, cytokine production is increased by about 95%. In one embodiment, cytokine production is increased by about 100%. In one embodiment, cytokine production is increased by about 125%. In one embodiment, cytokine production is increased by about 150%. In one embodiment, cytokine production is increased by about 175%. In one embodiment, cytokine production is increased by about 200%. In one embodiment, cytokine production is increased by about 250%. In one embodiment, cytokine production is increased by about 300%. In one embodiment, cytokine production is increased by about 400%. In one embodiment, cytokine production is increased by about 500%. In one embodiment, cytokine production is increased by about 600%. In one embodiment, cytokine production is increased by about 700%. In one embodiment, cytokine production is increased by about 800%. In one embodiment, cytokine production is increased by about 900%. In one embodiment, cytokine production is increased by about 1000%.

In some embodiments, provided herein is a method for increasing phagocytosis of diseased cells by a population of macrophages, comprising contacting the diseased cells, the macrophages, or both the diseased cells and the macrophage with therapeutic nucleic acid encoding a polypeptide comprising the KRAS domain according to the present disclosure. In some embodiments, provided herein is a method for increasing phagocytosis of diseased cells by a population of macrophages, comprising contacting the diseased cells, the macrophages, or both the diseased cells and the macrophage with a polypeptide comprising the KRAS domain according to the present disclosure.

In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 10-20%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 20-30%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 30-40%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 40-50%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 50-60%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 60-70%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 70-80%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 80-90%. In another embodiment, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 90-99%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 10%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 20%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 30%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 40%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 45%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 50%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 55%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 60%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 65%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 70%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 75%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 80%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 85%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 90%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about 95%. In some embodiments, a minimal percentage of phagocytotic macrophages in the population of macrophages is increased by about or 99%.

In some embodiments, the phagocytosis by macrophages is measured by co-culturing macrophages labeled with a first fluorescent dye and diseased cells labeled with a second fluorescent dye, wherein the first fluorescent dye and the second fluorescent dye are different. In some embodiments, the percentage of phagocytotic macrophages is measured by determining the percentage of macrophages comprising the diseased cells. In specific embodiments, the diseased cells are cancer cells expressing a mutant KRAS. In some embodiments, the cancer cell expressing a mutant KRAS comprising one or more mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.

In some embodiments, provided herein is a method for increasing presentation of a KRAS fragment by a population of dendritic cells, comprising contacting the dendritic cells with a therapeutic nucleic acid encoding a polypeptide comprising a KRAS domain according to the present disclosure. In some embodiments, provided herein is a method for increasing presentation of a KRAS fragment by a population of dendritic cells, comprising contacting the dendritic cells with a polypeptide comprising a KRAS domain according to the present disclosure.

In some embodiments, the antigen presentation by the dendritic cells is measured by co-culturing dendritic cells labeled with a first fluorescent dye and the antigen labeled with a second fluorescent dye, wherein the first fluorescent dye and the second fluorescent dye are different. In some embodiments, percentage of dendritic cells presenting the antigen is measured by determining the percentage of dendritic cells co-localizing with the antigen in the population of dendritic cells. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 10-20%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 20-30%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 30-40%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 40-50%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 50-60%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 60-70%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 70-80%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 80-90%. In another embodiment, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 90-99%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 10%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 20%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 30%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 40%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 45%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 50%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 55%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 60%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 65%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 70%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 75%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 80%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 85%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 90%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about 95%. In some embodiments, a minimal percentage of dendritic cells presenting the KRAS fragment in the population of dendritic cells is increased by about or 99%.

In some embodiments, provided herein is a method for forming a pro-inflammatory milieu in a tissue surrounding a population of diseased cells, comprising contacting the tissue with a therapeutic nucleic acid encoding a polypeptide comprising a KRAS domain according to the present disclosure. In some embodiments, provided herein is a method for forming a pro-inflammatory milieu in a tissue surrounding a population of diseased cells, comprising contacting the tissue with a polypeptide comprising a KRAS domain according to the present disclosure. In some embodiments, the diseased cells are cancer cells expressing a mutant KRAS. In some embodiments, the cancer cell expressing a mutant KRAS comprising one or more mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.

In some embodiments, infiltration of activated B cells, CD4+T cells, CD8+T cells, MAITs, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils and/or neutrophils in the tissue is increased. In some embodiments, infiltration of activated B cells is increased. In some embodiments, infiltration of CD4+T cells is increased. In some embodiments, infiltration of CD8+T cells is increased. In some embodiments, infiltration of dendritic cells is increased. In some embodiments, infiltration of macrophages is increased. In some embodiments, infiltration of natural killer cells is increased. In some embodiments, infiltration of monocytes is increased. In some embodiments, infiltration of granulocytes is increased. In some embodiments, infiltration of eosinophils is increased. In some embodiments, infiltration of neutrophils is increased.

In some embodiments, concentration of a pro-inflammatory cytokine is increased in the tissue. In some embodiments, the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1, or any combination thereof. In one embodiment, the cytokine is IL-1. In one embodiment, the cytokine is IL-2. In one embodiment, the cytokine is IL-6. In one embodiment, the cytokine is IL-12. In one embodiment, the cytokine is IL-17. In one embodiment, the cytokine is IL-22. In one embodiment, the cytokine is IL-23. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IFN-γ. In one embodiment, the cytokine is IP-10. In one embodiment, the cytokine is CXCL1. In one embodiment, the cytokine is MCP-1.

In some embodiments, presentation of KRAS fragments originated or derived from KRAS domain by antigen presentation cells is increased in the tissue. In some embodiments, phagocytosis of the diseased cells is increased in the tissue. In some embodiments, apoptosis of the diseased cells induced by cell-mediated cytotoxicity is increased in the tissue. In some embodiments, apoptosis of the diseased cells induced by antibody-dependent cellular cytotoxicity is increased in the tissue. In some embodiments, the population of the diseased cells is reduced in the tissue. In some embodiments, the population of the diseased cells is reduced by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%in the tissue. In one embodiment, the population of the diseased cells is reduced by about 10-20%. In another embodiment, the population of the diseased cells is reduced by about 20-30%. In another embodiment, the population of the diseased cells is reduced by about 30-40%. In another embodiment, the population of the diseased cells is reduced by about 40-50%. In another embodiment, the population of the diseased cells is reduced by about 50-60%. In another embodiment, the population of the diseased cells is reduced by about 60-70%. In another embodiment, the population of the diseased cells is reduced by about 70-80%. In another embodiment, the population of the diseased cells is reduced by about 80-90%. In another embodiment, the population of the diseased cells is reduced by about 90-99%. In one embodiment, the population of the diseased cells is reduced by about 10%. In one embodiment, the population of the diseased cells is reduced by about 20%. In one embodiment, the population of the diseased cells is reduced by about 30%. In one embodiment, the population of the diseased cells is reduced by about 40%. In one embodiment, the population of the diseased cells is reduced by about 45%. In one embodiment, the population of the diseased cells is reduced by about 50%. In one embodiment, the population of the diseased cells is reduced by about 55%. In one embodiment, the population of the diseased cells is reduced by about 60%. In one embodiment, the population of the diseased cells is reduced by about 65%. In one embodiment, the population of the diseased cells is reduced by about 70%. In one embodiment, the population of the diseased cells is reduced by about 75%. In one embodiment, the population of the diseased cells is reduced by about 80%. In one embodiment, the population of the diseased cells is reduced by about 85%. In one embodiment, the population of the diseased cells is reduced by about 90%. In one embodiment, the population of the diseased cells is reduced by about 95%. In one embodiment, the population of the diseased cells is reduced by about 99%. In one embodiment, the population of the diseased cells is reduced by about 100%. In some embodiments, the diseased cells are cancer cells expressing a mutant KRAS. In some embodiments, the cancer cell expressing a mutant KRAS comprising one or more mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.

According to the present disclosure, the pharmaceutical compositions (including lipid nanoparticles) comprising the therapeutic nucleic acid described herein may be suitable for a variety of modes of administration described herein, including for example systemic or localized administration.

In some embodiments, the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is suitable for administration to a rodent (e.g., mice, rats) or non-human primates (e.g., Cynomolgus monkey) . In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.

Also provided are unit dosage forms of any of the vaccines described herein, or compositions (such as pharmaceutical compositions) thereof. These unit dosage forms can be stored in a suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed. In some embodiments, the pharmaceutical composition is administered as a single dose. In some embodiments, the pharmaceutical composition is administered as multiple doses. In some embodiments, the pharmaceutical composition is administered no more than once a year.

In some embodiments, the pharmaceutical composition is administered in combination with one or more anti-cancer therapies. In some embodiments, the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery.

In some cases, a subject method involves administering to an individual in need thereof an effective amount of a pharmaceutical composition described herein. In some embodiments, the effective amount is an amount that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to prevent cancer associated with oncogenic mutations of KRAS or to reduce symptoms of said cancer in the individual by at least about 20%, at least about 25%, 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 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 5-fold, at least about 10-fold, or greater than 10-fold, compared to the individual in the absence of treatment with the pharmaceutical composition.

The amount of therapeutic nucleic acid or a composition thereof which will be effective in the management, prevention and/or treatment of infectious disease will depend on the nature of the disease being treated, the route of administration, the general health of the subject, etc. and should be decided according to the judgment of a medical practitioner. Standard clinical techniques, such as in vitro assays, may optionally be employed to help identify optimal dosage ranges.
5.6 Kits

The present application also provides kits comprising compositions (such as pharmaceutical compositions) described herein and may further comprise instruction (s) on methods of using the composition, such as uses described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, topical applicators, and package inserts with instructions for performing any methods described herein.

In some embodiments, provided herein is a kit comprising one or more containers filled with one or more of the ingredients of a composition described herein. In some embodiments, provided herein is a kit comprising a container containing a therapeutic nucleic acid (e.g., mRNA) molecule described herein. In some embodiments, provided herein is a kit comprising a container containing a composition described herein. In some embodiments, provided herein is a kit comprising a container containing a lipid nanoparticle described herein. In some embodiments, provided herein is a kit comprising two containers, wherein each container comprises a different lipid nanoparticle. In some embodiments, provided herein is a kit comprises a container containing a lipid nanoparticle composition described herein. In some embodiments, provided herein is a kit comprising two or more containers, wherein each container comprises a different composition described herein. In some embodiments, provided herein is a kit comprising two or more containers, wherein each container comprises a different lipid nanoparticle composition described herein. In some embodiments, provided herein is a kit comprising at least two containers, wherein one container comprises a therapeutic nucleic acid (e.g., mRNA) molecule described herein, and the second container comprises a one or more lipids described herein. For example, the second container may contain a cationic lipid, such as described herein. In some embodiments, the second container contains a compound of Formula 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulas thereof) as described herein. Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The kits encompassed herein can be used in accordance with the methods described herein.
EMBODIMENTS
1. A polypeptide comprising a trafficking peptide and a KRAS domain, wherein the KRAS domain comprises at
least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
2. The polypeptide of embodiment 1, wherein the trafficking peptide is derived from one or more polypeptides
selected from the group consisting of MHC Class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.
3. The polypeptide of embodiment 2, wherein the trafficking peptide is derived from MHC Class I.
4. The polypeptide of embodiment 2, wherein the trafficking peptide consists of, essentially consists of, or
comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 99.
5. The polypeptide of any one of embodiments 1 to 4, further comprising a helper T cell epitope.
6. The polypeptide of embodiment 5, wherein the helper T cell epitope is a universal CD4 epitope.
7. The polypeptide of any one of embodiments 5 or 6, wherein the helper T cell epitope is an epitope of tetanus and
diphtheria toxoids.
8. The polypeptide of any one of embodiments 5 to 7, wherein the helper T cell epitope comprises one or more
epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE.
9. The polypeptide of embodiment 5, wherein the helper T cell epitope consists of, essentially consists of, or
comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 94.
10. The polypeptide of any one of embodiments 1 to 9, further comprising an immunomodulator.
11. The polypeptide of embodiment 10, wherein the immunomodulator is selected from the group consisting of
GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof.
12. The polypeptide of embodiment 10 or 11, wherein the immunomodulator is GM-CSF, optionally wherein the
GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) .
13. The polypeptide of embodiment 10, wherein the immunomodulator consists of, essentially consists of, or
comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 or 115.
14. The polypeptide of any one of embodiments 10 to 13, wherein the immunomodulator is connected to the KRAS
domain via a cleavable linker.
15. The polypeptide of embodiment 14, wherein the cleavable liner is a 2A peptide.
16. The polypeptide of embodiment 14 or 15, wherein the 2A peptide is selected from the group consisting of P2A,
F2A, T2A, and E2A.
17. The polypeptide of embodiment 14, wherein the cleavable linker consists of, essentially consists of, or
comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 105 or 258.
18. The polypeptide of any one of embodiments 1 to 17, further comprising a signal peptide.
19. The polypeptide of embodiment 18, wherein the signal peptide consists of, essentially consists of, or comprises
an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 89.
20. A polypeptide comprising, in the N-to-C direction, a signal peptide, a KRAS domain, a helper T cell epitope,
a trafficking peptide, a cleavable linker and an immunomodulator, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
21. The polypeptide of embodiment 20, wherein the KRAS domain comprises the amino acid sequence set forth in
SEQ ID NO: 138, 161, 260, or 409.
22. The polypeptide of embodiment 20 or 21, wherein the trafficking peptide comprises the amino acid sequence
set forth in SEQ ID NO: 99.
23. The polypeptide of any one of embodiments 20 to 22, wherein the helper T cell epitope comprises the amino
acid sequence set forth in SEQ ID NO: 94.
24. The polypeptide of any one of embodiments 20 to 23, wherein the immunomodulator comprises the amino acid
sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 115.
25. The polypeptide of any one of embodiments 20 to 24, wherein the signal peptide comprises the amino acid
sequence set forth in SEQ ID NO: 89.
26. A polypeptide comprising a degradation domain and a KRAS domain,
wherein the degradation domain is configured to mark the KRAS domain for proteasomal degradation; and wherein the KRAS domain comprises at least one mutated fragment of human KRAS.
27. The polypeptide of embodiment 26, wherein the degradation domain comprises a ubiquitin peptide.
28. The polypeptide of embodiment 27, wherein the ubiquitin peptide is a ubiquitin monomer, a ubiquitin multimer,
a ubiquitin like domain (ULD) , or a functional derivative thereof.
29. The polypeptide of embodiment 28, wherein the polypeptide comprises a C-terminal portion fused to the C-
terminus of the degradation domain, and wherein the C-terminal portion has a length of at least about 15 amino acids.
30. The polypeptide of embodiment 29, wherein the KRAS domain is located in the C-terminal portion of the
polypeptide.
31. The polypeptide of embodiment 30, wherein the KRAS domain is fused directly to the C-terminus of the
degradation domain, and wherein the KRAS domain is at least about 10 amino acids in length.
32. The polypeptide of embodiment 30, wherein the KRAS domain comprises two or more mutated fragments of
human KRAS.
33. The polypeptide of embodiment 30, wherein a spacer peptide is located between the degradation domain and
the KRAS domain; optionally wherein the spacer peptide is at least about 15 amino acids in length; optionally wherein the spacer peptide is about 15-30 amino acids in length; optionally wherein the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids in length.
34. The polypeptide of any one of embodiments 26 to 33, wherein the degradation domain consists of, essentially
consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
35. The polypeptide of embodiment 34, wherein the degradation domain further comprises a spacer located
between the ubiquitin peptide and the KRAS domain, and wherein the spacer peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.
36. The polypeptide of any one of embodiments 26 to 35, wherein the KRAS domain comprises at least one mutated
fragment of human KRAS, wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
37. The polypeptide of embodiment 36, wherein the mutated fragment comprises one or more KRAS mutation
selected from any one of (a) to (r) :
(a) G12A;
(b) G12C;
(c) G12D;
(d) G12V;
(e) G13D;
(f) Q61R;
(g) G12A and G13D;
(h) G12C and G13D;
(i) G12D and G13D;
(j) G12V and G13D;
(k) G12A and Q61R;
(l) G12C and Q61R;
(m) G12D and Q61R;
(n) G12V and Q61R;
(o) G12A, G13D, and Q61R;
(p) G12C, G13D, and Q61R;
(q) G12D, G13D, and Q61R;
(r) G12V, G13D, and Q61R.
38. The polypeptide of embodiment 36 or 37, wherein the at least one mutated fragment of human KRAS is at least
about 10 amino acids in length; optionally wherein the mutated fragment of human KRAS is about 10 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, or about 35 amino acids in length.
39. The polypeptide of any one of embodiments 36 to 38, wherein the at least one mutated fragment of human
KRAS comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different.
40. The polypeptide of any one of embodiments 36 to 39, wherein the KRAS domain comprises two or more
mutated fragments of human KRAS, and wherein at least two of the two or more mutated fragments are fused directly or through a linker peptide.
41. The polypeptide of any one of embodiments 36 to 40, wherein the at least one mutated fragment of human
KRAS comprises a G12A fragment, wherein the G12A fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12A mutation.
42. The polypeptide of embodiment 41, wherein the KRAS domain comprises two or more identical copies of the
G12A fragment.
43. The polypeptide of embodiment 41 or 42, wherein the G12A fragment has the amino acid sequence of SEQ ID
NO: 7.
44. The polypeptide of any one of embodiments 36 to 43, wherein the at least one mutated fragment of human
KRAS comprises a G12C fragment, wherein the G12C fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12C mutation.
45. The polypeptide of embodiment 44, wherein the KRAS domain comprises two or more identical copies of the
G12C fragment.
46. The polypeptide of embodiment 44 or 45, wherein the G12C fragment has the amino acid sequence of SEQ ID
NO: 20.
47. The polypeptide of any one of embodiments 36 to 46, wherein the at least one mutated fragment of human
KRAS comprises an G12D fragment, wherein the G12D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12D mutation.
48. The polypeptide of embodiment 47, wherein the KRAS domain comprises two or more identical copies of the
G12D fragment.
49. The polypeptide of embodiment 47 or 48, wherein the G12D fragment has the amino acid sequence of SEQ ID
NO: 36.
50. The polypeptide of any one of embodiments 36 to 49, wherein the at least one mutated fragment of human
KRAS comprises a G12V fragment, wherein the G12V fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12V mutation.
51. The polypeptide of embodiment 50, wherein the KRAS domain comprises two or more identical copies of the
G12V fragment.
52. The polypeptide of embodiment 50 or 51, wherein the G12V fragment has the amino acid sequence of SEQ ID
NO: 52.
53. The polypeptide of any one of embodiments 36 to 52, wherein the at least one mutated fragment of human
KRAS comprises a G13D fragment, wherein the G13D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G13D mutation.
54. The polypeptide of embodiment 53, wherein the KRAS domain comprises two or more identical copies of the
G13D fragment.
55. The polypeptide of embodiment 53 or 54, wherein the G13D fragment has the amino acid sequence of SEQ ID
NO: 68.
56. The polypeptide of any one of embodiments 36 to 55, wherein the at least one mutated fragment of human
KRAS comprises a Q61R fragment, wherein the Q61R fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the Q61R mutation.
57. The polypeptide of embodiment 56, wherein the KRAS domain comprises two or more identical copies of the
Q61R fragment.
58. The polypeptide of embodiment 56 or 57, wherein the Q61R fragment has the amino acid sequence of SEQ ID
NO: 83.
59. The polypeptide of any one of embodiment 36 to 58, wherein the KRAS domain comprises:
(a) two of the G12A fragments, two of the G12C fragments, two of the G12D fragments, two of the G12V
fragments, two of the G13D fragments, and two of the Q61R fragments,
optionally wherein the KRAS domain comprises two repeating units each comprising in the N-to-C direction: the
G12C fragment, the G12V fragment, the G12D fragment, the G13D fragment, the G12A fragment, and the Q61R fragment;
optionally wherein each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7, optionally wherein
each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20, optionally wherein each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36, optionally wherein each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52; optionally wherein each of the G13D fragment has the amino acid sequence of SEQ ID NO: 68; optionally wherein each of the Q61R fragments has the amino acid sequence of SEQ ID NO: 83;
or
(b) three of the G12A fragments, three of the G12C fragments, three of the G12D fragments, three of the G12V
fragments, and three of the G13D fragments;
optionally wherein the KRAS domain comprises three repeating units each comprising in the N-to-C direction: a
copy of the G12C fragment, a copy of the G12V fragment, a copy of the G12D fragment, a copy of the G13D fragment and a copy of the G12A fragment;
optionally wherein each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7, optionally wherein
each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20, optionally wherein each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36, optionally wherein each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52; optionally wherein each of the G13D fragment has the amino acid sequence of SEQ ID NO: 68.
60. A polypeptide consists of, essentially consists of, or comprises the amino acid sequence selected from SEQ ID
NOS: 138, 141, 146, 153, 404, 406 or 408.
61. An mRNA molecule comprising the sequence selected from SEQ ID NOS: 139, 142, 144, 147, 149, 151, 154,
156, and 158.
62. A DNA molecule comprising the sequence selected from SEQ ID NOS: 140, 143, 145, 148, 150, 152, 155, 157,
and 159, or a transcribed RNA sequence thereof, optionally the transcribed RNA is mRNA.
63. A composition comprising a plurality of species of polypeptides, wherein each species of polypeptide is
selected from the polypeptide according to any one of embodiments 26 to 59, wherein at least two of the plurality of species of polypeptides are different.
64. A nucleic acid molecule comprising a coding sequence encoding the polypeptide of any one of embodiments 1
to 60.
65. The nucleic acid molecule of embodiment 64, wherein the nucleic acid molecule further comprises a 5’
untranslated region (UTR) ; optionally wherein the 5’ UTR comprises the sequence of SEQ ID NO: 136.
66. The nucleic acid molecule of embodiment 64 or 65, wherein the nucleic acid molecule further comprises a 3’
UTR, optionally wherein the 3’ UTR comprises the sequence of SEQ ID NO: 137.
67. The nucleic acid molecule of any one of embodiments 64 to 66, wherein the nucleic acid is a DNA molecule
or an RNA molecule.
68. The nucleic acid molecule of any one of embodiments 64 to 67, wherein the nucleic acid comprises one or more
functional nucleotide analogs that are selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.
69. A vector comprising the nucleic acid molecule of any one of embodiments 64 to 67.
70. A composition comprising the nucleic acid of any one of embodiments 64 to 68, and at least one lipid.
71. A composition comprising a first coding sequence encoding a fusion protein, the fusion protein comprising an
KRAS domain, and a second coding sequence encoding an immunomodulator, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
72. The composition of embodiment 71, wherein the first coding sequence comprises a nucleic acid sequence
selected from the sequences set forth in SEQ ID NOS: 8 to 19, 21 to 35, 37 to 51, 53 to 67, 69 to 82, 84 to 88, 160, 162 to 167, or a codon optimized variant thereof.
73. The composition of embodiment 71 or 72, wherein the fusion protein further comprises a helper T cell epitope.
74. The composition of embodiment 73, wherein the helper T cell epitope is a universal CD4 epitope.
75. The composition of embodiment 73 or 74, wherein the helper T cell epitope is an epitope of tetanus and
diphtheria toxoids.
76. The composition of any one of embodiments 73 to 75, wherein the helper T cell epitope comprises one or more
epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE.
77. The composition of embodiment 73, wherein the first coding sequence further comprises a nucleic acid
sequence set forth in SEQ ID NOS: 95 to 98, or a codon optimized variant thereof.
78. The composition of any one of embodiments 71 to 77, wherein the fusion protein further comprises a trafficking
peptide.
79. The composition of embodiment 78, wherein the trafficking peptide is derived from one or more polypeptides
selected from the group consisting of MHC Class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.
80. The composition of embodiment 78, wherein the trafficking peptide is derived from MHC Class I.
81. The composition of embodiment 78, wherein the first coding sequence further comprises a nucleic acid
sequence set forth in SEQ ID NOS: 101 to 104, or a codon-optimized variant thereof.
82. The composition of any one of embodiments 71 to 81, wherein the immunomodulator is selected from the
group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof.
83. The composition of embodiment 82, wherein the immunomodulator is GM-CSF, optionally wherein the GM-
CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) .
84. The composition of embodiment 83, wherein the second coding sequence comprises a nucleic acid sequence
set forth in SEQ ID NOS: 112 to 114, and 116 to 118, or a codon optimized variant thereof.
85. The composition of any one of embodiments 71 to 84, wherein the first coding sequence and the second coding
sequence are in one nucleic acid molecule.
86. The composition of any one of embodiments 71 to 81, wherein the first coding sequence and the second coding
sequence are in separate nucleic acid molecules.
87. The composition of any one of embodiments 71 to 86, wherein the nucleic acid is a DNA molecule or an RNA
molecule.
88. The composition of embodiments 71 to 86, wherein the nucleic acid comprises one or more functional
nucleotide analogs that are selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.
89. The composition of any one of embodiments 71 to 88, further comprising at least one lipid.
90. A vector comprising the nucleic acid molecule of embodiments 85.
91. A set of vectors comprising a first vector comprising the first coding sequence and a second vector comprising
the second coding sequence of the nucleic acid composition of embodiment 86.
92. A composition comprising a plurality of species of nucleic acid molecules each encoding an KRAS domain,
wherein each encoded KRAS domain comprises at least one mutated fragment of human KRAS, wherein the mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R, and wherein the KRAS domains encodedby at least two of the plurality of species of nucleic acid molecules are different.
93. The composition of embodiment 92, wherein the KRAS domain encoded by at least one of the plurality of
species of nucleic acid molecules comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different.
94. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules comprise KRAS mutations selected from any one of
(a) to (r) :
(a) G12A;
(b) G12C;
(c) G12D;
(d) G12V;
(e) G13D;
(f) Q61R;
(g) G12A and G13D;
(h) G12C and G13D;
(i) G12D and G13D;
(j) G12V and G13D;
(k) G12A and Q61R;
(l) G12C and Q61R;
(m) G12D and Q61R;
(n) G12V and Q61R;
(o) G12A, G13D, and Q61R;
(p) G12C, G13D, and Q61R;
(q) G12D, G13D, and Q61R;
(r) G12V, G13D, and Q61R.
95. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules are a C12A fragment, wherein the C12A fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the C12A mutation.
96. The composition of embodiment 95, wherein the plurality of species of nucleic acid molecules encoding the
G12A fragment each comprises a coding sequence selected from SEQ ID NOS: 8 to 19, or a codon optimized variant thereof.
97. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules are a G12C fragment, wherein the G12C fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12C mutation.
98. The composition of embodiment 97, wherein the plurality of species of nucleic acid molecules encoding the
G12C fragment each comprises a coding sequence selected from SEQ ID NOS: 21 to 35, or a codon optimized variant thereof.
99. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules are a G12D fragment, wherein the G12D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12D mutation.
100. The composition of embodiment 99, wherein the plurality of species of nucleic acid molecules encoding G12D
fragment each comprises a coding sequence selected from SEQ ID NOS: 37 to 51, or a codon optimized variant thereof.
101. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules are a G12V fragment, wherein the G12V fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12V mutation.
102. The composition of embodiment 101, wherein the plurality of species of nucleic acid molecules encoding the
G12V fragment each comprises a coding sequence selected from SEQ ID NOS: 53 to 67, or a codon optimized variant thereof.
103. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules are a G13D fragment, wherein the G13D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G13D mutation.
104. The composition of embodiment 103, wherein the plurality of species of nucleic acid molecules encoding the
G13D fragment each comprises a coding sequence selected from SEQ ID NOS: 69 to 82, or a codon optimized variant thereof.
105. The composition of embodiment 92 or 93, wherein one or more of the mutated fragments of human KRAS
encoded by the plurality of species of nucleic acid molecules are a Q61R fragment, wherein the Q61R fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the Q61R mutation.
106. The composition of embodiment 105, wherein the plurality of species of nucleic acid molecules encoding the
Q61R fragment each comprises a coding sequence selected from SEQ ID NOS: 84 to 86, or a codon optimized variant thereof.
107. The composition of embodiment 92 or 93, wherein the plurality of species of nucleic acid molecules further
comprises at least one first species of nucleic acid molecule encoding an KRAS domain that is longer than about 10 amino acids, and wherein the at least one first species of nucleic acid molecule further encodes a degradation domain, wherein the degradation domain is configured to mark the KRAS domain for proteasomal degradation.
108. The composition of embodiment 107, wherein the degradation domain comprises a ubiquitin peptide.
109. The composition of embodiment 108, wherein the ubiquitin peptide is a ubiquitin monomer, a ubiquitin
multimer, a ubiquitin like domain (ULD) , or a functional derivative thereof.
110. The composition of embodiment 109, wherein a spacer peptide is located between the degradation domain
and the KRAS domain; optionally wherein the spacer peptide is at least about 15 amino acids in length; optionally wherein the spacer peptide is about 15-30 amino acids in length; optionally wherein the spacer peptide is about 20 amino acids, about 25 amino acids, or about 30 amino acids in length.
111. The composition of any one of embodiments 108 to 110, wherein the at least one species of the nucleic acid
molecule comprises a coding sequence for the ubiquitin peptide selected from SEQ ID NOS: 2 and 3, or a codon optimized variant thereof.
112. The composition of any one of embodiments 107 to 111, wherein the plurality of species of nucleic acid
molecules further comprises at least one second species of nucleic acid molecule encoding a KRAS domain that is not longer than about 10 amino acids, and wherein the at least one second species of nucleic acid molecule is devoid of a coding sequence for a degradation domain.
113. The composition of any one of embodiments 92 to 112, wherein the plurality of species of nucleic acid
molecules comprise mRNA molecules.
114. The composition of any one of embodiments 92 to 113, wherein at least one of the plurality of species of
nucleic acid molecules further comprises a 3’-UTR and/or a 5’-UTR; optionally wherein the 5’-UTR comprises the sequence of SEQ ID NO: 136; optionally wherein the 3’-UTR comprises the sequence of SEQ ID NO: 137.
115. The composition of embodiment 92, wherein the plurality of species of nucleic acid molecules comprise RNA
molecules comprising the sequences selected from SEQ ID NOS: 139, 142, and 144.
116. The composition of embodiment 92, wherein the plurality of species of nucleic acid molecules comprise DNA
molecules comprising the sequences selected from SEQ ID NOS: 140, 143, and 145.
117. The composition of any one of embodiments 92 to 116, wherein at least one of the plurality of species of
nucleic acids comprises one or more functional nucleotide analogs that are selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.
118. The composition of any one of embodiments 92 to 117, further comprising at least one lipid.
119. The composition of any one of embodiments 92 to 118, wherein the composition is formulated as lipid
nanoparticles encompassing the nucleic acid.
120. A method of activating KRAS-mutant specific immune effector cells, comprising providing a starting
population of immune effector cells, and contacting the starting population of immune effector cells with the polypeptide of any one of embodiments 1 to 60, the mRNA molecule of embodiment 61, the DNA molecule of embodiment 62, the nucleic acid molecule of any one of embodiments 64 to 68, or the composition of any one of embodiment 63, 70 to 89 under a suitable condition, wherein upon the contacting, KRAS-mutant specific immune effector cells are activated.
121. The method of embodiment 120, wherein the contacting is performed in the presence of a proteosome system,
and wherein the proteosome system degrades the KRAS domain into one or more peptides of about 9 to 15 amino acids in length.
122. The method of embodiments 122 or 121, wherein the contacting is performed in the presence of antigen
presenting cells (APCs) , wherein the APCs present the KRAS domain or a degraded portion thereof on their surface.
123. The method of embodiment 122, wherein the APCs are selected from dendritic cells, macrophages,
Langerhans cells and B cells.
124. The method of embodiment 122 or 123, wherein the APCs express HLA class I and/or HLA class II molecules;
optionally wherein the HLA class I molecule is of the HLA-A*11: 01 subtype.
125. The method of any one of embodiments 120 to 124, wherein the immune effector cells are selected from T
cells, peripheral blood lymphocytes, and natural killer cells; optionally wherein the T cells comprises CD4+T cells, CD8+T cells, antigen-specific T cells, tumor infiltration T cells, γδT cells, cytotoxic T cells, natural killer T cells, and mucosal associated invariant T cells (MAIT) .
126. A method of forming a pro-inflammatory milieu in a tissue surrounding a population of diseased cells
expressing a mutant KRAS, comprising contacting the tissue with an effective amount of the polypeptide of any one of embodiments 1 to 60, the mRNA molecule of embodiment 61, the DNA molecule of embodiment 62, the nucleic acid molecule of any one of embodiments 64 to 68, or the composition of any one of embodiment 63, 70 to 89 under a suitable condition.
127. The method of embodiment 126, wherein infiltration of activated B cells, CD4+T cells, CD8+T cells,
dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophil and/or neutrophils in the tissue is increased.
128. The method of embodiment 127, wherein concentration of a pro-inflammatory cytokine is increased in the
tissue.
129. The method of embodiment 128, wherein the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-
22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1 or any combination thereof.
130. The method of any one of embodiments 126 to 129, wherein presentation of antigens originated or derived
from the diseased cells by antigen presentation cells is increased in the tissue.
131. The method of any one of embodiments 126 to 130, wherein phagocytosis of the diseased cells is increased
in the tissue.
132. The method of any one of embodiments 126 to 131, wherein apoptosis of the diseased cells induced by cell-
mediated cytotoxicity is increased in the tissue.
133. The method of any one of embodiments 126 to 132, wherein apoptosis of the diseased cells induced by
antibody-dependent cellular cytotoxicity is increased in the tissue.
134. The method of any one of embodiments 126 to 133, wherein the population of the diseased cells is reduced in
the tissue.
135. The method of embodiment 134, wherein the population of the diseased cells is reduced by about 10%, 20%,
30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%in the tissue.
136. The method of any one of embodiments 126 to 135, wherein the diseased cells are cancer cells expressing a
mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
137. The method of any one of embodiments 126 to 136, wherein the method is performed in vitro or in vivo.
138. A method for inducing an immune response against a target cell expressing a mutant KRAS in a subject,
wherein the method comprises administering to the subject the polypeptide of any one of embodiments 1 to 60, the mRNA molecule of embodiment 61, the DNA molecule of embodiment 62, the nucleic acid molecule of any one of embodiments 64 to 68, or the composition of any one of embodiment 63, 70 to 89, wherein upon the administration,
(a) the KRAS domain encoded by the nucleic acid molecules are expressed;
(b) the KRAS domain is degraded into fragments of about 9 to 15 amino acids;
(c) the KRAS domain or a degraded fragment thereof is associated with an HLA class I or HLA class II complex;
(d) innate immune response against the target cell is increased;
(e) pro-inflammatory cytokine secretion is increased;
(f) a percentage of phagocytotic macrophages in a population of macrophages is increased;
(g) a percentage of dendritic cells presenting the KRAS domain or degraded fragments thereof in a population of
dendritic cells is increased;
(h) a percentage of antigen-specific T cells specific for the KRAS domain or an epitope thereof is increased; or
(i)a percentage of B cells producing antibodies specific for the KRAS domain or an epitope thereof in a population
of B cells is increased.
139. The method of embodiment 138, wherein the innate immune response is measured by
(a) activation of the NF-kB signaling pathway; or
(b) increase in secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any
combination thereof.
140. The method of embodiment 138, wherein the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-
22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof.
141. The method of any one of embodiments 138 to 140, wherein the target cell is a diseased cell expresses a
mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
142. A method of eliminating a diseased cell expressing a mutant KRAS in a subject, comprising administering an
effective amount of the polypeptide of any one of embodiments 1 to 60, the mRNA molecule of embodiment 61, the DNA molecule of embodiment 62, the nucleic acid molecule of any one of embodiments 64 to 68, or the composition of any one of embodiment 63, 70 to 89.
143. The method of embodiment 142, wherein the subject is a human subject having the HLA subtype of HLA-
A*11: 01.
144. The method of embodiment 142 or 143, wherein the diseased cell is cancer cells expressing a mutant KRAS
with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
145. A method of treating a KRAS mutant cancer in a subject in need thereof, comprising administering to the
subject an effective amount of the polypeptide of any one of embodiments 1 to 60, the mRNA molecule of embodiment 61, the DNA molecule of embodiment 62, the nucleic acid molecule of any one of embodiments 64 to 68, or the composition of any one of embodiment 63, 70 to 89.
146. The method of embodiment 145, wherein the cancer cells express a mutant KRAS with mutations selected
from G12A, G12C, G12D, G12V, G13D, and Q61R.
147. The method of embodiment 145 or 146, wherein the cancer is selected from epithelial cancers, lung cancers,
particularly non-small cell lung cancers, colorectal cancers, and pancreatic cancers.
OTHER EMBODIMENTS
1. An immunomodulating nucleic acid system encoding a KRAS domain, wherein the system comprises a
first coding sequence encoding a polypeptide comprising a helper T cell epitope, wherein the helper T cell epitope is configured to enhance immunogenicity of the KRAS domain upon co-expression with the KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
2. The immunomodulating nucleic acid system of embodiment 1, wherein the system further comprises a
second coding sequence encoding a polypeptide comprising an immunomodulator, wherein the immunomodulator is configured to enhance immunogenicity of the KRAS domain upon co-expression with the KRAS domain.
3. The immunomodulating nucleic acid system of embodiment 1 or 2, wherein the system further comprises
a third coding sequence encoding a polypeptide comprising a trafficking peptide configured for intracellularly trafficking the target antigen towards proteosome upon expression.
4. The immunomodulating nucleic acid system of any one of embodiments 1 to 3, wherein the system further
comprises a fourth coding sequence encoding a polypeptide comprising a signal peptide.
5. The immunomodulating nucleic acid system of any one of embodiments 1 to 4, wherein the system further
comprises a fifth coding sequence encoding a polypeptide comprising a degradation domain, optionally wherein the degradation domain comprises a ubiquitin peptide.
6. The immunomodulating nucleic acid system of any one of embodiments 1 to 5, wherein the first coding
sequence encodes a polypeptide comprising the helper T cell epitope and the KRAS domain.
7. The immunomodulating nucleic acid system of any one of embodiments 2 to 6, wherein the second coding
sequence encodes a polypeptide comprising the immunomodulator and the KRAS domain; optionally wherein the immunomodulator is positioned N-terminal to the KRAS domain or is positioned C-terminal to the KRAS domain in the polypeptide.
8. The immunomodulating nucleic acid system of any one of embodiments 3 to 7, wherein the third coding
sequence encodes a polypeptide comprising the trafficking peptide and the KRAS domain; optionally wherein the trafficking peptide is positioned C-terminal to the KRAS domain in the polypeptide.
9. The immunomodulating nucleic acid system of any one of embodiments 4 to 8, wherein the fourth coding
sequence encodes a polypeptide comprising the signal peptide and the KRAS domain; optionally wherein the signal peptide is positioned N-terminal to the KRAS domain in the polypeptide.
10. The immunomodulating nucleic acid system of any one of embodiments 5 to 9, wherein the fifth coding
sequence encodes a polypeptide comprising the ubiquitin peptide and the KRAS domain; optionally wherein the ubiquitin peptide is positioned N-terminal to the KRAS domain in the polypeptide; optionally wherein polypeptide further comprises a spacer peptide positioned in between the ubiquitin peptide and the KRAS domain in the polypeptide; optionally the spacer peptide is at least about 25 amino acids in length.
11. The immunomodulating nucleic acid system of any one of embodiments 1 to 10, wherein:
(a) the first coding sequenc is in a first nucleic acid molecule;
(b) at least one of the first coding sequence and the second coding sequence is in a first nucleic acid
molecule;
(c) at least one of the first coding sequence, the second coding sequence and the third coding sequence is
in a first nucleic acid molecule;
(d) at least one of the first coding sequence, the second coding sequence, the third coding sequence and
the fourth coding sequence is in a first nucleic acid molecule; or
(e) at least one of the first coding sequence, the second coding sequence, the third coding sequence, the
fourth coding sequence and the fifth coding sequences is in a first nucleic acid molecule.
12. The immunomodulating nucleic acid system of embodiment 11, wherein:
(a) the second coding sequence is in the first nucleic acid molecule, and wherein the polypeptide comprises
the immunomodulatory and the KRAS domain;
(b) the first coding sequence and the second coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the helper T cell epitope, the immunomodulator and the KRAS domain;
(c) the first coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein
the polypeptide comprises the helper T cell epitope, the trafficking peptide and the KRAS domain;
(d) the first coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the helper T cell epitope, the signal peptide and the KRAS domain;
(e) the first coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein
the polypeptide comprises the helper T cell epitope, the ubiquitin peptide and the KRAS domain;
(f) the second coding sequence and the third coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the immunomodulator, the trafficking peptide and the KRAS domain;
(g) the second coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the immunomodulator, the signal peptide, and the KRAS domain;
(h) the second coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the immunomodulator, the ubiquitin peptide and the KRAS domain;
(i) the third coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the trafficking peptide, the signal peptide and the KRAS domain;
(j) the third coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein
the polypeptide comprises the trafficking peptide, the ubiquitin peptide and the KRAS domain;
(k) the fourth coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and
wherein the polypeptide comprises the signal peptide, the ubiquitin peptide and the KRAS domain;
(l) the first coding sequence, the second coding sequence, and the third coding sequence are in the first
nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, and the KRAS domain;
(m) the first coding sequence, the second coding sequence, and the fourth coding sequence are in the first
nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the signal peptide, and the KRAS domain;
(n) the first coding sequence, the second coding sequence, and the fifth coding sequence are in the first
nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the ubiquitin peptide, and the KRAS domain;
(o) the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first
nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide, the signal peptide, and the KRAS domain;
(p) the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first
nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the KRAS domain;
(q) the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first
nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the signal peptide, the ubiquitin peptide, and the KRAS domain;
(r) the first coding sequence, the second coding sequence, the third coding sequence, and the fourth coding
sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the KRAS domain;
(s) the first coding sequence, the second coding sequence, the third coding sequence, and the fifth coding
sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the KRAS domain; or
(t) the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding
sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the ubiquitin peptide, and the KRAS domain.
13. The immunomodulating nucleic acid system of embodiment 11, wherein at least one of the first coding
sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences is in a second nucleic acid molecule, and wherein the first nucleic acid molecule and the second nucleic acid molecule are different molecules.
14. The immunomodulating nucleic acid system of embodiment 13, wherein the second nucleic acid molecule
does not encode the KRAS domain.
15. The immunomodulating nucleic acid system of embodiment 13 or 14, wherein the first nucleic acid
molecule encodes the polypeptide comprising the KRAS domain, wherein the polypeptide does not comprise the immunomodulator; and wherein the second nucleic acid molecule comprises the second coding sequence encoding the immunomodulator.
16. The immunomodulating nucleic acid system of any one of embodiments 11 to 14, wherein at least the
second coding sequence is in the first nucleic acid molecule encoding the polypeptide comprising at least the immunomodulator and the KRAS domain; and wherein the first nucleic acid molecule further comprises a means for producing the immunomodulator and the KRAS domain as separate proteins or peptides.
17. The immunomodulating nucleic acid system of embodiment 16, wherein the means for producing the
immunomodulator and the KRAS domain as separate proteins or peptides comprises a sixth coding sequence encoding a cleavable linker in between the second coding sequence and a coding sequence for the KRAS domain in the first nucleic acid.
18. The immunomodulating nucleic acid system of embodiment 17, wherein the cleavable linker is a 2A
peptide, selected from the group consisting of P2A, F2A, T2A, and E2A; optionally wherein the cleavable linker consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 105.
19. The immunomodulating nucleic acid system of any one of embodiments 1 to 18, wherein the helper T cell
epitope is a universal CD4 epitope.
20. The immunomodulating nucleic acid system of any one of embodiments 1 to 18, wherein the helper T cell
epitope is an epitope of tetanus and diphtheria toxoids.
21. The immunomodulating nucleic acid system of any one of embodiments 1 to 18, wherein the helper T cell
epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827- ₈₄₁, pDT_331-350, TT_632-651, and PADRE; optionally wherein the helper T cell epitope consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 94.
22. The immunomodulating nucleic acid system of any one of embodiments 1 to 18, wherein the first coding
sequence encoding the helper T cell epitope comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 95-98 and 186.
23. The immunomodulating nucleic acid system of any one of embodiments 1 to 18, wherein the first coding
sequence encodes the helper T cell epitope that is a pan DR-binding epitope (PADRE) , and wherein the first coding sequence comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence of SEQ ID NO: 98 or 186.
24. The immunomodulating nucleic acid system of any one of embodiments 2 to 23, wherein the
immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof.
25. The immunomodulating nucleic acid system of any one of embodiments 2 to 23, wherein the
immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.
26. The immunomodulating nucleic acid system of any one of embodiments 2 to 23, wherein
(a) the first coding sequence encodes a polypeptide comprising the KRAS domain and a helper T cell epitope,
and
(b) the second coding sequence encodes an immunomodulator, wherein the immunomodulator comprises
GM-CSF, STING, or both GM-CSF and STING.
27. The isolated nucleic acid of any one of embodiments 24 to 26, wherein the GM-CSF is human GM-CSF
(hGM-CSF) or mouse GM-CSF (mGM-CSF) ; optionanlly wherein the immunomodulator consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268.
28. The isolated nucleic acid of any one of embodiments 24 to 26, wherein the GM-CSF is a full-length GM-
CSF polypeptide.
29. The isolated nucleic acid of any one of embodiments 24 to 26, wherein the GM-CSF comprises a
truncated GM-CSF or a mutant GM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268, and wherein the truncated GM-CSF or mutant GM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) .
30. The isolated nucleic acid of any one of embodiments 24 to 26, wherein the STING is human STING
(hSTING) (V155M) .
31. The isolated nucleic acid of embodiment 30, wherein the STING comprises a polypeptide comprising an
amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence of SEQ ID NO: 220 or 253.
32. The isolated nucleic acid of any one of embodiments 24 to 31, wherein the second coding sequence
encoding the immunomodulator comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 112-114, 215-217, 116-118, 218-219, 221, 254, 265, and 269-271.
33. The immunomodulating nucleic acid system of any one of embodiments 3 to 32, wherein the trafficking
peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.
34. The immunomodulating nucleic acid system of any one of embodiments 3 to 32, wherein the trafficking
peptide is MHC class I trafficking peptide; optionally wherein the trafficking peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 99.
35. The immunomodulating nucleic acid system of any one of embodiments 3 to 32, the trafficking peptide is
MHC class I trafficking peptide or LAMP3 transmembrane domain (LAMP3 TM) .
36. The immunomodulating nucleic acid system of any one of embodiments 3 to 32, wherein the third coding
sequence encoding the trafficking peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 100-104 and 198-202.
37. The immunomodulating nucleic acid system of any one of embodiments 4 to 36, wherein the fourth
coding sequence encoding the signal peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 90-93 and 171-175; optionally wherein the signal peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 89 or 170.
38. The immunomodulating nucleic acid system of any one of embodiments 5 to 37, wherein the ubiquitin
peptide comprises a ubiquitin monomer, a ubiquitin multimer, a ubiquitin like domain (ULD) , or a functional derivative thereof; optionally wherein the ubiquitin peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; optionally wherein the fifth coding sequence encoding the ubiquitin peptide comprises the nucleic acid sequence set forth in SEQ ID NOS: 2 and 3, or a codon optimized variant thereof.
39. The immunomodulating nucleic acid system of embodiment 38, wherein the ubiquitin peptide further
comprises a spacer located between the ubiquitin peptide and the KRAS domain, and wherein the spacer peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.
40. The immunomodulating nucleic acid system of any one of embodiments 11 to 39, wherein the first nucleic
acid molecule is DNA or RNA.
41. The immunomodulating nucleic acid system of any one of embodiments 13 to 40, wherein the second
nucleic acid molecule is DNA or RNA.
42. The immunomodulating nucleic acid system of embodiment 41, wherein the RNA is mRNA, self-
amplifying RNA, or circular RNA.
43. The immunomodulating nucleic acid system of any one of embodiments 1 to 42, wherein the one or more
coding sequences are codon optimized.
44. The immunomodulating nucleic acid system of any one of embodiments 1 to 43, wherein the one or more
coding sequences are in one or more mRNA molecule.
45. The immunomodulating nucleic acid system of embodiment 44, wherein the mRNA molecule further
comprises a 5’ untranslated region (UTR) , a 3’ UTR, or both a 5’ UTR and a 3’ UTR.
46. The immunomodulating nucleic acid system of embodiment 45, wherein the 5’ UTR comprises the
nucleic acid sequence set forth in SEQ ID NO: 136, 251 or 275.
47. The immunomodulating nucleic acid system of embodiment 45 or 46, wherein the 3’ UTR comprises the
nucleic acid sequence set forth in SEQ ID NO: 137 or 252.
48. The immunomodulating nucleic acid system of any one of embodiments 44 to 47, wherein the mRNA
molecule further comprises a 5’ Cap.
49. The immunomodulating nucleic acid system of any one of embodiments 44 to 48, wherein the mRNA
molecule further comprises a poly (A) sequence.
50. The immunomodulating nucleic acid system of embodiment 49, wherein the poly (A) sequence has a
length of about 50 nucleotides or longer.
51. The immunomodulating nucleic acid system of any one of embodiments 11 to 50, wherein the first nucleic
acid molecule comprises a chemical modification.
52. The immunomodulating nucleic acid system of any one of embodiments 13 to 51, wherein the second
nucleic acid molecule comprises a chemical modification.
53. The immunomodulating nucleic acid system of embodiment 51 or 52, wherein the chemical modification
comprises one or more functional nucleotide analogs that are selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.
54. The immunomodulating nucleic acid system of any one of embodiments 11 to 53, wherein the first nucleic
acid molecule is in a first vector.
55. The immunomodulating nucleic acid system of any one of embodiments 13 to 54, wherein the second
nucleic acid molecule is in a second vector.
56. An immunomodulating nucleic acid system encoding a polypeptide comprising, in the N-to-C direction, a
signal peptide, a KRAS domain, a helper T cell epitope, a trafficking peptide, a cleavable linker and an immunomodulator, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
57. The immunomodulating nucleic acid system of embodiment 56, wherein the KRAS domain comprises the
amino acid sequence set forth in SEQ ID NO: 138, 161, 260, or 409.
58. The immunomodulating nucleic acid system of embodiment 56 or 57, wherein the trafficking peptide
comprises the amino acid sequence set forth in SEQ ID NO: 99.
59. The immunomodulating nucleic acid system of any one of embodiments 56-58, wherein the helper T cell
epitope comprises the amino acid sequence set forth in SEQ ID NO: 94.
60. The immunomodulating nucleic acid system of any one of embodiments 56-59, wherein the
immunomodulator comprises the amino acid sequence set forth in SEQ ID NO: 111, 115, 334 or 338.
61. The immunomodulating nucleic acid system of any one of embodiments 56-60, wherein the signal peptide
comprises the amino acid sequence set forth in SEQ ID NO: 89.
62. The immunomodulating nucleic acid system of any one of embodiments 1-61, wherein the KRAS domain
comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from any one of (a) to (r) :
(a) G12A;
(b) G12C;
(c) G12D;
(d) G12V;
(e) G13D;
(f) Q61R;
(g) G12A and G13D;
(h) G12C and G13D;
(i) G12D and G13D;
(j) G12V and G13D;
(k) G12A and Q61R;
(l) G12C and Q61R;
(m) G12D and Q61R;
(n) G12V and Q61R;
(o) G12A, G13D, and Q61R;
(p) G12C, G13D, and Q61R;
(q) G12D, G13D, and Q61R;
(r) G12V, G13D, and Q61R.
63. The immunomodulating nucleic acid system of any one of embodiments 1-62, wherein the at least one
mutated fragment of human KRAS is at least about 10 amino acids in length; optionally wherein the mutated fragment of human KRAS is about 10 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, or about 35 amino acids in length.
64. The immunomodulating nucleic acid system of any one of embodiments 1-63, wherein the at least one
mutated fragment of human KRAS comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different.
65. The immunomodulating nucleic acid system of any one of embodiments 1-64, wherein the KRAS domain
comprises two or more mutated fragments of human KRAS, and wherein at least two of the two or more mutated fragments are fused directly or through a linker peptide.
66. The immunomodulating nucleic acid system of any one of embodiments 1-65, wherein the at least one
mutated fragment of human KRAS comprises a G12A fragment, wherein the G12A fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12A mutation.
67. The immunomodulating nucleic acid system of embodiment 66, wherein the KRAS domain comprises
two or more identical copies of the G12A fragment.
68. The immunomodulating nucleic acid system of embodiment 66 or 67, wherein the G12A fragment has the
amino acid sequence of SEQ ID NO: 7.
69. The immunomodulating nucleic acid system of any one of embodiments 1-68, wherein the at least one
mutated fragment of human KRAS comprises a G12C fragment, wherein the G12C fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12C mutation.
70. The immunomodulating nucleic acid system of embodiment 69, wherein the KRAS domain comprises
two or more identical copies of the G12C fragment.
71. The immunomodulating nucleic acid system of embodiment 69 or 70, wherein the G12C fragment has the
amino acid sequence of SEQ ID NO: 20.
72. The immunomodulating nucleic acid system of any one of embodiments 1-71, wherein the at least one
mutated fragment of human KRAS comprises an G12D fragment, wherein the G12D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12D mutation.
73. The immunomodulating nucleic acid system of embodiment 72, wherein the KRAS domain comprises
two or more identical copies of the G12D fragment.
74. The immunomodulating nucleic acid system of embodiment 72 or 73, wherein the G12D fragment has the
amino acid sequence of SEQ ID NO: 36.
75. The immunomodulating nucleic acid system of any one of embodiments 1-74, wherein the at least one
mutated fragment of human KRAS comprises a G12V fragment, wherein the G12V fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12V mutation.
76. The immunomodulating nucleic acid system of embodiment 75, wherein the KRAS domain comprises
two or more identical copies of the G12V fragment.
77. The immunomodulating nucleic acid system of embodiment 75 or 76, wherein the G12V fragment has the
amino acid sequence of SEQ ID NO: 52.
78. The immunomodulating nucleic acid system of any one of embodiments 1-77, wherein the at least one
mutated fragment of human KRAS comprises a G13D fragment, wherein the G13D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G13D mutation.
79. The immunomodulating nucleic acid system of embodiment 78, wherein the KRAS domain comprises
two or more identical copies of the G13D fragment.
80. The immunomodulating nucleic acid system of embodiment 78 or 79, wherein the G13D fragment has the
amino acid sequence of SEQ ID NO: 68.
81. The immunomodulating nucleic acid system of any one of embodiments 1-80, wherein the at least one
mutated fragment of human KRAS comprises a Q61R fragment, wherein the Q61R fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the Q61R mutation.
82. The immunomodulating nucleic acid system of embodiment 81, wherein the KRAS domain comprises
two or more identical copies of the Q61R fragment.
83. The immunomodulating nucleic acid system of embodiment 81 or 82, wherein the Q61R fragment has the
amino acid sequence of SEQ ID NO: 83.
84. The immunomodulating nucleic acid system of any one of embodiments 1-83, wherein the KRAS domain
comprises:
(a) two of the G12A fragments, two of the G12C fragments, two of the G12D fragments, two of the G12V
fragments, two of the G13D fragments, and two of the Q61R fragments,
optionally wherein the KRAS domain comprises two repeating units each comprising in the N-to-C direction: the
G12C fragment, the G12V fragment, the G12D fragment, the G13D fragment, the G12A fragment, and the Q61R fragment;
optionally wherein each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7, optionally wherein
each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20, optionally wherein each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36, optionally wherein each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52; optionally wherein each of the G13D fragment has the amino acid sequence of SEQ ID NO: 68; optionally wherein each of the Q61R fragments has the amino acid sequence of SEQ ID NO: 83;
or
(b) three of the G12A fragments, three of the G12C fragments, three of the G12D fragments, three of the G12V
fragments, and three of the G13D fragments;
optionally wherein the KRAS domain comprises three repeating units each comprising in the N-to-C direction: a
copy of the G12C fragment, a copy of the G12V fragment, a copy of the G12D fragment, a copy of the G13D fragment and a copy of the G12A fragment;
optionally wherein each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7, optionally
wherein each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20, optionally wherein each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36, optionally wherein each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52; optionally wherein each of the G13D fragment has the amino acid sequence of SEQ ID NO: 68.
85. The immunomodulating nucleic acid system of any one of embodiments 1-84, wherein the at least one
mutated fragment of human KRAS is encoded by a nucleic acid sequence selected from the sequences set forth in SEQ ID NOS: 8 to 19, 21 to 35, 37 to 51, 53 to 67, 69 to 82, 84 to 88, 160, 162 to 167, 261, or a codon optimized variant thereof.
86. The immunomodulating nucleic acid system of any one of embodiments 1-85, comprising a plurality of
species of nucleic acid molecules each encoding an KRAS domain, wherein each encoded KRAS domain comprises at least one mutated fragment of human KRAS, wherein the mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R, and wherein the KRAS domains encoded by at least two of the plurality of species of nucleic acid molecules are different.
87. The immunomodulating nucleic acid system of any one of embodiments 1-86, wherein the KRAS domain
encoded by at least one of the plurality of species of nucleic acid molecules comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different.
88. A polypeptide encoded by the immunomodulating nucleic acid system of any one of embodiments 1-87.
89. A polypeptide consists of, essentially consists of, or comprises the amino acid sequence selected from
SEQ ID NOS: 138, 141, 146, 153, 404, 406 or 408.
90. A non-naturally occurring nucleic acid comprising the nucleotide sequence that is at least 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to the nucleotide sequence set forth in SEQ ID NOs: 139, 142, 144, 147, 149, 151, 154, 156, or 158, optionally the nucleic acid comprises one or more functional nucleotide analogs.
91. A non-naturally occurring nucleic acid comprising the nucleotide sequence that is at least 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to SEQ ID NOs: 140, 143, 145, 148, 150, 152, 155, 157, or 159, or a transcribed RNA sequence thereof, optionally the transcribed RNA is mRNA.
92. A composition comprising a plurality of species of polypeptides, wherein each species of polypeptide is
selected from the polypeptides encoded by the immunomodulating nucleic acid system of any one of embodiments 1-87, wherein at least two of the plurality of species of polypeptides are different.
93. A vector comprising the immunomodulating nucleic acid system of any one of embodiments 1-87.
94. A set of vectors comprising two or more coding sequences of the immunomodulating nucleic acid system
of any one of embodiments 1-87 in separate nucleid acid molecules.
95. A composition comprising the immunomodulating nucleic acid system of any one of embodiments 1-87,
and at least one lipid.
96. The composition of embodiment 95, wherein the composition is formulated as lipid nanoparticles
encompassing the immunomodulating nucleic acid system.
97. A combination of (a) the immunomodulating nucleic acid system of any one of embodiments 1-87, and (b)
one or more anti-cancer therapies.
98. The combination of embodiment 97, wherein the one or more anti-cancer therapies are selected from a
chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery.
99. The combination of embodiment 97, wherein the one or more anti-cancer therapies comprise an anti-PD-1
or PDL1 antibody.
100. A method of activating KRAS-mutant specific immune effector cells, comprising providing a starting
population of immune effector cells, and contacting the starting population of immune effector cells with the immunomodulating nucleic acid system of any one of embodiments 1-87, the polypeptide of embodiment 88 or 89, the mRNA molecule of embodiment 90, the DNA molecule of embodiment 91, the composition of any one of embodiment 92 and 95 to 96, or the combination of any one of embodiments 97-99 under a suitable condition, wherein upon the contacting, KRAS-mutant specific immune effector cells are activated.
101. The method of embodiment 100, wherein the contacting is performed in the presence of a proteosome
system, and wherein the proteosome system degrades the KRAS domain into one or more peptides of about 9 to 15 amino acids in length.
102. The method of embodiments 100 or 101, wherein the contacting is performed in the presence of antigen
presenting cells (APCs) , wherein the APCs present the KRAS domain or a degraded portion thereof on their surface.
103. The method of embodiment 102, wherein the APCs are selected from dendritic cells, macrophages,
Langerhans cells and B cells.
104. The method of embodiment 102 or 103, wherein the APCs express HLA class I and/or HLA class II
molecules; optionally wherein the HLA class I molecule is of the HLA-A*11: 01 subtype.
105. The method of any one of embodiments 100 to 104, wherein the immune effector cells are selected from T
cells, peripheral blood lymphocytes, and natural killer cells; optionally wherein the T cells comprises CD4+T cells, CD8+T cells, antigen-specific T cells, tumor infiltration T cells, γδT cells, cytotoxic T cells, natural killer T cells, and mucosal associated invariant T cells (MAIT) .
106. A method of forming a pro-inflammatory milieu in a tissue surrounding a population of diseased cells
expressing a mutant KRAS, comprising contacting the tissue with an effective amount of the immunomodulating nucleic acid system of any one of embodiments 1-87, the polypeptide of embodiment 88 or 89, the mRNA molecule of embodiment 90, the DNA molecule of embodiment 91, the composition of any one of embodiment 92 and 95 to 96, or the combination of any one of embodiments 97-99 under a suitable condition.
107. The method of embodiment 106, wherein infiltration of activated B cells, CD4+T cells, CD8+T cells,
dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophil and/or neutrophils in the tissue is increased.
108. The method of embodiment 107, wherein concentration of a pro-inflammatory cytokine is increased in the
tissue.
109. The method of embodiment 108, wherein the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17,
IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1 or any combination thereof.
110. The method of any one of embodiments 106 to 109, wherein presentation of antigens originated or derived
from the diseased cells by antigen presentation cells is increased in the tissue.
111. The method of any one of embodiments 106 to 110, wherein phagocytosis of the diseased cells is
increased in the tissue.
112. The method of any one of embodiments 106 to 111, wherein apoptosis of the diseased cells induced by
cell-mediated cytotoxicity is increased in the tissue.
113. The method of any one of embodiments 106 to 112, wherein apoptosis of the diseased cells induced by
antibody-dependent cellular cytotoxicity is increased in the tissue.
114. The method of any one of embodiments 106 to 113, wherein the population of the diseased cells is
reduced in the tissue.
115. The method of embodiment 114, wherein the population of the diseased cells is reduced by about 10%,
20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%in the tissue.
116. The method of any one of embodiments 106 to 115, wherein the diseased cells are cancer cells expressing
a mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
117. The method of any one of embodiments 106 to 116, wherein the method is performed in vitro or in vivo.
118. A method for inducing an immune response against a target cell expressing a mutant KRAS in a subject,
wherein the method comprises administering to the subject the immunomodulating nucleic acid system of any one of embodiments 1-87, the polypeptide of embodiment 88 or 89, the mRNA molecule of embodiment 90, the DNA molecule of embodiment 91, the composition of any one of embodiment 92 and 95 to 96, or the combination of any one of embodiments 97-99, wherein upon the administration,
(a) the KRAS domain encoded by the nucleic acid molecules are expressed;
(b) the KRAS domain is degraded into fragments of about 9 to 15 amino acids;
(c) the KRAS domain or a degraded fragment thereof is associated with an HLA class I or HLA class II complex;
(d) innate immune response against the target cell is increased;
(e) pro-inflammatory cytokine secretion is increased;
(f) a percentage of phagocytotic macrophages in a population of macrophages is increased;
(g) a percentage of dendritic cells presenting the KRAS domain or degraded fragments thereof in a population
of dendritic cells is increased;
(h) a percentage of antigen-specific T cells specific for the KRAS domain or an epitope thereof is increased; or
(i) a percentage of B cells producing antibodies specific for the KRAS domain or an epitope thereof in a population of B cells is increased.
119. The method of embodiment 118, wherein the innate immune response is measured by
(a) activation of the NF-kB signaling pathway; or
(b) increase in secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any
combination thereof.
120. The method of embodiment 118, wherein the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17,
IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof.
121. The method of any one of embodiments 118 to 120, wherein the target cell is a diseased cell expresses a
mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
122. A method of eliminating a diseased cell expressing a mutant KRAS in a subject, comprising administering
an effective amount of the immunomodulating nucleic acid system of any one of embodiments 1-87, the polypeptide of embodiment 88 or 89, the mRNA molecule of embodiment 90, the DNA molecule of embodiment 91, the composition of any one of embodiment 92 and 95 to 96, or the combination of any one of embodiments 97-99.
123. The method of embodiment 122, wherein the subject is a human subject having the HLA subtype of HLA-
A*11: 01.
124. The method of embodiment 122 or 123, wherein the diseased cell is cancer cells expressing a mutant
KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
125. A method of treating a KRAS mutant cancer in a subject in need thereof, comprising administering to the
subject an effective amount of the immunomodulating nucleic acid system of any one of embodiments 1-87, the polypeptide of embodiment 88 or 89, the mRNA molecule of embodiment 90, the DNA molecule of embodiment 91, the composition of any one of embodiment 92 and 95 to 96, or the combination of any one of embodiments 97-99.
126. The method of embodiment 125, wherein the cancer cells express a mutant KRAS with mutations selected
from G12A, G12C, G12D, G12V, G13D, and Q61R.
127. The method of embodiment 125 or 126, wherein the cancer is selected from epithelial cancers, lung
cancers, particularly non-small cell lung cancers, colorectal cancers, and pancreatic cancers.
128. The method of any one of embodiments 100 to 127, wherein the method further comprises administering
to the subject one or more anti-cancer therapies.
129. The method of embodiment 128, wherein the one or more anti-cancer therapies are selected from a
chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery.
130. The method of embodiment 128, wherein the one or more anti-cancer therapies comprise an anti-PD-1 or
PDL1 antibody.
6. EXAMPLES

The examples in this section (i.e., Section 6) are offered by way of illustration, and not by way of limitation.
6.1 General Methods and Materials.

Preparation of mRNA Constructs

Efficiency of antigen presentation and downstream activation of target T cells can be influenced by specific molecules that regulate dendritic cell (DC) maturation and function. The purpose of this Example is to describe exemplary mRNA expression cassettes that include a KRAS domain comprising one or more mutant human KRAS peptide under the control of one or more additional elements configured for increasing efficiency of antigen presentation by DCs and thus increase T cell response. In various construct designs, the additional elements include one or more selected from a signal peptide, a degradation and/or trafficking peptide that marks the KRAS domain for processing including degradation proteosome, lysosome or exosome, an immunomodulator, a helper T cell epitope, and/or various non-cleavable linkers (e.g., Gly-Ser linker domain) or a cleavable linker (e.g., P2A linker) .

As shown in FIGS. 1A to 1D, the KRAS-001 construct contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a mutant KRAS polypeptide containing2 repeating units of tandem mutant KRAS peptides having point mutations G12C, G12V, G12D, G13D, G12A, and Q61R, respectively, and a 3’ poly-adenylation (poly-A) region (FIG. 1A) . The KRAS-002 and KRAS-003 constructs each contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein of a ubiquitin peptide fused to a mutant KRAS polypeptide via a 25 aa spacer, and a 3’ poly-adenylation (poly-A) region. The KRAS-002 and KRAS-003 both encode a mutant KRAS polypeptide containing 2 repeating units of tandem mutant KRAS peptides having point mutations G12C, G12V, G12D, G13D, G12A, and Q61R, respectively, and these constructs have different codon optimization in their respective coding regions (FIG. 1B) . The KRAS-004, KRAS-005, and KRAS-006 constructs each contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein, and a 3’ poly-adenylation (poly-A) region. The fusion protein contains a signal peptide (SP) , a mutant KRAS polypeptide, a helper T cell epitope (PADRE) , a MHC Class I trafficking peptide, a cleavable linker (p2A) and a mouse GM-CSF polypeptide. The KRAS-004, KRAS-005, and KRAS-006 constructs all encode a mutant KRAS polypeptide containing 3 repeating units of tandem mutant KRAS peptides having point mutations G12C, G12V, G12D, G13D, and G12A, respectively, and have different codon optimization in their respective coding regions (FIG. 1C) . The KRAS-007, KRAS-008, and KRAS-009 constructs each contains a 5’-Cap structure, untranslated regions located upstream (5’) and downstream (3’) of a coding region encoding a fusion protein, and a 3’ poly-adenylation (poly-A) region. The fusion protein contains a signal peptide (SP) , a mutant KRAS polypeptide, a helper T cell epitope (PADRE) , a MHC Class I trafficking peptide, a cleavable linker (p2A) and a human GM-CSF polypeptide. The KRAS-007, KRAS-008, and KRAS-009 constructs all encode a mutant KRAS polypeptide containing 3 repeating units of tandem mutant KRAS peptides having point mutations G12C, G12V, G12D, G13D, and G12A, respectively, and have different codon optimization in their respective coding regions. Exemplary sequences of the mRNA constructs are provided in Table 6.1 below.

In vitro transfection and expression detection. mRNA constructs were transfected into a target cell (e.g., cultured expi293F cells or dendritic cells) respectively with different mass gradients and using Lipo 2000 reagent, to detect the expression of encoded polypeptide. mRNA with the optimal expression was selected. The transfected cell supernatant was collected, to detect the expression of the encoded polypeptide in the supernatant can be assessed by enzyme-linked immunosorbent assay (ELISA) .

General Methods for Preparation and Characterization of Lipid Nanoparticle Formulations. The mRNA expression cassettes are packaged with lipid nanoparticles (LNPs) , thereby creating the final LNP composition used for in vitro or in vivo testing, as described below. Exemplary LNPs are described in Section 5.4.6 (Formulation) . Exemplary methods of preparation for the cationic lipids described in Section 5.4.1 (Cationic Lipids) can be found in, e.g., WO2021204175A1 , WO2023138611A1 , WO2022152109A1, and WO2022247755A1. Exemplary methods for the preparation of lipid nanoparticles described in Section 5.4.6 (Formulation) and mRNA synthesis can be found in, e.g., patent WO2023098842A1.
6.2 Example 2: Designs of mRNA encoding a KRAS polypeptide having tandem mutant
KRAS epitopes and codon optimization to enhance immunogenicity.

To evaluate activities of different KRAS polypeptide designs KRAS-001 and KRAS-002/003 and of different codon optimized variants KRAS-002 and KRAS-003 in inducing antigen-specific cellular immune response, HLA-A*11: 01 (purchased from TACONIC) transgenic mice were intramuscularly injected with PBS, a lipid nanoparticle (LNP) composition containing 20μg KRAS-001, 20μg KRAS-002 or 20μg KRAS-003 (each group 2 mice) at day 0, 7 and 14, respectively. The spleens of mice were collected and prepared into single cell suspension at day 3 after the last injection. In addition, peptide libraries of KRAS G12A, G12C, G12D, G12V, G13D, Q61R and corresponding WT peptides (all peptide libraries and WT peptides were synthesized by GenScript) were added into ELISPOT plate (Mabtech, cat#3321-4HST-2) , and then cultured for 48 hours in the incubator. After incubation, the secretion level of IFN-γ produced by KRAS mutation specific T cells were detected by ELISPOT assay.

As shown in FIG. 2, G12V, G12C, G12A and G12D fragments can induce antigen-specific T cell response in HLA-A*11: 01 transgenic mice. KRAS-002 induced stronger immune response than KRAS-003. Ubiquitin fusion design improved immunogenicity of the construct in HLA-A*11: 01 transgenic mice. However, G13D and Q61R did not reveal MHC class I mediated immunogenicity, the reason could be that these epitopes are presented by other types of HLA or even MHC class II. For example, KRAS G13D has been reported to be presented by HLA-DQB1, thus alternative models need to be applied to further verify immunogenicity of G13D and Q61R.
6.3 Example 3. Dose-dependency of antigen-specific T cell immunity induced by mRNA
encoding a mutant KRAS polypeptide.

To verify different doses of KRAS-002 can have immunogenicity in HLA-A*11: 01 transgenic mice, a lipid nanoparticle composition comprising 5μg or 20μg KRAS-002 molecule or negative control PBS were intramuscularly injected at days 0, 7 and 14, 3 mice per each treatment group. On Day 17, all spleens were collected, and single cell suspension were prepared. subsequently, cell suspension was incubated with 10μg/mL KRAS G12V (SEQ ID NO: 52) , G12D (SEQ ID NO: 36) , G12A (SEQ ID NO: 7) , G12C (SEQ ID NO: 20) and G13D (SEQ ID NO: 68) fragments pool or corresponding wild-type (WT) peptides in ELISPOT plate for 48 hours. After incubation, the specific T activation mediated IFN-γsecretion was detected by ELISPOT.

As shown in FIGS. 3A and 3B, KRAS-002 induced antigen-specific T cell response against the various mutant KRAS fragments in HLA-A*1101 transgenic mice.
6.4 Example 4. In vivo SW480-KRAS^G12V tumor growth inhibition mediated by
immunization using mRNA encoding a mutant KRAS polypeptide

To verify the effect of KRAS-002 induced G12V antigen-specific T cells activation and killing KRAS G12V mutant tumor in vivo, 5×10⁶ viable SW480-KRAS^G12Vcells (HLA-A*1101) (Kyinno, cat#KC-2831) was subcutaneously injected into each B-NDG immunodeficient mice (purchased from Biocytogen) to establish the tumor model. A lipid nanoparticle composition comprising 40μg KRAS-002 mRNA or PBS were injected intramuscularly into HLA-A*1101 transgenic mice at days 0, 7 and 14. And single cell suspension was obtained from the spleens on day 3 after the last injection. 2×10⁷ splenocytes were transplanted intravenously into SW480-KRAS^G12V tumor bearing mice. PBS control also injected group set as blank control simultaneously. Tumor size was measured periodically, and tumor growth inhibition rate (TGI) was calculated.

As shown in FIG. 4, KRAS-002 induced the generation of G12V antigen-specific T cells in HLA-A*1101 transgenic mice, and adoptive transfer immunized solenocytes led to complete tumor regression in the mouse model.
6.5 Example 5. In vivo Panc-1-KRAS^G12D tumor growth inhibition mediated by
immunization using mRNA encoding a mutant KRAS polypeptide.

To verify the effect of KRAS-002 induced G12D antigen-specific T cells on KRAS G12D mutant tumor in vivo, 5×10⁶ viable Panc-1-KRAS^G12Dcells (HLA-A*1101) was subcutaneously injected into each B-NDG immunodeficient mice (purchased from Biocytogen) to established tumor model. A lipid nanoparticle (LNP) composition containing 20μg KRAS-002 mRNA or PBS were intramuscularly immunized into HLA-A*1101 transgenic mice at days 0, 7 and 14. And single cell suspension was obtained from the spleens on days 3 after the last immunization. 2×10⁷ splenocytes were transplanted intravenously into Panc-1-KRAS^G12D tumor bearing mice. The group injected with only PBS was set as blank control. Tumor size was measured periodically, and tumor growth inhibition rate (TGI) was calculated.

As shown in FIG. 5, KRAS-002 induced generation of G12D antigen-specific T cells in HLA-A*1101 transgenic mice and adoptive transfer immunized solenocytes led to complete tumor regression in the mouse model.
6.6 Example 6. Backbone optimization of mRNA constructs encoding mutant KRAS
polypeptides

To compare activities of different codon optimized variants encoding a mutant KRAS polypeptide, we evaluated by the proteins expressed on expi293F cells. Recovered expi293F cells were cultured for 3 passages and transfected different doses of mRNA candidates (KRAS-004/005/006, KRAS-007/008/009) with lipofectamine 2000 into expi293F cells for 24 hrs. Collected cellular supernatant and measured the mouse GM-CSF by mGM-CSF Elisa kit (R&D, cat#DY415-05) and human GM-CSF by hGM-CSF Elisa kit (Thermofisher, cat#88-8337-88) .

As shown in FIGS. 6A and 6B, KRAS-004 expressed more mGM-CSF as compared to KRAS-005/006, and KRAS-007 expressed more hGM-CSF as compared to KRAS-008/009. Furthermore, both 1 μg and 5μg doses resulted in high expression levels of the GM-CSF protein encoded by the mRNA constructs.
6.7 Example 7. Immunogenicity evaluation of mutant KRAS polypeptides and mRNA
encoding mutant KRAS polypeptides in vitro

The immunogenicity of mRNA constructs encoding mutant KRAS polypeptides was measured by IFN-γElispot after up to two weeks of in vitro stimulation (IVS) . On day 0, monocyte-derived dendritic cells generated from healthy donor’s peripheral blood mononuclear cells (PBMC) , 5 x 10⁷ PBMC were plated in 6-well tissue culture plate at a density of 1x10⁷cells per well in 3ml AIM-V medium supplemented with 1%heat-inactivated human AB serum (AIM-V/1%AB) for 4 hours. Then non-adherent cells were removed by washing and cryopreserved, adherent cells were further cultured for 6 days in medium with 1000U/ml GM-CSF and 500U/ml IL4. On Day 5, cryopreserved non-adherent cells were recovered in RPMI 1640 with 10%human AB serum as effector cells for in vitro stimulation. On day 6, immature dendritic cells (iDC) were harvest and washed twice in serum-free AIM-V medium, resuspended cells at a density of 5x10⁶ cells/ml and mixed with 4ug mutant KRAS mRNA construct (KRAS-003) in a final volume of 20ul, then transferred to Amaxa cuvette for immediate electroporation using Lonza Amaxa 4D-Nucleofector system and run Program DJ108. Cells mixed with mock or mRNA encoding GFP were treated in a similar fashion and were included as negative and positive controls, respectively. After transfection, cells were resuspended at in AIM-V/1%AB medium with 1000U/ml GM-CSF, 200U/ml IL4, 1000U/ml IL6, 10ng/ml IL-1β, 10ng/ml TNFαand 10ug/ml PEG2 for 24 hours. On Day 7, for in vitro stimulation, effector cells were co-cultured with KRAS mRNA-electroporated mature DC for 14 days in presence of 50U/ml IL2, 10ng/ml IL7 and 10ng/ml IL21. On day 21, human IFNγ-Elispot Kit (Mabtech) was used to determine the frequency of cytokine-secreting T cells after IVS.

Effector cells were harvested to co-culture with mature DCs pulsed with 10ug/ml mutant KRAS G12V, G12C, G12D, G12V fragments, individually and added to duplicate wells for 18-20 hours. The plates were washed before the addition of the diluted detection antibody (1: 1000 dilution) and then incubated at room temperature (RT) for 1hour. After washing of the plates, streptavidin-HRP (1: 1000dulution) was added and incubated at RT for another 1 hours. TMB solution was added to each well and the plates were left in the dark for about 15-25 minutes at RT before deionized water was added to stop development. Plates were scanned by ELISPOT CTL Reader (Cellular Technology Inc. ) , and the results were analyzed with Elispot Software. Spots greater than twice the no-peptide control were considered positive for T cell reactivity. As shown in FIG. 7, both dendritic cells treated with mutant KRAS peptides and dendritic cells transfected with mutant KRAS encoding mRNA were able to activate effector cells (antigen specific T cells) . These data have confirmed the immunogenicity of mutated KRAS epitopes, and also cross-validated by using both methods.
6.8 Example 8. In vivo comparison of immunogenicity between old and new backbone KRAS
mRNA

To compare the immunogenicity between KRAS-002 and KRAS-004, HLA-A*1101 transgenic mice were intramuscularly injected with PBS and 20μg mRNA vaccine at day 0, 7 and 14. The spleen of mice was collected and prepared into single cell suspension at day 17. The peptide libraries of G12V (SEQ ID NO: 52) , G12D (SEQ ID NO: 36) , G12A (SEQ ID NO: 7) , G12C (SEQ ID NO: 20) and WT peptides were added into ELISPOT plate, and then cultured for 48 hours in the incubator. After incubation, the secretion level of IFN-γ induced by each antigen-specific T cells were detected by ELISPOT.

As shown in FIG. 8, KRAS-004 induced stronger antigen-specific T cell activation than KRAS-002. These data demonstrate that the KRAS-004 molecule is superior to the KRAS-002 molecule.
6.9 Example 9. Immunogenicity evaluation of unmodified and modified mRNA encoding
mutant KRAS polypeptides in vitro and in vivo

According to the results in FIG. 6A, KRAS-004 was selected to compare the GM-CSF expression in vitro and immunogenicity in vivo among unmodified, ψ-modified and M1-ψ-modified mRNAs. Recovered expi293F cells were mixed with 10μg different mRNAs, then transfected by using Lonza 4D-Nucleofector. After electroporation, cells were transferred to medium with 10%FBS for culture 24 hours. Collected cellular supernatant and measured the GM-CSF section by mGM-CSF Elisa kit (R&D, cat#DY415-05) . To determine which modification could induce the best immunogenicity, 20μg un-modified KRAS-004, KRAS-004-ψ and KRAS-004-M1-ψ mRNA vaccines were immunized HLA-A*1101 transgenic mice three times (d0, d7, d14) by intramuscular injection (i. m. ) . At day 17, all mice were sacrificed, and the spleens were collected for single cell suspension preparation. The cell suspension was stimulated by 5μg/mL G12V (SEQ ID NO: 52) , G12D (SEQ ID NO: 36) , G12A (SEQ ID NO: 7) , G12C (SEQ ID NO: 20) and G13D (SEQ ID NO: 68) fragments pool or corresponding wild-type (WT) peptides in ELISPOT plates for 48 hours. Then antigen-specific T cells response was measured by IFN-γ secretion.

As shown in FIG. 9 and FIG. 10, unmodified KRAS-004 showed the better immunogenicity than the other two modified molecules.
6.10 Example 10. Comparison of KRAS-004 and BMK for immunogenicity and efficacy in
vitro and in vivo

To verification the effect of KRAS-004 induced KRAS G12V antigen-specific T cells on G12V mutant tumor in vivo, 5E6 SW480-KRAS^G12V (HLA-A*1101) tumor cells were injected into M-NSG immunodeficient mice (SHANGHAI MODEL ORGANISIMS) to establish tumor-bearing model. 20μg KRAS-004, 20μg BMK and PBS were intramuscularly immunized HLA-A*1101 transgenic mice at days 0, 7 and 14. Single cell suspension was obtained from the spleen on days 3 after the last immunization. Three mice splenocytes from each group were randomly selected for immunogenicity detection in vitro. The rest cells of same group were mixed well, and 2E7 splenocytes were transplanted intravenously into tumor bearing M-NSG mice. Tumor size was measuredperiodically, and tumor growth inhibition rate (TGI) was calculated.

As shown in FIG. 11A, 20μg KRAS-004 showed strong immunogenicity than 20μg BMK. And in FIG. 11B, 20μg KRAS-004 can completely inhibit tumor growth (CR rate: 4/5) , while BMK can only inhibit tumor growth a little (CR rate: 0/5) . In summary, KRAS-004 is superior to BMK in both immunogenicity and efficacy.
6.11 Example 11. KRAS-004 and mPD-1 showed synergistic effect in SW480-KRAS^G12V
tumor model in vivo.

50mm³ SW480-KRAS^G12V (HLA-A*1101) tumor volume was used to evaluate the synergistic effect of KARS-004 with PD-1 blocking antibody. 10mpk mPD-1 antibody was injected during the second dose of KRAS-004 or PBS immunized HLA-A*1101 mice, twice a week. At 17 days, the mice were sacrificed to prepare splenocytes. The same group of cells were mixed and adopted into tumor bearing M-NSG mice according to the number of cells in 1E7. The inhibition of tumor volume was recorded.

As shown in FIG. 12and the Table 6.11 below, compared with PBS combo with mPD-1 group, KRAS-004 combo mPD-1 group showed 100%complete remission rate. While KRAS-004 group showed 40%CR rate, KRAS-004 combo mPD-1 group have a significant synergistic effect.
Table 6.11

6.12 Example 12. KRAS-007 induced KRAS antigen specific immunogenicity in PBMC.

To verify the KRAS-007 could induce immunogenicity in PBMC, we isolated CD14⁺monocytes from PBMC and incubated with different cytokines to obtained immature DCs (iDCs) . Then electroporation was carried out using the Lonza 4D-Nucleofector X-unit system. Finally, isolated CD14^-cells (mainly T cells) were incubated with iDCs for 24 hours with peptide pools together and assessed the specific IFN-γ secretion by ELISpot kit.

As shown in FIG. 13, the result shows that KRAS-007 can induce KRAS tumor antigen and PADRE specific immune response in PBMC.
6.13 Example 13. KRAS-007 induced specific T cell killing towards KRAS G13D tumor
positive tumor cell line in vitro.

The fact that the antigen-specific T cells can kill tumor antigen-positive tumor cells is critical to whether tumor vaccines can exert anti-tumor efficacy. We designed an in vitro specific T cell killing experiment to demonstrate. The CD14⁺monocytes isolated from PBMCs from different individuals were induced into immature DCs, and KRAS-007 was electroporated into iDCs, which were then co-cultured with CD14 negative cells (mainly T cells) to induce and activate specific T cells. At the same time, iDCs electroporated with only culture medium and T cells were incubated as non-specific control groups. After 10 days of induction, T cells were used as effector cells and CFSE-labeled HCT-116 KRAS-G13D tumor cells were used as target cells. They were added to 96-well plates at a 10: 1 effector-target ratio for specific killing. After 3 days, the proportion of HCT-116 live cells was detected.

As shown in FIG. 14, the results proved that this group of induced specific T cells can indeed kill tumor antigen-positive tumor cells, indicating that KRAS-007 has specific anti-tumor efficacy.
6.14 Example 14. KRAS-004 elicited mouse GM-CSF in HLA-A11 Tg mice serum.

To investigate the effect of KRAS-004 on mouse GM-CSF production in immunized mice, we immunized HLA-A*1101 transgenic mice with 20μg KRAS-004 vaccine. Serum samples were collected 6 hours after the 1^stimmunization to evaluate mouse GM-CSF release levels using the Legendplex kit. Changes in GM-CSF levels were also monitored.

As shown in FIG. 15, compared with PBS control, KRAS-004 can effectively elicite mouse GM-CSF in serum. Additionally, a dose-dependent increase in GM-CSF was observed which could originate from two sources: first, the expression of soluble GM-CSF encoded by KRAS-004; second, the elevation of endogenous GM-CSF induced by KRAS-004.
6.15 Example 15. KRAS-004 induced changes in multiple innate immunity-related cytokines
in mouse serum.

To study the changes in cytokine levels following vaccination with the KRAS-004 vaccine, serum samples of HLA-A*1101 transgenic mice were collected 6 h after the first intramuscular injection of 20μg KRAS-004 vaccine, and PBS using as a negative control.

As shown in FIG. 16, the results suggested that KRAS-004 triggered a robust immune activation in HLA-A11 transgenic mice, as evidenced various cytokines, including IFN-α, IFN-β, IFN-γand IL-6. These cytokines are typically associated with innate immune responses and the early phase of immune activation, particularly in response to pathogen-associated molecular patterns (PAMPs) , such as mRNA.
6.16 Example 16. KRAS-004 induced HLA-A11 Tg mouse splenocytes to specifically kill
KRAS G12A/G12C/G12D/G12V tumor cells.

To evaluate whether KRAS-004 induced specific T cells were capable of killing KRAS mutant tumor cells, HLA-A*1101 transgenic mice were intramuscularly immunized on days 0, 7, and 14 with 10μg KRAS-004 vaccine or a negative control (PBS) . 3 days after the final injection, single-cell suspensions were prepared from mouse spleen to serve as effector cells. HUH1-G12A (HLA-A*1101) , H2030-G12C (HLA-A*1101) , SW480-G12V (HLA-A*1101) and Panc-1-G12D (HLA-A*1101) tumor cell lines were labeled with CFSE as target cells and co-incubated with effector cells at 100: 1 E: T ratio. After 72 h, tumor cells viability was measured.

As shown in FIG. 17, the analysis revealed that splenocytes from KRAS-004 immunized mice exhibited antigen-specific T cell-mediated anti-tumor activity ex vivo.
6.17 Example 17. Enhancement of KRAS antigen immunogenicity by backbone elements.

To demonstrate the enhancement of KRAS antigen immunogenicity by backbone elements, the different arrangements and combinations of elements mRNA/LNP were designed to intramuscular injected HLAA*1101 transgenic mice three times (d0, d7, d14) , 5 mice each group. At day 17, all mice were sacrificed and the spleens were collected for single cell suspension preparation. The cell suspension was stimulated by the peptide libraries of G12A, G12C, G12D, G13D, WT and PADRE peptide for 24 hours in ELISPOT plates. Then antigen-specific T cells response were measured by IFN-γ secretion.

Table 6.17 Exemplary sequences.

SP: signal peptide; Ag: antigen

At the same dose, ABO-24 (PADRE+GM-CSF) immunized mice exhibited superior KRAS G12A and G12C specific immunogenicity to ABO-25 (PADRE+MITD) and ABO-26 (GM-CSF+MITD) in splenocytes, and also induced the strongest immunogenicity compared with other elements of the vaccines when stimulated by PADRE peptide.

In another experiment demonstrating the immunogenicity induced by combinations of different backbone elements in PBMC cells, we isolated CD14⁺monocytes from PBMC and incubated with different cytokines to obtained immature DCs. Then electroporation was carried out using the Lonza 4D-Nucleofector X-unit system. Finally, isolated CD14^-cells (mainly T cells) were incubated with iDCs for 24 hours with peptide pools together and assessed the specific IFN-γ secretion by ELISpot kit.

All references mentioned in the present invention are incorporated herein by reference as if each of those references has been incorporated by reference individually. Although the description referred to particular embodiments, it will be clear to a person skilled in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set forth herein.
7. SEQUENCE CHART

Claims

An immunomodulating nucleic acid system encoding a KRAS domain, wherein the system comprises a first coding sequence encoding a polypeptide comprising a helper T cell epitope, wherein the helper T cell epitope is configured to enhance immunogenicity of the KRAS domain upon co-expression with the KRAS domain, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
The immunomodulating nucleic acid system of claim 1, wherein the system further comprises a second coding sequence encoding a polypeptide comprising an immunomodulator, wherein the immunomodulator is configured to enhance immunogenicity of the KRAS domain upon co-expression with the KRAS domain.
The immunomodulating nucleic acid system of claim 1 or 2, wherein the system further comprises a third coding sequence encoding a polypeptide comprising a trafficking peptide configured for intracellularly trafficking the target antigen towards proteosome upon expression.
The immunomodulating nucleic acid system of any one of claims 1 to 3, wherein the system further comprises a fourth coding sequence encoding a polypeptide comprising a signal peptide.
The immunomodulating nucleic acid system of any one of claims 1 to 4, wherein the system further comprises a fifth coding sequence encoding a polypeptide comprising a degradation domain, optionally wherein the degradation domain comprises a ubiquitin peptide.
The immunomodulating nucleic acid system of any one of claims 1 to 5, wherein

(a) the first coding sequence encodes a polypeptide comprising the helper T cell epitope and the KRAS domain;

(b) the second coding sequence encodes a polypeptide comprising the immunomodulator and the KRAS domain; optionally wherein the immunomodulator is positioned N-terminal to the KRAS domain or is positioned C-terminal to the KRAS domain in the polypeptide;

(c) the third coding sequence encodes a polypeptide comprising the trafficking peptide and the KRAS domain; optionally wherein the trafficking peptide is positioned C-terminal to the KRAS domain in the polypeptide;

(d) the fourth coding sequence encodes a polypeptide comprising the signal peptide and the KRAS domain; optionally wherein the signal peptide is positioned N-terminal to the KRAS domain in the polypeptide; and/or

(e) the fifth coding sequence encodes a polypeptide comprising the ubiquitin peptide and the KRAS domain; optionally wherein the ubiquitin peptide is positioned N-terminal to the KRAS domain in the polypeptide; optionally wherein polypeptide further comprises a spacer peptide positioned in between the ubiquitin peptide and the KRAS domain in the polypeptide; optionally the spacer peptide is at least about 25 amino acids in length.
The immunomodulating nucleic acid system of any one of claims 1 to 6, wherein:

(a) the first coding sequenc is in a first nucleic acid molecule;

(b) at least one of the first coding sequence and the second coding sequence is in a first nucleic acid molecule;

(c) at least one of the first coding sequence, the second coding sequence and the third coding sequence is in a first nucleic acid molecule;

(d) at least one of the first coding sequence, the second coding sequence, the third coding sequence and the fourth coding sequence is in a first nucleic acid molecule; or

(e) at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences is in a first nucleic acid molecule.
The immunomodulating nucleic acid system of claim 7, wherein:

(a) the second coding sequence is in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulatory and the KRAS domain;

(b) the first coding sequence and the second coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator and the KRAS domain;

(c) the first coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the trafficking peptide and the KRAS domain;

(d) the first coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the signal peptide and the KRAS domain;

(e) the first coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the ubiquitin peptide and the KRAS domain;

(f) the second coding sequence and the third coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide and the KRAS domain;

(g) the second coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the signal peptide, and the KRAS domain;

(h) the second coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the ubiquitin peptide and the KRAS domain;

(i) the third coding sequence and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the signal peptide and the KRAS domain;

(j) the third coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the ubiquitin peptide and the KRAS domain;

(k) the fourth coding sequence and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the signal peptide, the ubiquitin peptide and the KRAS domain;

(l) the first coding sequence, the second coding sequence, and the third coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, and the KRAS domain;

(m) the first coding sequence, the second coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the signal peptide, and the KRAS domain;

(n) the first coding sequence, the second coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the ubiquitin peptide, and the KRAS domain;

(o) the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide, the signal peptide, and the KRAS domain;

(p) the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the KRAS domain;

(q) the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the trafficking peptide, the signal peptide, the ubiquitin peptide, and the KRAS domain;

(r) the first coding sequence, the second coding sequence, the third coding sequence, and the fourth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the KRAS domain;

(s) the first coding sequence, the second coding sequence, the third coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the ubiquitin peptide, and the KRAS domain; or

(t) the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence, and the fifth coding sequence are in the first nucleic acid molecule, and wherein the polypeptide comprises the helper T cell epitope, the immunomodulator, the trafficking peptide, the signal peptide, and the ubiquitin peptide, and the KRAS domain.
The immunomodulating nucleic acid system of claim 7, wherein at least one of the first coding sequence, the second coding sequence, the third coding sequence, the fourth coding sequence and the fifth coding sequences is in a second nucleic acid molecule, and wherein the first nucleic acid molecule and the second nucleic acid molecule are different molecules;

optionally wherein the second nucleic acid molecule does not encode the KRAS domain;

optionally wherein the first nucleic acid molecule encodes the polypeptide comprising the KRAS domain, wherein the polypeptide does not comprise the immunomodulator, and wherein the second nucleic acid molecule comprises the second coding sequence encoding the immunomodulator.
The immunomodulating nucleic acid system of any one of claims 8 to 9, wherein at least the second coding sequence is in the first nucleic acid molecule encoding the polypeptide comprising at least the immunomodulator and the KRAS domain; and wherein the first nucleic acid molecule further comprises a means for producing the immunomodulator and the KRAS domain as separate proteins or peptides.
The immunomodulating nucleic acid system of claim 10, wherein the means for producing the immunomodulator and the KRAS domain as separate proteins or peptides comprises a sixth coding sequence encoding a cleavable linker in between the second coding sequence and a coding sequence for the KRAS domain in the first nucleic acid; optionally wherein the cleavable linker is a 2A peptide, selected from the group consisting of P2A, F2A, T2A, and E2A; optionally wherein the cleavable linker consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 105.
The immunomodulating nucleic acid system of any one of claims 1 to 11, wherein the helper T cell epitope is a universal CD4 epitope;

wherein the helper T cell epitope is an epitope of tetanus and diphtheria toxoids; or

wherein the helper T cell epitope comprises one or more epitopes selected from the group consisting of P2, P16, P2P16, P65, TT_827-841, pDT_331-350, TT_632-651, and PADRE; optionally wherein the helper T cell epitope consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 94.
The immunomodulating nucleic acid system of any one of claims 1 to 12, wherein the first coding sequence encoding the helper T cell epitope comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 95-98 and 186; or

wherein the first coding sequence encodes the helper T cell epitope that is a pan DR-binding epitope (PADRE) , and wherein the first coding sequence comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence of SEQ ID NO: 98 or 186.
The immunomodulating nucleic acid system of any one of claims 2 to 13, wherein the immunomodulator is selected from the group consisting of GM-CSF, STING, FLT3L, c-FLIP, ΙKKβ, RIPKl, Btk, TAKl, TAK-TAB l, TBKl, MyD88, IRAKI, IRAK2, IRAK4, TAB2, TAB 3, TRAF6, TRAM, MKK3, MKK4, MKK6, type 1 IFN, and any combination thereof; or

wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING; or

wherein (a) the first coding sequence encodes a polypeptide comprising the KRAS domain and a helper T cell epitope, and (b) the second coding sequence encodes an immunomodulator, wherein the immunomodulator comprises GM-CSF, STING, or both GM-CSF and STING.
The isolated nucleic acid of claim 14, wherein the GM-CSF is human GM-CSF (hGM-CSF) or mouse GM-CSF (mGM-CSF) ;

optionanlly wherein the immunomodulator consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268;

optionally wherein the GM-CSF is a full-length GM-CSF polypeptide;

optionally wherein the GM-CSF comprises a truncated GM-CSF or a mutant GM-CSF comprising an amino acid sequence comprising one or more variations compared to the amino acid sequence set forth in SEQ ID NO: 111, 115, 264 or 268, and wherein the truncated GM-CSF or mutant GM-CSF is capable of stimulating macrophage differentiation and proliferation, and/or activating antigen presenting cells (APCs) .
The isolated nucleic acid of claim 14, wherein the STING is human STING (hSTING) (V155M) ; optionally wherein the STING comprises a polypeptide comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the amino acid sequence of SEQ ID NO: 220 or 253.
The isolated nucleic acid of any one of claims 14 to 16, wherein the second coding sequence encoding the immunomodulator comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 112-114, 215-217, 116-118, 218-219, 221, 254, 265, and 269-271.
The immunomodulating nucleic acid system of any one of claims 3 to 17, wherein the trafficking peptide is derived from one or more polypeptides selected from the group consisting of MHC class I, CD1b, CD1c, CD1d, HLA-E, HLA-DMB, LAMP1, LAMP-2a, LAMP3, CD63, GMP-17, Cystinosin, CTLA-4, CD4, NPC1, CIMPR, LRP3, Furin, VAMP4, VMAT1, VMAT2, PAM, CPD, PC7, Beta-secretase, Sortilin, GLUT4, TRP-1, LDL receptor, LRP1, Megalin, Integrin beta-1, APLP1, APP, Insulin receptor, and EGF receptor.
The immunomodulating nucleic acid system of any one of claims 3 to 18, wherein the trafficking peptide is MHC class I trafficking peptide or LAMP3 transmembrane domain (LAMP3 TM) ; optionally wherein the trafficking peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 99; optionally wherein the third coding sequence encoding the trafficking peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 100-104 and 198-202.
The immunomodulating nucleic acid system of any one of claims 4 to 19, wherein the fourth coding sequence encoding the signal peptide comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%or more identity to the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 90-93 and 171-175; optionally wherein the signal peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 89 or 170.
The immunomodulating nucleic acid system of any one of claims 5 to 20, wherein the ubiquitin peptide comprises a ubiquitin monomer, a ubiquitin multimer, a ubiquitin like domain (ULD) , or a functional derivative thereof; optionally wherein the ubiquitin peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; optionally wherein the fifth coding sequence encoding the ubiquitin peptide comprises the nucleic acid sequence set forth in SEQ ID NOS: 2 and 3, or a codon optimized variant thereof.
The immunomodulating nucleic acid system of claim 21, wherein the ubiquitin peptide further comprises a spacer located between the ubiquitin peptide and the KRAS domain, and wherein the spacer peptide consists of, essentially consists of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence set forth in SEQ ID NO: 4.
The immunomodulating nucleic acid system of any one of claims 7 to 22, wherein the first nucleic acid molecule is DNA or RNA; and/or wherein the second nucleic acid molecule is DNA or RNA; optionally wherein the RNA is mRNA, self-amplifying RNA, or circular RNA.
The immunomodulating nucleic acid system of any one of claims 1 to 23, wherein the one or more coding sequences are codon optimized; and/or wherein the one or more coding sequences are in one or more mRNA molecule, optionally wherein the mRNA molecule further comprises a 5’ untranslated region (UTR) , a3’ UTR, or both a 5’ UTR and a 3’ UTR, optionally wherein the mRNA molecule further comprises a 5’ Cap, optionally wherein the mRNA molecule further comprises a poly (A) sequence.
The immunomodulating nucleic acid system of claim 24, wherein the 5’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 136, 251 or 275; and/or wherein the 3’ UTR comprises the nucleic acid sequence set forth in SEQ ID NO: 137 or 252; and/or wherein the poly (A) sequence has a length of about 50 nucleotides or longer.
The immunomodulating nucleic acid system of any one of claims 7 to 25, wherein the first nucleic acid molecule comprises a chemical modification; and/or wherein the second nucleic acid molecule comprises a chemical modification; optionally wherein the chemical modification comprises one or more functional nucleotide analogs that are selected from pseudouridine, 1-methyl-pseudouridine and 5-methylcytosine.
The immunomodulating nucleic acid system of any one of claims 7 to 26, wherein the first nucleic acid molecule is in a first vector; and/or wherein the second nucleic acid molecule is in a second vector.
An immunomodulating nucleic acid system encoding a polypeptide comprising, in the N-to-C direction, a signal peptide, a KRAS domain, a helper T cell epitope, a trafficking peptide, acleavable linker and an immunomodulator, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R.
The immunomodulating nucleic acid system of claim 28, wherein the KRAS domain comprises the amino acid sequence set forth in SEQ ID NO: 138, 161, 260, or 409; and/or wherein the trafficking peptide comprises the amino acid sequence set forth in SEQ ID NO: 99; and/or wherein the helper T cell epitope comprises the amino acid sequence set forth in SEQ ID NO: 94; and/or wherein the immunomodulator comprises the amino acid sequence set forth in SEQ ID NO: 111, 115, 334 or 338; and/or wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 89.
The immunomodulating nucleic acid system of any one of claims 1-29, wherein the KRAS domain comprises at least one mutated fragment of human KRAS, and wherein each mutated fragment comprises one or more KRAS mutation selected from any one of (a) to (r) :

(a) G12A;

(b) G12C;

(c) G12D;

(d) G12V;

(e) G13D;

(f) Q61R;

(g) G12A and G13D;

(h) G12C and G13D;

(i) G12D and G13D;

(j) G12V and G13D;

(k) G12A and Q61R;

(l) G12C and Q61R;

(m) G12D and Q61R;

(n) G12V and Q61R;

(o) G12A, G13D, and Q61R;

(p) G12C, G13D, and Q61R;

(q) G12D, G13D, and Q61R; and

(r) G12V, G13D, and Q61R.
The immunomodulating nucleic acid system of any one of claims 1-30, wherein

(a) the at least one mutated fragment of human KRAS is at least about 10 amino acids in length; optionally wherein the mutated fragment of human KRAS is about 10 amino acids, 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, or about 35 amino acids in length; and/or

(b) wherein the at least one mutated fragment of human KRAS comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different; and/or

(c) wherein the KRAS domain comprises two or more mutated fragments of human KRAS, and wherein at least two of the two or more mutated fragments are fused directly or through a linker peptide.
The immunomodulating nucleic acid system of any one of claims 1-31, wherein the at least one mutated fragment of human KRAS comprises a G12A fragment, wherein the G12A fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12A mutation; optionally wherein the KRAS domain comprises two or more identical copies of the G12A fragment; optionally wherein the G12A fragment has the amino acid sequence of SEQ ID NO: 7.
The immunomodulating nucleic acid system of any one of claims 1-32, wherein the at least one mutated fragment of human KRAS comprises a G12C fragment, wherein the G12C fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12C mutation; optionally wherein the KRAS domain comprises two or more identical copies of the G12C fragment; optionally wherein the G12C fragment has the amino acid sequence of SEQ ID NO: 20.
The immunomodulating nucleic acid system of any one of claims 1-33, wherein the at least one mutated fragment of human KRAS comprises an G12D fragment, wherein the G12D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12D mutation; optionally wherein the KRAS domain comprises two or more identical copies of the G12D fragment; optionally wherein the G12D fragment has the amino acid sequence of SEQ ID NO: 36.
The immunomodulating nucleic acid system of any one of claims 1-34, wherein the at least one mutated fragment of human KRAS comprises a G12V fragment, wherein the G12V fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G12V mutation; optionally wherein the KRAS domain comprises two or more identical copies of the G12V fragment; optionally wherein the G12V fragment has the amino acid sequence of SEQ ID NO: 52.
The immunomodulating nucleic acid system of any one of claims 1-35, wherein the at least one mutated fragment of human KRAS comprises a G13D fragment, wherein the G13D fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the G13D mutation; optionally wherein the KRAS domain comprises two or more identical copies of the G13D fragment; optionally wherein the G13D fragment has the amino acid sequence of SEQ ID NO: 68.
The immunomodulating nucleic acid system of any one of claims 1-36, wherein the at least one mutated fragment of human KRAS comprises a Q61R fragment, wherein the Q61R fragment is a fragment of about 15 to 30 continuous amino acids of human KRAS that comprises the Q61R mutation; optionally wherein the KRAS domain comprises two or more identical copies of the Q61R fragment; optionally wherein the Q61R fragment has the amino acid sequence of SEQ ID NO: 83.
The immunomodulating nucleic acid system of any one of claims 1-37, wherein the KRAS domain comprises:

(a) two of the G12A fragments, two of the G12C fragments, two of the G12D fragments, two of the G12V fragments, two of the G13D fragments, and two of the Q61R fragments,

optionally wherein the KRAS domain comprises two repeating units each comprising in the N-to-C direction: the G12C fragment, the G12V fragment, the G12D fragment, the G13D fragment, the G12A fragment, and the Q61R fragment;

optionally wherein each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7, optionally wherein each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20, optionally wherein each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36, optionally wherein each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52; optionally wherein each of the G13D fragment has the amino acid sequence of SEQ ID NO: 68; optionally wherein each of the Q61R fragments has the amino acid sequence of SEQ ID NO: 83;

or

(b) three of the G12A fragments, three of the G12C fragments, three of the G12D fragments, three of the G12V fragments, and three of the G13D fragments;

optionally wherein the KRAS domain comprises three repeating units each comprising in the N-to-C direction: a copy of the G12C fragment, a copy of the G12V fragment, a copy of the G12D fragment, a copy of the G13D fragment and a copy of the G12A fragment;

optionally wherein each of the G12A fragments has the amino acid sequence of SEQ ID NO: 7, optionally wherein each of the G12C fragments has the amino acid sequence of SEQ ID NO: 20, optionally wherein each of the G12D fragments has the amino acid sequence of SEQ ID NO: 36, optionally wherein each of the G12V fragments has the amino acid sequence of SEQ ID NO: 52; optionally wherein each of the G13D fragment has the amino acid sequence of SEQ ID NO: 68.
The immunomodulating nucleic acid system of any one of claims 1-38, wherein the at least one mutated fragment of human KRAS is encoded by a nucleic acid sequence selected from the sequences set forth in SEQ ID NOS: 8 to 19, 21 to 35, 37 to 51, 53 to 67, 69 to 82, 84 to 88, 160, 162 to 167, 261, or a codon optimized variant thereof.
The immunomodulating nucleic acid system of any one of claims 1-39, comprising a plurality of species of nucleic acid molecules each encoding an KRAS domain, wherein each encoded KRAS domain comprises at least one mutated fragment of human KRAS, wherein the mutated fragment comprises one or more KRAS mutation selected from G12A, G12C, G12D, G12V, G13D and Q61R, and wherein the KRAS domains encoded by at least two of the plurality of species of nucleic acid molecules are different.
The immunomodulating nucleic acid system of any one of claims 1-40, wherein the KRAS domain encoded by at least one of the plurality of species of nucleic acid molecules comprises two or more mutated fragments of human KRAS, and wherein the two or more mutated fragments are identical or different.
A polypeptide encoded by the immunomodulating nucleic acid system of any one of claims 1-41.
A polypeptide consists of, essentially consists of, or comprises the amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to SEQ ID NOS: 138, 141, 146, 153, 404, 406 or408.
A non-naturally occurring nucleic acid comprising the nucleotide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to the nucleotide sequence set forth in SEQ ID NOs: 139, 142, 144, 147, 149, 151, 154, 156, or 158, optionally the nucleic acid comprises one or more functional nucleotide analogs.
A non-naturally occurring nucleic acid comprising the nucleotide sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to SEQ ID NOs: 140, 143, 145, 148, 150, 152, 155, 157, or 159, or a transcribed RNA sequence thereof.
A composition comprising a plurality of species of polypeptides, wherein each species of polypeptide is selected from the polypeptides encoded by the immunomodulating nucleic acid system of any one of claims 1-41, wherein at least two of the plurality of species of polypeptides are different.
A vector comprising the immunomodulating nucleic acid system of any one of claims 1-41.
A set of vectors comprising two or more coding sequences of the immunomodulating nucleic acid system of any one of claims 1-41 in separate nucleid acid molecules.
A composition comprising the immunomodulating nucleic acid system of any one of claims 1-41, and at least one lipid.
The composition of claim 49, wherein the composition is formulated as lipid nanoparticles encompassing the immunomodulating nucleic acid system.
A combination of (a) the immunomodulating nucleic acid system of any one of claims 1-41, and (b) one or more anti-cancer therapies.
The combination of claim 51, wherein the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery; optionally wherein the one or more anti-cancer therapies comprise an anti-PD-1 or PDL1 antibody.
A method of activating KRAS-mutant specific immune effector cells, comprising providing a starting population of immune effector cells, and contacting the starting population of immune effector cells with the immunomodulating nucleic acid system of any one of claims 1-41, the polypeptide of claim 42 or43, the non-naturally occurring nucleic acid of claim 44 or 45, the composition of any one of claim 46 and 49 to 50, or the combination of any one of claims 51-52 under a suitable condition, wherein upon the contacting, KRAS-mutant specific immune effector cells are activated.
The method of claim 53, wherein the contacting is performed in the presence of a proteosome system, and wherein the proteosome system degrades the KRAS domain into one or more peptides of about 9 to 15 amino acids in length; and/or

wherein the contacting is performed in the presence of antigen presenting cells (APCs) , wherein the APCs present the KRAS domain or a degraded portion thereof on their surface; optionally wherein the APCs are selected from dendritic cells, macrophages, Langerhans cells and B cells; optionally wherein the APCs express HLA class I and/or HLA class II molecules; optionally wherein the HLA class I molecule is of the HLA-A*11: 01 subtype.
The method of any one of claims 53 to 54, wherein the immune effector cells are selected from T cells, peripheral blood lymphocytes, and natural killer cells; optionally wherein the T cells comprises CD4+T cells, CD8+T cells, antigen-specific T cells, tumor infiltration T cells, γδT cells, cytotoxic T cells, natural killer T cells, and mucosal associated invariant T cells (MAIT) .
A method of forming a pro-inflammatory milieu in a tissue surrounding a population of diseased cells expressing a mutant KRAS, comprising contacting the tissue with an effective amount of the immunomodulating nucleic acid system of any one of claims 1-41, the polypeptide of claim 42 or 43, the non-naturally occurring nucleic acid of claim 44 or 45, the composition of any one of claim 46 and 49 to 50, or the combination of any one of claims 51-52 under a suitable condition.
The method of claim 56, wherein infiltration of activated B cells, CD4+T cells, CD8+T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophil and/or neutrophils in the tissue is increased; optionally wherein concentration of a pro-inflammatory cytokine is increased in the tissue; optionally wherein the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, IP-10, CXCL1, MCP-1 or any combination thereof.
The method of any one of claims 56 to 57, wherein presentation of antigens originated or derived from the diseased cells by antigen presentation cells is increased in the tissue; and/or wherein phagocytosis of the diseased cells is increased in the tissue; and/or wherein apoptosis of the diseased cells induced by cell-mediated cytotoxicity is increased in the tissue; and/or wherein apoptosis of the diseased cells induced by antibody-dependent cellular cytotoxicity is increased in the tissue; and/or wherein the population of the diseased cells is reduced in the tissue, optionally wherein the population of the diseased cells is reduced by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%in the tissue; and/or wherein the diseased cells are cancer cells expressing a mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R; and/or wherein the method is performed in vitro or in vivo.
A method for inducing an immune response against a target cell expressing a mutant KRAS in a subject, wherein the method comprises administering to the subject the immunomodulating nucleic acid system of any one of claims 1-41, the polypeptide of claim 42 or 43, the non-naturally occurring nucleic acid of claim 44 or 45, the composition of any one of claim 46 and 49 to 50, or the combination of any one of claims 51-52, wherein upon the administration,

(a) the KRAS domain encoded by the nucleic acid molecules are expressed;

(b) the KRAS domain is degraded into fragments of about 9 to 15 amino acids;

(c) the KRAS domain or a degraded fragment thereof is associated with an HLA class I or HLA class II complex;

(d) innate immune response against the target cell is increased;

(e) pro-inflammatory cytokine secretion is increased;

(f) a percentage of phagocytotic macrophages in a population of macrophages is increased;

(g) a percentage of dendritic cells presenting the KRAS domain or degraded fragments thereof in a population of dendritic cells is increased;

(h) a percentage of antigen-specific T cells specific for the KRAS domain or an epitope thereof is increased; or

(i) a percentage of B cells producing antibodies specific for the KRAS domain or an epitope thereof in a population of B cells is increased.
The method of claim 59, wherein the innate immune response is measured by activation of the NF-kB signaling pathway, or increase in secretion of cytokines selected from IFN-α, IP-10, TNF-α, CXCL1, MCP-1, IL-2, or any combination thereof; and/or

wherein the pro-inflammatory cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof; and/or

wherein the target cell is a diseased cell expresses a mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
A method of eliminating a diseased cell expressing a mutant KRAS in a subject, comprising administering an effective amount of the immunomodulating nucleic acid system of any one of claims 1-41, the polypeptide of claim 42 or 43, the non-naturally occurring nucleic acid of claim 44 or 45, the composition of any one of claim 46 and 49 to 50, or the combination of any one of claims 51-52.
The method of claim 61, wherein the subject is a human subject having the HLA subtype of HLA-A*11: 01; and/or wherein the diseased cell is cancer cells expressing a mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R.
A method of treating a KRAS mutant cancer in a subject in need thereof, comprising administering to the subject an effective amount of the immunomodulating nucleic acid system of any one of claims 1-41, the polypeptide of claim 42 or 43, the non-naturally occurring nucleic acid of claim 44 or 45, the composition of any one of claim 46 and 49 to 50, or the combination of any one of claims 51-52.
The method of claim 63, wherein the cancer cells express a mutant KRAS with mutations selected from G12A, G12C, G12D, G12V, G13D, and Q61R; and/or wherein the cancer is selected from epithelial cancers, lung cancers, particularly non-small cell lung cancers, colorectal cancers, and pancreatic cancers.
The method of any one of claims 53 to 64, wherein the method further comprises administering to the subject one or more anti-cancer therapies.
The method of claim 65, wherein the one or more anti-cancer therapies are selected from a chemotherapeutic agent, a cytokine, an immunotherapy, a radiotherapy, a therapeutic antibody, and surgery; optionally wherein the one or more anti-cancer therapies comprise an anti-PD-1 or PDL1 antibody.