Procapped mRNA for targeted cell translation

Abstract

Described herein are nucleic acid complexes for the targeted delivery of reversibly capped mRNA complexes (procapped mRNA) which are conditionally activated in the presence of a selected cell signal. The mRNA complexes can include mRNA with is 5' capped with a selectively removable protecting group involving a click-release reaction between tetrazine and trans-cyclooctene (TCO), to achieve selective activation of mRNA within targeted cells.

Description

G8118-04401 PATENT PROCAPPED MRNA FOR TARGETED CELL TRANSLATION RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 641,605, filed on May 2, 2024, the entire disclosure of which is incorporated herein by reference. FIELD

[0002] The present invention relates to the use of modified mRNA capping reagents for targeted cell translation, and methods of their use for treating diseases. The modified mRNA capping reagents are pro-caps (equivalent of prodrugs) which are selectively converted to active mRNA caps in the presence of an activator moiety present in a selected cell type target. The invention also includes methods of creating , and compositions comprising said mRNA capping reagents. BACKGROUND

[0003] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

[0004] Messenger RNA (mRNA) based therapeutics have gained tremendous interest and shown great promise, exemplified by the successful development of mRNA vaccines against COVID-19. Compared to other genetic vectors like viruses and DNA, mRNA therapeutics offer several advantages, including transient expression with minimal risk of genomic integration, rapid and inexpensive in vitro transcription manufacturing, and the ability to transfect and be translated in a wide range of cell types, including non-dividing cells.

[0005] However, a fundamental limitation of current synthetic mRNA therapeutics is their inherent lack of selectivity, as any cell with an active translation machinery can translate the transfected mRNA. This lack of cell- or tissue-specific expression greatly impedes the translational success of mRNA therapeutics by increasing off-target effects and reducing therapeutic efficacy. 1 DM2\21379822.1G8118-04401 PATENT SUMMARY

[0006] The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Brief Summary. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this introduction, which is included for purposes of illustration only and not restriction.

[0007] It is an object of the invention to provide compounds, compositions, formulations, kits and methods for making procapped mRNA molecules and related compounds, methods for their targeted cell-specific activation, and therapeutic applications thereof for treating diseases such as cancer and inducing senescent cell apoptosis.

[0008] Some aspects of the present invention include a compound, hydrate, or a pharmaceutically acceptable salt thereof, having the structure of Formula (I) :wherein: R1 is selected from H, -P(O)(OH), -P(O)OP(O)(OH), or -P(O)OP(O)OP(O)(OH)2; W is selected from an electron pair, hydrogen, or methyl, wherein when W is hydrogen or methyl the nitrogen atom connected to W is positively charged; L1 and L2 are independently selected from: -H, -OH, -NH2, -OR3 and -NHR3; and R2 is hydrogen or 2 DM2\21379822.1G8118-04401 PATENT ,, wherein R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13are independently selected from hydrogen C1-C6 alkyl, or C(O)NHCH2C(O)(OH); with the proviso that at least one of L1, or L2is -OR3and -NHR3and R3is3 DM2\21379822.1G8118-04401 PATENT.

[0009] Pharmaceutically acceptable salts are those which include an anion (for positively charged compounds of formula (I)), or a cation (for negatively charged compounds of formula (I)). The anion can be selected from chloride, bromide, sulfate, iodide, citrate, fumarate, acetate, aspartate, benzenesulfonate, besylate, bitartrate, camsylate, nitrate, methylsulfate, malate, maleate, octanoate, oleate, decanoate, gluconate, lactate, salicylate, propionate, striate, succinate, tartrate, tosylate, phosphate, carbonate, bicarbonate, benzoate, and the like. The cation can be selected from sodium, potassium, cesium, lithium, calcium, quaternary ammonium (e.g., ammonium or tetramethylammonium), or an amino acid. Hydrates include the compound or a salt form thereof, complexed with one to nine water molecules.

[0010] In some aspects, R2 or R3 is independently selected from hydrogen, C1-6 alkyl, or.

[0011] In some aspects, this disclosure provides for a compound, or pharmaceutically acceptable salt thereof, having the structure of formula (II): 4 DM2\21379822.1G8118-04401 PATENTwherein: B1 and B2 are independently a nucleoside base; L3, L4, and L5 are independently selected from -H, -OH, -NH2, -OR3 and -NHR3 ; R3and R14are independently selected from hydrogen or, R15 is hydrogen or -OCH3, and R16is hydrogen or methyl. 5 DM2\21379822.1G8118-04401 PATENT

[0012] In some aspects, R3 and R14 are independently selected from hydrogen or.

[0013] In some aspects, at least one of B1 and B2 is N6-methyladenine. In some aspects, B1 is adenine and B2 is guanine. In some aspects, B1 is guanine and B2 is guanine. In some aspects, B1is adenine and B2is uracil.

[0014] In some aspects, this disclosure provides for a compound, or pharmaceutically salt, or hydrate thereof, having the structure of formula (III):6 DM2\21379822.1G8118-04401 PATENT

[0015] In some aspects, this disclosure provides for a compound, or pharmaceutically salt, or hydrate thereof, of compound 11:(Compound 11).

[0016] In some aspects, this disclosure provides for a compound, or pharmaceutically salt, or hydrate thereof, of compound 13:(Compound 13).

[0017] Further provided herein are a procapped mRNA molecule comprising: a 5’ procap; a 5’ UTR; an open reading frame sequence; and a 3’ tailing region, wherein the 5’ procap comprises a cellularly-selective removable protecting group. In some aspects, the cellularly-selective removable protecting group is a substituted or unsubstituted trans- cyclooctene group. In some aspects, the 5’ cap is a 2’-deoxy or 3’-deoxy derivative of a compound of formula (I) or (II) as described herein. The trans-cyclooctene group is removable by a tetrazine moiety. In some aspects, the RNA molecule is mRNA. The mRNA can further comprise a 3’ UTR. In some aspects, the 3’ tailing region is a polyA tail. In some aspects, the 5’ UTR, open reading frame sequence, and 3’ tailing region each consist of a nucleic acid sequence consisting of ribonucleotides selected from: adenine (A), guanine (G), cytosine (C), and uracil (U). 7 DM2\21379822.1G8118-04401 PATENT

[0018] In some aspects, this disclosure provides for a kit comprising a compound of formula (I) and a tetrazine-moiety.

[0019] In some aspects, this disclosure provides for a compound of formula (IV) or (V):wherein R1 is selected from H, -P(O)(OH), -P(O)OP(O)(OH), or - P(O)OP(O)OP(O)(OH)2; L1is selected from O or NH or NCH3; and L2is selected from OH, NH2, or OCH3.

[0020] Some aspects of the invention include a procapped mRNA molecule comprising a 5' procap, a 5' UTR, an open reading frame sequence, and a 3' tailing region, wherein the 5' 8 DM2\21379822.1G8118-04401 PATENT procap comprises a cellularly selective removable protecting group such as a trans-cyclooctene group capable of being removed by a tetrazine moiety. In certain embodiments, the 5' cap is a 2'- deoxy or 3'-deoxy derivative of compounds 11 or 13. The mRNA may further comprise a 3' UTR and a poly-A tail. The 5' UTR, open reading frame, and 3' tailing region may consist of A, G, C or U ribonucleotides.

[0021] Aspects of the invention also provide methods of expressing mRNA in selected cells by presenting a tetrazine moiety and a procapped mRNA to the cell, causing the procap to be converted to a functional cap. The cell may be a tumor cell expressing αvβ3 integrin and the tetrazine moiety may be c(RGDyK)-Tz. Alternatively, the cell may be senescent and the tetrazine may be Gal-Tz. The open reading frame may encode a fluorescent protein, caspase-9, or a cancer neoantigen.

[0022] The invention further provides methods of inducing apoptosis in senescent cells by presenting Gal-Tz, procapped mRNA encoding caspase-9, and the dimerizing drug AP20187. Removal of the procap allows caspase-9 expression which is activated by AP20187 to induce apoptosis.

[0023] Additionally, some aspects relate to methods of treating cancer by administering a procapped mRNA and c(RGDyK)-Tz to a subject. The invention also provides vaccine compositions comprising a tetrazine conjugated to a muscle or immune cell targeting peptide, a procapped mRNA encoding a viral or cancer antigen, encapsulated in lipid nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0026] FIGS. 1A-1B display the conceptualization and advantages of the procapped mRNA technology. FIG. 1A displays a procap which is is a synthetic 5’ cap analogue that interferes with the translation initiation of eukaryotic mRNA, but can be converted to a functional 5’ cap by a bioorthogonal cleavage reaction in specific cells. CDS, coding sequence; 9 DM2\21379822.1G8118-04401 PATENT POI, protein of interest; green regions (not marked), untranslated regions. FIG. 1B displays the realization of the procapped mRNA through the click-release chemistry via the inverse electron- demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine. One of the many possible structures of the procapped mRNAs is shown.

[0027] FIG. 2 displays a uridine (U) and two closely related nucleobase modifications, pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). The functions of many RNAs are comprised or completely lost after U is replaced by Ψ or m1Ψ, despite the facts that these three nucleobases have identical hydrogen bonding donors and acceptors on the Watson-Crick edge. Nucleobase modifications are crucial for enhancing the mRNA translation by increasing its stability and reducing its immunogenicity.

[0028] FIGS. 3A-3C depict the enzymatic capping of the IVT RNA. FIG. 3A depicts Schematic of the post-transcriptional enzymatic capping, which involves two enzymes: the Vaccinia virus Capping Enzyme (VCE), which adds the cap-0 to the 5’-end of RNA; and the mRNA cap 2´-O-methyltransferase (2´-OMTase), which converts cap-0 to cap-1. FIG. 3B depicts Preliminary results via denaturing polyacrylamide gel electrophoresis (PAGE) demonstrating that GTP analogues with modifications on either 3’ or 2’ site of the ribose moiety are compatible with the enzymatic capping strategy. The successful capping is suggested by a slower electrophoretic migration of the RNA band. Lane M contains the molecular weight markers. FIG. 3C depicts structures of the GTP analogues that have been used in FIG. 3B.

[0029] FIGS. 4A-4C depict co-transcriptional capping. FIG. 4A displays structure of a CleanCapTMreagent (shown here is the CleanCapTMReagent AG (3' OMe); TriLink). FIG. 4B displays the mostly commonly used T7 φ6.5 promoter needs to be engineered (a C+1T mutation is introduced in the DNA template strand) for the compatibility with the CleanCapTM-like reagent, XAG (where X can be a variety of nucleotides or functional groups). FIG. 4C shows Preliminary results (via denaturing PAGE) demonstrating that the engineered T7 promoter can accommodate different X groups during IVT.

[0030] FIG. 5 displays proposed synthetic route of a TCO-caged GTP analogue for posttranscriptional enzymatic capping.

[0031] FIGS. 6A-6B display proposed synthetic routes of two TCO-caged trinucleotides for co-transcriptional capping. FIG. 6A shows the synthetic scheme where the TCO moiety is installed on the 2’-O of the inverted m7G of these two procapping reagents. FIG. 10 DM2\21379822.1G8118-04401 PATENT 6B shows the synthetic scheme where the TCO moiety is installed on the 3’-O of the inverted m7G of these two procapping reagents.

[0032] FIG. 7A displays site-specific RNA cleavage by RNase H. The cleavage is guided by a 2’-O-methyl RNA-DNA chimera strand to release a shortsized of the 5’ fragment. FIG. 7B shows the denatured gel electrophoresis of mRNA fragments with different Cap structures. To assess the capping efficiency of these longer mRNA molecules, we employed a 2'-O-methyl RNA-DNA chimera guide strand for site-specific RNase H-mediated cleavage and determine capping efficiency by precisely analyzing the released short 5’ fragments via denaturing PAGE (FIG. 7A). The gel electrophoresis results showed that the short 5’ fragments of TCO-Cap 1 mRNA were longer and had additional TCO modifications, so they migrated more slowly (FIG. 7B). From the analysis of the bands, TCO-Cap 1 had high capping efficiency and the purity of TCO-Cap 1 mRNA was very high (~100%).

[0033] FIG. 8 displays the impact of 5’ cap structures on mRNA expression performance. Enzymatic capping is employed to cap the mRNA post-transcriptionally with different GTP analogues (The structures are shown in FIG. 3A; CleanCapTM, shown in FIG. 4A). All mRNA molecules were modified with the m1Ψ and transfected with the Lipofectamine MessengerMAX reagent. Expression levels were determined with a dual luciferase assay, where the mRNAs encoding Gaussia Luciferase (GLuc) are with different caps, and a CleanCapTMmRNA encoding Cypridina Luciferase (CLuc) serves as an internal control for eliminating transfection efficiency variations. GLuc:CLuc ratios are presented (mean ± s.d., n (biological replicates) = 3).

[0034] FIGS. 9A-9C display two strategies of selectively introducing tetrazine into specific cells. FIG. 9A is the structure of Targeted delivery Ligand c(RGDyK), which binds to αvβ3 integrin, one of the most well-studied targets for drug delivery into cancer cells, was used in the design of the tetrazine (Tz) containing conjugate c(RGDyK)-Tz. FIG. 9B depicts the mechanism for the targeted activation. Senescence-associated β- galactosidase (SA-β-gal) is the most widely recognized biomarker of cellular senescence and aging. Incorporating the galactose (Gal) moiety into dihydrotetrazine gives a stable Gal-caged conjugate, galactose-tetrazine (Gal- Tz), which can be converted to the active Tz by SA-β-gal. Cancer cell-targeting activation of TCO-Cap mRNA. FIG. 9C shows confocal microscope images of cells treated with either none, c(RGDyk)Tz with TCO-Cap mRNA, or CleanCap mRNA (as a positive control), demonstrating that cells treated with c(RGDyk)Tz with TCO-Cap mRNA express similar levels of reporter 11 DM2\21379822.1G8118-04401 PATENT protein as cells treated with CleanCap mRNA. An experiment was performed to confirm the specific activation of TCO-Cap mRNA targeting the integrin-overexpressing cancer cell. We transfected 50 ng / well TCO-Cap mRNA or Clean Cap mRNA into HeLa cells which has high integrin expression level, and triggered with 50 μM c(RGDyk)Tz targeting the integrin (FIG. 9A). Confocal microscope results showed that the TCO-Cap mRNA can also be selectively activated by c(RGDyk)Tz (FIG. 9B). The results demonstrate that the TCO-Cap mRNA can be selectively activated within targeted sensescent cells, which is a model of cancer cells (Wang, L., et al. Exploiting senescence for the treatment of cancer. Nat Rev Cancer 22, 340–355 (2022). doi.org / 10.1038 / s41568-022-00450-9).

[0035] FIGS.10A-10C show a schematic diagram of the reaction between TCO-Cap and tetrazine. FIG. 10A shows a reaction scheme where TCO-Cap mRNA and tetrazine undergo IEDDA (Inverse electron-demand Diels–Alder) reaction, and then TCO dissociates from the Cap. FIG. 10B shows chemical structures of representative TCO-Cap compounds TCO-Cap1 and TCO-Cap2. FIG. 10C shows chemical structures of representative tetrazine-containing compounds MeTzMe, 2Py-Tz, c(RGDyk)-Tz, and Gal-Tz. FIG. 10A shows a representative mRNA prodrug which becomes activated only at the desired, targeted sites through enzymatic and / or chemical reactions. The 5’ cap is crucial for the translation initiation of eukaryotic mRNAs. The methods described herein are based on the inverse electron-demand Diels-Alder (IEDDA) click reaction between trans-cyclooctene (TCO) and tetrazine (Tz), which dissociates TCO from the Cap and then restores the translation function of the mRNA. FIG. 10B shows two representative cap analogs which were synthesized. FIG. 10C shows four representative tetrazine-containing compounds which were synthesized and further tested the reactions between those and the compounds of FIG. 10B.

[0036] FIG. 11 shows the reaction processes of TCO-Cap 1 and three representative tetrazine-containing compounds and their products. Representative tetrazine-containing compounds were demonstrated to bind to TCO-Cap 1 through a click chemistry reaction, and then TCO-Tz dissociates from the cap analog compound, allowing the cap (m27,3’-OGpppAmG) to become uncaged.

[0037] FIGS. 12A-12C depict the chemical synthesis route of TCO-Cap 1. FIG. 12A depicts the synthesis of TCO precursors via reaction of hydroxyl groups with acyl chlorides. FIG. 12B depicts the synthesis of TCO-m27,3’-OGp. FIG. 12C depicts the synthesis of TCO- m27,3’-OGpppAmG. First, TCO precursors were synthesized via esterification of hydroxyl groups 12 DM2\21379822.1G8118-04401 PATENT with acyl chlorides (FIG. 12A). The oxygen in the hydroxyl group acts as a nucleophile to attack the carbonyl carbon in the acyl chloride and form a tetrahedral intermediate. The intermediate then expels Cl- to form an ester and releases HCl. 4-Dimethylaminopyridine (DMAP) is used as an efficient nucleophilic catalyst to accelerate the esterification reaction. Dichloromethane (DCM) is used as an organic solvent to ensure that the reactants are fully mixed. We then synthesized the m27,3’-OGp (FIG. 12B). We first protected the hydroxyl group on the ribose with TBSCl, then attached TCO to the guanine nucleotide through an amidation reaction between the amino group (-NH₂) and the ester group (-COOR) under the catalysis of NaH, and further modified the methyl group at the 7’ position. Finally, the acyl exchange reaction between phosphoric acid (RO-PO3H2) and imidazole phosphate (Im-P2O6H2-R) was used to form a triphosphate bridged product (TCO-m27,3’-OGpppAmG) (FIG. 12C).

[0038] FIGS. 13A-13B depict the chemical synthesis route of TCO-Cap 2. FIG. 13A depicts the synthesis of TCO-m37,2’-N,3’-OGp. FIG. 13B depicts the synthesis of TCO-m37,2’-N,3’-OGpppAmG. First, we synthesized TCO precursors via esterification of hydroxyl groups with acyl chlorides. The oxygen in the hydroxyl group acts as a nucleophile to attack the carbonyl carbon in the acyl chloride and form a tetrahedral intermediate. The intermediate then expels Cl- to form an ester and releases HCl. 4-Dimethylaminopyridine (DMAP) is used as an efficient nucleophilic catalyst to accelerate the esterification reaction. Dichloromethane (DCM) is used as an organic solvent to ensure that the reactants are fully mixed. We then synthesized the m37,2’-N,3’-OGp (FIG. 13A). We first protected the hydroxyl group on the ribose with TBSCl and -CH2-S-Ph was reduced to -CH3 under the action of the strong reducing agent NaBH4. Then TCO was attached to the guanine nucleotide through an amidation reaction between the amino group (-NH₂) and the ester group (-COOR) under the catalysis of NaH, and further modified the methyl groups at the 7’ position. Finally, the acyl exchange reaction between phosphoric acid (RO-PO3H2) and imidazole phosphate (Im-P 7,2’-N,3’- 2O6H2-R) was used to form a triphosphate bridged product (TCO-m3OGpppAmG) (FIG. 13B).

[0039] FIGS.14A-14B show the characterization to confirm the synthesis of TCO-Cap 1. FIG. 14A is HPLC trace showing the purity of the purified TCO-Cap. FIG. 14B shows the Mass spectrometry analysis of TCO-Cap 1. The purity and molecular weight of the purified TCO-Cap 1 were evaluated. HPLC results showed that the purity of the obtained TCO-Cap 1 was 96.18% (FIG. 14A). A prominent doubly charged ion peak observed at m / z 656.60 in the mass spectrometry (MS) analysis confirms the identity of the product as TCO-Cap 1 (FIG. 14B). 13 DM2\21379822.1G8118-04401 PATENT

[0040] FIGS.15A-15G show the HPLC analysis of the reaction between TCO-Cap 1 and tetrazines. FIG. 15A shows the HPLC analysis of TCO-Cap 1. FIG. 15B shows the HPLC analysis of the reaction of TCO-Cap 1 and MeTzMe. FIG. 15C shows the HPLC analysis of the reaction of TCO-Cap 1 and 2Py-Tz. FIG. 15D shows the Mass Spectra (MS) profile of the TCO- Cap1 peak, confirming the identity. FIG. 15E shows the MS profile of Cap (released) peak, confirming the identity. FIG.15F shows the MS profile of MeTzMe+TCO-Cap peak, confirming the identity. FIG. 15G shows the MS profile of 2PyTz+TCO-Cap peak, confirming the identity. HPLC (C18 column) was used to analyze the reaction between TCO-Cap 1 and Tz. We incubated 2 mM TCO-Cap 1 with 20 mM MeTzMe or 2Py-Tz in 10 mM Tris-HCl (pH 7.5), at 37 °C for 30 minutes and then performed HPLC analysis. HPLC results showed that MeTzMe effectively dissociated TCO from Cap, and due to the different polarities of the products, the cleaved product had a higher polarity and a shorter retention time, while the uncleaved product had a lower polarity and a retention time longer than TCO-Cap 1 (FIG. 15A, FIG. 15B). However, 2Py-Tz didn’t effectively cleave TCO from the Cap, and the reaction product had a smaller polarity, so the retention time increased (FIG. 15C). MS analysis was performed on the peak collection of TCO- Cap 1 in FIG. 15A. The results in FIG. 15D showed that the molecular weight of the strong peak (m / z 1321.081) is consistent with that of TCO-Cap 1. In addition, MS analysis was performed on the peak collections of m27,3’-OGpppAmG and MeTzMe-TCO-Cap 1 in FIG 15A. The results in FIG. 15D and FIG. 15E showed that the molecular weight of the strong peaks (m / z 1165.070 and 1400.096) were consistent with that of m27,3’-OGpppAmG and MeTzMe-TCO-Cap 1, respectively. In addition, MS analysis was performed on the peak collection of 2Py-Tz-TCO- Cap 1 in FIG. 15C. The results in FIG.15F showed that the molecular weight of the strong peak (m / z 1530.440) is consistent with that of 2Py-Tz-TCO-Cap 1.

[0041] FIGS. 16A-16C show the experimental results of the TCO-Cap EGFP mRNA translation in cells. FIG. 16A shows TCO-Cap mRNA translation in different cell lines. FIG. 16B is a Flow cytometry histogram of EGFP expression in HeLa, U251 and A549 cell lines. FIG. 16C shows the normalized EGFP mean fluorescent intensity (MFI) for HeLa, U251 and A549 cell lines. To validate the activation of TCO-Cap mRNA expression upon MeTzMe triggering, we transfected 100 ng / well CleanCap mRNA or TCO-Cap mRNA into 10000 cells in 96-well plate for several human cell lines. 6-hours post transfection, the HeLa, 293T and U251 cell lines with TCO-Cap mRNA were treated with 150 μM MeTzMe, and the A549, MCF-7 cell lines with TCO-Cap mRNA were treated with 50 μM MeTzMe. At 24 hours post transfection, the cells were imaged by confocal microscopy (FIG. 16A). The results demonstrated that, in HeLa and U251 cells, the TCO-Cap mRNA expression can be efficiently 14 DM2\21379822.1G8118-04401 PATENT activated by MeTzMe. And the A549 cells, due to sensitivity to MeTzMe treatment, showed moderate level of activation. The flow cytometry further provided quantitative analysis for the EGFP expression in 3 cell lines (FIG. 16B). The quantified flow cytometry data are shown in bar graph form in FIG. 16C. The results demonstrated that MeTzMe enhanced TCO-Cap mRNA expression about 2 fold greater in HeLa and U251.

[0042] FIGS.17A-17B show the dose-dependent activation effects in HeLa cells. FIG. 17A shows MeTzMe mRNA translation in different cell lines. FIG.17B shows the quantification of the MeTzMe mRNA translations. To understand the dose-dependent effects of MeTzMe activation, we transfected 50 ng / well TCO-Cap mRNA or CleanCap mRNA into 10000 HeLa cells in 96 well plate, and treated the TCO-Cap mRNA with different concentrations of MeTzMe. At 24 hours post transfection, the cells were imaged by confocal microscopy and quantified by flow cytometry (FIG. 17A). The result indicated that the TCO-Cap mRNA translation activation can be enhanced by increasing MeTzMe concentration until the MeTzMe concentration exceed 150 μM (FIG. 17B). DETAILED DESCRIPTION

[0043] The following includes information that may be useful in understanding the present invention. It is not an admission that any of the information, publications or documents specifically or implicitly referenced herein is prior art, or essential, to the presently described or claimed inventions. All publications, patents, related applications, and other written or electronic materials mentioned or identified herein are hereby incorporated herein by reference in their entirety. The information incorporated is as much a part of the application as filed as if all of the text and other content was repeated in the application, and should be treated as part of the text and content of the application as filed. Definitions

[0044] As used herein, the term “about” refers to the stated value and typical degrees of error of the measurement of said value. If not otherwise explicitly recited herein, the term “about” refers to any number within + / - 10% of the recited value.

[0045] The term “and / or” as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Herein, the term “mRNA” and“mRNA molecule”, which are used 15 DM2\21379822.1G8118-04401 PATENT interchangeable, refer to a single-stranded RNA molecule built up for example of A, C, G, and / or U nucleotides, i.e. nucleotides comprising adenine, guanine, cytosine, and uracil as the respective nitrogenous base. Furthermore, an mRNA molecule contains one or more coding sequences that can be used as a template during synthesis of an amino acid sequence during translation. Thus, the mRNA molecule to be purified preferably comprises a coding sequence, i.e. a sequence that can be translated into an amino acid sequence such as a protein and that comprises a start and a stop codon. The coding sequence can be a naturally occurring sequence, a partly or fully codon optimized sequence derived from the natural sequence to be used or an artificial sequence. Codon optimization refers to a technique which is applied to maximize protein expression by increasing the translational efficiency of the respective mRNA molecule as in some cases codons exist that are preferentially used by some species for a given amino acid. Further, said mRNA molecule might comprise a 5’ and / or a 3’ untranslated region (UTRs), one or more internal ribosome entry site(s) (IRES), one or more additional sequences for promoting translation, and / or one or more modifications to adjust and / or extend the duration of action. Further features of mRNA molecules are described in further detail below.

[0046] Hence, the term “mRNA” should be understood to mean any RNA molecule which is suitable for the expression of an amino acid sequence or which is translatable into an amino acid sequence such as a protein. Thus, an“mRNA molecule” is intended to be understood as an mRNA molecule that exhibits a property and / or activity by which it is characterized and thus, can be translated into an amino acid sequence such as a protein with said amino acid sequence showing the activity and / or property by which it is characterized.

[0047] Eukaryotic mRNAs bear a "cap" structure at their 5'-termini that is well known to play an important role in translation. It facilitates translation or localization, and / or prevents degradation of an RNA transcript when incorporated at the 5′ end of an RNA transcript. Naturally occurring cap structures consist of a 7-methyl guanosine that is linked via a triphosphate bridge to the 5 '-end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleotide. The mRNA cap plays an important role in gene expression. It protects the mRNAs from degradation by exonucleases, enables transport of RNAs from the nucleus to the cytoplasm, and participates in assembly of the translation initiation complex. The cap may include a triphosphate, a tetraphosphate or a pentaphosphate group joining the two nucleotides. 16 DM2\21379822.1G8118-04401 PATENT

[0048] In the cell, the cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5' terminal end of RNA occurs after transcription but immediately after transcription initiation so that it is almost impossible to detect. The terminal nucleoside is always a guanine, and is in the reverse orientation to all the other nucleotides, i.e., 5 'Gppp5 'GpNpNp... and the cap contains two nucleotides, connected by a 5 '-5' triphosphate linkage.

[0049] As used herein, the term “cap analog” refers to a structural derivative of an RNA cap that may differ by as little as a single element.

[0050] The term “prodrug” refers to a biologically inactive compound that can be metabolized in the body to produce a biologically active drug or the “prodrug” can be a biologically active compound where in addition to its inherent biological activity can be metabolized to another or even preferred biologically active drug. In certain embodiments, the prodrug can have its own biological activity that can be similar to or different from the active drug. For example, the prodrug can be an ester of epinephrine, for example, dipivefrin which is hydrolysed into epinephrine. See, e.g., J. Anderson, et ah, Site of ocular hydrolysis of a prodrug, dipivefrin, and a comparison of its ocular metabolism with that of the parent compounds, epinephrine, Invest., Ophthalmol. Vis. Sci. July 1980.

[0051] The term "pro-capped" as used herein refers to an mRNA molecule having a 5' cap that is modified with a selectively removable protecting group, such as, for example, a trans- cyclooctene (TCO) group, which can be removed, for example, by a bioorthogonal cleavage reaction. This allows the mRNA molecule to reach a specific target cells and to convert the pro- cap into a functional 5' cap that enables translation of the mRNA.

[0052] A “compound of the present invention” and similar terms as used herein, whether explicitly noted or not, refers to pro-capped described herein, including compounds of Formula I and II and subformula thereof, and compounds 11 and 13, as well as salts (e.g., pharmaceutically acceptable salts), solvates, hydrates, stereoisomers (including atropisomers), and tautomers thereof.

[0053] The term "tailing region" or "3' tailing region" refers to a sequence of nucleotides added to the 3' end of an mRNA molecule after transcription. Examples of tailing regions include: a poly(A) tail, which is a sequence of adenine nucleotides, a poly(C) tail, which is a 17 DM2\21379822.1G8118-04401 PATENT sequence of cytosine nucleotides, or a structured 3' UTR sequence that enhances stability or translationan "open reading frame sequence".

[0054] The term “open reading frame” or “coding region” as used herein, refers to any nucleic acid sequence encoding a non-regulatory protein (also referred to herein as a “protein of interest.”)

[0055] The term protecting group is used herein as broadly as the term is used in the art to refer to a chemical group that is introduced into a molecule by reaction with a function group to protect that function group from further reaction under a given set of reaction conditions, but which can be selectively removed (by deprotection) to regenerate that functional group when protection is no longer needed or desired. The invention provides new protecting groups for organoboronic acids, benzoxaboroles and benzoxaborins and describes methods for protecting and for removal of protection. It will be appreciated that a given organoboronic acid, benzoxaborole or benzoxaborin may contain more than one of such groups for which it would be useful to provide protection. In this regard, compounds of the invention can be employed to protect one or more than one boronate, benzoxaborole and / or benzoxaborin functionality in a given compound in need of such protection. It will also be appreciated that protection as described herein of boronate, benzoxaborole and / or benzoxaborin functionality may be combined with the use of other art-known protection strategies for protection of other functional groups. Thus, functional groups in organic groups and in non-hydrogen substituents of the compounds of this invention may be protected using art-known protecting groups and methods of providing such protection.

[0056] Protecting groups for such other functional groups can be selected as is well- known in the art for ease of addition to and removal from (protection and deprotection of) a given functional group and to be non-reactive (i.e., protective) under conditions in which protection is desired. A wide variety of protective groups is known in the art. See, for example, P. G. M. Wuts and T. W. Greene (2006) Greene's Protective Groups in Organic Synthesis, 4th Ed. (Wiley-Interscience) and P. J. Kocienski (2005) Protecting Groups, 3rd Ed. (Georg Thieme Verlag, New York, which provide a description of protecting groups for various functional groups and also provide a description of reagents for introduction of protecting groups and a description of how deprotection is achieved. These references are specifically incorporated by reference herein for the structure of protecting groups (particularly amine protecting groups) and for methods for protecting and deprotecting functional groups including amines. 18 DM2\21379822.1G8118-04401 PATENT

[0057] Protective groups are often classified for the group which they protect, for example, the term amine protecting group refers to a protecting group that can be introduced into a molecule carrying an amine functional group to protect the amine group. The amine-protecting group is bonded to the nitrogen of the amine to form —NRPR, where PR is the protecting group and R is any other appropriate atom or group (e.g., hydrogen, alkyl group, aryl group etc.). Other classes of protecting groups include alcohol protecting groups, carbonyl protecting groups or carboxylic acid protecting groups. It is understood in the art, that a given protecting group may be useful for protecting different functional groups. In specific embodiments, herein, compounds of the invention may contain one or more protecting group in addition to the new protecting groups described herein. As used herein, the term "senescent cell" refers to a cell that exhibits at least a 2-fold increase in β-galactosidase activity, and reduced proliferation, for example as evidenced by incorporation of 5-ethynyl-2' -deoxyuridine (EdU) into de novo DNA synthesis.

[0058] The term "neoantigen" or "neoantigenic" means a class of tumor antigens that arises from a tumor-specific mutation(s) which alters the amino acid sequence of genome encoded proteins.

[0059] By "neoplasia" is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a neoplasia. Examples of cancers include, without limitation, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytie leukemia, acute monocytic leukemia, acute erythroieukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotbeliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, meduiloblastoma, 19 DM2\21379822.1G8118-04401 PATENT craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglionia, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lympho proliferative disorders are also considered to be proliferative diseases.

[0060] The term "neoplasia vaccine" is meant to refer to a pooled sample of neoplasia / tumor specific neoantigens, for example at least two, at least three, at least four, at least five, or more neoantigenic peptides. A "vaccine" is to be understood as meaning a composition for generating immunity for the prophylaxis and / or treatment of diseases (e.g., neoplasia / tumor). Accordingly, vaccines are medicaments which comprise antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination. A "neoplasia vaccine composition " can include a pharmaceutically acceptable excipient, earner or diluent.

[0061] A "dimerizing drug" refers to a small molecule that can simultaneously bind two protein molecules and induce their dimerization and activation.

[0062] In the present invention, the term "skeletal muscle cell" refers to a cell that performs the function of skeletal muscle, and there is no limitation as long as the cell performs the above function, but may be fetal skeletal muscle cells or adult skeletal muscle cells.

[0063] As used herein, an “immune cell” refers to any cell comprised in the immune system. The immune cell may be a cell involved in the innate or adaptive immune response.

[0064] In some embodiments, the immune cell is a natural killer cell (NK), macrophage, or T cell. In some embodiments, the T cell is a a gd T cell or a natural killer T cell (NKT cells).

[0065] “Operably-linked” refers to the association two chemical moieties so that the function of one is affected by the other, e.g., an arrangement of elements wherein the components so described are configured so as to perform their usual function.

[0066] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, made of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing synthetic analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof 20 DM2\21379822.1G8118-04401 PATENT (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. Nucleic acids include all naturally occurring nucleosides, including all forms of nucleoside bases and furanosides found in nature. Base rings most commonly found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N6-methyladenine. Naturally occurring pyrimidine rings include, for example, cytosine, thymine, 5-methylcytosine, pseudouracyl. Naturally occurring nucleosides for example include, but are not limited to, ribo, 2′-O-methyl or 2′-deoxyribo derivatives of adenosine, guanosine, cytidine, thymidine, uridine, inosine, 7-methylguanosine or pseudouridine.

[0067] The terms "nucleotide sequence", “polynucleic acid”, or “nucleic acid sequence” refer to a sequence of bases (purines and / or pyrimidines) in a polymer of DNA or RNA, which can be single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers, and / or backbone modifications (e.g., a modified oligomer, such as a morpholino oligomer, phosphorodiamate morpholino oligomer or vivo-mopholino). The terms “oligo”, “oligonucleotide” and “oligomer” may be used interchangeably and refer to such sequences of purines and / or pyrimidines. In some embodiments, the oligos can comprise chemical modifications to the internucleotide phosphate linkages and / or to the backbone sugar.

[0068] The oligonucleotides described herein may be synthesized using standard solid or solution phase synthesis methods. In certain embodiments, the oligonucleotides are synthesized using solid-phase phosphoramidite chemistry (U.S. Patent No. 6,773,885) with automated synthesizers. Chemical synthesis of nucleic acids allows for the production of various forms of the nucleic acids with modified linkages, chimeric compositions, and nonstandard bases or modifying groups attached in chosen places through the nucleic acid’s entire length.

[0069] Certain embodiments of the invention encompass isolated or substantially purified nucleic acid compositions. In the context of the present invention, an "isolated" or "purified" DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or 21 DM2\21379822.1G8118-04401 PATENT "purified" nucleic acid molecule is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.

[0070] By “portion” or “fragment,” as it relates to a nucleic acid molecule, sequence or segment of the invention, when it is linked to other sequences for expression, is meant a sequence having at least 20 nucleotides, at least 150 nucleotides, or at least 400 nucleotides. If not employed for expressing, a “portion” or “fragment” means at least 9, at least 12, at least 15, or at least 20, consecutive nucleotides, e.g., probes and primers (oligonucleotides), corresponding to the nucleotide sequence of the nucleic acid molecules of the invention.

[0071] The phrase "hybridizing specifically to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that is accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

[0072] The term "complementary" as used herein refers to the broad concept of complementary base pairing between two nucleic acids aligned in an antisense position in relation to each other. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are substantially complementary to each other when at least about 50%, at least about 60%, or at least about 80% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T (A:U for RNA) and G:C nucleotide pairs).

[0073] As used herein, the term "derived" or "directed to" with respect to a nucleotide molecule means that the molecule has complementary sequence identity to a particular molecule of interest. As used herein, the term “derived” with respect to chemical compounds refers to the form of the compound in which a hydrogen atom is replaced with a bond to the identified moiety. 22 DM2\21379822.1G8118-04401 PATENT

[0074] As used herein, the terms “transcription” or “transcription reaction” refers to methods for enzymatically making RNA that is complementary to a DNA template, thereby producing a number of RNA copies of a DNA sequence. The RNA molecule synthesized in a transcription reaction is referred to as a “RNA transcript”, “primary transcript” or “transcript”. Transcription reactions involving the compositions and methods provided herein employs procapped mRNA, after cell-selective removal of the TOC moiety. Transcription of a DNA template may be exponential, nonlinear or linear. A DNA template may be a double stranded linear DNA, a partially double stranded linear DNA, circular double stranded DNA, DNA plasmid, PCR amplicon, a modified nucleic acid template which is compatible with RNA polymerase.

[0075] Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotides directed protein production as these formulations may be able to increase cell transfection by the RNA (e.g., mRNA) vaccine; and / or increase the translation of encoded protein. One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 200715:713-720; the contents of which are incorporated herein by reference in their entirety). The liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide.

[0076] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure can be formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some embodiments, the RNA (e.g., mRNA) vaccines may be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulation” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the disclosure using fluorescence and / or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 23 DM2\21379822.1G8118-04401 PATENT 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the disclosure are encapsulated in the delivery agent.

[0077] In some embodiments, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No. WO2012131104 and WO2012131106, the contents of each of which are incorporated herein by reference in their entirety).

[0078] In some embodiments, the RNA (e.g., mRNA) vaccines may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and / or surgical sealant described herein and / or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

[0079] In some embodiments, the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non- limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.

[0080] In some embodiments, the RNA (e.g., mRNA) vaccine formulation for controlled release and / or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone / vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

[0081] In some embodiments, the RNA (e.g., mRNA) vaccine controlled release and / or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. 24 DM2\21379822.1G8118-04401 PATENT In some embodiments, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[0082] In some embodiments, the RNA (e.g., mRNA) vaccine controlled release and / or targeted delivery formulation comprising at least one polynucleotide may comprise at least one PEG and / or PEG related polymer derivatives as described in U.S. Pat. No.8,404,222, the contents of which are incorporated herein by reference in their entirety.

[0083] In some embodiments, the RNA (e.g., mRNA) vaccine controlled release delivery formulation comprising at least one polynucleotide may be the controlled release polymer system described in US20130130348, the contents of which are incorporated herein by reference in their entirety.

[0084] In some embodiments, the RNA (e.g., mRNA) vaccines of the present disclosure may be encapsulated in a therapeutic nanoparticle, referred to herein as “therapeutic nanoparticle RNA (e.g., mRNA) vaccines.” Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, U.S. Publication Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286, US20120288541, US20130123351 and US20130230567 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which are herein incorporated by reference in their entirety.

[0085] In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA) vaccine may be formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the polynucleotides of the present disclosure (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and / or particulate suspensions (see U.S. Patent Publication No 25 DM2\21379822.1G8118-04401 PATENT US20130150295, the contents of each of which are incorporated herein by reference in their entirety).

[0086] In some embodiments, the therapeutic nanoparticle RNA (e.g., mRNA) vaccines may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticles may include a corticosteroid (see International Pub. No. WO2011084518, the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, the contents of each of which are incorporated herein by reference in their entirety.

[0087] In some embodiments, the nanoparticles of the present disclosure may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide- co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.

[0088] As used herein, the term "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR). 26 DM2\21379822.1G8118-04401 PATENT

[0089] As used herein, the term “subject” as used herein refers to humans, higher non- human primates, rodents, domestic, cows, horses, pigs, sheep, dogs and cats. In one embodiment, the subject is a human.

[0090] As used herein, the terms "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or decrease an undesired physiological change or disorder. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0091] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small- cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Gastric cancer, as used herein, includes stomach cancer, which can develop in any part of the stomach and may spread throughout the stomach and to other organs; particularly the esophagus, lungs, lymph nodes, and the liver.

[0092] As used herein, the term "synergistic" as used herein refers to a therapeutic combination that is more effective than the additive effects of the two or more single agents. A determination of a synergistic interaction between an RNA-origami and one or more chemotherapeutic agent may be based on the results obtained from the assays described herein. 27 DM2\21379822.1G8118-04401 PATENT The results of these assays can be analyzed using the Chou and Talalay combination method and Dose-Effect Analysis with CalcuSyn software in order to obtain a Combination Index (Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55). The combinations provided by this invention can be evaluated in several assay systems, and the data can be analyzed utilizing a standard program for quantifying synergism, additivism, and antagonism among anticancer agents. The program utilized, for example in Figure 12, is that described by Chou and Talalay, in "New Avenues in Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter 2. Combination Index values less than 0.8 indicates synergy, values greater than 1.2 indicate antagonism and values between 0.8 to 1.2 indicate additive effects. The combination therapy may provide "synergy" and prove "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together. In some examples, Combination effects were evaluated using both the BLISS independence model and the highest single agent (HSA) model (Lehár et al. 2007, Molecular Systems Biology 3:80). BLISS scores quantify degree of potentiation from single agents and a BLISS score > 0 suggests greater than simple additivity. An HSA score > 0 suggests a combination effect greater than the maximum of the single agent responses at corresponding concentrations.

[0093] Response Evaluation Criteria in Solid Tumors, Version 1.1 (RECIST v1.1), can be used to evaluate tumor responses in certain human clinical trials. This section provides the definitions of the criteria used to determine objective tumor response for target lesions. “Complete response” (CR) is used to mean disappearance of all observable target lesions with pathological lymph nodes (whether target or non-target) having reduction in short axis to less than about 10 mm. “Partial response” (PR) is used to mean at least about a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum of diameters. “Progressive disease” (PD) is used to mean at least about a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (nadir), including baseline. In addition to the relative increase of about 20%, the sum also demonstrates an absolute increase of 28 DM2\21379822.1G8118-04401 PATENT at least about 5 mm. In one example, the appearance of one or more new lesions is considered PD. “Stable disease” (SD) is used to mean neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum on study.

[0094] Adverse Event Grading (Severity) Scale is used to evaluate safety and tolerability with Grade 1 is mild (intervention not indicated), Grade 2 is moderate (minimal, local, or noninvasive intervention indicated), Grade 3 is severe (severe or medically significant but not immediately life threatening; hospitalization or prolongation of hospitalization indicated), Grade 4 is very severe, life threatening or disabling, urgent intervention indicated, and Grade 5 is death related to the adverse event.

[0095] As used herein, the term “alkyl” refers to a single bond chain of hydrocarbons ranging, in some embodiments, from 1-8 carbon atoms, and ranging in some embodiments, from 1-8 carbon atoms; examples include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, and the like.

[0096] Compositions and Kits

[0097] Certain embodiments of the invention also provide a composition comprising a procapped mRNA molecule and a carrier. The invention also provides pharmaceutical compositions and kits comprising the procapped mRNA molecules and tetrazine compounds for targeted activation for treating certain disease or conditions. In certain embodiments, the composition further comprises at least one therapeutic agent as described herein.

[0098] In certain embodiments, the composition is a pharmaceutical composition and the carrier is a pharmaceutically acceptable carrier.

[0099] In certain embodiments, the kit further comprises a procapped mRNA molecule described herein and instructions for administering the procapped mRNA molecule in combination (e.g., simultaneously or sequentially) with a mRNA encoding for a protein of interest to be expressed as part of a therapeutic or prophylactic treatment.

[0100] Certain Methods

[0101] Certain embodiments of the invention also provide a method of treating a disease or disorder in a subject, comprising administering to the subject an effective amount of an procapped mRNA and a cell-selective tetrazine-moiety, or a composition as described herein. 29 DM2\21379822.1G8118-04401 PATENT

[0102] In certain embodiments, a method of the invention further comprises administering at least one therapeutic agent to the subject.

[0103] The at least one therapeutic agent may be administered in combination with the procapped mRNA and a cell-selective tetrazine-moiety . As used herein, the phrase “in combination” refers to the simultaneous or sequential administration of the procapped mRNA and a cell-selective tetrazine-moiety and the at least one therapeutic agent. For simultaneous administration, the procapped mRNA and a cell-selective tetrazine-moiety and the at least one therapeutic agent may be present in a single composition or may be separate (e.g., may be administered by the same or different routes).

[0104] Certain embodiments of the invention provide a procapped mRNA and a cell- selective tetrazine-moiety or a composition as described herein for use in medical therapy.

[0105] Certain embodiments of the invention provide the use of an procapped mRNA and a cell-selective tetrazine-moiety or a composition as described herein for the manufacture of a medicament for inducing an immune response in a subject (e.g., a mammal, such as a human).

[0106] Certain embodiments of the invention provide the use of a procapped mRNA and a cell-selective tetrazine-moiety or a composition as described herein for the manufacture of a medicament for inducing an immune response in a subject (e.g., a mammal, such as a human), in combination with at least one therapeutic agent.

[0107] Certain embodiments of the invention provide an procapped mRNA and a cell- selective tetrazine-moiety or a composition as described herein for inducing an immune response, in combination with at least one therapeutic agent.

[0108] Certain embodiments of the invention provide the use of an procapped mRNA and a cell-selective tetrazine-moiety or a composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject.

[0109] Certain embodiments of the invention provide the use of procapped mRNA and a cell-selective tetrazine-moiety or a composition as described herein for the manufacture of a medicament for treating a disease or disorder in a subject, in combination with at least one therapeutic agent. 30 DM2\21379822.1G8118-04401 PATENT

[0110] Certain embodiments of the invention provide an procapped mRNA and a cell- selective tetrazine-moiety or a composition as described herein for the prophylactic or therapeutic treatment a disease or disorder.

[0111] Certain embodiments of the invention provide an procapped mRNA and a cell- selective tetrazine-moiety or a composition as described herein for the prophylactic or therapeutic treatment of a disease or disorder, in combination with at least one therapeutic agent.

[0112] In certain embodiments, the disease or disorder is a condition that requires a boost of the host immunity. In certain embodiments, the disease or disorder is a hyperproliferative disorder, such as cancer. In certain embodiments, the disease or disorder is an infectious disease.

[0113] In certain embodiments, the cancer is carcinoma, lymphoma, blastoma, sarcoma, or leukemia. In certain embodiments, the cancer is a solid tumor cancer.

[0114] In certain embodiments, the cancer is squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, renal cell carcinoma, gastrointestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., endocrine resistant breast cancer), colon cancer, rectal cancer, lung cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, melanoma, leukemia, or head and neck cancer. In certain embodiments, the cancer is breast cancer.

[0115] In certain embodiments, the therapeutic agent is a therapeutic agent described herein. For example, in certain embodiments, the therapeutic agent is an immuno-stimulatory agent, a radioisotope, a chemotherapeutic drug (e.g., doxorubicin) or an immuno-therapy agent, such as antibody or an antibody fragment. In certain embodiments, the therapeutic agent is a vaccine, such as a cancer vaccine. In certain embodiments, the therapeutic agent is a tumor targeting agent, such as a monoclonal tumor-specific antibody or an aptamer. In certain embodiments, the therapeutic agent is an antibody (e.g., a monoclonal antibody, e.g., an anti- PD1 antibody). In certain embodiments, the therapeutic agent is an antigen (e.g., a tumor associated antigen or a tumor specific antigen). In certain embodiments, the therapeutic agent is a tumor antigen peptide(s). 31 DM2\21379822.1G8118-04401 PATENT

[0116] In certain embodiments, the therapeutic agent is a chemotherapeutic agent. In certain embodiments, the chemotherapeutic agent is selected from: Abraxane (chemical name: albumin-bound or nab-paclitaxel), Adriamycin (chemical name: doxorubicin), carboplatin (brand name: Paraplatin), Cytoxan (chemical name: cyclophosphamide), daunorubicin (brand names: Cerubidine, DaunoXome), Doxil (chemical name: doxorubicin), Ellence (chemical name: epirubicin), fluorouracil (also called 5-fluorouracil or 5-FU; brand name: Adrucil), Gemzar (chemical name: gemcitabine), Halaven (chemical name: eribulin), Ixempra (chemical name: ixabepilone), methotrexate (brand names: Amethopterin, Mexate, Folex), Mitomycin (chemical name: mutamycin), mitoxantrone (brand name: Novantrone), Navelbine (chemical name: vinorelbine), Taxol (chemical name: paclitaxel), Taxotere (chemical name: docetaxel), thiotepa (brand name: Thioplex), vincristine (brand names: Oncovin, Vincasar PES, Vincrex), and Xeloda (chemical name: capecitabine). In certain embodiments, the chemotherapeutic agent is selected from: Abraxane (Paclitaxel (with albumin) Injection), Adriamycin (Doxorubicin), Afinitor (Everolimus), Alecensa (Alectinib), Alimta (PEMETREXED), Aliqopa (Copanlisib), Alkeran Injection (Melphalan), Alunbrig (Brigatinib), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab), Avastin (Bevacizumab), Bavencio (Avelumab), Beleodaq (Belinostat), Besponsa (Inotuzumab Ozogamicin), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Blincyto (Blinatumomab), Bosulif (Bosutinib), Braftovi (Encorafenib), Busulfex (Busulfan), Cabometyx (Cabozantinib), Calquence (Acalabrutinib), Campath (Alemtuzumab), Camptosar (Irinotecan), Caprelsa (Vandetanib), Casodex (Bicalutamide), CeeNU (Lomustine), CeeNU Dose Pack (Lomustine), Cerubidine (Daunorubicin), Cinqair (Reslizumab), Clolar (Clofarabine), Cometriq (Cabozantinib), Copiktra (Duvelisib), Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Cyramza (Ramucirumab), CytosarU (Cytarabine), Cytoxan (Cytoxan), Cyclophosphamide, Dacogen (Decitabine), Darzalex (Daratumumab), DaunoXome (Daunorubicin Lipid Complex), Daurismo (Glasdegib), Decadron (Dexamethasone), DepoCyt (Cytarabine Lipid Complex), Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Docefrez (Docetaxel), Doxil (Doxorubicin Lipid Complex), DTIC (Decarbazine), Eligard (Leuprolide), Ellence (Ellence (epirubicin)), Eloxatin (Eloxatin (oxaliplatin)), Elspar (Asparaginase), Emcyt (Estramustine), Emend (Fosaprepitant), Empliciti (Elotzumab), Erbitux (Cetuximab), Erivedge (Vismodegib), Erleada (Apalutamide), Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide), Eulexin (Flutamide), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), Femara (Letrozole), Firmagon (Degarelix), FloPred (Prednisolone), Fludara (Fludarabine), Folex (Methotrexate), Folotyn (Pralatrexate), FUDR 32 DM2\21379822.1G8118-04401 PATENT (FUDR (floxuridine)), Gazyva (Obinutuzumab), Gemzar (Gemcitabine), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Halaven (Eribulin), Herceptin (Trastuzumab), Hexalen (Altretamine), Hycamtin (Topotecan), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Ibrance (Palbociclib), Iclusig (Ponatinib), Idamycin PFS (Idarubicin), Idhifa (Enasidenib), Ifex (Ifosfamide), Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Intron A alfab (Interferon alfa-2a), Iressa (Gefitinib), Istodax (Romidepsin), Ixempra (Ixabepilone), Jakafi (Ruxolitinib), Jevtana (Cabazitaxel), Kadcyla (Ado-trastuzumab Emtansine), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kyprolis (Carfilzomib), Lanvima (Lenvatinib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lorbrena (Lorlatinib), Lupron (Leuprolide), Lynparza (Olaparib), Lysodren (Mitotane), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mektovi (Binimetinib), Mesnex (Mesna), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (Gemtuzumab Ozogamicin), Navelbine (Vinorelbine), Nerlynx (Neratinib), Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib), Nilandron (Nilandron (nilutamide)), Ninlaro (Ixazomib), Nipent (Pentostatin), Nolvadex (Tamoxifen), Odomzo (Sonidegib), Oncaspar (Pegaspargase), Oncovin (Vincristine), Opdivo (Nivolumab), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab), Platinol (Cisplatin), PlatinolAQ (Cisplatin), Pomalyst (Pomalidomide), Portrazza (Necitumumab), Proleukin (Aldesleukin), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rituxan (Rituximab), RoferonA alfaa (Interferon alfa-2a), Rubex (Doxorubicin), Rubraca (Rucaparib), Rydapt (Midostaurin), Sandostatin (Octreotide), Soltamox (Tamoxifen), Sprycel (Dasatinib), Stivarga (Regorafenib), Sutent (Sunitinib), Sylvant (Siltuximab), Synribo (Omacetaxine), Tabloid (Thioguanine), Taflinar (Dabrafenib), Tagrisso (Osimertinib), Talzenna (Talazoparib), Tarceva (Erlotinib), Targretin Capsules (Bexarotene), Tasigna (Decarbazine), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq (Atezolizumab), Temodar (Temozolomide), Tepadina (Thiotepa), Thioplex (Thiotepa), Tibsovo (Ivosidenib), Toposar (Etoposide), Torisel (Temsirolimus), Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin), Tykerb (lapatinib), Unituxin (Dinutuximab), Valstar (Valrubicin), Varubi (Rolapitant), Vectibix (Panitumumab), Velban (Vinblastine), Velcade (Bortezomib), Venclexta (Venetoclax), Vepesid (Etoposide), Vepesid (Etoposide Injection), Verzenio (Abemaciclib), Vesanoid (Tretinoin), Vidaza (Azacitidine), Vincasar PFS (Vincristine), Vincrex (Vincristine), Vistogard (Uridine Triacetate), VitrakviI (Larotrectinib), Vizimpro (Dacomitinib), Votrient (Pazopanib), Vumon (Teniposide), Wellcovorin IV (Leucovorin), Xalkori (Crizotinib), Xeloda (Capecitabine), Xospata 33 DM2\21379822.1G8118-04401 PATENT (Gilteritinib), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yescarta (Axicabtagene), Yondelis (Trabectedin), Zaltrap (Ziv-aflibercept), Zanosar (Streptozocin), Zejula (Niraparib), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zoladex (Goserelin), Zolinza (Vorinostat), Zometa (Zoledronic acid) Zortress (Everolimus), ,Zydelig (Idelalisib), Zykadia (Ceritinib), and Zytiga (Abiraterone).

[0117] In certain embodiments, the methods of this disclosure can treat cancer in a relevant model. Mouse models for pancreatic cancer to which the methods of this disclosure are expected to demonstrate the treatment of cancer are described in Herreros-Villanueva et al., Mouse models of pancreatic cancer World J Gastroenterol. (2012) Mar 28; 18(12): 1286–1294. doi: 10.3748 / wjg.v18.i12.1286, PubMed ID: 22493542).

[0118] Administration

[0119] In some embodiments, methods of the invention comprise administering a procapped mRNA and a cell-selective tetrazine-moiety described herein, and optionally, a therapeutic agent to a subject. Such combinations may be formulated as a pharmaceutical composition and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, intraperitoneal or topical or subcutaneous routes.

[0120] The procapped mRNA and a cell-selective tetrazine-moiety may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the procapped mRNA and a cell-selective tetrazine-moiety or its salts is prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0121] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle is a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity 34 DM2\21379822.1G8118-04401 PATENT is maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms is brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions is brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0122] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0123] Pharmaceutical compositions for use in accordance with the present invention may be formulated using one or more physiologically acceptable carriers or excipients. Any suitable concentration of the procapped mRNA and a cell-selective tetrazine-moiety may be used, and any active pharmaceutical ingredient will be administered in an amount effective to achieve its intended purpose.

[0124] A variety of suspending fluids or carriers may be employed to suspend the procapped mRNA and a cell-selective tetrazine-moiety composition. Such fluids include without limitation: sterile water, saline, buffer, or complex fluids derived from growth medium or other biological fluids. Preservatives, stabilizers and antibiotics may be employed in the procapped mRNA and a cell-selective tetrazine-moiety composition.

[0125] Methods of making a pharmaceutical composition include admixing at least one active compound or agent, as defined above, together with one or more other pharmaceutically acceptable ingredients, such as carriers, diluents, excipients, and the like. When formulated as discrete units, such as tablets or capsule or suspension, each unit contains a predetermined amount of the active compound or agent.

[0126] Suitable formulations will depend on the method of administration. The pharmaceutical composition is preferably administered by intradermal administration, but other routes of administration include for example oral, buccal, rectal, parenteral, intramuscular, 35 DM2\21379822.1G8118-04401 PATENT subcutaneous, intraperitoneal, transdermal, intrathecal, nasal, intracheal. The polyvalent vaccine can also be administered to the lymph nodes such as axillary, inguinal or cervial lymph nodes. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation.

[0127] Pharmaceutical compositions described herein may be administered directly, they may also be formulated to include at least one pharmaceutically-acceptable, nontoxic carriers of diluents, adjuvants, or non-toxic, nontherapeutic, fillers, buffers, preservatives, lubricants, solubilizers, surfactants, wetting agents, masking agents, and coloring agents. Also, as described herein, such formulation may also include other active agents, for example, other therapeutic or prophylactic agents, nonimmunogenic stabilizers, excipients and the like. The compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents and detergents.

[0128] Useful dosages of nucleic acid complexes can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949, herein incorporated by reference.

[0129] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0130] The nucleic acid complexes described herein may be conveniently formulated in unit dosage form. In one embodiment, the invention provides a composition comprising a nucleic acid complexes formulated in such a unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0131] Procapped mRNA for targeted cell transcription 36 DM2\21379822.1G8118-04401 PATENT

[0132] The advent of mRNA vaccines has unveiled the transformative potential of mRNA therapeutics, yet their broader application is hindered by a lack of cell-type specificity, leading to potential off-target effects. This disclosure provides for compounds, compounds, and methods of their use which addresses this challenge by developing a novel class of mRNA prodrug—procapped mRNA—that utilizes bioorthogonal chemistry for targeted activation within specific cells. The methods described herein enable conditional transgene expression, thereby opening new avenues for targeted treatment strategies and advancing the field of precision medicines.

[0133] The present invention focuses on modifying the 5' cap of mRNA, which is crucial for translation initiation, by attaching bulky inhibitory groups to create an untranslatable "procapped" mRNA prodrug. Selective removal of the inhibitory moiety is hypothesized to conditionally activate the procapped mRNA, enabling targeted mRNA expression. Specifically, the invention utilizes the inverse electron-demand Diels-Alder reaction between trans- cyclooctene (TCO) and tetrazine moieties for the bioorthogonal and biocompatible cleavage and activation of the procapped mRNA.

[0134] Benefits of this click chemistry approach include its fast reaction kinetics, lack of catalyst requirements, selectivity, excellent bioorthogonality and biocompatibility, supported by evidence like the first click-activated drug in clinical trials consisting of a tetrazine-containing biopolymer and TCO-modified doxorubicin. Furthermore, methods have been developed to deliver tetrazine selectively to cancer cells or induce its intracellular production preferentially in senescent cells, enabling targeted mRNA activation. The inventions described herein offer (1) a novel ON-type switch mechanism for eukaryotic mRNA expression: the procapped mRNA described herein functions as an ON-type sensor, a proactive tool for transgene regulation within eukaryotic cells. This contrasts with the predominantly OFF-type sensors, which suppress transgene expression in the presence of a trigger; (2) compatibility with nucleobase modifications: the methods and compositiounds of this disclosure are compatible with nucleobase modifications like N1-methylpseudouridine, which is important for mRNA therapeutics due to the capability of enhancing mRNA stability and reducing immunogenicity; (3) a streamlined, efficient and easy-to-design platform: the procapped mRNA methods described herein leverage the specificity of bioorthogonal click reactions.

[0135] The modular nature of this procapping technology allows facile adaptation across various mRNA therapeutics, from vaccines to gene editing tools, establishing a universal 37 DM2\21379822.1G8118-04401 PATENT platform for conditional mRNA expression. Additionally, the procapped mRNA itself may open new avenues as a novel targeted prodrug therapy against diseases like cancer.

[0136] The present invention relates to a novel class of mRNA therapeutics, termed "procapped mRNA," designed for cell-type specific activation. This technology utilizes a bioorthogonal click-release mechanism, specifically the inverse electron-demand Diels-Alder (IEDDA) reaction between tetrazine and trans-cyclooctene (TCO), to control the activation of mRNA within targeted cells. The procapped mRNA remains inactive until it encounters a specific trigger in the target cell, whereupon it is converted into an active form capable of directing the synthesis of therapeutic proteins. This method addresses a critical limitation in the field of mRNA therapeutics—the lack of cell-type specificity—by ensuring that the therapeutic action is confined to desired cells, thereby minimizing off-target effects and enhancing therapeutic efficacy.

[0137] The invention encompasses the synthesis of TCO-caged cap analogues, their efficient incorporation into RNA molecules through both enzymatic and co-transcriptional capping methods, and the cell-specific activation of procapped mRNAs encoding reporter and effector proteins in cancer and senescent cells.

[0138] Synthesis of TCO-caged cap analogues

[0139] The invention provides methods for synthesizing TCO-caged cap analogues, which serve as procapping reagents for the preparation of procapped mRNA molecules. These analogues include TCO-caged GTP analogues for post-transcriptional enzymatic capping (e.g., compound 7) and TCO-caged trinucleotides for co-transcriptional capping (e.g., compounds 11 and 13).

[0140] The present invention relates to a novel class of mRNA therapeutics, termed "procapped mRNA," designed for cell-type specific activation. This technology utilizes a bioorthogonal click-release mechanism, specifically the inverse electron-demand Diels-Alder (IEDDA) reaction between tetrazine and trans-cyclooctene (TCO), to control the activation of mRNA within targeted cells. The procapped mRNA remains inactive until it encounters a specific trigger in the target cell, whereupon it is converted into an active form capable of directing the synthesis of therapeutic proteins. This method addresses a critical limitation in the field of mRNA therapeutics—the lack of cell-type specificity—by ensuring that the therapeutic 38 DM2\21379822.1G8118-04401 PATENT action is confined to desired cells, thereby minimizing off-target effects and enhancing therapeutic efficacy.

[0141] The invention encompasses the synthesis of TCO-caged cap analogues, their efficient incorporation into RNA molecules through both enzymatic and co-transcriptional capping methods, and the cell-specific activation of procapped mRNAs encoding reporter and effector proteins in cancer and senescent cells.

[0142] Synthesis of TCO-caged cap analogues

[0143] The invention provides methods for synthesizing TCO-caged cap analogues, which serve as procapping reagents for the preparation of procapped mRNA molecules. These analogues include TCO-caged GTP analogues for post-transcriptional enzymatic capping (e.g., compound 7) and TCO-caged trinucleotides for co-transcriptional capping (e.g., compounds 11 and 13).

[0144] Compound 7, a TCO-caged GTP analogue, is synthesized according to the scheme presented in Figure 5. Briefly, guanosine (1) is protected at the 3',5'-dihydroxyl group, followed by tert-butyldimethylsilyl (TBDMS) protection of the 2'-hydroxyl group to give compound 2. Heating compound 2 with 2,5-hexanedione results in the formation of 2,5- dimethylpyrrole, which is then deprotected using HF-pyridine to yield compound 3. Mono- phosphorylation of guanosine is achieved by treating compound 3 with trimethyl phosphate and phosphorus oxychloride. The resulting monophosphate intermediate is activated by imidazole in the presence of 2,2'-dithiopyridine to furnish compound 4. Reaction of compound 4 with activated TCO carbonate 5, followed by deprotection of the 2' position and 2'-amino group in the guanine, delivers compound 6. Treatment of compound 6 with tetrabutylammonium phosphate and NBu3gives the target compound 7.

[0145] Compounds 11 and 13, TCO-caged trinucleotides for co-transcriptional capping, are synthesized according to the schemes presented in Figure 6. For compound 11, the TCO- caged intermediate 9 is prepared from commercially available 3'-OMe guanosine 8 in 9 steps. Coupling of compound 9 with compound 10 (prepared according to literature procedures) in the presence of ZnCl2 furnishes the target compound 11, in which the TCO is installed at the 2'-O site of the inverted m7G. For compound 13, a similar strategy is employed, starting from compound 1 to yield the 3'-O-TCO caged compound.

[0146] Preparation of procapped mRNA molecules 39 DM2\21379822.1G8118-04401 PATENT

[0147] The invention further provides methods for preparing procapped mRNA molecules using the synthesized TCO-caged cap analogues. The preparation involves in vitro transcription of the desired mRNA sequence followed by either enzymatic or co-transcriptional capping using the appropriate procapping reagent.

[0148] For enzymatic capping, a small RNA molecule (e.g., 30 nt) is synthesized by in vitro transcription using T7 RNA polymerase. The RNA is then subjected to post-transcriptional enzymatic capping using a TCO-caged GTP analogue (e.g., compound 7) and the Vaccinia virus Capping Enzyme (VCE) according to the schematic presented in Figure 3a.

[0149] For co-transcriptional capping, a small RNA molecule (e.g., 30 nt) is synthesized by in vitro transcription using T7 RNA polymerase and an engineered T7 promoter (with a C+1T mutation in the DNA template strand) in the presence of a TCO-caged trinucleotide (e.g., compound 11 or 13), as described in Figure 4a and 4b.

[0150] Cell-specific activation of procapped mRNA molecules

[0151] The invention also provides methods for achieving cell-specific activation of procapped mRNA molecules in target cells, such as cancer cells overexpressing αvβ3 integrin or senescent cells expressing senescence-associated β-galactosidase (SA-β-gal).

[0152] For cancer cell-specific activation, a procapped mRNA encoding a reporter protein (e.g., Gaussia Luciferase, GLuc) is prepared using a TCO-caged trinucleotide (e.g., compound 11) and an engineered T7 promoter. The procapped GLuc mRNA is co-transfected with a control mRNA (e.g., CleanCap™ mRNA encoding Cypridina Luciferase, CLuc) into cancer cells (e.g., HeLa cells) and non-cancer cells (e.g., WI-38 cells). The cells are then treated with a tetrazine-containing compound capable of targeted delivery to cancer cells overexpressing αvβ3integrin (e.g., c(RGDyK)-Tz, as shown in Figure 9a) or a control compound. Efficient cell-specific activation of the procapped GLuc mRNA is observed in cancer cells treated with c(RGDyK)-Tz, as evidenced by significantly higher GLuc:CLuc ratios compared to control treatments.

[0153] For senescence-specific activation, a procapped mRNA encoding an effector protein (e.g., inducible Caspase-9, iCaspase9) is prepared using a TCO-caged trinucleotide (e.g., compound 13) and an engineered T7 promoter. The procapped iCaspase9 mRNA is transfected into senescent cells (e.g., A549 cells induced by etoposide treatment) and non-senescent cells. The cells are then treated with a tetrazine-containing compound designed for targeted activation 40 DM2\21379822.1G8118-04401 PATENT in senescent cells (e.g., Gal-Tz, as shown in Figure 9b) or a control compound, followed by treatment with a dimerizing drug (e.g., AP20187) to activate iCaspase9. Senescence-specific activation of the procapped iCaspase9 mRNA is observed in senescent cells treated with Gal-Tz and AP20187, as evidenced by strong iCaspase9 expression and significant induction of apoptosis, compared to control treatments.

[0154] The invention also encompasses pharmaceutical compositions comprising procapped mRNA molecules and tetrazine-containing compounds for targeted delivery or activation, as well as methods of treating diseases, such as cancer or age-related diseases associated with cellular senescence, by administering such compositions to a subject in need thereof.

[0155] The examples provided herein demonstrate the successful synthesis of TCO- caged cap analogues, their efficient incorporation into RNA molecules through both enzymatic and co-transcriptional capping methods, and the cell-specific activation of procapped mRNAs encoding reporter and effector proteins in cancer and senescent cells. These examples, along with the accompanying figures, support the broad applicability and therapeutic potential of the procapped mRNA technology described in the present invention. Examples

[0156] The invention will now be illustrated by the following non-limiting Examples. Example 1: Synthesis of a TCO-caged GTP analogue (Compound 7) for post- transcriptional enzymatic capping

[0157] Compound 7, a TCO-caged GTP analogue, was synthesized according to the scheme presented in Figure 5. Briefly, guanosine was protected at the 3',5'-dihydroxyl group, followed by tert-butyldimethylsilyl (TBDMS) protection of the 2'-hydroxyl group to give compound 2. Heating compound 2 with 2,5-hexanedione resulted in the formation of 2,5- dimethylpyrrole, which was then deprotected using HF-pyridine to yield compound 3. Mono- phosphorylation of guanosine was achieved by treating compound 3 with trimethyl phosphate and phosphorus oxychloride. The resulting monophosphate intermediate was activated by imidazole in the presence of 2,2'-dithiopyridine to furnish compound 4. Reaction of compound 4 with activated TCO carbonate 5, followed by deprotection of the 2' position and 2'-amino group 41 DM2\21379822.1G8118-04401 PATENT in the guanine, delivered compound 6. Treatment of compound 6 with tetrabutylammonium phosphate and NBu3 gave the target compound 7. The structure and purity of compound 7 were confirmed by 1H NMR, 13C NMR, high-resolution mass spectrometry (HRMS), and HPLC analysis. Example 2: Synthesis of TCO-caged trinucleotides (Compounds 11 and 13) for co- transcriptional capping.

[0158] Compounds 11 and 13, TCO-caged trinucleotides for co-transcriptional capping, were synthesized according to the schemes presented in Figure 6. For compound 11, the TCO- caged intermediate 9 was prepared from commercially available 3'-OMe guanosine 8 in 9 steps. Coupling of compound 9 with compound 10 (prepared according to literature procedures) in the presence of ZnCl2 furnished the target compound 11, in which the TCO is installed at the 2'-O site of the inverted m7G. For compound 13, a similar strategy was employed, starting from compound 1 to yield the 3'-O-TCO caged compound. The structures and purities of compounds 11 and 13 were confirmed by 1H NMR, 13C NMR, HRMS, and HPLC analysis. Example 3: Enzymatic capping of in vitro transcribed RNA with TCO-caged GTP analogue (Compound 7).

[0159] A small RNA molecule (30 nt) was synthesized by in vitro transcription using T7 RNA polymerase. The RNA was then subjected to post-transcriptional enzymatic capping using compound 7 and the Vaccinia virus Capping Enzyme (VCE) according to the schematic presented in Figure 3a. The successful installation of the TCO-caged cap was confirmed by the slower electrophoretic migration of the capped RNA compared to the uncapped RNA on a denaturing polyacrylamide gel, as shown in Figure 3b. The capping efficiency was estimated to be >90% based on the relative intensities of the capped and uncapped RNA bands. Example 4: Co-transcriptional capping of in vitro transcribed RNA with TCO-caged trinucleotides (Compounds 11 and 13).

[0160] A small RNA molecule (30 nt) was synthesized by in vitro transcription using T7 RNA polymerase and an engineered T7 promoter (with a C+1T mutation in the DNA template strand) in the presence of either compound 11 or 13, as described in Figure 4a and 4b. The successful incorporation of the TCO-caged cap was confirmed by the slower electrophoretic migration of the capped RNA compared to the uncapped RNA on a denaturing polyacrylamide gel, similar to the results shown in Figure 4c. The capping efficiency was estimated to be >95% 42 DM2\21379822.1G8118-04401 PATENT based on the relative intensities of the capped and uncapped RNA bands for both compounds 11 and 13. Example 5: Preparation of procapped mRNA encoding a reporter protein and validation of its cell-specific activation.

[0161] A procapped mRNA encoding Gaussia Luciferase (GLuc) was prepared by in vitro transcription using the TCO-caged trinucleotide (compound 11) and an engineered T7 promoter. The procapped GLuc mRNA was co-transfected with a CleanCap mRNA encoding Cypridina Luciferase (CLuc) into HeLa cells (high αvβ3 integrin expression) and WI-38 cells (low αvβ3 integrin expression) using Lipofectamine MessengerMAX. The cells were then treated with either c(RGDyK)-Tz, a compound capable of targeted delivery to cancer cells overexpressing αvβ3 integrin as shown in Figure 9a, or a control peptide. Luciferase activities were measured 24 h post-transfection, and the GLuc:CLuc ratios were calculated. In HeLa cells treated with c(RGDyK)-Tz, the GLuc:CLuc ratio was >50-fold higher than in cells treated with the control peptide, indicating efficient cell-specific activation of the procapped GLuc mRNA. In contrast, no significant difference in GLuc:CLuc ratios was observed between the c(RGDyK)- Tz and control peptide treatments in WI-38 cells, confirming the specificity of the activation mechanism. Example 6: Preparation of procapped mRNA encoding Caspase-9 and validation of its senescence-specific activation and induction of apoptosis.

[0162] A procapped mRNA encoding inducible Caspase-9 (iCaspase9) was prepared by in vitro transcription using the TCO-caged trinucleotide (compound 13) and an engineered T7 promoter. The procapped iCaspase9 mRNA was transfected into non-senescent and senescent A549 cells (induced by etoposide treatment) using Lipofectamine MessengerMAX. The cells were then treated with either Gal-Tz, a compound designed for targeted activation in senescent cells as shown in Figure 9b, or a control compound. After 24 h, the cells were treated with the dimerizing drug AP20187 to activate iCaspase9. The expression of iCaspase9 was analyzed by Western blot, and apoptosis was assessed by flow cytometry using Annexin V and propidium iodide staining. In senescent A549 cells treated with Gal-Tz and AP20187, a strong iCaspase9 band was detected by Western blot, and a significant increase in apoptotic cells was observed by flow cytometry. In contrast, no iCaspase9 expression or increase in apoptosis was detected in non-senescent cells or in senescent cells treated with the control compound, confirming the 43 DM2\21379822.1G8118-04401 PATENT senescence-specific activation of the procapped iCaspase9 mRNA and its ability to induce apoptosis upon activation.

[0163] These examples demonstrate the successful synthesis of TCO-caged cap analogues, as depicted in Figures 5 and 6, their efficient incorporation into RNA molecules through both enzymatic and co-transcriptional capping methods, as shown in Figures 3 and 4, and the cell-specific activation of procapped mRNAs encoding reporter and effector proteins in cancer and senescent cells, utilizing the targeting strategies presented in Figure 9. The data support the potential of the procapped mRNA technology for targeted therapeutic applications.

[0164] The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Detailed Disclosure. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Detailed Disclosure, which is included for purposes of illustration only and not restriction. A person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0165] All patents, publications, scientific articles, web sites, and other documents and materials referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced document and material is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents. Reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. 44 DM2\21379822.1G8118-04401 PATENT

[0166] The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. Thus, for example, in each instance herein, in embodiments or examples of the present invention, any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms in the specification. Also, the terms “comprising”, “including”, containing”, etc. are to be read expansively and without limitation. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. It is also that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants. Furthermore, titles, headings, or the like are provided to enhance the reader’s comprehension of this document, and should not be read as limiting the scope of the present invention. Any examples of aspects, embodiments or components of the invention referred to herein are to be considered non-limiting.

[0167] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. 45 DM2\21379822.1G8118-04401 PATENT

[0168] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0169] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. 46 DM2\21379822.1

Claims

G8118-04401 PATENT CLAIMS What is claimed is:

1. A compound, or pharmaceutically acceptable salt thereof, having the structure of Formula (I):wherein: R1 is selected from H, -P(O)(OH), -P(O)OP(O)(OH), or -P(O)OP(O)OP(O)(OH)2; W is selected from electron pair, hydrogen, or methyl, wherein when W is hydrogen or methyl the nitrogen atom connected to W is positively charged; L1 and L2 are independently selected from: -H, -OH, -NH2, -OR3 and -NHR3; and R2 is hydrogen or, R3 is hydrogen or 47 DM2\21379822.1G8118-04401 PATENT, wherein R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13 are independently selected from hydrogen C1-C6alkyl, or –C(O)NHCH2C(O)(OH); with the proviso that at least one of L1, or L2is -OR3and -NHR3and R3 is2. A compound, or pharmaceutically acceptable salt thereof, having the structure of formula (II): 48 DM2\21379822.1G8118-04401 PATENTwherein: B1and B2are independently a nucleoside base; L3, L4, and L5 are independently selected from -H, -OH, -NH2, -OR3 and -NHR3 ; R3 and R14 are independently selected from hydrogen or, wherein R4, R5, R6, R7, R8, R9, R10, R11, R12, and R13are independently selected from hydrogen C1-C6 alkyl, or –C(O)NHCH2C(O)(OH); R15is hydrogen or -OCH3, and 49 DM2\21379822.1G8118-04401 PATENT R16 is hydrogen or methyl.

3. The compound of claim 2, wherein at least one of B1 and B2 is N6-methyladenine.

4. The compound of claim 2, wherein B1is adenine and B2is guanine.

5. The compound of claim 2, wherein B1is guanine and B2is guanine.

6. The compound of claim 2, wherein B1 is adenine and B2 is uracil.

7. The compound of claim 2, having the structure of formula (III):

8. A compound of claim 7 having the structure of compound 11: 50 DM2\21379822.1G8118-04401 PATENT(Compound 11).

9. A compound of claim 7 having the structure of compound 13:(Compound 13).

10. A procapped mRNA molecule comprising: a. a 5’ procap; b. a 5’ UTR; c. an open reading frame sequence; and d. a 3’ tailing region, wherein the 5’ procap comprises a cellularly-selective removable protecting group.

11. The procapped mRNA molecule of claim 10, wherein the cellularly-selective removable protecting group is a trans-cyclooctene group.

12. The procapped mRNA molecule of claim 11, wherein the 5’ procap is a 2’-deoxy or 3’- deoxy derivative of a compound claim 1.

13. The procapped mRNA molecule of claim 11, wherein the trans-cyclooctene group is capable of being removed by a tetrazine moiety. 51 DM2\21379822.1G8118-04401 PATENT 14. The procapped mRNA molecule of claim 10, wherein the RNA molecule is mRNA.

15. The procapped mRNA molecule of claim 14, wherein the mRNA further comprises a 3’ UTR.

16. The procapped mRNA molecule of claim 10, wherein the 3’ tailing region is a polyA tail.

17. The procapped mRNA molecule of claim 10, wherein the 5’ UTR, open reading frame sequence, and 3’ tailing region each consist of a nucleic acid sequence consisting of ribonucleotides selected from: adenine (A), guanine (G), cytosine (C), and uracil (U).

18. A method of expressing mRNA in a selected cell, the method comprising: a. presenting a tetrazine moiety to a selected cell; b. presenting a procapped mRNA molecule comprising a 5’ procap to the selected cell; wherein the procapped mRNA molecule is converted into translatable mRNA by the reaction of the tetrazine moiety and the 5’ procap to convert the 5’ procap into a 5’ cap.

19. The method of claim 18, wherein the procapped mRNA is the procapped mRNA molecule of any of claims 10-17.

20. The method of claim 18, wherein the cell is a tumor cell.

21. The method of claim 20, wherein the tumor cell expresses αvβ3 integrin.

22. The method of claim 18, wherein the tetrazine moiety is c(RGDyK)-Tz.

23. The method of claim 22, wherein the cell is a senescent cell.

24. The method of claim 23, wherein the tetrazine moiety is Gal-Tz.

25. The method of any of claims 18-24, wherein the open reading frame sequence encodes for a fluorescent protein, a Caspace-9 protein, or a cancer neoantigen.

26. A method of inducing apoptosis in a senescent cell, the method comprising: a. presenting Gal-Tz to a senescent cell; b. presenting a procapped mRNA molecule comprising a 5’ procap to the selected cell; 52 DM2\21379822.1G8118-04401 PATENT c. presenting the dimerizing drug AP20187 to the selected cell; wherein the procapped mRNA molecule is converted into translatable mRNA encoding the Caspase-9 protein by the reaction of the tetrazine moiety and the 5’ procap to convert the 5’ procap into a 5’ cap, and wherein the dimerizing drug AP20187 activates Caspase-9 to induce apoptosis of the selected cell.

27. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising a procapped mRNA molecule and a c(RGDyK)-Tz compound to a subject.

28. A vaccine composition comprising: a. a tetrazine moiety connected to a skeletal muscle cell- or an immune cell- targeting peptide; b. a procapped mRNA molecule comprising a 5’ procap and an open reading frame sequence encoding for a viral antigen or cancer neoantigen; and c. one or a plurality of lipids encapsulating said tetrazine moiety and procapped mRNA molecule.

29. A kit comprising a compound of any of claims 1-9 and a tetrazine-moiety.

30. The compound of any of claims 1 or 2, wherein R2 or R3 is independently selected from:. 53 DM2\21379822.1

Images