WO2025259745A1 - Lnp-rna-based antigen presentation platform - Google Patents

Lnp-rna-based antigen presentation platform

Info

Publication number
WO2025259745A1
WO2025259745A1 PCT/US2025/033132 US2025033132W WO2025259745A1 WO 2025259745 A1 WO2025259745 A1 WO 2025259745A1 US 2025033132 W US2025033132 W US 2025033132W WO 2025259745 A1 WO2025259745 A1 WO 2025259745A1
Authority
WO
WIPO (PCT)
Prior art keywords
lnp
composition
rna
spike
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/033132
Other languages
French (fr)
Inventor
Yonger XUE
Xucheng HOU
Yichen Zhong
Yizhou Dong
Darrell J. Irvine
Ron Weiss
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Massachusetts Institute of Technology
Icahn School of Medicine at Mount Sinai
Original Assignee
Massachusetts Institute of Technology
Icahn School of Medicine at Mount Sinai
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Massachusetts Institute of Technology, Icahn School of Medicine at Mount Sinai filed Critical Massachusetts Institute of Technology
Publication of WO2025259745A1 publication Critical patent/WO2025259745A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • LNP-mRNA vaccines demonstrated extraordinary abilities to elicit protective immunity against SARS-CoV-2 and its multiple variants.
  • Both vaccines encapsulating mRNA encoding a full-length spike protein induce robust spike-specific antibodies and T-cell responses, achieving immune protection against the virus.
  • a significant portion of the global population has been Attorney Docket No.766501:MTST-288PC (240503G-P) immunized with LNP-mRNA vaccines.
  • TAAs tumor-associated antigens
  • MHC major histocompatibility complex
  • CMV cytomegalovirus
  • MHC class I molecules MHC class I molecules
  • CTLs cytotoxic T lymphocytes
  • the spike-derived epitopes may serve as a new universal antigen that can be leveraged to redirect spike-specific T-cell immunity against tumors.
  • LNP-RNA-mediated antigen presentation platform that redirects spike-specific T-cell immunity against cancer cells.
  • One aspect of the present disclosure is polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6.
  • SE-SCT spike epitope-loaded single-chain trimer
  • A] P] Pb_TRc' cWT J (J;K E@; A ⁇ [TRd[T R ⁇ _aXbTb cWT bXV]P[ bT ⁇ dT]RT ⁇ U t2m, the ⁇ PcdaT _ ⁇ acX ⁇ ] ⁇ U ⁇ daX]T t2m, and the H-2kb heavy chain.
  • the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers.
  • the polynucleotide comprises DNA or RNA.
  • the DNA is plasmid DNA (pDNA) or complementary DNA (cDNA).
  • the RNA is messenger ribonucleic acid (mRNA), self-amplifying RNA (saRNA), small RNA (sRNA), micro RNA (miRNA), or circular RNA (circRNA).
  • RNA molecule comprising a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike Attorney Docket No.766501:MTST-288PC (240503G-P) epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, wherein the RNA molecule is mRNA or saRNA.
  • SE-SCT spike epitope-loaded single-chain trimer
  • the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers.
  • One aspect of the disclosure is an expression construct comprising the polynucleotide or the RNA molecule.
  • the expression construct further includes a 3’ UTR, 5’ UTR, a 5’ Cap, a replicase and/or Poly(A) sequence.
  • One aspect of the disclosure is a composition comprising the polynucleotide, the RNA molecule, or the expression construct.
  • the polynucleotide is formulated, in communication with, or encapsulated with a delivery vehicle.
  • the delivery vehicle is selected from the group consisting of amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate.
  • the delivery vehicle has an encapsulation efficiency of about 50% to about 100%.
  • the delivery vehicle comprises an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid.
  • the ionizable lipid is an amino alcohol-derived lipid.
  • the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers.
  • the ionizable lipid comprises a carboxylic acid group.
  • the ionizable lipid comprises two hydroxyl groups introduced to the head group via an ethylidene linker.
  • the ionizable lipid further comprises a hydrophobic tail selected from the group consisting of non-biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups.
  • the disclosure is a biological preparation comprising the composition.
  • the biological preparation is a vaccine, an immunotherapy, or a therapeutic composition.
  • the biological preparation is configured to be administered to a subject known or suspected to have cancer, or for use in an anti-cancer therapy or cancer immunotherapy.
  • the biological preparation is configured to be administered as an injectable preparation.
  • FIG. 1 is an Illustration of LNP-RNA-mediated antigen presentation. E, spike epitope; TM, transmembrane domain.
  • FIG. 2A is the design and structure of AA ionizable lipids.
  • FIG. 2B shows a general synthesis route of exemplary AA lipids.
  • FIGS. 3A and 3B are graphs depicting the characterizations of AA LNP-FLuc mRNA.
  • FIG. 3A is a graph showing the particle size and PDI of AA LNP-FLuc mRNA.
  • FIG. 3B is a graph showing encapsulation efficiency and Zeta potential of AA LNP-FLuc mRNA.
  • FIGs. 4A-4E show the results of an investigation of AA LNPs for Spike mRNA vaccinations.
  • FIG. 4A is the luminescence intensity of AA LNP-FLuc mRNA treated C2C12 cells.
  • FIG. 4B is the luminescence intensity in JAWSII cells.
  • FIG. 4C is the luminescence intensity of the muscle in the lead AA LNP-FLuc mRNA-treated C57BL/6 mice. The intensity was normalized to the ALC-0315 LNP group.
  • FIG.4D are representative images of C57BL/6 mice i.m. treated with AA2 LNP-FLuc mRNA, ALC-0315 LNP-FLuc mRNA, and SM-102 LNP-FLuc mRNA.
  • FIG. 4A is the luminescence intensity of AA LNP-FLuc mRNA treated C2C12 cells.
  • FIG. 4B is the luminescence intensity in JAWSII cells.
  • FIG. 4C is the luminescence intensity of the muscle in the lead AA LNP-FLuc mRNA-treated C57BL/6 mice. The intensity was normalized to
  • FIG. 4E shows spike-specific IgG titer in blood drawn from C57BL/6 mice vaccinated with AA2 LNP-spike mRNA, ALC-0315 LNP-spike mRNA or SM-102 LNP- spike mRNA.
  • Data in (FIGs. 4A-4D) are from nw7w.
  • FIG. 7A and 7B show flow cytometry gating schemes for AIM assay.
  • FIG. 7A shows a flow cytometry gating scheme of activation markers for CD4+ and CD8+ T cells from blood.
  • FIG.7B shows a flow gating scheme of activation marker for CD4+ T cells and CD8+ T cells from spleen.
  • FIGs. 8A-8H depict treatment with AA15V LNP-sSE-SCTs enhances spike-specific T cell-mediated killing of cancer cells.
  • FIG. 8A is a screening of AA LNPs in B16F10 cells.
  • FIG. 8B shows construction of saRNA encoding MHC I single-chain trimer (SCT) containing spike epitopes.
  • SCT single-chain trimer
  • FIG. 8C shows dynamic expression of H- 2Kb + t- ⁇ + in B16F10 cells.
  • FIG.8D shows expression of OVA-H-2Kb + in B16F10 cells after a single i.t. injection of AA15V LNP-sOP-SCTs. OP, OVA 257-264 peptide (SIINFEKL).
  • FIG. 8E shows percentage of apoptotic B16F10 cells in the T cell-mediated killing assay.
  • FIG. 8F shows percentage of dead B16F10 cells in the T cell-mediated killing assay.
  • FIGs.8G and 8H show spike epitope-specific activation of CD4 + T cells (FIG.8G) and CD8 + T cells (FIG.8H) isolated from B16F10 tumors in mice vaccinated with AA2 LNP-spike mRNA.
  • CE control epitope SE spike epitope.
  • Data in (FIG. 8A) and (FIG. 8C) are from nw7w.
  • FIGs. 8D are from nw7w/ QX ⁇ [ ⁇ VXRP[[h X]ST_T]ST]c bP ⁇ _[Tb) ⁇ PcP Attorney Docket No.766501:MTST-288PC (240503G-P) in (FIGs. 8E-8H) are from nw7w0 QX ⁇ [ ⁇ VXRP[[h X]ST_T]ST]c bP ⁇ _[Tb) ⁇ PcP PaT _aTbT]cTS Pb ⁇ TP]wkwJ ⁇ ) JcPcXbcXRP[ bXV]XUXRP]RT X] #FIGs. 8A, 8C) and (FIGs.
  • FIGs. 9A – 9F depict formulation optimization of AA15 LNPs in B16F10 cells.
  • FIG. 9A is a table for the three rounds of AA15 LNP-FLuc mRNA optimization. Chol, cholesterol.
  • FIG.9B is an orthogonal assay to determine the impact trend of each lipid component in AA15 formulation at four levels.
  • FIG.9C shows luminescence intensity fold changes of the three rounds of optimization.
  • FIG.9D shows particle size and PDI of AA15V LNP.
  • FIG.9E shows encapsulation efficiency and zeta potential of AA15V LNP.
  • FIGs. 10A – 10D show AA15V LNP-sSE-SCTs delivery in B16F10 cells.
  • FIG. 10A shows expression of SCTs with different epitopes in B16F10 cells.
  • FIG. 10B shows flow cytometry gating scheme of expression of SE-SCTs on B16F10 cells.
  • FIG. 10C shows flow cytometry gating scheme of T cell killing assay.
  • FIG. 11 shows the flow cytometry gating schemes for intratumoral delivery efficiency of AA15V LNP-sOP-SCTs.
  • the B16F10-FLuc cells were used for tumor model establishment. Live tumor cells were identified by gating for CD45- CD31- FLuc+ cell population.
  • FIGs. 12A – 12L depict how AA15V LNP-sSE-SCTs redirects spike-specific T-cell immunity to treat tumors and reprogram TME.
  • FIG. 12A is a schematic of the treatment regimen in the B16F10 tumor model. Created in BioRender. Xue, Y. (2025) https://BioRender.com/b49g753.
  • FIG. 12B shows tumor volumes in different groups.
  • FIG. 12C shows survival rates of the mice in the B16F10 tumor model.
  • FIG. 12D bW ⁇ fb Tg_aTbbX ⁇ ] ⁇ U Rhc ⁇ ZX]Tb P]S RWT ⁇ ZX]Tb X] cd ⁇ a bP ⁇ _[Tb Pc -/wW _ ⁇ bc(caTPc ⁇ T]c)
  • FIG. 12E shows expression level of representative cytokines and chemokines in tumor samples from Attorney Docket No.766501:MTST-288PC (240503G-P) (FIG. 12D).
  • FIG. 12D shows expression level of representative cytokines and chemokines in tumor samples from Attorney Docket No.766501:MTST-288PC (240503G-P) (FIG. 12D).
  • FIG.12G shows expression level of representative cytokines and chemokines in blood samples from (FIG. 12F).
  • FIGs. 12H-12L show immune cell populations in tumor tissues. Populations of FIG. 12I activated DCs, FIG. 12J activated macrophages, FIG. 12K primed CD4 + T cells, and FIG.12L primed CD8 + T cells in tumor tissues. Data in (FIG. 12B, 12C) and (FIGs.
  • 12H-12L are from nw7w0 QX ⁇ [ ⁇ VXRP[[h X]ST_T]ST]c bP ⁇ _[Tb) ⁇ PcP X] (FIGs. 12D-12G) are from nw7w.
  • FIGs. 14A – 14G show the therapeutic effects of AA15V LNP-sSE SCTs in B16F10 tumor model with T cell depletion.
  • FIG. 14A shows a schematic of the treatment regimen in the B16F10 tumor model. The tumor-bearing mice were intraperitoneally injected with T cell depletion antibodies.
  • FIG. 14B shows tumor volumes in different groups.
  • FIG. 14C shows survival rates of the mice in different groups. Tumor volume of individual mice in (FIG.14D) PBS group, (FIG. 14E) sSE-SCT + CD4 Ab group, (FIG. 14F) sSE-SCT + CD8 Ab group, and (FIG.
  • FIG. 15A and 15B depict a gating strategy for tumor-infiltrating immune cells.
  • FIG.15A shows flow cytometry gating schemes of macrophages and conventional type 1 and type 2 dendritic cells (cDC1s and cDC2s).
  • FIG.15B shows flow cytometry gating schemes of CD4+ and CD8+ T cells.
  • FIGs. 16A-16J depict the applicability of AA15V LNP-sSE-SCTs in multiple tumor models and human tumor samples.
  • FIG. 16A shows a schematic of the treatment regimen in B16F10 tumor model treated with the combination of AA15V LNP-sSE-SCTs and ICI.
  • FIG. 16B shows tumor volumes over time.
  • FIG.16C shows survival rates of mice bearing B16F10 tumor.
  • FIG.16D shows a schematic of the treatment regimen in A20 tumor model.
  • FIG. 16E shows tumor volumes over time.
  • FIG. 16F shows survival rates of the mice bearing A20 tumor.
  • FIG. 16G shows a schematic depicting ex vivo AA15V LNP-sSE-SCTs delivery in human tumor tissues. Created in BioRender. Xue, Y. (2025) https://BioRender.com/q02v567.
  • FIGs. 16H shows expression of H- 2Kb + t- ⁇ + expression in CD45 o cells from pediatric glioma dissections after ex vivo treatment with AA15V LNP-sSE-SCTs.
  • FIGs.16I and 16J show H-2Kb + t- ⁇ + in CD45 o cells from two separate lung left lower lobe (LLL) adenocarcinoma specimens after ex vivo treatment with AA15V LNP-sSE-SCTs.
  • Data in (FIGs. 16B, 16C) are from nw7w1 #H:J Va ⁇ d_$ P]S 3 #J;K groups) biologically independent samples, respectively.
  • 16E, 16F are from nw7w1 #H:J Va ⁇ d_ P]S L]MPR Va ⁇ d_b$ P]S 2 #MPR Va ⁇ d_b$ QX ⁇ [ ⁇ VXRP[[h X]ST_T]ST]c samples, respectively.
  • Data in (FIG. 16H) and (FIG. 16J) are from nw7w. X]SXeXSdP[ cXbbdT slices. Data in (Fig.
  • FIGS. 16I are from nw7w/ X]SXeXSdP[ cXbbdT b[XRTb) ⁇ PcP PaT _aTbT]cTS Pb ⁇ TP]wkwJ ⁇ ) JcPcXbcXRP[ bXV]XUXRP]RT X] #FIGs. 16B, 16E) and (FIGs. 16H-16J) was determined by the two-tailed Student’s t-test. Statistical significance in (FIG. 16C) and (FIG. 16F) was determined by the log-rank (Mantel–Cox) test.
  • FIGs. 17A – 17D depict tumor volumes in the B16F10 tumor model treated with combination of sSE-SCT and ICI.
  • FIG. 18A – 18E depict tumor volumes in the A20 tumor model.
  • Tumor volume of individual mice in (FIG. 18A) PBS group, (FIG. 18B) sSE-SCT-treated unvaccinated mice group, (FIG. 18C) sSE-SCT + ICI-treated unvaccinated mice group, (FIG. 18D) sSE-SCT- treated vaccinated mice group, and (FIG.18E) sSE-SCT + ICI-treated vaccinated mice group. Data in a-e are from n 6 or 7 biologically independent samples.
  • FIG. 19 depicts a gating strategy for human tumor tissues.
  • the CD45- cells were bT[TRcTS U ⁇ a ⁇ dP]cXUhX]V Tg_aTbbX ⁇ ] ⁇ U @(-ZQ P]S t- ⁇ ) Attorney Docket No.766501:MTST-288PC (240503G-P) DETAILED DESCRIPTION I. Introduction [0063] Humans have encountered a multitude of viral infections. Following recovery, the body harbors memory T cells dispersed throughout its entirety. These antiviral memory T cells exhibit notable characteristics: heightened vigilance, rapid response, and cytotoxic abilities. Upon encountering virus-specific epitopes, indicating potential reinfection, these memory T cells become primed, efficiently orchestrating immune defenses at the reinfection site.
  • the epitopes recognized by virus-specific T cells are renowned for their universality and immunogenicity. Therefore, redirecting established antiviral immunity may offer a therapeutic avenue for cancer immunotherapy, although more studies need to determine if the antitumor activity of vaccine-induced T cells mirrors that of T cells generated through natural viral infections.
  • the widespread use of mRNA vaccines against SARS-CoV-2 has elicited anti-spike T- cell immunity on a global scale, establishing spike epitopes as a viable antigenic target.
  • RNA-encoding spike epitope-loaded SCTs SE-SCT
  • the inventors synthesized and evaluated a library of amino alcohol- or amino acid-derived (AA) ionizable lipid materials, identifying AA2, the optimal LNP formulation for spike mRNA vaccination, and AA15V, the optimal LNP formulation for RNA delivery in cancer cells.
  • AA15V LNP-sSE-SCTs enabled the presentation of SE-SCT on cancer cell surface, expediting the recognition by spike-specific T cells induced through AA2 LNP-spike mRNA vaccination.
  • Such redirection of spike-specific T-cell immunity facilitated cancer cell death induced by T-cell-mediated cytotoxicity.
  • i.t. administration of AA15V LNP-sSE-SCTs reprogrammed the TME by eliciting antitumor phenotypes of immune cells and pro-inflammatory cytokines as well as chemokines.
  • a single treatment significantly suppressed the growth of melanoma and lymphoma tumors and extended the survival of AA2 LNP-spike mRNA-vaccinated mice, particularly when in combination with ICI treatments.
  • the single-chain trimer (SCT) MHC class I molecule which consists of a polymorphic WTPeh RWPX] #@;$' P t- ⁇ XRa ⁇ V[ ⁇ Qd[X] #t- ⁇ $ [XVWc RWPX]' P]S P R ⁇ eP[T]c[h Q ⁇ d]S T_Xc ⁇ _T connected by a linker, has been developed to induce antigen-specific immune responses.
  • the Attorney Docket No.766501:MTST-288PC (240503G-P) covalent binding of an epitope in the SCT allows stable expression of defined epitope-loaded MHC class I molecules on the cell surface.
  • RNA encoding spike epitope-loaded SCTs may retarget the anti-spike immunity against tumors in mice immunized by SARS-CoV-2 vaccines (FIG. 1).
  • SE-SCT spike epitope-loaded SCTs
  • the inventors designed a series of mRNAs and self-amplifying RNAs (saRNAs) encoding SE-SCT.
  • saRNAs self-amplifying RNAs
  • the inventors synthesized a library of amino alcohol- or amino acid-derived ionizable lipid materials (AA lipids) containing various headgroup structures (differ in types and quantities of hydrogen-donors) and types of hydrophobic tails (variable in length, biodegradability, and location of biodegradable groups).
  • AA2 and AA15V Two lead LNP formulations, AA2 and AA15V.
  • the FDA-approved LNP-mRNA vaccines, Moderna’s mRNA-1273 and Pfizer/BioNTech’s BNT162b2 utilize SM-102 and ALC-0315 as ionizable lipids, respectively.
  • AA2 LNP showed higher Spike mRNA delivery efficiency post intramuscular (i.m.) injection, leading to stronger vaccination efficacy against SARS-CoV-2.
  • the mice vaccinated with a prime-boost regimen generated robust spike-specific CD8 + T cell immunity.
  • AA15V LNP demonstrated more potent RNA delivery efficiency to both mouse and human cancer cells than the FDA-approved LNPs.
  • a single i.t. treatment with AA15V LNP-saRNA encoding SE-SCT significantly inhibited tumor progression and extended the overall survival of the tumor-bearing mice that were immunized by AA2 LNP- Spike mRNA vaccines.
  • AA15V LNP-sSE-SCTs facilitated SE-SCT expression in ex vivo human cancer samples, suggesting the potential for future clinical applications.
  • the inventors have developed an LNP-RNA-based antigen presentation platform designed to redirect spike-specific T-cell immunity against cancer.
  • AA2 LNP demonstrates superior efficacy for mRNA vaccine delivery in vivo compared to FDA-approved LNPs
  • AA15V LNP facilitates the delivery of sSE-SCTs to human cancer samples.
  • x, y, and/or z means any element of the seven-element set ⁇ (x), (y), (z), (x, y), (x, z), (y, z), (x, y, z) ⁇ .
  • x, y and/or z means "one or more of x, y and z”.
  • a "disease”, as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate.
  • a “disorder” is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.
  • a “syndrome” is a recognizable complex of symptoms and physical findings that occur together and suggest the presence of a certain disease or disorder or an increased chance of developing the disease or disorder.
  • a disease, disorder, or syndrome is "alleviated” if the severity of a sign or symptom of the disease, disorder, or syndrome, or the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.
  • the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals.
  • mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos.
  • the term “animal” as used herein Attorney Docket No.766501:MTST-288PC (240503G-P) includes humans.
  • subject may also include a patient, i.e., an animal, having a disease.
  • a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).
  • the terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered.
  • amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure.
  • treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and/or decrease the number, frequency, or severity of clinical symptoms and/or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder.
  • treatment of a disease and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.
  • prophylactic refers to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject.
  • the prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.
  • the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable.
  • the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.
  • Attorney Docket No.766501:MTST-288PC (240503G-P) [0086]
  • administered means administration of an effective amount of, for example, at least one nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule and any another other additional agent for treatment.
  • an “effective amount” includes a “therapeutically effective amount” and a “prophylactically effective amount.”
  • therapeutically effective amount refers to an amount effective in treating and/or ameliorating a disease or condition in a subject.
  • prophylactically effective amount refers to an amount effective in preventing and/or substantially lessening the chances of a disease or condition in a subject.
  • nucleic acid or “polynucleotide” refers to any polymeric chain of nucleotides.
  • polynucleotide refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”).
  • a nucleic acid comprises of one or more nucleic acid analogs.
  • nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues).
  • a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.
  • polynucleotide includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows Attorney Docket No.766501:MTST-288PC (240503G-P) for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • the polynucleotide comprises an mRNA.
  • Nucleotides are referred to by their commonly accepted single-letter codes. Unless ⁇ cWTafXbT X]SXRPcTS' ]dR[TXR PRXSb PaT faXccT] [TUc c ⁇ aXVWc X] 0s c ⁇ .s ⁇ aXT]cPcX ⁇ ]) FdR[T ⁇ cXSTb PaT referred to herein by their commonly known one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission.
  • A represents adenine
  • C represents cytosine
  • G represents guanine
  • T represents thymine
  • U represents uracil.
  • mRNA messenger RNA
  • the term “payload” maybe used herein may consist of nucleic acids and/or proteins.
  • a delivery molecule is used to deliver a nucleic acid payload (e.g., DNA and/or RNA).
  • the nucleic acid payload can be any nucleic acid of interest, for example, the nucleic acid payload can be linear or circular, a plasmid, a viral genome, RNA or DNA.
  • the DNA may be plasmid DNA (pDNA) or complementary DNA (cDNA). It may be coding RNA or non-coding RNA.
  • the RNA may be mRNA, guide RNA, self-amplifying RNA (saRNA),small interfering RNA or short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA),piwi RNA (piRNA), small nuclear RNA or U-rich snRNA (snRNA), Small nucleolar RNA (snoRNA), signal recognition particle RNA (7SL), Y RNAs, small Cajal body-specific RNA (scaRNA), long intergenic ncRNA (lincRNA), natural antisense transcript (NAT), circular RNA (circRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA), etc.
  • saRNA self-amplifying RNA
  • siRNA small interfering RNA or short interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • piwi RNA piwi RNA
  • small nuclear RNA or U-rich snRNA s
  • the nucleic acid payload is an RNA interference (RNAi) agent or a DNA template encoding an RNAi agent.
  • the nucleic acid payload is a locked nucleic acid (LNA) molecule.
  • the nucleic acid payload may encode a protein of interest.
  • Calculation of the percent identity of two polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned Attorney Docket No.766501:MTST-288PC (240503G-P) for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.
  • compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
  • delivery vehicle refers to any substance that facilitates, at least in part, the in vivo, in vitro, or ex vivo delivery of a polynucleotide (e.g., therapeutic polynucleotide) to cells or tissues (e.g., tumors, etc.).
  • a delivery vehicle need not exclude the possibility of the delivery vehicle also having therapeutic effects.
  • Some versions of a delivery vehicle may provide additional therapeutic effects.
  • a delivery vehicle may be a peptoid molecule, such as an amino-lipidated peptoid molecule, that may be used to at least partially encapsulate mRNA.
  • the term “DV” may also be used herein as a shorthand for “delivery vehicle.”
  • the mRNA for use in the delivery vehicle complexes herein comprise an mRNA comprising at least one region encoding a peptide (e.g., a polypeptide), or protein, or functional fragment of the foregoing.
  • “functional fragment” refers to a fragment of a peptide, (e.g., a polypeptide), or protein that retains the ability to induce an immune response.
  • amphipathic molecules refers to molecules comprising both hydrophilic and hydrophobic regions, enabling interaction with both aqueous and lipid-based environments.
  • amino-lipidated peptides refers to peptides that have been chemically conjugated with lipid moieties via amino groups.
  • lipid nanoparticle refers to a nanoscale structure composed of lipids, which may encapsulate or associate with the composition.
  • polymeric compound refers to a chemical compound comprising repeating monomeric units.
  • proteoid refers to a synthetic oligomer or polymer structurally analogous to peptides, wherein side chains are attached to the nitrogen atom of the backbone rather than the alpha-carbon.
  • lipoid refers to lipid-like substances, including but not limited to phospholipids, sterols, glycolipids, and other fat-associated compounds.
  • liposome refers to a vesicular structure composed of one or more concentric phospholipid bilayers encapsulating an aqueous core.
  • lipoplex refers to a complex formed between cationic lipids and nucleic acids.
  • conjugate refers to a molecular entity formed by the chemical linkage of two or more distinct components.
  • delivery refers to the physical introduction of an agent such as the composition disclosed herein into a subject or cell, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration known to those skilled in the art for the formulations disclosed herein to a subject include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the formulation is administered via a non-parenteral Attorney Docket No.766501:MTST-288PC (240503G-P) route, e.g., orally.
  • non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • biological preparation refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • vaccine refers to a biological preparation that induces or enhances an immune response.
  • the term “immunotherapy” refers to therapeutic interventions that modulate the immune system for the treatment or prevention of disease
  • the term “therapeutic composition” refers to a biologically active formulation comprising one or more therapeutic agents designed to prevent, treat, or manage disease conditions.
  • the terms “efficacy,” “efficiency,” or grammatical equivalents refers to an assessment of a biologically relevant endpoint. Specifically, efficiency herein relates to the encapsulation of the composition.
  • the invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein. III.
  • One aspect of the disclosure relates to a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule.
  • a spike epitope refers to a specific part of the spike protein of a virus that is recognized by the immune system.
  • Table 1 lists exemplary spike epitopes.
  • Table 1 Spike epitopes and spike derived epitopes Attorney Docket No.766501:MTST-288PC (240503G-P)
  • the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:1.
  • the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:2. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:3. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:4. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:5. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:6. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:7.
  • the SE-SCT MHC I molecule also comprises the signal sequence of t2 ⁇ ' cWT ⁇ PcdaT _ ⁇ acX ⁇ ] ⁇ U ⁇ daX]T t2m, and the H-2kb heavy chain.
  • the SE- SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers.
  • FIG. 8B is an exemplary SE-SCT.
  • the polynucleotide may be configured for administration directly, configured for administration in a composition including other polynucleotides, be formulated with a delivery vehicle, be encoded in one or more polynucleotides for expression in a cell, and/or may be encoded in DNA, RNA, or mRNA for administration.
  • the polynucleotide comprises DNA or RNA. In some embodiments, the polynucleotide comprises DNA, wherein the DNA is pDNA or cDNA. In some embodiments, the polynucleotide comprises RNA, wherein the RNA is mRNA, siRNA, miRNA, saRNA, or circRNA. [0119]
  • One aspect of the disclosure includes an RNA molecule comprising a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, wherein the RNA molecule is mRNA or saRNA.
  • SE-SCT spike epitope-loaded single-chain trimer
  • an expression construct including the polynucleotide sequence or the RNA molecule according to an aspect of the disclosure.
  • “Expression construct” refers to an engineered DNA or RNA molecule designed to drive the production of a specific protein or RNA in a host cell. It typically contains regulatory elements such as a promoter to initiate transcription, an enhancer to increase expression levels, a coding sequence for the gene of interest, and a terminator to end transcription.
  • an expression construct includes a 3’ UTR, an effector region encoding a protein (e.g., mRNA), and a 5’ UTR.
  • the expression construct may have the following formula: 5’UTR—replicase—coding region—3’ UTR—Poly(A) [0123] where “UTRs” are the untranslated regions located at the 5’ and 3’ ends of an RNA construct, and “PolyA” refers to the polyadenylation site of the RNA. [0124] In some embodiments, the expression construct may have the following formula: 5’ cap -5’UTR—replicase—coding region—3’ UTR—Poly(A).
  • the term “5’ UTR” refers to a part of the nucleic acid molecule which is located 5' (upstream) of the open reading frame of the RNA.
  • the RNA may include mRNA, siRNA, miRNA, saRNA, or circRNA.
  • the open reading frame encodes the viral non-structural proteins while the sequence of interest is encoded in the subgenomic fragment of the viral RNA.
  • the 5’UTR is upstream of the nsP1 open reading frame.
  • the subgenomic RNA of the saRNA has a 5’UTR.
  • the subgenomic RNA containing a sequence of interest encoding a protein of interest contains a 5’UTR.
  • the 5’UTR starts with the transcriptional start site and ends with one nucleotide before the start codon of the open reading frame.
  • the 5’UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract.
  • the 5’UTR may be post transcriptionally modified, for example by addition of a 5'-CAP.
  • 5'-CAP (or a 5'-CAP-Structure) is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide, added to the 5' end of an mRNA molecule.
  • CAP analogue particularly a guanine nucleotide
  • the 5'-CAP is added using a 5'-5'-triphosphate linkage (also named m7GpppN).
  • a 5' CAP structure may also be formed in Attorney Docket No.766501:MTST-288PC (240503G-P) chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using cap analogues, or a cap structure may be formed in vitro using capping enzymes (e.g., commercially available capping kits).
  • capping enzymes e.g., commercially available capping kits.
  • 3’UTR refers to a part of the nucleic acid molecule which is located 3' (i.e. "downstream") of an open reading frame and which is not translated into protein.
  • a 3’UTR is the part of an RNA which is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA.
  • the term 3’UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence.
  • a 3’UTR of the RNA is not translated into an amino acid sequence.
  • the 3’UTR sequence is generally encoded by the viral genomic RNA, which is transcribed into the respective mRNA during the gene expression process.
  • Poly(A) or “Poly(A) tail” refers to the polyadenylation site of the mRNA.
  • the poly(A) tail is a stretch of adenine nucleotides added to the 3' end of eukaryotic messenger RNA (mRNA) molecules during post-transcriptional processing. This modification is catalyzed by the enzyme poly(A) polymerase and plays a critical role in mRNA stability, nuclear export, translation efficiency, and protection from exonucleases.
  • poly(A) tail can influence gene expression, as longer tails typically enhance translation, while shorter tails may signal mRNA decay. Additionally, the poly(A) tail interacts with poly(A)- binding proteins (PABPs), which help mediate its functions.
  • PABPs poly(A)- binding proteins
  • “Replicase” refers to an enzyme that catalyzed the synthesis of a complementary RNA molecule using an RNA template.
  • Ionizable lipids play a pivotal role in the encapsulation of mRNA molecules by delivery vehicles such as lipid nanoparticles (LNPs). Their positive head groups interact electrostatically with the negatively charged phosphate groups of the mRNA, facilitating the complexation of the lipid with the mRNA and the formation of nanoparticles.
  • AA1-AA26 lipids are amino alcohol-type lipids featuring a hydroxyl group connected with the amino head groups via varying carbon spacer.
  • AA27-AA45 lipids are amino acid-type lipids incorporating a carboxylic acid group. While, in AA46-AA50 lipids, two hydroxyl groups were introduced to the head group via an ethylidene linker.
  • hydrophobic tails were attached to the amino head groups, including non-biodegradable hydrocarbon tails (tail R1), and biodegradable tails containing ester (tail R2), carbonate ester (tail R3-R6), and acid-labile acetal groups (tail R7-R9).
  • tail R1 non-biodegradable hydrocarbon tails
  • tail R2 biodegradable tails containing ester
  • tail R3-R6 carbonate ester
  • acid-labile acetal groups tail R7-R9
  • delivery vehicles including, but not limited to amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a cationic lipid nanoparticle, a polymeric compound, or a conjugate.
  • the polynucleotide, the RNA molecule, and/or the expression construct is formulated, in communication with, or encapsulated with a delivery vehicle.
  • the delivery vehicle partially encapsulates the polynucleotide, the RNA molecule, and/or the expression construct. In other aspects, the delivery vehicle fully encapsulates the polynucleotide, the RNA molecule, and/or the expression construct. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 50%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 55%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 60%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 65%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 70%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 75%.
  • the delivery vehicle has an encapsulation efficiency of about 80%. In some Attorney Docket No.766501:MTST-288PC (240503G-P) embodiments, the delivery vehicle has an encapsulation efficiency of about 85%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 90%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 95%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 95%. [0133] Delivery vehicle encapsulation efficiency refers to the ability of the delivery vehicles to successfully encapsulate and protect the polynucleotide, RNA molecule, or expression construct.
  • the delivery vehicle comprises an ionizable lipid, one or more of an anionic or zwitterionic component, such as a phospholipid; a neutral lipid, such as a sterol; and a shielding lipid, such as a PEGylated lipid.
  • the delivery vehicle compositions further comprise an additional anionic or zwitterionic component (e.g., a phospholipid), neutral lipid (e.g., a sterol), or shielding lipid (e.g., a PEGylated lipid).
  • an additional anionic or zwitterionic component e.g., a phospholipid
  • neutral lipid e.g., a sterol
  • shielding lipid e.g., a PEGylated lipid.
  • the ionizable lipid is an amino alcohol-derived lipid.
  • the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers.
  • the ionizable lipid comprises a carboxylic acid group.
  • the ionizable lipid comprises two hydroxyl groups introduced to the head group via an ethylidene linker. [0138] In some embodiments, the ionizable lipid further comprises a hydrophobic tail selected from the group consisting of non-biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups. [0139] In some embodiments, the ionizable lipid has a structure of
  • AA LNPs were formulated with 1,2-dioleoyl-sn-glycerol-3- phosphoethanolamine (DOPE), cholesterol, and DMG-PEG2k with firefly luciferase (FLuc) ⁇ IF9) KWT 99 DFH(>DdR ⁇ IF9 SXb_[PhTS _PacXR[T bXiTb QTcfTT] 31)/wkw/)1w] ⁇ c ⁇ ,0/)+wkw/)0w] ⁇ ' fXcW P _ ⁇ [hSXb_TabXch X]STg #H ⁇ A$w6w+).
  • DOPE 1,2-dioleoyl-sn-glycerol-3- phosphoethanolamine
  • FLuc firefly luciferase
  • lipids with branched ester tails (tail R2), especially lipid AA2, exhibited superior mRNA delivery efficiency compared to other AA lipids and clinically approved SM-102 and ALC- 0315 LNPs at an equivalent mRNA dose (FIGs.4A and 4B).
  • This enhanced performance may be attributed to the hydrogen bond-forming capability of the hydroxyl group in the amino head groups of AA lipids.
  • the increased delivery efficiency of lipids with branched tails R2 may be attributed to their potential to adopt a more cone-shaped structure, facilitating improved endosomal escape compared to AA lipids with linear tails (such as hydrocarbonic, carbonated, and acetal tails).
  • the inventors selected 18 lead AA LNPs with over 2-fold higher mRNA delivery efficiency than the SM-102 LNP in both cell lines for in vivo evaluations. These AA LNPs encapsulating FLuc mRNA were i.m.
  • the inventors selected AA2 LNP for detailed characterizations.
  • AA2 LNP generated 4.7-fold and 3.4-fold higher anti-spike IgG antibody titer than ALC-0315 LNP and SM-102 LNP, respectively (FIG.2E).
  • AIM activation- induced marker
  • the inventors stimulated peripheral blood mononuclear cells (PBMCs) with a mixture of spike epitopes (SE; SEQ ID NO: 1 SGWTFGAGAALQIPF and SEQ ID NO: 2 VTWFHAIHVSGTNGT).
  • PBMCs peripheral blood mononuclear cells
  • SE spike epitopes
  • SEQ ID NO: 1 SGWTFGAGAALQIPF SEQ ID NO: 2 VTWFHAIHVSGTNGT
  • the random peptide sequence SEQ ID NO: 7 AAAAFAAL was used as a negative control epitope (CE).
  • CE negative control epitope
  • spike epitopes significantly increased activation markers of CD4 + T cells from blood (FIG.6B; FIG. 7A).
  • spike epitopes elicited higher percentages of CD137 + CD134 + and CD154 + CD134 + in CD4 + T cells from the spleen (FIG. 6D; FIG. 7B).
  • the CD4 + T cells isolated from the blood and spleen of mice vaccinated with SM-102 LNP also showed increased activation markers following stimulation with spike epitopes (FIGs. 6D, 6B).
  • the activation efficiency was lower than those observed with AA2 LNP.
  • ICS intracellular cytokine staining
  • AIM and ICS detected obvious CD8 + T cell responses to spike epitopes, with AA2 LNP-spike mRNA vaccination inducing increased levels of CD69+, CD69+CD137+' A>F(u+, KF>(q + , and granzyme B + compared to SM102 LNP (FIG. 6C and 6E; FIGs. 7A and 7B). These results are consistent with the in vivo mRNA delivery efficiency and spike-specific IgG titers, indicating that vaccinations with AA2 LNP-spike mRNA generate stronger spike- specific T-cell immunity compared to SM-102 LNP-spike mRNA.
  • the inventors fine-tuned the molar ratios for each formulated lipid using an L16 orthogonal table (FIG. 9A). The inventors also optimized the mass ratio of AA15 lipid to mRNA in the formulation.
  • the lead formulation AA15V LNP demonstrated 1.5-fold and 3.1-fold higher luminescence intensity compared to the 1st-round top orthogonal formulation AA15I and the original formulation AA15, respectively (FIGs.9B, 9C$) KWT WhSa ⁇ Sh]P ⁇ XR SXP ⁇ TcTa ⁇ U 99,0M DFH fPb ,+-).wkw/),w] ⁇ fXcW P H ⁇ Aw6w+),0) GeTa 85% mRNA was encapsulated in AA15V LNP, and the particle exhibited a slightly positive charge and spherical morphology (FIGs. 9D-9F).
  • AA15V LNP was chosen for mRNA delivery to cancer cells in the following studies.
  • SCT single-chain trimer
  • SE-SCT spike epitope-loaded single-chain trimer
  • the inventors refer to mRNAs and Attorney Docket No.766501:MTST-288PC (240503G-P) saRNAs encoding SE-SCTs as mSE-SCTs and sSE-SCTs, respectively.
  • the inventors constructed saRNAs encoding OVA257-264 peptide-SCTs (OP-SCTs), enabling the quantification of OVA-H-2Kb+ live tumor cells using established antibodies.
  • the inventors established the tumor model using B16F10-FLuc cells and found a single i.t. injection of 99,0M DFH(bGH(J;Kb aTbd[cTS X] -2)3wkw2)2" ⁇ U GM9(@(-CQ& [XeT :,1>,+ RT[[b fXcWX] tumor tissues (FIG.8D; FIG.11).
  • Cytotoxicity of spike-specific CD8+ T cells was evaluated by isolating CD8+ T cells from the spleens of vaccinated mice, which were then co-cultured with B16F10 cells that had been pretreated in vitro with PBS, AA15V LNP-sCE-SCTs, or AA15V LNP-sSE-SCTs. These CD8+ T cells exhibited cytotoxicity against B16F10 cells pretreated with AA15V LNP-sSE- SCTs (FIGs. 8E, 8F; FIG. 10C).
  • AIM assays revealed that specific epitopes targeting MHC II and MHC I stimulated tumor- infiltrating T cells, resulting in increased levels of CD69+CD134+ in CD4+ T cells and CD69+CD137+ in CD8+ T cells, respectively (FIG. 8G, 8H). These findings indicate the potential of AA15V LNP-sSE-SCTs to retarget spike-specific T-cell immunity against tumors in vaccinated mice.
  • AA15V LNP-sCE-SCTs encoding control epitopes demonstrated measurable antitumor efficacy (FIGs. 12B, 12C), potentially due to the immunogenic properties of saRNA.
  • saRNA forms double-stranded RNA intermediates that activate innate immune responses.
  • AA15V LNP- sSE-SCTs recruited more immune cells to the TME and enhanced immune cell activation, evidenced by increased levels of CD80+/86+ ⁇ ;b Pb fT[[ Pb A>F(u+' KF>(q+, and granzyme B + CD8 + T cells (FIGs. 12I-12L). Furthermore, depletion of CD8 + T cells significantly diminished the therapeutic effects of AA15V LNP-sSE-SCTs (FIGs. 14A-14G). All these results underscore the crucial roles of both saRNA-mediated innate immune responses and spike-specific T-cell immunity in the antitumor efficacy of AA15V LNP-sSE-SCTs.
  • AA15V LNP-mSE-SCTs Compared to PBS, AA15V LNP-mSE-SCTs, and AA15V LNP-sCE-SCTs treatment, AA15V LNP-sSE-SCTs more effectively suppressed the cd ⁇ a Va ⁇ fcW Qh --)+(U ⁇ [S' ,3)-(U ⁇ [S' P]S 2)0(U ⁇ [S #,/wS _ ⁇ bc(X] ⁇ Rd[PcX ⁇ ]$' aTb_TRcXeT[h' P]S substantially extended the overall survival time (FIG. 12B, 12C; FIGs. 13A – 13D).
  • AA15V LNP-mSE-SCTs Relative to PBS treatment, AA15V LNP-mSE-SCTs, AA15V LNP-sCE-SCTs, and AA15V LNP-sSE-SCTs all stimulated immune responses, as indicated by elevated levels of pro- inflammatory cytokines and chemokines in both tumor and blood samples (FIG. 12D-12G).
  • AA15V LNP-sCE-SCTs induced comparable or even higher levels of multiple pro- inflammatory cytokines and chemokines compared to AA15V LNP-mSE-SCTs. This may be attributed to the immunogenicity of saRNA which can form double-stranded RNA intermediates during translation, triggering innate immune responses.
  • AA15V LNP-sCE-SCTs demonstrated an equal or greater capacity of recruiting and activating immune cells within tumor tissues including macrophages and CD80 + cDC1s (FIG.12H-12L). Consistent with the trends observed in cytokine and chemokine levels, AA15V LNP-sSE-SCTs treatment recruited more immune cells to the tumor tissues and achieved stronger immune cell activation than other treatments.
  • AA15V LNP-sSE-SCTs treatment increased the populations of activated APCs, including CD80 + /86 + DCs and CD80 + /86 + macrophages (FIG. 12I-12J), as well as activated CD4 + T cells expressing CD137 + CD134 + and CD154 + CD134 + within tumor tissues (FIG.12K).
  • the treatment also upregulated the expression of Ki-67, IFN- u' KF>(q' P]S VaP]ih ⁇ T : X] ; ⁇ 3+ T cells compared to other groups, indicating activation of cytotoxic responses (FIG. 12L).
  • AA15V LNP-sSE-SCTs treatment showed obvious antitumor effects, the incomplete eradication of the tumor necessitates boosting the efficacy of primed T-cell responses.
  • Immune checkpoint inhibitors ICIs
  • the inventors incorporate the ICI combination, anti-PD-1 and anti-CTLA-4 Abs, in our strategy. The inventors intraperitoneally injected one dose of ICI prior to the AA15V LNP-sSE-SCTs treatment, followed by three subsequent doses given at three-day intervals throughout the treatment (FIG.
  • H-2Kd-based SE-SCTs were constructed to match the MHC I haplotype of BALB/C mice (FIG. 16D). Additionally, the inventors incorporated control groups of unvaccinated mice to assess the role of spike-specific T-cell immunity in the antitumor efficacy of our strategy.
  • the AA15V LNP-sSE-SCTs + ICI treatment induced completed tumor rejection and long-term survival in over 28.6% of the vaccinated mice (FIG.16E, 16F; FIGs. 18A – 18E). However, such treatment in the unvaccinated mice failed to achieve a complete response, and all the mice reached the end point criteria before day 31, indicating the importance of spike-specific T-cell immunity in suppressing tumors expressing SE-SCTs.
  • AA15V LNP-sSE-SCTs To further explore the clinical translatability of AA15V LNP-sSE-SCTs, the inventors examined AA15V LNP for the delivery of sSE-SCTs to human tumor tissues ex vivo (FIG. 16G). Glioma samples from one patient were treated with AA15V LNP-sSE-SCTs. Quantitative analysis revealed H-2Kb+t- ⁇ + Tg_aTbbX ⁇ ] X] 2),wkw+)-" ⁇ U ; ⁇ /0o cells in the glioma samples while the untreated slices exhibited no detectable expression (FIG.16H; FIG. 19).
  • excipients for use with the biological preparations disclosed herein include maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N- dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and surfactant polyoxyethylene-sorbitan monooleate.
  • solutions or suspensions used for parenteral application include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • pH is adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Biological preparations for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
  • the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), or suitable mixtures thereof.
  • Fluidity is maintained, in some embodiments, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.
  • Isotonic agents for example, sugars; polyalcohols such as mannitol or sorbitol; or sodium chloride, in some embodiments, are included in the composition.
  • an agent which delays absorption in some embodiments, for example, aluminum monostearate or gelatin prolongs absorption of injectable compositions.
  • sterile injectable formulations are prepared by incorporating the active composition in the required amount in an appropriate solvent with one or a combination Attorney Docket No.766501:MTST-288PC (240503G-P) of above ingredients.
  • dispersions are prepared by incorporating the active composition into a sterile vehicle containing a basic dispersion medium and any other ingredient.
  • methods of preparation include, for example, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously prepared solution thereof.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • transmucosal administration is accomplished through the use of nasal sprays, inhalation devices (e.g., aspirators) or suppositories.
  • the active compounds are formulated into ointments, salves, gels, creams or patches.
  • the biological preparations are prepared with carriers that protect against rapid elimination from the body, such as a controlled release formulation or a time delay material such as glyceryl monostearate or glyceryl stearate.
  • the formulations in some embodiments, are also delivered using articles of manufacture such as implants and microencapsulated delivery systems to achieve local, regional or systemic delivery or controlled or sustained release.
  • Any suitable route of administration is contemplated for the biological preparations disclosed herein.
  • the biological preparations disclosed are administered by intravenous administration.
  • the biological preparations disclosed are administered by subcutaneous administration.
  • the biological preparations disclosed are administered locally.
  • the biological preparations disclosed is administered systemically (e.g., intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, sublingually).
  • the biological preparations disclosed are formulated as a salve, lotion or emulsion.
  • the biological preparations disclosed are formulated as a solution.
  • the biological preparations disclosed are formulated for topical, oral, buccal, or nasal administration.
  • an individual is monitored prior to administration of the biological preparations disclosed. Symptoms are identified and their severity is assessed.
  • the Attorney Docket No.766501:MTST-288PC (240503G-P) biological preparation as described herein may be administered alone or in combination with additional treatments, singly or multiply over time as discussed herein or known to one of skill in the art.
  • the individual is monitored such that the efficacy of the treatment regimen is determined.
  • a treatment regimen is modified in response to preliminary treatment outcomes, such that treatment dose or frequency or dose and frequency is altered so as to attain a desired level of subject response in light of symptom alleviation, side effect reduction, or a combination of symptom alleviation and side effect reduction.
  • the biological preparations may be used to treat a disease or disorder in a subject.
  • the biological preparations may be used to prevent a disease or disorder in a subject.
  • the disease or disorder is cancer.
  • the disclosure includes methods for inducing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of the delivery vehicle composition or biological preparation of the disclosure.
  • An "effective amount” includes a “therapeutically effective amount” and a “prophylactically effective amount.”
  • therapeutically effective amount refers to an amount effective in treating and/or ameliorating a disease or condition in a subject.
  • prolactically effective amount refers to an amount effective in preventing and/or substantially lessening the chances of a disease or condition in a subject.
  • cancers may be treated with the nucleic acids delivered by the compositions of the present disclosure.
  • cancer refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths.
  • Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid, uterus, vagina, and vulva.
  • lymphomas/leukemias such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx
  • the subject may be known or suspects to have cancer or a disease associated with MHC I.
  • Attorney Docket No.766501:MTST-288PC (240503G-P) [0173]
  • the biological preparation is a vaccine or a cancer therapeutic.
  • a vaccine may be referred to as a substance used to stimulate the production of antibodies and provide immunity against one or several diseases, prepared from the causative agent of a disease, its products, or a synthetic substitute.
  • the vaccine may further comprise one or more immunologic adjuvants.
  • immunogen refers to a compound or a mixture of compounds that acts to accelerate, prolong, enhance or modify immune responses when used in conjugation with an immunogen (e.g., neoantigens).
  • Adjuvant may be non-immunogenic when administered to a host alone, but that augments the host's immune response to another antigen when administered conjointly with that antigen.
  • adjuvant and “immunologic adjuvant” are used interchangeably in the present disclosure.
  • Adjuvant-mediated enhancement and/or extension of the duration of the immune response can be assessed by any method known in the art including without limitation one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant/antigen combination versus those produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells recognizing the antigen or the adjuvant; and (iii) an increase in the level of one or more cytokines.
  • Adjuvants may be aluminum based adjuvants including but not limiting to aluminum hydroxide and aluminum phosphate; saponins such as steroid saponins and triterpenoid saponins; bacterial flagellin and some cytokines such as GM-CSF. Adjuvants selection may depend on antigens, vaccines, and routes of administrations. [0174] In some aspects, adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation. Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs).
  • APCs antigen presenting cells
  • DCs dendritic cells
  • Adjuvants may be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity.
  • adjuvants may signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation.
  • This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures.
  • the composition further comprises pidotimod as an adjuvant.
  • the composition further comprises CpG as an adjuvant.
  • the disclosed compositions and biological preparations may be part of a kit.
  • the kit may include a pharmaceutically acceptable carrier and/or a package insert Attorney Docket No.766501:MTST-288PC (240503G-P) comprising instructions for intratumoral administration (e.g., injection) of the nucleic acid- based therapeutic composition.
  • the kit may further include instructions for treating or delaying progression of cancer in a subject.
  • the package insert further comprises instructions for administration of the composition by intratumoral administration in combination with injection in another site (e.g., systemic injection) for treating or delaying progression of cancer in a subject.
  • the kit may also include additional therapeutic nucleic acids, drug, therapeutic agent, diagnostic agent, prophylactic agent, and/or any other agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • BAIB (1.77 g, 5.5 mmol) was added to a suspension of 20 (1.23 g, 5.0 mmol), TEMPO (78.2 mg, 0.5 mmol) and NaHCO 3 (924 mg, 11.0 mmol) in 20 mL of DCM.
  • the reaction mixture was stirred for 3 h till TLC showed A was totally consumed.
  • the mixture was quenched with saturated aqueous solution of Na2S2O3 (30 mL) and extracted with DCM (3*20 mL).
  • the DCM phase was combined and washed with aqueous NaHCO3 (20 mL) and brine (20 mL), dried over Na2SO4, filtrated and concentrated under reduced pressure.
  • Amine 4 (2.61 g, 15.0 mmol) was dissolved in 20 mL of dry MeCN. Potassium carbonate was added (1.59 g, 15.0 mmol) to the solution. A solution of 5-m (10.0 mmol) in 10 mL of dry MeCN was added dropwise to the above solution over 1 hour at RT. The solution was then stirred at RT for 24 h. Solid Na 2 CO 3 was removed via filtration, and the solvent was removed under vacuum. The residue was purified via silica gel chromatography.
  • Example 2 In vivo studies [0226] Reagents [0227] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from Avanti Polar Lipids. ALC-0315, ALC-0159 and SM-102 were obtained from MedKoo Biosciences.
  • DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPC 1,2-distearoyl-sn- glycero-3-phosphocholine
  • cholesterol 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from Avanti Polar Lipids.
  • Mass spectrometry analyses were executed using Acquity SQD UPLC/MS (Waters), LTQ Orbitrap XL mass spectrometer (Thermo Scientific), and the ultrafleXtreme MALDI-TOF mass spectrometer (Bruker) at The Ohio State University.
  • Murine myoblast C2C12 cell line (CRL-1772), murine JAWSII dendritic cell line (CRL-3612), murine melanoma B16F10 cell line (CRL-6475), and murine B cell lymphoma A20 cell line (TIB-208) were all obtained from American Type Culture Attorney Docket No.766501:MTST-288PC (240503G-P) Collection (ATCC).
  • C2C12 cells and B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 U/mL).
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • penicillin-streptomycin 50 U/mL
  • JAWSII cells were cultured in RPMI-1640 medium with L-Glutamine, 20% FBS, penicillin-streptomycin (50 U/mL), and 5 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • LNP formulation and characterization [0233] LNPs were formulated using Rapid Nanomedicine System INano L+ (Micro&Nano biologics Technology Ltd.) by mixing an ethanol solution comprising ionizable lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2K ), fXcW P ⁇ dT ⁇ db RXcaPcT b ⁇ [dcX ⁇ ] R ⁇ ]cPX]X]V cWT ⁇ IF9b #,+w ⁇ E' _@ .)+$) > ⁇ a cWT _aT[X ⁇ X]Pah screening phase, LNPs were formulated with newly synthesized ionizable lipids, DO
  • FLuc mRNA was introduced at the mass ratio of ionizable lipid: mRNA set at 10:1.
  • an L16 (4)4 orthogonal array was constructed to optimize the LNP formulations.
  • the lipid ratios and the mRNA to lipid mass ratio remained consistent with previously published formulations.
  • the lipid ratios and the mRNA-to-lipid mass ratio for FDA-approved LNPs remained consistent with those as previously published.41
  • LNP-mRNA formulation was added to cells at the dose of 50ng mRNA per 2!104 cells.
  • mRNA delivery efficiency was evaluated 18 hours after treatment by exposing cells to Bright-Glo luciferase substrate (Promega), followed by quantification of luminescence intensity using Cytation 5 (Biotek). Hydrodynamic diameter, zeta potential, and polydispersity index (PDI) were assessed by NanoZS Zetasizer (Malvern, USA). Encapsulation efficiency was evaluated using Ribogreen assay, while the morphology of LNPs was visualized by Glacios Cryo-TEM (Thermo Scientific, USA).
  • mice were intramuscularly injected with AA LNP-FLuc ⁇ IF9 Pc +)+20 ⁇ V*ZV ⁇ IF9 S ⁇ bT) JXg W ⁇ dab _ ⁇ bc(X]YTRcX ⁇ ]' ,0+v[ ⁇ U ⁇ ([dRXUTaX] substrate (30 mg/mL) was intraperitoneally injected into the mice. Following an eight- minute interval, the mice were imaged using a Xenogen IVIS imaging system to quantify the luminescence signals at the injection sites.
  • mice were intramuscularly immunized by AA2 LNP-Spike mRNA at the mRNA dose of 0.3 mg/kg on day 0.
  • Booster immunization with the same dose was conducted on day 21.
  • mouse blood was collected with anticoagulant (3.8% Sodium citrate) and centrifugated at 1500 x g for 10 min at 4°C to obtain plasma before being stored at -80°C until subsequent analysis.
  • ELISA assay was used to detect the plasma titer of Delta SARS-CoV-2 spike-specific antibodies.
  • Luminex analysis of cytokines and chemokines [0238] Tumoral tissues and blood samples from mice were collected at 6h and 24h post- intratumoral injection of PBS, mRNA-LNP, or replicon-LNP. Tumoral tissues were promptly flash-frozen in liquid nitrogen, followed by pulverization and extraction using RIPA lysis buffer (Thermo Scientific, 89900) supplemented with protease inhibitors (Thermo Scientific, 87785). Murine whole blood was collected in sodium citrate- containing tubes, followed by centrifugation to isolate the serum.
  • Single-cell suspension was prepared from mouse spleen and pan T cells were isolated according to the manufacturer’s instructions (T cell isolation kit, Miltenyi Biotec, Catalog # 130-096-130).
  • Peripheral blood mononuclear cells (PMBCs) from the whole blood were Xb ⁇ [PcTS Qh RT]caXUdVPcX ⁇ ] P]S aTbdb_T]STS X] K RT[[ ⁇ TSXd ⁇ U ⁇ a 1wW) :,1>,+ cd ⁇ ab Ua ⁇ ePRRX]PcTS ⁇ XRT fTaT WPaeTbcTS fWT] cWT [PaVTbc cd ⁇ a SXP ⁇ TcTa TgRTTSTS +)3wR ⁇ P]S dissociated into a single-cell suspension for T-cell isolation (Miltenyi Biotec, Catalog #130-096-730).
  • pan T cells were resuspended in T cell medium and rested for 1wW) > ⁇ [[ ⁇ fX]V cWXb' cWT K RT[[b fTaT bcX ⁇ d[PcTS fXcW R ⁇ ]ca ⁇ [' ; ⁇ 3' ⁇ a ; ⁇ / T_Xc ⁇ _Tb) 9UcTa ,+m,-wW ⁇ U Rd[cdaT' 9AE Tg_aTbbX ⁇ ] fPb cWT] PbbTbbTS Qh U[ ⁇ f Rhc ⁇ Tcah) > ⁇ a ; ⁇ 3+ T cell killing assay, B16F10 cells receiving AA15V LNP-sSE-SCTs or controls were co-cultured with CD8 + T cells isolated from the spleen of the mice vaccinated with AA2 LNP-spike mRNA.
  • mice All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University (2014A00000106) and Icahn School of Medicine at Mount Sinai (IPROTO202200000134), complied with local, state, and federal regulations. In this study, a maximum of five mice were accommodated in each cage Attorney Docket No.766501:MTST-288PC (240503G-P) within a barrier environment, maintaining conditions of approximately 20°C, 45% humidity, and a 12-hour light/12-hour dark cycle.
  • IACUC Institutional Animal Care and Use Committee
  • T cell depletion tumor model 1 ⁇ 10 5 B16F10 cells s.c. inoculated on the right flank of the mice.
  • the tumor-bearing mice were intraperitoneally injected with anti-mouse ; ⁇ 3q' P]cX( ⁇ dbT ; ⁇ /' ⁇ a P]cX(aPc AV?-Q Xb ⁇ ch_T P]cXQ ⁇ SXTb' aTb_TRcXeT[h' ⁇ ] SPh 1 _ ⁇ bc( tumor inoculation.
  • Each antibody was administered every 3 days.
  • One day after the first antibody depletion treatment (Day 7) the tumor-bearing mice received a single i.t. dose of AA15V LNP-sSE-SCTs.
  • mice fTaT X]YTRcTS X]caP_TaXc ⁇ ]TP[[h fXcW P]cX(H ⁇ , #,++wlV* ⁇ dbT' R[ ⁇ ]T5 IEH,(,/' :X ⁇ ORT[[$ P]S P]cX(;KD9/ P]cXQ ⁇ SXTb #,++wlV* ⁇ dbT' R[ ⁇ ]T5 4 ⁇ 4' :X ⁇ ORT[[$) PRW P]cXQ ⁇ Sh fPb administered every 3 days for four doses.
  • Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine an d attenuated peripheral CD8 + T cell responses post SARS-CoV-2 infection. Immunity 56, 864-878.e864 (2023). Collier, A.-r.Y. et al. Differential Kinetics of Immune Responses Elicited by Covid-19 Vaccines. New England Journal of Medicine 385, 2010-2012 (2021). Liu, J. et al. Vaccines elicit highly conserved cellular immunity to SARS-CoV-2 Omicron. Nature 603, 493-496 (2022). Lauring, A.S. et al.
  • Various embodiments of the disclosure include the following: [0252] 1. A polynucleotide encoding a spike epitope-loaded single-chain trimer (SE- SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6. Attorney Docket No.766501:MTST-288PC (240503G-P) [0253] 2.
  • SE- SCT spike epitope-loaded single-chain trimer
  • RNA messenger ribonucleic acid (mRNA), self-amplifying RNA (saRNA), small RNA (sRNA), micro RNA (miRNA), or circular RNA (circRNA).
  • mRNA messenger ribonucleic acid
  • saRNA self-amplifying RNA
  • sRNA small RNA
  • miRNA micro RNA
  • circRNA circular RNA
  • SE-SCT spike epitope- loaded single-chain trimer
  • An expression construct comprising the polynucleotide of any one of claims 1 to 6 or the RNA molecule of any one of claims 7 to 9.
  • 12. A composition comprising the polynucleotide of any one of claims 1 to 6, the RNA molecule of any one of claims 7 to 9, or the expression construct of embodiment 10 or embodiment 11.
  • Attorney Docket No.766501:MTST-288PC (240503G-P) [0264] 13. The composition of embodiment 12, wherein the polynucleotide is formulated, in communication with, or encapsulated with a delivery vehicle.
  • composition of embodiment 13, wherein the delivery vehicle is selected from the group consisting of amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate.
  • the delivery vehicle has an encapsulation efficiency of about 50% to about 100%.
  • composition of any one of claims 13 to 15, wherein the delivery vehicle comprises an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid.
  • the delivery vehicle comprises an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid.
  • the ionizable lipid is an amino alcohol-derived lipid.
  • 18 The composition of embodiment 17, wherein the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers.
  • 19 The composition of embodiment 17, wherein the ionizable lipid comprises a carboxylic acid group.
  • a hydrophobic tail selected from the group consisting of non- biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups.
  • n 1, 2, 3, 4, or 5
  • R R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein Attorney Docket No.766501:MTST-288PC (240503G-P) [0275] 23.
  • the biological preparation of embodiment 29 or embodiment 30, wherein the biological preparations is configured to be administered as an injectable preparation.
  • a kit comprising the biological preparation of any one of claims 28 to 32 and instructions for use.
  • 36 A cell comprising the polynucleotide of any one of claims 1 to 6, the RNA molecule of any one of claims 7 to 9, or the expression construct of embodiment 10 or embodiment 11.
  • 37 An isolated polynucleotide comprising a nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule.
  • 38 The isolated polynucleotide of embodiment 37, wherein the nucleic acid is messenger ribonucleic acid (mRNA) or self-amplifying RNA (saRNA).
  • mRNA messenger ribonucleic acid
  • saRNA self-amplifying RNA
  • composition of embodiment 39 wherein the nucleic acid is saRNA.
  • nucleic acid is saRNA.
  • a method comprising, administering to a subject in need thereof, a therapeutically effective amount of a composition of any one of claims 39 to 47.
  • 50. A kit comprising the composition of any one of claims 39 to 47 and instructions for use.
  • 51. A vector comprising the isolated polynucleotide of embodiment 37 or embodiment 38.
  • a host cell comprising the isolated polynucleotide of embodiment 37 or embodiment 38.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The disclosure provides a polynucleotide comprising a nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule, delivery vehicles for encapsulating the polynucleotide, and methods of treatment.

Description

Attorney Docket No.766501: MTST-288PC (240503G-P) LNP-RNA-BASED ANTIGEN PRESENTATION PLATFORM CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Application 63/658,904, titled “LNP-RNA-MEDIATED ANTIGEN PRESENTATION LEVERAGES SARS-COV-2- SPECIFIC IMMUNITY FOR CANCER TREATMENT”, filed on June 12, 2024. The aforementioned application is incorporated by reference in its entirety, STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with government support under grant no. R35GM144117 awarded by National Institute of General Medical Sciences. The Government has certain rights in the invention. INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING [0003] This application contains a Sequence listing that has been submitted electronically in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, 766501_MTST-288PC.xml, created 11 June 2025 and is 6,895bytes in size. BACKGROUND [0004] The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly transmissible RNA virus, has posed a significant threat to global public health since the outbreak in late 2019. To counteract its morbidity and mortality, a variety of vaccines have been developed. Among them, lipid nanoparticle (LNP)-mRNA vaccines, Pfizer/BioNTech’s BNT162b2 and Moderna’s mRNA-1273, demonstrated extraordinary abilities to elicit protective immunity against SARS-CoV-2 and its multiple variants. Both vaccines encapsulating mRNA encoding a full-length spike protein induce robust spike-specific antibodies and T-cell responses, achieving immune protection against the virus. Notably, to prevent the spread of SARS-CoV-2, a significant portion of the global population has been Attorney Docket No.766501:MTST-288PC (240503G-P) immunized with LNP-mRNA vaccines. Thus, this widely prevalent anti-spike T-cell immunity may be reprogrammed for broader applications, like cancer immunotherapy. [0005] Immunotherapy has highlighted the benefit of harnessing the immune system for cancer treatment. Effective anti-tumor immunity requires the priming of T cells against tumor- associated antigens (TAAs) presented by the major histocompatibility complex (MHC). However, due to tumor heterogeneity and individual differences, it is challenging to identify TAAs displaying high universality, specificity, and immunogenicity. To tackle this challenge, redirecting pre-existent immunity against cancer cells has emerged as a feasible and promising strategy. For example, cytomegalovirus (CMV) epitopes were conjugated with tumor-targeting antibodies, which facilitated their accumulation on cancer cell surface and presentation by MHC class I molecules. This approach enabled to retarget CMV-specific cytotoxic T lymphocytes (CTLs), already primed to attack CMV, to recognize and destroy cancer cells instead. Given the prevalence and efficacy of LNP-mRNA vaccine-induced anti-spike T-cell immunity, the spike-derived epitopes may serve as a new universal antigen that can be leveraged to redirect spike-specific T-cell immunity against tumors. A need still exists for an LNP-RNA-mediated antigen presentation platform that redirects spike-specific T-cell immunity against cancer cells. BRIEF SUMMARY [0006] One aspect of the present disclosure is polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6. [0007] A] P] Pb_TRc' cWT J=(J;K E@; A \^[TRd[T R^\_aXbTb cWT bXV]P[ bT`dT]RT ^U t2m, the \PcdaT _^acX^] ^U \daX]T t2m, and the H-2kb heavy chain. [0008] In an aspect, the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. [0009] In an aspect, the polynucleotide comprises DNA or RNA. In an aspect, the DNA is plasmid DNA (pDNA) or complementary DNA (cDNA). In an aspect, the RNA is messenger ribonucleic acid (mRNA), self-amplifying RNA (saRNA), small RNA (sRNA), micro RNA (miRNA), or circular RNA (circRNA). [0010] One aspect of the disclosure is an RNA molecule comprising a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike Attorney Docket No.766501:MTST-288PC (240503G-P) epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, wherein the RNA molecule is mRNA or saRNA. [0011] A] P] Pb_TRc' cWT J=(J;K E@; A \^[TRd[T R^\_aXbTb cWT bXV]P[ bT`dT]RT ^U t2m, the \PcdaT _^acX^] ^U \daX]T t2m, and the H-2kb heavy chain. [0012] In an aspect, the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. [0013] One aspect of the disclosure is an expression construct comprising the polynucleotide or the RNA molecule. [0014] In an aspect, the expression construct further includes a 3’ UTR, 5’ UTR, a 5’ Cap, a replicase and/or Poly(A) sequence. [0015] One aspect of the disclosure is a composition comprising the polynucleotide, the RNA molecule, or the expression construct. [0016] In an aspect, the polynucleotide is formulated, in communication with, or encapsulated with a delivery vehicle. [0017] In an aspect, the delivery vehicle is selected from the group consisting of amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate. [0018] In an aspect, the delivery vehicle has an encapsulation efficiency of about 50% to about 100%. [0019] In an aspect, the delivery vehicle comprises an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid. [0020] In an aspect, the ionizable lipid is an amino alcohol-derived lipid. [0021] In an aspect, the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers. [0022] In an aspect, the ionizable lipid comprises a carboxylic acid group. [0023] In an aspect, the ionizable lipid comprises two hydroxyl groups introduced to the head group via an ethylidene linker. [0024] In an aspect, the ionizable lipid further comprises a hydrophobic tail selected from the group consisting of non-biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups. Attorney Docket No.766501:MTST-288PC (240503G-P) [0025] In an aspect, the ionizable lipid has a structure of , wherein n = 1, 2, 3, 4, or 5 and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein [0027] In an aspect, n=1 and R=R1, n=1 and R=R2, n=1 and R =R3, n=1 and R=R7, n=1 and R =R9, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=3 and R=R4, n=3 and R=R9, n=4 and R=R2, n=4 and R=R3, n=4 and R=R7, n=4 and R=R9, n=5 and R=R1, n=5 and R=R2, n=5 and R=R3, n=5 and R=R5, n=5 and R=R6, n=5 and R=R7, n=5 and R=R8, or n=5 and R=R9. [0028] In an aspect, the ionizable lipid has a structure of , wherein n = 1, 2, 3, 4, or 5 and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein Attorney Docket No.766501:MTST-288PC (240503G-P) [0030] In an aspect, n=1 and R=R1, n=1 and R=R2, n=1 and R =R6, n=1 and R=R8, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=4 and R=R1, n=4 and R=R2, n=5 and R=R1, n=5 and R=R2, n=5 and R=R6, n=5 and R=R7, or n=5 and R=R9. [0031] In an aspect, the ionizable lipid has a structure of , wherein and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein
Attorney Docket No.766501:MTST-288PC (240503G-P) [0032] . [0033] In an aspect, R =R1, R=R2, R= R6, R=R7, or R=R9. [0034] One aspect of the disclosure is a biological preparation comprising the composition. [0035] In an aspect, the biological preparation is a vaccine, an immunotherapy, or a therapeutic composition. [0036] In an aspect, the biological preparation is configured to be administered to a subject known or suspected to have cancer, or for use in an anti-cancer therapy or cancer immunotherapy. [0037] In an aspect, the biological preparation is configured to be administered as an injectable preparation. [0038] In an aspect, the biological preparation further comprises one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients or other therapeutic agents. [0039] One aspect of the disclosure is a method comprising, administering to a subject in need thereof, a therapeutically effective amount of the biological preparation. [0040] In an aspect, the subject has or is suspected to have cancer. [0041] One aspect of the disclosure is a kit comprising the biological preparation and instructions for use. [0042] One aspect of the disclosure is a cell comprising the polynucleotide, the RNA, or the expression construct. Attorney Docket No.766501:MTST-288PC (240503G-P) BRIEF DESCRIPTION OF THE DRAWINGS [0043] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. [0044] FIG. 1 is an Illustration of LNP-RNA-mediated antigen presentation. E, spike epitope; TM, transmembrane domain. [0045] FIG. 2A is the design and structure of AA ionizable lipids. FIG. 2B shows a general synthesis route of exemplary AA lipids. [0046] FIGs. 3A and 3B are graphs depicting the characterizations of AA LNP-FLuc mRNA. FIG. 3A is a graph showing the particle size and PDI of AA LNP-FLuc mRNA. FIG. 3B is a graph showing encapsulation efficiency and Zeta potential of AA LNP-FLuc mRNA. Data in P P]S Q PaT Ua^\ ] 7 . QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) [0047] FIGs. 4A-4E show the results of an investigation of AA LNPs for Spike mRNA vaccinations. FIG. 4A is the luminescence intensity of AA LNP-FLuc mRNA treated C2C12 cells. FIG. 4B is the luminescence intensity in JAWSII cells. FIG. 4C is the luminescence intensity of the muscle in the lead AA LNP-FLuc mRNA-treated C57BL/6 mice. The intensity was normalized to the ALC-0315 LNP group. FIG.4D are representative images of C57BL/6 mice i.m. treated with AA2 LNP-FLuc mRNA, ALC-0315 LNP-FLuc mRNA, and SM-102 LNP-FLuc mRNA. FIG. 4E shows spike-specific IgG titer in blood drawn from C57BL/6 mice vaccinated with AA2 LNP-spike mRNA, ALC-0315 LNP-spike mRNA or SM-102 LNP- spike mRNA. Data in (FIGs. 4A-4D) are from nw7w. QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP X] (FIG. 4E ) are from nw7w1 QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. *Pw6w+)+0' %%Pw6w+)+,' %%%%Pw6w+)+++,) [0048] FIGs. 5A – 5G depict examination of AA LNPs for mRNA delivery. FIG. 5A shows the luminescence intensity of muscle in the mice i.m. injected with AA2 LNP-FLuc mRNA, ALC-0315 LNP-FLuc mRNA, and SM-102 LNP-FLuc mRNA at 6h after treatment. FIG.5B shows the luminescence intensity of major organs at 6h after treatment. FIG. 5C shows the luminescence intensity of major organs at 24h after treatment. FIG. 5D shows representative images of major organs from FIG.5B and FIG.5C. FIG.5E shows the particle size and PDI of AA2 LNP. f, Encapsulation efficiency and zeta potential of AA2 LNP. FIG. 5G shows aT_aTbT]cPcXeT ;ah^(K=E X\PVT ^U 99- DFH Ua^\ ]w7w. X]ST_T]ST]c Tg_TaX\T]cb) JRP[T QPa Attorney Docket No.766501:MTST-288PC (240503G-P) = 100 nm. Data in a-g are from n = 3 biologically independent samples. Data are presented as \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT fPb STcTa\X]TS Qh ^]T(fPh 9FGM9 U^[[^fTS Qh Dunnett’s multiple comparison test. *P < 0.05, **P < 0.0.1. [0049] FIGs. 6A-6E show AA2 LNP-spike mRNA vaccination generates spike-specific T-cell immunity. FIG.6A is a schematic depicting activation-induced marker (AIM) assay of T cells isolated from spleen and blood of mice vaccinated with AA2 LNP-spike mRNA or SM-102 LNP-spike mRNA. Created in BioRender. Xue, Y. (2025) https://BioRender.com/z43s143. FIGs. 6B and 6D show spike epitope-specific activation of CD4+ T cells isolated from the blood (FIG.6B) and spleen (FIG.6D) of the mice vaccinated with AA2 LNP-spike mRNA or SM-102 LNP-spike mRNA. CE control epitope, SE spike epitope. FIGs. 6C and 6E show epitope-specific activation of CD8+ T cells isolated from the blood (FIG.6C) and spleen (FIG. 6E) of the mice vaccinated with AA2 LNP-spike mRNA or SM-102 LNP-spike mRNA. Gzmb VaP]ih\T :' ;= R^]ca^[ T_Xc^_T' J= b_XZT T_Xc^_T) <PcP PaT Ua^\ ]w7w0 QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb P]S PaT _aTbT]cTS Pb \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT fPb STcTa\X]TS Qh cWT cf^(cPX[TS JcdST]cnb c(cTbc) ])b) ]^c bXV]XUXRP]c Hw8w+)+0' %Hw6w+)+0' %%Hw6w+)+,' %%%Hw6w+)++,' %%%%Hw6w+)+++,) [0050] FIGs. 7A and 7B show flow cytometry gating schemes for AIM assay. FIG. 7A shows a flow cytometry gating scheme of activation markers for CD4+ and CD8+ T cells from blood. FIG.7B shows a flow gating scheme of activation marker for CD4+ T cells and CD8+ T cells from spleen. [0051] FIGs. 8A-8H depict treatment with AA15V LNP-sSE-SCTs enhances spike-specific T cell-mediated killing of cancer cells. FIG. 8A is a screening of AA LNPs in B16F10 cells. FIG. 8B shows construction of saRNA encoding MHC I single-chain trimer (SCT) containing spike epitopes. E spike epitope, TM transmembrane domain. Created in BioRender. Xue, Y. (2025) https://BioRender.com/k91i086. FIG. 8C shows dynamic expression of H- 2Kb+t-\+ in B16F10 cells. FIG.8D shows expression of OVA-H-2Kb+ in B16F10 cells after a single i.t. injection of AA15V LNP-sOP-SCTs. OP, OVA257-264 peptide (SIINFEKL). FIG. 8E shows percentage of apoptotic B16F10 cells in the T cell-mediated killing assay. FIG. 8F shows percentage of dead B16F10 cells in the T cell-mediated killing assay. FIGs.8G and 8H show spike epitope-specific activation of CD4+ T cells (FIG.8G) and CD8+ T cells (FIG.8H) isolated from B16F10 tumors in mice vaccinated with AA2 LNP-spike mRNA. CE control epitope, SE spike epitope. Data in (FIG. 8A) and (FIG. 8C) are from nw7w. QX^[^VXRP[[h independent samples. Data in (FIG. 8D) are from nw7w/ QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP Attorney Docket No.766501:MTST-288PC (240503G-P) in (FIGs. 8E-8H) are from nw7w0 QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT X] #FIGs. 8A, 8C) and (FIGs. 8E, 8F) was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. Statistical significance in (FIG. 8D) and (FIGs. 8G, 8H) was determined by the two-tailed Student’s t-test. *Pw6w+)+0' **Pw6w+)+,' +++,) [0052] FIGs. 9A – 9F depict formulation optimization of AA15 LNPs in B16F10 cells. FIG. 9A is a table for the three rounds of AA15 LNP-FLuc mRNA optimization. Chol, cholesterol. FIG.9B is an orthogonal assay to determine the impact trend of each lipid component in AA15 formulation at four levels. FIG. 9C shows luminescence intensity fold changes of the three rounds of optimization. FIG.9D shows particle size and PDI of AA15V LNP. FIG.9E shows encapsulation efficiency and zeta potential of AA15V LNP. FIG.9F is a representative Cryo- TEM image of AA15V LNP. Scale bar = 100 nm. Data in c-f are from n = 3 biologically X]ST_T]ST]c bP\_[Tb) <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT fPb STcTa\X]TS by one-way ANOVA followed by Dunnett’s multiple comparison test. *P < 0.05, ***P < 0.001, ****P < 0.0001. [0053] FIGs. 10A – 10D show AA15V LNP-sSE-SCTs delivery in B16F10 cells. FIG. 10A shows expression of SCTs with different epitopes in B16F10 cells. FIG. 10B shows flow cytometry gating scheme of expression of SE-SCTs on B16F10 cells. FIG. 10C shows flow cytometry gating scheme of T cell killing assay. FIG. 10D shows a schematic depicting activation-induced marker assay of T cells isolated from B16F10 tumors of the mice vaccinated with AA2 LNP-spike mRNA. Created in BioRender. Xue, Y. (2025) https://BioRender.com/z43s143. Data in a are from n = 3 biologically independent samples. <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) [0054] FIG. 11 shows the flow cytometry gating schemes for intratumoral delivery efficiency of AA15V LNP-sOP-SCTs. The B16F10-FLuc cells were used for tumor model establishment. Live tumor cells were identified by gating for CD45- CD31- FLuc+ cell population. [0055] FIGs. 12A – 12L depict how AA15V LNP-sSE-SCTs redirects spike-specific T-cell immunity to treat tumors and reprogram TME. FIG. 12A is a schematic of the treatment regimen in the B16F10 tumor model. Created in BioRender. Xue, Y. (2025) https://BioRender.com/b49g753. FIG. 12B shows tumor volumes in different groups. FIG. 12C shows survival rates of the mice in the B16F10 tumor model. FIG. 12D bW^fb Tg_aTbbX^] ^U Rhc^ZX]Tb P]S RWT\^ZX]Tb X] cd\^a bP\_[Tb Pc -/wW _^bc(caTPc\T]c) FIG. 12E shows expression level of representative cytokines and chemokines in tumor samples from Attorney Docket No.766501:MTST-288PC (240503G-P) (FIG. 12D). FIG. 12F bW^fb Tg_aTbbX^] ^U Rhc^ZX]Tb P]S RWT\^ZX]Tb X] Q[^^S bP\_[Tb Pc -/wW post-treatment. FIG.12G shows expression level of representative cytokines and chemokines in blood samples from (FIG. 12F). FIGs. 12H-12L show immune cell populations in tumor tissues. Populations of FIG. 12I activated DCs, FIG. 12J activated macrophages, FIG. 12K primed CD4+ T cells, and FIG.12L primed CD8+ T cells in tumor tissues. Data in (FIG. 12B, 12C) and (FIGs. 12H-12L) are from nw7w0 QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP X] (FIGs. 12D-12G) are from nw7w. QX^[^VXRP[[h X]ST_T]ST]c bP\_[Tb) <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT X] #FIG. 12B) was determined by two-tailed Student’s t- test. Statistical significance in (FIG.12C) was determined by the log-rank (Mantel–Cox) test. Statistical significance in (FIGs.12E-12L) was determined by one-way ANOVA followed by Dunnett’s multiple comparison test. n.s. not significant Pw8w+)+0' %Pw6w+)+0' %%Pw6w+)+,' +++,) [0056] FIGs. 13A – 13D, show tumor volumes in the B16F10 tumor model. Tumor volume of individual mice in (FIG.13A) PBS group, (FIG.13B) mSE-SCT group, (FIG.13C) sCE-SCT group and (FIG.13D) sSE-SCT group. Data are from n = 5 biologically independent samples. [0057] FIGs. 14A – 14G show the therapeutic effects of AA15V LNP-sSE SCTs in B16F10 tumor model with T cell depletion. FIG. 14A shows a schematic of the treatment regimen in the B16F10 tumor model. The tumor-bearing mice were intraperitoneally injected with T cell depletion antibodies. FIG. 14B shows tumor volumes in different groups. FIG. 14C shows survival rates of the mice in different groups. Tumor volume of individual mice in (FIG.14D) PBS group, (FIG. 14E) sSE-SCT + CD4 Ab group, (FIG. 14F) sSE-SCT + CD8 Ab group, and (FIG. 14G) sSE-SCT + isotype Ab group. Data in b-g are from n = 6 biologically X]ST_T]ST]c bP\_[Tb) <PcP X] Q PaT _aTbT]cTS Pb \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT X] Q fPb determined by two-tailed Student’s t-test. Statistical significance in c was determined by the log-rank (Mantel–Cox) test. n.s. not significant P > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001. [0058] FIGs. 15A and 15B depict a gating strategy for tumor-infiltrating immune cells. FIG.15A shows flow cytometry gating schemes of macrophages and conventional type 1 and type 2 dendritic cells (cDC1s and cDC2s). FIG.15B shows flow cytometry gating schemes of CD4+ and CD8+ T cells. [0059] FIGs. 16A-16J depict the applicability of AA15V LNP-sSE-SCTs in multiple tumor models and human tumor samples. FIG. 16A shows a schematic of the treatment regimen in B16F10 tumor model treated with the combination of AA15V LNP-sSE-SCTs and ICI. FIG. Attorney Docket No.766501:MTST-288PC (240503G-P) 16B shows tumor volumes over time. FIG.16C shows survival rates of mice bearing B16F10 tumor. FIG.16D shows a schematic of the treatment regimen in A20 tumor model. FIG. 16E shows tumor volumes over time. FIG. 16F shows survival rates of the mice bearing A20 tumor. FIG. 16G shows a schematic depicting ex vivo AA15V LNP-sSE-SCTs delivery in human tumor tissues. Created in BioRender. Xue, Y. (2025) https://BioRender.com/q02v567. FIG. 16H shows expression of H- 2Kb+t-\+ expression in CD45o cells from pediatric glioma dissections after ex vivo treatment with AA15V LNP-sSE-SCTs. FIGs.16I and 16J show H-2Kb+t-\+ in CD45o cells from two separate lung left lower lobe (LLL) adenocarcinoma specimens after ex vivo treatment with AA15V LNP-sSE-SCTs. Data in (FIGs. 16B, 16C) are from nw7w1 #H:J Va^d_$ P]S 3 #J;K groups) biologically independent samples, respectively. Data in (FIGs. 16E, 16F) are from nw7w1 #H:J Va^d_ P]S L]MPR Va^d_b$ P]S 2 #MPR Va^d_b$ QX^[^VXRP[[h X]ST_T]ST]c samples, respectively. Data in (FIG. 16H) and (FIG. 16J) are from nw7w. X]SXeXSdP[ cXbbdT slices. Data in (Fig. 16I) are from nw7w/ X]SXeXSdP[ cXbbdT b[XRTb) <PcP PaT _aTbT]cTS Pb \TP]wkwJ<) JcPcXbcXRP[ bXV]XUXRP]RT X] #FIGs. 16B, 16E) and (FIGs. 16H-16J) was determined by the two-tailed Student’s t-test. Statistical significance in (FIG. 16C) and (FIG. 16F) was determined by the log-rank (Mantel–Cox) test. *Pw6w+)+0' %%Pw6w+)+,' %%%Pw6w+)++,' ****Pw6w+)+++,) [0060] FIGs. 17A – 17D depict tumor volumes in the B16F10 tumor model treated with combination of sSE-SCT and ICI. Tumor volume of individual mice in (FIG.17A) PBS group, (FIG. 17B) sCE-SCT group, (FIG. 17C) sSE-SCT group, and (FIG. 17D) sSE-SCT + ICI group. Data in a-d are from n = 6 or 8 biologically independent samples. [0061] FIGs. 18A – 18E depict tumor volumes in the A20 tumor model. Tumor volume of individual mice in (FIG. 18A) PBS group, (FIG. 18B) sSE-SCT-treated unvaccinated mice group, (FIG. 18C) sSE-SCT + ICI-treated unvaccinated mice group, (FIG. 18D) sSE-SCT- treated vaccinated mice group, and (FIG.18E) sSE-SCT + ICI-treated vaccinated mice group. Data in a-e are from n = 6 or 7 biologically independent samples. [0062] FIG. 19 depicts a gating strategy for human tumor tissues. The CD45- cells were bT[TRcTS U^a `dP]cXUhX]V Tg_aTbbX^] ^U @(-ZQ P]S t-\) Attorney Docket No.766501:MTST-288PC (240503G-P) DETAILED DESCRIPTION I. Introduction [0063] Humans have encountered a multitude of viral infections. Following recovery, the body harbors memory T cells dispersed throughout its entirety. These antiviral memory T cells exhibit notable characteristics: heightened vigilance, rapid response, and cytotoxic abilities. Upon encountering virus-specific epitopes, indicating potential reinfection, these memory T cells become primed, efficiently orchestrating immune defenses at the reinfection site. In contrast to human tumor antigens, which may be patient-specific and non-immunogenic, the epitopes recognized by virus-specific T cells are renowned for their universality and immunogenicity. Therefore, redirecting established antiviral immunity may offer a therapeutic avenue for cancer immunotherapy, although more studies need to determine if the antitumor activity of vaccine-induced T cells mirrors that of T cells generated through natural viral infections. [0064] The widespread use of mRNA vaccines against SARS-CoV-2 has elicited anti-spike T- cell immunity on a global scale, establishing spike epitopes as a viable antigenic target. Thus, the inventors constructed RNA-encoding spike epitope-loaded SCTs (SE-SCT) and explored the therapeutic potential of leveraging LNP-RNA formulation-mediated antigen presentation to redirect spike-specific T-cell immunity against cancer. The inventors synthesized and evaluated a library of amino alcohol- or amino acid-derived (AA) ionizable lipid materials, identifying AA2, the optimal LNP formulation for spike mRNA vaccination, and AA15V, the optimal LNP formulation for RNA delivery in cancer cells. AA15V LNP-sSE-SCTs enabled the presentation of SE-SCT on cancer cell surface, expediting the recognition by spike-specific T cells induced through AA2 LNP-spike mRNA vaccination. Such redirection of spike-specific T-cell immunity facilitated cancer cell death induced by T-cell-mediated cytotoxicity. Moreover, i.t. administration of AA15V LNP-sSE-SCTs reprogrammed the TME by eliciting antitumor phenotypes of immune cells and pro-inflammatory cytokines as well as chemokines. As a result, a single treatment significantly suppressed the growth of melanoma and lymphoma tumors and extended the survival of AA2 LNP-spike mRNA-vaccinated mice, particularly when in combination with ICI treatments. [0065] The single-chain trimer (SCT) MHC class I molecule, which consists of a polymorphic WTPeh RWPX] #@;$' P t- \XRa^V[^Qd[X] #t-\$ [XVWc RWPX]' P]S P R^eP[T]c[h Q^d]S T_Xc^_T connected by a linker, has been developed to induce antigen-specific immune responses. The Attorney Docket No.766501:MTST-288PC (240503G-P) covalent binding of an epitope in the SCT allows stable expression of defined epitope-loaded MHC class I molecules on the cell surface. Intratumoral (i.t.) delivery of RNA encoding spike epitope-loaded SCTs (SE-SCT) may retarget the anti-spike immunity against tumors in mice immunized by SARS-CoV-2 vaccines (FIG. 1). The inventors designed a series of mRNAs and self-amplifying RNAs (saRNAs) encoding SE-SCT. To effectively deliver RNA in vivo, the inventors synthesized a library of amino alcohol- or amino acid-derived ionizable lipid materials (AA lipids) containing various headgroup structures (differ in types and quantities of hydrogen-donors) and types of hydrophobic tails (variable in length, biodegradability, and location of biodegradable groups). Through comprehensive explorations, the inventors identified two lead LNP formulations, AA2 and AA15V. The FDA-approved LNP-mRNA vaccines, Moderna’s mRNA-1273 and Pfizer/BioNTech’s BNT162b2, utilize SM-102 and ALC-0315 as ionizable lipids, respectively. Compared with the FDA-approved LNPs (SM-102 and ALC-0315), AA2 LNP showed higher Spike mRNA delivery efficiency post intramuscular (i.m.) injection, leading to stronger vaccination efficacy against SARS-CoV-2. Meanwhile, the mice vaccinated with a prime-boost regimen generated robust spike-specific CD8+ T cell immunity. Moreover, AA15V LNP demonstrated more potent RNA delivery efficiency to both mouse and human cancer cells than the FDA-approved LNPs. As a result, a single i.t. treatment with AA15V LNP-saRNA encoding SE-SCT significantly inhibited tumor progression and extended the overall survival of the tumor-bearing mice that were immunized by AA2 LNP- Spike mRNA vaccines. Additionally, AA15V LNP-sSE-SCTs facilitated SE-SCT expression in ex vivo human cancer samples, suggesting the potential for future clinical applications. [0066] The inventors have developed an LNP-RNA-based antigen presentation platform designed to redirect spike-specific T-cell immunity against cancer. The clinical potential of this platform is highlighted by two key aspects: first, AA2 LNP demonstrates superior efficacy for mRNA vaccine delivery in vivo compared to FDA-approved LNPs; second, AA15V LNP facilitates the delivery of sSE-SCTs to human cancer samples. Considering that a significant portion of the global population has already developed SARS-CoV-2 T cell memory, this strategy provides a new avenue for cancer immunotherapy. [0067] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. Attorney Docket No.766501:MTST-288PC (240503G-P) II. Definitions [0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated. [0069] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. [0070] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. [0071] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention. [0072] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. [0073] By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements. [0074] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and/or" unless the Attorney Docket No.766501:MTST-288PC (240503G-P) content clearly dictates otherwise. The term "and/or" means any one or more of the items in the list joined by "and/or". As an example, "x and/or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and/or y" means "one or both of x and y". As another example, "x, y, and/or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and/or z" means "one or more of x, y and z". [0075] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range. [0076] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects. [0077] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is +/-10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents Attorney Docket No.766501:MTST-288PC (240503G-P) to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. [0078] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. [0079] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations. [0080] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest. [0081] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. A "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A “syndrome” is a recognizable complex of symptoms and physical findings that occur together and suggest the presence of a certain disease or disorder or an increased chance of developing the disease or disorder. A disease, disorder, or syndrome is "alleviated" if the severity of a sign or symptom of the disease, disorder, or syndrome, or the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced. [0082] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein Attorney Docket No.766501:MTST-288PC (240503G-P) includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child). [0083] The terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and/or decrease the number, frequency, or severity of clinical symptoms and/or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms “treatment of a disease” and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder. [0084] The terms “prophylactic”, “preventive”, “preventing”, and “prevention” refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods. [0085] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms. Attorney Docket No.766501:MTST-288PC (240503G-P) [0086] The term “administered” as used herein, means administration of an effective amount of, for example, at least one nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule and any another other additional agent for treatment. [0087] An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and/or ameliorating a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially lessening the chances of a disease or condition in a subject. [0088] The term "nucleic acid" or “polynucleotide” refers to any polymeric chain of nucleotides. The term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). In some embodiments, a nucleic acid comprises of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. [0089] More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows Attorney Docket No.766501:MTST-288PC (240503G-P) for base pairing and base stacking, such as is found in DNA and RNA. In particular aspects, the polynucleotide comprises an mRNA. [0090] Nucleotides are referred to by their commonly accepted single-letter codes. Unless ^cWTafXbT X]SXRPcTS' ]dR[TXR PRXSb PaT faXccT] [TUc c^ aXVWc X] 0s c^ .s ^aXT]cPcX^]) FdR[T^cXSTb PaT referred to herein by their commonly known one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil. [0091] Messenger RNA (mRNA): As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and is capable of being translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. [0092] The term “payload” maybe used herein may consist of nucleic acids and/or proteins. A delivery molecule is used to deliver a nucleic acid payload (e.g., DNA and/or RNA). The nucleic acid payload can be any nucleic acid of interest, for example, the nucleic acid payload can be linear or circular, a plasmid, a viral genome, RNA or DNA. The DNA may be plasmid DNA (pDNA) or complementary DNA (cDNA). It may be coding RNA or non-coding RNA. In some embodiments, the RNA may be mRNA, guide RNA, self-amplifying RNA (saRNA),small interfering RNA or short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA),piwi RNA (piRNA), small nuclear RNA or U-rich snRNA (snRNA), Small nucleolar RNA (snoRNA), signal recognition particle RNA (7SL), Y RNAs, small Cajal body-specific RNA (scaRNA), long intergenic ncRNA (lincRNA), natural antisense transcript (NAT), circular RNA (circRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA), etc. In some cases, the nucleic acid payload is an RNA interference (RNAi) agent or a DNA template encoding an RNAi agent. In some cases, the nucleic acid payload is a locked nucleic acid (LNA) molecule. In some cases, the nucleic acid payload may encode a protein of interest. [0093] Identity: As used herein, the term “identity” refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned Attorney Docket No.766501:MTST-288PC (240503G-P) for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. [0094] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. [0095] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls. [0096] As used herein, “delivery vehicle” refers to any substance that facilitates, at least in part, the in vivo, in vitro, or ex vivo delivery of a polynucleotide (e.g., therapeutic polynucleotide) to cells or tissues (e.g., tumors, etc.). Referring to something as a delivery vehicle need not exclude the possibility of the delivery vehicle also having therapeutic effects. Some versions of a delivery vehicle may provide additional therapeutic effects. In some versions, a delivery vehicle may be a peptoid molecule, such as an amino-lipidated peptoid molecule, that may be used to at least partially encapsulate mRNA. The term “DV” may also be used herein as a shorthand for “delivery vehicle.” In some aspects, the mRNA for use in the delivery vehicle complexes herein comprise an mRNA comprising at least one region encoding a peptide (e.g., a polypeptide), or protein, or functional fragment of the foregoing. As used herein, “functional fragment” refers to a fragment of a peptide, (e.g., a polypeptide), or protein that retains the ability to induce an immune response. Attorney Docket No.766501:MTST-288PC (240503G-P) [0097] The term "amphipathic molecules" refers to molecules comprising both hydrophilic and hydrophobic regions, enabling interaction with both aqueous and lipid-based environments. [0098] The term "amino-lipidated peptides" refers to peptides that have been chemically conjugated with lipid moieties via amino groups. [0099] The term "lipid nanoparticle" (LPN) refers to a nanoscale structure composed of lipids, which may encapsulate or associate with the composition. [0100] The term "polymeric compound" refers to a chemical compound comprising repeating monomeric units. [0101] The term "peptoid" refers to a synthetic oligomer or polymer structurally analogous to peptides, wherein side chains are attached to the nitrogen atom of the backbone rather than the alpha-carbon. [0102] The term "lipoid" refers to lipid-like substances, including but not limited to phospholipids, sterols, glycolipids, and other fat-associated compounds. [0103] The term "liposome" refers to a vesicular structure composed of one or more concentric phospholipid bilayers encapsulating an aqueous core. [0104] The term "lipoplex" refers to a complex formed between cationic lipids and nucleic acids. [0105] The term "conjugate" refers to a molecular entity formed by the chemical linkage of two or more distinct components. [0106] The term “delivery” or "administering" as used herein, refers to the physical introduction of an agent such as the composition disclosed herein into a subject or cell, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration known to those skilled in the art for the formulations disclosed herein to a subject include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a non-parenteral Attorney Docket No.766501:MTST-288PC (240503G-P) route, e.g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. [0107] As used herein, the term “biological preparation” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. [0108] As used herein, the term “vaccine” refers to a biological preparation that induces or enhances an immune response. [0109] The term "immunotherapy" refers to therapeutic interventions that modulate the immune system for the treatment or prevention of disease [0110] The term “therapeutic composition” refers to a biologically active formulation comprising one or more therapeutic agents designed to prevent, treat, or manage disease conditions. [0111] As used herein, the terms “efficacy,” “efficiency,” or grammatical equivalents refers to an assessment of a biologically relevant endpoint. Specifically, efficiency herein relates to the encapsulation of the composition. [0112] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein. III. Encapsulated Spike Protein Encoding Nucleic Acids [0113] One aspect of the disclosure relates to a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule. A spike epitope refers to a specific part of the spike protein of a virus that is recognized by the immune system. Table 1 lists exemplary spike epitopes. [0114] Table 1: Spike epitopes and spike derived epitopes Attorney Docket No.766501:MTST-288PC (240503G-P) [0115] In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:1. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:2. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:3. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:4. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:5. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:6. In an embodiment, the SE-SCT MHC I molecule includes the spike epitope of SEQ ID NO:7. [0116] In some aspects, the SE-SCT MHC I molecule also comprises the signal sequence of t2\' cWT \PcdaT _^acX^] ^U \daX]T t2m, and the H-2kb heavy chain. In another aspect, the SE- SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. [0117] FIG. 8B is an exemplary SE-SCT. Beginning with the amino terminus, the SE-SCT X]R[dSTb cWT bXV]P[ bT`dT]RT ^U t2m, the spike epitope, a first linker, the mature portion of \daX]T t2m, the second linker, and the H-2kb heavy chain. [0118] In some embodiments, the polynucleotide may may be configured for administration directly, configured for administration in a composition including other polynucleotides, be formulated with a delivery vehicle, be encoded in one or more polynucleotides for expression in a cell, and/or may be encoded in DNA, RNA, or mRNA for administration. In some embodiments, the polynucleotide comprises DNA or RNA. In some embodiments, the polynucleotide comprises DNA, wherein the DNA is pDNA or cDNA. In some embodiments, the polynucleotide comprises RNA, wherein the RNA is mRNA, siRNA, miRNA, saRNA, or circRNA. [0119] One aspect of the disclosure includes an RNA molecule comprising a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, wherein the RNA molecule is mRNA or saRNA. Attorney Docket No.766501:MTST-288PC (240503G-P) [0120] One aspect of the disclosure includes an expression construct including the polynucleotide sequence or the RNA molecule according to an aspect of the disclosure. “Expression construct” refers to an engineered DNA or RNA molecule designed to drive the production of a specific protein or RNA in a host cell. It typically contains regulatory elements such as a promoter to initiate transcription, an enhancer to increase expression levels, a coding sequence for the gene of interest, and a terminator to end transcription. [0121] In example aspects, an expression construct includes a 3’ UTR, an effector region encoding a protein (e.g., mRNA), and a 5’ UTR. Additional features like selectable markers, tags for protein purification, or sequences for proper localization can also be included. [0122] In some embodiments, the expression construct may have the following formula: 5’UTR—replicase—coding region—3’ UTR—Poly(A) [0123] where “UTRs” are the untranslated regions located at the 5’ and 3’ ends of an RNA construct, and “PolyA” refers to the polyadenylation site of the RNA. [0124] In some embodiments, the expression construct may have the following formula: 5’ cap -5’UTR—replicase—coding region—3’ UTR—Poly(A). [0125] Generally the term “5’ UTR” refers to a part of the nucleic acid molecule which is located 5' (upstream) of the open reading frame of the RNA. In certain aspect, the RNA may include mRNA, siRNA, miRNA, saRNA, or circRNA. In the case of saRNA, the open reading frame encodes the viral non-structural proteins while the sequence of interest is encoded in the subgenomic fragment of the viral RNA. Thus, the 5’UTR is upstream of the nsP1 open reading frame. In addition, the subgenomic RNA of the saRNA has a 5’UTR. Thus, the subgenomic RNA containing a sequence of interest encoding a protein of interest contains a 5’UTR. Typically, the 5’UTR starts with the transcriptional start site and ends with one nucleotide before the start codon of the open reading frame. The 5’UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract. The 5’UTR may be post transcriptionally modified, for example by addition of a 5'-CAP. [0126] As used herein “5'-CAP” (or a 5'-CAP-Structure) is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide, added to the 5' end of an mRNA molecule. In certain implementations, the 5'-CAP is added using a 5'-5'-triphosphate linkage (also named m7GpppN). In the context of the present disclosure, a 5' CAP structure may also be formed in Attorney Docket No.766501:MTST-288PC (240503G-P) chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using cap analogues, or a cap structure may be formed in vitro using capping enzymes (e.g., commercially available capping kits). [0127] Generally, the term "3’UTR " refers to a part of the nucleic acid molecule which is located 3' (i.e. "downstream") of an open reading frame and which is not translated into protein. Typically, a 3’UTR is the part of an RNA which is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA. In the context of the present disclosure, the term 3’UTR may also comprise elements, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence. A 3’UTR of the RNA is not translated into an amino acid sequence. With respect to srRNA, the 3’UTR sequence is generally encoded by the viral genomic RNA, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. [0128] “Poly(A)” or “Poly(A) tail” refers to the polyadenylation site of the mRNA. The poly(A) tail is a stretch of adenine nucleotides added to the 3' end of eukaryotic messenger RNA (mRNA) molecules during post-transcriptional processing. This modification is catalyzed by the enzyme poly(A) polymerase and plays a critical role in mRNA stability, nuclear export, translation efficiency, and protection from exonucleases. The length of the poly(A) tail can influence gene expression, as longer tails typically enhance translation, while shorter tails may signal mRNA decay. Additionally, the poly(A) tail interacts with poly(A)- binding proteins (PABPs), which help mediate its functions. [0129] “Replicase” refers to an enzyme that catalyzed the synthesis of a complementary RNA molecule using an RNA template. [0130] Ionizable lipids play a pivotal role in the encapsulation of mRNA molecules by delivery vehicles such as lipid nanoparticles (LNPs). Their positive head groups interact electrostatically with the negatively charged phosphate groups of the mRNA, facilitating the complexation of the lipid with the mRNA and the formation of nanoparticles. Furthermore, it has been observed that the establishment of hydrogen bonds between the hydroxyl or amide groups present in these lipids and mRNA molecules can significantly enhance the entrapment of mRNA, thereby leading to improved mRNA expression. The state-of-art ionizable lipids Attorney Docket No.766501:MTST-288PC (240503G-P) developed for COVID-19 vaccines, ALC-0315 and SM-102, incorporate hydroxyl groups in their structure, demonstrating superior mRNA delivery efficacy. In this work, the inventors designed a library of amino alcohol- or amino acid-derived ionizable lipids, which contain hydroxyl or carboxylic moieties as the hydrogen bond donor. To enhance the chemical diversity of AA lipids, the inventors fine-tuned the quantities of hydrogen-bond donors in the head groups, adjusted the length of carbon spacer between hydrogen-bond donors and head group, and varied the types of hydrophobic tails (FIG.2A). Particularly, AA1-AA26 lipids are amino alcohol-type lipids featuring a hydroxyl group connected with the amino head groups via varying carbon spacer. AA27-AA45 lipids are amino acid-type lipids incorporating a carboxylic acid group. While, in AA46-AA50 lipids, two hydroxyl groups were introduced to the head group via an ethylidene linker. To further augment the chemical diversity of the lipid library, four classes of hydrophobic tails were attached to the amino head groups, including non-biodegradable hydrocarbon tails (tail R1), and biodegradable tails containing ester (tail R2), carbonate ester (tail R3-R6), and acid-labile acetal groups (tail R7-R9). The presence of these diverse hydrophobic tails in lipids can greatly affect their biodegradability, particle formation, and interactions with biological membranes. The general synthesis routes of AA lipids are shown in FIG.2B. [0131] Depending on the delivery methods desired, other delivery vehicles may be used including, but not limited to amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a cationic lipid nanoparticle, a polymeric compound, or a conjugate. In some embodiments, the polynucleotide, the RNA molecule, and/or the expression construct is formulated, in communication with, or encapsulated with a delivery vehicle. [0132] In some aspects, the delivery vehicle partially encapsulates the polynucleotide, the RNA molecule, and/or the expression construct. In other aspects, the delivery vehicle fully encapsulates the polynucleotide, the RNA molecule, and/or the expression construct. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 50%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 55%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 60%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 65%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 70%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 75%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 80%. In some Attorney Docket No.766501:MTST-288PC (240503G-P) embodiments, the delivery vehicle has an encapsulation efficiency of about 85%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 90%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 95%. In some embodiments, the delivery vehicle has an encapsulation efficiency of about 95%. [0133] Delivery vehicle encapsulation efficiency refers to the ability of the delivery vehicles to successfully encapsulate and protect the polynucleotide, RNA molecule, or expression construct. High encapsulation efficiency is critical for maximizing the delivery of the active compound to target cells and minimizing the loss of payload during formulation or storage. Several factors influence encapsulation efficiency, including the ratio of ionizable lipids to other lipids, the method of nanoparticle preparation (e.g., ethanol injection or microfluidics), and the physicochemical properties of the payload, such as size and charge. [0134] In some aspects, the delivery vehicle comprises an ionizable lipid, one or more of an anionic or zwitterionic component, such as a phospholipid; a neutral lipid, such as a sterol; and a shielding lipid, such as a PEGylated lipid. In various aspects, the delivery vehicle compositions further comprise an additional anionic or zwitterionic component (e.g., a phospholipid), neutral lipid (e.g., a sterol), or shielding lipid (e.g., a PEGylated lipid). [0135] In an aspect, the ionizable lipid is an amino alcohol-derived lipid. In some embodiments, the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers. [0136] In some embodiments, the ionizable lipid comprises a carboxylic acid group. [0137] In some embodiments, the ionizable lipid comprises two hydroxyl groups introduced to the head group via an ethylidene linker. [0138] In some embodiments, the ionizable lipid further comprises a hydrophobic tail selected from the group consisting of non-biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups. [0139] In some embodiments, the ionizable lipid has a structure of
Attorney Docket No.766501:MTST-288PC (240503G-P) [0141] In some embodiments n=1 and R=R1, n=1 and R=R2, n=1 and R =R3, n=1 and R=R7, n=1 and R =R9, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=3 and R=R4, n=3 and R=R9, n=4 and R=R2, n=4 and R=R3, n=4 and R=R7, n=4 and R=R9, n=5 and R=R1, n=5 and R=R2, n=5 and R=R3, n=5 and R=R5, n=5 and R=R6, n=5 and R=R7, n=5 and R=R8, or n=5 and R=R9. [0142] In some embodiments, the ionizable lipid has a structure of , wherein n = 1, 2, 3, 4, or 5 and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein
Attorney Docket No.766501:MTST-288PC (240503G-P) [0144] In some embodiments, n=1 and R=R1, n=1 and R=R2, n=1 and R =R6, n=1 and R=R8, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=4 and R=R1, n=4 and R=R2, n=5 and R=R1, n=5 and R=R2, n=5 and R=R6, n=5 and R=R7, or n=5 and R=R9. [0145] In some embodiments, the ionizable lipid has a structure of , wherein and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein
Attorney Docket No.766501:MTST-288PC (240503G-P) [0147] In some embodiments, R =R1, R=R2, R= R6, R=R7, or R=R9. [0148] To assess AA lipids, AA LNPs were formulated with 1,2-dioleoyl-sn-glycerol-3- phosphoethanolamine (DOPE), cholesterol, and DMG-PEG2k with firefly luciferase (FLuc) \IF9) KWT 99 DFH(>DdR \IF9 SXb_[PhTS _PacXR[T bXiTb QTcfTT] 31)/wkw/)1w]\ c^ ,0/)+wkw/)0w]\' fXcW P _^[hSXb_TabXch X]STg #H<A$w6w+). #FIG. 3A). These LNPs were slightly _^bXcXeT[h RWPaVTS P]S ^QcPX]TS \IF9 T]RP_bd[PcX^] TUUXRXT]RXTb aP]VX]V Ua^\ -4)2wkw0)." c^ 34)3wkw,)4" #FIG. 3B). C2C12 myoblasts and JAWSII dendritic cells (DCs) were treated with AA LNPs to evaluate their mRNA delivery efficiency. These two cell lines were selected because i.m. vaccination tends to target muscle cells and antigen-presenting cells (APCs), including DCs for antigen presentation and immune stimulation. Among all these AA lipids, lipids with branched ester tails (tail R2), especially lipid AA2, exhibited superior mRNA delivery efficiency compared to other AA lipids and clinically approved SM-102 and ALC- 0315 LNPs at an equivalent mRNA dose (FIGs.4A and 4B). This enhanced performance may be attributed to the hydrogen bond-forming capability of the hydroxyl group in the amino head groups of AA lipids. Moreover, the increased delivery efficiency of lipids with branched tails R2 may be attributed to their potential to adopt a more cone-shaped structure, facilitating improved endosomal escape compared to AA lipids with linear tails (such as hydrocarbonic, carbonated, and acetal tails). Conversely, the reduced mRNA delivery efficiency observed among AA46-AA50 lipids could be explained by the fact that their two-tailed core structure is less likely to adopt a cone-shaped structure compared to the three-tailed lipids. Additionally, Attorney Docket No.766501:MTST-288PC (240503G-P) the diminished mRNA delivery efficiency of AA27-AA50 lipids may be associated with the deprotonation of their carboxylic acid group under physiological pH conditions, leading to decreased hydrogen bond donation capacity of lipids and limited mRNA entrapment (FIG. 3B). Based on the in vitro results, the inventors selected 18 lead AA LNPs with over 2-fold higher mRNA delivery efficiency than the SM-102 LNP in both cell lines for in vivo evaluations. These AA LNPs encapsulating FLuc mRNA were i.m. administered into mice, P]S cWT [d\X]TbRT]RT X]cT]bXch Pc cWT X]YTRcX^] bXcT fPb \TPbdaTS PUcTa 1wW) 9\^]V P[[ cWT 99 LNPs tested, AA2 LNP displayed the most potent mRNA delivery efficiency with 5.4-fold and 2.4-fold higher luminescence intensity than ALC-0315 LNP and SM-102 LNP, respectively (FIG. 4C). Meanwhile, AA2 LNP showed lower off-target delivery to liver and spleen compared to ALC-0315 LNP and SM-102 LNP (FIGs. 5B-5D). Notably, the luminescence X]cT]bXch ^U 99- DFH R^d[S bcX[[ QT ^QbTaeTS Pc cWT X]YTRcX^] bXcT PUcTa -/wW #FIG. 4D; FIG. 5A). Based on the above results, the inventors selected AA2 LNP for detailed characterizations. KWT 99- DFH TgWXQXcTS P _PacXR[T bXiT ^U ,+3)1wkw.)2w]\ P]S P] \IF9 T]RP_bd[PcX^] TUUXRXT]Rh ^U 34)+wkw,)/" #FIGs. 5E and 5F). The particle was slightly positively charged and displayed a spherical morphology in Cryo-TEM images (FIGs.5F and 5G). [0149] To further evaluate the vaccination efficacy of AA2 LNP, the inventors synthesized the mRNA encoding the full-length spike protein from SARS-CoV-2 and vaccinated mice via a prime-boost regimen. AA2 LNP generated 4.7-fold and 3.4-fold higher anti-spike IgG antibody titer than ALC-0315 LNP and SM-102 LNP, respectively (FIG.2E). Importantly, activation- induced marker (AIM) assays revealed that AA2 LNP vaccination elicited spike-specific T cell populations in the blood and spleen (FIG.6A). Particularly, to measure spike-specific CD4+ T cells in the vaccinated mice, the inventors stimulated peripheral blood mononuclear cells (PBMCs) with a mixture of spike epitopes (SE; SEQ ID NO: 1 SGWTFGAGAALQIPF and SEQ ID NO: 2 VTWFHAIHVSGTNGT). The random peptide sequence SEQ ID NO: 7 AAAAFAAL was used as a negative control epitope (CE). Compared to treatment with CE, spike epitopes significantly increased activation markers of CD4+ T cells from blood (FIG.6B; FIG. 7A). Similarly, spike epitopes elicited higher percentages of CD137+CD134+ and CD154+CD134+ in CD4+ T cells from the spleen (FIG. 6D; FIG. 7B). The CD4+ T cells isolated from the blood and spleen of mice vaccinated with SM-102 LNP also showed increased activation markers following stimulation with spike epitopes (FIGs. 6D, 6B). However, the activation efficiency was lower than those observed with AA2 LNP. These results demonstrated the effective development of spike-specific CD4+ T cells induced by AA2 Attorney Docket No.766501:MTST-288PC (240503G-P) LNP-spike mRNA vaccinations. To detect spike-specific CD8+ T cells, the inventors included one additional methodology, intracellular cytokine staining (ICS). Four spike-derived epitopes (SE; SEQ ID NO: 3 VNFNFNGL, SEQ ID NO: 4 SIIAYTMSL, SEQ ID NO: 5 VVFLHVTYV, SEQ ID NO: 6 VVLSFELL) were selected for CD8+ T cell activation based on their algorithm-predicted MHC I binding affinity and reported applications. In both blood and spleen, AIM and ICS detected obvious CD8+ T cell responses to spike epitopes, with AA2 LNP-spike mRNA vaccination inducing increased levels of CD69+, CD69+CD137+' A>F(u+, KF>(q+, and granzyme B+ compared to SM102 LNP (FIG. 6C and 6E; FIGs. 7A and 7B). These results are consistent with the in vivo mRNA delivery efficiency and spike-specific IgG titers, indicating that vaccinations with AA2 LNP-spike mRNA generate stronger spike- specific T-cell immunity compared to SM-102 LNP-spike mRNA. This provides a solid foundation for the utilization of SE-SCTs to redirect spike-specific T-cell immune responses against tumors. [0150] Next, the inventors evaluated the delivery efficiency of AA LNP-FLuc mRNA in B16F10 melanoma cells. Similarly, AA lipids with branched ester tails (tail R2) exhibited higher mRNA delivery efficiency than other tail types of AA lipids in B16F10 cells. The lead material, AA15 LNP, induced the highest luminescence intensity, which was over 25.6-fold and 5.3-fold greater than ALC-0315 LNP and SM-102 LNP, respectively (FIG.8A). To further increase mRNA delivery capacity of AA15 LNP, the inventors fine-tuned the molar ratios for each formulated lipid using an L16 orthogonal table (FIG. 9A). The inventors also optimized the mass ratio of AA15 lipid to mRNA in the formulation. The lead formulation AA15V LNP demonstrated 1.5-fold and 3.1-fold higher luminescence intensity compared to the 1st-round top orthogonal formulation AA15I and the original formulation AA15, respectively (FIGs.9B, 9C$) KWT WhSa^Sh]P\XR SXP\TcTa ^U 99,0M DFH fPb ,+-).wkw/),w]\ fXcW P H<Aw6w+),0) GeTa 85% mRNA was encapsulated in AA15V LNP, and the particle exhibited a slightly positive charge and spherical morphology (FIGs. 9D-9F). Accordingly, AA15V LNP was chosen for mRNA delivery to cancer cells in the following studies. [0151] The covalent binding of an epitope in a single-chain trimer (SCT) MHC I molecule enables stable expression of defined epitope-loaded MHC I molecules on the cell surface. To express spike epitope (SE)-loaded single-chain trimer (SE-SCT) MHC I molecule on B16F10 cells, the inventors constructed mRNAs and saRNAs encoding four distinct SE-SCTs with spike epitopes (SEQ ID NO: 3 VNFNFNGL, SEQ ID NO: 4 SIIAYTMSL, SEQ ID NO: 5 VVFLHVTYV, SEQ ID NO: 6 VVLSFELL, FIG. 8B). The inventors refer to mRNAs and Attorney Docket No.766501:MTST-288PC (240503G-P) saRNAs encoding SE-SCTs as mSE-SCTs and sSE-SCTs, respectively. The saRNA encoding SCT with SEQ ID NO: 7 AAAAFAAL, the control epitope, was named sCE-SCTs. Both mRNAs encoding SE-SCTs (mSE-SCTs) and saRNAs encoding SE-SCTs (sSE-SCTs) ST[XeTaTS Qh 99,0M T]PQ[TS ^eTa 3+" :,1>,+ RT[[b c^ Tg_aTbb @(-CQ&t-\& fXcW X]SXeXSdP[ T_Xc^_Tb Pc -/wW #FIG. 10A). To evaluate dynamic expression of SE-SCTs, B16F10 cells were treated with AA15V containing the four-mixed mSE-SCTs or sSE-SCTs. Although both caTPc\T]cb X]SdRTS ^eTa 4+" Tg_aTbbX^] ^U J=(J;Kb Pc -/wW #FIG. 8C; FIG. 10B), the sSE- J;Kb Va^d_ aTcPX]TS 14)+wkw-)4" @(-CQ&t-\& :,1>,+ RT[[b ^] SPh . fWX[T cWT \J=(J;Kb Va^d_ bW^fTS P bWPa_[h STRaTPbTS [TeT[ c^ 04),wkw-)0" ^] SPh - P]S UdacWTa c^ 0)3wkw,)3" ^] day 3. To assess the expression of antigen-specific H-2Kb molecules on tumor cells following i.t. injection of AA15V LNP encapsulating SCT-saRNAs in vivo, the inventors constructed saRNAs encoding OVA257-264 peptide-SCTs (OP-SCTs), enabling the quantification of OVA-H-2Kb+ live tumor cells using established antibodies. The inventors established the tumor model using B16F10-FLuc cells and found a single i.t. injection of 99,0M DFH(bGH(J;Kb aTbd[cTS X] -2)3wkw2)2" ^U GM9(@(-CQ& [XeT :,1>,+ RT[[b fXcWX] tumor tissues (FIG.8D; FIG.11). This result indicated our AA15V LNP-SCT saRNAs could effectively induce expression of antigen-specific H-2Kb molecules on cancer cells in vivo. [0152] Cytotoxicity of spike-specific CD8+ T cells was evaluated by isolating CD8+ T cells from the spleens of vaccinated mice, which were then co-cultured with B16F10 cells that had been pretreated in vitro with PBS, AA15V LNP-sCE-SCTs, or AA15V LNP-sSE-SCTs. These CD8+ T cells exhibited cytotoxicity against B16F10 cells pretreated with AA15V LNP-sSE- SCTs (FIGs. 8E, 8F; FIG. 10C). In contrast, no notable T cell-mediating cell killing was observed in the AA15V LNP-sCE-SCTs group compared to PBS control. To assess whether spike-specific T cells could infiltrate tumors, C57BL/6 mice vaccinated with AA2 LNP-spike mRNA were inoculated with B16F10 cells, after which T cells were isolated from melanoma tissues and later co-cultured with CD4- and CD8-specific spike epitopes (FIG. 10D). AIM assays revealed that specific epitopes targeting MHC II and MHC I stimulated tumor- infiltrating T cells, resulting in increased levels of CD69+CD134+ in CD4+ T cells and CD69+CD137+ in CD8+ T cells, respectively (FIG. 8G, 8H). These findings indicate the potential of AA15V LNP-sSE-SCTs to retarget spike-specific T-cell immunity against tumors in vaccinated mice. [0153] Compared to AA15V LNP-mSE-SCTs, the enhanced antitumor effects observed with AA15V LNP-sSE-SCTs are likely due to the prolonged translation and increased Attorney Docket No.766501:MTST-288PC (240503G-P) immunogenicity of saRNAs. First, treatment with AA15V LNP-sSE-SCTs resulted in approximately 69.0% of B16F10 cells expressing SE-SCTs by day 3, in contrast to just 5.8% expression observed with AA15V LNP-mSE-SCTs (FIG. 8C), highlighting the extended translation capacity of saRNA. Second, AA15V LNP-sCE-SCTs encoding control epitopes demonstrated measurable antitumor efficacy (FIGs. 12B, 12C), potentially due to the immunogenic properties of saRNA. During translation, saRNA forms double-stranded RNA intermediates that activate innate immune responses. As shown in FIGs. 12D, 12E, AA15V DFH(b;=(J;Kb X]SdRTS WXVWTa [TeT[b ^U AD(,q' A>F(u' P]S KF>(q X] cd\^a cXbbdTb R^\_PaTS to AA15V LNP-mSE-SCTs. Such nonspecific immune responses benefiting cancer treatment have also been reported in previous studies, where i.t. injection of LNP-saRNA encoding mCherry as a reporter gene showed significant antitumor efficacy in mouse models. However, the antitumor effects of LNP-sCE-SCTs were limited relative to AA15V LNP-sSE-SCTs, likely due to the absence of spike-specific CD8+ T cell responses. As shown in FIG.8H, spike- specific CD8+ T cells were detected in tumor tissues following vaccination. Moreover, i.t. injection of AA15V LNP-sSE-SCTs led to significantly elevated levels of pro-inflammatory Rhc^ZX]Tb P]S RWT\^ZX]Tb' bdRW Pb AD(,q' AD(1' AD(2' AD(,-_2+' AD(,0' A>F(u' KF>(q' P]S CXCL9 (FIGs.12E, 12G) compared to AA15V LNP-sCE-SCTs. Consistently, AA15V LNP- sSE-SCTs recruited more immune cells to the TME and enhanced immune cell activation, evidenced by increased levels of CD80+/86+ <;b Pb fT[[ Pb A>F(u+' KF>(q+, and granzyme B+ CD8+ T cells (FIGs. 12I-12L). Furthermore, depletion of CD8+ T cells significantly diminished the therapeutic effects of AA15V LNP-sSE-SCTs (FIGs. 14A-14G). All these results underscore the crucial roles of both saRNA-mediated innate immune responses and spike-specific T-cell immunity in the antitumor efficacy of AA15V LNP-sSE-SCTs. [0154] To evaluate the antitumor effects of spike-specific T-cell immunity against cancer cells expressing SE-SCT, the inventors performed a single i.t. treatment in a B16F10 melanoma mouse model, where the mice received prime-boost immunizations of AA2 LNP-spike mRNA before tumor inoculation (FIG. 12A). Compared to PBS, AA15V LNP-mSE-SCTs, and AA15V LNP-sCE-SCTs treatment, AA15V LNP-sSE-SCTs more effectively suppressed the cd\^a Va^fcW Qh --)+(U^[S' ,3)-(U^[S' P]S 2)0(U^[S #,/wS _^bc(X]^Rd[PcX^]$' aTb_TRcXeT[h' P]S substantially extended the overall survival time (FIG. 12B, 12C; FIGs. 13A – 13D). To evaluate the function of CD4+ and CD8+ T cells in AA15V LNP-sSE-SCTs treatment, tumor- bearing mice were treated with isotype control, anti-CD4, or anti-CD8 antibodies prior to receiving AA15V LNP-sSE-SCTs (FIG. 14A). Depletion of CD8+ T cells markedly Attorney Docket No.766501:MTST-288PC (240503G-P) diminished the therapeutic effects of AA15V LNP-sSE-SCTs, as evidenced by reduced tumor regression and lower survival rates (FIGs.14B-14G). In contrast, while CD4+ T cell depletion moderately compromised tumor suppression, it did not lead to significant differences in overall survival between mice pretreated with isotype control or anti-CD4 antibodies (FIGs. 14B- 14G). These results indicate that CD8+ T cells play a more crucial role than CD4+ T cells in the antitumor effects of AA15V LNP-sSE-SCTs treatment. [0155] Such enhanced therapeutic effects may be attributed to the durable expression of SE- SCT on cancer cell surface and the innate immune stimulatory properties of the saRNA. To profile the changes in the tumor microenvironment (TME) after a single treatment, the inventors examined the expression of cytokines and chemokines in tumor tissues and blood. Relative to PBS treatment, AA15V LNP-mSE-SCTs, AA15V LNP-sCE-SCTs, and AA15V LNP-sSE-SCTs all stimulated immune responses, as indicated by elevated levels of pro- inflammatory cytokines and chemokines in both tumor and blood samples (FIG. 12D-12G). Notably, AA15V LNP-sCE-SCTs induced comparable or even higher levels of multiple pro- inflammatory cytokines and chemokines compared to AA15V LNP-mSE-SCTs. This may be attributed to the immunogenicity of saRNA which can form double-stranded RNA intermediates during translation, triggering innate immune responses. Compared to these treatments, AA15V LNP-sSE-SCTs significantly increased levels of pro-inflammatory Rhc^ZX]Tb P]S RWT\^ZX]Tb' X]R[dSX]V AD(,q' AD(1' AD(2' AD(,-_2+' AD(,0' A>F(u' KF>(q' P]S ;O;D4' X] Q^cW cd\^a cXbbdTb P]S Q[^^S -/wW _^bc(PS\X]XbcaPcX^] #FIG. 12D-12G). Next, the inventors detected the infiltration and activation of immune cells in tumors including conventional dendritic cells (cDC1 and cDC2), macrophages, CD4+, and CD8+ T cells (FIG. 12H-12L; FIGs. 15A-15B). Compared to AA15V LNP-mSE-SCTs, AA15V LNP-sCE-SCTs demonstrated an equal or greater capacity of recruiting and activating immune cells within tumor tissues including macrophages and CD80+ cDC1s (FIG.12H-12L). Consistent with the trends observed in cytokine and chemokine levels, AA15V LNP-sSE-SCTs treatment recruited more immune cells to the tumor tissues and achieved stronger immune cell activation than other treatments. Specifically, AA15V LNP-sSE-SCTs treatment increased the populations of activated APCs, including CD80+/86+ DCs and CD80+/86+ macrophages (FIG. 12I-12J), as well as activated CD4+ T cells expressing CD137+CD134+ and CD154+CD134+ within tumor tissues (FIG.12K). Importantly, the treatment also upregulated the expression of Ki-67, IFN- u' KF>(q' P]S VaP]ih\T : X] ;<3+ T cells compared to other groups, indicating activation of cytotoxic responses (FIG. 12L). These findings suggest that both saRNA-mediated innate Attorney Docket No.766501:MTST-288PC (240503G-P) immune responses and spike-specific T-cell immunity contribute to the antitumor effects of AA15V LNP-sSE-SCTs Although AA15V LNP-sSE-SCT treatment showed significant antitumor effects, the incomplete eradication of the tumor burden necessitates boosting the efficacy of primed T-cell responses. Sufficient T cell proliferation and population in the TME before presenting SE-SCT on tumor cell surface is essential to initiate robust cancer cell death. [0156] Although AA15V LNP-sSE-SCTs treatment showed obvious antitumor effects, the incomplete eradication of the tumor necessitates boosting the efficacy of primed T-cell responses. Immune checkpoint inhibitors (ICIs), including antibodies that target PD-1 and CTLA-4, enable enhanced T lymphocyte survival and efficacy in cancer treatments. To further improve the antitumor effects of AA15V LNP-sSE-SCTs, the inventors incorporate the ICI combination, anti-PD-1 and anti-CTLA-4 Abs, in our strategy. The inventors intraperitoneally injected one dose of ICI prior to the AA15V LNP-sSE-SCTs treatment, followed by three subsequent doses given at three-day intervals throughout the treatment (FIG. 16A). The combination of AA15V LNP-sSE-SCTs (single dose) and ICI (four doses) dramatically inhibited tumor growth and prolonged survival of the tumor-bearing vaccinated mice in comparison to those treated with AA15V LNP-sSE-SCTs alone or AA15V LNP-sCE-SCTs (FIG. 16B, 16C; FIGs. 17A – 17D). Moreover, 12.5% of the mice treated with the AA15V LNP-sSE-SCTs + ICI showed complete regression (FIG.16C). To explore the applicability of AA15V LNP-sSE-SCTs, the inventors assessed the antitumor effects on the A20 tumor model in BALB/C mice. H-2Kd-based SE-SCTs were constructed to match the MHC I haplotype of BALB/C mice (FIG. 16D). Additionally, the inventors incorporated control groups of unvaccinated mice to assess the role of spike-specific T-cell immunity in the antitumor efficacy of our strategy. The AA15V LNP-sSE-SCTs + ICI treatment induced completed tumor rejection and long-term survival in over 28.6% of the vaccinated mice (FIG.16E, 16F; FIGs. 18A – 18E). However, such treatment in the unvaccinated mice failed to achieve a complete response, and all the mice reached the end point criteria before day 31, indicating the importance of spike-specific T-cell immunity in suppressing tumors expressing SE-SCTs. [0157] To further explore the clinical translatability of AA15V LNP-sSE-SCTs, the inventors examined AA15V LNP for the delivery of sSE-SCTs to human tumor tissues ex vivo (FIG. 16G). Glioma samples from one patient were treated with AA15V LNP-sSE-SCTs. Quantitative analysis revealed H-2Kb+t-\+ Tg_aTbbX^] X] 2),wkw+)-" ^U ;</0o cells in the glioma samples while the untreated slices exhibited no detectable expression (FIG.16H; FIG. 19). Furthermore, in one lung left lower lobe (LLL) adenocarcinoma specimen from a patient, Attorney Docket No.766501:MTST-288PC (240503G-P) 99,0M DFH(bJ=(J;Kb caTPc\T]c aTbd[cTS X] 0)3wkw-)-" ^U ;</0o cells expressing H- 2Kb+t-\+ (FIG.16I). In the LLL adenocarcinoma sample from another adult human subject, AA15V LNP-sSE-SCTs treatment induced H-2Kb+t-\+ Tg_aTbbX^] X] 3)2wkw-)+" ^U CD45o cells (FIG.16J). Given the cellular heterogeneity within the tumor microenvironment of human tumors, the expression of H-2Kb+t-\+ could be present in both cancer cells and tumor stromal cells. These results demonstrate the ability of AA15V LNP for delivery of sSE- SCTs to primary human tumor samples. [0158] Recent studies have explored the feasibility of using i.t. injection of viral epitopes or intravenous injection of antibody-conjugated viral epitopes to repurpose anti-virus T-cell immunity for cancer treatment. However, certain tumors exhibit impaired antigen presentation due to the downregulation or complete loss of MHC I23. Additionally, competition between exogenously introduced epitopes and those generated endogenously can further reduce the efficiency of MHC I presentation. The LNP-sSE-SCTs treatment restores MHC I presentation in cancer cells, potentially enhancing the immunogenicity of cancer cells. Moreover, the expression of sSE-SCTs can facilitate epitope loading onto MHC I molecules, circumventing competition with endogenous tumor epitopes with low immunogenicity. [0159] IV. Biological Preparations [0160] In some aspects, the composition disclosed herein may be formulated as a biological preparation. In some embodiments, the biological preparation is a vaccine, an immunotherapy, a pharmaceutical formulation, or a therapeutic composition. In alternatively embodiments, the biological preparation includes one or more therapeutically or pharmaceutically acceptable carriers, diluents, or excipients such as salts, buffering agents, preservatives, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants, lubricants, sorbents, suspending or dispersing agents, sweeteners, waters of hydration, and/or other therapeutic agents. [0161] In some embodiments, excipients for use with the biological preparations disclosed herein include maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N- dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and surfactant polyoxyethylene-sorbitan monooleate. Attorney Docket No.766501:MTST-288PC (240503G-P) [0162] Biological preparations, in some embodiments, are made to be compatible with a particular local, regional or systemic administration or delivery route. Thus, pharmaceutical formulations include carriers, diluents, or excipients suitable for administration by particular routes. Specific non-limiting examples of routes of administration for compositions herein are parenteral, e.g., intravenous, intra-arterial, intradermal, intramuscular, subcutaneous, intra- pleural, transdermal (topical), transmucosal, intra-cranial, intra-spinal, intra-ocular, rectal, oral (alimentary), mucosal administration, and any other formulation suitable for the treatment method or administration protocol. [0163] In some embodiments, solutions or suspensions used for parenteral application include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. In some embodiments, pH is adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. [0164] Biological preparations for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), or suitable mixtures thereof. Fluidity is maintained, in some embodiments, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars; polyalcohols such as mannitol or sorbitol; or sodium chloride, in some embodiments, are included in the composition. In some cases, also included is an agent which delays absorption, in some embodiments, for example, aluminum monostearate or gelatin prolongs absorption of injectable compositions. [0165] In some embodiments, sterile injectable formulations are prepared by incorporating the active composition in the required amount in an appropriate solvent with one or a combination Attorney Docket No.766501:MTST-288PC (240503G-P) of above ingredients. Generally, dispersions are prepared by incorporating the active composition into a sterile vehicle containing a basic dispersion medium and any other ingredient. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include, for example, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously prepared solution thereof. [0166] For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. In some embodiments, transmucosal administration is accomplished through the use of nasal sprays, inhalation devices (e.g., aspirators) or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, creams or patches. [0167] In some embodiments, the biological preparations are prepared with carriers that protect against rapid elimination from the body, such as a controlled release formulation or a time delay material such as glyceryl monostearate or glyceryl stearate. The formulations, in some embodiments, are also delivered using articles of manufacture such as implants and microencapsulated delivery systems to achieve local, regional or systemic delivery or controlled or sustained release. [0168] Any suitable route of administration is contemplated for the biological preparations disclosed herein. In some embodiments, the biological preparations disclosed are administered by intravenous administration. In some embodiments, the biological preparations disclosed are administered by subcutaneous administration. In some embodiments, the biological preparations disclosed are administered locally. In some embodiments, the biological preparations disclosed is administered systemically (e.g., intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, sublingually). In some embodiments, the biological preparations disclosed are formulated as a salve, lotion or emulsion. In some embodiments, the biological preparations disclosed are formulated as a solution. In some embodiments, the biological preparations disclosed are formulated for topical, oral, buccal, or nasal administration. [0169] In some embodiments, an individual is monitored prior to administration of the biological preparations disclosed. Symptoms are identified and their severity is assessed. The Attorney Docket No.766501:MTST-288PC (240503G-P) biological preparation as described herein may be administered alone or in combination with additional treatments, singly or multiply over time as discussed herein or known to one of skill in the art. In some embodiments, the individual is monitored such that the efficacy of the treatment regimen is determined. In some embodiments, a treatment regimen is modified in response to preliminary treatment outcomes, such that treatment dose or frequency or dose and frequency is altered so as to attain a desired level of subject response in light of symptom alleviation, side effect reduction, or a combination of symptom alleviation and side effect reduction. [0170] In some aspects, the biological preparations may be used to treat a disease or disorder in a subject. In some aspects, the biological preparations may be used to prevent a disease or disorder in a subject. In some aspects, the disease or disorder is cancer. [0171] The disclosure includes methods for inducing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of the delivery vehicle composition or biological preparation of the disclosure. An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective in treating and/or ameliorating a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially lessening the chances of a disease or condition in a subject. [0172] Various cancers may be treated with the nucleic acids delivered by the compositions of the present disclosure. As used herein, the term "cancer" refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid, uterus, vagina, and vulva. In non- limiting aspects, the subject may be known or suspects to have cancer or a disease associated with MHC I. Attorney Docket No.766501:MTST-288PC (240503G-P) [0173] In some aspects, the biological preparation is a vaccine or a cancer therapeutic. A vaccine may be referred to as a substance used to stimulate the production of antibodies and provide immunity against one or several diseases, prepared from the causative agent of a disease, its products, or a synthetic substitute. The vaccine may further comprise one or more immunologic adjuvants. As used herein, the term "immunologic adjuvant" refers to a compound or a mixture of compounds that acts to accelerate, prolong, enhance or modify immune responses when used in conjugation with an immunogen (e.g., neoantigens). Adjuvant may be non-immunogenic when administered to a host alone, but that augments the host's immune response to another antigen when administered conjointly with that antigen. Specifically, the terms "adjuvant" and "immunologic adjuvant" are used interchangeably in the present disclosure. Adjuvant-mediated enhancement and/or extension of the duration of the immune response can be assessed by any method known in the art including without limitation one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant/antigen combination versus those produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells recognizing the antigen or the adjuvant; and (iii) an increase in the level of one or more cytokines. Adjuvants may be aluminum based adjuvants including but not limiting to aluminum hydroxide and aluminum phosphate; saponins such as steroid saponins and triterpenoid saponins; bacterial flagellin and some cytokines such as GM-CSF. Adjuvants selection may depend on antigens, vaccines, and routes of administrations. [0174] In some aspects, adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation. Adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs). Adjuvants may be ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity. In other instances, adjuvants may signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant. [0175] In some aspects, the disclosed compositions and biological preparations may be part of a kit. The kit may include a pharmaceutically acceptable carrier and/or a package insert Attorney Docket No.766501:MTST-288PC (240503G-P) comprising instructions for intratumoral administration (e.g., injection) of the nucleic acid- based therapeutic composition. The kit may further include instructions for treating or delaying progression of cancer in a subject. In some aspects, the package insert further comprises instructions for administration of the composition by intratumoral administration in combination with injection in another site (e.g., systemic injection) for treating or delaying progression of cancer in a subject. The kit may also include additional therapeutic nucleic acids, drug, therapeutic agent, diagnostic agent, prophylactic agent, and/or any other agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect. [0176] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims. [0177] EXAMPLES [0178] Example 1: Synthetic Schemes [0179] The general synthetic route of amino alcohol lipids and amino acid lipids is depicted below. [0180] Synthesis of aldehydes [0181] To a suspension of paraformaldehyde (1.5 g, 50 mmol) in TMSCl (25 mL, 197 mmol) was added 1-hexanol 10 (5.1 g, 50 mmol) dropwise at RT. The reaction mixture was allowed to stir for 2 h at room temperature.s The clean solution was concentrated Attorney Docket No.766501:MTST-288PC (240503G-P) under reduce pressure to give 1-chloromethoxy-hexane 11 as a colorless oil that was used directly without further purification. [0182] The above chloromethyl ether was added dropwise to a solution of 1,6-hexanediol 12 (11.8 g, 100 mmol) and i-Pr2NEt (17.45 mL, 100 mmol) in CH2Cl2 (150 mL). The reaction mixture was stirred at room temperature for 24 h and was subsequently quenched by the addition of a saturated solution of NH4Cl (80 mL). Extraction with CH2Cl2 (60 mL*2 times) was performed and the combined organic layers were washed with water (30 mL) and brine (30 mL) and dried over Na2SO4. The organic phase was filtered and concentrated under reduced pressure, the residue was purified via silica gel flash chromatography (20% EtOAc in hexane). 6.74 g of 13 was obtained as a colorless oil, hXT[S 03)+") ,@ FEI #.++ E@i' ;W[^a^U^a\(S$ r /)10 #b' -@$' .)1. #`' B 71)/ @i' -@$' 3.51 (td, J = 6.6, 2.9 Hz, 4H), 1.57 (dq, J = 13.5, 6.8 Hz, 6H), 1.50 – 1.21 (m, 10H), 0.94 – 0.82 (m, 3H). HRMS (m/z): [M+Na]+ calcd. For C13H28NaO3, 255.1931; found: 255.1941. [0183] The synthetic scheme is depicted below. [0184] (Diacetoxyiodo)benzene (0.79 g, 2.45 mmol) was added to a suspension of 14 (518 mg, 2.23 mmol), TEMPO (34.9 mg, 0.22 mmol) and NaHCO3 (412 mg, 4.9 mmol) in 20 mL of dry DCM at room temperature. The reaction mixture was stirred for 3 h and TLC showed 14 was totally consumed. Then the mixture was quenched with saturated aqueous solution of Na2S2O3 (30 mL) and extracted with DCM (3*20 mL). The DCM phase was combined and washed with aqueous NaHCO3 (20 mL) and brine (20 mL), dried over Na2SO4, filtrated and concentrated under reduced pressure. The residue was purified via silica gel flash chromatography (10% EtOAc in hexane). 510 mg of 15 was obtained as a colorless oil, quant. 1H NMR (300 MHz, Chloroform-d$ r 4)21 #b' ,@$' /)1/ #b' -@$' .)0, (q, J = 5.9 Hz, 4H), 2.43 (t, J = 7.4 Hz, 2H), 1.60 (ddt, J = 17.8, 12.9, 7.3 Hz, 6H), 1.46 – 1.23 (m, 8H), 0.95 – 0.81 (m, 3H). [0185] The synthetic scheme is depicted below. [0186] To a solution of 16 (5.0 g, 30.4 mmol) in DCM (30 mL) was added dropwise into a solution of 1,5-hexanediol 12 and TEA (16.9 mL, 121 mmol) in DCM (100 mL) at 0 °C Attorney Docket No.766501:MTST-288PC (240503G-P) over 30 min. After stirring for 3 h, the reaction was quenched with 50 mL of water and extracted with DCM (50 mL*3), then the organic phase was combined and washed with water, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified via silica gel chromatography (20% EtOAc in hexanes).3.2 g of 17 was obtained as a colorless oil, yield 45.3%. 1H NMR (300 MHz, Chloroform-d$ r /),, #cS' J = 6.7, 2.6 Hz, 4H), 3.62 (td, J = 6.5, 3.1 Hz, 3H), 1.65 (p, J = 7.0 Hz, 4H), 1.56 (dq, J = 6.5, 3.3 Hz, 2H), 1.43 – 1.33 (m, 6H), 1.29 (dd, J = 6.8, 3.8 Hz, 4H), 0.91 – 0.84 (m, 3H). HRMS (m/z): [M+Na]+ calcd. For C13H26NaO4, 269.1723; found: 269.1735. [0187] The synthetic scheme is depicted below. [0188] BAIB (1.41 g, 4.38 mmol) was added to a suspension of 17 (0.98 g, 3.98 mmol), TEMPO (62.2 mg, 0.4 mmol) and NaHCO3 (736 mg, 8.6 mmol) in 20 mL of DCM. The reaction mixture was stirred for 3 h till TLC showed A was totally consumed. Then the mixture was quenched with saturated aqueous solution of Na2S2O3 (30 mL) and extracted with DCM (3*20 mL). The DCM phase was combined and washed with aqueous NaHCO3 (20 mL) and brine (20 mL), dried over Na2SO4, filtrated and concentrated under reduced pressure. The residue was purified via silica gel flash chromatography (10% EtOAc in hexane).0.78 g of 18 was obtained as a light yellow oil, yield 80.2%.1H NMR (300 MHz, Chloroform-d$ r 4)22 #`' J = 1.6 Hz, 1H), 4.13 (td, J = 6.6, 2.6 Hz, 5H), 2.45 (td, J = 7.2, 1.7 Hz, 2H), 1.78 – 1.59 (m, 7H), 1.49 – 1.24 (m, 10H), 0.95 – 0.84 (m, 4H). [0189] The synthetic scheme is depicted below. [0190] A To a solution of acetaldehyde (1.76 g, 40 mmol) in TMSCl (20 mL, 157 mmol) was added 1-hexanol 10 (5.1 g, 50 mmol) dropwise at RT. The reaction mixture was allowed to stir for 2 h at room temperature. The clean solution was concentrated under reduce pressure to give 19 as a colorless oil that was used directly without further purification. [0191] The above chloromethyl ether was added dropwise to a solution of 1,6-hexanediol 12 (9.44 g, 80 mmol) and i-Pr2NEt (13.96 mL, 80 mmol) in CH2Cl2 (150 mL). The reaction mixture was stirred at room temperature for 24 h and was subsequently quenched by the Attorney Docket No.766501:MTST-288PC (240503G-P) addition of a saturated solution of NH4Cl (80 mL). Extraction with CH2Cl2 (60 mL*2 times) was performed and the combined organic layers were washed with water (30 mL) and brine (30 mL) and dried over Na2SO4. The organic phase was filtered and concentrated under reduced pressure, the residue was purified via silica gel flash chromatography (20% EtOAc in hexane).3.45 g of 20 was obtained as a colorless oil, yield 35.0%.1H NMR (300 MHz, Chloroform-d$ r /)1/ #`' J = 5.3 Hz, 1H), 3.66 – 3.47 (m, 4H), 3.38 (dtd, J = 9.2, 6.6, 2.5 Hz, 2H), 1.65 – 1.48 (m, 7H), 1.42 – 1.22 (m, 12H), 0.92 – 0.81 (m, 3H). HRMS (m/z): [M+Na]+ calcd. For C14H30NaO3, 269.2087; found: 269.2075. [0192] The synthetic scheme is depicted below. [0193] BAIB (1.77 g, 5.5 mmol) was added to a suspension of 20 (1.23 g, 5.0 mmol), TEMPO (78.2 mg, 0.5 mmol) and NaHCO3 (924 mg, 11.0 mmol) in 20 mL of DCM. The reaction mixture was stirred for 3 h till TLC showed A was totally consumed. Then the mixture was quenched with saturated aqueous solution of Na2S2O3 (30 mL) and extracted with DCM (3*20 mL). The DCM phase was combined and washed with aqueous NaHCO3 (20 mL) and brine (20 mL), dried over Na2SO4, filtrated and concentrated under reduced pressure. The residue was purified via silica gel flash chromatography (10% EtOAc in hexane).1.0 g of 21 was obtained as a light yellow oil, yield 81.8%. 1H NMR (300 MHz, Chloroform-d$ r 4)23 #b' ,@$' /)11 #`' J = 5.3 Hz, 1H), 3.57 (dq, J = 9.2, 6.8 Hz, 2H), 3.41 (dtd, J = 9.7, 6.5, 3.3 Hz, 2H), 2.45 (td, J = 7.3, 1.7 Hz, 2H), 1.73 – 1.51 (m, 6H), 1.48 – 1.25 (m, 11H), 0.95 – 0.82 (m, 3H). HRMS (m/z): [M+Na]+ calcd. For C13H28NaO3, 255.1931; found: 255.1941. [0194] The synthetic scheme is depicted below.
Attorney Docket No.766501:MTST-288PC (240503G-P) [0197] General Procedure for the synthesis of alcohol 3-n. [0198] To a solution of 1,3-propyldiamine 1 (2.22 g, 30.0 mmol) and 2-n (10 mmol) in n- butanol (10 mL) was added potassium iodide (84 mg,0.5 mmol) and potassium carbonate (0.69 g, 5.0 mmol). The resulting mixture was refluxed for 24 h. Then the mixture was slowly cooled to room temperature, filtered, and concentrated under vacuum. The resulting mixture was dissolved in 10 mL of MeOH and treated with Boc2O (8.3 g, 60 mmol) for 30 min at RT. MeOH was removed under reduced pressure. The residue was purified via CombiFlash system to give the desired product (Hexane/Ethyl Acetate = 1:1). [0202] 1H NMR (300 MHz, Chloroform-d$ r /)++ #c' J = 6.0 Hz, 2H), 3.59 (t, J = 6.9 Hz, 2H), 3.52 (d, J = 7.2 Hz, 2H), 3.44 (t, J = 6.6 Hz, 2H), 2.13 (br s, 1H), 2.05 – 1.84 (m, 6H), 1.80 (s, 9H), 1.78 (s, 9H). [0206] 1H NMR (400 MHz, Chloroform-d$ r .)1/ #c' J = 6.6 Hz, 2H), 3.33 – 3.21 (m, 2H), 3.20 – 3.04 (m, 4H), 1.89 – 1.75 (m, 1H), 1.71 – 1.62 (m, 2H), 1.60 – 1.50 m, 4H), 1.47 (s, 9H), 1.44 (s, 9H), 1.39 (q, J = 7.6 Hz, 2H), 1.34 – 1.28 (m, 2H). MS (m/z): [M+Na]+ calcd. For C14H30NaO3, 275.2; found: 275.3. [0207] General Procedure for the synthesis of alcohol 6-m. [0208] Amine 4 (2.61 g, 15.0 mmol) was dissolved in 20 mL of dry MeCN. Potassium carbonate was added (1.59 g, 15.0 mmol) to the solution. A solution of 5-m (10.0 mmol) in 10 mL of dry MeCN was added dropwise to the above solution over 1 hour at RT. The solution was then stirred at RT for 24 h. Solid Na2CO3 was removed via filtration, and the solvent was removed under vacuum. The residue was purified via silica gel chromatography. Attorney Docket No.766501:MTST-288PC (240503G-P) [0209] (t, J = 6.6 Hz, 2H), 2.69 (t, J = 6.6 Hz, 2H), 2.44 (t, J = 6.6 Hz, 2H), 1.98 (br s, 1H), 1.67 (p, J = 6.6 Hz, 2H), 1.45 (s, 9H), 1.44 (s, 9H). [0217] 1H NMR (400 MHz, Chloroform-d$ r 0),3 #Qa b' ,@$' .)1. #b' ,@$' .)-, m .),- (m, 2H), 2.65 (t, J = 6.4 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.21 (t, J = 7.2 Hz, 2H), 1.66 – 1.57 (m, 5H), 1.50 (q, J = 7.4 Hz, 2H), 1.45 – 1.40 (m, 18H). [0218] General Procedure for the synthesis of amino alcohol lipids and amino acid lipids. [0219] To a solution of 3-n or 6-m (0.1 mmol) in CH2Cl2 (0.46 mL) was added trifluoroacetic acid (TFA, 0.46 mmol) at 0 °C. The mixture was allowed to warm to RT, stirred at RT for 3-4 h and monitored with thin layer chromatography (TLC). Upon completion of the reaction, the solvent was evaporated and the residue was dissolved in MeOH and concentrated. The residue was dissolved in 2 mL of THF and TEA (0.028 mL, 0.2 mmol) was added, and stirred for 10 min at RT. Then aldehyde (0.4 mmol) was added followed by NaBH(OAc)3 (95.4 mg, 0.45 mmol). The obtained solution was stirred for 48 h at RT. Saturated aq. NaHCO3 was added to quench the reaction, THF was removed under reduced pressure, the aqueous phase was extracted with DCM (10 mL* 3 times), the organic phase was combined and dried over anhydrous Na2SO4, then the solution was filtered and concentrated, the residues was purified by silica gel chromatography to give desired product. Attorney Docket No.766501:MTST-288PC (240503G-P) [0222] 1H NMR (300 MHz, Chloroform-d$ r /)3+ #_' J = 6.3 Hz, 3H), 3.52 (t, J = 5.1 Hz, 2H), 2.61 – 2.32 (m, 12H), 2.27 (t, J = 7.4 Hz, 6H), 1.67 – 1.35 (m, 26H), 1.32 – 1.17 (m, 42H), 0.92 – 0.80 (m, 18H). [0224] 1H NMR (300 MHz, Chloroform-d$ r /)3+ #_' J = 6.3 Hz, 3H), 3.62 (t, J = 6.6 Hz, 2H), 2.43 – 2.35 (m, 12H), 2.27 (t, J = 7.5 Hz, 6H), 1.67 – 1.37 (m, 30H), 1.35 – 1.20 (m, 44H), 0.93 – 0.79 (m, 18H). [0225] Example 2: In vivo studies [0226] Reagents [0227] 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from Avanti Polar Lipids. ALC-0315, ALC-0159 and SM-102 were obtained from MedKoo Biosciences. The purification of AA lipids was achieved via column chromatography using a RediSep Gold Resolution silica column (Teledyne Isco) with a CombiFlash Rf system employing VaPSXT]c T[dcX^]) p@ FEI b_TRcaP[ P]P[hbTb fTaT R^]SdRcTS Qh P :adZTa 9eP]RT /++wE@i device. Mass spectrometry analyses were executed using Acquity SQD UPLC/MS (Waters), LTQ Orbitrap XL mass spectrometer (Thermo Scientific), and the ultrafleXtreme MALDI-TOF mass spectrometer (Bruker) at The Ohio State University. [0228] Cell culture [0229] Murine myoblast C2C12 cell line (CRL-1772), murine JAWSII dendritic cell line (CRL-3612), murine melanoma B16F10 cell line (CRL-6475), and murine B cell lymphoma A20 cell line (TIB-208) were all obtained from American Type Culture Attorney Docket No.766501:MTST-288PC (240503G-P) Collection (ATCC). C2C12 cells and B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 U/mL). JAWSII cells were cultured in RPMI-1640 medium with L-Glutamine, 20% FBS, penicillin-streptomycin (50 U/mL), and 5 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF). [0230] RNA synthesis [0231] The linear dsDNA of firefly luciferase (FLuc), the spike protein of SARS-CoV-2 Delta variant, and spike epitope single-chain trimer MHC class I molecules (SE-SCTs) were obtained from Integrated DNA Technologies. Corresponding plasmids were generated from pUC19 vector via NEBuilder® HiFi DNA assembly. All mRNAs used were chemically modified with N1-methylpseudouridine. [0232] LNP formulation and characterization [0233] LNPs were formulated using Rapid Nanomedicine System INano L+ (Micro&Nano biologics Technology Ltd.) by mixing an ethanol solution comprising ionizable lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K), fXcW P`dT^db RXcaPcT b^[dcX^] R^]cPX]X]V cWT \IF9b #,+w\E' _@ .)+$) >^a cWT _aT[X\X]Pah screening phase, LNPs were formulated with newly synthesized ionizable lipids, DOPE, cholesterol, and DMG-PEG2K at the molar ratios of 20:30:40:0.75, respectively. Concurrently, FLuc mRNA was introduced at the mass ratio of ionizable lipid: mRNA set at 10:1. Subsequently, an L16 (4)4 orthogonal array was constructed to optimize the LNP formulations. For FDA-approved LNPs, the lipid ratios and the mRNA to lipid mass ratio remained consistent with previously published formulations. The lipid ratios and the mRNA-to-lipid mass ratio for FDA-approved LNPs remained consistent with those as previously published.41 For in vitro screening, LNP-mRNA formulation was added to cells at the dose of 50ng mRNA per 2!104 cells. mRNA delivery efficiency was evaluated 18 hours after treatment by exposing cells to Bright-Glo luciferase substrate (Promega), followed by quantification of luminescence intensity using Cytation 5 (Biotek). Hydrodynamic diameter, zeta potential, and polydispersity index (PDI) were assessed by NanoZS Zetasizer (Malvern, USA). Encapsulation efficiency was evaluated using Ribogreen assay, while the morphology of LNPs was visualized by Glacios Cryo-TEM (Thermo Scientific, USA). Attorney Docket No.766501:MTST-288PC (240503G-P) [0234] Intramuscular injection and vaccination [0235] For in vivo screening, mice were intramuscularly injected with AA LNP-FLuc \IF9 Pc +)+20 \V*ZV \IF9 S^bT) JXg W^dab _^bc(X]YTRcX^]' ,0+v[ ^U <([dRXUTaX] substrate (30 mg/mL) was intraperitoneally injected into the mice. Following an eight- minute interval, the mice were imaged using a Xenogen IVIS imaging system to quantify the luminescence signals at the injection sites. [0236] For spike-mRNA vaccinations, mice were intramuscularly immunized by AA2 LNP-Spike mRNA at the mRNA dose of 0.3 mg/kg on day 0. Booster immunization with the same dose was conducted on day 21. Five days after the booster doses, mouse blood was collected with anticoagulant (3.8% Sodium citrate) and centrifugated at 1500 x g for 10 min at 4°C to obtain plasma before being stored at -80°C until subsequent analysis. ELISA assay was used to detect the plasma titer of Delta SARS-CoV-2 spike-specific antibodies. Specifically, 250 ng of Delta SARS-CoV-2 spike S1 subunit peptide was applied to coat a 96-well plate, after which diluted mouse plasma was added to the wells. Goat anti-mouse IgG linked to HRP (abcam, ab7068, 1:5000 dilution) was then added, and the mean optical density at 492 nm (OD492) was recorded using a Cytation 5 cell imaging multi-mode reader (Biotek). The anti-S1 antibody titer in the mouse plasma was quantified against a standard curve employing a recombinant anti-spike monoclonal antibody (Sino Biological, 40591-MM43). [0237] Luminex analysis of cytokines and chemokines [0238] Tumoral tissues and blood samples from mice were collected at 6h and 24h post- intratumoral injection of PBS, mRNA-LNP, or replicon-LNP. Tumoral tissues were promptly flash-frozen in liquid nitrogen, followed by pulverization and extraction using RIPA lysis buffer (Thermo Scientific, 89900) supplemented with protease inhibitors (Thermo Scientific, 87785). Murine whole blood was collected in sodium citrate- containing tubes, followed by centrifugation to isolate the serum. Both tumoral lysate and serum samples were kept at o3+wj; d]cX[ UdacWTa P]P[hbXb) E^dbT Rhc^ZX]T P]S RWT\^ZX]T levels were evaluated using mouse cytokine/chemokine discovery assay (Eve Technologies). [0239] Expression of activation-induced markers (AIM) of T cells from SARS-CoV-2 vaccinated mice Attorney Docket No.766501:MTST-288PC (240503G-P) [0240] Mouse spleens and blood samples were collected 5 days post-boost vaccination. Single-cell suspension was prepared from mouse spleen and pan T cells were isolated according to the manufacturer’s instructions (T cell isolation kit, Miltenyi Biotec, Catalog # 130-096-130). Peripheral blood mononuclear cells (PMBCs) from the whole blood were Xb^[PcTS Qh RT]caXUdVPcX^] P]S aTbdb_T]STS X] K RT[[ \TSXd\ U^a 1wW) :,1>,+ cd\^ab Ua^\ ePRRX]PcTS \XRT fTaT WPaeTbcTS fWT] cWT [PaVTbc cd\^a SXP\TcTa TgRTTSTS +)3wR\ P]S dissociated into a single-cell suspension for T-cell isolation (Miltenyi Biotec, Catalog #130-096-730). The isolated pan T cells were resuspended in T cell medium and rested for 1wW) >^[[^fX]V cWXb' cWT K RT[[b fTaT bcX\d[PcTS fXcW R^]ca^[' ;<3' ^a ;</ T_Xc^_Tb) 9UcTa ,+m,-wW ^U Rd[cdaT' 9AE Tg_aTbbX^] fPb cWT] PbbTbbTS Qh U[^f Rhc^\Tcah) >^a ;<3+ T cell killing assay, B16F10 cells receiving AA15V LNP-sSE-SCTs or controls were co-cultured with CD8+ T cells isolated from the spleen of the mice vaccinated with AA2 LNP-spike mRNA. The apoptosis of B16F10 cells was detected by dead cell apoptosis kit (V13242 Invitrogen™). [0241] Intratumoral delivery of AA15V LNP-SCT saRNAs in vivo [0242] C57BL/6 mice were subcutaneously (s.c.) inoculated with 1 × 105 B16F10-FLuc RT[[b ^] cWT aXVWc U[P]Z) NWT] cWT [PaVTbc cd\^a SXP\TcTa TgRTTSTS +)3wR\' cWT \XRT were i.t) X]YTRcTS fXcW 99,0M DFH(bGH J;Kb Pc ,+wvV IF9 S^bT) 9UcTa -/wW' cWT cd\^a tissues were dissected and dissociated into a single-cell suspension using tumor dissociation kit (Miltenyi Biotec, Catalog # 130-096-730). Erythrocytes were removed with Red Blood Cell Lysis Solution (10×, Miltenyi Biotec, Catalog # 130-094-183). The cells were then stained with LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit (Invitrogen™, L34955). After washing with PBS, the cells were stained with antibodies followed by flow cytometry analysis. [0243] Tumor models and treatment regimens [0244] C57BL/6 and BALB/c mice (male and female, 6-8 weeks) were purchased from the Jackson Laboratory and housed in the Icahn School of Medicine at Mount Sinai. All mouse studies were approved by the Institutional Animal Care and Use Committee (IACUC) at The Ohio State University (2014A00000106) and Icahn School of Medicine at Mount Sinai (IPROTO202200000134), complied with local, state, and federal regulations. In this study, a maximum of five mice were accommodated in each cage Attorney Docket No.766501:MTST-288PC (240503G-P) within a barrier environment, maintaining conditions of approximately 20°C, 45% humidity, and a 12-hour light/12-hour dark cycle. [0245] KWT \XRT aTRTXeTS QX(S^bT ePRRX]PcX^]b ^U 99- DFH(b_XZT \IF9 Pc +).w\V*ZV mRNA dose. Five days after the booster doses, around 1 × 105 B16F10 cells or 5 × 106 A20 cells were s.c. inoculated on the right flank of the mice. The A20 tumor model included unvaccinated mice as control groups. On day 6 post-tumor inoculation, mice with tumor bXiT PQ^dc ^a ^eTa +)0wR\ ^U cWT [PaVTbc SXP\TcTa fTaT aP]S^\[h bT_PaPcTS X]c^ SXUUTaT]c groups. For T cell depletion tumor model, 1 × 105 B16F10 cells s.c. inoculated on the right flank of the mice. The tumor-bearing mice were intraperitoneally injected with anti-mouse ;<3q' P]cX(\^dbT ;</' ^a P]cX(aPc AV?-Q Xb^ch_T P]cXQ^SXTb' aTb_TRcXeT[h' ^] SPh 1 _^bc( tumor inoculation. Each antibody was administered every 3 days. One day after the first antibody depletion treatment (Day 7), the tumor-bearing mice received a single i.t. dose of AA15V LNP-sSE-SCTs. For the B16F10 tumor model groups without combinatorial therapy of ICIs (anti-PD1+ anti-CTLA4 antibodies), a single dose of various treatments #,+wvV IF9 S^bT$ fPb i.t. administered to the tumor-bearing mice on day 6 following tumor inoculation. For the combination therapy involving ICIs, starting from day 6, mice fTaT X]YTRcTS X]caP_TaXc^]TP[[h fXcW P]cX(H<, #,++wlV*\^dbT' R[^]T5 IEH,(,/' :X^ORT[[$ P]S P]cX(;KD9/ P]cXQ^SXTb #,++wlV*\^dbT' R[^]T5 4<4' :X^ORT[[$) =PRW P]cXQ^Sh fPb administered every 3 days for four doses. One day after the first ICI treatment (Day 7), the tumor-bearing mice received a single i.t. dose of various treatments. [0246] Ex vivo saRNA delivery in human tumor tissues [0247] Lung tumor samples were obtained from surgical specimens of patients undergoing resection at Mount Sinai Hospital (New York, NY) after obtaining informed consent in accordance with a protocol reviewed and approved by the Institutional Review Board at the Icahn School of Medicine at Mount Sinai (IRB Human Subjects Electronic Research 9__[XRPcX^] ,+(++/2-w9$ P]S X] R^[[PQ^aPcX^] fXcW cWT :X^aT_^bXc^ah P]S <T_Pac\T]c ^U Pathology. All glioma tissue was banked, de-identified under approved institutional IRB protocol (STUDY-18-00983), and informed consent was secured from all participants. The obtained human tumor tissue biopsies were promptly placed on ice in PBS and sectioned X]c^ 1++ v\ cWXRZ b[XRTb dbX]V DTXRP MK ,-++J \XRa^c^\T fXcWX] .+w\X]) KWT b[XRTb fTaT cWT] Rd[cdaTS X] IHEA(,1/+ \TSXd\ R^]cPX]X]V -+" >:J U^a ,wW QTU^aT QTX]V caTPcTS fXcW ,wlV 99,0M DFH(bJ=(J;Kb) 9UcTa -/wW' cWT b[XRTb fTaT SXbb^RXPcTS X]c^ bX]V[T(RT[[ suspension and prepared for immunofluorescence staining with specific cell markers. Attorney Docket No.766501:MTST-288PC (240503G-P) [0248] Statistics and reproducibility [0249] For the comparison of multiple data sets, one-way ANOVA with Dunnett’s multiple comparison test was used, whereas the two-tailed Student’s t-test was used for comparing two groups. In the mouse tumor model, tumor sizes were compared using two- way ANOVA with Dunnett’s multiple comparison, and survival rates were analyzed with the log-rank (Mantel–Cox) test. P-values of <0.05 were considered statistically significant. All data adhered to the assumptions of the statistical tests applied, including normal distribution and equal variances, and underwent formal testing to validate the statistical analysis. The sample size was not predetermined by any statistical method. [0250] References Guan, W.J. et al. Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med 382, 1708-1720 (2020). Huang, C. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497-506 (2020). Krammer, F. SARS-CoV-2 vaccines in development. Nature 586, 516-527 (2020). Barbier, A.J., Jiang, A.Y., Zhang, P., Wooster, R. & Anderson, D.G. The clinical progress of mRNA vaccines and immunotherapies. Nature Biotechnology 40, 840-854 (2022). Baden Lindsey, R. et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. New England Journal of Medicine 384, 403-416 (2021). Polack Fernando, P. et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. New England Journal of Medicine 383, 2603-2615 (2020). Goel, R.R. et al. mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern. Science 374, abm0829 (2021). Wang, L. et al. T cell immune memory after covid-19 and vaccination. BMJ Med 2, e000468 (2023). Gao, F. et al. Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine an d attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection. Immunity 56, 864-878.e864 (2023). Collier, A.-r.Y. et al. Differential Kinetics of Immune Responses Elicited by Covid-19 Vaccines. New England Journal of Medicine 385, 2010-2012 (2021). Liu, J. et al. Vaccines elicit highly conserved cellular immunity to SARS-CoV-2 Omicron. Nature 603, 493-496 (2022). Lauring, A.S. et al. Clinical severity of, and effectiveness of mRNA vaccines against, covid-19 from omicron, delta, and alpha SARS-CoV-2 variants in the United States: prospective observational study. BMJ 376, e069761 (2022). Attorney Docket No.766501:MTST-288PC (240503G-P) Waldman, A.D., Fritz, J.M. & Lenardo, M.J. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nature Reviews Immunology 20, 651-668 (2020). Demaria, O. et al. Harnessing innate immunity in cancer therapy. Nature 574, 45-56 (2019). Schumacher, T.N. & Schreiber, R.D. Neoantigens in cancer immunotherapy. Science 348, 69-74 (2015). Yarchoan, M., Johnson III, B.A., Lutz, E.R., Laheru, D.A. & Jaffee, E.M. Targeting neoantigens to augment antitumour immunity. Nature Reviews Cancer 17, 209-222 (2017). Blass, E. & Ott, P.A. Advances in the development of personalized neoantigen-based therapeutic cancer vaccines. Nature reviews Clinical oncology 18, 215-229 (2021). Lawrence, M.S. et al. Mutational heterogeneity in cancer and the search for new cancer- associated genes. Nature 499, 214-218 (2013). Hu, Z., Ott, P.A. & Wu, C.J. Towards personalized, tumour-specific, therapeutic vaccines for cancer. Nature Reviews Immunology 18, 168-182 (2018). Newman, J.H. et al. Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer. Proceedings of the National Academy of Sciences 117, 1119-1128 (2020). Çuburu, N. et al. Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors. Proceedings of the National Academy of Sciences 119, e2116738119 (2022). Millar, D.G. et al. Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy. Nature Biotechnology 38, 420-425 (2020). Garrido, F., Aptsiauri, N., Doorduijn, E.M., Lora, A.M.G. & Van Hall, T. The urgent need to recover MHC class I in cancers for effective immunotherapy. Current opinion in immunology 39, 44-51 (2016). Sharma, P., Hu-Lieskovan, S., Wargo, J.A. & Ribas, A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 168, 707-723 (2017). Sade-Feldman, M. et al. Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nature Communications 8, 1136 (2017). Neefjes, J., Jongsma, M.L.M., Paul, P. & Bakke, O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nature Reviews Immunology 11, 823-836 (2011). Rosenblum, D., Joshi, N., Tao, W., Karp, J.M. & Peer, D. Progress and challenges towards targeted delivery of cancer therapeutics. Nat Commun 9, 1410 (2018). Cruz, F.M., Colbert, J.D., Merino, E., Kriegsman, B.A. & Rock, K.L. The Biology and Underlying Mechanisms of Cross-Presentation of Exogenous Antigens on MHC-I Molecules. Annual Review of Immunology 35, 149-176 (2017). Attorney Docket No.766501:MTST-288PC (240503G-P) Yu, Y.Y., Netuschil, N., Lybarger, L., Connolly, J.M. & Hansen, T.H. Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells. J Immunol 168, 3145-3149 (2002). Huang, C. et al. Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope. Gene therapy 12, 1180-1186 (2005). Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials 6, 1078-1094 (2021). Chen, S. et al. Nanotechnology-based mRNA vaccines. Nature Reviews Methods Primers 3, 63 (2023). Tilstra, G. et al. Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery. Journal of the American Chemical Society 145, 2294-2304 (2023). Dangi, T., Class, J., Palacio, N., Richner, J.M. & Penaloza MacMaster, P. Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV-2. Cell Reports 36 (2021). Poluektov, Y., George, M., Daftarian, P. & Delcommenne, M.C. Assessment of SARS- CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers. Vaccine 39, 2110-2116 (2021). Ahmed, S.F., Quadeer, A.A. & McKay, M.R. in Viruses, Vol. 12 (2020). Davenport, B.J., Morrison, T.E., Kedl, R.M. & Klarquist, J. Conserved and Novel Mouse CD8 T Cell Epitopes within SARS-CoV-2 Spike Receptor Binding Domain Protein Identified following Subunit Vaccination. The Journal of Immunology 206, 2503-2507 (2021). Parn, S., Savsani, K. & Dakshanamurthy, S. SARS-CoV-2 Omicron (BA.1 and BA.2) specific novel CD8+ and CD4+ T cell epitopes targeting spike protein. ImmunoInformatics 8 (2022). Son, E.T. et al. The self-peptide repertoire plays a critical role in transplant tolerance induction. The Journal of Clinical Investigation 131 (2021). Xue, Y. et al. LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing. Nature Communications 15, 739 (2024). Schoenmaker, L. et al. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. International Journal of Pharmaceutics 601, 120586 (2021). [0251] Various embodiments of the disclosure include the following: [0252] 1. A polynucleotide encoding a spike epitope-loaded single-chain trimer (SE- SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6. Attorney Docket No.766501:MTST-288PC (240503G-P) [0253] 2. The polynucleotide of embodiment 1, wherein the SE-SCT MHC I molecule R^\_aXbTb cWT bXV]P[ bT`dT]RT ^U t2\' cWT \PcdaT _^acX^] ^U \daX]T t2m, and the H-2kb heavy chain. [0254] 3. The polynucleotide of embodiment 1 or embodiment 2, wherein the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. [0255] 4. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide comprises DNA or RNA. [0256] 5. The polynucleotide of embodiment 4, wherein the DNA is plasmid DNA (pDNA) or complementary DNA (cDNA). [0257] 6. The polynucleotide of embodiment 4, wherein the RNA is messenger ribonucleic acid (mRNA), self-amplifying RNA (saRNA), small RNA (sRNA), micro RNA (miRNA), or circular RNA (circRNA). [0258] 7. An RNA molecule comprising a polynucleotide encoding a spike epitope- loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, wherein the RNA molecule is mRNA or saRNA. [0259] 8. The RNA molecule of embodiment 7, wherein the SE-SCT MHC I molecule R^\_aXbTb cWT bXV]P[ bT`dT]RT ^U t-\' cWT \PcdaT _^acX^] ^U \daX]T t-\' P]S cWT @(-ZQ heavy chain. [0260] 9. The RNA molecule of embodiment 7 or embodiment 8, wherein the SE- SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. [0261] 10. An expression construct comprising the polynucleotide of any one of claims 1 to 6 or the RNA molecule of any one of claims 7 to 9. [0262] 11. The expression construct of embodiment 10, further comprising a 3’ UTR, 5’ UTR, a 5’ Cap, a replicase and/or Poly(A) sequence. [0263] 12. A composition comprising the polynucleotide of any one of claims 1 to 6, the RNA molecule of any one of claims 7 to 9, or the expression construct of embodiment 10 or embodiment 11. Attorney Docket No.766501:MTST-288PC (240503G-P) [0264] 13. The composition of embodiment 12, wherein the polynucleotide is formulated, in communication with, or encapsulated with a delivery vehicle. [0265] 14. The composition of embodiment 13, wherein the delivery vehicle is selected from the group consisting of amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate. [0266] 15. The composition of embodiment 13 or clam 14, wherein the delivery vehicle has an encapsulation efficiency of about 50% to about 100%. [0267] 16. The composition of any one of claims 13 to 15, wherein the delivery vehicle comprises an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid. [0268] 17. The composition of embodiment 16, wherein the ionizable lipid is an amino alcohol-derived lipid. [0269] 18. The composition of embodiment 17, wherein the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers. [0270] 19. The composition of embodiment 17, wherein the ionizable lipid comprises a carboxylic acid group. [0271] 20. The composition of embodiment 17, wherein the ionizable lipid comprises two hydroxyl groups introduced to the head group via an ethylidene linker. [0272] 21. The composition of any one of claims 17 to 20, wherein the ionizable lipid further comprises a hydrophobic tail selected from the group consisting of non- biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups. [0273] 22. The composition of embodiment 21, wherein the ionizable lipid has a structure of , wherein n = 1, 2, 3, 4, or 5 and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein Attorney Docket No.766501:MTST-288PC (240503G-P) [0275] 23. The composition of embodiment 22, wherein n=1 and R=R1, n=1 and R=R2, n=1 and R =R3, n=1 and R=R7, n=1 and R =R9, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=3 and R=R4, n=3 and R=R9, n=4 and R=R2, n=4 and R=R3, n=4 and R=R7, n=4 and R=R9, n=5 and R=R1, n=5 and R=R2, n=5 and R=R3, n=5 and R=R5, n=5 and R=R6, n=5 and R=R7, n=5 and R=R8, or n=5 and R=R9. [0276] 24. The composition of embodiment 21, wherein the ionizable lipid has a structure of , wherein n = 1, 2, 3, 4, or 5 and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein
Attorney Docket No.766501:MTST-288PC (240503G-P) [0278] 25. The composition of embodiment 24, wherein n=1 and R=R1, n=1 and R=R2, n=1 and R =R6, n=1 and R=R8, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=4 and R=R1, n=4 and R=R2, n=5 and R=R1, n=5 and R=R2, n=5 and R=R6, n=5 and R=R7, or n=5 and R=R9. [0279] 26. The composition of embodiment 21, wherein the ionizable lipid has a structure of , wherein and R =R1, R2, R3, R4, R5, R6, R7, R8, or R9, wherein
Attorney Docket No.766501:MTST-288PC (240503G-P) [0281] 27. The composition of embodiment 26, wherein R =R1, R=R2, R= R6, R=R7, or R=R9. [0282] 28. A biological preparation comprising the composition of any one of claims 12 to 27. [0283] 29. The biological preparation of embodiment 28, wherein the biological preparation is a vaccine, an immunotherapy, or a therapeutic composition. [0284] 30. The biological preparation of embodiment 29, wherein the biological preparations is configured to be administered to a subject known or suspected to have cancer, or for use in an anti-cancer therapy or cancer immunotherapy. [0285] 31. The biological preparation of embodiment 29 or embodiment 30, wherein the biological preparations is configured to be administered as an injectable preparation. [0286] 32. The biological preparation of any one of claims 28 to 31, wherein the biological preparations further comprises one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients or other therapeutic agents. [0287] 33. A method comprising, administering to a subject in need thereof, a therapeutically effective amount of a biological preparation of any one of claims 28 to 32. Attorney Docket No.766501:MTST-288PC (240503G-P) [0288] 34. The method of embodiment 33, wherein the subject has or is suspected to have cancer. [0289] 35. A kit comprising the biological preparation of any one of claims 28 to 32 and instructions for use. [0290] 36. A cell comprising the polynucleotide of any one of claims 1 to 6, the RNA molecule of any one of claims 7 to 9, or the expression construct of embodiment 10 or embodiment 11. [0291] 37. An isolated polynucleotide comprising a nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule. [0292] 38. The isolated polynucleotide of embodiment 37, wherein the nucleic acid is messenger ribonucleic acid (mRNA) or self-amplifying RNA (saRNA). [0293] 39. A composition comprising the isolated polynucleotide of embodiment 37 or embodiment 38. [0294] 40. The composition of embodiment 39, wherein the nucleic acid is saRNA. [0295] 41. The composition of embodiment 39 or embodiment 40, wherein the isolated polynucleotide is formulated, in communication with, or encapsulated with a delivery vehicle. [0296] 42. The composition of embodiment 41, wherein the delivery vehicle is selected from the group consisting of amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate. [0297] 43. The composition of embodiment 41 or embodiment 42, wherein the delivery vehicle comprises a phospholipid, a sterol, and a PEGylated lipid. [0298] 44. The composition of any one of claims 39 to 43, wherein the composition is a therapeutic composition or a vaccine. [0299] 45. The composition of embodiment 44, wherein the composition is configured to be administered to a subject known or suspected to have cancer, or for use in an anti- cancer therapy or cancer immunotherapy. Attorney Docket No.766501:MTST-288PC (240503G-P) [0300] 46. The composition of embodiment 44 or embodiment 45, wherein the composition is configured to be administered as an injectable preparation. [0301] 47. The composition of any one of claims 44 to 46, wherein the composition further comprises one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients or other therapeutic agents. [0302] 48. A method comprising, administering to a subject in need thereof, a therapeutically effective amount of a composition of any one of claims 39 to 47. [0303] 49. The method of embodiment 48, wherein the subject has or is suspected to have cancer. [0304] 50. A kit comprising the composition of any one of claims 39 to 47 and instructions for use. [0305] 51. A vector comprising the isolated polynucleotide of embodiment 37 or embodiment 38. [0306] 52. A host cell comprising the isolated polynucleotide of embodiment 37 or embodiment 38. [0307] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.766501:MTST-288PC (240503G-P) CLAIMS 1. A polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6. 2. The polynucleotide of claim 1, wherein the SE-SCT MHC I molecule comprises the bXV]P[ bT`dT]RT ^U t2\' cWT \PcdaT _^acX^] ^U \daX]T t2m, and the H-2kb heavy chain. 3. The polynucleotide of claim 1 or claim 2, wherein the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. 4. The polynucleotide of any one of claims 1 to 3, wherein the polynucleotide comprises DNA or RNA. 5. The polynucleotide of claim 4, wherein the DNA is plasmid DNA (pDNA) or complementary DNA (cDNA). 6. The polynucleotide of claim 4, wherein the RNA is messenger ribonucleic acid (mRNA), self-amplifying RNA (saRNA), small RNA (sRNA), micro RNA (miRNA), or circular RNA (circRNA). 7. An RNA molecule comprising a polynucleotide encoding a spike epitope-loaded single-chain trimer (SE-SCT) MHC I molecule, wherein the spike epitope is selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, wherein the RNA molecule is mRNA or saRNA. 8. The RNA molecule of claim 7, wherein the SE-SCT MHC I molecule comprises the bXV]P[ bT`dT]RT ^U t2\' cWT \PcdaT _^acX^] ^U \daX]T t2m, and the H-2kb heavy chain. 9. The RNA molecule of claim 7 or claim 8, wherein the SE-SCT MHC I molecule further comprises at least one linker, alternatively at least two linkers. 10. An expression construct comprising the polynucleotide of any one of claims 1 to 6 or the RNA molecule of any one of claims 7 to 9. 11. The expression construct of claim 10, further comprising a 3’ UTR, 5’ UTR, a 5’ Cap, a replicase and/or Poly(A) sequence. Attorney Docket No.766501:MTST-288PC (240503G-P) 12. A composition comprising the polynucleotide of any one of claims 1 to 6, the RNA molecule of any one of claims 7 to 9, or the expression construct of claim 10 or claim 11. 13. The composition of claim 12, wherein the polynucleotide is formulated, in communication with, or encapsulated with a delivery vehicle. 14. The composition of claim 13, wherein the delivery vehicle is selected from the group consisting of amphipathic molecules, amino-lipidated peptides, tertiary amino lipidated cationic peptides, a cationic component, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate. 15. The composition of claim 13 or clam 14, wherein the delivery vehicle has an encapsulation efficiency of about 50% to about 100%. 16. The composition of any one of claims 13 to 15, wherein the delivery vehicle comprises an ionizable lipid, a phospholipid, a sterol, and a PEGylated lipid. 17. The composition of claim 16, wherein the ionizable lipid is an amino alcohol-derived lipid. 18. The composition of claim 17, wherein the ionizable lipid comprises a hydroxyl group connected with the amino head groups via varying carbon spacers. 19. The composition of claim 17, wherein the ionizable lipid comprises a carboxylic acid group. 20. The composition of claim 17, wherein the ionizable lipid comprises two hydroxyl groups introduced to the head group via an ethylidene linker. 21. The composition of any one of claims 17 to 20, wherein the ionizable lipid further comprises a hydrophobic tail selected from the group consisting of non-biodegradable hydrocarbon tails, branched ester tails, carbonate ester, and acid-labile acetal groups. Attorney Docket No.766501:MTST-288PC (240503G-P) 22. The composition of claim 21, wherein the ionizable lipid has a structure of or n=5 and R=R9. Attorney Docket No.766501:MTST-288PC (240503G-P) 24. The composition of claim 21, wherein the ionizable lipid has a structure of . 25. The composition of claim 24, wherein n=1 and R=R1, n=1 and R=R2, n=1 and R =R6, n=1 and R=R8, n=2 and R =R1, n=2 and R =R2, n=2 and R =R6, n=2 and R =R8, n=2 and R =R9, n=3 and R=R1, n=3 and R=R2, n=4 and R=R1, n=4 and R=R2, n=5 and R=R1, n=5 and R=R2, n=5 and R=R6, n=5 and R=R7, or n=5 and R=R9. 26. The composition of claim 21, wherein the ionizable lipid has a structure of Attorney Docket No.766501:MTST-288PC (240503G-P) . 27. The composition of claim 26, wherein R =R1, R=R2, R= R6, R=R7, or R=R9. 28. A biological preparation comprising the composition of any one of claims 12 to 27. 29. The biological preparation of claim 28, wherein the biological preparation is a vaccine, an immunotherapy, or a therapeutic composition. 30. The biological preparation of claim 29, wherein the biological preparations is configured to be administered to a subject known or suspected to have cancer, or for use in an anti-cancer therapy or cancer immunotherapy. 31. The biological preparation of claim 29 or claim 30, wherein the biological preparations is configured to be administered as an injectable preparation. 32. The biological preparation of any one of claims 28 to 31, wherein the biological preparations further comprises one or more one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients or other therapeutic agents. 33. A method comprising, administering to a subject in need thereof, a therapeutically effective amount of a biological preparation of any one of claims 28 to 32. Attorney Docket No.766501:MTST-288PC (240503G-P) 34. The method of claim 33, wherein the subject has or is suspected to have cancer. 35. A kit comprising the biological preparation of any one of claims 28 to 32 and instructions for use. 36. A cell comprising the polynucleotide of any one of claims 1 to 6, the RNA molecule of any one of claims 7 to 9, or the expression construct of claim 10 or claim 11.
PCT/US2025/033132 2024-06-12 2025-06-11 Lnp-rna-based antigen presentation platform Pending WO2025259745A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463658904P 2024-06-12 2024-06-12
US63/658,904 2024-06-12

Publications (1)

Publication Number Publication Date
WO2025259745A1 true WO2025259745A1 (en) 2025-12-18

Family

ID=96498490

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/033132 Pending WO2025259745A1 (en) 2024-06-12 2025-06-11 Lnp-rna-based antigen presentation platform

Country Status (1)

Country Link
WO (1) WO2025259745A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022236102A1 (en) * 2021-05-07 2022-11-10 Institute For Systems Biology Single chain trimer mhc class i nucleic acids and proteins and methods of use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022236102A1 (en) * 2021-05-07 2022-11-10 Institute For Systems Biology Single chain trimer mhc class i nucleic acids and proteins and methods of use

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
AHMED, S.F.QUADEER, A.A.MCKAY, M.R., VIRUSES, vol. 12, 2020
BADEN LINDSEY, R. ET AL.: "Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine", NEW ENGLAND JOURNAL OF MEDICINE, vol. 384, 2021, pages 403 - 416, XP093093433, DOI: 10.1056/NEJMoa2035389
BARBIER, A.J.JIANG, A.Y.ZHANG, P.WOOSTER, R.ANDERSON, D.G: "The clinical progress of mRNA vaccines and immunotherapies", NATURE BIOTECHNOLOGY, vol. 40, 2022, pages 840 - 854, XP037897817, DOI: 10.1038/s41587-022-01294-2
BLASS, E.OTT, P.A: "Advances in the development of personalized neoantigen-based therapeutic cancer vaccines", NATURE REVIEWS CLINICAL ONCOLOGY, vol. 18, 2021, pages 215 - 229, XP038001836, DOI: 10.1038/s41571-020-00460-2
C-H HUANG ET AL: "Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope", GENE THERAPY, vol. 12, no. 15, 1 August 2005 (2005-08-01), GB, pages 1180 - 1186, XP055712997, ISSN: 0969-7128, DOI: 10.1038/sj.gt.3302519 *
CHEN, S. ET AL.: "Nanotechnology-based mRNA vaccines", NATURE REVIEWS METHODS PRIMERS, vol. 3, 2023, pages 63
COLLIER, A.-R.Y. ET AL.: "Differential Kinetics of Immune Responses Elicited by Covid-19 Vaccines", NEW ENGLAND JOURNAL OF MEDICINE, vol. 385, 2021, pages 2010 - 2012
CRUZ, F.M.COLBERT, J.D.MERINO, E.KRIEGSMAN, B.A.ROCK, K.L: "The Biology and Underlying Mechanisms of Cross-Presentation of Exogenous Antigens on MHC-I Molecules", ANNUAL REVIEW OF IMMUNOLOGY, vol. 35, 2017, pages 149 - 176
CUBURU, N. ET AL.: "Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 119, 2022, pages e2116738119
DANGI, T.CLASS, J.PALACIO, N.RICHNER, J.M.PENALOZA MACMASTER, P: "Combining spike- and nucleocapsid-based vaccines improves distal control of SARS-CoV", CELL REPORTS, 2021, pages 36
DAVENPORT, B.J.MORRISON, T.E.KEDL, R.M.KLARQUIST, J: "Conserved and Novel Mouse CD8 T Cell Epitopes within SARS-CoV-2 Spike Receptor Binding Domain Protein Identified following Subunit Vaccination", THE JOURNAL OF IMMUNOLOGY, vol. 206, 2021, pages 2503 - 2507
DEMARIA, O. ET AL.: "Harnessing innate immunity in cancer therapy", NATURE, vol. 574, 2019, pages 45 - 56, XP037070569, DOI: 10.1038/s41586-019-1593-5
GAO, F. ET AL.: "Spheromers reveal robust T cell responses to the Pfizer/BioNTech vaccine an d attenuated peripheral CD8+ T cell responses post SARS-CoV-2 infection", IMMUNITY, vol. 56, 2023, pages 864 - 878
GARRIDO, F.APTSIAURI, N.DOORDUIJN, E.M.LORA, A.M.G.VAN HALL, T: "The urgent need to recover MHC class I in cancers for effective immunotherapy", CURRENT OPINION IN IMMUNOLOGY, vol. 39, 2016, pages 44 - 51, XP029463514, DOI: 10.1016/j.coi.2015.12.007
GOEL, R.R. ET AL.: "mRNA vaccines induce durable immune memory to SARS-CoV-2 and variants of concern", SCIENCE, vol. 374, 2021, pages abm0829
GUAN, W.J. ET AL.: "Clinical Characteristics of Coronavirus Disease 2019 in China", N ENGL J MED, vol. 382, 2020, pages 1708 - 1720, XP009532528, DOI: 10.1056/NEJMoa2002032
HOU, X.ZAKS, T.LANGER, R.DONG, Y: "Lipid nanoparticles for mRNA delivery", NATURE REVIEWS MATERIALS, vol. 6, 2021, pages 1078 - 1094, XP037634156, DOI: 10.1038/s41578-021-00358-0
HU, Z.OTT, P.A.WU, C.J: "Towards personalized, tumour-specific, therapeutic vaccines for cancer", NATURE REVIEWS IMMUNOLOGY, vol. 18, 2018, pages 168 - 182, XP055602258, DOI: 10.1038/nri.2017.131
HUANG, C. ET AL.: "Cancer immunotherapy using a DNA vaccine encoding a single-chain trimer of MHC class I linked to an HPV-16 E6 immunodominant CTL epitope", GENE THERAPY, vol. 12, 2005, pages 1180 - 1186, XP037774086, DOI: 10.1038/sj.gt.3302519
HUANG, C. ET AL.: "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China", LANCET, vol. 395, 2020, pages 497 - 506, XP086050317, DOI: 10.1016/S0140-6736(20)30183-5
JOS� PABLO ROMERO-L�PEZ ET AL: "A bioinformatic prediction of antigen presentation from SARS-CoV-2 spike protein revealed a theoretical correlation of HLA-DRB1*01 with COVID-19 fatality in Mexican population: An ecological approach", JOURNAL OF MEDICAL VIROLOGY, JOHN WILEY & SONS, INC, US, vol. 93, no. 4, 10 October 2020 (2020-10-10), pages 2029 - 2038, XP072669111, ISSN: 0146-6615, DOI: 10.1002/JMV.26561 *
KRAMMER, F: "SARS-CoV-2 vaccines in development", NATURE, vol. 586, 2020, pages 516 - 527, XP093222110, DOI: 10.1038/s41586-020-2798-3
LAURING, A.S. ET AL.: "Clinical severity of, and effectiveness of mRNA vaccines against, covid-19 from omicron, delta, and alpha SARS-CoV-2 variants in the United States: prospective observational study", BMJ, vol. 376, 2022, pages e069761
LAWRENCE, M.S. ET AL.: "Mutational heterogeneity in cancer and the search for new cancer-associated genes", NATURE, vol. 499, 2013, pages 214 - 218, XP055251629, DOI: 10.1038/nature12213
LIU, J. ET AL.: "Vaccines elicit highly conserved cellular immunity to SARS-CoV-2 Omicron", NATURE, vol. 603, 2022, pages 493 - 496, XP037725571, DOI: 10.1038/s41586-022-04465-y
MAO TIANYANG ET AL: "Unadjuvanted intranasal spike vaccine elicits protective mucosal immunity against sarbecoviruses", SCIENCE - AUTHOR MANUSCRIPT, vol. 378, no. 6622, 25 November 2022 (2022-11-25), US, XP093137515, ISSN: 0036-8075, DOI: 10.1126/science.abo2523 *
MILLAR DAVID G ET AL: "Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 38, no. 4, 10 February 2020 (2020-02-10), pages 420 - 425, XP037086855, ISSN: 1087-0156, [retrieved on 20200210], DOI: 10.1038/S41587-019-0404-8 *
MILLAR, D.G. ET AL.: "Antibody-mediated delivery of viral epitopes to tumors harnesses CMV-specific T cells for cancer therapy", NATURE BIOTECHNOLOGY, vol. 38, 2020, pages 420 - 425, XP037086855, DOI: 10.1038/s41587-019-0404-8
NEEFJES, J.JONGSMA, M.L.M.PAUL, P.BAKKE, O: "Towards a systems understanding of MHC class I and MHC class II antigen presentation", NATURE REVIEWS IMMUNOLOGY, vol. 11, 2011, pages 823 - 836, XP055057595, DOI: 10.1038/nri3084
NEWMAN, J.H. ET AL.: "Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 117, 2020, pages 1119 - 1128, XP055879826, DOI: 10.1073/pnas.1904022116
PAMELA C. ROSATO ET AL: "Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy", NATURE COMMUNICATIONS, vol. 10, no. 1, 1 February 2019 (2019-02-01), XP055569651, DOI: 10.1038/s41467-019-08534-1 *
PARN, S.SAVSANI, K.DAKSHANAMURTHY, S: "SARS-CoV-2 Omicron (BA.1 and BA.2) specific novel CD8+ and CD4+ T cell epitopes targeting spike protein", IMMUNOLNFORMATICS, 2022, pages 8
POLACK FERNANDO, P. ET AL.: "Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine", NEW ENGLAND JOURNAL OF MEDICINE, vol. 383, 2020, pages 2603 - 2615, XP055820495, DOI: 10.1056/NEJMoa2034577
POLUEKTOV YURI ET AL: "Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers", VACCINE, vol. 39, no. 15, 1 April 2021 (2021-04-01), AMSTERDAM, NL, pages 2110 - 2116, XP093005957, ISSN: 0264-410X, DOI: 10.1016/j.vaccine.2021.03.008 *
POLUEKTOV, Y.GEORGE, M.DAFTARIAN, P.DELCOMMENNE, M.C: "Assessment of SARS-CoV-2 specific CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers", VACCINE, vol. 39, 2021, pages 2110 - 2116, XP093005957, DOI: 10.1016/j.vaccine.2021.03.008
ROSENBLUM, D.JOSHI, N.TAO, W.KARP, J.M.PEER, D: "Progress and challenges towards targeted delivery of cancer therapeutics", NAT COMMUN, vol. 9, 2018, pages 1410, XP055914762, DOI: 10.1038/s41467-018-03705-y
SADE-FELDMAN, M. ET AL.: "Resistance to checkpoint blockade therapy through inactivation of antigen presentation", NATURE COMMUNICATIONS, vol. 8, 2017, pages 1136, XP055514594, DOI: 10.1038/s41467-017-01062-w
SCHOENMAKER, L. ET AL.: "mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 601, 2021, pages 120586, XP093270453, DOI: 10.1016/j.ijpharm.2021.120586
SCHUMACHER, T.N.SCHREIBER, R.D: "Neoantigens in cancer immunotherapy", SCIENCE, vol. 348, 2015, pages 69 - 74, XP055185153, DOI: 10.1126/science.aaa4971
SHARMA, P.HU-LIESKOVAN, S.WARGO, J.A.RIBAS, A: "Primary, adaptive, and acquired resistance to cancer immunotherapy", CELL, vol. 168, 2017, pages 707 - 723, XP029935389, DOI: 10.1016/j.cell.2017.01.017
SON, E.T. ET AL.: "The self-peptide repertoire plays a critical role in transplant tolerance induction", THE JOURNAL OF CLINICALINVESTIGATION, 2021, pages 131
TILSTRA, G. ET AL.: "Iterative Design of Ionizable Lipids for Intramuscular mRNA Delivery", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 145, 2023, pages 2294 - 2304, XP093031017, DOI: 10.1021/jacs.2c10670
WALDMAN, A.D.FRITZ, J.M.LENARDO, M.J: "A guide to cancer immunotherapy: from T cell basic science to clinical practice", NATURE REVIEWS IMMUNOLOGY, vol. 20, pages 651 - 668
WANG, L. ET AL.: "T cell immune memory after covid-19 and vaccination", BMJ MED, vol. 2, 2023, pages e000468
XUE YONGER ET AL: "LNP-RNA-mediated antigen presentation leverages SARS-CoV-2-specific immunity for cancer treatment", NATURE COMMUNICATIONS, vol. 16, no. 1, 4 March 2025 (2025-03-04), UK, XP093317299, ISSN: 2041-1723, DOI: 10.1038/s41467-025-57149-2 *
XUE, Y. ET AL.: "LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing", NATURE COMMUNICATIONS, vol. 15, 2024, pages 739
YARCHOAN, M.JOHNSON III, B.A.LUTZ, E.R.LAHERU, D.A.JAFFEE, E.M: "Targeting neoantigens to augment antitumour immunity", NATURE REVIEWS CANCER, vol. 17, 2017, pages 209 - 222, XP055507793, DOI: 10.1038/nrc.2016.154
YU Y Y ET AL: "Yu, Y. Y., Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells. J. Immunol. 168, 3145-3149", J. IMMUNOL., vol. 168, 1 January 2002 (2002-01-01), pages 3145 - 3149, XP093317366 *
YU, Y.Y.NETUSCHIL, N.LYBARGER, L.CONNOLLY, J.M.HANSEN, T.H: "Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells", J IMMUNAL, vol. 168, 2002, pages 3145 - 3149, XP002433697

Similar Documents

Publication Publication Date Title
Liu et al. mRNA cancer vaccines: construction and boosting strategies
AU2019345104B2 (en) Therapeutic nanoparticles and methods of use thereof
JP7181880B2 (en) A core/shell structural platform for immunotherapy
Malla et al. mRNA vaccines and their delivery strategies: a journey from infectious diseases to cancer
Mockey et al. mRNA-based cancer vaccine: prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes
US20240033364A1 (en) Castration resistant prostate cancer
Karimi-Sani et al. Personalized mRNA vaccines in glioblastoma therapy: from rational design to clinical trials
CN110392577A (en) RNA vaccines and immune checkpoint inhibitors for combination anticancer therapy
Yang et al. Synthetic multiepitope neoantigen DNA vaccine for personalized cancer immunotherapy
IL260030B2 (en) Neoepitope therapeutic vaccine against cancer
US20140086828A1 (en) Modified gold nanoparticles for therapy
AU2020234003A1 (en) Nucleic acid vaccination using neo-epitope encoding constructs
Song et al. Design and evaluation of synthetic delivery formulations for peptide-based cancer vaccines
JP2023533871A (en) APC targeting unit for immunotherapy
WO2020225779A1 (en) Rig-i agonists for cancer treatment and immunotherapy
EP4132959A1 (en) Neoepitope immunotherapy with apc targeting unit
JP2024539435A (en) Spherical nucleic acids for modulation of the cGAS-STING and STAT3 pathways for the immunotherapeutic treatment of cancer - Patents.com
WO2025259745A1 (en) Lnp-rna-based antigen presentation platform
KR20220007850A (en) mRNA vaccine
Zhou et al. Engineering cGAS-agonistic oligonucleotides as therapeutics and vaccine adjuvants for cancer immunotherapy
Jarzebska et al. Protamine for RNA transfection: from heparin antagonist to RNA delivery
Ko et al. Lipid nanoparticle encapsulated TLR3 agonist adjuvant elicits potent T cell immunity against cancer and viruses
US20240382429A1 (en) Polymeric nanoparticle genetic vaccines
Lee et al. A synthetic cationic helical polypeptide as a multipotent innate immune activator for cancer immunotherapy
Del Bene et al. From genetic code to global health: the impact of nucleic acid vaccines on disease prevention and treatment

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25743633

Country of ref document: EP

Kind code of ref document: A1