mRNA therapeutic components

Abstract

The presently disclosed and described technology relates to multimodal mRNA-based immunotherapy that delivers both antigens and immunomodulators. Related formulations, administration methods, and kits are disclosed and described.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This patent application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 416,241, filed October 14, 2022, U.S. Provisional Patent Application No. 63 / 434,219, filed December 21, 2022, U.S. Provisional Patent Application No. 63 / 442,566, filed February 1, 2023, U.S. Provisional Patent Application No. 63 / 444,195, filed February 8, 2023, and U.S. Provisional Patent Application No. 63 / 495,412, filed April 11, 2023, the contents of each of which are incorporated by reference in their entirety into this disclosure. [Background technology]

[0002] Human papillomaviruses (HPVs) are infectious causes of anogenital and oropharyngeal cancers that arise from persistently infected and subsequently transformed basal keratinocytes of mucosal epithelium. More than 90% of cervical cancers and precancerous cervical intraepithelial neoplasia (CIN) are caused by infection with high-risk HPVs, of which more than 50% are attributed to HPV16 (1, 2). Other cancers caused by HPV include head and neck, oropharyngeal, anal, penile, and vulvar cancers. In the case of cervical cancer, at least 25% of women with high-grade CIN lesions will progress to intraepithelial or invasive cancer if untreated (3). Current treatments for HPV-associated cancers can remove abnormal tissue but do not address the underlying HPV infection. In the case of cervical cancer, 15% of treated women develop residual or recurrent high-grade CIN or cervical cancer. (4) Long-term efficacy of HPV cancer treatments may require reduction of local immunosuppression and increased tumor immune cell infiltration, in addition to induction of tumor-specific T cell responses.

[0003] Infection with high-risk HPV types is the primary etiology of cervical cancer and its precancerous lesions (Walboomers, 1999), with types 16 and 18 accounting for approximately 70% of cervical cancers and approximately 50% of high-grade lesions (CIN2 / 3) (Smith, 2007). Persistent high-grade HPV infection is thought to be a factor promoting progression from premalignant stages to invasive cancer (Moscicki, 2006). While preventive HPV vaccines are highly effective, many women remain at risk for CIN because they are ineligible for vaccination or choose not to be vaccinated. In the United States, the annual incidence of CIN2 / 3 is approximately 200,000 (CDC, 2019). Loop electrosurgical excision procedure (LEEP) is an effective treatment for many patients with CIN2 / 3, but the recurrence rate is 20% (Young, 2010). An observational study of 110 patients with high-grade CIN lesions after LEEP between January 2010 and June 2015 showed that LEEP does not completely eradicate HPV infection. The rate of HPV persistence after LEEP was higher in women with HPV type 16 infection and women aged 36.5 years or older (Pirtea, 2016). Furthermore, treatment with major excision procedures, including LEEP, was associated with an increased risk of preterm birth and spontaneous abortion (Noehr, 2009; Bjorge, 2016). The risk of preterm birth was highest during the first trimester, and the amount of tissue removed was the greatest (Bjorge, 2016). Therefore, there is an unmet medical need for non-surgical therapeutic agents for the treatment of patients with high-grade CIN.

[0004] Women with CIN3 have a two-fold increased risk of developing cervical cancer, even after treatment, and this risk increases the older the patient is diagnosed (Loopik, 2020). In 2018, cervical cancer caused an estimated 570,000 new cancer cases and 311,000 deaths worldwide (Arbyn, 2020). More than 50% of cervical cancers are the result of infection with HPV16 (Mirabello, 2018). The standard treatment for early-stage cervical cancer consists of radical hysterectomy with or without lymphadenectomy, followed by adjuvant chemotherapy in intermediate- to high-risk patients. For women at low risk of recurrence, fertility-sparing surgery may be considered. For high-risk women, adjuvant chemotherapy, such as single-agent cisplatin along with radiation therapy, is used as primary treatment for locally advanced cervical cancer (NCCN, 2022). Immunotherapy such as KEYTRUDA® (pembrolizumab) in combination with chemotherapy has shown improved overall survival in patients with persistent, recurrent, or metastatic cervical cancer whose tumors express PD-L1 (CPS ≥ 1), with or without bevacizumab. However, when KEYTRUDA® is used as a single agent to treat recurrent or metastatic cervical cancer whose tumors express PD-L1 (CPS ≥ 1) that has progressed after or during chemotherapy, only approximately 14% of patients have an objective response (KEYNOTE-158) (KEYTRUDA, 2022). Therefore, new treatments for cervical cancer remain needed.

[0005] While cervical cancer is the most common HPV-associated cancer among women, oropharyngeal cancer (cancer of the back of the throat, including the base of the tongue and tonsils) is most common among men. An estimated 36,500 cancers were attributed to HPV annually between 2014 and 2018. Of these, 29,500 were caused by HPV types 16 and 18, including squamous cell cancers of the cervix, oropharynx, anus, penis, vagina, and vulva (CDC, 2021). Currently, there is a lack of approved treatment options for HPV-associated cancers. Summary of the Invention

[0006] Multimodal mRNA-based immunotherapy, which delivers both antigens and immunomodulators in a single therapeutic, represents a promising new approach for the treatment of HPV-associated cancers or HPV-associated diseases, which may address the current disease as well as the underlying cause (HPV infection).

[0007] One aspect of the disclosure is an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, or SEQ ID NO: 17. Another aspect of the disclosure is an isolated polynucleotide encoding a polypeptide of the disclosure.

[0008] One embodiment of the present disclosure is an isolated polynucleotide comprising a first isolated messenger ribonucleic acid (mRNA), at least a portion of which encodes membrane-stabilizing LIGHT. One embodiment is an isolated polynucleotide comprising the formula: 5'UTR-signal / leader-mRNA coding region-3'UTR-polyA, wherein the mRNA coding region encodes membrane-stabilizing LIGHT. In one embodiment, the isolated polynucleotide comprises a modified 3'UTR, a modified 5'UTR, one or more modifications to the nucleobase-sugar-internucleoside linkage, or a combination thereof.

[0009] In one aspect, the isolated polynucleotide comprises a nucleobase-sugar-internucleoside linkage selected from the group consisting of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, 2-aminopurine-riboside-MP, pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 5-bromo-cytidine-MP, and combinations thereof.

[0010] In one aspect, the 5' UTR comprises a sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52. In one aspect, the 3' UTR has a sequence comprising SEQ ID NO:53.

[0011] In one embodiment, the membrane-stabilized LIGHT is membrane-stabilized human LIGHT. In another embodiment, the membrane-stabilized LIGHT comprises replacing a section of the transmembrane domain of soluble LIGHT with a linker. In one embodiment, the linker is an antibody variable region linker or a peptide linker. In another embodiment, the linker comprises (Gly4Ser)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In yet another embodiment, the linker comprises the sequence SSASTDKTHT (SEQ ID NO: 54).

[0012] In one aspect, membrane-stabilized LIGHT has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17.

[0013] In another embodiment, membrane-stabilized LIGHT has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% amino acid sequence identity to SEQ ID NO:11 or SEQ ID NO:17.

[0014] In some embodiments, the first isolated mRNA has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18.

[0015] In another aspect, the first isolated mRNA has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% nucleic acid sequence identity to SEQ ID NO:12 or SEQ ID NO:18.

[0016] One aspect of the present disclosure is a composition comprising the described isolated polynucleotide. In some embodiments, the composition further comprises at least a second isolated mRNA, at least a portion of which encodes a checkpoint inhibitor, an immunosuppressant antagonist, a pro-inflammatory agent, or a pro-inflammatory cytokine. In another embodiment, the pro-inflammatory cytokine is interleukin-12. In yet another embodiment, the pro-inflammatory cytokine has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:21.

[0017] In one aspect of the composition, the second isolated mRNA has the formula: 5'UTR-signal / leader-mRNA coding region-3'UTR-polyA, wherein the mRNA coding region encodes human interleukin-12. In another aspect, the second isolated mRNA comprises a modified 3'UTR, a modified 5'UTR, one or more modifications to the nucleobase-sugar-internucleoside linkage, or a combination thereof.

[0018] In one aspect, the second isolated mRNA comprises a nucleobase-sugar-internucleoside linkage selected from the group consisting of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, 2-aminopurine-riboside-MP, pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 5-bromo-cytidine-MP, and combinations thereof.

[0019] In another embodiment, the mRNA coding region encodes at least two heterodimers of human interleukin-12. In a further embodiment, a first interleukin-12 heterodimer is linked to a second interleukin-12 heterodimer via a linker. In yet another embodiment, the linker is an antibody variable region linker, a peptide linker, and / or a (Gly4Ser)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a particular embodiment, the linker is a (Gly4Ser)n linker where n is 3.

[0020] In one embodiment, the second isolated mRNA has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:20.

[0021] In another embodiment of the present disclosure, the composition further comprises at least a third isolated mRNA, wherein the third isolated mRNA comprises a first region encoding a first antigen and a second region encoding a second antigen. In one embodiment, the first region and / or the second region encode all or a portion of an antigen specific to cervical cancer, an HPV-associated cancer, or an HPV-associated disease. In another embodiment, the third isolated mRNA encodes HPV16 E6 E7 or HPV18 E6 E7. In yet another embodiment, the third isolated mRNA encodes HPV16 E6 E7.

[0022] In one aspect, the third isolated mRNA has the formula 5'UTR--signal / leader-(An1)n-Xo-(An2)p-3'UTR-polyA, where An1 encodes HPV16 E6 and An2 encodes HPV16 E7, X is a spacer or linker, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another aspect, the linker is an antibody variable region linker, a peptide linker, and / or a (Gly4Ser)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the linker is a (Gly4Ser)n linker, where n is 3.

[0023] In one aspect, the third isolated mRNA comprises a modified 3'UTR, a modified 5'UTR, one or more modifications to the nucleobase-sugar-internucleoside linkage, or a combination thereof.

[0024] In one aspect, the third isolated mRNA comprises a nucleobase-sugar-internucleoside linkage selected from the group consisting of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, 2-aminopurine-riboside-MP, pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 5-bromo-cytidine-MP, and combinations thereof.

[0025] In one aspect, the third isolated mRNA encodes an antigen, wherein the antigen has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:24 or SEQ ID NO:27.

[0026] In another embodiment, the third isolated mRNA has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:23 or SEQ ID NO:26.

[0027] In one embodiment, at least the first isolated mRNA, the second isolated mRNA, and / or the third isolated mRNA are formulated and / or mediated by a delivery vehicle. In another embodiment, at least the first isolated mRNA, the second isolated mRNA, and / or the third isolated mRNA are at least partially encapsulated in a delivery vehicle. In one embodiment, the delivery vehicle is selected from the group comprising amphiphilic molecules, aminolipidated peptides, and tertiary aminolipidated cationic peptides. In another embodiment, the delivery vehicle has a particle size of about 200 nm or less.

[0028] In certain embodiments, the polynucleotides, mRNAs, and / or compositions described herein are formulated with a delivery agent, delivery vehicle, or delivery vehicle composition to create a delivery vehicle complex or pharmaceutical preparation. Such polyanionic compounds may also be referred to as polyanionic cargo compounds or cargos of a delivery vehicle complex (also referred to as a multi-component delivery system), which complex or system also includes a delivery vehicle composition.

[0029] In some aspects, the delivery vehicle or delivery vehicle composition comprises a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a polymeric compound, or a conjugate.

[0030] In some embodiments, the delivery vehicle or delivery vehicle composition comprises a lipid nanoparticle (LNP), such as a cationic lipid nanoparticle. Exemplary cationic lipid nanoparticles are described, for example, in PCT International Publication Nos. WO 2020 / 219941 and WO 2020 / 097548. In some embodiments, the delivery vehicle or delivery vehicle composition comprises a peptoid, for example, a tertiary amino lipidated and / or PEGylated cationic peptoid. Exemplary cationic peptoids are described, for example, in PCT International Publication Nos. WO 2020 / 069442, WO 2020 / 069445, WO 2021 / 030218, and WO 2022 / 32058, each of which is incorporated herein by reference.

[0031] In some embodiments, the delivery vehicle or delivery vehicle composition is a cationic peptoid. In some embodiments, the cationic peptoid is a hydroxyethyl-capped, tertiary amino-lipidated cationic peptoid. In some embodiments, the cationic peptoid forms a complex with a polyanionic compound, such as a nucleic acid, including but not limited to, an mRNA (including but not limited to the first isolated mRNA, second isolated mRNA, or third isolated mRNA described herein), an isolated polynucleotide, a polynucleotide encoding a polypeptide, a polynucleotide, and a nucleic acid encoding a polypeptide, including those described herein.

[0032] In some embodiments, the delivery vehicle has formula (I):

[0033] [ka] and a compound having the formula: n is 1, 2, 3, 4, 5, or 6; R 1 is H, C 1~3 alkyl, or hydroxyethyl, and each R 2 independently, C 8~24 Alkyl or C 8~24 In some embodiments, n is 3. In various embodiments, n is 4. In some embodiments, R 1 is H. In some embodiments, R 1 is ethyl or hydroxyethyl. In various cases, R 2 independently, C 8~18 Alkyl or C 8~18 In some embodiments, each R is an alkenyl. 2 teeth,

[0034] [ka] In various embodiments, each R 2 is, independently,

[0035] [ka] In some embodiments, R 2 is, independently,

[0036] [ka] In various embodiments, each R 2 teeth,

[0037] [ka] In some embodiments, the compound of formula (I) is

[0038] [ka]

[0039] [ka] In various embodiments, the compound of formula (I) has the structure:

[0040] [ka] It has.

[0041] In some embodiments, the delivery vehicle comprises at least one pharmaceutically acceptable salt of the compound and / or the compound of formula (I).

[0042] One aspect of the present disclosure is a therapeutic composition comprising a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding interleukin-12, and a third isolated mRNA encoding an oncoprotein, anti-cancer antigen, tumor-specific antigen, or tumor-associated antigen. In one embodiment, the oncoprotein or antigen is HPV16 E6 E7.

[0043] In one aspect, a composition of the present invention comprises a first isolated mRNA, a second isolated mRNA, and a third isolated mRNA as separate polynucleotide strands. In one aspect, the separate polynucleotide strands are formulated in separate delivery vehicles, and / or mediated by separate delivery vehicles, and / or at least partially encapsulated in separate delivery vehicles. In another aspect, the separate polynucleotide strands are formulated in the same delivery vehicle, and / or mediated by the same delivery vehicle, and / or at least partially encapsulated in the same delivery vehicle. In one aspect, the first isolated mRNA, the second isolated mRNA, and the third isolated mRNA are part of a single polynucleotide strand. In one aspect, the first isolated mRNA and the second isolated mRNA are part of a single polynucleotide strand. In one aspect, the first isolated mRNA and the third isolated mRNA are part of a single polynucleotide strand. In one aspect, the second isolated mRNA and the third isolated mRNA are part of a single polynucleotide strand. In one embodiment, any of the above polynucleotide chains may be formulated in a delivery vehicle, mediated by a delivery vehicle, and / or at least partially encapsulated in a delivery vehicle. In any of the above embodiments, the delivery vehicle may be selected from the group consisting of amphiphilic molecules, aminolipidated peptides, and tertiary aminolipidated cationic peptides. In another embodiment, the delivery vehicle has a particle size of about 200 nm or less.

[0044] One aspect of the disclosure is a composition comprising a first isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 12 or SEQ ID NO: 18, a second isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 20, and a third isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26, wherein the first isolated mRNA, the second isolated mRNA, and the third isolated mRNA are at least partially encapsulated in a delivery vehicle. In another aspect of the composition, the first isolated mRNA has at least about 80% nucleic acid sequence identity to SEQ ID NO: 18, and the third isolated mRNA has at least about 80% nucleic acid sequence identity to SEQ ID NO: 26.

[0045] One aspect of the present disclosure is a composition comprising a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding interleukin-12, and a third isolated mRNA encoding HPV16 E6 E7, wherein the first isolated mRNA, the second isolated mRNA, and the third isolated mRNA are at least partially encapsulated in a delivery vehicle.

[0046] In one embodiment, the composition is a therapeutic composition or vaccine. In another embodiment, the composition is a human papillomavirus (HPV) mRNA vaccine. In yet another embodiment, the composition is used in cancer immunotherapy. In one embodiment, the composition is configured to be administered to a subject known to have cervical cancer, an HPV-associated cancer, or an HPV-associated disease.

[0047] Another aspect of the present disclosure provides an mRNA therapeutic formulation in a delivery vehicle composition comprising the above-disclosed compound or a pharmaceutically acceptable salt thereof. In some embodiments, the delivery vehicle composition further comprises one or more of a phospholipid, a sterol, and a PEGylated lipid. In some embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 30 mol% to about 60 mol%. In some embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 35 mol% to about 55 mol%. In various embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 30 mol% to about 45 mol%. In various embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 35 mol% to about 39 mol%. In some embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 39 mol% to about 52 mol%. In various embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 30 mol% to about 35 mol%. In various embodiments, the compound or salt of Formula (I) is present in the delivery vehicle composition in an amount of about 40 mol% to about 45 mol%. In various embodiments, the compound or salt of Formula (I) is present in an amount of about 42 mol% to about 49 mol%. In some embodiments, the compound or salt of Formula (I) is present in an amount of about 50 mol% to about 52 mol%.

[0048] In various embodiments, the delivery vehicle composition comprises a phospholipid, a sterol, and a PEGylated lipid. In some cases, the delivery vehicle composition comprises a compound disclosed herein or a salt thereof, a phospholipid, a sterol, and a PEGylated lipid. In some cases, the delivery vehicle composition comprises about 30 mol% to about 60 mol% of a compound of Formula (I), about 3 mol% to about 20 mol% of a phospholipid, about 25 mol% to about 60 mol% of a sterol, and about 1 mol% to about 5 mol% of a PEGylated lipid. In various embodiments, the delivery vehicle composition comprises about 35 mol% to about 55 mol% of a compound or salt of Formula (I), about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some embodiments, the delivery vehicle composition comprises about 38 mol% to about 52 mol% of the compound or salt of Formula (I), about 9 mol% to about 12 mol% of a phospholipid, about 35 mol% to about 50 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid. In various embodiments, the delivery vehicle composition comprises about 30 mol% to about 49 mol% of the compound of Formula (I), about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some embodiments, the delivery vehicle composition comprises about 35 mol% to about 49 mol% of the compound or salt of Formula (I), about 7 mol% to about 12 mol% of a phospholipid, about 35 mol% to about 50 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid. In some cases, the delivery vehicle composition comprises about 30 mol% to about 45 mol% of the compound or salt of Formula (I), about 7 mol% to about 12 mol% of a phospholipid, about 40 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some cases, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of the compound or salt of Formula (I), about 7 mol% to about 12 mol% of a phospholipid, about 50 mol% to about 55 mol% of a sterol, and about 2 mol% to about 3 mol% of a PEGylated lipid. In some cases, the delivery vehicle composition comprises about 40 mol% to about 45 mol% of the compound or salt of Formula (I), about 7 mol% to about 12 mol% of a phospholipid, about 40 mol% to about 45 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid.In some embodiments, the phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleo ... Hemisuccinoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16.0PE), 1,2-distearoyl-sn-glycero-3 phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and combinations thereof. In some embodiments, the phospholipid is DOPE, DSPC, or a combination thereof. In various embodiments, the phospholipid is DSPC.In some embodiments, the sterol is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some cases, the sterol is cholesterol. In some embodiments, the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified sterol, and PEG-modified phospholipid. In various embodiments, the PEG-modified lipid is selected from the group consisting of PEG-modified cholesterol, N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}, N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}, PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), PEG-modified DOPE (DOPE-PEG), dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), dioleoylglycerol-polyethylene glycol (DOG-PEG), and combinations thereof. In some embodiments, the PEG-modified lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000). In various cases, the delivery vehicle composition comprises about 38.2 mol% Compound 140, about 11.8 mol% DSPC, about 48.2 mol% cholesterol, and about 1.9 mol% DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises about 42.6 mol% Compound 140, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000.In some embodiments, the delivery vehicle composition comprises about 48.2 mol% Compound 140, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG 2000. In various embodiments, the delivery vehicle composition comprises about 51.3 mol% Compound 140, about 9.3 mol% DSPC, about 38 mol% cholesterol, and about 1.5 mol% DMG-PEG 2000. In various embodiments, the delivery vehicle composition comprises about 44.4 mol% Compound 140, about 10.6 mol% DSPC, about 43.3 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000. In various embodiments, the delivery vehicle composition comprises about 44.4 mol% Compound 140, about 10.6 mol% DSPC, about 43.4 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000. In various embodiments, the delivery vehicle composition comprises about 33.1 mol% Compound 140, about 10.6 mol% DSPC, about 53.8 mol% cholesterol, and about 2.5 mol% DMG-PEG 2000.

[0049] Further disclosed herein are therapeutic formulations comprising one or more polyanionic compounds (e.g., mRNA (including, but not limited to, a first isolated mRNA, a second isolated mRNA, or a third isolated mRNA described herein), an isolated polynucleotide, or a polynucleotide encoding a polypeptide described herein) in combination with a delivery vehicle composition described herein and a delivery vehicle complex comprising a polyanionic compound. In some embodiments, the compound of Formula (I) or a salt thereof is complexed with a polyanionic compound. In various embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a mass ratio of about 5:1 to about 25:1. In some embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a mass ratio of about 7:1 to about 20:1. In various embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a mass ratio of about 10:1 to about 17:1. In some embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 19:1. In some embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 20:1. In some embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 10:1. In various embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 12:1. In other embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 13:1. In some embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 15:1. In various embodiments, the compound or salt of Formula (I) and the polyanionic compound are present in a weight ratio of about 17:1. In some embodiments, the phospholipid and polyanionic compound are present in a weight ratio of about 2:1 to about 10:1. In some embodiments, the phospholipid and polyanionic compound are present in a weight ratio of about 2:1 to about 4:1. In various embodiments, the phospholipid and polyanionic compound are present in a weight ratio of about 2:1 to about 3:1. In various embodiments, the phospholipid and polyanionic compound are present in a weight ratio of about 4.0:1. In various embodiments, the phospholipid and polyanionic compound are present in a weight ratio of about 2.7:1.In some embodiments, the sterol and polyanionic compound are present in a mass ratio of about 5:1 to about 8:1. In some embodiments, the sterol and polyanionic compound are present in a mass ratio of about 5:1 to about 6:1. In various embodiments, the sterol and polyanionic compound are present in a mass ratio of about 5.4:1. In some embodiments, the sterol and polyanionic compound are present in a mass ratio of about 8.1:1. In some embodiments, the sterol and polyanionic compound are present in a mass ratio of about 6.7:1. In some embodiments, the PEGylated lipid and polyanionic compound are present in a mass ratio of about 0.5:1 to about 2.5:1. In various embodiments, the PEGylated lipid and polyanionic compound are present in a mass ratio of about 1:1 to about 2:1. In some embodiments, the phospholipid and polyanionic compound are present in a mass ratio of about 2.1:1. In some embodiments, the phospholipid and polyanionic compound are present in a mass ratio of about 1.4:1. In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 10:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 in a mass ratio of about 1.4:1 to the polyanionic compound. In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 12:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 in a mass ratio of about 1.4:1 to the polyanionic compound. In some embodiments, the delivery vehicle complex comprises Compound 140 having a mass ratio of about 15:1 to the polyanionic compound, DSPC having a mass ratio of about 2.7:1 to the polyanionic compound, and cholesterol having a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 having a mass ratio of about 1.4:1 to the polyanionic compound.In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 17:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 in a mass ratio of about 1.4:1 to the polyanionic compound. In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 13:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 in a mass ratio of about 1.4:1 to the polyanionic compound. In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 19:1 to the polyanionic compound, DSPC in a mass ratio of about 4.0:1 to the polyanionic compound, cholesterol in a mass ratio of about 5.4:1 to the polyanionic compound, and DMG-PEG 2000 in a mass ratio of about 2.1:1 to the polyanionic compound. In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 9.7:1 to the polyanionic compound, DSPC in a mass ratio of about 2.7:1 to the polyanionic compound, cholesterol in a mass ratio of about 6.7:1 to the polyanionic compound, and DMG-PEG 2000 in a mass ratio of about 2.1:1 to the polyanionic compound.

[0050] In some embodiments, the pharmaceutical formulation comprises one or more mRNAs (including, but not limited to, a first isolated mRNA, a second isolated mRNA, or a third isolated mRNA described herein), an isolated polynucleotide, a polynucleotide encoding a polypeptide, a polynucleotide, and a nucleic acid encoding a polypeptide (including those described herein), and a delivery vehicle composition, the delivery vehicle composition comprising Compound 140, DSPC, cholesterol, and DMG-PEG 2000, suspended in a sucrose-containing citrate buffer at a pH of 5.0 to 6.0, e.g., pH 5.5. In some embodiments, the pharmaceutical formulation comprises the above-described delivery vehicle composition and an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17. In some embodiments, a pharmaceutical formulation comprises the delivery vehicle composition described above and an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17. In some embodiments, a pharmaceutical formulation comprises the delivery vehicle composition described above and an isolated mRNA comprising a sequence having at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18.

[0051] In some embodiments, the mRNA encodes a human papillomavirus (HPV) protein or a functional fragment thereof. In various cases, the mRNA encodes an HPV E6 protein and / or an HPV E7 protein, a variant thereof, or a functional fragment of any of the foregoing. In some embodiments, the HPV protein is derived from HPV subtypes HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and / or 68. In various embodiments, the HPV protein is derived from HPV subtypes HPV 16 and / or HPV 18.

[0052] In some embodiments, a pharmaceutical formulation comprises the delivery vehicle composition described above and a first isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 12 or SEQ ID NO: 18, a second isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 20, and a third isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26. In another embodiment, the first isolated mRNA has at least about 80% nucleic acid sequence identity to SEQ ID NO: 18 and the third isolated mRNA has at least about 80% nucleic acid sequence identity to SEQ ID NO: 26.

[0053] In some embodiments, the pharmaceutical formulation comprises the delivery vehicle composition described above and a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding interleukin-12, and a third isolated mRNA encoding HPV16 E6 E7.

[0054] Further disclosed herein are pharmaceutical compositions or formulations comprising the delivery vehicle complex or multi-component delivery system described herein and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical compositions are suitable for intramuscular (IM), intratumoral (IT), intracervical, pericervical, and / or intracervical administration, e.g., E-CERVIC routes of administration, as well as intravulvar, intravaginal, intrapenile, intraanal, and intraocular administration.

[0055] Also disclosed herein are methods of delivering one or more polyanionic compounds, such as one or more mRNAs (including, but not limited to, a first isolated mRNA, a second isolated mRNA, or a third isolated mRNA described herein), isolated polynucleotides, polynucleotides encoding polypeptides, polynucleotides, and nucleic acids encoding polypeptides (including those described herein) to a cell, the method comprising contacting the cell with a pharmaceutical formulation or multi-component delivery system comprising a delivery vehicle complex described herein. In some embodiments, the cell is a muscle cell, an epithelial cell, a tumor cell, or a combination of two or all three. In some embodiments, the cell is a cervical cell. In some embodiments, the cell is a cervical epithelial cell. In some embodiments, the cell is a cervical squamous intraepithelial neoplasia cell. In some embodiments, the cell is a cervical dysplasia. In some embodiments, the polyanionic compound is an mRNA encoding a peptide, a protein, or a fragment of any of the foregoing, and the cell expresses the peptide, protein, or fragment after contact with the delivery vehicle complex.

[0056] Also disclosed herein is a method of forming a delivery vehicle complex as disclosed herein, comprising contacting a compound or salt of Formula (I) with a polyanionic compound (such as mRNA). Optionally, the method comprises mixing a solution comprising the compound or salt of Formula (I) with a solution comprising the polyanionic compound (such as mRNA).

[0057] Also disclosed herein are vaccines comprising the delivery vehicle complexes disclosed herein or the pharmaceutical compositions disclosed herein. Also disclosed are vaccines comprising the delivery vehicle complexes disclosed herein or the pharmaceutical compositions disclosed herein for use in treating or preventing cancer. Also disclosed are methods of treating or preventing cancer in a patient, comprising administering to the patient a delivery vehicle complexes disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the administration is for treating cervical dysplasia (cervical intraepithelial neoplasia, or CIN), a precancerous condition in which abnormal cells grow on the surface of the cervix. In some cases, the cancer is cervical cancer, head and neck cancer, B-cell lymphoma, T-cell lymphoma, or prostate cancer.

[0058] In another embodiment, the composition is configured to be administered as an injectable formulation. In another embodiment, the composition further comprises one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients, or other therapeutic agents. In yet another embodiment, the therapeutically acceptable excipient is selected from the group including salts, buffers, preservatives, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film-forming agents, coating agents, flavors, fragrances, glidants, lubricants, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration.

[0059] One aspect of the present disclosure is a method of administering a therapeutically effective amount of a composition of the present disclosure to a subject in need thereof. In one aspect, the composition is administered as an injection. In another aspect, the composition is configured for intratumoral administration to a subject in need thereof.

[0060] One aspect of the present disclosure is a kit comprising any one of the disclosed compositions and instructions for use. In another aspect, the instructions include instructions for intratumoral administration and / or intratumoral administration in combination with injection at another site. In yet another aspect, the kit further comprises at least one of a therapeutic nucleic acid, a drug, a therapeutic agent, a diagnostic agent, or a prophylactic agent. [Brief explanation of the drawings]

[0061] Various aspects of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1A] 1 shows the inhibition of activation of membrane-stabilized LIGHT by soluble LIGHT. [Figure 1B] 1 shows the inhibition of activation of membrane-stabilized LIGHT by soluble LIGHT. [Figure 2A] 1 is a graph illustrating membrane-bound proteins produced by membrane-stabilized LIGHT produced according to embodiments of the present disclosure compared to soluble LIGHT. [Figure 2B] 1 is a graph illustrating membrane-bound proteins produced by membrane-stabilized LIGHT produced according to embodiments of the present disclosure compared to soluble LIGHT. [Figure 2C] 1 is a graph illustrating membrane-bound proteins produced by membrane-stabilized LIGHT produced according to embodiments of the present disclosure compared to soluble LIGHT. [Figure 2D] 1 is a graph illustrating membrane-bound proteins produced by membrane-stabilized LIGHT produced according to embodiments of the present disclosure compared to soluble LIGHT. [Figure 3A] FIG. 1 is a diagram of isolated mRNA according to an embodiment of the present disclosure. [Figure 3B] FIG. 1 is a diagram of an isolated mRNA that can encode a tumor-associated antigen according to one embodiment of the present disclosure. [Figure 3C] FIG. 1 is a diagram of an isolated mRNA that can encode a tumor-associated antigen according to one embodiment of the present disclosure. [Figure 3D] FIG. 1 is a diagram of an isolated mRNA that can encode a human LIGHT polypeptide according to one embodiment of the present disclosure. [Figure 3E] FIG. 1 is a diagram of an isolated mRNA that can encode a human IL-12 polypeptide or fusion protein according to one embodiment of the present disclosure. [Figure 3F] The sequences of the template DNA and synthetic RNA are shown, with the T7 promoter sequence (underlined) and the 5' cap initiation sequence (AGG). The arrow indicates the transcription start site with the nucleotide position indicated above. [Figure 4A] Comparison of antitumor efficacy with mRNA-based therapeutics containing soluble or membrane-stabilized LIGHT. [Figure 4B] Comparison of antitumor efficacy with mRNA-based therapeutics containing soluble or membrane-stabilized LIGHT. [Figure 4C] Comparison of antitumor efficacy with mRNA-based therapeutics containing soluble or membrane-stabilized LIGHT. [Figure 4D] Comparison of antitumor efficacy with mRNA-based therapeutics containing soluble or membrane-stabilized LIGHT. [Figure 4E] 1 shows the in vivo dose response of mNTX-250 (containing either WT or ENG mLIGHT) on growth inhibition of C3.43 tumors. [Figure 4F] 1 shows the in vivo dose response of mNTX-250 (containing either WT or ENG mLIGHT) on growth inhibition of C3.43 tumors. [Figure 4G] 1 shows the effect of mNTX-250 (containing either WT or ENG mLIGHT) on survival in a C3.43 tumor mouse model. [Figure 5] 1 shows the in vivo dose response of mNTX-250 (containing either WT or ENGmLIGHT) on the generation of HPV16 tetramer-positive T cells. [Figure 6A] 1 shows that a composition comprising membrane-stabilized LIGHT and IL-12 produced according to one embodiment of the present disclosure promotes an increase in inflammatory cytokines in the tumor microenvironment compared to soluble murine LIGHT+IL-12. [Figure 6B] 1 shows that a composition comprising membrane-stabilized LIGHT and IL-12 produced according to one embodiment of the present disclosure promotes an increase in inflammatory cytokines in the tumor microenvironment compared to soluble murine LIGHT+IL-12. [Figure 6C] 1 shows that a composition comprising membrane-stabilized LIGHT and IL-12 produced according to one embodiment of the present disclosure promotes an increase in inflammatory cytokines in the tumor microenvironment compared to soluble murine LIGHT+IL-12. [Figure 6D] Cytokine levels in the tumor microenvironment are compared between mice treated with ENG mLIGHT and mIL-12 and mice treated with WT mLIGHT and mIL-12. [Figure 7A] FIG. 1 is a diagram of a composition comprising three isolated mRNAs formulated in a delivery vehicle and subsequent administration according to an embodiment of the present disclosure. [Figure 7B] FIG. 1 is a diagram of a composition comprising three isolated mRNAs formulated into separate delivery vehicles and subsequent administration according to one embodiment of the present disclosure. [Figure 7C] FIG. 1 is a diagram of a composition comprising an isolated polynucleotide, including three mRNAs, formulated into a delivery vehicle and subsequent administration according to an embodiment of the present disclosure. [Figure 8A] 1 is a graph comparing CD8 proliferation of soluble and membrane-stabilized LIGHT T cells. [Figure 8B] 1 is a graph comparing CD8 proliferation of soluble and membrane-stabilized LIGHT T cells. [Figure 9A] Flow cytometry visualization of T cell proliferation for compositions containing soluble LIGHT or membrane-stabilized LIGHT. [Figure 9B] Flow cytometry visualization of T cell proliferation for compositions containing soluble LIGHT or membrane-stabilized LIGHT. [Figure 10A] T cell proliferation is compared for compositions containing soluble LIGHT and IL-12 or membrane-stabilized LIGHT and IL-12. The mitotic index is the total number of divisions divided by the number of cells at the start of the culture, the proliferation index is the total number of divisions divided by the number of cells that underwent division, the expansion index is the total number of cells divided by the number of cells at the start of the culture, and the replication index is the total number of dividing cells divided by the number of dividing cells. [Figure 10B]T cell proliferation is compared for compositions containing soluble LIGHT and IL-12 or membrane-stabilized LIGHT and IL-12. The mitotic index is the total number of divisions divided by the number of cells at the start of the culture, the proliferation index is the total number of divisions divided by the number of cells that underwent division, the expansion index is the total number of cells divided by the number of cells at the start of the culture, and the replication index is the total number of dividing cells divided by the number of dividing cells. [Figure 11] CD8 proliferation is compared for compositions containing soluble LIGHT and IL-12 or membrane-stabilized LIGHT and IL-12. [Figure 12] Pre-blocking of soluble LIGHT in HVEM co-HEK transfected cells. [Figure 13A] Comparison of HVEM activation by supernatant (blue) or cells (orange) for soluble or membrane-stabilized LIGHT. [Figure 13B] Comparison of HVEM activation by supernatant (blue) or cells (orange) for soluble or membrane-stabilized LIGHT. [Figure 13C] Comparison of HVEM activation by supernatant (blue) or cells (orange) for soluble or membrane-stabilized LIGHT. [Figure 14A] 1 shows the assessment of surface expression of soluble LIGHT or membrane-stabilized LIGHT by flow cytometry using HVEM binding. [Figure 14B] 1 shows the assessment of surface expression of soluble LIGHT or membrane-stabilized LIGHT by flow cytometry using HVEM binding. [Figure 15] 1 is a study design according to one aspect of the present disclosure. [Figure 16] Comparison of the percentage of viable CD4 and CD8 T cells during proliferation by flow cytometry for control mRNA, antigen mRNA, or mRNA-based therapeutic mRNA. [Figure 17A] HPV16+ donor proliferation by flow cytometry is shown. [Figure 17B] HPV16+ donor proliferation by flow cytometry is shown. [Figure 17C] HPV16+ donor proliferation by flow cytometry is shown. [Figure 17D] HPV16+ donor proliferation by flow cytometry is shown. [Figure 18A] 1 shows the cellular response of a delivery vehicle complex comprising DV-140-F2 according to an embodiment of the present disclosure. [Figure 18B] 1 shows the humoral response of a delivery vehicle complex comprising DV-140-F2 according to one embodiment of the present disclosure. [Figure 19A] 1 is a graph showing that the CD1d endolysosomal transport domain significantly improves antigen-specific immune responses in vivo. 1 shows IFNg ELISpot responses in HPV16E7 peptide mix-pulsed splenocytes from mice vaccinated with nanoparticle-formulated HPV16E6E7 mRNA vaccine. [Figure 19B] 1 is a graph showing that the CD1d endolysosomal trafficking domain significantly improves antigen-specific immune responses in vivo. The graph shows the frequency of tetramer-positive (E7-specific) CD8 T cells in splenocytes from mice vaccinated with nanoparticle-formulated HPV16 E6E7 mRNA vaccine. [Figure 19C] 1 is a graph showing that the CD1d endolysosomal trafficking domain significantly improves antigen-specific immune responses in vivo.

[0023] Figure 1 shows anti-E7 immunoglobulin responses in mice vaccinated with the HPV16E6E7 mRNA vaccine. DETAILED DESCRIPTION OF THE INVENTION

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press, The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press, and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0063] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or more (i.e., at least one). The term "and / or" means any one or more of the items in the list joined by "and / or." As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y." As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z."

[0064] The term "about" as used in connection with numerical values ​​throughout this specification and claims indicates an interval of accuracy well known and accepted by those skilled in the art. Generally, such an interval of accuracy is + / - 10%.

[0065] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can, in different embodiments of the present disclosure, assume any specific value or subrange within the stated range, to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0066] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As used herein, the words "eg" and "for example" refer to a list of one or more non-limiting aspects, examples, instances, or illustrations.

[0067] As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculations of percent similarity of polymer molecules relative to each other can be performed similarly to calculations of percent identity, except that percent similarity calculations take into account conservative substitutions.

[0068] As used herein, the term "substantially" refers to a qualitative condition indicating the entire or nearly entire extent or degree of a feature or characteristic of interest. Biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" can refer to within at least about 20%, or at least about 10%, or at least about 5% of the feature or characteristic of interest.

[0069] The terms "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence" are used interchangeably and refer to a contiguous nucleic acid sequence, which can be either single- or double-stranded DNA or RNA (e.g., mRNA).

[0070] The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Examples of nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0071] The phrases "nucleotide sequence encoding" or "encoding" refer to a nucleic acid (e.g., an mRNA or DNA molecule) coding sequence that encodes a polypeptide. As used herein, the terms "coding region" and "coding sequence" refer to an open reading frame (ORF) in a polynucleotide that, upon expression, produces a polypeptide or protein. A coding sequence may further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. A coding sequence may further include a sequence encoding a signal peptide.

[0072] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. Embodiments of the present disclosure include polynucleotides having lengths of 18 to 25 nucleotides (e.g., 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, 25-mer), or intermediate polynucleotides having lengths of 26 nucleotides or more (e.g., 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250 The present invention also includes compositions containing polynucleotides having a length of about 50, 260, 270, 280, 290, or 300 nucleotides, or longer polynucleotides having a length of more than about 300 nucleotides (e.g., polynucleotides having a length of about 300 to about 400 nucleotides, about 400 to about 500 nucleotides, about 500 to about 600 nucleotides, about 600 to about 700 nucleotides, about 700 to about 800 nucleotides, about 800 to about 900 nucleotides, about 900 to about 1000 nucleotides, about 300 to about 500 nucleotides, about 300 to about 600 nucleotides, about 300 to about 700 nucleotides, about 300 to about 800 nucleotides, about 300 to about 900 nucleotides, about 1000 nucleotides, or longer than about 1000 nucleotides).

[0073] When a polynucleotide is double-stranded, its length can also be described in terms of base pairs. This term refers to the primary structure of the molecule. Thus, this term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) (including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing non-nucleotide backbones, such as polyamides (e.g., peptide nucleic acids "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA. In certain embodiments, the polynucleotide comprises mRNA.

[0074] Nucleotides are referred to by their generally accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in a 5' to 3' orientation. Nucleotides are referred to herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and U represents uracil.

[0075] Although T bases in the codon maps disclosed herein are present in DNA, the T bases may be replaced by U bases in the corresponding RNA. For example, a codon-nucleotide sequence disclosed herein in DNA form, such as a vector or in vitro translation (IVT) template, may have its T base transcribed as a U base in its corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (containing T) and their corresponding RNA sequences (containing U) are considered codon-optimized nucleotide sequences of the present disclosure. Equivalent codon maps may be generated by substituting one or more bases with unnatural bases. Thus, for example, a TTC codon (DNA map) may correspond to a UUC codon (RNA map), which in turn may correspond to a "P"C codon (RNA map in which U is substituted with pseudouridine).

[0076] Standard AT and GC base pairs are formed under conditions that allow hydrogen bond formation between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2 of adenosine, respectively, and between the C2-oxy, N3, and C4-NH2 of cytidine and the C2-NH2, N'-H, and C6-oxy of guanosine, respectively. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modifications result in nucleoside bases that no longer effectively form standard base pairs with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) results in a modified nucleotide that does not base pair efficiently with guanosine but does base pair with isoguanosine.

[0077] Typically, RNA can be obtained by transcription of a DNA sequence, for example, within a cell. In eukaryotic cells, transcription typically occurs in the nucleus or mitochondria. In vivo, transcription of DNA usually produces so-called immature RNA (also called pre-mRNA, precursor mRNA, or heteronuclear RNA), which must usually be processed into so-called messenger RNA, abbreviated as mRNA. For example, the processing of immature RNA in eukaryotes involves a variety of different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, and export from the nucleus or mitochondria. The sum of these processes is also called RNA maturation. Mature messenger RNA usually provides a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mature mRNA includes a 5'CAP, an optional 5'untranslated region ("5'UTR"), an open reading frame, an optional 3'untranslated region ("3'UTR"), and a poly(A) tail.

[0078] As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0079] In addition to messenger RNA, there are several non-coding types of RNA that may be involved in regulating transcription and / or translation, and immune stimulation. Within the present disclosure, the term "RNA" further encompasses any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulatory / immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).

[0080] As used herein, a 5'-CAP (or 5'-CAP structure) is typically a modified nucleotide (CAP analog), particularly a guanine nucleotide, added to the 5' end of an mRNA molecule. In certain implementations, the 5'-CAP is added using a 5'-5'-triphosphate linkage (also referred to as m7GpppN). Further examples of 5'-CAP structures include glyceryl, inverted deoxyabasic residues (moieties), 4',5' methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, and the like. Modified 5'-CAP structures include nucleotides such as 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, or bridged or non-bridged methylphosphonate moieties. These modified 5'-CAP structures may be used in the context of the present disclosure to modify the RNA sequences of the present disclosure. Further modified 5'-CAP structures that may be used in the context of the present disclosure are CAP1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), CAP2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), CAP3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), CAP4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse CAP analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0081] In the context of the present disclosure, the 5' CAP structure can also be formed during chemical RNA synthesis or in vitro transcription of RNA (co-transcriptional capping) using a cap analog, or the cap structure can be formed in vitro using a capping enzyme (e.g., a commercially available capping kit).

[0082] A cap analog refers to a non-polymerizable dinucleotide that possesses a cap functionality in that, when incorporated at the 5' end of an RNA molecule, it facilitates translation or localization and / or prevents degradation of the RNA molecule. Non-polymerizable means that the cap analog is incorporated only at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase.

[0083] Cap analogs include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC, unmethylated cap analogs (e.g., GpppG), dimethylated cap analogs (e.g., m2,7 GpppG), trimethylated cap analogs (e.g., m2,2,7 GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G), or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG, and their tetraphosphate derivatives). The synthesis of N7-(4-chlorophenoxyethyl)-substituted dinucleotide cap analogs has recently been described.

[0084] As used herein, a "poly(A) tail," also referred to as a "3'-poly(A) tail" or "poly(A) tail," is typically a long homopolymeric sequence of adenosine nucleotides, e.g., about 25 to about 400, about 50 to about 400, about 50 to about 300, about 50 to about 250, or about 60 to about 250 adenosine nucleotides, added to the 3' end of an mRNA. In certain implementations of the present disclosure, the poly(A) tail of an mRNA or srRNA is derived from a DNA template by in vitro transcription of the RNA. Alternatively, the poly(A) sequence may also be obtained in vitro by common methods of chemical synthesis, without necessarily being transcribed from a DNA precursor. Furthermore, the poly(A) sequence or poly(A) tail may be generated by enzymatic polyadenylation of RNA.

[0085] Stabilized nucleic acids typically exhibit modifications that increase resistance to in vivo degradation (e.g., degradation by exonucleases or endonucleases) and / or ex vivo degradation (e.g., degradation due to manufacturing processes prior to administration of the composition, e.g., degradation during preparation of the composition to be administered). RNA stabilization can be achieved, for example, by providing a 5'-CAP structure, a poly(A) tail, or any other UTR modification. Stabilization can also be achieved by backbone modifications (e.g., use of synthetic backbones such as phosphorothioates) or modifying the G / C or C content of the nucleic acid. A variety of other methods for stabilizing nucleic acids or otherwise improving their function are known in the art and are contemplated in the context of the present disclosure. Thus, provided herein are polynucleotides designed to improve one or more of: tissue stability and / or clearance, receptor uptake and / or kinetics, cellular access, engagement with the translation machinery, RNA half-life, translation efficiency, immune evasion, immune induction (in the case of vaccines), protein production capacity, secretion efficiency (where applicable), accessibility to circulation, protein half-life, and / or modulation of cellular state, function and / or activity.

[0086] As used herein, "therapeutic polynucleotide" refers to a polynucleotide (e.g., mRNA) that can be part of a therapeutic polynucleotide composition for delivery to a subject to treat a symptom, disease, or condition in the subject, prevent a symptom, disease, or condition in the subject, or improve or otherwise alter the health of the subject.

[0087] As used herein, a "therapeutic polynucleotide composition" (or simply "therapeutic composition") may refer to a composition comprising one or more therapeutic polynucleotides (e.g., mRNA) encapsulated by a delivery vehicle, which may be administered to a subject in need thereof using any suitable route of administration, such as intratumoral injection, intramuscular injection, etc. An example of a therapeutic polynucleotide composition is an mRNA (therapeutic) nanoparticle comprising at least one mRNA encapsulated by a delivery vehicle molecule. An mRNA vaccine is an example of a therapeutic polynucleotide composition. A therapeutic composition may be administered in an "effective amount." An "effective amount" includes a "therapeutically effective amount" and a "prophylactically effective amount." The term "therapeutically effective amount" refers to an amount effective to treat and / or ameliorate a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective to prevent and / or substantially reduce the likelihood of a disease or condition in a subject.

[0088] As used herein, a "delivery vehicle" refers to any substance that at least partially facilitates in vivo, in vitro, or ex vivo delivery of a polynucleotide (e.g., a therapeutic polynucleotide) to a target cell or tissue (e.g., a tumor, etc.). Referring to something as a delivery vehicle need not exclude the possibility that the delivery vehicle may also have a therapeutic effect. Some modified forms of a delivery vehicle may provide additional therapeutic effects. In some modified forms, the delivery vehicle may be a peptoid molecule (e.g., an amino-lipidated peptoid molecule) that can be used to at least partially encapsulate an mRNA. The term "DV" may also be used herein as shorthand for "delivery vehicle." In some aspects, mRNA for use in the delivery vehicle complexes herein includes mRNA that includes at least one region encoding a peptide (e.g., a polypeptide), or protein, or a functional fragment of the foregoing. As used herein, a "functional fragment" refers to a fragment of a peptide (e.g., a polypeptide) or protein that retains the ability to induce an immune response.

[0089] As used herein, "multimodal" refers to a therapeutic composition that includes at least two different therapeutic polynucleotides, or alternatively, at least three different therapeutic polynucleotides.

[0090] As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in a first sequence is occupied by the same nucleotide as the corresponding position in a second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0091] Suitable software programs are available from a variety of sources for aligning both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI).

[0092] Sequence alignment can be performed using methods such as, but not limited to, MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), or MUSCLE.

[0093] Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity.

[0094] As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it is associated (whether in nature or in an experimental setting). Isolated substances (e.g., nucleotide sequences or protein sequences) can have various levels of purity with respect to the materials with which they are associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, an isolated agent is greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. The term "substantially isolated" means that a compound is substantially separated from the environment in which it was formed or detected. Partial isolation can include, for example, compositions enriched in a compound of the present disclosure. Substantial isolation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of a compound of the present disclosure or a salt thereof.

[0095] An "isolated" polynucleotide, vector, polypeptide, cell, or any composition disclosed herein is a polynucleotide, vector, polypeptide, cell, or composition that is in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some aspects, an isolated polynucleotide, vector, polypeptide, or composition is substantially pure.

[0096] As used herein, "native" or "naturally occurring" polynucleotide sequence refers to a polynucleotide sequence that exists in nature without artificial assistance. A synthetic polynucleotide sequence that is identical to a wild-type polynucleotide sequence is considered a naturally occurring sequence for purposes of this disclosure. For example, but not limited to, a wild-type polynucleotide sequence is a naturally occurring polynucleotide sequence. A naturally occurring polynucleotide sequence also refers to variant polynucleotide sequences found in nature that differ from the wild type, for example, but not limited to, allelic variants and naturally occurring recombinant polynucleotide sequences due to hybridization or horizontal gene transfer. The term "wild type," as used herein with respect to a polynucleotide, refers to the naturally occurring, non-mutated form of the polynucleotide.

[0097] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. Polymers can contain modified amino acids. These terms also include amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine) are also included within this definition. As used herein, the terms refer to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single polypeptide or a multimolecular complex, such as a dimer, trimer, or tetramer. They can also include single-chain or multi-chain polypeptides. Most commonly, disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be about 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0098] As used herein, the term "variant" refers to a polynucleotide or polypeptide sequence that differs from a specifically identified sequence by having one or more deleted, substituted, or added nucleotide or amino acid residues. Variants may be naturally occurring allelic variants or non-naturally occurring variants. Variants may be from the same species or from other species and may include homologs, paralogs, and orthologs.

[0099] LIGHT is a multidomain protein belonging to the TNF superfamily, consisting of an intracellular domain, a transmembrane domain, a receptor-binding domain, and a linker region connecting the transmembrane domain to the receptor-binding domain, and plays an important role in innate and adaptive immunity.

[0100] LIGHT is expressed by activated T cells, NK cells, and immature dendritic cells. Herpes virus entry mediator (HVEM) and lymphotoxin-β receptor (LTβR) are two distinct receptors to which LIGHT binds and elicits distinct functions, primarily in a cell-type-dependent manner. LIGHT binds to HVEM and LTβR via its homotrimeric form and also functions in soluble and membrane-stabilized forms. These proteases have been reported to be overexpressed in many tumors, including HPV-associated cancers (e.g., cervical cancer).

[0101] Surprisingly, the novel membrane-stabilized LIGHT constructs described herein were found to have superior activity compared to wild-type ("soluble") LIGHT in activating HVEM in a dose-dependent manner. Therapeutics containing membrane-stabilized LIGHT ("engineered" or "ENG") also cause increased T cell proliferation compared to soluble (wild-type) LIGHT and are more functional as costimulators. As shown in Figures 1A and 1B, the presence of soluble LIGHT reduces the ability of membrane-stabilized LIGHT to activate HVEM. Incorporation of modified membrane-stabilized LIGHT into cancer immunotherapy allows for effective targeting of cancers, such as HPV-associated cancers.

[0102] In some aspects of the present disclosure, suitable membrane-stabilized LIGHT proteins, protein fragments, peptides, or polypeptides (collectively "membrane-stabilized LIGHT") can be generated by modifying soluble LIGHT. In a non-limiting example, a section of the transmembrane region of soluble LIGHT can be replaced with a linker or modified trimerization domain to generate membrane-stabilized LIGHT. In one aspect, the membrane-stabilized LIGHT comprises a modified trimerization domain, such as collagen, foldon, and leucine zipper. In some aspects, the linker can be, but is not limited to, an antibody linker or a peptide linker.

[0103] In some embodiments, to generate the novel membrane-stabilized LIGHT described herein, the primary amino acid sequence of soluble LIGHT was examined for potential transmembrane regions to identify positions occupied by glycines that could function as helix-terminating residues. This left potential sites for the N-terminus of a linker at either position 66 or 75. To identify the C-terminus of the linker region, the crystal structure of the receptor-binding domain of LIGHT was examined. Although the protein sequence used for the crystal structure determination begins at residue 83, the structured region in the structure begins only at residue 92. Positions 83 and 92 were therefore identified as the C-terminus of the linker region connecting the transmembrane domain and the receptor-binding domain. Using this linker region prediction information, the segment in LIGHT from residue 66 or 75 to residue 83 or 92 was replaced with a linker. RNA-1495 corresponds to a single mRNA sequence (SEQ ID NO: 18) encoding native secreted human LIGHT engineered by replacing amino acids 66-92 with a novel 10-amino acid linker sequence to enhance membrane stability and expression, and is represented herein as SEQ ID NO: 17. This construct, also referred to as "ENG hLIGHT," is designed to be a surface-associated type II membrane protein. It is understood that any suitable polynucleotide sequence encoding SEQ ID NO: 17 is contemplated as useful in the embodiments described herein.

[0104] As used herein, "linker" refers to a group of atoms, e.g., 10 to 1,000 atoms, which may be composed of atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. Linkers may also be composed of amino acids, peptides, proteins, antibodies, and / or polynucleotides.

[0105] Examples of chemical groups that can be incorporated into a linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted as described herein. Other examples include, but are not limited to, cleavable moieties within the linker, such as a disulfide bond (-SS-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis.

[0106] In some embodiments, a (Gly4Ser)n linker was used. In some examples, n is an integer greater than 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, n can be greater than 10. In some examples, the linker sequence is an antibody combining an IgG1 variable domain and constant domain (CH1) and constant domains of CH1 and CH2. In some embodiments, the linker comprises the sequence SSASTDKTHT (SEQ ID NO: 54).

[0107] Non-limiting examples of membrane-stabilized human LIGHT made in accordance with the present disclosure are disclosed in Table 1. Wild-type soluble human LIGHT (SEQ ID NO: 1) is also included for reference.

[0108] [Table 1-1]

[0109] [Table 1-2] ICD intracellular domain of LIGHT TM transmembrane region

[0110] As shown in Figures 2A-2D, it was surprisingly found that membrane-stabilized LIGHT ("engineered LIGHT") produced in accordance with the present disclosure has significantly higher cell surface expression of membrane-associated proteins and a significantly lower soluble fraction compared to soluble (wild-type) LIGHT. This enhanced membrane stability for the disclosed membrane-stabilized LIGHT may increase the beneficial activities of LIGHT.

[0111] In some examples, the isolated polynucleotide may encode a membrane-stabilized LIGHT according to embodiments of the present disclosure. In a non-limiting example, the isolated polynucleotide comprises at least a first isolated messenger ribonucleic acid (mRNA), at least a portion of which encodes membrane-stabilized LIGHT.

[0112] The isolated polynucleotide may be configured for direct administration, may be configured for administration in a composition comprising other isolated mRNAs, may be encoded in one or more polynucleotides for expression in a cell, and / or may be encoded in DNA, RNA, or mRNA for administration. According to the present disclosure, a first isolated mRNA (FIG. 3A) has the following formula: It may have a structure of 5'UTR-signal / leader-mRNA coding region-3'UTR-polyA. In the formula, "UTR" refers to the untranslated region located at the 5' and 3' ends of the mRNA construct, and "PolyA" refers to the polyadenylation site of the mRNA.

[0113] A 5'-UTR is typically understood to be a specific section of RNA. It is located 5' to the open reading frame of an mRNA. In the case of srRNA, the open reading frame encodes a viral nonstructural protein, but the sequence of interest is encoded in a subgenomic fragment of the viral RNA. Thus, the 5'-UTR is upstream of the nsP1 open reading frame. Furthermore, subgenomic RNAs of srRNA have a 5'-UTR. Thus, a subgenomic RNA containing a sequence of interest encoding a protein of interest contains a 5'-UTR. Typically, the 5'-UTR begins at the transcription initiation site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR may contain elements for controlling gene expression (also called regulatory elements). Such regulatory elements may be, for example, a ribosome binding site or a 5'-terminal oligopyrimidine tract. The 5'-UTR may be post-transcriptionally modified, for example, by the addition of a 5'-CAP. In the context of the present disclosure, the 5'-UTR corresponds to the sequence of a mature mRNA or srRNA located between the 5'-CAP and the start codon. In one implementation, the 5'-UTR corresponds to the sequence extending from the nucleotide located 3' to the 5'-CAP, and in certain implementations, from the nucleotide located immediately 3' to the 5'-CAP, to the nucleotide located 5' to the start codon of the protein-coding region, and in some cases, to the nucleotide located immediately 5' to the start codon of the protein-coding region. The nucleotide located immediately 3' to the 5'-CAP of a mature mRNA or srRNA typically corresponds to the transcription start site. The term "corresponds to" means that the 5'-UTR sequence can be an RNA sequence, such as an mRNA sequence, used to define the 5'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence.In the context of the present disclosure, the term "a 5'-UTR of a gene," such as "a 5'-UTR of a NYESO1 gene," is the sequence corresponding to the 5'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the premature mRNA. The term "5'-UTR of a gene" encompasses the DNA and RNA sequences of the 5'-UTR.

[0114] Generally, the term "3'-UTR" refers to a portion of a nucleic acid molecule that is located 3' (i.e., "downstream") of an open reading frame and is not translated into a protein. Typically, a 3'-UTR is a portion of an RNA that is located between the protein-coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of an mRNA. In the context of the present disclosure, the term 3'-UTR can also include elements that are not encoded in the template from which the RNA is transcribed, but are added post-transcriptionally during maturation, such as poly(A) sequences. The 3'-UTR of an RNA is not translated into an amino acid sequence.

[0115] With respect to srRNA, the 3'-UTR sequence is generally encoded by the viral genomic RNA, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into a premature mRNA. The premature mRNA is then further processed into a mature mRNA in a maturation process, which includes 5'-capping. In the context of the present disclosure, the 3'-UTR corresponds to a sequence of a mature mRNA or srRNA (and srRNA subgenomic RNA), which is located between the stop codon of the protein-coding region, preferably the stop codon of the protein-coding region of the sequence of interest, and the poly(A) sequence of the mRNA. The term "corresponds to" means that the 3'-UTR sequence can be an RNA sequence, such as an mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to such an RNA sequence. In the context of the present disclosure, the term "a 3'-UTR of a gene" is the sequence corresponding to the 3'-UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the premature mRNA. The term "3'-UTR of a gene" encompasses the DNA and RNA sequences of the 3'-UTR (both sense and antisense strands, and both mature and premature).

[0116] In some embodiments, the 3'UTR is a modified 3'UTR and refers to a 3'UTR sequence resulting from sequence optimization. In some embodiments, the 5'UTR is a modified 5'UTR and refers to a 5'UTR sequence resulting from sequence optimization. As used herein, "sequence optimization" refers to a process or set of processes in which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties.

[0117] Non-limiting examples of modified 3'UTRs (wild-type and modified) are listed in Table 2.

[0118] [Table 2]

[0119] Non-limiting examples of 5'UTRs (wild-type and modified) are listed in Table 3.

[0120] [Table 3]

[0121] Signal / Leader refers to a suitable signal sequence, leader sequence, or sorting sequence that is in frame with and upstream of the mRNA coding region.

[0122] As used herein, the terms "mRNA coding region" and "mRNA coding sequence" refer to an open reading frame (ORF) (a sequence that does not contain a stop codon in a given reading frame) in a polynucleotide that, upon expression, gives rise to a polypeptide or protein. The mRNA coding sequence may further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide. In some embodiments, the formula may further include a 5' cap. In some embodiments, the formula may further include a polyA tail.

[0123] In a non-limiting example, an RNA sequence for encoding membrane-stabilized human LIGHT has the following formula (see, eg, Figure 3D): 5'UTR-signal / leader-mRNA membrane-stabilizing human Light coding region-3'UTR-poly(A)

[0124] In one example, the 3'UTR is modified. In another example, the 5'UTR is modified. In a further example, both the 3'UTR and the 5'UTR are modified. In one example, the signal / leader is a secretion signal.

[0125] Non-limiting examples of RNA sequences encoding membrane-stabilized human LIGHT made in accordance with the present disclosure are disclosed in Table 4. Wild-type soluble human LIGHT (SEQ ID NO: 2) is also included for reference.

[0126] [Table 4-1]

[0127] [Table 4-2]

[0128] [Table 4-3]

[0129] [Table 4-4]

[0130] [Table 4-5] ICD intracellular domain of LIGHT TM transmembrane region

[0131] In some non-limiting embodiments, the isolated mRNA encodes membrane-stabilized LIGHT that has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO: 17. In other non-limiting embodiments, the membrane-stabilized LIGHT has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% amino acid sequence identity to SEQ ID NO:11 or SEQ ID NO:17.

[0132] In other non-limiting examples, the isolated mRNA encoding membrane-stabilized LIGHT has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO: 18. In another embodiment, the isolated mRNA has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% nucleic acid sequence identity to SEQ ID NO:12 or SEQ ID NO:18.

[0133] A characteristic of the universal genetic code is its redundancy: most amino acids are coded for by more than one codon (the exceptions being the amino acids methionine and tryptophan, each of which has only one corresponding codon). Thus, within the constraints of this degeneracy, a polynucleotide sequence can be altered without affecting the sequence of amino acids in the encoded polypeptide.

[0134] For example, the amino acid alanine has four possible codons (GCT, GCC, GCA, and GCG) that differ from each other at only one nucleotide position, meaning that every time an alanine codon appears in a polynucleotide, the third nucleotide in the codon can be replaced with another nucleotide without changing the encoded polypeptide.

[0135] mRNA-based therapeutics combining mRNA-encoded membrane-stabilized LIGHT with mRNA-encoded effector molecules, mRNA-encoded immunomodulators, and / or mRNA-encoded tumor-associated antigens may function as effective cancer immunotherapies. Prevention or treatment of disease with substances that stimulate the immune response is generally referred to as immunotherapy. Immunotherapy (sometimes referred to as immuno-oncology) introduces therapies that target the host immune system rather than the tumor. These treatments may have unique pharmacological response profiles and therefore represent potentially curative therapies for many different types of cancer. In one example, lung, kidney, bladder, and skin cancers are among those that derive substantial benefit from immuno-oncology treatment in terms of survival or tumor response, as is melanoma in particular. Immunotherapy is often characterized by checkpoint inhibitor treatment with biological drugs known as checkpoint inhibitor antibodies.

[0136] Because this technology provides for the delivery of both tumor-specific antigen mRNA and one or more mRNAs encoding, for example, immunomodulators or agents, enabling de novo synthesis of functional proteins within target cells, e.g., tumors, the disclosed mRNA-based therapeutics are particularly well suited for cancer immunotherapy. Immunomodulators or agents include, but are not limited to, oncology-related polypeptides, checkpoint inhibitors, immunosuppressive antagonists, pro-inflammatory agents, pro-inflammatory cytokines, and other agents useful in immuno-oncology ("immuno-oncology"). These cancer immunotherapies leverage the ability of mRNA to deliver genetic information and initiate immune stimulatory activity. The disclosed mRNA-based therapeutics may contain modified nucleotides to minimize unwanted immune activation (e.g., innate immune responses associated with in vivo introduction of foreign nucleic acids) and optimize the efficiency of mRNA translation into protein. As shown in Figures 4A-4D, membrane-stabilized LIGHT (ENG-LIGHT) exhibits antitumor efficacy.

[0137] A non-limiting embodiment of the present disclosure is an mRNA-based therapeutic agent capable of targeting cervical cancer, HPV-associated cancer, or HPV-associated disease. HPV-associated cancers include, but are not limited to, cervical cancer, oropharyngeal cancer, which typically develops in the throat (usually the tonsils or underside of the tongue), anal cancer, penile cancer, vaginal cancer, and vulvar cancer. Cervical cancer is the leading reproductive cancer among women worldwide, with nearly 500,000 new cases per year (GLOBOCAN, 2012). In 2015, 526,000 women worldwide developed cervical cancer, and 239,000 died. In addition to the risk of death, cervical cancer is associated with increased morbidity, including bleeding, pain, and renal failure, which are difficult to treat.

[0138] In a non-limiting embodiment, an mRNA-based therapeutic capable of targeting cervical cancer, an HPV-associated cancer, or an HPV-associated disease may include a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding an immunomodulator, and a third isolated mRNA encoding at least one tumor-associated antigen. As shown in Figure 5, membrane-stabilized LIGHT generates an HPV-specific T cell response.

[0139] In some examples, the mRNA-based therapeutic may further comprise an additional isolated mRNA encoding an additional immunomodulatory agent, including an immune checkpoint modulator, a checkpoint inhibitor, and / or another antibody therapeutic.

[0140] Non-limiting examples of checkpoint inhibitors include proteins that target and inhibit one or more immune system checkpoint proteins. Examples include proteins that inhibit one or more of CTLA4, PD-1, and PD-L1. In some variations, a checkpoint inhibitor refers to a protein that inhibits CTLA-4. For example, a checkpoint inhibitor protein may include a protein that binds to CTLA-4, such as an antibody or antibody fragment, such as an antigen-binding fragment (Fab), single-chain variable fragment (scFv), aptamer, etc. An mRNA encoding a checkpoint inhibitor may encode, for example, an anti-PD-L1 antibody (such as atezolizumab, avelumab, or durvalumab), an anti-CTLA-4 antibody (such as tremelimumab or ipilimumab), an anti-PD-1 antibody (such as nivolumab or pembrolizumab), or a combination thereof.

[0141] Immunosuppressive antagonists may include proteins that prevent or limit immunosuppression and block pathways for immune system suppression. For example, antagonists against transforming growth factor β (TGF-β-RII) are cytokines that can act as immunosuppressive antagonists. Proinflammatory agents typically induce the inflammatory response itself and may cause local inflammation. For example, interleukin-12 is a proinflammatory pleiotropic cytokine widely accepted as a key regulator of Th1 responses. It also promotes the proliferation and survival of activated T cells and NK cells and regulates the cytotoxic activity of CTL and NK cells. Therefore, as used herein, immunomodulators (immunomodulatory drugs) may include interleukins (e.g., IL-2, IL-7, IL-12), other cytokines (interferons, GM-CSF), chemokines (CCL3, CCL26, CXCL-7), and the like.

[0142] In some instances, the additional isolated mRNA has the following formula: It may have a structure of 5'UTR-signal / leader-mRNA coding region-3'UTR-polyA.

[0143] In a non-limiting example, the isolated mRNA encoding human IL-12 has the following formula: It has 5'UTR-signal / leader-mRNA human IL-12 coding region-3'UTR-polyA.

[0144] In one example, the 3'UTR is modified. In another example, the 5'UTR is modified. In a further example, both the 3'UTR and the 5'UTR are modified. In one example, the signal / leader is a secretion signal.

[0145] In some embodiments, the mRNA human IL-12 coding region can encode at least two heterodimers of IL-12, where a first IL-12 heterodimer is linked to a second IL-12 heterodimer via a linker. See, e.g., Figure 3E. The linker can be any linker known in the art, including an antibody variable region linker, a peptide linker, and / or a (Gly4Ser)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0146] As shown in Figures 6A-6D, mRNA-based therapeutics containing both membrane-stabilized LIGHT and a pro-inflammatory cytokine such as IL-12 can promote increased beneficial Th1, M1, and overall pro-inflammatory tumor microenvironment changes compared to soluble LIGHT.

[0147] Non-limiting examples of isolated mRNA sequences encoding human IL-12 fusions and human IL-12 fusion amino acid sequences are provided in Table 5.

[0148] [Table 5-1]

[0149] [Table 5-2]

[0150] In one embodiment, the second isolated mRNA encodes a pro-inflammatory cytokine having at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO: 21. In a different non-limiting embodiment, the pro-inflammatory cytokine has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% amino acid sequence identity to SEQ ID NO: 21.

[0151] In one embodiment, the second isolated mRNA has at least 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO: 20. In a different non-limiting embodiment, the second isolated mRNA has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% nucleic acid sequence identity to SEQ ID NO: 20.

[0152] The most oncogenic HPV type is HPV16, with 50% of all cervical cancers associated with HPV16. The second most oncogenic HPV type is HPV18, with 15-20% of all cervical cancers associated with HPV18. HPV replication involves the viral E region, which encodes the viral genes E6 and E7, among others, which are associated with deregulated cell proliferation, the initiation of tumorigenesis, and ultimately the progression of cancer in the host. Furthermore, the E6 and E7 viral genes are retained, integrated into the host genome, and constitutively expressed, targeting specific antigens in HPV-infected cells and the major histocompatibility complex class I-restricted CD8 receptor in cervical cancer patients. + Provides a T cell response.

[0153] In some embodiments, the third isolated mRNA has the following formula: It may encode a tumor-associated antigen that may have 5'UTR--Signal / Leader-(An1)n-Xo-(An2)p-3'UTR-polyA. where "UTR" refers to the untranslated region located at the 5' and 3' ends of the mRNA construct, and "polyA" refers to the polyadenylation site of the mRNA. In one example, the 3'UTR is modified. In another example, the 5'UTR is modified. In a further example, both the 3'UTR and the 5'UTR are modified.

[0154] Signal / Leader refers to a suitable signal sequence, leader sequence, or sorting sequence that is in frame with and upstream of the antigenic region. In one example, the signal / leader is a secretion signal and / or a helper epitope.

[0155] (An1)n-Xo-(An2)p refers to any suitable antigen region comprising a first antigen (An1), a spacer or linker region (X), and a second antigen (An2). In some examples, n is an integer greater than 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, n can be greater than 10. In some examples, o is 0. In other examples, o is an integer greater than 0. For example, o can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, o can be greater than 10. In some examples, p is an integer greater than 0. For example, p can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, p can be greater than 10.

[0156] There are several HPV-associated antigens, including HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68. HPV16 and HPV18 are responsible for most HPV-associated diseases. In a non-limiting example, a polynucleotide comprising an mRNA encoding a tumor-associated antigen encodes the HPV16 E6 and E7 or HPV18 E6 and E7 oncoproteins, which are linked by a short Gly4Ser linker to create a single continuous protein antigen. See, for example, Figures 3B and 3C.

[0157] In a non-limiting example, the third isolated mRNA has the following formula: 5'UTR--Signal / Leader-(An1)n-Xo-(An2)p-3'UTR-polyA, wherein An1 encodes HPV16 E6, An2 encodes HPV16 E7, and X is a spacer or linker; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0158] Some mutations in the E6 and E7 sequences may be made to reduce the oncogenic function of the E6 and E7 proteins. In some embodiments, the mRNA-encoding tumor-associated antigen cassette may further comprise a PADRE T helper epitope to enhance adaptive immune responses and / or an hCD1d scaffold to enhance CD4 T cell responses and antigen cross-presentation.

[0159] After synthesis in the endoplasmic reticulum (ER), CD1d passes through the secretory pathway (Trans-Golgi Network (TGM)) to reach the cell surface or directly to multivesicular bodies / antigen processing compartments. The cytoplasmic tails of CD1d isoforms interact with adaptor-protein complexes, resulting in distinct patterns of steady-state distribution in subcompartments of the endosomal network.

[0160] By piggybacking onto the intracellular trafficking of the CD1d molecule, we designed an mRNA encoding an antigen flanked by the CD1d signal peptide (Sec) and the CD1d cytoplasmic tail (antigen-CD1d). Upon cell transfection, the mRNA is translated, and the resulting antigen protein is directed to the antigen processing compartment by the CD1d endolysosomal trafficking signal. The endolysosomal trafficking domain, CD1d, enhances antigen reactivity and cross-presentation. Utilizing the CD1d endolysosomal trafficking signal promotes antigen processing and cross-presentation in both MHC class I (endogenously derived antigens) and MHC class II (exogenously derived antigens).

[0161] CD1d endolysosomal transport domain-linked antigens exhibit sustained intracellular expression in multivesicular bodies, resulting in improved CD4 and CD8 antigen-specific binding. In vivo, antigen-CD1d mRNA significantly improves antigen-specific CD8 T cell responses and enhances immunoglobulin responses (Figures 19A-19C). Improved immunoglobulin responses indicate more productive antigen presentation to both B cells and T cells. This further demonstrates how vaccine antigenicity can be improved by utilizing the unique features of mRNA drugs to promote intracellular expression of intracellular proteins.

[0162] Non-limiting examples of nucleic acid sequences of isolated mRNAs encoding tumor-associated antigens and amino acid sequences of tumor-associated antigens according to one embodiment of the present disclosure are listed in Table 6.

[0163] [Table 6-1]

[0164] [Table 6-2]

[0165] [Table 6-3]

[0166] In one aspect, the third isolated mRNA encodes an antigen having at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO: 24 or SEQ ID NO: 27. In a different non-limiting aspect, the antigen has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% amino acid sequence identity to SEQ ID NO: 24 or SEQ ID NO: 27.

[0167] In one embodiment, the third isolated mRNA has at least about 50%, alternatively at least about 60%, alternatively at least about 65%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26. In a different non-limiting embodiment, the third isolated mRNA has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% nucleic acid sequence identity to SEQ ID NO: 23 or SEQ ID NO: 26.

[0168] Methods for producing polynucleotides of a predetermined sequence are well known. Solid-phase synthesis methods are well known for both polyribonucleotides and polydeoxyribonucleotides (the well-known methods for synthesizing DNA are also useful for synthesizing RNA). Polyribonucleotides can also be prepared enzymatically. Non-naturally occurring nucleobases can be incorporated into polynucleotides as well.

[0169] Any method known in the art for making RNA is contemplated herein, including, but not limited to, making mRNA. Exemplary methods for making RNA include, but are not limited to, chemical synthesis and in vitro transcription.

[0170] In certain embodiments, RNA for use in the methods herein is chemically synthesized. Chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structures provides rapid and inexpensive access to custom oligonucleotides of any desired sequence. While enzymes synthesize DNA, RNA, and mRNA only in the 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, but is most often performed in the opposite direction, i.e., 3' to 5'. In certain implementations, this process is implemented as solid-phase synthesis using the phosphoramidite method and phosphoramidite building blocks derived from protected nucleosides (A, C, G, and U) or chemically modified nucleosides.

[0171] In some embodiments, modifications are included in the modified nucleic acid or in one or more individual nucleosides or nucleotides. For example, modifications to a nucleoside may include one or more modifications to the nucleobase, sugar, and / or internucleoside linkage. In some implementations, a polynucleotide having at least one modification includes a backbone portion containing the nucleobase, sugar, and internucleoside linkage of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, or 2-aminopurine-riboside-MP.

[0172] In other embodiments, polynucleotides having at least one modification comprise a backbone moiety containing nucleobases, sugars, and internucleoside linkages of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, or 5-bromo-cytidine-MP. Examples of such nucleoside and nucleotide modifications contemplated for use in this disclosure are provided in PCT International Publication No. WO 2022 / 232087, which is incorporated by reference in its entirety.

[0173] To obtain the desired oligonucleotide, building blocks are sequentially coupled to a growing oligonucleotide chain on a solid phase in the order required by the product sequence in a fully automated process. Once chain assembly is complete, the product is released from the solid phase into solution, deprotected, and recovered. Because the number of errors increases with the length of the synthesized oligonucleotide, the occurrence of side reactions sets a practical limit to the length of synthetic oligonucleotides (up to approximately 200 nucleotide residues). The product is often isolated by HPLC, yielding the desired oligonucleotide in high purity.

[0174] In certain embodiments, RNA is produced using in vitro transcription. The terms "RNA in vitro transcription" or "in vitro transcription" refer to a process in which RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as a template for the production of RNA and / or mRNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which in certain implementations is a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent mRNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, and SP6 RNA polymerases. DNA templates for in vitro RNA transcription can be obtained by cloning nucleic acids, particularly cDNAs corresponding to the respective RNAs to be in vitro transcribed, and introducing them into an appropriate vector for in vitro transcription, such as plasmid DNA. In one embodiment of the present disclosure, the DNA template is linearized with a suitable restriction enzyme before being transcribed in vitro. cDNA can be obtained by reverse transcription of mRNA or by chemical synthesis. Additionally, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis.

[0175] Methods for in vitro transcription are well known in the art, for example, in PCT International Publication Nos. WO 2022 / 170228 and WO 2022 / 232087, which are incorporated herein by reference in their entireties. The reagents used in this method typically include: 1) a linearized DNA template having a promoter sequence with high binding affinity for its respective RNA polymerase, such as a bacteriophage-encoded RNA polymerase; 2) ribonucleoside triphosphates (NTPs) for four bases (adenine, cytosine, guanine, and uracil); 3) optionally, a cap analog as defined above (e.g., m7G(5')ppp(5')G(m7G)); 4) a DNA-dependent RNA polymerase (e.g., T7, T3, or SP6) capable of binding to the promoter sequence in the linearized DNA template. RNA polymerase), 5) optionally an RNase inhibitor to inactivate any contaminating ribonucleases (RNases), 6) optionally a pyrophosphatase to degrade pyrophosphates that may inhibit transcription, 7) MgCl2 to provide Mg2+ ions as a cofactor for the polymerase, and 8) a buffer to maintain an appropriate pH value, which may also contain optimal concentrations of antioxidants (e.g., DTT) and / or polyamines such as spermidine.

[0176] In some embodiments, all or part of the processing steps (including, but not limited to, template generation, mRNA synthesis, and formulation) can be performed in a continuous fluid processing pathway, which can be configured as one or a series of consumable microfluidic pathway device(s)—sometimes referred to as a process chip or biochip (although chips do not necessarily have to be used in bio-related applications). The entire manufacturing process can proceed as a closed-loop process that is intentionally sterile and without contact with the atmosphere. All manufacturing operations can be automated and controlled by a control system to achieve a copy-exact process regardless of the attributes of the facility housing the system. Production parameters, raw materials, and environmental data (including complete visual records) can be secured in the cloud and become part of a large-scale encrypted electronic file associated with each production run. Furthermore, purification operations, as well as many QC assays, can be performed inline during the manufacturing process in a single fluid flow, allowing anomalies to be detected at an early stage through process control concepts developed in the semiconductor industry.

[0177] In one particular embodiment, the mRNA-based therapeutic composition comprises a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding interleukin-12, and a third isolated mRNA encoding HPV16 E6 E7. In another particular embodiment, the mRNA-based therapeutic composition is a multimodal mRNA therapeutic.

[0178] In some embodiments, mRNA-based therapeutic compositions are formulated with and / or mediated by a delivery vehicle. A "delivery vehicle" refers to any substance that at least partially facilitates in vivo, in vitro, or ex vivo delivery of a polynucleotide to a target cell or tissue (e.g., tumor, etc.). The reference to something as a delivery vehicle does not imply that the delivery vehicle may not also have a therapeutic effect. Delivery vehicles include, but are not limited to, viral vectors and particles (e.g., lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, retrovirus, etc.). Other modalities (e.g., mRNA, plasmids, and recombinant proteins) may also be used.

[0179] Disclosed herein are delivery vehicle compositions containing hydroxyethyl-capped cationic peptoids, including, for example, hydroxyethyl-capped tertiary amino lipidated cationic peptoids. The delivery vehicle compositions of the present disclosure can form electrostatic interactions between the hydroxyethyl-capped tertiary amino lipidated cationic peptoids of the delivery vehicle composition and polyanionic compounds, such as nucleic acids, to form delivery vehicle complexes, in which the polyanionic compounds function as the cargo of the complexes. The delivery vehicle complexes are useful for delivering polyanionic compounds, such as nucleic acids (e.g., mRNA), to cells. The delivery vehicle complexes of the present disclosure, which contain mRNA as the polyanionic cargo, unexpectedly exhibit excellent mRNA expression both in vitro and in vivo. When the mRNA of the delivery vehicle complex encodes, for example, a viral antigen, the delivery vehicle complexes can elicit humoral and cellular immune responses in vivo, thereby functioning as vaccines. The delivery vehicle complexes disclosed herein are further advantageous in that they are stable, well tolerated, and exhibit low toxicity.

[0180] As used herein, "peptoid" refers to a peptidomimetic compound in which one or more nitrogen atoms of the peptide backbone are replaced by a side chain. As used herein, "lipidated peptoid" refers to a peptoid in which one or more of the side chains on the nitrogen atoms contain a lipid. As used herein, "polyanionic" refers to a compound (e.g., a nucleic acid) having at least two negative charges.

[0181] Some exemplary delivery vehicle compositions of the present disclosure comprise one or more hydroxyethyl-capped tertiary amino lipidated cationic peptoids. These positively charged peptoids can be associated with polyanionic compounds (e.g., nucleic acids) to form delivery vehicle complexes. In some embodiments, the delivery vehicle composition further comprises one or more anionic or zwitterionic components (e.g., phospholipids), neutral lipids (e.g., sterols), and shielding lipids (e.g., PEGylated lipids). In various embodiments, the delivery vehicle composition further comprises anionic or zwitterionic components (e.g., phospholipids), neutral lipids (e.g., sterols), and shielding lipids (e.g., PEGylated lipids). In some embodiments, the delivery vehicle composition consists essentially of the hydroxyethyl-capped tertiary amino lipidated cationic peptoids, anionic or zwitterionic components (e.g., phospholipids), neutral lipids (e.g., sterols), and shielding lipids (e.g., PEGylated lipids).

[0182] Hydroxyethyl-capped tertiary aminolipidated cationic peptoid components The delivery vehicle compositions of the present disclosure comprise a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (sometimes referred to as a "cationic component" or "ionizable lipid"). In some embodiments, the hydroxyethyl-capped tertiary amino lipidated cationic peptoid is a compound of formula (I):

[0183] [ka] (wherein n is 1, 2, 3, 4, 5, or 6; R 1 is H, C 1~3 Alkyl or C 2~3 hydroxyalkyl, and each R 2 independently, C 8~24 Alkyl or C 8~24 As used herein, "alkyl" refers to straight- and branched-chain saturated hydrocarbon groups containing 1 to 30 carbon atoms, e.g., 1 to 4 (e.g., 1, 2, 3, or 4) carbon atoms. n The term "alkyl" means that the alkyl group has "n" carbon atoms. For example, C3 alkyl refers to an alkyl group having 3 carbon atoms. C 1~4 Alkyl refers to alkyl groups having a number of carbon atoms inclusive of the entire range (i.e., 1 to 4 carbon atoms) and all subgroups (e.g., 1 to 2, 1 to 3, 2 to 3, 2 to 4, 1, 2, 3, and 4 carbon atoms). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl (2-methylpropyl), and t-butyl (1,1-dimethylethyl). Unless otherwise indicated, an alkyl group can be an unsubstituted alkyl group or a substituted alkyl group. As used herein, "hydroxyalkyl" refers to an alkyl group, as defined herein, that is substituted with a hydroxyl group. For example, a "C2 hydroxyalkyl" or "hydroxyethyl" refers to a group having the structure:

[0184] [ka] As used herein, "alkenyl" refers to straight and branched chain hydrocarbon groups having a double bond and containing 2 to 30 carbon atoms, e.g., 2 to 4 carbon atoms (e.g., 2, 3, or 4). nThe term "alkenyl" means that the alkenyl group has "n" carbon atoms. For example, C3 alkenyl refers to an alkenyl group having 3 carbon atoms. C2-C4 alkenyl refers to alkenyl groups having numbers of carbon atoms throughout the entire range (i.e., 2-4 carbon atoms) and all subgroups (e.g., 2-3, 2-4, 2, 3, and 4 carbon atoms). Non-limiting examples of alkenyl groups include ethenyl, propenyl, and butenyl. Unless otherwise specified, alkenyl groups can be unsubstituted or substituted.

[0185] In some embodiments, n is 2 to 5. In various embodiments, n is 3 to 4. In some embodiments, n is 1. In various embodiments, n is 2. In some embodiments, n is 3. In various embodiments, n is 4. In some embodiments, n is 5. In various embodiments, n is 6.

[0186] In some embodiments, R 1 is H. In various embodiments, R 1 is C 1~3 In some embodiments, R 1 is methyl or ethyl. In some embodiments, R 1 is ethyl. In various embodiments, R 1 is C 2~3 In some embodiments, R 1 teeth,

[0187] [ka] In various embodiments, R 1 is ethyl or hydroxyethyl.

[0188] In some embodiments, each R 2 independently, C 8~18 Alkyl or C 8~18 alkenyl. In various embodiments, each R 2 independently, C 8~16Alkyl or C 10~18 In some embodiments, each R is an alkenyl. 2 independently, C 10~12 Alkyl or C 10~18 In some embodiments, each R is an alkenyl. 2 independently, C 8~18 Alkyl, C 8~16 Alkyl, C 8~14 Alkyl or C 8~12 In various embodiments, each R 2 is, independently,

[0189] [ka] In some embodiments, each R 2 is, independently,

[0190] [ka] In various embodiments, each R 2 is, independently,

[0191] [ka] In some embodiments, each R 2 is, independently,

[0192] [ka] is.

[0193] In some embodiments, n is 4 and R 1 is H, and each R 2 teeth,

[0194] [ka] is.

[0195] Contemplated compounds of Formula (I) include, but are not limited to, those listed in Table 7.

[0196] [Table 7-1]

[0197] [Table 7-2]

[0198] In some embodiments, the compound of Formula (I) is Compound 140.

[0199] The compounds of the present disclosure are defined herein by their chemical structure and / or chemical name. If a compound is referred to by both its chemical structure and chemical name, and the chemical structure and chemical name conflict, the chemical structure shall be determinative of the identity of the compound.

[0200] Unless otherwise indicated, structures depicted herein are also intended to include all isomeric (e.g., enantiomers, diastereomers, cis-trans, conformational, and rotamer) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in the disclosure unless only one of the isomers is specifically indicated. Thus, single stereochemical isomers of the present compounds as well as mixtures of enantiomers, diastereoisomers, cis / trans isomers, conformational, and rotamer forms are within the scope of the disclosure. In some embodiments, the compounds disclosed herein are stereoisomers. "Stereoisomer" refers to a compound that differs in the chirality of one or more stereocenters. Stereoisomers include enantiomers and diastereomers. The compounds disclosed herein can exist as a single stereoisomer or as a mixture of stereoisomers. The stereochemistry of the compounds shown herein indicates relative, not absolute, stereochemistry unless otherwise stated. As used herein, a single stereoisomer, a single diastereomer, or a single enantiomer refers to a compound that is at least 50% or more of the indicated stereoisomer, diastereomer, or enantiomer, and in some embodiments, at least 90% or 95% of the indicated stereoisomer, diastereomer, or enantiomer.

[0201] The compounds described herein may exist in free form or, where appropriate, as pharmaceutically acceptable salts. As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue adverse effects (e.g., toxicity, irritation, allergic response, etc.) and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (e.g., acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or by using other methods used in the art, such as, for example, ion exchange.Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutamate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and 2-hydroxy-ethanesulfonate. Salts of compounds containing carboxylic acid or other acidic functional groups can be prepared by reacting them with a suitable base. Examples of such salts include alkali metal salts, alkaline earth metal salts, aluminum salts, ammonium salts, and N-methyl-N ... + (C 1~4Examples of suitable salts include, but are not limited to, salts of alkyl (alkyl) 4 salts and salts of organic bases, such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine. The present disclosure also contemplates the quaternization of any basic nitrogen-containing group of the compounds disclosed herein. Water- or oil-soluble or dispersible products may be obtained by such quaternization. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0202] In embodiments, the delivery vehicle composition comprises about 25 mol% to about 70 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I), such as Compound 140), based on the total moles of components in the delivery vehicle composition. The unit "mol%" or "molar percentage" refers to the number of moles of a particular component of the delivery vehicle composition divided by the total number of moles of all components in the delivery vehicle composition multiplied by 100%. Polyanionic cargo is not calculated as part of the total moles of the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 60 mol%, or about 35 mol% to about 55 mol%, or about 30 mol% to about 45 mol%, or about 35 mol% to about 40 mol%, or about 45 mol% to about 60 mol%, or about 50 mol% to about 55 mol%, or about 38 mol% to about 52 mol%, or about 38 mol%, or about 52 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I), such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises less than about 50 mol%, e.g., less than about 49 mol%, less than about 48 mol%, less than about 47 mol%, less than about 46 mol%, less than about 45 mol%, less than about 44 mol%, less than about 43 mol%, less than about 42 mol%, less than about 41 mol%, less than about 40 mol%, less than about 39 mol%, less than about 38 mol%, less than about 37 mol%, less than about 36 mol%, less than about 35 mol%, less than about 34 mol%, less than about 33 mol%, less than about 32 mol%, less than about 31 mol%, less than about 30 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic surfactant. and greater than about 20 mol%, e.g., greater than about 21 mol%, greater than about 22 mol%, greater than about 23 mol%, greater than about 24 mol%, greater than about 25 mol%, greater than about 26 mol%, greater than about 27 mol%, greater than about 28 mol%, greater than about 29 mol%, greater than about 30 mol%, greater than about 31 mol%, greater than about 33 mol%, greater than about 34 mol%, greater than about 35 mol%, greater than about 36 mol%, greater than about 38 mol%, greater than about 39 mol%, greater than about 40 mol%, greater than about 41 mol%, greater than about 42 mol%, greater than about 43 mol%, or greater than about 44 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid.In some embodiments, the delivery vehicle composition comprises less than about 50 mol%, e.g., less than about 49 mol%, less than about 48 mol%, less than about 47 mol%, less than about 46 mol%, less than about 45 mol%, less than about 44 mol%, less than about 43 mol%, less than about 42 mol%, less than about 41 mol%, less than about 40 mol%, less than about 39 mol%, less than about 38 mol%, less than about 37 mol%, less than about 36 mol%, less than about 35 mol%, less than about 34 mol%, less than about 33 mol%, less than about 32 mol%, less than about 31 mol%, less than about 30 mol% hydroxyethyl ester. and greater than about 20 mol%, greater than about 21 mol%, greater than about 22 mol%, greater than about 23 mol%, greater than about 24 mol%, greater than about 25 mol%, greater than about 26 mol%, greater than about 27 mol%, greater than about 28 mol%, greater than about 29 mol%, greater than about 30 mol%, greater than about 31 mol%, greater than about 33 mol%, greater than about 34 mol%, greater than about 35 mol%, greater than about 36 mol%, greater than about 38 mol%, greater than about 39 mol%, or greater than about 40 mol% of hydroxyethyl-capped tertiary amino lipidated cationic peptoid. In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 49.5 mol%, or about 30 mol% to about 45 mol%, or about 30 mol% to about 35 mol%, or about 40 mol% to about 45 mol%, or about 35 mol% to about 49 mol%, or about 36 mol% to about 48 mol%, or about 38 mol% to about 45 mol%, or about 38 mol% to about 42 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I), such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises, based on the total number of moles of components in the delivery vehicle composition, about 30 mol% to about 49.5 mol%, or about 35 mol% to about 49 mol%, or about 36 mol% to about 48 mol%, or about 38 mol% to about 45 mol%, or about 38 mol% to about 42 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140). In some embodiments, the delivery vehicle composition comprises, based on the total number of moles of components in the delivery vehicle composition, about 30 mol% to about 35 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140).In some embodiments, the delivery vehicle composition comprises about 40 mol% to about 45 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 35 mol% to about 39 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In various embodiments, the delivery vehicle composition comprises about 39 mol% to about 52 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 42 mol% to about 49 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In various embodiments, the delivery vehicle composition comprises about 50 mol% to about 52 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I) such as Compound 140), based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, or about 45 mol% of a hydroxyethyl-capped tertiary amino lipidated cationic peptoid (e.g., a compound of Formula (I), such as Compound 140), based on the total number of moles of components in the delivery vehicle composition.

[0203] Anionic / Zwitterionic Ingredients In some embodiments, the delivery vehicle composition further comprises a component that is anionic or zwitterionic ("anionic component / zwitterionic component"). The anionic component / zwitterionic component can buffer the zeta potential of the particle or delivery vehicle complex formed from the delivery vehicle composition without affecting the cargo ratio and / or contributing to particle or delivery vehicle endosomal escape via protonation at low endosome pH. The zwitterionic component can further function to hold the particle tightly by interacting with both the hydroxyethyl-capped tertiary amino lipidated cationic peptoid and the polyanionic cargo compound. The anionic component can also enable the formation of a core-shell structure of the particle or delivery vehicle, where a net positive zeta potential particle is first created (e.g., by mixing the hydroxyethyl-capped tertiary amino lipidated cationic peptoid and the cargo at a positive + / - charge ratio) and then coated with the anionic component. These negatively charged multicomponent particles avoid reticuloendothelial system (RES) clearance better than their positively charged counterparts.

[0204] Examples of suitable anionic and zwitterionic components of the delivery vehicle composition are described in PCT International Publication Nos. WO 2020 / 069442 and WO 2020 / 069445, each of which is incorporated herein by reference in its entirety. In some embodiments, the zwitterionic component comprises one or more phospholipids. Phospholipids can provide additional stabilization to the complex in solution and can facilitate cellular endocytosis due to their amphiphilic properties and ability to disrupt cell membranes.

[0205] In some embodiments, the one or more phospholipids are selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (ChemsPC), 16LysoPC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, the phospholipid is DSPC, DOPE, or a combination thereof. In various embodiments, the phospholipid is DSPC. In various embodiments, the phospholipid is DOPE.

[0206] In embodiments, the delivery vehicle composition comprises about 1 mol% to about 40 mol% of a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 3 mol% to about 30 mol%, or about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol%, or about 10 mol% to about 15 mol%, or about 9 mol% to about 12 mol%, or about 7 mol% to about 11 mol%, or about 7 mol% to about 12 mol%, or about 10 mol% to about 14 mol%, or about 9 mol%, or about 12 mol% of a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 10 mol% to about 11 mol% of a phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 10.0 mol%, about 10.1 mol%, about 10.2 mol%, about 10.3 mol%, about 10.4 mol%, about 10.5 mol%, about 10.6 mol%, about 10.7 mol%, about 10.8 mol%, about 10.9 mol%, or about 11.0 mol% phospholipid (e.g., DSPC or DOPE) based on the total number of moles of components in the delivery vehicle composition.

[0207] Neutral lipid components In some embodiments, the delivery vehicle composition further comprises a component that is a neutral lipid (a "neutral lipid component"). The neutral lipid component can be designed to degrade or hydrolyze to facilitate in vivo clearance of the multi-component delivery system. Contemplated neutral lipid components include, for example, naturally occurring lipids and lipidated peptoids that comprise a lipid moiety at the N-position of the peptoid. Further examples of lipidated petoids are described in PCT International Publication Nos. WO 2020 / 069442 and WO 2020 / 069445, each of which is incorporated herein by reference in its entirety.

[0208] In some embodiments, the neutral lipid component of the delivery vehicle composition comprises one or more sterols. In some embodiments, the one or more sterols are selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the sterol comprises cholesterol. In embodiments, the delivery vehicle composition comprises about 10 mole % to about 80 mole % of a sterol (e.g., cholesterol) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 20 mol% to about 70 mol%, or about 25 mol% to about 60 mol%, or about 30 mol% to about 55 mol%, or about 35 mol% to about 50 mol%, or about 25 mol% to about 45 mol%, or about 40 mol% to about 60 mol%, or about 30 mol% to about 40 mol%, or about 45 mol% to about 55 mol%, or about 35 mol%, or about 50 mol% of a sterol (e.g., cholesterol) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 40 mol% to about 55 mol%, or about 40 mol% to about 45 mol%, or about 50 mol% to about 55 mol% of a sterol (e.g., cholesterol) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, or about 55 mol% sterol (e.g., cholesterol) based on the total number of moles of components in the delivery vehicle composition.

[0209] shielding component In some embodiments, the delivery vehicle composition further comprises a shielding component. The shielding component acts as a steric barrier, thereby increasing the stability of the particle or delivery vehicle in vivo and thereby improving its circulation half-life. Examples of suitable shielding components are described in PCT International Publication Nos. WO2020 / 069442 and WO2020 / 069445, each of which is incorporated herein by reference in its entirety.

[0210] In some embodiments, the shielding component comprises one or more PEGylated lipids. As used herein, "PEGylated lipid" includes any lipid or lipid-like compound covalently attached to a polyethylene glycol moiety. Lipid moieties suitable for PEGylated lipids can include, for example, branched or straight-chain aliphatic moieties, which can be unsubstituted or substituted, or moieties derived from natural lipid compounds, including fatty acids, sterols, and isoprenoids, which can be unsubstituted or substituted.

[0211] In some embodiments, the lipid moiety may comprise a branched or straight-chain aliphatic moiety having about 6 to about 50 carbon atoms or about 10 to about 50 carbon atoms. The aliphatic moiety may, in some embodiments, comprise one or more heteroatoms and / or one or more double or triple bonds (i.e., saturated or mono- or polyunsaturated). In some embodiments, the lipid moiety may comprise an aliphatic straight- or branched-chain moiety, wherein each hydrophobic tail independently has about 8 to about 30 carbon atoms or about 6 to about 30 carbon atoms, and the aliphatic moiety may be unsubstituted or substituted. In various embodiments, the lipid moiety may comprise aliphatic carbon chains derived from, for example, fatty acids and fatty alcohols. In some embodiments, each lipid moiety independently has a carbon number between C8 and C9. 24 Alkyl or C8-C 24 alkenyl, where C-C 24 Alkenyls can be monounsaturated or polyunsaturated in some aspects.

[0212] Natural lipid moieties used in implementations of the present disclosure can be derived from, for example, phospholipids, glycerides (e.g., diglycerides or triglycerides), glycosylglycerides, sphingolipids, ceramides, as well as saturated and unsaturated sterols, isoprenoids, and other similar natural lipids.

[0213] Other suitable lipid moieties may include lipophilic aromatic groups such as optionally substituted aryl or arylalkyl moieties, including, for example, naphthalenyl or ethylbenzyl, or lipids containing ester functional groups, such as sterol esters and wax esters.

[0214] In some embodiments, the one or more PEGylated lipids are selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and any combination thereof. In some embodiments, the PEGylated lipid comprises a PEG-modified sterol. In various embodiments, the PEGylated lipid comprises a PEG-modified cholesterol. In some embodiments, the PEGylated lipid is a PEG-modified ceramide lipid. In some embodiments, the PEG-modified ceramine is selected from the group consisting of N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]} and N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)]}, and any combination thereof.

[0215] In some embodiments, the PEGylated lipid is a PEG-modified phospholipid, which phospholipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (D SPC), 1,2-diundecanoyl-sn-glycero-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (ChemsPC), 16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In various embodiments, the phospholipid is DOPE.

[0216] In some embodiments, the one or more PEGylated lipids comprise a PEG-modified phosphatidylethanol, hi some embodiments, the PEGylated lipid is a PEG-modified phosphatidylethanol selected from the group consisting of PEG-modified DMPE (DMPE-PEG), PEG-modified DSPE (DSPE-PEG), PEG-modified DPPE (DPPE-PEG), and PEG-modified DOPE (DOPE-PEG).

[0217] In various embodiments, the PEGylated lipid is selected from the group consisting of dimyristoylglycerol-polyethylene glycol (DMG-PEG), distearoylglycerol-polyethylene glycol (DSG-PEG), dipalmitoylglycerol-polyethylene glycol (DPG-PEG), and dioleoylglycerol-polyethylene glycol (DOG-PEG). In some embodiments, the PEG lipid is DMG-PEG.

[0218] The molecular weight of the PEG chain in the PEGylated lipid can be adjusted as desired to optimize the properties of the delivery vehicle composition. In some embodiments, the PEG chain has a molecular weight of 350-6,000 g / mol, 1,000-5,000 g / mol, 2,000-5,000 g / mol, about 1,000-3,000 g / mol, or about 1,500-4,000 g / mol. In some embodiments, the PEG chain of the PEG lipid has a molecular weight of about 350 g / mol, 500 g / mol, 600 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, 3,000 g / mol, 5,000 g / mol, or 10,000 g / mol. In some embodiments, the PEG chain of the PEGylated lipid has a molecular weight of about 500 g / mol, 750 g / mol, 1,000 g / mol, 2,000 g / mol, or 5,000 g / mol. The PEG chain can be branched or linear. In some embodiments, the PEGylated lipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000).

[0219] In embodiments, the delivery vehicle composition comprises about 1 mol% to about 5 mol% of PEGylated lipid (e.g., DMG-PEG 2000) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 1 mol% to about 3 mol%, or about 1 mol% to about 2 mol%, or about 2 mol% to about 5 mol%, or about 0.5 mol% to about 1.5 mol%, or about 1.5 mol% to about 2.5 mol%, or about 1.5 mol% to about 2.0 mol%, or about 2.0 mol% to about 2.5 mol%, or about 1 mol%, or about 1.5 mol%, or about 2 mol%, or about 2.5 mol%, or about 3 mol%, or about 3.5 mol%, or about 4 mol%, or about 5 mol% of PEGylated lipid (e.g., DMG-PEG 2000) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 1 mol% to about 3 mol%, or about 1 mol% to about 2 mol%, or about 2 mol% to about 5 mol%, or about 0.5 mol% to about 1.5 mol%, or about 1.5 mol% to about 2.5 mol%, or about 1 mol%, or about 1.5 mol%, or about 2 mol%, or about 2.5 mol%, or about 3 mol%, or about 3.5 mol%, or about 4 mol%, or about 5 mol% of a PEGylated lipid (e.g., DMG-PEG 2000) based on the total number of moles of components in the delivery vehicle composition. In some embodiments, the delivery vehicle composition comprises about 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, or about 2.5 mol% PEGylated lipid (e.g., DMG-PEG 2000) based on the total number of moles of components in the delivery vehicle composition.

[0220] Representative examples Non-limiting delivery vehicle combinations are listed below: As noted above, the unit "mol %" or "molar percentage" refers to the number of moles of a particular component of a delivery vehicle composition divided by the total number of moles of all components in the delivery vehicle composition multiplied by 100%.

[0221] In some embodiments, the delivery vehicle composition comprises at least 99 mol% cationic component and less than about 1 mol% shielding component (e.g., Formula F1A in Table 2). In some embodiments, the delivery vehicle composition comprises less than about 20 mol% cationic component, less than about 5 mol% shielding component, and more than about 75 mol% mixture of anionic / zwitterionic component and neutral lipid component (e.g., Formulas F2A and F4A in Table 2). In some embodiments, the delivery vehicle composition comprises about 30 to about 45 mol% cationic component, about 50 to about 70 mol% mixture of anionic / zwitterionic component and neutral lipid component, and about 1.5 to about 4.5 mol% shielding component (e.g., Formulas F3A and F5A in Table 2). In various embodiments, the delivery vehicle composition comprises about 15 to about 35 mol % of a cationic component, about 60 to about 80 mol % of a mixture of anionic / zwitterionic and neutral lipid components, and about 1.5 to about 3.0 mol % of a shielding component (e.g., Formulas F2A and F3A in Table 2). In some embodiments, the delivery vehicle composition comprises about 15 to about 35 mol % of a cationic component, about 10 to about 20 mol % of anionic / zwitterionic components, about 50 to about 65 mol % of a neutral lipid component, and about 1.5 to about 3.0 mol % of a shielding component (e.g., Formulas F2A and F3A in Table 2). In various embodiments, the delivery vehicle composition comprises about 10 to about 20 mol% of a cationic component, about 75 to about 89 mol% of a lipid component, and about 1 to about 5 mol% of a shielding component (e.g., Formula F4A in Table 2). In some embodiments, the delivery vehicle composition comprises about 40 to about 50 mol% of a cationic component, about 50 to about 59 mol% of an anionic / zwitterionic component, and about 1 to about 5 mol% of a shielding component (e.g., Formula F5A in Table 2). In various embodiments, the delivery vehicle composition comprises about 30 to about 50 mol% of a cationic component, about 50 to about 70 mol% of a neutral lipid component, and about 1 to about 5 mol% of a shielding component (e.g., Formula F6A in Table 2). In various embodiments, the delivery vehicle composition comprises about 40 to about 45 mole % of a cationic component, about 50 to about 60 mole % of a mixture of an anionic / zwitterionic component and a neutral lipid component, and about 1.5 to about 2.0 mole % of a shielding component (e.g., Formulas F6.1 and F6.2 in Table 2).In some embodiments, the delivery vehicle composition comprises about 40 to about 45 mol% of a cationic component, about 10 to about 15 mol% of an anionic / zwitterionic component, about 40 to about 45 mol% of a neutral lipid component, and about 1.5 to about 2.0 mol% of a shielding component (e.g., Formulas F6.1 and F6.2 in Table 2). In various embodiments, the delivery vehicle composition comprises about 30 to about 35 mol% of a cationic component, about 60 to about 70 mol% of a mixture of anionic / zwitterionic and neutral lipid components, and about 2.0 to about 3.0 mol% of a shielding component (e.g., Formula F6.3 in Table 2). In some embodiments, the delivery vehicle composition comprises about 30 to about 35 mol % of a cationic component, about 10 to about 15 mol % of an anionic / zwitterionic component, about 50 to about 55 mol % of a neutral lipid component, and about 2.0 to about 3.0 mol % of a shielding component (e.g., Formula F6.3 in Table 2). The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 7, such as Compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid (e.g., sterol) described herein. In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component (e.g., PEGylated lipid) described herein. In some embodiments, the shielding component is DMG-PEG 2000.

[0222] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 60 mol% (e.g., about 35 mol% to about 39 mol%, or about 39 mol% to about 52 mol%, or about 42 mol% to about 49 mol%, or about 50 mol% to about 52 mol%) of a cationic component, about 3 mol% to about 20 mol% of an anionic / zwitterionic component, about 25 mol% to about 60 mol% of a neutral lipid compound, and about 1 mol% to about 5 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 35 to about 55 mol% of a cationic component, about 5 mol% to about 15 mol% of an anionic / zwitterionic component, about 30 mol% to about 55 mol% of a neutral lipid compound, and about 1 mol% to about 3 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 38 to about 52 mol% of a cationic component, about 9 to about 12 mol% of an anionic / zwitterionic component, about 35 mol% to about 50 mol% of a neutral lipid compound, and about 1 mol% to about 2 mol% of a shielding component. In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 49 mol% of a compound of Formula (I), about 5 mol% to about 15 mol% of a phospholipid, about 30 mol% to about 55 mol% of a sterol, and about 1 mol% to about 3 mol% of a PEGylated lipid. In some embodiments, the composition comprises about 35 mol% to about 49 mol% of a compound or salt of Formula (I), about 7 mol% to about 12 mol% of a phospholipid, about 35 mol% to about 50 mol% of a sterol, and about 1 mol% to about 2 mol% of a PEGylated lipid. The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., the compounds listed in Table 7, such as compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid).In some embodiments, the shielding component is DMG-PEG2000.

[0223] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 45 mol% of a cationic component, about 5 mol% to about 15 mol% of an anionic / zwitterionic component, about 40 mol% to about 60 mol% of a neutral lipid compound, and about 1 mol% to about 5 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 35 mol% to about 40 mol% of a cationic component, about 8 mol% to about 12 mol% of an anionic / zwitterionic component, about 45 mol% to about 50 mol% of a neutral lipid compound, and about 1 mol% to about 3 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 38.2 mol% of a cationic component, about 11.8 mol% of an anionic / zwitterionic component, about 48.2 mol% of a neutral lipid compound, and about 1.9 mol% of a shielding component ("Form F2"). The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., the compounds listed in Table 7, such as Compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some embodiments, the shielding component is DMG-PEG-2000. In some embodiments, the delivery vehicle composition comprises Formulation F2 shown in Table 8 below. In some embodiments, the delivery vehicle composition comprises about 38.2 mol% Compound 140, about 11.8 mol% DSPC, about 48.2 mol% cholesterol, and about 1.9 mol% DMG-PEG-2000 ("DV-140-F2").

[0224] In some embodiments, the delivery vehicle composition comprises about 45 to about 55 mol% cationic component, about 5 mol% to about 15 mol% anionic / zwitterionic component, about 35 mol% to about 55 mol% neutral lipid compound, and about 1 mol% to about 5 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 48 mol% to about 52 mol% cationic component, about 5 mol% to about 12 mol% anionic / zwitterionic component, about 38 mol% to about 42 mol% neutral lipid compound, and about 1 mol% to about 3 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 51.3 mol% cationic component, about 9.3 mol% anionic / zwitterionic component, about 38.0 mol% neutral lipid compound, and about 1.5 mol% shielding component ("F6 / 17 type"). The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., the compounds listed in Table 7, such as Compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some embodiments, the shielding component is DMG-PEG2000. In some embodiments, the delivery vehicle composition comprises Formulation F6 / 17, shown in Table 8 below. In some embodiments, the delivery vehicle composition comprises about 51.3 mol% Compound 140, about 9.3 mol% DSPC, about 38.0 mol% cholesterol, and about 1.5 mol% DMG-PEG2000 ("DV-140-F6 / 17").

[0225] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 49 mol% cationic component, about 5 mol% to about 15 mol% anionic / zwitterionic component, about 30 mol% to about 55 mol% neutral lipid compound, and about 1 mol% to about 3 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 48 mol% to about 52 mol% cationic component, about 5 mol% to about 12 mol% anionic / zwitterionic component, about 38 mol% to about 42 mol% neutral lipid compound, and about 1 mol% to about 3 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 42.6 mol% cationic component, about 10.0 mol% anionic / zwitterionic component, about 44.7 mol% neutral lipid compound, and about 1.7 mol% shielding component. The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 7, such as compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some embodiments, the shielding component is DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises Formulation F6 / 12 or Formulation F6 / 15 shown in Table 8 below. In some embodiments, the delivery vehicle composition comprises about 42.6 mol% Compound 140, about 10.9 mol% DSPC, about 44.7 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000 ("DV-140-F6 / 12"). In some embodiments, the delivery vehicle composition comprises about 48.1 mol% Compound 140, about 9.9 mol% DSPC, about 40.4 mol% cholesterol, and about 1.6 mol% DMG-PEG 2000 ("DV-140-F6 / 15").

[0226] In some embodiments, the delivery vehicle composition comprises about 40 mol% to about 49 mol% cationic component, about 5 mol% to about 15 mol% anionic / zwitterionic component, about 30 mol% to about 55 mol% neutral lipid compound, and about 1 mol% to about 3 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 42 mol% to about 46 mol% cationic component, about 7 mol% to about 12 mol% anionic / zwitterionic component, about 41 mol% to about 45 mol% neutral lipid compound, and about 1 mol% to about 2 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 44.4 mol% cationic component, about 10.6 mol% anionic / zwitterionic component, about 43.3 mol% neutral lipid compound, and about 1.7 mol% shielding component. In various embodiments, the delivery vehicle composition comprises about 44.4 mol% of a cationic component, about 10.6 mol% of an anionic / zwitterionic component, about 43.4 mol% of a neutral lipid compound, and about 1.7 mol% of a shielding component. The cationic component can be any cationic component described herein, such as any of the compounds of Formula (I) (e.g., compounds listed in Table 7, such as compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some embodiments, the shielding component is DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises F6.1 or F6.2 as shown in Table 8 below. In some embodiments, the delivery vehicle composition comprises about 44.4 mol% Compound 140, about 10.6 mol% DSPC, about 43.3 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000 ("DV-140-F6.1").In some embodiments, the delivery vehicle composition comprises about 44.4 mol% Compound 140, about 10.6 mol% DSPC, about 43.4 mol% cholesterol, and about 1.7 mol% DMG-PEG 2000 ("DV-140-F6.2").

[0227] In some embodiments, the delivery vehicle composition comprises about 30 mol% to about 39 mol% of a cationic component, about 5 mol% to about 15 mol% of an anionic / zwitterionic component, about 30 mol% to about 55 mol% of a neutral lipid compound, and about 1 mol% to about 3 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 30 mol% to about 35 mol% of a cationic component, about 7 mol% to about 12 mol% of an anionic / zwitterionic component, about 50 mol% to about 55 mol% of a neutral lipid compound, and about 2 mol% to about 3 mol% of a shielding component. In various embodiments, the delivery vehicle composition comprises about 33.1 mol% of a cationic component, about 10.5 mol% of an anionic / zwitterionic component, about 53.8 mol% of a neutral lipid compound, and about 2.5 mol% of a shielding component. The cationic component can be any cationic component described herein, such as, for example, any of the compounds of Formula (I) (e.g., compounds listed in Table 7, such as compounds 140, 146, 151, 152, 160, 161, and 162). In some embodiments, the cationic compound is Compound 140. The anionic / zwitterionic component can be any anionic / zwitterionic component described herein (e.g., a phospholipid). In some embodiments, the anionic / zwitterionic component is DSPC or DOPE. The neutral lipid component can be any neutral lipid described herein (e.g., a sterol). In some embodiments, the neutral lipid component is cholesterol. The shielding component can be any shielding component described herein (e.g., a PEGylated lipid). In some embodiments, the shielding component is DMG-PEG 2000. In some embodiments, the delivery vehicle composition comprises F6.3 as shown in Table 8 below. In some embodiments, the delivery vehicle composition comprises about 33.1 mol% Compound 140, about 10.5 mol% DSPC, about 53.8 mol% cholesterol, and about 2.5 mol% DMG-PEG 2000 ("DV-140-F6.1").

[0228] [Table 8]

[0229] In some embodiments, the delivery vehicle composition is F6.1, F6.2, or F6.3. In some embodiments, the delivery vehicle composition is F1A, F2A, F3A, F4A, F5A, F6A, F1, F2, F3, F4, F5, F6 / 12, F6 / 15, or F6 / 17.

[0230] The delivery vehicle compositions disclosed herein can form complexes with one or more polyanionic compounds (e.g., nucleic acids) through electrostatic interactions between the cationic components of the delivery vehicle composition and the polyanionic compounds. Thus, a delivery vehicle complex refers to a mixture comprising a delivery vehicle composition disclosed herein and a polyanionic compound. The complexes can, in some cases, enable high cargo loading, are stable, and exhibit excellent in vivo efficacy and tolerability. Thus, the delivery vehicle complexes are useful as delivery vehicles for transporting polyanionic cargoes encapsulated therein to target cells. Additionally or alternatively, the delivery vehicle complexes can contain non-anionic cargoes. Thus, another aspect of the present disclosure relates to a delivery vehicle complex comprising (1) the delivery vehicle composition described herein above and (2) a polyanionic compound (or cargo). In some aspects, the delivery vehicle composition forms a complex with one polyanionic compound (e.g., one RNA). In various embodiments, the delivery vehicle composition is complexed with two different polyanionic compounds (e.g., two different RNAs or RNA and DNA). In some embodiments, the delivery vehicle composition is complexed with three or more different polyanionic compounds (e.g., three, four, or five different RNAs).

[0231] The delivery vehicle complexes described herein may be characterized by the relative mass ratio of one of the components of the delivery vehicle composition to the cargo (e.g., polyanionic compound) in the complex. The mass ratio of components in a delivery vehicle complex can be easily calculated based on the known concentrations and volumes of stock solutions of each component used in preparing the complex. Furthermore, when a non-anionic cargo is present in the delivery vehicle complex, the mass ratio can more accurately represent the relative amount of the delivery vehicle component to the total cargo than the cation-to-anion ratio (which does not take non-anionic materials into account). Specifically, the mass ratio of components refers to the ratio of the mass of this particular component in the system to the mass of the "cargo" in the system. "Cargo" can refer to the total polyanionic compound(s) present in the system. In one example, the polyanionic compound(s) can refer to nucleic acid(s). In one example, the polyanionic compound refers to mRNA encoding at least one protein.

[0232] In some embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 0.5:1 to about 20:1, about 0.5:1 to about 10:1, about 0.5:1 to about 5:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 2:1 to about 20:1, about 2:1 to about 10:1, or about 2:1 to about 5:1. In some embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 2:1 to about 5:1. In still other embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 3:1. In other embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 19:1. In other embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 20:1. In other embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 13:1. In other embodiments, the cationic component and polyanionic compound of the delivery vehicle complex have a mass ratio of about 10:1. In some embodiments, the cationic component can be a compound of Formula (I), such as a compound listed in Table 7 (e.g., Compound 140).

[0233] In certain embodiments in which the delivery vehicle complex comprises a polyanionic compound or a nucleic acid as cargo, the mass ratio of cationic component to nucleic acid is about 0.5:1 to about 20:1, or about 0.5:1 to about 10:1, or about 0.5:1 to about 5:1, or about 1:1 to about 20:1, or about 1:1 to about 10:1, or about 1:1 to about 5:1, or about 2:1 to about 20:1, or about 2:1 to about 10:1, or about 2:1 to about 5:1. In certain embodiments, the mass ratio of cationic component to nucleic acid is about 2:1 to about 5:1. In still other embodiments, the mass ratio of cationic component to nucleic acid is about 3:1. In other embodiments, the mass ratio of cationic component to nucleic acid is about 19:1. In other embodiments, the mass ratio of cationic component to nucleic acid is about 20:1. In other embodiments, the mass ratio of cationic moiety to nucleic acid is about 13: 1. In other embodiments, the mass ratio of cationic moiety to nucleic acid is about 10: 1. In some embodiments, the cationic moiety can be a compound of Formula (I), e.g., a compound listed in Table 7 (e.g., Compound 140).

[0234] In some embodiments, the mass ratio of cationic component to nucleic acid is about 5:1 to about 25:1, or about 7:1 to about 20:1, or about 10:1 to about 17:1, or about 9.5:1 to about 10.5:1, or about 11:1 to about 17:1. In various embodiments, the mass ratio of cationic component to nucleic acid is about 20:1. In various embodiments, the mass ratio of cationic component to nucleic acid is about 19:1. In some embodiments, the mass ratio of cationic component to nucleic acid is about 17:1. In various embodiments, the mass ratio of cationic component to nucleic acid is about 15:1. In various embodiments, the mass ratio of cationic component to nucleic acid is about 13:1. In various embodiments, the mass ratio of cationic component to nucleic acid is about 12:1. In various embodiments, the mass ratio of cationic component to nucleic acid is about 10:1. In some embodiments, the cationic moiety can be a compound of formula (I) described hereinabove, such as the compounds listed in Table 7. In various embodiments, the cationic moiety is compound 140. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA.

[0235] In some embodiments, the mass ratio of the anionic component / zwitterionic component to the polyanionic compound is about 2:1 to about 10:1, or about 2:1 to about 3:1, or about 2:1 to about 4:1, or about 5:1 to about 10:1. In some embodiments, the mass ratio of the anionic component / zwitterionic component to the polyanionic compound is about 2:1 to about 10:1, or about 2:1 to about 3:1, or about 5:1 to about 10:1. In some embodiments, the mass ratio of the anionic component / zwitterionic component to the polyanionic compound is about 4:1. In some embodiments, the mass ratio of the anionic component / zwitterionic component to the polyanionic compound is about 2.7:1. In some embodiments, the anionic component / zwitterionic component can be a phospholipid, as described herein above. In various embodiments, the anionic / zwitterionic component is DOPE, DSPC, or a combination thereof. In some embodiments, the anionic / zwitterionic component is DSPC. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA.

[0236] In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 5:1 to about 8:1, or about 4:1 to about 7:1, or about 5:1 to about 6:1, or about 1:1 to about 5:1. In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 4:1 to about 7:1, or about 5:1 to about 6:1, or about 1:1 to about 5:1. In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 5.4:1. In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 8.1:1. In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 6.7:1. In some embodiments, the neutral lipid component can be a sterol as described herein above. In various embodiments, the neutral lipid component is cholesterol. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA.

[0237] In some embodiments, the weight ratio of the shielding component to the polyanionic compound is about 0.5:1 to about 2.5:1, or about 1:1 to about 2:1, or about 2:1 to about 3:1. In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 2.1:1. In some embodiments, the weight ratio of the neutral lipid component to the polyanionic compound is about 1.4:1. In some embodiments, the shielding component can be a PEGylated lipid, as described previously herein. In various embodiments, the shielding component is DMG-PEG 2000. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA.

[0238] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 10:1, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 1.4:1 ("Formulation F2"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic compound is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises compound 140 in a mass ratio of about 10:1 to nucleic acid, DSPC in a mass ratio of about 2.7:1 to nucleic acid, cholesterol in a mass ratio of about 5.4:1 to nucleic acid, and DMG-PEG2000 in a mass ratio of about 1.4 to nucleic acid ("DV-140-F2").

[0239] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 17:1, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 1.4:1 ("Form F6 / 17"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 17:1 to nucleic acid, DSPC in a mass ratio of about 2.7:1 to nucleic acid, cholesterol in a mass ratio of about 5.4:1 to nucleic acid, and DMG-PEG 2000 in a mass ratio of about 1.4 to nucleic acid ("DV-140-F6 / 17").

[0240] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 12:1, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 1.4:1 ("Form F6 / 12"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 12:1 to nucleic acid, DSPC in a mass ratio of about 2.7:1 to nucleic acid, cholesterol in a mass ratio of about 5.4:1 to nucleic acid, and DMG-PEG 2000 in a mass ratio of about 1.4 to nucleic acid ("DV-140-F6 / 12").

[0241] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 15:1, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 1.4:1 ("Form F6 / 15"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 15:1 to nucleic acid, DSPC in a mass ratio of about 2.7:1 to nucleic acid, cholesterol in a mass ratio of about 5.4:1 to nucleic acid, and DMG-PEG 2000 in a mass ratio of about 1.4 to nucleic acid ("DV-140-F6 / 15").

[0242] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 13:1, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 5.4:1, and a shielding component and a polyanionic cargo in a mass ratio of about 1.4:1 ("F6.1"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 13:1 to nucleic acid, DSPC in a mass ratio of about 2.7:1 to nucleic acid, cholesterol in a mass ratio of about 5.4:1 to nucleic acid, and DMG-PEG 2000 in a mass ratio of about 1.4 to nucleic acid ("DV-140-F6.1").

[0243] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 19:1, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 4.0:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 8.1:1, and a shielding component and a polyanionic cargo in a mass ratio of about 2.1:1 ("F6.2"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 19:1 to nucleic acid, DSPC in a mass ratio of about 4.0:1 to nucleic acid, cholesterol in a mass ratio of about 8.1:1 to nucleic acid, and DMG-PEG 2000 in a mass ratio of about 2.1 to nucleic acid ("DV-140-F6.2").

[0244] In some embodiments, the delivery vehicle complex comprises a cationic component and a polyanionic cargo in a mass ratio of about 9.7, an anionic component / zwitterionic component and a polyanionic cargo in a mass ratio of about 2.7:1, a neutral lipid component and a polyanionic cargo in a mass ratio of about 6.7:1, and a shielding component and a polyanionic cargo in a mass ratio of about 2.1:1 ("F6.3"). In some embodiments, the cationic component is a compound of Formula (I), the anionic component / zwitterionic component is a phospholipid, the neutral lipid component is cholesterol, and the shielding component is a PEGylated lipid. In some embodiments, the polyanionic cargo is a nucleic acid, such as RNA. In various embodiments, the delivery vehicle complex comprises Compound 140 in a mass ratio of about 9.7:1 to nucleic acid, DSPC in a mass ratio of about 2.7:1 to nucleic acid, cholesterol in a mass ratio of about 6.7:1 to nucleic acid, and DMG-PEG 2000 in a mass ratio of about 2.1 to nucleic acid ("DV-140-F6.3").

[0245] In yet other embodiments, the amount of polyanionic cargo present in a delivery vehicle complex may be characterized by the mass ratio of the delivery vehicle composition (e.g., the total combined hydroxyethyl-capped lipidated cationic peptoid, phospholipid, cholesterol, and / or shielding component) to one or more polyanionic cargo compounds. In some embodiments, the mass ratio of the delivery vehicle composition to one or more polyanionic cargo compounds is about 0.5:1 to about 20:1, about 0.5:1 to about 10:1, about 0.5:1 to about 5:1, about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 2:1 to about 20:1, about 2:1 to about 10:1, or about 2:1 to about 5:1. In certain embodiments, the mass ratio of the delivery vehicle composition to one or more polyanionic cargo compounds is about 5:1 to about 8:1 or about 6:1 to about 7:1.

[0246] In one non-limiting example, the mRNA-based therapeutic composition may comprise a first isolated mRNA, a second isolated mRNA, and / or a third isolated mRNA at least partially encapsulated by a delivery vehicle molecule having a formulation that may be, but is not limited to, a poly(lactic-co-glycolic acid) (PLGA) microsphere, a lipidoid, a lipoplex, a liposome, a polymer, a carbohydrate (including a monosaccharide), a cationic lipid, and combinations thereof.

[0247] In one embodiment, the delivery vehicle molecular formulation may include at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and PEGylated lipids. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.

[0248] In one embodiment, the delivery vehicle molecule may have the shape of a nanoparticle. The delivery vehicle may be, for example, an aminolipidated peptide, which may include a tertiary aminolipidated cationic peptide, such as those described in PCT International Publication No. WO / US 19 / 53661, entitled "LIPID NANOPARTICLE FORMULATIONS COMPRISING LIPIDATED CATIONIC PEPTIDE COMPOUNDS FOR NUCLEIC ACID DELIVERY," filed September 27, 2019, and PCT International Publication No. WO / US 19 / 53655, entitled "TERTIAL AMINO LIPIDATED CATIONIC PEPTIDES FOR NUCLEIC ACID DELIVERY," filed September 27, 2019, the contents of each of which are incorporated herein by reference in their entirety. The nanoparticle delivery vehicle may also include additional lipids / components. For example, the aminolipidated peptide may include one or more phospholipids, such as MSPC or DSPC. The lipid composition may also include a quaternary amine compound, such as DOTAP. In some embodiments, the delivery vehicle has a particle size of about 200 nm or less.

[0249] The mRNA-based therapeutic may be formulated using, for example, any of the delivery vehicles taught in U.S. Patent Application Publication No. 2018 / 0028688, the entire contents of which are incorporated herein by reference.

[0250] The components of the delivery vehicle complex can be prepared by a variety of physical and / or chemical methods to adjust their physical, chemical, and biological properties. These can include rapidly combining a hydroxyethyl-capped, tertiary amino-lipidated cationic peptoid in water or a water-miscible organic solvent with a desired polyanionic cargo compound (e.g., an oligonucleotide or nucleic acid) in water or an aqueous buffer. These methods can include simple mixing of the components by pipetting or microfluidic mixing processes, such as those involving a T-mixer, vortex mixer, or other chaotic mixing structure. In some embodiments, the multi-component delivery system is prepared on a microfluidic platform.

[0251] It should be understood that the specific process conditions for preparing the delivery vehicle complexes described herein can be adjusted or selected as appropriate to provide the desired physical properties of the complex. For example, parameters for mixing the components of the delivery system complex that can affect the final composition can include, but are not limited to, the order of mixing, the temperature of mixing, the speed / rate of mixing, the flow rate, the physical dimensions of the mixing structure, the concentration of the starting solution, the molar ratio of the components, and the solvent used.

[0252] Formulation of the delivery vehicle complex can be accomplished in a number of ways: in some aspects, all components can be premixed prior to addition of the nucleic acid cargo, which can result in a uniform distribution of the components throughout the delivery particle.

[0253] Exemplary mixing methods are detailed, for example, in US Pat. Nos. 11,278,895 and 11,325,122, which are incorporated herein by reference.

[0254] In other embodiments, components can be added sequentially to generate a core-shell structure. For example, a cationic component can be added first to initiate particle condensation, followed by a lipid component to associate the particle surface with target cells, followed by a shielding component to prevent particle aggregation. For example, a hydroxyethyl-capped tertiary amino lipidated cationic peptoid can be premixed with a nucleic acid cargo to form the core structure. Next, a lipid component (e.g., a lipid component comprising phospholipids and cholesterol) can be added to affect cell / endosomal membrane binding. Because the shielding component is primarily useful on the outside of a multi-component delivery system, this component can be introduced last, so that it does not disrupt the internal structure of the system but rather provides a coating for the system after it has formed.

[0255] Additional components in the complexes and compositions (e.g., additional components of polymers, surfactants, targeting moieties, and / or excipients) may be mixed and combined with the remaining components before, during, or after the main component of the nucleic acid cargo, the cationic component, the lipid component, and the shielding component are combined.

[0256] Accordingly, also provided herein is a method of forming a delivery vehicle complex disclosed herein, comprising contacting a compound or salt of Formula (I) with a polyanionic compound. In some embodiments, the method comprises mixing a solution comprising a compound or salt of Formula (I) with a solution comprising a polyanionic compound.

[0257] The delivery vehicle complexes disclosed herein can be used to deliver complex (or cargo) polyanionic compounds to cells. Accordingly, disclosed herein are methods for delivering polyanionic compounds, such as nucleic acids (e.g., RNA), to cells, comprising contacting the cells with a delivery vehicle complex or pharmaceutical composition disclosed herein. In some embodiments, the cells are obtained from a subject. In some embodiments, the cells are tumor cells. In some embodiments, the cells are muscle cells.

[0258] In some embodiments, one or more polyanionic cargo compounds may be delivered for therapeutic uses, including, but not limited to, as a vaccine, CIN, and cancer.

[0259] The delivery vehicle complexes of the present disclosure are useful as vaccines, in which the polyanionic compound is RNA encoding a polypeptide described herein. The host immune system provides a means for rapidly and specifically mounting a defensive response against pathogenic microorganisms and for contributing to the rejection of malignant tumors. The immune response has generally been described as comprising a humoral response, in which antigen-specific antibodies are produced by differentiated B lymphocytes, and a cell-mediated response, in which various types of T lymphocytes eliminate antigens by various mechanisms. For example, CD4 (also called CD4+) helper T cells, which can recognize specific antigens, may respond by releasing soluble mediators, such as cytokines, to recruit additional cells of the immune system to participate in the immune response. CD8 (also called CD8+) cytotoxic T cells are also capable of recognizing specific antigens and may bind to and destroy or damage antigen-bearing cells or particles. In particular, cell-mediated immune responses, including cytotoxic T lymphocyte (CTL) responses, can be important for the elimination of tumor cells and cells infected with microorganisms (e.g., viruses, bacteria, or parasites). Delivery vehicle complexes of the present disclosure have been shown to induce immune responses when one or more of the polyanionic compounds of the complex encodes a viral peptide (e.g., a viral polypeptide), a viral protein, or a functional fragment of the foregoing. For example, delivery vehicle complexes containing either DV-140-F2 or DV-140-F6 / 17 complexed with mRNA encoding the HPV E6 / E7 oncogene (e.g., from HPV 16 and / or HPV 18) elicited strong humoral and cellular immune responses. See, e.g., Example 7 and Figures 18A and 18B.

[0260] Accordingly, the present disclosure includes a method for inducing an immune response in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle complex of the present disclosure (e.g., formulated as an antigenic composition). Also disclosed herein are methods for treating CIN, squamous cell carcinoma, and adenocarcinoma, such as cervical, vulvar, vaginal, penile, anal, oropharyngeal, and head and neck cancers in a subject in need thereof, comprising administering to the subject an effective amount of a delivery vehicle complex of the present disclosure. In some aspects, administration is by intramuscular, intratumoral, intravenous, intraperitoneal, or subcutaneous delivery.

[0261] In various aspects, administering a delivery vehicle complex of the present disclosure (e.g., formulated as a composition, pharmaceutical preparation, or antigenic composition) to a subject can result in an increase in the amount of antibodies (e.g., neutralizing antibodies) produced against a viral antigen in the subject compared to the amount of antibodies (e.g., neutralizing antibodies) produced in a subject that was not administered the delivery vehicle complex. In some aspects, the increase is a 2-fold increase, a 5-fold increase, a 10-fold increase, a 50-fold increase, a 100-fold increase, a 200-fold increase, a 500-fold increase, a 700-fold increase, or a 1000-fold increase.

[0262] The immune response elicited by the methods of the present disclosure generally includes antibody responses, preferably neutralizing antibody responses, T cell and B cell maturation and memory, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and T cell-mediated responses such as CD4+ and CD8+. The immune response generated by the delivery vehicle complexes comprising RNA encoding a viral antigen disclosed herein generates an immune response that recognizes and preferably ameliorate and / or neutralize viral infection, as described herein. Methods for assessing antibody responses following administration (immunization or vaccination) of an antigenic composition are well known in the art and / or described herein. In some embodiments, the immune response includes a T cell-mediated response (e.g., a peptide-specific response, such as a proliferative response or a cytokine response). In some embodiments, the immune response includes both a B cell response and a T cell response. The antigenic composition may be administered in several suitable ways, including intramuscular injection, intratumoral injection, subcutaneous injection, intradermal administration, and mucosal administration, e.g., oral or intranasal. Additional modes of administration include, but are not limited to, intravenous, intraperitoneal, intranasal, intravaginal, intrarectal, and oral administration. Combinations of different routes of administration in an immunized subject, e.g., simultaneous intramuscular and intranasal administration, are also contemplated by the present disclosure.

[0263] Various cancers (e.g., cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, and head and neck cancer), as well as CIN, may be treated with polyanionic cargo compounds delivered by the delivery vehicle complexes of the present disclosure. As shown in Example 7, DV-140-F2 complexed with mRNA encoding HPV E6 / E7 (from HPV 16 and / or HPV 18) elicited both strong cellular and humoral immune responses, demonstrating that the delivery vehicle complexes of the present disclosure have the ability to treat cancer. As used herein, the term "cancer" refers to any of a variety of malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to pathological conditions characterized by such malignant neoplastic growth. The cancer may be a tumor or hematological malignancy, examples of which include, but are not limited to, all types of lymphoma / leukemia, carcinomas and sarcomas, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, bone marrow, tailbone, testicles, thyroid, uterus, vagina, and vulva.

[0264] As non-limiting examples, cancers that may be treated may be anal cancer, cervical cancer, esophageal cancer, head and neck cancer, laryngeal cancer, lip cancer, metastatic squamous cell cervical cancer, oral cancer, nasal cancer, nasopharyngeal cancer, cervical cancer, oral cancer, oral cancer, oropharyngeal cancer, penile cancer, pharyngeal cancer, rectal cancer, squamous cell carcinoma, tongue cancer, tonsil cancer, vaginal cancer, and vulvar cancer.

[0265] In some aspects, the delivery vehicle complexes of the present disclosure are used to treat a cancer selected from the group consisting of cervical cancer, head and neck cancer, B cell lymphoma, T cell lymphoma, prostate cancer, and lung cancer. In some aspects, the delivery vehicle complexes may be used to treat cervical cancer.

[0266] In some embodiments, the mRNA-based composition is configured to be administered to a subject in need thereof. "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sport animals, pet animals (e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows), primates such as apes, monkeys, orangutans, and chimpanzees, canines such as dogs and wolves, felines such as cats, lions, and tigers, equines such as horses, donkeys, and zebras, food animals such as cows, pigs, and sheep, ungulates such as deer and giraffes, and rodents, e.g., mice, rats, hamsters, guinea pigs, etc. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. As used herein, "patient" refers to a subject who may seek or need treatment, who is in need of treatment, who is undergoing treatment, who is receiving treatment, or who is under the care of a professional trained in a particular disease or condition.

[0267] In some non-limiting embodiments, the composition is a therapeutic composition, such as an mRNA-based therapeutic composition. Therapeutic compositions may optionally include one or more therapeutically acceptable carriers, diluents, or excipients such as salts, buffers, preservatives, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film-forming or coating agents, flavors, fragrances, glidants, lubricants, adsorbents, suspending or dispersing agents, sweeteners, water for hydration, and / or other therapeutic agents. As used herein, the term "excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than (and typically in addition to) the active pharmaceutical ingredient (API), appropriately selected with respect to the intended administration form and consistent with conventional pharmaceutical practice. The disclosed compounds can be administered to a subject or patient in a therapeutically effective amount. The conjugate can be administered alone or as part of a pharmaceutically acceptable composition or formulation. Furthermore, the composition can be administered all at once, for example by bolus injection, in multiple doses, or can be delivered substantially uniformly over a period of time. It should also be noted that the dose of the compound can vary over time.

[0268] In some embodiments, the composition is a vaccine. A vaccine refers to a substance used to stimulate antibody production and provide immunity against one or several diseases, and may be prepared from the causative agent of the disease, its products, or synthetic substitutes. In a non-limiting example, the composition may be a human papillomavirus (HPV) mRNA vaccine. The vaccine may further comprise one or more immunological adjuvants. As used herein, the term "immunologic adjuvant" refers to a compound or mixture of compounds that, when used in conjugation with an immunogen (e.g., a neoantigen), acts to accelerate, prolong, enhance, or modify the immune response. An adjuvant may be non-immunogenic when administered alone to a host, but enhances the host's immune response to that antigen when administered in conjunction with another antigen. Specifically, the terms "adjuvant" and "immunological adjuvant" are used interchangeably in this disclosure. Adjuvant-mediated enhancement and / or prolongation of the duration of an immune response can be assessed by any method known in the art, including, but not limited to, one or more of the following: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant / antigen combination relative to the number of antibodies produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells that recognize the antigen or adjuvant; and (iii) an increase in the level of one or more cytokines. Adjuvants may be aluminum-based adjuvants, including, but not limited to, aluminum hydroxide and aluminum phosphate, saponins, such as steroidal saponins and triterpenoid saponins, bacterial flagellins, and some cytokines, such as GM-CSF. The choice of adjuvant may depend on the antigen, vaccine, and route of administration.

[0269] In some embodiments, adjuvants improve adaptive immune responses to vaccine antigens by modulating innate immunity or facilitating delivery and presentation. Adjuvants act directly or indirectly on antigen-presenting cells (APCs), including dendritic cells (DCs). Adjuvants may be ligands for Toll-like receptors (TLRs) and directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity. In other examples, adjuvants may signal through pro-inflammatory pathways to promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes inorganic salts, oil emulsions, nanoparticles, and polyelectrolytes, including colloids and molecular aggregates exhibiting complex heterogeneous structures. In one example, the composition further comprises pidotimod as an adjuvant. In another example, the composition further comprises CpG as an adjuvant.

[0270] In non-limiting embodiments, the subject may be known to have cervical cancer, HPV-associated cancer, or HPV-associated disease. Figures 7A-7C illustrate the formulation and subsequent administration of multiple isolated mRNAs using a delivery vehicle according to embodiments of the present disclosure. The mRNA-based therapeutic may be administered to a subject in need thereof by any route to achieve a therapeutically effective result, including, but not limited to, an injectable formulation. In some embodiments, the injectable formulation is configured for intratumoral administration. The exact amount required to achieve a therapeutically effective result will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc. The term "therapeutically effective amount" refers to an amount effective to treat and / or ameliorate a disease or condition in a subject.

[0271] Some non-limiting embodiments include an expression cassette. As used herein, an expression cassette includes a promoter sequence, an open reading frame, and a termination sequence. The expression cassette may be configured for direct administration or may be encoded by one or more polynucleotides for expression in a cell, and may be encoded by DNA, RNA, or mRNA for administration. In some examples, the cassette may include a first mRNA encoding membrane-stabilizing LIGHT, a second mRNA encoding interleukin-12, and / or a third mRNA encoding HPV16 E6 E7. In some examples, the cassette may include a first isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 18, a second isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 20, and a third isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO: 26. In some embodiments, the cassette containing the first isolated mRNA, the second isolated mRNA, or the third isolated mRNA is at least partially encapsulated in a delivery vehicle. In another example, a multimodal mRNA therapeutic may contain at least two or three of the disclosed cassettes.

[0272] The delivery vehicle complexes disclosed herein and the pharmaceutically active compounds described herein can be administered to a subject or patient by any suitable route, for example, by parenteral injection(s), for example, intramuscular (IM), intratumoral (IT), intracervical, pericervical, intracervical, intravulvar, intravaginal, intrapenile, intraanal, and oropharyngeal. All methods that can be used by those skilled in the art to administer a pharmaceutically active agent are contemplated.

[0273] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to well-known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, United States Pharmacopeia (USP) solution, and isotonic sodium chloride solution. Sterile, fixed oils are conventionally used as solvents or suspending media. Any bland, fixed oil, including synthetic mono- or diglycerides, may be used for this purpose. Fatty acids, such as oleic acid, may be used in the preparation of injectable preparations. Exemplary buffers include citrate, succinate, acetate, malate, succinate, and histidine. Sterile stabilizers, such as sucrose, may also be included. For example, the therapeutic composition may be suspended in a sucrose-containing citrate buffer at a pH of between pH 5 and pH 6, for example, about pH 5.5.

[0274] The injectable preparations may be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium before use. Adjuvants such as local anesthetics, preservatives, and buffering agents can also be added to the compositions.

[0275] To prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the active ingredient depends on its rate of dissolution, which may in turn depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. Microbial contamination can be prevented by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like.

[0276] The compounds and / or compositions of the present disclosure can be administered to a subject or patient at dosage levels ranging from about 0.1 to about 100 mg per day. When administered on a weight basis, a dosage of about 0.001 to about 1 mg per kg of body weight is believed to be sufficient for a normal adult human weighing approximately 70 kg. The particular dosage and dosage range used can potentially depend on several factors, including the requirements of the subject or patient, the severity of the condition or disease being treated, and the pharmacological activity of the compound being administered. Determination of dosage ranges and optimal dosages for a particular subject or patient is within the skill of one of ordinary skill in the art.

[0277] In some embodiments, the mRNA-based therapeutic may be part of a kit. The kit may include a pharmaceutically acceptable carrier and / or a package insert containing instructions for intratumoral administration (e.g., injection) of the mRNA-based therapeutic composition. The kit may further include instructions for treating or delaying the progression of cancer in a subject. In some embodiments, the package insert further includes instructions for administering the composition by intratumoral administration in combination with injection at another site (e.g., systemic injection) to treat or delay the progression of cancer in a subject. The kit may also include additional therapeutic nucleic acids, drugs, therapeutic agents, diagnostic agents, prophylactic agents, and / or any other agents that have a therapeutic, diagnostic, and / or prophylactic effect and / or induce a desired biological and / or pharmacological effect when administered to a subject.

[0278] In some aspects, mRNA-based therapeutics comprising isolated mRNA encoding membrane-stabilized LIGHT may result in durable tumor regression and long-term CD8+ T cell-mediated cancer immunity compared to wild-type (soluble) LIGHT.

[0279] In some aspects, embodiments of the present invention include RNA sequences encoding human papillomavirus 16 (hHPV16) E6 / E7 antibody, human interleukin 12 (hIL-12), and modified human LIGHT (ENG hLIGHT) described herein, which is based on wild-type hLIGHT, also known as tumor necrosis factor superfamily member 14 (TNFSF14).

[0280] Figures 3C, 3D, and 3E are schematic diagrams showing the arrangement of RNA elements that, in some embodiments, are part of the RNA sequence encoding each of the hHPV16 E6 / E7 antibodies, ENGhLIGHT, and hIL-12 (fusions), respectively. These elements include the 5' cap, 5' UTR, the coding sequence for the hHPV16 E6 / E7 antibodies, ENGhLIGHT, or hIL-12 fusions, the 3' UTR, and the polyA tail.

[0281] In some aspects, the 5' cap consists of a naturally occurring 5'N 7-methylguanosine linked to the first two transcribed nucleotides by a 5'→5' triphosphate bridge, a methylated A at the ribose O-2 position, and guanine (G) nucleotides at positions +1 and +2. Embodiments of template DNA and synthetic RNA with a T7 promoter sequence (underlined) and a 5' cap initiation sequence are shown in Figure 3F.

[0282] In one embodiment, designated "NTX-250," the hHPV16 antigen is encoded by RNA-795 (SEQ ID NO: 26), hIL-12 is encoded by RNA-683 (SEQ ID NO: 20), and ENGhLIGHT is encoded by RNA-1495 (SEQ ID NO: 18).

[0283] hHPV16 antigen mRNA (RNA-795) RNA-795 corresponds to a single mRNA sequence encoding a protein consisting of a vaccine scaffold, a pan-human leukocyte antigen (HLA) DR-binding epitope (PADRE), and a derivative of the E6 and E7 (E6 / E7) antigen sequences from HPV16. The nucleotide sequence, codon-optimized for increased expression, consists of 1,321 nucleotides and has a molecular weight of 429,037 g / mol. The construct follows the schematic diagram shown in Figure 3C.

[0284] The vaccine scaffold consists of the signal sequence and C-terminal membrane and intracellular domains of the cluster of differentiation 1 (CD1d) gene, a member of a family of glycoproteins expressed on the surface of various antigen-presenting cells. Linking of antigens to the CD1d trafficking signal sequence delivers the antigen to processing compartments, resulting in improved T cell responses. The PADRE epitope is a peptide sequence that has been shown to activate antigen-specific CD4+ T cells (Wieking, 2012).

[0285] The antigenic E6 / E7 sequences are connected by a flexible glycine-serine (G4S)3 linker to allow expression of full-length fusion proteins. Note that the following mutations were made to the native human papillomavirus (HPV) E6 / E7 protein sequence to attenuate the known oncogenic functions of E6 / E7:

[0286] Leucine (UUA) to glycine (GGC) in construct nucleotide sequence #301-303 encompasses the p53 binding and telomerase activation sites.

[0287] Four mutations were made in the C-terminal PDZ-binding domain of E6: glutamic acid (Glu, GAG), threonine (ACU), glutamine (CGA), and leucine (CUU) in construct nucleotide sequence #595-606, which were mutated to four alanines (GCU-GCU-GCA-GCU). The PDZ-binding domain is involved in transformation by inactivating the tumor suppressor protein PTPN13 and other proteins with PDZ domain interactions (Spanos, 2008).

[0288] Three single nucleotide mutations in HPV16 E7 at the retinoblastoma (Rb) protein binding site, namely:

[0289] Mutation of histidine (CAA) to proline (CCU) in construct nucleotide sequence #655-657.

[0290] Mutation of cysteine ​​(UGC) to glycine (GGC) in construct nucleotide sequence #721-723.

[0291] Mutation of glutamic acid (GAG) to alanine (GCU) in construct nucleotide sequence #787-789.

[0292] These mutations prevent E7 from binding to and inactivating Rb and other proteins, and from associating with Mi2β, which enhances cell proliferation ( Narisawa-Saito, 2007 ).

[0293] A single base mutation of leucine (CUG) to arginine (AGA) in construct nucleotide sequence #850-853 was generated to disrupt Mi2β binding.

[0294] hIL-12 mRNA (RNA-683) RNA-683 corresponds to a single mRNA sequence encoding the hIL-12 protein, in which the two subunits p40 and p35 are linked into a single-chain construct using a flexible (G4S)3 linker. The nucleotide sequence, codon-optimized for increased expression, consists of 1936 nucleotides and has a molecular weight of 628,446 g / mol. The construct follows the schematic diagram shown in Figure 3E.

[0295] ENG hLIGHT mRNA (RNA-1495) RNA-1495 corresponds to a single mRNA sequence encoding native secreted LIGHT engineered by replacing amino acids 66-92 with a novel 10-amino acid linker sequence to enhance membrane stability and expression. ENG hLIGHT is designed to be a surface-associated type II membrane protein. The nucleotide sequence, codon-optimized for increased expression, consists of 994 nucleotides and has a molecular weight of 323,476 g / mol. The construct follows the schematic shown in Figure 3D.

[0296] formulation In some aspects, the formulated drug product is presented as a white to off-white colloidal frozen suspension for injection. In one aspect, a vial of the formulated product may contain 1.0 mg of mRNA (1.0 mg / mL) containing hHPV16 antigen, hIL-12, and ENG hLIGHT mRNA in a 1:1:1 mass ratio. For example, in one embodiment, the final NTX-250 product may contain the aforementioned mRNA encapsulated in a proprietary lipid nanoparticle (LNP) formulation dispersed in a sucrose-containing citrate buffer at pH 5.5. In some aspects, the sucrose-containing citrate buffer contains 14.07 mM sodium citrate dihydrate, 5.93 mM citric acid, 300 mM sucrose, pH 5.5. In some embodiments, the four lipids utilized to generate the LNPs are the proprietary peptoid lipid N-(2-amino-2-oxoethyl)-N-decyl-2-(N-decyl-2-(N-decyl-2-((2-hydroxyethyl)amino)acetamido)acetamido)acetamido)acetamide (DVI-0140, described in detail above), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). In some embodiments, the formulation is the 140-F2 formulation. In some embodiments, the formulation is the 140-F6.1 formulation. In some embodiments, the formulation is the 140-F6.2 formulation. In some embodiments, the formulation is the 140-F6.3 formulation.

[0297] Preclinical studies of NTX-250 Nonclinical pharmacology studies conducted in support of the present invention included in vitro and in vivo pharmacology studies to characterize the biological activity and antitumor effects of NTX-250 or its murine surrogate, mNTX-250, and their respective mRNA components. A series of nonclinical pharmacology studies included NTX-250 containing either native wild-type (WT) LIGHT mRNA or ENG LIGHT mRNA. In vitro, treatment with NTX-250 or a combination of its mRNA components effectively induced IFN-γ secretion and T cell activation in HPV16-positive CIN and healthy donor PBMCs. Compared to WT LIGHT mRNA, ENG LIGHT mRNA resulted in higher surface expression and biological activity in transfected cells, leading to increased T cell proliferation. In vivo, mNTX-250 effectively eradicated C3.43 tumors and improved survival in a dose-dependent manner in a mouse syngeneic tumor model, demonstrating the synergistic antitumor effects of the three mRNA components of NTX-250, regardless of whether WT or ENG LIGHT mRNA was administered. mNTX-250 was also associated with HPV16-specific T cell generation, T cell infiltration into tumors, and pro-inflammatory cytokine induction. Rechallenge of HPV16 E7-expressing TC-1 tumors in mNTX-250-treated tumor-free mice pre-inoculated with C3.43 tumors demonstrated immune memory and sustained tumor growth inhibition (TGI). In a subcutaneous (SC) MC38 colon cancer model in mice, treatment with ENG LIGHT resulted in higher levels of cytokines in the tumor microenvironment (TME) compared with WT LIGHT. HPV16-specific T cell immune responses were observed in both rats and monkeys administered intramuscular (IM) doses of mNTX-250 or NTX-250, respectively. [Example]

[0298] The technology described herein and its advantages will be better understood by referring to the following examples. These examples are provided to illustrate specific implementations of the technology. Providing these specific examples is not intended to limit the scope and spirit of the technology.

[0299] Some of the experiments described below are performed using human sequence constructs, which are typically identified with an "h" before the construct name, e.g., hLIGHT for human LIGHT. Some of the experiments described below are performed using mouse surrogate constructs, which are typically identified with an "m" before the construct name, e.g., mLIGHT for mouse LIGHT.

[0300] Example 1: Comparative analysis of soluble and membrane-stabilized LIGHT. LIGHT variants were formulated in MessengerMax mRNA transfection reagent. Human embryonic kidney (HEK) cells were transfected with LIGHT variant constructs for cell surface expression of LIGHT. CellTrace Violet (Thermo Fischer Scientific) was used to identify CD3 IgG from peripheral blood mononuclear cells (PBMCs). + T cells were labeled. 100 ng / mL of soluble CD3 antibody was added to the resulting labeled CD3 + T cells were added to the soluble (wild-type) LIGHT solution. T cell proliferation and interferon-gamma (IFN-γ) secretion were assessed after approximately 5 days of coculture. As shown in Figures 8A and 8B, membrane-stabilized LIGHT induced greater CD8+ T cell proliferation than wild-type LIGHT. T cell proliferation induced by soluble (wild-type) LIGHT and membrane-stabilized (1356, 1359) LIGHT is shown in Figures 9A and 9B, demonstrating the increased T cell proliferation by membrane-stabilized LIGHT.

[0301] Example 1A: Evaluation of human T cell proliferation in response to ENG or WT hLIGHT mRNA with the presence of hIL-12 mRNA in human PBMCs. This study was designed to compare the ability of ENG and WT hLIGHT mRNA in combination with hIL-12 mRNA to stimulate primary human T cell proliferation. Human T cells were isolated from PBMCs from a single healthy donor and stained with CellTrace Violet. HEK293 cells were transfected with hIL-12 mRNA (RNA-683) and either WT hLIGHT (RNA-786) or ENG hLIGHT (RNA-1359) mRNA formulated in Lipofectamine MessengerMAX (0.5 ng of each mRNA per 1000 cells). Control cells were transfected in the same manner with a non-coding mRNA control. Transfected HEK293 cells were co-cultured with T cells at a 1:5 ratio in the presence of 30 ng / mL or 100 ng / mL anti-CD3 antibody to mimic antigen-specific TCR binding for 4 days at 37°C in a 5% CO2 environment. CD8+ T cell proliferation was assessed by flow cytometry, and a proliferation index was calculated. Coculture of T cells with HEK293 cells expressing IL-12 and ENG hLIGHT in the presence of anti-CD3 resulted in increased CD8+ T cell proliferation and survival compared with coculture of T cells with HEK293 cells expressing IL-12 and WT hLIGHT in the presence of anti-CD3. The results are shown in Figure 10A, where ENG = engineered, HEK = human embryonic kidney, IL-12 = interleukin-12, PBMC = peripheral blood mononuclear cells, and WT = wild-type. Data in Figure 10A represent the mean ± standard deviation (n = 4). Statistical analysis was performed by unpaired Student's t-test (P < 0.05).

[0302] There was a biologically significant increase in T cell proliferation with membrane-stabilized LIGHT compared to soluble LIGHT, which is further supported by the data shown in Figure 10B and Figure 11.

[0303] Soluble LIGHT was also shown to compete for HVEM binding and reduce signaling by membrane-stabilized LIGHT. Figure 12 shows increased function of membrane-stabilized LIGHT in cells using an HVEM reporter cell line, with details shown in Figures 13A-C, and Figures 14A-B showing increased cellular expression of membrane-stabilized LIGHT.

[0304] Example 2: Tumor Model The well-established, clinically relevant C3.43 tumor model (5) was used to test mRNA-based therapeutic compositions containing a first isolated mRNA encoding membrane-stabilizing LIGHT, a second isolated mRNA encoding HPV16 E6 E7, and a third isolated mRNA encoding interleukin-12 for preclinical efficacy. C3.43 is an advanced subclone of the C3, HPV16-transformed B6 mouse embryonic cell line, expressing HPV16 E6 and E7 antigens under their native promoters (5).

[0305] Pathogen-free 6- to 8-week-old C57BL / 6J female mice were inoculated with 1 × 10 HIV-1 strains in 100 mL of HBSS. 5 C3.43 tumor cells were inoculated subcutaneously into the right flank. Mice bearing tumors large enough for intratumoral (IT) injection were randomized to treatment groups on study day 0, 15 days after tumor inoculation. Intratumoral injections were performed at 7-day intervals for a total of three injections with 20 mL of the assigned treatment. Satellite groups of animals from each treatment arm were euthanized 3 days after dose 1. Satellite group tumors were harvested and processed for isolation of tumor-infiltrating lymphocytes (TILs). The remaining animals followed the dosing schedule and had tumor volumes measured every two weeks. Blood was drawn for PBMC collection on day 13. Survival was analyzed through day 84. See Figure 15 for a study schematic.

[0306] The therapeutic efficacy of mRNA-based therapeutics was evaluated by tracking tumor growth and survival over time across treatment groups. Tumor growth was measured biweekly with manual calipers to measure tumor volume. Satellite groups of animals from each treatment arm were euthanized by asphyxiation or cervical dislocation on day 3 after Dose 1 for mechanistic studies and immune response profiling. Right flank tumors were harvested, and half of the tumor was formalin-fixed and paraffin-embedded for histological analysis, while the other half was processed for cytokine analysis and isolation of tumor-infiltrating lymphocytes (TILs) for flow cytometry. Immunogenicity was assessed by flow cytometry of peripheral blood mononuclear cells (PBMCs). The frequency of HPV16 E7-specific CD8+ T cells was compared across all treatment groups. Mechanistic studies were performed by characterizing the tumor microenvironment after treatment. Data are shown in Figure 4A, Figure 4B, Figure 4C, and Figure 4D.

[0307] Example 2A: Repeated-dose, intratumoral injection, efficacy and pharmacodynamic studies in a C3.43 tumor mouse model using mNTX-250 containing mHPV16 and mIL-12, and either ENG or WT mLIGHT mRNA This study compared the in vivo effects of increasing doses of mNTX-250 with mHPV16 antibody, mIL-12, and WT mLIGHT or ENG mLIGHT on tumor growth inhibition (TGI) and induction of tumor antigen-specific T cells in a syngeneic C3.43 tumor model in female C57BL / 6 mice.

[0308] C3.43 tumors were implanted into female C57BL / 6 mice as described above. Tumor volumes were approximately 180 mm 3On day 18 post-inoculation (day 0), when tumor size reached 18 days, mice were randomly assigned to treatment groups (n=10 per group) according to the study design in Table 9 below. On days 0 and 7, mice were treated by IT injection with increasing doses of mNTX-250 containing WT mLIGHT or ENG mLIGHT formulated in 140-F6.3 in a 20 μL dose volume. Control mice were similarly administered citrate buffer. Tumor volume was measured twice weekly using calipers. Blood samples were collected on day 12 for quantification of HPV16 E7 tetramer-positive T cells by flow cytometry.

[0309] [Table 9] ENG = engineered, mIL-12 = murine surrogate for interleukin-12, mLIGHT = murine surrogate for LIGHT, N / A = not applicable, WT = wild type

[0310] Treatment with mNTX-250 containing WT or ENG mLIGHT resulted in comparable dose-dependent TGI (Figures 4E and 4F) and improved survival (Figure 4G) compared with control mice. Similarly, treatment with mNTX-250 containing WT or ENG mLIGHT resulted in comparable dose-dependent generation of HPV16 tetramer-positive T cells (Figure 5). Together, these results demonstrate comparable efficacy of WT and ENG mLIGHT in the C3.43 syngeneic tumor mouse model.

[0311] Example 2B: Comparison of cytokine induction in the tumor microenvironment using ENG mLIGHT mRNA or WT mLIGHT mRNA in combination with IL-12 mRNA in the MC38 transplantable mouse tumor model The purpose of this study was to evaluate the activity of ENG mLIGHT mRNA compared to WT mLIGHT mRNA in combination with mIL-12 mRNA within the TME of a subcutaneous syngeneic MC38 model. MC38 is a murine adenocarcinoma cell line. The MC38 model is an immunocompetent cancer model with a moderate growth rate (4-day doubling time), allowing for an extended treatment schedule and reflecting the complex TME of adenocarcinoma and squamous cell carcinoma. To establish the MC38 tumor model, 2 × 10 5 MC38 tumor cells were inoculated into the flank of female C57BL / 6 mice. Tumor volumes were approximately 100 mm 3 Upon reaching 100 mg / kg / day, mice received intratumoral (20 μL) injections on days 0 and 3 according to the study protocol in Table 10. Twelve hours after the second dose, mice were euthanized and tumors were harvested for cytokine profiling by Luminex assay.

[0312] [Table 10] ENG = engineered, IT = intratumoral, mIL-12 = mouse interleukin-12, mLIGHT = mouse LIGHT, WT = wild type. All mRNAs were formulated in 140-F6.3.

[0313] Quantification of cytokines induced within the TME revealed that mice treated with mIL-12 + ENG mLIGHT mRNA had significantly higher levels of TNF-α, IFN-γ, and IL-15 compared with mice treated with mIL-12 mRNA + WT mLIGHT mRNA (Figure 6D). Increased T helper 1 and macrophage 1 cytokine-related responses (TNF-α, IFN-γ, and IL-15) have repeatedly been shown to be important for antitumor immune responses (Pan, 2012).

[0314] Example 2C: Efficacy in Cynomolgus Monkeys The functionality of mRNA-based therapeutics in non-human primates was evaluated in cynomolgus monkeys. Monkeys were randomized to receive either a low-dose (n=2) or a high-dose (n=2) mRNA-based therapeutic regimen via intramuscular injection. Each animal received three injections, spaced 7 days apart. Blood was collected for collection of PBMCs and evaluation of HPV16 E6- and E7-specific T cell responses by ELISpot assay. The tumor microenvironment was evaluated to determine the type of immune response generated by the mRNA-based therapeutics.

[0315] Example 3: HPV16+ donor proliferation CIN PBMCs from two independent donors were treated with control mRNA, antigen mRNA, or mRNA-based therapeutic mRNA formulated in a delivery vehicle (n=3). T cell proliferation was measured by dilution of CellTrace Violet dye (VTD) Tracking. Data are presented as the percentage of viable CD4 and CD8 T cells during proliferation by flow cytometry (Figure 16). T cell proliferation was measured using the same method for independent donors treated with control, antigen, antigen + soluble LIGHT + IL-12, and several membrane-stabilized LIGHT mRNA-based therapeutics. Data are presented as the percentage of viable CD4 T cells during proliferation by flow cytometry (Figures 17A and 17B), CD8 T cells during proliferation by flow cytometry (Figure 17C), and NK cells during proliferation by flow cytometry (Figure 17D).

[0316] Example 4: General synthesis of tertiary amino lipidated cationic peptoids General protocols for synthesizing the tertiary amino lipidated cationic peptoids disclosed herein can be found in International Patent Applications Nos. 2020 / 069442 and 2020 / 069445, each of which is incorporated herein by reference in its entirety. The following examples illustrate general protocols for the synthesis of tertiary amino lipidated cationic peptoids.

[0317] All polymers were synthesized using bromoacetic acid and primary amines. Fmoc-Rink amide resin was used as the solid support. The Fmoc group on the resin was deprotected with 20% (v / v) piperidine-dimethylformamide (DMF). The amino resin was then amidated with bromoacetic acid. Following amidation, amination of the α-carbon was carried out by nucleophilic displacement of the bromide with a primary amine. The two steps were repeated sequentially to generate the desired cationic peptide sequence.

[0318] All reactions and washes were performed at room temperature unless otherwise noted. Resin washing refers to adding the wash solvent (usually DMF or dimethyl sulfoxide (DMSO)) to the resin, stirring the resin to obtain a homogeneous slurry, and then allowing the solvent to completely drain from the resin. Solvent was removed by vacuum filtration through the fritted bottom of the reaction vessel until the resin appeared dry. In all syntheses, the resin slurry was stirred by bubbling argon through the bottom of the fritted vessel.

[0319] Initial resin deprotection. A fritted reaction vessel was loaded with Fmoc-Rink amide resin. DMF was added to the resin, and the solution was agitated to swell the resin. The DMF was then drained. The Fmoc group was removed by adding 20% ​​piperidine in DMF to the resin, agitating the resin, and draining the resin. 20% piperidine in DMF was added to the resin, agitated for 15 minutes, and then drained. The resin was then washed six times with DMF.

[0320] Acylation / amidation. The deblocked amine was then acylated by adding bromoacetic acid in DMF to the resin, followed by N,N'-diisopropylcarbodiimide (DIC) in DMF. The solution was stirred at room temperature for 30 minutes and then drained. This process was repeated twice. The resin was then washed twice with DMF and once with DMSO. This was one completed reaction cycle.

[0321] Nucleophilic Substitution / Amination. The acylated resin was treated with the desired primary or secondary amine to effect nucleophilic substitution at the bromine leaving group on the α-carbon. This acylation / substitution cycle was repeated until the desired peptide sequence was obtained.

[0322] Peptide cleavage from resin. The dried resin was placed in a glass scintillation vial containing a Teflon-coated microstir bar, and 95% trifluoroacetic acid (TFA) in water was added. The solution was stirred for 20 minutes and then filtered through a solid-phase extraction (SPE) column fitted with a polyethylene frit into a polypropylene conical centrifuge tube. The resin was washed with 1 mL of 95% TFA. The combined filtrate was then lyophilized three times from 1:1 acetonitrile:water. The lyophilized peptide was redissolved in 5% acetonitrile in water to a concentration of 5 mM.

[0323] Purification and characterization. The redissolved crude peptide was purified by preparative HPLC. The purified peptide was characterized by LC-MS analysis.

[0324] Example 5: Synthesis of hydroxyethyl-capped tertiary amino-lipidated cationic peptoids Hydroxyethyl-capped lipidated peptoids were synthesized using bromoacetic acid and N,N'-diisopropylcarbodiimide (DIC) by the submonomer method described in Example 1. Polystyrene-supported MBHA Fmoc-protected Rink amide resin (200 mg typical scale, 0.64 mmol / g loading, Protein Technologies) was used as the solid support. For bromoacetylation, the resin was combined with a 1:1 mixture of 0.8 M bromoacetic acid and 0.8 M N,N'-diisopropylcarbodiimide (DIC) for 15 min. Amine substitution was performed using a 1 M solution of the amine in DMF for 45 min. After synthesis, the crude peptoid was cleaved from the resin using 5 mL of a 95:2.5:2.5 mixture of trifluoroacetic acid (TFA):water:triisopropylsilane at room temperature for 40 min. The resin was removed by filtration, and the filtrate was concentrated using a vacuum centrifuge. The crude peptoids were further purified by reversed-phase flash chromatography (Biotage Selekt) using a C4 column and a gradient of 60-95% ACN / HO + 0.1% TFA. The resulting product was assayed for identity and purity using a Waters Acquity UPLC system equipped with an Acquity Diode Array UV detector and a Waters SQD2 mass spectrometer, running a 5-95% gradient on a Waters Acquity UPLC Peptide BEH C4 column. Selected peptoids were further purified on a Waters XBridge BEH300 Prep C4 column using a 40-85% acetonitrile gradient in water with 0.1% TFA over 30 minutes using a Waters 2489 UV / Visible detector.

[0325] Example 6: Synthesis of delivery vehicle complexes Synthesis. Hydroxyethyl-capped tertiary amino-lipidated peptoids can be combined with polyanionic compounds, such as the mRNA polynucleotides described herein, to form delivery vehicle complexes that can be administered in vitro or in vivo for therapeutic and / or prophylactic purposes. Without being bound by any particular theory, the cationic moiety(s) of the amino-lipidated peptoid bind to the negatively charged phosphodiester backbone of the polyanionic cargo (e.g., nucleic acid cargo) primarily through electrostatic interactions, forming a mixed coacervate complex. Hydrophobic interactions between the lipid chains on the hydroxyethyl-capped tertiary amino-lipidated peptoid may act to stabilize microparticle formation and assist membrane association.

[0326] Delivery vehicle complexes can be prepared by any physical and / or chemical method known in the art to adjust their physical, chemical, and biological properties. These methods typically involve rapidly combining a hydroxyethyl-capped tertiary amino lipidated peptoid in water or a water-miscible organic solvent with an oligonucleotide in water or an aqueous buffer. These methods can involve simple mixing of the components by pipetting, or microfluidic mixing processes such as those involving a T-mixer, vortex mixer, or other chaotic mixing structures. Exemplary mixing methods are described in detail, for example, in U.S. Patent Nos. 11,278,895 and 11,325,122, which are incorporated herein by reference.

[0327] In a standard formulation, a hydroxyethyl-capped tertiary amino-lipidated peptoid and additional lipid are dissolved in absolute ethanol at a concentration of 10 mg / mL, resulting in a solution that is stable at room temperature. In some embodiments, the solution is stored at -20°C. The nucleic acid cargo is dissolved in DNAse- or RNAse-free water to a final concentration of 1-2 mg / mL. These solutions can be stored long-term at -20°C or -78°C.

[0328] To prepare the delivery vehicle compositions disclosed herein, the hydroxyethyl-capped tertiary amino-lipidized peptoid and additional lipid components are first premixed in an ethanol phase at the required mass ratio. The nucleic acid cargo is diluted with ethanol and an acidic buffer (e.g., 10 mM phosphate / citrate, pH 5.0). The ethanol and aqueous phases are mixed in a 3:1 volume ratio and then immediately diluted with PBS to a 1:1 volume ratio to obtain a final mRNA concentration of 0.1 μg / uL. Non-limiting exemplary delivery vehicle compositions prepared by the above method include those listed in Table 8 above (e.g., compositions F2, F6 / 17, F6 / 12, and F6 / 15).

[0329] The delivery vehicle compositions were combined with a polyanionic compound, such as an E6 / E7 oncogene (e.g., from HPV16, HPV18, functional fragments thereof, and / or variants thereof) in the ratios shown in Table 11 to form a delivery vehicle complex, where w / w is the mass ratio of the indicated component to mRNA.

[0330] [Table 11]

[0331] Example 7: RNA-based vaccine for cervical cancer The efficacy of the delivery vehicle complexes of the present disclosure to act as RNA-based vaccines against cancer was evaluated.

[0332] Cellular Responses. The efficacy of the delivery vehicle complexes described herein in disease models was evaluated by formulating mRNA encoding either ovalbumin (OVA) or HPV E6 / E7 oncogenes (from HPV16 and / or HPV18) with representative peptoids and administering the vaccine to C57B1 / 6 mice. The vaccine candidate was administered twice: a prime on day 0 and a boost on day 7. On day 14, the resulting immune responses against the characterized epitopes were determined by measuring the levels of antigen-specific CD8+ T cells in the peripheral blood and spleen using fluorescent MHC-I tetramer conjugates (MBL International). The DV-140-F2 complex elicited a stronger cellular response than the other complexes tested. See Figure 18A.

[0333] Humoral responses. Humoral responses to the vaccine candidates were evaluated by E7-IgG ELISA. Briefly, MaxiSorp ELISA plates (Thermo Scientific) were coated with 1 μg / mL E7-his protein (Abcam) overnight at 4°C. The plates were then washed and blocked with 10% FBS. Plasma samples were diluted 1:5 in blocking buffer (10% FCS), and five 10-fold dilutions were added to the plates. The samples were added to the plates and incubated overnight at 4°C. Detection was performed using 1:1000 donkey anti-mouse IgG-HRP (Jackson Immunology) in blocking buffer for 1 hour, followed by detection with HRP substrate and reading at 450 nm. The DV-140-F2 conjugate induced a strong IgGr response compared to the other conjugates tested. See Figure 18B.

[0334] Example 8: Efficacy and Toxicity Studies of Compound 140 Compound 140 was found to be well tolerated in efficacy studies in mice and toxicity studies in rats. Briefly, Sprague-Dawley rats (n=8) were treated with 0.03 or 0.3 mg / kg of DV-140-F2 or control mRNA formulated in PBS vehicle. Four injections were administered intramuscularly in the hind leg over a 13-day period. Six hours after the first and final doses, and two weeks after the final dose, whole blood was collected for hematology, and serum was collected for clinical chemistry and cytokine analysis. Two animals were sacrificed six hours after the final dose and two weeks after the final dose for gross necropsy. Tissue samples were retained, and selected organs were subjected to histopathological examination. The results are summarized in Table 12 below.

[0335] [Table 12]

[0336] standard (1)https: / / www.who.int / health-topics / cervical-cancer#tab=tab_1 (2) da Silva, RL, da Silva Batista, Z., Bastos, GR et al., Role of HPV 16 variants among cervical carcinoma samples from Northeastern Brazil. BMC Women's Health 20, 162 (2020). (3). Tao, L., Han, L., Li, X. et al., Prevalence and risk factors for cervical neoplasia: a cervical cancer screening program in Beijing. BMC Public Health 14, 1185 (2014). (4) Risk of recurrent high-grade cervical intraepithelial neoplasia after successful treatment: A longitudinal multicohort study. Marielle Kocken 1, Theo JM Helmerhorst, Johannes Berkhof, Jacqueline A Louwers, Marielle AE Nobbenhuis, Aagje G Bais, Cornelis JA Hogewoning, Afra Zaal, Rene HM Verheijen, Peter JF Snijders, Chris JLM Meijer. Lancet Oncol 2011 May, 12(5):441-50 (5) Feltkamp MC, Smits HL, Vierboom MP et al., Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 1993, 23:2242-9. Arbyn M, Weiderpass E, Bruni L et al., Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis. Lancet Glob Health 2020, 8:e191-203. Bjorge T, Skare GB, Bjorge L, et al. Adverse pregnancy outcomes after treatment for cervical intraepithelial neoplasia. Obstetrics and Gynecology. 2016, 128(6):1265-1273. Centers for Disease Control and Prevention (CDC). Estimated cases of high-grade cervical lesions diagnosed among women - United States, 2008 and 2016. MMWR Morb Mortal Wkly Rep. 2019, 68(15):337-343. Keytruda® (pembrolizumab) prescribing information. August 2022. https: / / dailymed.nlm.nih.gov / dailymed / drugInfo.cfm?setid=9333c79b-d487-4538-a9f0-71b91a02b287. Loopik DL, IntHout J, Ebisch RMF et al. The risk of cervical intraepithelial neoplasia grade 3: A population-based cohort study of 80,442 women. Gynecol Oncol. 2020, 157(1):195-201. Mirabello L., Clarke MA, Nelson CW et al., "The intersection of HPV epidemiology, genomics and mechanistic studies of HPV-mediated carcinogenesis." Viruses. 2018, 10(2). Moscicki AB, Schiffman M, Kjaer S, et al., Chapter 5: Updating the natural history of HPV and anogenital cancer. Vaccine. 2006, 24 Suppl 3:S 3 / 42-S 3 / 51. Narisawa-Saito M, Kiyono T. Basic mechanisms of high-risk human papillomavirus-induced carcinogenesis: role of E6 and E7 proteins. Cancer Sci. 2007, 98(10):1505-1511. National Comprehensive Cancer Network (NCCN), Clinical Practice Guidelines in Oncology (NCCN Guidelines®). Cervical Cancer. Version 1. 2022. Noehr B, Jensen A, Frederiksen K, Tabor A, Kjaer SK. Loop electrosurgical excision of the cervix and subsequent risk for spontaneous preterm delivery: a population-based study of singleton deliveries during a 9-year period. Am J Obstet Gynecol. 2009, 201(1):33.e 1-33.e336. Pan XQ. The mechanism of the anticancer function of M1 macrophages and their use in the clinic. Chin J Cancer. 2012, 31(12):557-563. Smith JS, Lindsay L, Hoots B, et al., Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int J Cancer. 2007, 121(3):621-632. Spanos WC, Hoover A, Harris GF, et al., "The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with RAS for invasive growth." J. Virol. 2008, 82(5):2493-2500. Walboomers JM, Jacobs MV, Manos MM et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 1999, 189(1):12-19. Wieking BG, Vermeer DW, Spanos WC, et al., A non-oncogenic HPV 16 E6 / E7 vaccine enhances treatment of HPV-expressing tumors. Cancer Gene Ther. 2012, 19(10):667-674. Young N., The effect of loop electrosurgical excision procedure on the subsequent risk of preterm delivery. [scholarly project]. Toledo, OH: University of Toledo, 2010.

[0337] All features disclosed in this specification, including the claims, abstract, and drawings, and all steps in any disclosed method or process, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification, including the claims, abstract, and drawings, may be replaced by an alternative feature serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0338] While the present invention has been described in conjunction with its detailed description, it will be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0339] The technology described herein is described in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains to practice the same. The foregoing describes preferred embodiments of the invention, and it is to be understood that modifications can be made without departing from the spirit or scope of the invention.

Claims

1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:

17.

2. An isolated polynucleotide encoding the polypeptide of claim 1.

3. An isolated polynucleotide comprising a first isolated messenger ribonucleic acid (mRNA), at least a portion of which encodes membrane-stabilized LIGHT.

4. 1. An isolated polynucleotide having the formula: 5'UTR-signal / leader-mRNA coding region-3'UTR-polyA, An isolated polynucleotide, wherein said mRNA coding region encodes a membrane-stabilized LIGHT.

5. 5. The isolated polynucleotide of claim 4, wherein the isolated polynucleotide comprises a modified 3'UTR, a modified 5'UTR, one or more modifications to the nucleobase-sugar-internucleoside linkage, or a combination thereof.

6. 6. The isolated polynucleotide of claim 5, wherein the isolated polynucleotide comprises a nucleobase-sugar-internucleoside linkage selected from the group consisting of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, 2-aminopurine-riboside-MP, pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 5-bromo-cytidine-MP, and combinations thereof.

7. The isolated polynucleotide of any one of claims 3 to 6, wherein said membrane-stabilized LIGHT is membrane-stabilized human LIGHT.

8. 8. The isolated polynucleotide of any one of claims 3 to 7, wherein said membrane-stabilized LIGHT comprises a replacement of a section of the transmembrane domain of soluble LIGHT with a linker.

9. The isolated polynucleotide of claim 7 , wherein the linker is an antibody variable region linker or a peptide linker.

10. 10. The isolated polynucleotide of claim 8 or 9, wherein the linker comprises (Gly4Ser)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

11. 10. The isolated polynucleotide of claim 8 or 9, wherein the linker comprises the sequence SSASTDKTHT.

12. 12. The isolated polynucleotide of any one of claims 3 to 11, wherein the membrane-stabilized LIGHT has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:

17.

13. 13. The isolated polynucleotide of claim 12, wherein the membrane-stabilized LIGHT has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% amino acid sequence identity to SEQ ID NO:11 or SEQ ID NO:

17.

14. 14. The isolated polynucleotide of any one of claims 3 to 13, wherein the first isolated mRNA has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:

18.

15. 15. The isolated polynucleotide of claim 14, wherein the first isolated mRNA has at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 93%, alternatively at least about 95%, alternatively at least about 96%, alternatively at least about 97%, alternatively at least about 98%, alternatively at least about 99% nucleic acid sequence identity to SEQ ID NO: 12 or SEQ ID NO:

18.

16. A composition comprising the isolated polynucleotide of any one of claims 2 to 15.

17. 17. The composition of claim 16, further comprising at least a second isolated mRNA, at least a portion of which encodes a checkpoint inhibitor, an immunosuppressant antagonist, a pro-inflammatory agent, or a pro-inflammatory cytokine.

18. 18. The composition of claim 17, wherein the pro-inflammatory cytokine is interleukin-12.

19. 18. The composition of claim 17, wherein the pro-inflammatory cytokine has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:

21.

20. The second isolated mRNA has the formula: 5'UTR-signal / leader-mRNA coding region-3'UTR-polyA, The composition of any one of claims 17 to 19, wherein the mRNA coding region encodes human interleukin-12.

21. 21. The composition of claim 20, wherein the second isolated mRNA comprises a modified 3'UTR, a modified 5'UTR, one or more modifications to the nucleobase-sugar-internucleoside linkage, or a combination thereof.

22. 22. The composition of claim 21, wherein the second isolated mRNA comprises a nucleobase-sugar-internucleoside linkage selected from the group consisting of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, 2-aminopurine-riboside-MP, pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 5-bromo-cytidine-MP, and combinations thereof.

23. The composition of any one of claims 20 to 22, wherein the mRNA coding region encodes a heterodimer of at least two of human interleukin-12.

24. 24. The composition of claim 23, wherein the first interleukin-12 heterodimer is linked to the second interleukin-12 heterodimer via a linker.

25. 25. The composition of claim 24, wherein the linker is an antibody variable region linker, a peptide linker, and / or a (Gly4Ser)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

26. 26. The composition of any one of claims 17-25, wherein the second isolated mRNA has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:

20.

27. 27. The composition of any one of claims 16 to 26, further comprising at least a third isolated mRNA, said third isolated mRNA comprising a first region encoding a first antigen and a second region encoding a second antigen.

28. 28. The composition of claim 27, wherein the first region and / or the second region encodes all or a portion of an antigen specific for cervical cancer, an HPV-associated cancer, or an HPV-associated disease.

29. 29. The composition of claim 27 or 28, wherein the third isolated mRNA encodes HPV16 E6 E7 or HPV18 E6 E7.

30. 30. The composition of claim 29, wherein the third isolated mRNA encodes HPV16 E6 E7.

31. The third isolated mRNA has the formula: 5'UTR--Signal / Leader-(An1)n-Xo-(An2)p-3'UTR-polyA, wherein An1 encodes HPV16 E6, An2 encodes HPV16 E7, and X is a spacer or linker; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; o is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; 31. The composition of claim 30, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

32. 32. The composition of claim 31 , wherein the linker is an antibody variable region linker, a peptide linker, and / or a (Gly4Ser)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

33. 33. The composition of claim 32, wherein the linker is a (Gly4Ser)n linker, wherein n is 3.

34. 32. The composition of claim 31 , wherein the third isolated mRNA comprises a modified 3′ UTR, a modified 5′ UTR, one or more modifications to the nucleobase-sugar-internucleoside linkage, or a combination thereof.

35. 35. The composition of claim 34, wherein the third isolated mRNA comprises a nucleobase-sugar-internucleoside linkage selected from the group consisting of pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 1-ethyl-pseudouridine-MP, 1-propyl-pseudouridine-MP, 1-(2,2,2-trifluoroethyl)-pseudouridine-MP, 2-amino-adenine-MP, xanthosine-MP, 5-bromo-cytidine-MP, 5-aminoallyl-cytidine-MP, 2-aminopurine-riboside-MP, pseudouridine-α-thio-MP, 1-methyl-pseudouridine-α-thio-MP, 5-bromo-cytidine-MP, and combinations thereof.

36. 36. The composition of any one of claims 27 to 35, wherein the third isolated mRNA encodes an antigen, wherein the antigen has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% amino acid sequence identity to SEQ ID NO:24 or SEQ ID NO:

27.

37. 36. The composition of any one of claims 27-35, wherein the third isolated mRNA has at least about 70%, alternatively at least about 75%, alternatively at least about 80%, alternatively at least about 85%, alternatively at least about 90%, alternatively at least about 95% nucleic acid sequence identity to SEQ ID NO:23 or SEQ ID NO:

26.

38. 38. The composition of any one of claims 27 to 37, wherein at least the first isolated mRNA, the second isolated mRNA, and / or the third isolated mRNA are formulated and / or delivered in a delivery vehicle.

39. 39. The composition of claim 38, wherein at least the first isolated mRNA, the second isolated mRNA, and / or the third isolated mRNA are at least partially encapsulated in the delivery vehicle.

40. 40. The composition of claim 38 or 39, wherein the delivery vehicle is selected from the group consisting of an amphiphilic molecule, an aminolipidated peptide, a tertiary aminolipidated cationic peptide, a cationic moiety, a peptoid, a lipoid, a liposome, a lipoplex, a lipid nanoparticle, a cationic lipid nanoparticle, a polymeric compound, and a conjugate.

41. 41. The composition of any one of claims 38 to 40, wherein the delivery vehicle has a particle size of about 200 nm or less.

42. The delivery vehicle has formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3, 4, 5, or 6; and R １ But H, C １～３ alkyl, or hydroxyethyl, and each R ２ are independently ８～２４ Alkyl or C ８～２４ The composition of any one of claims 38 to 41, wherein the alkyl group is alkenyl.

43. The compound is 【Chemistry 2】 【Transformation 3】 and a pharmaceutically acceptable salt of said compound. 【Request Item 44】 【Chemistry 4】 44. The composition of claim 43, wherein the compound is, or a pharmaceutically acceptable salt of the compound.

45. A composition comprising a first isolated mRNA, a second isolated mRNA, and a third isolated mRNA, wherein the first isolated mRNA, the second isolated mRNA, and / or the third isolated mRNA are part of a single polynucleotide strand.

46. 46. ​​The composition of claim 45, wherein the polynucleotide strand is formulated in a delivery vehicle and / or mediated by a delivery vehicle and / or at least partially encapsulated in a delivery vehicle.

47. 1. A composition comprising: a first isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO:12 or SEQ ID NO:18; a second isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO:20; and a third isolated mRNA having at least about 80% nucleic acid sequence identity to SEQ ID NO:23 or SEQ ID NO:26, wherein the first isolated mRNA, the second isolated mRNA, and the third isolated mRNA are at least partially encapsulated in a delivery vehicle.

48. 48. The composition of claim 47, wherein the first isolated mRNA has at least about 80% nucleic acid sequence identity to SEQ ID NO: 18 and the third isolated mRNA has at least about 80% nucleic acid sequence identity to SEQ ID NO:

26.

49. 1. A composition comprising a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding interleukin-12, and a third isolated mRNA encoding HPV16 E6 E7, wherein said first isolated mRNA, said second isolated mRNA, and said third isolated mRNA are at least partially encapsulated in a delivery vehicle.

50. 50. The composition of any one of claims 47 to 49, wherein the delivery vehicle is selected from the group consisting of amphiphilic molecules, aminolipidated peptides, tertiary aminolipidated cationic peptides, cationic moieties, peptoids, lipoids, liposomes, lipoplexes, lipid nanoparticles, cationic lipid nanoparticles, polymeric compounds, and conjugates.

51. 51. The composition of any one of claims 47 to 50, wherein the delivery vehicle has a particle size of about 200 nm or less.

52. The delivery vehicle has formula (I): 【Transformation 5】 or a pharmaceutically acceptable salt thereof, wherein n is 1, 2, 3, 4, 5, or 6; and R １ is H, C １～３ alkyl, or hydroxyethyl, and each R ２ are independently ８～２４ Alkyl or C ８～２４ The composition of any one of claims 47 to 51, wherein the alkyl group is alkenyl.

53. The compound is 【Transformation 6】 【Transformation 7】 【Transformation 8】 and a pharmaceutically acceptable salt of said compound. 【Request Item 54】 【Chemistry 9】 54. The composition of claim 53, wherein the compound is, or a pharmaceutically acceptable salt of the compound.

55. The composition of any one of claims 16 to 54, wherein the composition is a therapeutic composition or a vaccine.

56. 56. The composition of claim 55, wherein the composition is a human papillomavirus (HPV) mRNA vaccine.

57. 57. The composition of claim 55 or 56, wherein the composition is used in cancer immunotherapy.

58. 58. The composition of any one of claims 55 to 57, wherein the composition is configured to be administered to a subject known to have cervical cancer, an HPV-associated cancer, or an HPV-associated disease.

59. 59. The composition of any one of claims 55 to 58, wherein the composition is adapted to be administered as an injectable formulation.

60. 60. The composition of any one of claims 55 to 59, further comprising one or more therapeutically acceptable carriers, therapeutically acceptable diluents, therapeutically acceptable excipients, or other therapeutic agents.

61. 61. The composition of claim 60, wherein the therapeutically acceptable excipient is selected from the group consisting of salts, buffers, preservatives, anti-adherents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film-forming agents, coating agents, flavors, fragrances, glidants, lubricants, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration.

62. 62. A method comprising administering a therapeutically effective amount of the composition of any one of claims 16 to 61 to a subject in need thereof.

63. 63. The method of claim 62, wherein the composition is administered as an injection.

64. 64. The method of claim 62 or 63, wherein the composition is configured for intratumoral administration to the subject in need thereof.

65. An expression cassette comprising a first isolated mRNA encoding membrane-stabilized LIGHT, a second isolated mRNA encoding interleukin-12, or a third isolated mRNA encoding HPV16 E6 E7.

66. A kit comprising the composition of any one of claims 16 to 65 and instructions for use.

67. 67. The kit of claim 66, wherein the instructions for use include instructions for intratumoral administration and / or intratumoral administration in combination with injection at another site.

68. 68. The kit of claim 66 or 67, wherein the kit further comprises at least one of a therapeutic nucleic acid, a drug, a therapeutic agent, a diagnostic agent, or a prophylactic agent.

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