Compositions and methods for delivery of RNA

Abstract

The lipid nanoparticle compositions provided herein are preferentially delivered and / or transfected to the lung. Also provided herein are therapeutic polynucleotides, such as TERT mRNA, that can be delivered with the LNP formulations for the treatment of pulmonary disease and fibrosis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 169,118, filed March 31, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporating sequence tables The contents of the text file named "REJU_005_01WO_SeqList_ST25.txt", created on March 29, 2022 and having a size of 205KB, are incorporated herein by reference in their entirety.

[0003] Field of Disclosure The present disclosure relates generally to compositions and methods for delivering ribonucleic acid (RNA) therapeutics in the treatment of pulmonary and fibrotic diseases. [Background technology]

[0004] There are many lung diseases that have no treatment today.Lung diseases, such as pulmonary fibrosis, interstitial lung disease, and lung cancer, often induce fibrosis as part of disease progression, which further limits the extent to which patients can recover.Delivery of polynucleotides to the lung for the treatment of lung diseases is one of the treatment methods under development, but the compositions and methods for administering these treatments need to be improved, and no treatment has been developed to treat the resulting fibrosis.

[0005] Thus, there remains a need in the art for delivery formulations capable of delivering polynucleotides, such as mRNA, to the lung to treat pulmonary disease and / or pulmonary fibrosis. The following disclosure addresses this need. Summary of the Invention

[0006] Provided herein are delivery vehicles and compositions thereof for delivering mRNA to lung cells with high transfection rates. In some embodiments, the mRNA is delivered to lung cells with low toxicity. In some embodiments, the lung cells include alveolar epithelium and vascular endothelium, and the delivery vehicles disclosed herein are useful for delivery of mRNA useful for treating or preventing lung diseases and disorders. In some embodiments, the mRNA delivered to the lung cells encodes a protein useful for treating lung diseases or disorders. In some embodiments, the mRNA encodes a TERT protein. In some embodiments, the protein is an antigen of a pathogen. In some embodiments, the lung diseases and disorders include, but are not limited to, pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, interstitial lung disease, chronic obstructive pulmonary disease (COPD), lung infections, pneumonia, tuberculosis, gastric reflux, lung cancer, cystic fibrosis, dyskeratosis congenita, alpha-1 antitrypsin deficiency, and other acquired or inherited lung diseases. The present disclosure relates to telomerase reverse transcriptase (TERT) messenger ribonucleic acid (mRNA) therapy for treating, for example, pulmonary, fibrotic diseases and conditions, and pulmonary diseases and conditions. Treatment with a composition comprising TERT mRNA can prevent, reverse, or treat fibrosis and other pathological features of fibrotic and / or pulmonary diseases, leading to improved overall organ function and subject health. Thus, in some embodiments, compositions are provided herein that comprise one or more synthetic messenger ribonucleic acids (mRNAs) encoding telomerase reverse transcriptase (TERT).

[0007] In some embodiments, a composition comprises (i) a ribonucleic acid (RNA) encoding telomerase reverse transcriptase (TERT) and (ii) a delivery vehicle, wherein the RNA encoding TERT comprises one or more modified nucleotides, and the delivery vehicle of (ii) is operably linked to the RNA encoding TERT.

[0008] In some embodiments of the compositions of the present disclosure, the delivery vehicle comprises one or more of a nanoparticle, a liposome, a cationic lipid, an exosome, an extracellular vesicle, a lipid nanoparticle, a natural lipoprotein particle, and an artificial lipoprotein particle.

[0009] Provided herein are lipid nanoparticles (LNPs) capable of transfecting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a lung cell population.

[0010] In some embodiments of the compositions of the present disclosure, the delivery vehicle comprises a lipid nanoparticle (LNP). In some embodiments, the delivery vehicle comprises a cationic lipid.

[0011] In some embodiments, the delivery vehicle comprises a targeting moiety. In some embodiments, the targeting lipid causes the delivery vehicle to specifically or selectively interact with lung cells. In some embodiments, the targeting moiety comprises cholesterol. In some embodiments, the targeting moiety is a lipid that specifically or selectively interacts with lung cells. In some embodiments, the targeting lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylammonium bromide (DABB), or 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

[0012] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the delivery vehicle comprises a compound of formula I: [ka] (In the formula, R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms; X a and X bare each independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one to two tertiary amino groups; R 2a and R 2b each independently represents an alkylene or oxydialkylene group having 8 or fewer carbon atoms; Y a and Y b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond; Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom; R 3a and R 3b each independently represents a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride, or a residue derived from a reaction product with glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms).

[0013] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the compound of formula I is: [ka]

[0014] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the compound of formula I is: [ka]

[0015] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the compound of formula I is: [ka]

[0016] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the compound of formula I is: [ka]

[0017] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the compound of formula I is: [ka]

[0018] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the compound of formula I is: [ka]

[0019] In some embodiments, the RNA comprises a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0020] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the RNA comprises a 5' cap. In some embodiments, the 5' cap comprises an anti-reverse cap analog (ARCA). In some embodiments, the ARCA comprises a 3'-O-Me-m7G(5')ppp(5')G structure. In some embodiments, the 5' cap comprises m7G(5')ppp(5')(2'OMeA)pG. In some embodiments, the 5' cap comprises m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0021] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the RNA further comprises at least one untranslated region (UTR). The UTR may comprise a sequence of SEQ ID NOs: 32-36, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, at least one UTR is located 5' to the RNA encoding TERT. In some embodiments, at least one UTR is located 3' to the RNA encoding TERT. In some embodiments, the UTR comprises a human sequence. In some embodiments, the UTR comprises a non-human or synthetic sequence. In some embodiments, the UTR comprises a chimeric sequence. In some embodiments, the UTR increases the stability, increases the half-life, increases the transcription rate, or decreases the time to transcription initiation of the RNA encoding TERT. In some embodiments, the UTR comprises a sequence having at least 70% identity to a UTR sequence isolated or derived from one or more of α-globin, β-globin, c-fos, and tobacco etch virus.

[0022] In some embodiments of the compositions of the present disclosure, including compositions in which the delivery vehicle is a lipid nanoparticle (LNP), the one or more modified nucleotides of the RNA encoding TERT include one or more of modified adenine or analogs thereof, modified cytidine or analogs thereof, modified guanosine or analogs thereof, and modified uridine or analogs thereof. In some embodiments, the one or more modified nucleotides of the RNA encoding TERT include one or more of 1-methylpseudouridine, also known as N1-methylpseudouridine, pseudouridine (N1m), 2-thiouridine, and 5-methylcytidine. In some embodiments, the one or more modified nucleotides of the RNA encoding TERT include 5-methoxyuridine (5-moU). In some embodiments, the one or more modified nucleotides of the RNA encoding TERT include one or more of the following: m1A 1-methyladenosine, m6A N6-methyladenosine, Am 2'-O-methyladenosine, i6A N6-isopentenyladenosine, io6A N6-(cis-hydroxyisopentenyl)adenosine, ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, g6A N6-glycinylcarbamoyladenosine, t6A N6-threonylcarbamoyladenosine, ms2t6A 2-methylthio-N6-threonylcarbamoyladenosine, Ar(p)2'-O-ribosyladenosine (phosphate), m6 2A N6,N6-dimethyladenosine, m6Am N6,2'-O-dimethyladenosine, m6 2Am N6,N6,2'-O-trimethyladenosine, m1Am 1,2'-O-dimethyladenosine, m3C 3-methylcytidine, m5C 5-methylcytidine, Cm 2'-O-methylcytidine, ac4C N4-acetylcytidine, f5C 5-formylcytidine, m4C N4-methylcytidine, hm5C 5-hydroxymethylcytidine, f5Cm 5-formyl-2'-O-methylcytidine, m1G 1-methylguanosine, m2G N2-methylguanosine, m7G 7-methylguanosine, Gm 2'-O-methylguanosine, m2 2G N2,N2-dimethylguanosine, Gr(p) 2'-O-ribosylguanosine (phosphate), yW wibtosine,o2yW peroxywybutosine, OHyW hydroxywybutosine, OHyW* hypomodified hydroxywybutosine, imG wybutosine, m2,7G N2,7-dimethylguanosine, m2,2,7G N2,N2,7-trimethylguanosine, I inosine, m1I 1-methylinosine, Im 2'-O-methylinosine, Q queuosine, galQ galactosyl-queuosine, manQ mannosyl-queuosine, Ψ pseudouridine, D dihydrouridine, m5U 5-methyluridine, Um 2'-O-methyluridine, m5Um 5,2'-O-dimethyluridine, m1Ψ 1-methylpseudouridine, Ψm 2'-O-methylpseudouridine, s2U 2-thiouridine, ho5U 5-hydroxyuridine, chm5U 5-(Carboxyhydroxymethyl)uridine, mchm5U 5-(carboxyhydroxymethyl)uridine, methyl ester mcm5U 5-methoxycarbonylmethyluridine, mcm5Um 5-methoxycarbonylmethyl-2'-O-methyluridine, mcm5s2U 5-methoxycarbonylmethyl-2-thiouridine, ncm5U 5-carbamoylmethyluridine, ncm5Um 5-carbamoylmethyl-2'-O-methyluridine, cmnm5U 5-carboxymethylaminomethyluridine, m3U 3-methyluridine, m1acp3Ψ 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, cm5U 5-carboxymethyluridine, m3Um 3,2'-O-dimethyluridine, m5D 5-methyldihydrouridine, τm5U 5-taurinomethyluridine, τm5s2U 5-taurinomethyl-2-thiouridine, 2-aminoadenosine, 2-amino-6-chloropurine riboside, 8-azaadenosine, 6-chloropurine riboside, 5-iodocytidine, 5-iodouridine, inosine, 2'-O-methylinosine, xanthosine, 4-thiouridine, O6-methylguanosine, 5,6-dihydrouridine, 2-thiocytidine, 6-azacytidine, 6-azauridine, 2'-O-methyl-2-aminoadenosine, 2'-O-methylpseudouridine, N1-methyladenosine, 2'-O-methyl-5-methyluridine, 7-deazaguanosine, 8-azidoadenosine, 5-bromocytidine, 5-bromouridine,7-Deazaadenosine, 5-aminoallyl uridine, 5-aminoallyl lysine, 8-oxoguanosine, 2-aminopurine riboside, pseudoisocytidine, N1-methylpseudouridine, 5,6-dihydro-5-methyluridine, N6-methyl-2-aminoadenosine, 5-carboxycytidine, 5-hydroxymethyluridine, thienoguanosine, 5-hydroxycytidine, 5-formyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-methoxycytidine, thienouridine, 5-carboxymethylesteruridine, thienosine Cytidine, 8-oxoadenosine, isoguanosine, N1-ethylpseudouridine, N1-methyl-2'-O-methylpseudouridine, N1-methoxymethylpseudouridine, N1-propylpseudouridine, 2'-O-methyl-N6-methyladenosine, 2-amino-6-Cl-purine-2'-deoxyriboside, 2-amino-2'-deoxyadenosine, 2-aminopurine-2'-deoxyriboside, 5-bromo-2'-deoxycytidine, 5-bromo-2'-deoxyuridine, 6-chloropurine-2'-deoxyriboside, 7-deaza-2 '-Deoxyadenosine, 7-deaza-2'-deoxyguanosine, 2'-deoxyinosine, 5-propynyl-2'-deoxycytidine, 5-propynyl-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-iodo-2'-deoxycytidine, 5-iodo-2'-deoxyuridine, N6-methyl-2'-deoxyadenosine, 5-methyl-2'-deoxycytidine, O6-methyl-2'-deoxyguanosine, N2-methyl-2'-deoxyguanosine, 8-oxo-2'-deoxyadenosine, 8-oxo-2'-deoxyguanosine nosine, 2-thiothymidine, 2'-deoxy-P-nucleoside, 5-hydroxy-2'-deoxycytidine, 4-thiothymidine, 2-thio-2'-deoxycytidine, 6-aza-2'-deoxyuridine, 6-thio-2'-deoxyguanosine, 8-chloro-2'-deoxyadenosine, 5-aminoallyl-2'-deoxycytidine, 5-aminoallyl-2'-deoxyuridine, N4-methyl-2'-deoxycytidine, 2'-deoxyzebularine, 5-hydroxymethyl-2'-deoxyuridine, 5-hydroxymethyl-2'-deoxycytidine,5-propargylamino-2'-deoxycytidine, 5-propargylamino-2'-deoxyuridine, 5-carboxy-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-[(3-indolyl)propionamido-N-allyl]-2'-deoxyuridine, 5-carboxy-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, 7-deaza-7-propargylamino-2'-deoxyadenosine, 7-deaza-7-propargylamino-2'-deoxyguanosine, biotin-16-aminoallyl-2'-dU TP, biotin-16-aminoallyl-2'-dCTP, biotin-16-aminoallylcytidine, N4-biotin-OBEA-2'-deoxycytidine, biotin-16-aminoallyluridine, dabsyl-5-3-aminoallyl-2'-dUTP, desthiobiotin-6-aminoallyl-2'-deoxycytidine, desthiobiotin-16-aminoallyl-uridine, biotin-16-7-deaza-7-propargylamino-2'-deoxyguanosine, cyanine 3-5-propargylamino-2'-deoxycytidine, cyanine 3-6-propargylamino-2'-deoxycytidine 2'-fluoro-2'-deoxyuridine, cyanine 5-6-propargylamino-2'-deoxycytidine, cyanine 5-6-propargylamino-2'-deoxyuridine, cyanine 3-aminoallylcytidine, cyanine 3-aminoallyluridine, cyanine 5-aminoallylcytidine, cyanine 5-aminoallyluridine, cyanine 7-aminoallyluridine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-O -methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methyluridine, puromycin, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine, aracytidine, arauridine, 2'-azido-2'-deoxyadenosine, 2'-amino-2'-deoxyadenosine, araadenosine, 2'-fluoro-thymidine, 3'-O-methyladenosine, 3'-O-methylcytidine, 3'-O-methylguanosine,3'-O-methyluridine, 2'-azido-2'-deoxyguanosine, araguanosine, 2'-deoxyuridine, 3'-O-(2-nitrobenzyl)-2'-deoxyadenosine, 3'-O-(2-nitrobenzyl)-2'-deoxyinosine, 3'-deoxyadenosine, 3'-deoxyguanosine, 3'-deoxycytidine, 3'-deoxy-5-methyluridine, 3'-deoxyuridine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyuridine, 2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 3'-azido-2',3'-dideoxyadenosine, 3'-azido-2',3'-dideoxythymidine, 3'-amino-2',3'-dideoxyadenosine, 3'-amino-2',3'-dideoxycytidine, 3'-amino-2',3'-dideoxyguanosine, 3'-amino-2',3'-dideoxythymidine, 3'-azido-2',3'-dideoxycytidine, 3'-azido-2',3'-dideoxyuridine, 5-bromo-2',3'-dideoxyuridine, 2',3'-dideoxyinosine, 2'-deoxy Cyadenosine-5'-O-(1-thiophosphate), 2'-deoxycytidine-5'-O-(1-thiophosphate), 2'-deoxyguanosine-5'-O-(1-thiotriphosphate), 2'-deoxythymidine-5'-O-(1-thiophosphate), adenosine-5'-O-(1-thiophosphate), cytidine-5'-O-(1-thiophosphate), guanosine-5'-O-(1-thiophosphate), uridine-5'-O-(1-thiophosphate), 2',3'-dideoxyadenosine-5'-O-(1-thiophosphate), 2',3'-dideoxycytidine-5'-O-(1-thiophosphate) acid), 2',3'-dideoxyguanosine-5'-O-(1-thiophosphate), 3'-deoxythymidine-5'-O-(1-thiophosphate), 3'-azido-2',3'-dideoxythymidine-5'-O-(1-thiophosphate), 2',3'-dideoxyuridine-5'-O-(1-thiophosphate), 2'-deoxyadenosine-5'-O-(1-boranophosphate), 2'-deoxycytidine-5'-O-(1-boranophosphate), 2'-deoxyguanosine-5'-O-(1-boranophosphate), and 2'-deoxythymidine-5'-O-(1-boranophosphate).

[0023] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the delivery vehicle comprises an RNA encoding TERT. In some embodiments, one or more of the surface, layer, or volume of the delivery vehicle comprises an RNA encoding TERT. In some embodiments, the surface comprises an outer surface or an inner surface. In some embodiments, the layer comprises a lipid monolayer or lipid bilayer. In some embodiments, the volume comprises an inner volume.

[0024] In some embodiments, the disclosure provides a composition comprising (i) a ribonucleic acid (RNA) encoding telomerase reverse transcriptase (TERT); and (ii) a delivery vehicle, wherein the RNA encoding TERT comprises one or more modified nucleotides, and the delivery vehicle of (ii) is operably linked to the RNA encoding TERT.

[0025] In some embodiments of the compositions of the present disclosure, including those in which the delivery vehicle is a lipid nanoparticle (LNP), the composition further comprises a ribonucleic acid (RNA) encoding a telomerase RNA component (TERC). In some embodiments, the delivery vehicle is operably linked to a ribonucleic acid (RNA) encoding a telomerase RNA component (TERC). In some embodiments, the delivery vehicle comprises an RNA encoding TERC. In some embodiments, one or more of a surface, layer, or volume of the delivery vehicle comprises an RNA encoding TERC. In some embodiments, the surface comprises an outer surface or an inner surface. In some embodiments, the layer comprises a lipid monolayer or lipid bilayer. In some embodiments, the volume comprises an inner volume.

[0026] In some embodiments, the RNA encoding TERT comprises a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, of SEQ ID NOs: 1-5, 7, 9, 14-17, 19, 21, 23, 25, 27, 29-31, 37-40. In some embodiments, the RNA encoding TERT comprises a UTR sequence of SEQ ID NOs: 32-34, 35, and 36, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0027] In some embodiments, the RNA comprises self-replicating RNA. In some embodiments, the RNA comprises circular RNA.

[0028] The present disclosure provides methods of increasing telomerase activity in a cell, the method comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is in vivo, ex vivo, or in vitro.

[0029] The present disclosure provides methods of extending telomeres in a cell, the methods comprising contacting the cell with a composition of the present disclosure. In some embodiments, the cell is in vivo, ex vivo, or in vitro.

[0030] The present disclosure provides a cell comprising a composition of the present disclosure.

[0031] The present disclosure provides a formulation comprising a cell of the present disclosure, comprising a composition of the present disclosure. In some embodiments of the formulation, the plurality of cells comprises a cell of the present disclosure, comprising a composition of the present disclosure. In some embodiments of the formulation, each of the plurality of cells is a cell of the present disclosure, comprising a composition of the present disclosure.

[0032] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject an effective amount of a composition of the present disclosure.

[0033] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject an effective amount of a cell of the present disclosure, including a composition of the present disclosure.

[0034] The present disclosure provides a method of treating a disease or disorder comprising administering to a subject an effective amount of a formulation of the present disclosure.

[0035] The present disclosure provides a method of delaying the onset of a disease comprising administering to a subject an effective amount of a composition of the present disclosure.

[0036] The present disclosure provides a method of delaying the onset of a disease comprising administering to a subject an effective amount of a cell of the present disclosure, including a composition of the present disclosure.

[0037] The present disclosure provides a method of delaying the onset of a disease comprising administering to a subject an effective amount of a formulation of the present disclosure.

[0038] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0039] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief description of the drawings]

[0040] [Figure 1]Schematic diagram showing the long-term therapeutic effect of transient rapid telomere elongation via telomerase reverse transcriptase (TERT) mRNA. In particular, the speed of telomere elongation enabled by TERT mRNA treatment allows for the maintenance of telomeres with infrequent administration of TERT mRNA. The telomerase activity provided by TERT mRNA delivery rapidly elongates telomeres in a short period of time before the mRNA is turned over, thereby allowing the protective anti-cancer mechanism of telomere shortening to function most of the time. During treatment, normal telomerase activity and telomere shortening are present, and therefore the anti-cancer safety mechanism of telomere shortening that prevents uncontrolled proliferation is maintained, while the risk of short telomere-related diseases remains low. In contrast, the best existing small molecule treatments for lengthening telomeres require long-term delivery, which poses the risk of chronic cancer, and even after that, the effect on telomere length is small and inconsistent, with about half of patients having no detectable effect on telomere length at all. [Diagram 2] 1 shows a representative dynamic light scattering (DLS) plot of mRNA-LNPs made using exemplary lipid components disclosed herein. [Diagram 3] 1 shows whole organ bioluminescence imaging of mice injected with mRNA-LNPs. [Figure 4] A-B show immunohistochemical staining of tdTomato in lung cells from mice treated with the mRNA reporter (FIG. 4A) and untreated control mice (FIG. 4B). [Diagram 5] A-B show measurements of telomerase activity in mouse lungs following TERT mRNA-LNP delivery (FIG. 5B) and in untreated control mouse lungs (FIG. 5A). [Figure 6A] 1 is a bar graph showing transfection efficiency of exemplary pulmonary delivery vehicle formulations. [Figure 6B] Representative images of lung sections taken from mice as described above are shown, with the reporter protein shown as darkened staining. [Figure 6C]Representative images of lung sections taken from mice as described above are shown, with the reporter protein shown as darkened staining. [Figure 6D] Representative images of lung sections taken from mice as described above are shown, with the reporter protein shown as darkened staining. [Figure 6E] Representative images of lung sections taken from mice as described above are shown, with the reporter protein shown as darkened staining. [Figure 6F] Representative images of lung sections taken from mice as described above are shown, with the reporter protein shown as darkened staining. [Figure 7A] 1 shows computed tomography (CT) X-ray scans of mouse lungs examined for pulmonary fibrosis. [Figure 7B] 1 shows computed tomography (CT) X-ray scans of mouse lungs examined for pulmonary fibrosis. [Figure 7C] 1 shows computed tomography (CT) X-ray scans of mouse lungs examined for pulmonary fibrosis. [Figure 7D] FIG. 1 is a bar graph quantifying the results shown in AC. [Figure 8] 6A shows a graph depicting the mortality rate of mice administered the formulations of Table 6A compared to other formulations of lung-targeted LNP. [Figure 9] A-D show various lung samples from mice treated with bleomycin to induce pulmonary fibrosis and treated with CRE mRNA or saline to demonstrate delivery of mRNA to alveolar cells. [Figure 10] FIG. 13 shows pulmonary luminescence of intravenous (IV) and intratracheal (OA) delivery of luciferase mRNA to the lung using SS-OP DOTAP and cKK DOTAP LNP formulations. [Figure 11]Figure 1 shows preferential delivery of intravenously administered SS-OP DOTAP formulations to the lungs. From left to right, the LNP formulations in the dishes are (1) orally delivered 75:1 with SS-OP DOTAP; (2) intravenously delivered 75:1 with SS-OP DOTAP; (3) PBS control (no LNPs); (4) orally delivered 40:1 with cKK DOTAP. [Figure 12] The percentage of Tomato(+) cells in the lung parenchyma following intravenous delivery of a CRE mRNA-containing LNP formulation is shown. [Figure 13] 1 shows lung luminescence versus flow rate used to formulate LNP compositions that deliver firefly luciferase mRNA. [Figure 14] We demonstrated ex vivo bioluminescence of an LNP formulation delivering luciferase RNA, without the structural lipids or cholesterol required for RNA delivery. [Figure 15] Ex vivo lung mean radiance of LNP formulations delivering luciferase mRNA is shown, with the RNA to LNP ratio varied according to the indicated ratio. [Figure 16] Figure 1 shows the ex vivo lung average radiance of LNP formulations delivering luciferase mRNA, where the RNA to LNP ratio was varied according to the ratios shown and no DOPC structured lipids were used in the formulation. [Figure 17] A and B show the ex vivo lung average radiance of LNP formulations delivering luciferase mRNA where the percentage of PEGylated lipid is varied in the formulation. [Figure 18] A and B show the ex vivo lung average radiance of LNP formulations delivering luciferase mRNA where the percentage of PEGylated lipid is less than 1.5% or absent in the formulation. [Figure 19] Figure 1 shows the ex vivo lung average radiance of LNP formulations delivering luciferase mRNA with varying percentages of DOTAP in the formulations. [Figure 20]A and B show preferential ex vivo delivery to the lungs of exemplary LNP formulations with varying percentages of DOTAP. A shows the liver mean radiance of LNP formulations delivering luciferase mRNA as a function of percentage of DOTAP. B shows the lung / liver delivery ratio of LNP formulations as a function of percentage of DOTAP. [Figure 21] A and B show transfection of LNP formulations in alveolar epithelial cells. Immunofluorescence staining of induced tdTomato expression in tomato fl / fl mice administered LNPs containing Cre mRNA is shown. AT1 and AT2 lung epithelial cell markers were also stained. [Figure 22] SS-OP DOTAP delivery of therapeutic mRNA as a disease treatment using SS-OP DOTAP LNPs of Table 6A containing TERT mRNA. LNP TERT mRNA formulations were delivered to a bleomycin-induced pulmonary fibrosis mouse model on day 0. Compared to control mRNA, delivery of TERT mRNA extended mouse survival by 210% at the endpoint. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The compositions and methods of the present disclosure provide lipid nanoparticles (LNPs) comprising SS-OP lipids or analogs thereof, such as compounds of formula I as described herein, and cationic lipids for intravenous delivery to the lung, such as for treating pulmonary disease and / or pulmonary fibrosis. In some variations, the SS-OP lipids or analogs thereof may be substituted for or combined with cKK lipids or analogs thereof. As disclosed herein, the LNPs may have improved lung transduction efficiency and / or lung specificity compared to known LNP formulations.

[0042] Also provided herein is a ribonucleic acid (RNA) encoding telomerase reverse transcriptase (TERT) delivered to the lung, for example to treat pulmonary fibrosis. The RNA-encoded TERT can optionally be delivered using the SS-OP LNP compositions described above, or other LNPs known in the art.

[0043] Telomerase reverse transcriptase (TERT) is an enzyme known to maintain and extend chromosome ends (telomeres). The TERT enzyme is the catalytic subunit of the ribonucleoprotein telomerase. TERT adds simple sequence repeats to telomeres by copying the template sequence 5'-GGTTAG-3' within the RNA component of telomerase. This addition of repetitive deoxyribonucleic acid (DNA) sequences helps slow telomere shortening, which occurs over time due to, for example, incomplete DNA replication during mitosis.

[0044] TERT translocates between the nucleus and cytoplasm and has been shown to be an important factor in many other biological processes, including cell proliferation and cancer metastasis, and thus, nuclear TERT levels may be an important step in regulating cell and organism health.

[0045] Telomerase reverse transcriptase (TERT) is also known in the art as TRT, cutaneous malignant melanoma 9 (CMM9), autosomal dominant dyskeratosis congenita type 2 (DKCA2), autosomal recessive dyskeratosis congenita 4 (DKCB4), human evershort telomere 2 (HEST2), pulmonary fibrosis / bone marrow failure telomere associated 1 (PFBMFT1), telomerase catalytic subunit (TCS1), and telomerase associated protein 2 (TP2).

[0046] In some embodiments, the treatments described herein may halt, slow, or reverse the progression of a fibrotic disease, such as a pulmonary disease, or other pulmonary disease.

[0047] TERT mRNA is transient and only requires a few hours to extend telomeres in human cells before being degraded.Therefore, TERT mRNA still does not impair protective anti-cancer telomere shortening mechanisms.The present disclosure provides compositions and methods for the delivery of TERT mRNA and the treatment of fibrotic and pulmonary diseases.

[0048] In normal aging, telomeres shorten by approximately 30-100 base pairs per year due to oxidation and incomplete DNA replication during the S phase of the cell cycle (Kurenova EV,et al.Telomere functions.A review.Biochemistry(Mosc)1997;62:1242-53). Telomerase, which consists of the TERT protein and the polynucleotide template (TERC), extends telomeres, but in humans, it is inactive in most somatic cell types and has low levels of activity that are insufficient to prevent net telomere shortening in many progenitor cell types. The exception is the spermatogenic lineage, where telomerase is active enough to maintain telomere length throughout the human lifespan (Takubo K,Aida J,Izumiyama-Shimomura N,et al.Changes of telomere length with aging.Geriatric Gerontology Int 2010;10 Suppl 1:S197-206). TERC elements are present at high levels in all cell types, usually exceeding 10,000 copies / cell, making TERT the limiting element. Short telomeres limit the cells' ability to proliferate and reproduce, and are therefore associated with aging, premature death, and a vast number of diseases and conditions.

[0049] Telomeres comprise repeated DNA sequences at the ends of linear chromosomes and, if long enough, can cause the end of each chromosome to form a loop that prevents the end from functioning as a double- or single-stranded DNA break. Telomeres can shorten over time, due in part to oxidative damage and incomplete DNA replication, ultimately resulting in extremely short telomeres that cannot form protective loops, exposed chromosome ends, chromosome-chromosome fusions, DNA damage responses, and cellular senescence, apoptosis, or malignancy.

[0050] Maintenance of telomere length may play a role in preventing cellular senescence and apoptosis, and the resulting cellular and organ dysfunction. In many diseases, the need for cell replication to replace cells damaged or killed by the underlying disease mechanism shortens telomeres more rapidly than normal, exhausting the cell's replicative capacity and resulting in tissue dysfunction, worsening or additional symptoms, disorders, or death. In addition, genetic mutations in the telomerase enzyme (TERT) may be associated with dyskeratosis congenita and fatal inherited disorders of insufficient telomere maintenance, including forms of pulmonary fibrosis, lung disease, and aplastic anemia. Cellular senescence due to interchromosomal fusions and short telomeres may increase the risk of cancer. Short telomeres are also associated with deleterious conditions and diseases of aging, as well as poor outcomes of many diseases. Lung diseases that cause pulmonary fibrosis can include, but are not limited to, pulmonary fibrosis, lung cancer, familial pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary fibrosis with congenital dyskeratosis, interstitial lung disease, pneumonia, interstitial pneumonia, tuberculosis, bronchitis, emphysema, lung cancer, chronic obstructive pulmonary disease (COPD), aging-related fibrosis, pulmonary hypertension, asthma, and cystic fibrosis.

[0051] The prospect of preventing, delaying or treating dysfunctions, symptoms and diseases by telomere elongation stimulates the need for safe and effective treatments for elongating telomeres of animal cells in vivo and / or in vitro, as well as safe and effective compositions and methods for administering therapy to animal cells to lengthen telomeres. Furthermore, there is a need for safe and rapid elongation of telomeres in cells used in cell therapy, cell and tissue engineering and regenerative medicine. At the same time, however, there may be dangers in constitutive rather than transient activation of telomerase activity. Indeed, cell therapy applications require avoiding cell immortalization. To this end, transient rather than constitutive telomerase activity may be advantageous for safety, for example, if the increase in telomerase activity is not only brief, but also elongates telomeres rapidly enough that treatment does not need to be continually repeated.

[0052] Thus, there is a need for therapies that safely lengthen telomeres to potentially prevent, delay, ameliorate, or treat these and other conditions and diseases, to do the same in the gradual decline in physical form and function and mental function that accompanies chronological aging, and to enable cell therapy and regenerative medicine. Additionally, there is a need for improved methods of delivering these therapies, e.g., nucleic acid molecules encoding telomerase, to cells.

[0053] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature and techniques used in connection with the chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0054] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "drug candidates" refers to one or a mixture of such candidates, reference to a "method" includes references to equivalent steps and methods known to those skilled in the art, and so forth.

[0055] The term "approximately" or "about" as used herein, when applied to one or more values ​​of interest, refers to a value that is similar in magnitude to and / or within a similar range to a stated reference value. In certain embodiments, the term "approximately" or "about" may refer to a range of values ​​that is 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) from a stated reference value, unless otherwise stated or otherwise clear from the context (unless such number exceeds 100% of possible values).

[0056] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the nearest tenth of the lower limit between the upper and lower limits of that range, as well as any other stated or intervening value within that stated range, is encompassed in the disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges that are also encompassed within the disclosure, subject to any specifically excluded end, within the stated range. Where the stated range includes one or both of the ends, ranges excluding either or both of those included ends are also included in the disclosure.

[0057] "G", "C", "A", "T", and "U" represent bases, which are usually guanine, cytosine, adenine, thymidine, and uracil, respectively. Nucleobases can form nucleosides by addition of a pentose sugar. When the sugar is ribose, the nucleoside is a ribonucleoside. Nucleosides can in turn form nucleotides by addition of one or more linker groups, such as phosphate groups. Nucleotides can in turn form polymers (polynucleotides), including short polymers (oligonucleotides). However, it will be understood that the terms "base", "nucleobase", "nucleoside", "ribonucleoside", "nucleotide", and "ribonucleotide" can also refer to modified bases, nucleobases, nucleosides, ribonucleosides, nucleotides, or ribonucleotides, or alternative replacement moieties (see, e.g., Table 2 and elsewhere herein), as further detailed below. It is well known to those skilled in the art that guanine, cytosine, adenine, thymidine, uracil can be substituted with other moieties without substantially impairing one or more of the specific properties (e.g., base pairing properties, translatability, or protein binding properties) of an oligonucleotide or polynucleotide containing a nucleotide having such a substituted moiety. Sequences containing such a substituted moiety are suitable for the compositions and methods featured in this disclosure. Similarly, it is well known to those skilled in the art that ribose can be substituted with other moieties without impairing the specific properties (e.g., base pairing properties, translatability, or protein binding properties) of an oligonucleotide or polynucleotide containing a nucleotide having such a substituted moiety. Sequences containing such a substituted moiety are suitable for the compositions and methods featured in this disclosure. Similarly, it is well known to those skilled in the art that phosphate can be substituted with other moieties without impairing the specific properties (e.g., base pairing properties, translatability, or protein binding properties) of an oligonucleotide or polynucleotide containing a nucleotide having such a substituted moiety. Sequences containing such a substituted moiety are suitable for the compositions and methods featured in this disclosure.

[0058] As used herein, the terms "polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length. The terms also encompass amino acid polymers that have been modified to include, for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety.

[0059] As used herein, the terms "identity" and "identical" refer to the percentage of exact matching residues in an alignment of a target sequence to a reference sequence, e.g., an alignment generated by the BLAST algorithm, for a polypeptide or polynucleotide target sequence. Identity is calculated over the entire length of the reference sequence, unless otherwise specified. Thus, when a reference sequence (as a query sequence) is aligned with a target sequence (as a target sequence), if at least x% (a truncation) of the residues in the target sequence align as exact matches with the corresponding residues in the query sequence, the target sequence "shares at least x% identity" with the reference sequence (the denominator being the entire length of the reference sequence plus the length of any gaps inserted in the reference sequence by aligning the reference sequence to the target sequence). If the target sequence has variable positions (e.g., residues designated by X), alignments to any residues in the query sequence are counted as matches. Sequence alignment can be performed using the NCBI Blast service (BLAST+ version 2.12.0) or another program that produces the same results.

[0060] The term "natural" or "wild type" as used herein refers to a nucleotide sequence, e.g., a gene, or a gene product, e.g., an RNA or a polypeptide, present in a wild-type cell, tissue, organ, or organism. The term "variant" as used herein refers to a variant of a reference polynucleotide or polypeptide sequence, e.g., a naturally occurring polynucleotide or polypeptide sequence, i.e., having less than 100% sequence identity with the reference polynucleotide or polypeptide sequence. In other words, a variant comprises at least one nucleotide difference (e.g., a nucleotide substitution, a nucleotide insertion, a nucleotide deletion) or one amino acid difference (e.g., an amino acid substitution, an amino acid insertion, an amino acid deletion) compared to a reference polynucleotide sequence, e.g., a naturally occurring polynucleotide or polypeptide sequence. For example, a variant may be a polynucleotide having 50% or more, 60% or more, or 70% or more sequence identity with a full-length naturally occurring polynucleotide sequence, e.g., 75% or 80% or more identity, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a full-length naturally occurring polynucleotide sequence. As another example, a variant may be a polypeptide having 70% or more sequence identity with a full-length native polypeptide sequence, e.g., 75% or 80% or more sequence identity, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a full-length native polypeptide sequence. Variants may also include variant fragments of a reference sequence, e.g., a native sequence, that share 70% or more sequence identity, e.g., 75% or 80% or more identity, e.g., 85%, 90%, or 95% or more, e.g., 98% or 99% identity with a reference sequence, e.g., a fragment of a native sequence.

[0061] As used herein, the term "codon optimization" refers to any process used to improve gene expression, increase the translation efficiency of a gene of interest, and / or reduce the immunogenicity of a polynucleotide by adapting to the codon bias of a host organism.

[0062] The term "treat" or "treatment" is used herein generally to mean obtaining a desired pharmacological and / or physiological effect with a therapeutic agent. The effect may be prophylactic in that it completely or partially prevents the disease or its symptoms, e.g., reduces the likelihood that the disease or its symptoms will occur in a subject, and / or it may be therapeutic in that it completely or partially reduces the symptoms or partially or completely cures the disease and / or the adverse reactions caused by the disease. "Treatment" as used herein encompasses any treatment of a mammalian disease, including: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not been diagnosed as having it; (b) inhibiting or slowing the onset or progression of the disease; or (c) relieving the disease, e.g., causing a diminution of the disease or symptoms associated with the disease. The therapeutic agent may be administered before, during, or after the onset of the disease. Of particular interest may be the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. In some embodiments, the treatment is performed before the function of the affected tissue is completely lost. In some embodiments, the subject therapy will be administered before a symptomatic stage of the disease; in some embodiments, during a symptomatic stage of the disease; in some embodiments, after a symptomatic stage of the disease.

[0063] In some embodiments, the therapeutic methods described herein treat a fibrotic or pulmonary disease, including but not limited to, fibrotic pulmonary disease.

[0064] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any subject for whom treatment or therapy is desired. The subject may be a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, rodents (e.g., rats, mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, e.g., a cynomolgus monkey. In some embodiments, the subject is a pet or service animal (e.g., a cat or dog).

[0065] As used herein, a subject "in need thereof" refers to any subject identified as suffering from or at risk of developing a fibrotic or pulmonary disease.

[0066] It is to be understood that the disclosure is not limited to the particular methodology, products, devices, and components described, as such methods, devices, and formulations may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is limited only by the appended claims.

[0067] I. Synthetic mRNA Synthetic ribonucleic acid (RNA) as used herein may refer to any RNA sequence that contains mutations (points or deletions) or additional nucleotides not found in the wild-type sequence. For example, synthetic TERT messenger RNA (mRNA) may refer to a wild-type sequence encoding the human TERT sequence flanked by the addition of 1, 2, 3, 10, 100 or more nucleotides. Similarly, the nucleotides themselves may be modified to encode amino acids different from the wild-type or to reduce immunogenicity of cells or tissues. The mRNA sequence in some embodiments may contain any of the following modifications, including, but not limited to, untranslated regions (UTRs), 5' caps, and polyadenosine tails. In some embodiments, the RNA may be circular and / or self-replicating.

[0068] Illustrative methods for producing circular mRNA are provided in: Chen et al. Science. 1995 Apr 21;268(5209):415-7; Perriman R.(2002)Circular mRNA Encoding for Monomeric and Polymeric Green Fluorescent Protein. In:Hicks BW(eds)Green Fluorescent Protein. Methods in Molecular Biology,vol 183.Humana Press; Wang et al. RNA. 2015 Feb;21(2):172-9.doi:10.1261 / rna.048272.114.Epub 2014 Dec 1; Wesselhoeft et al. Nat Commun. 2018 Jul 6;9(1):2629; and Wesselhoeft et al. Mol Cell. 2019 May 2;74(3):508-520.e4. Examples of methods for producing self-replicating mRNA are provided in: Tews BA, Meyers G. (2017) Self-Replicating RNA. In: Kramps T., Elbers K. (eds) RNA Vaccines. Methods in Molecular Biology, vol 1499. Humana Press; Leyman et al. Mol Pharm. 2018 Feb 5; 15(2): 377-384; and Huysmans et al. Mol Ther Nucleic Acids. 2019 Sep 6; 17: 388-395.

[0069] TERT mRNA In some embodiments, the composition may comprise a reverse transcriptase telomerase (TERT) mRNA sequence that treats one or more phenotypes or symptoms associated with fibrotic or pulmonary disease. In some embodiments, TERT mRNA refers to an mRNA that encodes any full-length functional fragment or portion of the TERT protein, including the wild-type sequence or a variant thereof.

[0070] In some embodiments, the TERT mRNA may comprise a codon-optimized sequence. In some embodiments, the TERT mRNA may comprise a uridine-depleted human TERT sequence. In some embodiments, the codon-optimized sequence may comprise SEQ ID NO: 1 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0071] In some embodiments, the TERT mRNA may comprise a mutant human TERT sequence. In some embodiments, the mutant human TERT mRNA may code for a Y707F mutation in the resulting peptide sequence. In some embodiments, the mutation in the TERT mRNA sequence codes for a mutation in a nuclear export signal that may result in nuclear retention of the TERT peptide. In some embodiments, the mutant TERT mRNA sequence may comprise SEQ ID NO: 2 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0072] In some embodiments, the mouse TERT mRNA may comprise a codon-optimized sequence. In some embodiments, the TERT mRNA may comprise a uridine-depleted mouse TERT sequence. In some embodiments, the codon-optimized sequence may comprise SEQ ID NO: 3, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0073] In some embodiments, the mouse TERT mRNA may comprise a mutant mouse TERT sequence. In some embodiments, the mutant mouse TERT mRNA may encode a Y707F mutation in the resulting peptide sequence. In some embodiments, the mutation in the TERT mRNA sequence encodes a mutation in a nuclear export signal that may result in nuclear retention of the TERT peptide. In some embodiments, the mutant mouse TERT mRNA sequence may comprise SEQ ID NO: 4, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0074] In some embodiments, the mouse TERT mRNA may comprise a mutant mouse TERT sequence. In some embodiments, the mutant mouse TERT mRNA may code for a Y697F mutation in the resulting peptide sequence. In some embodiments, the mutation in the TERT mRNA sequence codes for a mutation in a nuclear export signal that may result in nuclear retention of the TERT peptide. In some embodiments, the mutant mouse TERT mRNA sequence may comprise the sequence of SEQ ID NO:5 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0075] The compositions of the present disclosure may include a ribonucleic acid, for example, a synthetic ribonucleic acid encoding telomerase reverse transcriptase (TERT), and telomeres are extended in cells treated with the compound. The ribonucleic acid used for transient expression of TERT may include a ribonucleic acid encoding a TERT protein. The ribonucleic acid may further include one or more sequences that affect the expression and / or stability of the ribonucleic acid in the cell. For example, the ribonucleic acid may include a 5' cap and an untranslated region (UTR) 5' and / or 3' of the coding sequence. The ribonucleic acid may further include a 3' tail, such as a polyA tail. For example, the polyA tail may increase the stability of the ribonucleic acid. In some embodiments, the polyA tail includes at least 25 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 125 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 225 nucleotides, at least 250 nucleotides. In some embodiments, the polyA tail comprises between 1 and 25 nucleotides, between 25 and 50 nucleotides, between 50 and 75 nucleotides, between 75 and 100 nucleotides, between 100 and 125 nucleotides, between 125 and 150 nucleotides, between 150 and 175 nucleotides, between 175 and 200 nucleotides, between 200 and 225 nucleotides, or between 225 and 250 nucleotides, inclusive of the endpoints of each range. In some embodiments, the polyA tail comprises between 100 and 200 nucleotides, inclusive of the endpoints.

[0076] In some embodiments, the 5' cap of the ribonucleic acid is a non-immunogenic cap. In some embodiments, the 5' cap may increase translation of the ribonucleic acid. In some embodiments, the 5' cap may be treated with a phosphatase to modulate the natural immunogenicity of the ribonucleic acid. In some embodiments, the 5' cap is an anti-reverse cap analog ("ARCA"), such as a 3'-O-Me-m7G(5')ppp(5')G RNA cap structure analog. In some embodiments, the 5' cap is m7G(5')ppp(5')(2'OmeA)pG (also known as CleanCap® AG). In some embodiments, the 5' cap is m7(3'OmeG)(5')ppp(5')(2'OmeA)pG (also known as CleanCap® AG(3'OMe)).

[0077] The above features or other features may increase the translation of the TERT protein encoded by the ribonucleic acid, or may increase or decrease the stability of the ribonucleic acid itself in a cell type specific or cell type independent manner, or both. In some embodiments, the 5'UTR and / or 3'UTR are from genes with highly stable and / or rapidly translated mRNAs, such as alpha-globin or beta-globin, c-fos, or tobacco etch virus. In some embodiments, the 5'UTR and 3'UTR are from different genes or from a species different from the species to which the composition is delivered. The UTR may also be a collection of portions of UTRs from mRNAs of different genes, which portions are selected to achieve a particular combination of translational stability and efficiency. The UTR may also include engineered sequences that confer properties to the RNA, such as cell type specific or cell type independent stability.

[0078] The ribonucleic acid of the present disclosure may contain one or more modified nucleosides and / or a primary sequence of nucleosides that modulate the translation, stability, or immunogenicity of the RNA. Most mature RNA molecules in eukaryotic cells contain modified versions of the standard unmodified RNA nucleosides, adenine, cytidine, guanosine, and uridine. For example, the 5' cap of the mature RNA contains modified nucleosides, and other modified nucleosides are often present elsewhere in the RNA. These modifications may prevent the RNA from being recognized as a foreign RNA. Synthetic RNA molecules made using certain nucleosides are much less immunogenic than unmodified RNA. Immunogenicity can be reduced by further purifying the synthetic mRNA, for example, by using high performance liquid chromatography (HPLC). The modified nucleosides may be selected, for example, from the nucleosides listed below. The nucleoside is, in some embodiments, pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methoxyuridine, or 5-methylcytidine. The primary sequence can be modified in a way that increases or decreases immunogenicity. Depending on some circumstances, it may be desirable for the modified RNA to retain some immunogenicity.

[0079] Thus, in some embodiments, the ribonucleic acid of the composition comprises 1-methylpseudouridine, pseudouridine, 5-methoxyuridine (5-moU), 2-thiouridine, 5-methylcytidine, or another modified nucleoside. Modified nucleosides found in eukaryotic cells include: m1A 1-methyladenosine, m6A N6-methyladenosine, Am2'-O-methyladenosine, i6A N6-isopentenyladenosine, io6A N6-(cis-hydroxyisopentenyl)adenosine, ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, g6A N6-glycinylcarbamoyladenosine, t6A N6-threonylcarbamoyladenosine, ms2t6A 2-methylthio-N6-threonylcarbamoyladenosine, Ar(p)2'-O-ribosyladenosine (phosphate), m6 2A N6,N6-dimethyladenosine, m6Am N6,2'-O-dimethyladenosine, m6 2Am N6,N6,2'-O-trimethyladenosine, m1Am 1,2'-O-dimethyladenosine, m3C 3-methylcytidine, m5C 5-methylcytidine, Cm 2'-O-methylcytidine, ac4C N4-acetylcytidine, f5C 5-formylcytidine, m4C N4-methylcytidine, hm5C 5-hydroxymethylcytidine, f5Cm 5-formyl-2'-O-methylcytidine, m1G 1-methylguanosine, m2G N2-methylguanosine, m7G 7-methylguanosine, Gm 2'-O-methylguanosine, m2 2G N2,N2-dimethylguanosine, Gr(p)2'-O-ribosylguanosine (phosphate), yWwybutosine, o2yWperoxywybutosine, OhyWhydroxywybutosine, OhyW*hypomodifiedhydroxywybutosine, imGwybutosine, m2,7G N2,7-dimethylguanosine, m2,2,7G N2,N2,7-trimethylguanosine, Iinosine, m1I1-methylinosine, Im2'-O-methylinosine, Qqueuosine, galQgalactosyl-queuosine, manQmannosyl-queuosine, Ψpseudouridine, Ddihydrouridine, m5U5-methyluridine, Um2'-O-methyluridine, m5Um5,2'-O-dimethyluridine,m1Ψ 1-methylpseudouridine, Ψm2'-O-methylpseudouridine, s2U 2-thiouridine, ho5U 5-hydroxyuridine, chm5U 5-(carboxyhydroxymethyl)uridine, mchm5U 5-(carboxyhydroxymethyl)uridine, methyl ester mcm5U 5-methoxycarbonylmethyluridine, mcm5Um 5-methoxycarbonylmethyl-2'-O-methyluridine, mcm5s2U 5-methoxycarbonylmethyl-2-thiouridine, ncm5U 5-carbamoylmethyluridine, ncm5Um 5-carbamoylmethyl-2'-O-methyluridine, cmnm5U 5-carboxymethylaminomethyluridine, m3U 3-methyluridine, m1acp3Ψ 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, cm5U 5-Carboxymethyluridine, m3Um 3,2'-O-dimethyluridine, m5D 5-methyldihydrouridine, τm5U 5-taurinomethyluridine, τm5s2U 5-Taurinomethyl-2-thiouridine, 2-Aminoadenosine, 2-Amino-6-chloropurine riboside, 8-Azaadenosine, 6-Chloropurine riboside, 5-Iodocytidine, 5-Iodouridine, Inosine, 2'-O-Methylinosine, Xanthosine, 4-Thiouridine, O6-Methylguanosine, 5,6-Dihydrouridine, 2-Thiocytidine, 6-Azacytidine, 6-Azauridine, 2'-O-Methyl-2-aminoadenosine, 2'-O-Methylpseudouridine, N1-Methyladenosine, 2'-O-Methyl-5-methyluridine, 7-Deazaguanosine, 8-Azidoadenosine, 5-Bromo Bromocytidine, 5-bromouridine, 7-deazaadenosine, 5-aminoallyluridine, 5-aminoallylcytidine, 8-oxoguanosine, 2-aminopurine-riboside, pseudoisocytidine, N1-methylpseudouridine, 5,6-dihydro-5-methyluridine, N6-methyl-2-aminoadenosine, 5-carboxycytidine, 5-hydroxymethyluridine, thienoguanosine, 5-hydroxycytidine, 5-formyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-methoxycytidine, thienouridine, 5-carboxymethylesteruridine, thienocytidine,8-Oxoadenosine, Isoguanosine, N1-Ethylpseudouridine, N1-Methyl-2'-O-Methylpseudouridine, N1-Methoxymethylpseudouridine, N1-Propylpseudouridine, 2'-O-Methyl-N6-Methyladenosine, 2-Amino-6-Cl-purine-2'-deoxyriboside, 2-Amino-2'-deoxyadenosine, 2-Aminopurine-2'-deoxyriboside, 5-Bromo-2'-deoxycytidine, 5-Bromo-2'-deoxyuridine, 6-Chloropurine-2'-deoxyriboside, 7-Deaza-2'-deoxy Oxyadenosine, 7-deaza-2'-deoxyguanosine, 2'-deoxyinosine, 5-propynyl-2'-deoxycytidine, 5-propynyl-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-iodo-2'-deoxycytidine, 5-iodo-2'-deoxyuridine, N6-methyl-2'-deoxyadenosine, 5-methyl-2'-deoxycytidine, O6-methyl-2'-deoxyguanosine, N2-methyl-2'-deoxyguanosine, 8-oxo-2'-deoxyadenosine, 8-oxo-2'-deoxyguanosine , 2-thiothymidine, 2'-deoxy-P-nucleoside, 5-hydroxy-2'-deoxycytidine, 4-thiothymidine, 2-thio-2'-deoxycytidine, 6-aza-2'-deoxyuridine, 6-thio-2'-deoxyguanosine, 8-chloro-2'-deoxyadenosine, 5-aminoallyl-2'-deoxycytidine, 5-aminoallyl-2'-deoxyuridine, N4-methyl-2'-deoxycytidine, 2'-deoxyzebularine, 5-hydroxymethyl-2'-deoxyuridine, 5-hydroxymethyl-2'-deoxycytidine, 5 -propargylamino-2'-deoxycytidine, 5-propargylamino-2'-deoxyuridine, 5-carboxy-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-[(3-indolyl)propionamido-N-allyl]-2'-deoxyuridine, 5-carboxy-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, 7-deaza-7-propargylamino-2'-deoxyadenosine, 7-deaza-7-propargylamino-2'-deoxyguanosine, biotin-16-aminoallyl-2'-dUTP,Biotin-16-aminoallyl-2'-dCTP, Biotin-16-aminoallylcytidine, N4-Biotin-OBEA-2'-deoxycytidine, Biotin-16-aminoallyluridine, Dabsyl-5-3-aminoallyl-2'-dUTP, Desthiobiotin-6-aminoallyl-2'-deoxycytidine, Desthiobiotin-16-aminoallyl-uridine, Biotin-16-7-deaza-7-propargylamino-2'-deoxyguanosine, Cyanine 3-5-propargylamino-2'-deoxycytidine, Cyanine 3-6-propargyl Amino-2'-deoxyuridine, Cyanine 5-6-propargylamino-2'-deoxycytidine, Cyanine 5-6-propargylamino-2'-deoxyuridine, Cyanine 3-aminoallylcytidine, Cyanine 3-aminoallyluridine, Cyanine 5-aminoallylcytidine, Cyanine 5-aminoallyluridine, Cyanine 7-aminoallyluridine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-O -methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methyluridine, puromycin, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine, aracytidine, aurauridine, 2'-azido-2'-deoxyadenosine, 2'-amino-2'-deoxyadenosine, araadenosine, 2'-fluoro-thymidine, 3'-O-methyladenosine, 3'-O-methylcytidine, 3'-O-methylguanosine , 3'-O-methyluridine, 2'-azido-2'-deoxyguanosine, araguanosine, 2'-deoxyuridine, 3'-O-(2-nitrobenzyl)-2'-deoxyadenosine, 3'-O-(2-nitrobenzyl)-2'-deoxyinosine, 3'-deoxyadenosine, 3'-deoxyguanosine, 3'-deoxycytidine, 3'-deoxy-5-methyluridine, 3'-deoxyuridine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyuridine, 2',3'-dideoxythymidine,2',3'-dideoxycytidine, 3'-azido-2',3'-dideoxyadenosine, 3'-azido-2',3'-dideoxythymidine, 3'-amino-2',3'-dideoxyadenosine, 3'-amino-2',3'-dideoxycytidine, 3'-amino-2',3'-dideoxyguanosine, 3'-amino-2',3'-dideoxythymidine, 3'-azido-2',3'-dideoxycytidine, do-2',3'-dideoxyuridine, 5-bromo-2',3'-dideoxyuridine, 2',3'-dideoxyinosine, 2'-deoxyadenosine-5'-O-(1-thiophosphate), 2'-deoxycytidine-5'-O-(1-thiophosphate), 2'-deoxyguanosine-5'-O-(1-thiophosphate), 2'-deoxythymidine-5'-O-(1-thiophosphate), adenosine-5'-O-(1-thiophosphate) ), cytidine-5'-O-(1-thiophosphate), guanosine-5'-O-(1-thiophosphate), uridine-5'-O-(1-thiophosphate), 2',3'-dideoxyadenosine-5'-O-(1-thiophosphate), 2',3'-dideoxycytidine-5'-O-(1-thiophosphate), 2',3'-dideoxyguanosine-5'-O-(1-thiophosphate), 3'-deoxythymidine-5'-O-(1-thiophosphate), 3'-azido-2',3'-dideoxythymidine-5'-O-(1-thiophosphate), 2',3'-dideoxyuridine-5'-O-(1-thiophosphate), 2'-deoxyadenosine-5'-O-(1-boranophosphate), 2'-deoxycytidine-5'-O-(1-boranophosphate), 2'-deoxyguanosine-5'-O-(1-boranophosphate), and 2'-deoxythymidine-5'-O-(1-boranophosphate).

[0080] Without being bound by theory, the presence of modified nucleosides and / or sequences of nucleosides that alter the secondary structure of the RNA and / or the binding of the RNA to RNA-binding proteins or microRNAs may allow the mRNA to avoid activation of immune responses mediated by various receptors, including Toll-like receptors and RIG-1. Non-immunogenic mRNAs have been used as therapeutics in mice via localized delivery. Kormann et al. (2011) Nature Biotechnology 29:154-157. In some embodiments, the ribonucleic acid comprises two or more of the above nucleosides, or a combination of the above nucleosides. In some embodiments, the ribonucleic acid comprises 1-methylpseudouridine, 5-methoxyuridine, or pseudouridine and 5-methylcytidine.

[0081] In some embodiments, an immune response to the mRNA may be desired, and the RNA may be modified to induce an optimal level of natural immunity. In other embodiments, an immune response to the mRNA may be undesirable, and the RNA may be modified to minimize such a response. The RNA may be modified in either situation.

[0082] The ribonucleic acid molecule may be a synthetic ribonucleic acid. As used herein, the term "synthetic" may in some embodiments mean that the ribonucleic acid is prepared using molecular biology tools under human direction, for example, as described below. Synthetic ribonucleic acids may be prepared by in vitro synthesis, for example, using cell extracts or purified enzymes and nucleic acid templates. Synthetic ribonucleic acids may in some embodiments be prepared by partial or complete chemical synthesis. Alternatively, or in addition, in some embodiments, synthetic ribonucleic acids may be prepared by engineering expression in a cell, followed by disruption of the cell and at least partial purification of the ribonucleic acid.

[0083] The ribonucleic acids of the present disclosure may be prepared using a variety of techniques, as will be appreciated by those of skill in the art. In some embodiments, the ribonucleic acids may be prepared by in vitro synthesis. In some embodiments, the ribonucleic acids may be prepared by chemical synthesis. In some embodiments, the ribonucleic acids may be prepared by a combination of in vitro and chemical synthesis. As noted above, the term "synthetic" should be understood to include ribonucleic acids prepared by chemical synthesis, by in vitro synthesis, by in vivo expression and at least partial purification, or by a combination of such or other chemical or molecular biological methods.

[0084] In some embodiments, the ribonucleic acid may be purified. As mentioned above, purification may reduce the immunogenicity of the ribonucleic acid, which may be advantageous in some circumstances. In some embodiments, the ribonucleic acid is purified by one or more of HPLC, DNAse treatment, protease treatment, or affinity capture and elution.

[0085] The protein structure of TERT may include at least three distinct domains: a long extension at the amino terminus (N-terminal extension, NTE) that contains a conserved domain and an unstructured linker region; a catalytic reverse transcriptase domain in the middle of the primary sequence that contains seven conserved reverse transcriptase (RT) motifs; and a short extension at the carboxyl terminus. In some embodiments, the ribonucleic acid encodes the full-length TERT. In some embodiments, the ribonucleic acid encodes the catalytic reverse transcriptase domain of TERT. In some embodiments, the ribonucleic acid encodes a polypeptide having TERT activity. TERT activity may be measured using known methods, including the telomerase repeat amplification protocol (TRAP).

[0086] The TERT encoded by the ribonucleic acid of the present disclosure can be mammalian, avian, reptile, or fish TERT.In some embodiments, TERT is mammalian TERT, such as human TERT.Meyerson et al.(1997)Cell 90:785-795;Nakamura et al.(1997)Science 277:955-959;Wick et al.(1999)Gene 232:97-106.

[0087] The amino acid sequences of the two human TERT isoforms are available as NCBI reference sequences: NP_937983.2 and NP_001180305.1.

[0088] The amino acid sequence of human TERT isoform 1 may comprise or consist of the sequence of SEQ ID NO: 6 (also set forth in GenBank Accession No. NP_937983.2).

[0089] The nucleic acid sequence of human TERT isoform 1 may comprise or consist of the sequence of SEQ ID NO: 7 (also set forth in GenBank Accession No. NM_198253.3).

[0090] The amino acid sequence of human TERT isoform 2 may comprise or consist of the sequence of SEQ ID NO: 8 (also set forth in GenBank Accession No. NP_001180305.1).

[0091] The amino acid sequence of human TERT isoform 2 may comprise or consist of the sequence of SEQ ID NO: 9 (also set forth in GenBank Accession No. NM_001193376.3).

[0092] In some embodiments, the human TERT mRNA may comprise a wild-type TERT sequence, which may comprise the sequence of SEQ ID NO: 30, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0093] In some embodiments, the mouse TERT mRNA may comprise a wild-type TERT sequence, which may comprise SEQ ID NO: 31, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0094] In some embodiments, the TERT mRNA may comprise a nucleic acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1-5, 7, 9, or 30.

[0095] In some embodiments, a TERT mRNA may encode a modified TERT protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to SEQ ID NO:6 or 8 while retaining substantial TERT activity. In some embodiments, a TERT mRNA may encode an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6 or SEQ ID NO:8.

[0096] In other embodiments, the TERT mRNA may encode an amino acid sequence having the following mutations relative to the amino acid sequence of SEQ ID NO:6: L55Q, P65A, V70M, A202T, A279T, V299M, H412Y, a deletion of residue 441, R522K, K570N, R631Q, G682D, V694M, Y697F, P704S, Y707F, A716T, P721R, T726M, Y772C, P785L, V791I, R811C, L841F, R865H, V867M, R901W, K902N, P923L, S948R, R979W, V1025F, A1062T, V1090M, T1110M, and / or F1127L. In some embodiments, the TERT mRNA can encode a TERT isoform in which the translated protein lacks amino acid residues 711-722, 764-807, 808-1132, or 885-947 relative to the amino acid sequence of SEQ ID NO: 6. In some embodiments, about 1, about 5, about 10, about 20, or about 100 amino acids before and after the domain are also deleted from the amino acid sequence of SEQ ID NO: 6.

[0097] In some embodiments, the TERT The mRNA may encode an amino acid sequence in which one or more of the protein regions have been deleted or repeated relative to the amino acid sequence of SEQ ID NO:6, as follows: residues 1-230 corresponding to RNA interaction domain 1, residues 58-197 corresponding to a "GQ" residue motif, residues 137-141 associated with telomeric DNA specificity and primer extension, residues 210-320 corresponding to a disordered region, residues 231-324 associated with a linker sequence, residues 301-538 associated with oligomerization, residues 325-550 or 460-594 corresponding to an RNA interaction domain, residues 376-521 corresponding to a "QFP" residue motif, residues 397-417 corresponding to a "CP" residue motif, residues 825-884 corresponding to a DNA repeat template, residues 618-729 corresponding to a reverse transcriptase-like element, residues 914-928 associated with oligomerization, residues 930-934 associated with a primer grip sequence, and / or residues 936-1132 corresponding to the C-terminus. In some embodiments, about 1, about 5, about 10, about 20, or about 100 amino acids before and after the domain are also deleted or repeated.

[0098] In some embodiments, TERT mRNA can comprise or consist of a nucleotide sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to any subsequence of the disclosed nucleic acid sequences, e.g., any 100 base pairs (bp), 200 bp, 300 bp, 400 bp, 500 bp or more of the disclosed nucleic acid sequences. In some embodiments, the TERT mRNA can encode an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the disclosed polypeptide sequences, or any subsequence of the disclosed polypeptide sequences, e.g., any one of any 50 amino acids (aa), 100 aa, 200 aa, 300 aa, 400 aa, 500 aa or more of the disclosed polypeptide sequences.

[0099] Non-limiting TERT sequences of the present disclosure include the TERT nucleic acid and amino acid sequences listed in Table 1. [Table 1]

[0100] In some embodiments of the compositions and methods of the present disclosure, the amino acid sequence of TERT may comprise or consist of an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, of SEQ ID NOs: 6-8 or 10-13. In some embodiments of the compositions and methods of the present disclosure, some amino acid sequences of functional or non-functional TERT may comprise or consist of a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% identity to one or more of SEQ ID NOs: 6-8 or 10-13.

[0101] In some embodiments of the compositions and methods of the present disclosure, the nucleic acid sequence of TERT may comprise or consist of a sequence of SEQ ID NO: 1-5, 7, 9, 14-17, 30, or 31, or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0102] The amino acid sequence of non-human primate TERT isoform 1 may comprise or consist of the sequence of SEQ ID NO: 18 (also set forth in GenBank Accession No. XP_016808391.2).

[0103] The nucleic acid sequence of non-human primate TERT isoform 1 may comprise or consist of the sequence of SEQ ID NO: 19 (also set forth in GenBank Accession No. XM_016952902.2).

[0104] The amino acid sequence of non-human primate TERT isoform 2 may comprise or consist of the sequence of SEQ ID NO: 20 (GenBank Accession No. PNI27662.1).

[0105] The nucleic acid sequence of non-human primate TERT isoform 2 may comprise or consist of the sequence of SEQ ID NO: 21 (reverse machine translation of GenBank Accession No. PNI27662.1).

[0106] The amino acid sequence of non-human primate TERT isoform 3 may comprise or consist of the sequence of SEQ ID NO: 22 (also set forth in GenBank Accession No. PNI27663.1).

[0107] The nucleic acid sequence of non-human primate TERT isoform 3 may comprise or consist of the sequence of SEQ ID NO: 23 (reverse machine translation of GenBank Accession No. PNI27663.1).

[0108] The amino acid sequence of non-human primate TERT isoform 4 may comprise or consist of the sequence of SEQ ID NO: 24 (also set forth in GenBank Accession No. PNI27664.1).

[0109] The nucleic acid sequence of non-human primate TERT isoform 4 may comprise or consist of the sequence of SEQ ID NO: 25 (reverse machine translation of GenBank Accession No. PNI27664.1).

[0110] The amino acid sequence of non-human primate TERT isoform 5 may comprise or consist of the sequence of SEQ ID NO: 26 (also set forth in GenBank Accession No. PNI27665.1).

[0111] The nucleic acid sequence of non-human primate TERT isoform 5 may comprise or consist of the sequence of SEQ ID NO: 27 (reverse machine translation of GenBank Accession No. PNI27665.1).

[0112] The amino acid sequence of non-human primate TERT isoform 6 may comprise or consist of the sequence of SEQ ID NO: 28 (also set forth in GenBank Accession No. PNI27666.1).

[0113] The nucleic acid sequence of non-human primate TERT isoform 6 may comprise or consist of the sequence of SEQ ID NO: 29 (reverse machine translation of GenBank Accession No. PNI27666.1).

[0114] In some embodiments of the compositions and methods of the present disclosure, the amino acid sequence of TERT may comprise or consist of the sequence of SEQ ID NO: 6, 8, 10-13, 18, 20, 22, 24, 26, or 28, or an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0115] In some embodiments of the compositions and methods of the present disclosure, the nucleic acid sequence of TERT may comprise or consist of the sequence of SEQ ID NO: 1-5, 7, 9, 14-17, 19, 21, 23, 25, 27, 29, 30, or 31, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the ribonucleic acid may correspond to a naturally occurring gene sequence encoding the TERT protein described above, or may correspond to a variant permitted by redundancy in the genetic code, as will be understood by those of skill in the art. In some embodiments, codon selection may be optimized to optimize protein expression and / or reduce or increase immunogenicity using algorithms and methods known to those of skill in the art.

[0116] In some embodiments, the mRNA sequence may be synthesized as unmodified or modified mRNA. The mRNA may be modified to increase stability and / or avoid immune detection and degradation. The modified mRNA may include, for example, one or more of nucleotide modifications, nucleoside modifications, backbone modifications, sugar modifications, and / or base modifications. In some embodiments, the modified nucleoside is pseudouridine or a pseudouridine analog. In some embodiments, the pseudouridine analog is N-1-methylpseudouridine. In some embodiments, the modified nucleoside is 5-methoxyuridine. In some embodiments, the modified nucleoside used herein may include any of the moieties listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

[0117] In some embodiments, RNA, e.g., mRNA, can be synthesized from naturally occurring bases and / or base analogs (modified bases), including purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as analogs and derivatives thereof, e.g., 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl ... Isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), pseudouridine, N-1-methyl-pseudouridine, dihydro-uracil, 2-thio-uracil, 4-thio-uracil , 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil These include, but are not limited to, uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queuosine, beta-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0118] In some embodiments, RNA, e.g., mRNA, can be synthesized from naturally occurring nucleosides and / or nucleoside analogs (modified nucleosides), including nucleosides containing adenosine (A), guanosine (G), or pyrimidines (thymine (T), cytidine (C), uridine (U)), as well as nucleosides including analogs and derivatives thereof, e.g., 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2'-deoxypyridine ... ',3'-dideoxynucleosides, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, 2'-deoxynucleosides, -O-methylnucleosides, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine Tosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouridine, N-1-methyl-pseudouridine, dihydro-uracil, 2-thio-uracil, 4-thio-uridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5-fluorouridine, 5-bromo-uridine, 5-carboxymethylaminomethyl-uridine, 5-methyl-2-thiouridine, These include, but are not limited to, lysine, 5-methyl-uridine, N-uridine-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine, 5-methoxyaminomethyl-2-thiouridine, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouridine, queosine, beta-D-mannosyl-queosine, wybutoxosine, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0119] The preparation of such bases, nucleosides, nucleotides, and backbone analogs, modifications, and derivatives is known to those of skill in the art, e.g., U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. No. 5,262,530, and U.S. Pat. No. 5,700,642, all of which are incorporated by reference in their entireties.

[0120] In some embodiments, the uracil nucleosides of the mRNA are about 80%, about 90%, 95%, 99%, or 100% depleted and replaced with uracil nucleoside analogs, such as pseudouridine, 5-methoxyuridine, or N-1-methyl-pseudouridine.

[0121] In some embodiments, RNA may contain RNA backbone modification.Normally, backbone modification is a modification in which the phosphate of the backbone of the nucleotide contained in RNA is chemically modified.Exemplary backbone modifications may include, but are not limited to, the modification in which phosphodiester bond is replaced with a member of the group consisting of peptide, methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (e.g., cytidine 5'-O-(1-thiophosphate)), boranophosphate, and / or positively charged guanidinium group, or other means of replacing phosphodiester bond.

[0122] In some embodiments, the RNA may contain sugar modifications, including 2'O-methyl sugar modifications, 2'fluoro sugar modifications (e.g., 2'-fluororibose), 3'amino sugar modifications, 2'thio sugar modifications, 2'-O-alkyl sugar modifications, 5-methylthioribose, and sugar modifications of 2'-deoxy-2'-fluoro-ribonucleotides (2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine), 2'-deoxy-2'-deamine ribonucleotides (2'-amino-2'-deoxycytidine, These may include, but are not limited to, 2'-amino-2'-deoxyuridine), 2'-O-alkylribonucleotides, 2'-deoxy-2'-C-alkylribonucleotides (2'-O-methylcytidine, 2'-methyluridine), 2'-C-alkylribonucleotides and their isomers (2'-aracytidine, 2'-arauridine), or azidophosphates (2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine).

[0123] In some embodiments, the RNA may be synthesized from any of the nucleosides and nucleotides disclosed herein, or one or more of the nucleotide triphosphates, including any of the following nucleoside triphosphates: 2'-deoxyadenosine-5'-O-(1-thiotriphosphate), 2'-deoxycytidine-5'-O-(1-thiotriphosphate), 2'-deoxyguanosine-5'-O-(1-thiotriphosphate), 2'-deoxythymidine-5'-O-(1-thiotriphosphate), adenosine-5'-O-(1-thiotriphosphate), cytidine-5'-O-(1-thiotriphosphate), guanosine-5'-O-(1-thiotriphosphate), uridine-5'-O-(1-thiotriphosphate), 2',3'-dideoxy Cyadenosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxycytidine-5'-O-(1-thiotriphosphate), 2',3'-dideoxyguanosine-5'-O-(1-thiotriphosphate), 3'-deoxythymidine-5'-O-(1-thiotriphosphate), 3'-azido-2',3'-dideoxythymidine-5'-O-(1-thiotriphosphate) ), 2',3'-dideoxyuridine-5'-O-(1-thiotriphosphate), 2'-deoxyadenosine-5'-O-(1-boranotriphosphate), 2'-deoxycytidine-5'-O-(1-boranotriphosphate), 2'-deoxyguanosine-5'-O-(1-boranotriphosphate), and 2'-deoxythymidine-5'-O-(1-boranotriphosphate).

[0124] In some embodiments, the mRNA may include the addition of a "cap" at the N-terminal (5') end and a "tail" at the C-terminal (3') end. The presence of the cap may confer resistance to nucleases found in eukaryotic cells. The presence of the "tail" may protect the mRNA from exonuclease degradation.

[0125] Cap Construction In some embodiments, the mRNA may include a 5' cap structure. The 5' cap may include, for example, a triphosphate linkage and a guanine nucleotide in which the 7-nitrogen is methylated. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G. Naturally occurring cap structures initially include a 7-methylguanosine attached to the 5' end of the transcribed nucleotide via a triphosphate bridge, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added in the nucleus by the enzyme guanylyltransferase shortly after transcription initiation.

[0126] In some embodiments, the 5' cap may comprise m7(3'OmeG)(5')ppp(5')(2'OmeA)pG or (CleanCap™ 3'OMe) structure. In some embodiments, the 5' cap may comprise m7G(5')ppp(5')G. In some embodiments, an anti-reverse cap analog ("ARCA") or modified ARCA is a 5' cap in which the 2' or 3' OH group is replaced with -OCH3. In some embodiments, an ARCA comprises a 3'-O-Me-m7G(5')ppp(5')G structure. In some embodiments, the 5' cap comprises m7G(5')ppp(5')(2'OmeA)pG. Additional mRNA caps can include, but are not limited to, chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated caps (e.g., GpppG); methylated caps (e.g., m2'7GpppG), trimethylated cap analogs, or anti-reverse cap analogs (e.g., ARCA; m7,2'0meGpppG, m72'dGpppG, m7'3'0meGpppG, m7,3dGpppG, and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al, 'Wove anti-reverse cap analogs with superior translational properties', RNA, 9:1108-1122 (2003)).

[0127] In some embodiments, a suitable cap is 7-methylguanylic acid ("m7G") linked to the 5' end of the originally transcribed nucleotide via a triphosphate bridge, resulting in m7G(5')ppp(5')N, where N is any nucleoside. An embodiment of the m7G cap utilized in embodiments of the present disclosure is m7G(5')ppp(5')G. In some embodiments, the cap is a Cap0 structure. The Cap0 structure lacks a 2'-O-methyl residue on the ribose attached to bases 1 and 2. In some embodiments, the cap is a Cap1 structure. The Cap1 structure has a 2'-O-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. The Cap2 structure has a 2'-O-methyl residue attached to both bases 2 and 3.

[0128] A variety of m7G cap analogs are known in the art, many of which are commercially available. These include the m7 GpppG described above, as well as the ARCA 3'-OCH3 and 2'-OCH3 cap analogs (Jemielity, J. et al., RNA, 9:1108-1122 (2003)). Additional cap analogs that may be used in embodiments of the present disclosure include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10:1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E., et al., RNA, 13:1745-1755 (2007)), and cap analogs described in U.S. Patent Nos. 8,093,367 and 8,304,529 (herein incorporated by reference), including biotinylated cap analogs.

[0129] In some embodiments, the 5' cap is inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, CleanCap™, m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0130] In some embodiments, the 5' cap comprises or consists of an internal ribosome entry site (IRES). In some embodiments, the IRES is in the 5'UTR. In some embodiments, the 5' cap comprises or consists of a 2A self-cleaving peptide, e.g., one or more of P2A, T2A, E2A, and F2A.

[0131] Tail Structure The presence of a "tail" may help protect the mRNA from exonuclease degradation. PolyA tails are believed to stabilize natural messenger and synthetic sense RNA. Thus, in certain embodiments, long polyA tails can be added to mRNA molecules, thereby making the RNA more stable. PolyA tails can be added using a variety of techniques recognized in the art. For example, polyA polymerase can be used to add long polyA tails to synthetic or in vitro transcribed RNA (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Transcription vectors can also encode long polyA tails. Additionally, polyA tails can be added by direct transcription from PCR products. PolyA can also be ligated to the 3' end of sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2001). ndEd., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0132] In some embodiments, the mRNA may include a 3' poly(A) tail structure. The length of the polyA tail may be at least about 10, 50, 100, 200, 300, 400, or at least about 500 nucleotides. In some embodiments, the polyA tail at the 3' end of the mRNA may include about 10-300 adenosine nucleotides (e.g., about 10-200 adenosine nucleotides, about 10-150 adenosine nucleotides, about 10-100 adenosine nucleotides, about 20-70 adenosine nucleotides, or about 20-60 adenosine nucleotides). In some embodiments, the polyA tail is 120 adenosine nucleotides.

[0133] In some embodiments, the mRNA may include a 3' poly-C tail structure. The poly-C tail at the 3' end of the mRNA may include about 10-200 cytosine nucleotides (e.g., about 10-150 cytosine nucleotides, about 10-100 cytosine nucleotides, about 20-70 cytosine nucleotides, about 20-60 cytosine nucleotides, or about 10-40 cytosine nucleotides). The poly-C tail may be added to or replace the poly-A tail. In some embodiments, the length of the poly-A tail or poly-C tail is related to the stability of the modified sense mRNA and thus to the transcription of the protein. For example, since the length of the poly-A tail can affect the half-life of the sense mRNA molecule, the length of the poly-A tail may be adjusted to alter the level of resistance of the mRNA to nucleases, thereby providing more control over the time course of polynucleotide expression and / or polypeptide production.

[0134] 5' and 3' untranslated regions (UTRs) In some embodiments, the mRNA may include a 5' untranslated region (UTR) and / or a 3' UTR. In some embodiments, the 5' UTR may include one or more elements that affect mRNA stability or translation. In some embodiments, for example, the 5' UTR may include an iron-responsive element. In some embodiments, the 5' UTR may be about 50 to about 100, or about 50 to about 500 nucleotides in length. In some embodiments, the 3' UTR includes one or more of a polyA signal, a binding site for a protein that may affect mRNA stability or localization, or one or more binding sites for an miRNA. In some embodiments, the 3' UTR may be about 0 to about 50 nucleotides in length, or about 50 to about 100 nucleotides in length.

[0135] Exemplary 3' and 5' UTR sequences may be derived from mRNAs with relatively long half-lives (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) to enhance the stability of the sense mRNA molecule. For example, the 5' UTR sequence may include a partial sequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene, or a fragment thereof, to improve nuclease resistance and / or improve the half-life of the polynucleotide. In general, these modifications include modifications made to improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to its unmodified counterpart, e.g., to improve the resistance of such polynucleotides to nuclease digestion in vivo.

[0136] In some embodiments, the UTR may improve tissue-specific expression, for example in the lung. In some embodiments, the 3'UTR is a mouse alpha globin 3'UTR. In some embodiments, the 3'UTR comprises a sequence of SEQ ID NO: 32 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0137] In some embodiments, the 3'UTR is a wild-type human β-globin 3'UTR. In some embodiments, the 3'UTR comprises a sequence of SEQ ID NO: 33, or a nucleic acid at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0138] In some embodiments, the 3'UTR is a variant human beta globin 3'UTR. In some embodiments, the 3'UTR comprises a nucleic acid sequence of SEQ ID NO: 34, or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0139] In some embodiments, the 5'UTR is a synthetic 5'UTR. In some embodiments, the 5'UTR comprises a sequence of SEQ ID NO: 35, or a nucleic acid at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0140] In some embodiments, the 5'UTR is a human beta globin 5'UTR. In some embodiments, the 5'UTR comprises a sequence of SEQ ID NO: 36, or a nucleic acid at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0141] In some embodiments, the UTRs can be any of those described in PCT Application No. WO2017053297A1 and Patent No. US10519189B2, or functional variations thereof, both of which are incorporated herein in their entireties.

[0142] Exemplary Therapeutic TERT mRNA Sequences In some embodiments, TERT mRNA can refer to the full length mRNA sequence, i.e., coding and non-coding, that is delivered to a tissue, e.g., the lung. Exemplary sequences include sequences comprising mouse TERT in SEQ ID NOs: 37 and 38, and sequences comprising human TERT in SEQ ID NOs: 39 and 40.

[0143] In some embodiments, the mouse TERT mRNA comprises the sequence of SEQ ID NO:37 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0144] In some embodiments, the mouse TERT mRNA comprises the sequence of SEQ ID NO:38 or a nucleic acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0145] In some embodiments, the human TERT mRNA comprises the sequence of SEQ ID NO:39 or a nucleic acid that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto.

[0146] In some embodiments, the human TERT mRNA comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto, as set forth in SEQ ID NO:40.

[0147] In some embodiments, the TERT mRNA may comprise a nucleic acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 38-40.

[0148] The present disclosure provides a composition for extending telomeres in cells, the composition comprising a compound of the present disclosure as described above and an additional component. In some embodiments, the additional component comprises a telomerase RNA component (TERC). In some embodiments, the composition further comprises a telomerase RNA component (TERC). In some embodiments, the composition further comprises one or more additional components that can facilitate delivery of RNA to cells in vitro and / or in vivo. In some embodiments, the one or more additional components comprise a nanoparticle. In some embodiments, the nanoparticle comprises a lipid. In some embodiments, the nanoparticle or lipid comprises a coatosome-like lipid or compound of the present disclosure. In some embodiments, the nanoparticle or lipid comprises a compound of the present disclosure according to formula I.

[0149] II. Delivery Vehicles In some embodiments, one or more mRNAs can be delivered to cells or tissues via a delivery vehicle. In some embodiments, the delivery vehicle can be a nanoparticle. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP), including but not limited to nanoparticles containing lipids and / or polymers, liposomes, liposomal nanoparticles, cationic lipids, or exosomes. As used herein, a liposomal nanoparticle can be characterized as a microscopic vesicle having an internal aqueous space separated from the external medium by one or more bilayer membranes.

[0150] In some embodiments, the nanoparticles are polymeric nanoparticles. In some embodiments, the nanoparticles are metal nanoparticles. In other embodiments, the delivery vehicle comprises or consists of a recombinant virus or virus-like particle, such as an adenovirus, an adeno-associated virus (AAV), a herpes virus, or a retrovirus, such as a lentivirus. In some embodiments, the delivery vehicle comprises or consists of a modified viral vector, such as an adenovirus dodecahedron or a recombinant adenovirus assembly. In other embodiments, the delivery vehicle may comprise or consist of calcium phosphate nucleotides, aptamers, cell-penetrating peptides, or other vector tags.

[0151] In some embodiments, a suitable delivery vehicle is a lipid nanoparticle (LNP). Exemplary LNPs may include one or more different lipids and / or polymers. In some embodiments, LNPs include one or more of ionic lipids, cationic lipids, structural lipids, cholesterol, and / or insulating lipids (e.g., PEGylated lipids).

[0152] The composition of the present disclosure may include one or more components that can facilitate the delivery of RNA to cells. Collectively or in part, the components of the composition may include a delivery vehicle. In some embodiments, the delivery vehicle facilitates the targeting and uptake of the ribonucleic acid of the composition of the present disclosure into target cells. Exemplary delivery vehicles include, but are not limited to, nanoparticles, lipid nanoparticles (LNPs), liposomes, micelles, exosomes, cationic lipids, and natural or artificial lipoprotein particles.

[0153] Ionic Lipids In some embodiments of the present disclosure, the LNP may include an ionizable lipid, such as SS-OP or an analog thereof. The charge of the lipid depends on the pH of the surrounding solution, and may be an ionizable lipid. The ionizable lipid may also be cleavable. The ionizable lipid may be cationic in the pH range found in the endosomes or lysosomes of mammalian cells.

[0154] Ionizable lipids may refer to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH. In some embodiments, the LNPs may include ionizable lipids as disclosed in either WO2010 / 053572 or WO2012 / 170930, or variations thereof, both of which are incorporated by reference in their entireties.

[0155] In some embodiments, LNPs for pulmonary delivery of TERT mRNA may include one or more of the following: MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), DLin-MC3-DMA 4-(dimethylamino)-butanoate, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester, and / or cKK-E12. 3,6-Bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione. In some embodiments, the LNPs comprise 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA,1) and / or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate.

[0156] In some embodiments, the ionizable lipid may comprise SS-OP or an analog thereof. In some embodiments, the ionizable lipid is a compound of formula (1): [ka]

[0157] In equation (1), R 1a and R 1b R each independently represents an alkylene group having 1 to 6 carbon atoms, and may be linear or branched. The alkylene group may have 1 to 4 carbon atoms, or may have 1 to 2 carbon atoms. Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, and a neopentylene group. 1a and R 1b may each independently be a methylene group, an ethylene group, a trimethylene group, an isopropylene group, or a tetramethylene group, or may be an ethylene group.

[0158] R 1a is R 1b It may be different or the same.

[0159] X a and X b are each independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups, and / or a cyclic alkylene having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups and an alkylene tertiary amino group.

[0160] In the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, the alkyl group having 1 to 6 carbon atoms may be linear or branched. The alkyl group may be cyclic. The alkyl group may have 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, and an isopentyl group. Examples of the alkyl group having 1 to 6 carbon atoms include a neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group.

[0161] The specific structure of the non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group is X 1 It is expressed as: [ka]

[0162] X 1 R 5 represents an alkyl group having 1 to 6 carbon atoms, which may be linear, branched, or cyclic. The alkyl group may have 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, and an isopentyl group. Examples of the alkyl group having 1 to 6 carbon atoms include a neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, and a cyclohexyl group.

[0163] The number of carbon atoms in the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups may be 4 to 5. Specific examples of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups include an aziridylene group, an azetidylene group, a pyrrolidine group, a piperidylene group, an imidazolidylene group, a piperaziylene group, and optionally a pyrrolidine group, a piperidylene group, or a piperaziylene group.

[0164] The number is 2 to 5 carbon atoms, and X 2 This is a specific structure of an alkylene tertiary amino group containing one cyclic tertiary amino group represented by the following formula: [ka]

[0165] X 2 In the formula, p is 1 or 2. When p is 1, X 2 is a pyrrolidine group, and when p is 2, X 2 is a piperidylene group.

[0166] A specific structure of a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X 3 It is expressed as: [ka]

[0167] X 3 In this case, W is either 1 or 2. When w is 1, X 3 is an imidazolidylene group, and when w is 2, X 3 is a piperazylene group.

[0168] X a X b It may be different from or the same as.

[0169] R 2a and R 2beach independently represent an alkylene group or an oxydialkylene group having up to 8 carbon atoms, optionally each independently an alkylene group having up to 8 carbon atoms.

[0170] Alkylene groups having 8 or fewer carbon atoms may be linear or branched, but are optionally linear. The number of carbon atoms contained in the alkylene group is optionally 6 or fewer, and optionally 4 or fewer. Specific examples of alkylene groups having 8 or fewer carbon atoms include methylene, ethylene, propylene, isopropylene, tetramethylene, isobutylene, pentamethylene, hexamethylene, heptamethylene, octamethylene, and the like. Some embodiments include methylene, ethylene, propylene, and tetramethylene groups.

[0171] An oxydialkylene group having 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) via an ether bond, in which the total number of carbon atoms of the two alkylene groups is 8 or less. Here, the two alkylenes may be the same or different, but are optionally the same. Specific examples of oxydialkylene groups having 8 or less carbon atoms include oxydimethylene, oxydiethylene, oxydipropylene, and oxydibutylene groups.

[0172] R 2a and R 2b may be the same or different.

[0173] Y a and Y b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, and optionally are each independently an ester bond, an amide bond, or a carbamate bond. Y a and Y b The Y bond direction is not limited, but a and Y b is an ester bond, optionally, -Z a -CO---R 2a - and -Zb -CO-OR 2b -Structure.

[0174] Y a is Y b It may be different or the same as.

[0175] Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom. Represents the following. The number of carbon atoms contained in the aromatic compound is optionally 6 to 12 or 6 to 7. Furthermore, the number of aromatic rings contained in the aromatic compound is optionally 1.

[0176] The types of aromatic rings contained in aromatic compounds having 3 to 16 carbon atoms include, for aromatic hydrocarbon rings, benzene rings, naphthalene rings, and anthracene rings, and for aromatic heterocycles, imidazole rings, pyrazole rings, oxazole rings, isoxazole rings, thiazole rings, isothiazole rings, triazine rings, pyrrole rings, furanthiophene rings, pyrimidine rings, pyridazine rings, pyrazine rings, pyridine rings, purine rings, pteridine rings, benzimidazole rings, indole rings, benzofuran rings, quinazoline rings, phthalazine rings, quinoline rings, isoquinoline rings, coumarin rings, chromone rings, benzodiazepine rings, phenoxazine rings, phenothiazine rings, and acridine rings, and optionally, benzene rings, naphthalene rings, and anthracene rings. The aromatic rings may have a substituent. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Some examples include acetyl, methoxycarbonyl, methyl carbonate, moyl, acetoxy, acetamido, methoxycarbonylamino, fluorine, chlorine, bromine, iodine, methylsulfanyl, phenylsulfonyl, nitro, trifluoromethyl, cyano, methyl, ethyl, propyl, isopropyl, t-butyl, ureido, methoxy, ethoxy, propoxy, isopropoxy, t-butoxy, phenyl, and phenoxy groups.

[0177] Z aand Z b The specific structure of Z 1 Examples include: [ka]

[0178] s represents an integer of 0 to 3, t represents an integer of 0 to 3, u represents an integer of 0 to 4, and R4 is independently a substituent.

[0179] Z 1 In the formula, S is optionally an integer from 0 to 1.

[0180] Z 1 In the formula, T is optionally an integer from 0 to 2.

[0181] Z 1 In the formula, U is optionally an integer from 0 to 2.

[0182] Z 1In the formula, R4 is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms that does not inhibit the reaction in the synthesis process of the ionic lipid. Examples of the substituent include an acyl group having 2 to 4 carbon atoms, an alkoxycarbonyl group having 2 to 4 carbon atoms, a carbamoyl group having 2 to 4 carbon atoms, an acyloxy group having 2 to 4 carbon atoms, an acylamino group having 2 to 4 carbon atoms, an alkoxycarbonylamino group having 2 to 4 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, an alkylsulfanyl group having 1 to 4 carbon atoms, an alkylsulfonyl group having 1 to 4 carbon atoms, an arylsulfonyl group having 6 to 10 carbon atoms, a nitro group, a trifluoromethyl group, a cyano group, an alkyl group having 1 to 4 carbon atoms, a ureido group having 1 to 4 carbon atoms, an alkoxy group having 1 to 4 carbon atoms, an aryl group having 6 to 10 carbon atoms, and an aryloxy group having 6 to 10 carbon atoms. Examples of the substituent include acetyl, methoxycarbonyl, methylcarbamoyl, acetoxy, mido group (Mido group), group), a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylsulfanyl group, a phenylsulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a t-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, a isopropoxy group, a t-butoxy group, a phenyl group, and a phenoxy group. 4 If present, then each R 4 may be the same or different.

[0183] Z a is Z b It may be different from or the same as.

[0184] R 3a and R 3bare each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride or glutaric anhydride. Anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and optionally, each independently, a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a C12-22 aliphatic hydrocarbon group, and optionally, each independently, an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0185] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotaxolol, tocopherol, and tocotrienol. The fat-soluble vitamin having a hydroxyl group is optionally a tocopherol.

[0186] Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, and the like, and optionally, cholesterol or cholestanol.

[0187] The aliphatic hydrocarbon group having 12 to 22 carbon atoms may be linear or branched. The aliphatic hydrocarbon group may be saturated or unsaturated. In the case of an unsaturated aliphatic hydrocarbon group, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is usually 1 to 6, optionally 1 to 3, or 1 to 2. The unsaturated bonds include carbon-carbon double bonds and carbon-carbon triple bonds. The number of carbon atoms contained in the aliphatic hydrocarbon group is optionally 13 to 19 or 13 to 17. Examples of the aliphatic hydrocarbon group include an alkyl group, an alkenyl group, an alkynyl group, and the like, and optionally includes an alkyl group or an alkenyl group. Specific examples of aliphatic hydrocarbon groups having 12 to 22 carbon atoms include dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, heicosyl, docosyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, henicosenyl, docosenyl, dodecadienyl, tridecadienyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, henicosenyl, docosenyl, dodecadienyl, tridecadienyl, tetradecenyl, hexadecenyl, octa ... Examples of such groups include a cadienyl group, a pentadecadienyl group, a hexadecadienyl group, a heptadecadienyl group, an octadecadienyl group, a nonadecadienyl group, an icosadenyi group, a henicosadienyl group, a docosadienyl group, an octadecatrienyl group, an icosatrienyl group, a cosatetraenyl group, an icosapentaenyl group, a docosahexaenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, and a 1-decylundecyl group. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is optionally a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, or a tridecyl group, a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group.

[0188] In one embodiment of the present disclosure, R 3a and R 3bThe aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by the formula (I) is derived from a fatty acid. In this case, the carbonyl carbon derived from the fatty acid is contained in -CO-O- in formula (1). Specific examples of the aliphatic hydrocarbon group include a heptadecenyl group when linoleic acid is used as the fatty acid, and a heptadecenyl group when oleic acid is used as the fatty acid.

[0189] R 3a is R 3b It may be different from or the same as.

[0190] In one embodiment of the present disclosure, R 1a is R 1b is the same as X a X b is the same as R 2a is R 2b is the same as Y a is Y b is the same as Z a is Z b is the same as R 3a is R 3b is the same as:

[0191] Preferred examples of the ionic lipid represented by formula (1) include the following ionic lipids: ionic lipid (1-1); R1a and R1b are each independently an alkylene group having 1 to 6 carbon atoms (e.g., a methylene group, an ethylene group); Xa and Xb are each independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group (e.g., -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one to two tertiary amino groups (e.g., a piperidylene group); R 2a and R 2b are each independently an alkylene group having 8 or fewer carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y b each independently represents an ester bond or an amide bond; Z a and Z bare each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom (e.g., -CH-CH-, -CH-CH-CH-); R 3a and R 3b are each independently a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride or glutaric anhydride, or a hydrocarbon group which is an aliphatic group having 12 to 22 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0192] Ionic lipid (1-2); R 1a and R 1b are each independently an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); Xa and Xb are each independently a non-cyclic alkyl tertiary amino group having 1 to 3 carbon atoms and one tertiary amino group (e.g., -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one tertiary amino group (e.g., a piperidylene group); R 2a and R 2b are each independently an alkylene group having 6 or fewer carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y b each independently represents an ester bond or an amide bond; Za and Zb each independently represent a divalent group derived from an aromatic compound having 6 to 12 carbon atoms, one aromatic ring, and, optionally, a heteroatom (e.g., -CH-CH-, -CH-CH-CH-); R 3a and R 3b are each independently a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 19 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl). Ionic lipid (1-3); R 1a and R 1bare each independently an alkylene group having 1 to 2 carbon atoms (e.g., a methylene group, an ethylene group); a and X b are each independently 1 and [ka] (In the formula, R 5 is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X 2 : [ka] (wherein p is 1 or 2), R 2a and R 2b are each independently an alkylene group having 4 or fewer carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y bは each independently represents an ester bond or an amide bond; Z a and Z b are each independently 1 and [ka] In the formula, s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (optionally 0), and (R 4 ) u each independently represent a substituent; R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group).

[0193] Specific examples of ionic lipids according to formula 1 of the present disclosure include the following O-Ph-P3C1, O-Ph-P4C1, O-Ph-P4C2, O-Bn-P4C2, E-Ph-P4C2, L-Ph-P4C2, HD-Ph-P4C2, O-Ph-amide-P4C2, and O-Ph-C3M, as shown in Tables 3, 4, and 5. Table 3: Ionic lipids [Table 3-1] [Table 3-2] [Table 4]

[0194] In some embodiments, the delivery vehicle is an LNP capable of transfecting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a lung cell population, and the ionic lipid is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the molar percentage of the LNP.

[0195] In some embodiments, ionizable lipids are no more than 20%, no more than 30%, no more than 40%, no more than 50%, no more than 60%, no more than 70%, or no more than 90% of the molar percentage of the LNP. Examples of ionic lipids include, but are not limited to, (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

[0196] Lipids having the structure of formula I are shown in Table 5 below. For example, SS-OP is also called 0-Ph-P4C2. As used herein, the term "SS-OP analog" refers to a compound of formula I. [Table 5]

[0197] Cationic lipids In some embodiments, the lipid nanoparticles (LNPs) of the present disclosure include cationic lipids, such as DOTAP or variations thereof. The cationic lipids may be "permanently cationic lipids". The term cationic lipids may be cationic in the pH range found in mammalian physiological environments, such as blood or interstitial fluid. The cationic lipids may be composed of cationic amine moieties and lipid moieties, and the cationic amine moieties and polyanionic nucleic acids may interact to form positively charged liposomes or lipid membrane structures. Thus, cellular uptake may be facilitated and nucleic acids may be delivered into cells.

[0198] In some embodiments, the cationic lipid may be selected from one or more of the following: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylammonium bromide (DABB), or 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC). In some embodiments, the LNPs comprise an ionic lipid, the ionic lipid being selected from N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DOTAP ... N-Dioleyloxy-N,N-dimethyl-3-aminopropane (DODAP), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecylammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-Dioleyl-N,N-dimethyl Dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholestane) 1,2-N,N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), ...N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

[0199] In some embodiments, cationic lipid refers to cationic cholesterol lipid. In some embodiments of the present disclosure, the LNP comprises an imidazole cholesterol ester (ICE). In some embodiments, the ICE structure is substantially similar to the following: [ka]

[0200] In some embodiments of the present disclosure, the LNPs comprise 25-hydroxycholesterol (25OH Chol). In some embodiments, the 25OH Chol structure is substantially similar to the following: [ka]

[0201] In some embodiments of the present disclosure, the LNP comprises 20α-hydroxycholesterol 5-cholestene-3α.

[0202] In some embodiments, the 20α-hydroxycholesterol 5-cholestene-3α (also known as 20α-diol or 20α chol structure) is substantially similar to the following: [ka]

[0203] In some embodiments, the cationic lipid refers to dimethyldioctadecylammonium bromide (DDAB). In some embodiments of the present disclosure, the LNP comprises dimethyldioctadecylammonium bromide (DDAB). In some embodiments, the dimethyldioctadecylammonium bromide (DDAB) structure is substantially similar to the following: [ka]

[0204] structured lipids In some embodiments, the LNP comprises structured lipid. As used herein, structured lipid is lipid that imparts physical or chemical properties to the LNP in addition to or independent of charge. By way of example, structured lipid may tend to have shape, size, rigidity, hydrophobicity, or other properties that increase the therapeutic usefulness of the LNP, for example, by increasing stability, half-life, deformability, transfection efficiency, directionality, thermal stability, resistance to aggregation, membrane fluidity, or other parameters. In some embodiments, structured lipid is charge neutral, either because it lacks charged moieties, or because it is a zwitterion whose balanced charges sum to a net charge of zero.

[0205] In some embodiments, the LNPs are selected from the group consisting of 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol-monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG ...choline (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phos In some embodiments, the lipids may comprise a structured lipid selected from one or more of 1-stearoyl-2-oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or variants thereof.

[0206] In some embodiments, the LNPs may comprise one or more phosphatidyl lipids, such as phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine). In some embodiments, the LNPs may comprise sphingolipids, such as, but not limited to, sphingosine, ceramide, sphingomyelin, cerebrosides, and gangliosides. In some embodiments, the above-mentioned "structural" lipids contribute to the stability and / or specificity of the LNP composition.

[0207] Cholesterol-Based Lipids In some embodiments, the LNPs may include one or more cholesterol-based lipids, which may include, but are not limited to, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine.

[0208] PEGylated lipids In some embodiments of the present disclosure, the LNPs may include one or more PEGylated lipids. For example, polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, including derivatized ceramides (PEG-CER) (N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8PEG-2000 ceramide)), are contemplated by the present disclosure in combination with one or more ionic lipids and / or other lipids. In some embodiments, the PEGylated lipid includes a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18). In some embodiments, the PEGylated lipid DSPE-PEG-maleimide-lectin may be used. Other contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently attached to lipids with alkyl chain(s) C6-C20 in length. Without being bound to a particular theory, it is contemplated that the addition of PEGylated lipids may prevent complex aggregation, increase circulatory lifetime, and facilitate delivery of liposome-encapsulated mRNA to target cells.

[0209] In some embodiments, the lipid nanoparticle formulation may comprise, consist essentially of, or consist of any of those described in U.S. Patent Nos. 11,185,595; 9,868,693; 10,195,156; 9,877,919; 9,738,593; 10,399,937; 10,106,490; 9,738,593; 10,821,186; or 8 No. 5,058,069, each of which is incorporated by reference in its entirety; or as described in U.S. Patent Application Publication Nos. US20180085474A1, US20210259980A1, US20200206362A1, US20210267895A1, US20200283372A1, or US20200163878A1, each of which is incorporated by reference in its entirety.

[0210] Lipid Nanoparticle (LNP) Composition The following exemplary LNP formulations are not intended to be limiting. In some embodiments of the present disclosure, the LNPs may comprise an ionic lipid, e.g., SS-OP or an SS-OP analog, at a molar percentage of about 20, about 25, about 30, about 35, about 40, about 45, about 55, or about 60 relative to total lipid; and a cationic lipid, e.g., DOTAP, ICE, DDAB, or cationic cholesterol lipid, at a molar percentage of about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 relative to total lipid.

[0211] In some embodiments of the present disclosure, the LNPs comprise about 25% to about 35% molar percentage of cationic lipids, hi some embodiments, the LNPs comprise about 30% molar percentage of cationic lipids.

[0212] In some embodiments of the present disclosure, the LNP comprises a structured lipid. In some embodiments, the structured lipid is DOPC. In some embodiments, the LNP comprises DOPC at a molar ratio of about 1%, about 2%, about 3%, about 4%, or about 5% of the total lipid.

[0213] In some embodiments of the present disclosure, the LNPs are substantially free of structured lipids and / or contain up to 1% structured lipids.

[0214] In some embodiments of the present disclosure, the LNPs comprise cholesterol. In some embodiments, the LNPs comprise about 20% to about 40% molar percentage of cholesterol relative to total lipids. In some embodiments, the LNPs are substantially free of cholesterol.

[0215] In some embodiments of the present disclosure, the LNPs comprise an insulator lipid. In some embodiments, the LNPs comprise an insulator lipid at a molar ratio of about 0.1% to about 2%. In some embodiments, the LNPs comprise an insulator lipid at a molar ratio of about 0.5% to about 1.5%. In some embodiments, the LNPs are substantially free of insulator lipid.

[0216] Polymer Nanoparticles In some embodiments, suitable delivery vehicles are formulated using polymers as carriers alone or in combination with other carriers, including various lipids, as described herein. Thus, in some embodiments, liposomal delivery vehicles as used herein also encompass polymers, including nanoparticles. Suitable polymers include, for example, polyacrylates, polyaralkylcyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrin, protamine, polyethylene glycol (PEG)-modified (PEGylated) protamine, poly-D-lysine (PLL), PEGylated PLL, and polyethyleneimine (PEI). When PEI is present, it can be linear or branched PEI with a molecular weight in the range of 10-40 kDA, for example, branched PEI of 25 kDa (Sigma No. 408727). In some embodiments, the PEGylated lipid is 14:0 PEG2000PE and / or DMG-PEG2000.

[0217] lung targeting The delivery vehicles disclosed herein preferentially target the lung. In various embodiments, the delivery vehicles deliver polynucleotides, e.g., mRNA, to lung cells at a rate of 10, 10 or 10% relative to the liver. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 The LNP compositions provided herein can preferentially deliver and / or transfect polynucleotides, e.g., RNA, to the lung compared to the liver.

[0218] However, it will be understood that a certain level of delivery to non-target cells / organs can be tolerated without reducing efficacy in target organs / cells. In some embodiments, the lipid composition of the delivery vehicle enhances delivery to the lung compared to other lipid compositions known in the art. In other embodiments, the lipid composition of the delivery vehicle enhances delivery to the lung compared to other lipid compositions. In some embodiments, the presence or level of cholesterol enhances the delivery of the delivery vehicle, for example, LNP or extracellular vesicles, to the lung. In some embodiments, the delivery vehicle comprises an organ-specific targeting ligand that enhances delivery to a particular organ, for example, the lung.

[0219] III. Formulation of mRNA and Nanoparticle Delivery Vehicle Compositions Methods for the synthesis of mRNA and lipid nanoparticles (LNPs) are well established. For example, synthetic mRNAs containing a 5' cap, 5' and 3' UTR coding sequences, and a polyA tail can be synthesized from modified and unmodified nucleotides by in vitro transcription of a DNA template using an RNA polymerase, e.g., T7 RNA polymerase. The DNA template can be generated, for example, by PCR or plasmid amplification and restriction digestion, followed by purification.

[0220] Lipid nanoparticles (LNPs), liposomes, or polymeric nanoparticle delivery vehicles carrying mRNAs can be produced, for example, by mixing lipids or polymers in an organic solvent, such as ethanol, with one or more mRNAs in an aqueous buffer, followed by buffer exchange and concentration. In some embodiments, LNPs, liposomes, or polymeric nanoparticle delivery vehicles can be produced using a microfluidic device to rapidly mix reagents and form monodisperse particles of controlled size. For example, the microfluidic mixer can be a staggered herringbone mixer (SHM). For example, the microfluidic mixer can be manufactured by Precision Nanosystems (PNI) NanoAsssemblr. In other embodiments, LNPs, liposomes, or polymeric nanoparticle delivery vehicles can be produced in a T-mixer. In some embodiments, LNPs, liposomes, or polymeric nanoparticles can encapsulate mRNAs and / or bind to one or more mRNAs through electrostatic interactions. Buffer exchange and concentration of LNPs, liposomes, or polymeric nanoparticles can be performed with tangential flow filtration. In other embodiments, buffer exchange and concentration of the LNPs, liposomes, or polymeric nanoparticles can be performed by centrifugal ultrafiltration using membranes with a nominal molecular weight cut-off of 500,000 Da or less, e.g., 100,000 Da.

[0221] In some embodiments, the lipid nanoparticle (LNP) formulations provided herein are capable of transfecting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a lung cell population.

[0222] The form of the lipid membrane structure of the present disclosure is not particularly limited. For example, the form in which the lipid of the present disclosure is dispersed in an aqueous solvent may be a liposome (e.g., a single-layer liposome, a multi-layer liposome, etc.), a spherical micelle, a worm-like micelle, a lipid nanoparticle (LNP), or an unspecified layered structure.

[0223] The lipid membrane structure of the present disclosure may further contain other components. Examples of other components include lipids (phospholipids (e.g., phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylcholine), glycolipids, peptide lipids, cholesterol, ionic lipids, cationic lipids, PEGylated lipids, etc.), surfactants (e.g., 3[(3-cholamidopropyl)dimethylammonio]propanesulfonate, cholic acid sodium salt, octyl glycoside, ND-gluco-N-methylalkanamide), polyethylene glycol, proteins, etc. The content of other components in the lipid membrane structure of the present disclosure is usually 5 to 95 mol%, optionally 10 to 90 mol%, or 30 to 80 mol%.

[0224] The lipid membrane structure of the present disclosure is prepared by suspending the lipid and other components (such as lipids) of the present disclosure, and, if necessary, a tissue, in a suitable solvent or dispersion medium, for example, an aqueous solvent or an alcoholic solvent. It can be prepared by carrying out a procedure that induces crystallization.

[0225] Examples of the "operation for inducing organization" include an ethanol dilution method using a microchannel or vortex, a simple hydration method, such as ultrasonic treatment, heating, vortexing, ether injection, French press method, and cholic acid method. Examples of the operation include, but are not limited to, methods known per se, such as Ca2+ fusion method, freeze-thaw method, and reverse phase evaporation method.

[0226] Nucleic acids can be introduced into cells in vivo and / or in vitro by encapsulating the nucleic acids in a lipid membrane structure containing the ionic lipid of the present disclosure and contacting the structure with cells. Thus, the present disclosure provides a nucleic acid introduction agent comprising the ionic lipid or lipid membrane structure of the present disclosure.

[0227] The nucleic acid transfer agent of the present disclosure may transfer any nucleic acid into a cell. Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acid, DNA / RNA hybrid, and the like. The nucleic acid may be any one of 1 to 3 strands, but is optionally single-stranded or double-stranded. The nucleic acid may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers with non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs), etc.) or other oligomers with special bonds. The oligomers may contain nucleotides with configurations that allow base pairing or base bonding as found in DNA or RNA. Additionally, the nucleic acids may be substituted with, for example, known modified nucleic acids, labeled nucleic acids, capped nucleic acids, methylated nucleic acids, or one or more naturally occurring nucleotides known in the art, intramolecular nucleotide modified nucleic acids, nucleic acids with uncharged linkages (e.g., methylsulfonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing linkages (e.g., phosphorothioates), side groups such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (e.g., monosaccharides), nucleic acids including nucleic acids and mutual compounds (e.g., acridine, psoralen, etc.), nucleic acids containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), nucleic acids containing alkylating agents, and nucleic acids with modified linkages (e.g., alpha anomeric nucleic acids, etc.).

[0228] The type of DNA that can be used in the present disclosure is not particularly limited and can be appropriately selected according to the purpose of use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, and CpG oligo, and optionally, plasmid DNA, cDNA, and antisense DNA, or plasmid DNA. Circular DNA such as plasmid DNA can be appropriately digested with restriction enzymes and used as linear DNA.

[0229] The type of RNA that can be used in the present disclosure is not particularly limited and can be appropriately selected according to the purpose of use. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., optionally, siRNA, miRNA, shRNA, mRNA, antisense RNA, RNA replicon.

[0230] The nucleic acids used in the present disclosure are optionally purified by methods commonly used by those of skill in the art.

[0231] The nucleic acid transfer agent of the present disclosure encapsulating nucleic acid can be administered in vivo, for example, for the purpose of preventing and / or treating disease. Therefore, the nucleic acid used in the present disclosure is optionally a nucleic acid having preventive activity and / or therapeutic activity against a given disease (prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0232] In order to introduce nucleic acid into cells using the nucleic acid introduction agent of the present disclosure, when forming the lipid membrane structure of the present disclosure, the nucleic acid is encapsulated by coexisting with the target nucleic acid. The lipid membrane structure of the present disclosure is formed. For example, when the liposome is formed by the ethanol dilution method, the aqueous solution of nucleic acid and the ethanol solution of the components (such as lipids) of the lipid membrane structure of the present disclosure are vigorously mixed by a vortex or a microchannel. The solution is diluted with an appropriate buffer. When the liposome is formed by the simple hydration method, the components (such as lipids) of the lipid membrane structure of the present disclosure are dissolved in an appropriate organic solvent, the solution is placed in a glass container, the solvent is retained by drying under reduced pressure, and the solution is left to obtain a lipid film. Here, an aqueous solution of nucleic acid is added, hydrated, and then ultrasonicated with a sonicator. The present disclosure also provides the above lipid membrane structure in which such nucleic acid is encapsulated.

[0233] An example of a lipid membrane structure in which nucleic acids are encapsulated is LNPs in which nucleic acids are encapsulated by forming electrostatic complexes between nucleic acids and ionic lipids. These LNPs can be used as drug delivery systems to selectively deliver nucleic acids, etc., to specific cells. For example, DNA vaccines that introduce antigen genes, gene therapy drugs for tumors, and RNA into dendritic cells. It is useful for nucleic acid drugs that use interference to suppress the expression of target genes.

[0234] The particle size of the lipid membrane structure encapsulating the nucleic acid of the present disclosure is not particularly limited, but is optionally 10 nm to 500 nm or 20 nm to 200 nm. The particle size can be measured using a particle size distribution measuring device such as Zetasizer Nano (Malvern). The particle size of the lipid membrane structure can be appropriately adjusted depending on the preparation method of the lipid membrane structure.

[0235] The surface potential (zeta potential) of the lipid membrane structure of the present disclosure that encapsulates nucleic acid is not particularly limited, and may be -60 to +60 mV, -45 to 45 mV, -30 to +30 mV, -15 to +15 mV, or -10 to +10 mV. The zeta potential of the lipid membrane structure may be positive (>0 mV), +5 mV to +60 mV, +30 mV to +45 mV, or +10 mV to +45 mV. In conventional gene transfer, particles with a positive surface potential are mainly used. This is useful as a method for promoting electrostatic interaction with heparin sulfate on the negatively charged cell surface and promoting uptake into cells, but the surface positive charge is delivered into the cells. Release of nucleic acid from the carrier due to interaction with nucleic acid may be suppressed, and protein synthesis due to interaction with mRNA and delivered nucleic acid may be suppressed. This problem can be solved by adjusting the surface charge within the above range. The surface charge can be measured by using a zeta potential measuring device such as Zetasizer Nano. The surface charge of the lipid membrane structure can be adjusted by the composition of the components of the lipid membrane structure.

[0236] The lipid membrane surface pKa of the lipid membrane structure of the present disclosure (hereinafter referred to as liposome pKa) is not particularly limited, and may have a pKa of 5.5 to 7.2 or a pKa of 6.0 to 6.8. The pKa of the liposome is used as an index indicating that the lipid membrane structure taken up by endocytosis is susceptible to protonation of the lipid membrane structure in the weakly acidic environment in the endosome. The pKa of the liposome can be adjusted by the composition of the components of the lipid membrane structure.

[0237] The hemolytic activity (membrane fusion ability) of the lipid membrane structure of the present disclosure is not particularly limited, but may have no hemolytic activity (less than 5%) at physiological pH (pH 7.4) and may be endosomal. The higher the hemolytic activity, the more efficiently the nucleic acid can be delivered to the cytoplasm. However, if hemolytic activity exists at physiological pH, the nucleic acid will be delivered to unintended cells while present in the blood, resulting in reduced targeting and toxicity. Therefore, it is preferable to have hemolytic activity only in the endosomal environment as described above. The hemolytic activity can be adjusted by the composition of the components of the lipid membrane structure.

[0238] The nucleic acid encapsulated in the lipid membrane structure of the present disclosure can be brought into contact with cells, so that the encapsulated nucleic acid can be introduced into the cells. The cells can be cultured cell lines containing cancer cells, cells isolated from individuals or tissues, or tissue or tissue fragments of cells. Furthermore, the cells can be either adherent or non-adherent cells.

[0239] The step of contacting the lipid membrane structure encapsulating nucleic acid of the present disclosure with cells in vitro will be specifically described below.

[0240] The cells are suspended in an appropriate medium and cultured under appropriate conditions several days before contacting with the lipid membrane structures. When contacted with the lipid membrane structures, the cells may or may not be in a growth phase.

[0241] The medium at the time of contact may be a serum-containing medium or a serum-free medium, and the serum concentration in the medium may be 30% by weight or less, or 20% by weight or less. If the medium contains an excess of proteins such as serum, the contact between the lipid membrane structure and the cells may be inhibited.

[0242] The cell density at the time of contact is not particularly limited and can be appropriately set in consideration of the cell type. Generally, the cell density is 1×10 4 ~1×10 7 in the range of cells / mL.

[0243] For example, a suspension of the lipid membrane structure of the present disclosure in which the nucleic acid is encapsulated is added to the cells thus prepared. The amount of the suspension added is not particularly limited and can be appropriately set in consideration of the number of cells, etc. The concentration of the lipid membrane structure when contacted with the cells is not particularly limited as long as it can achieve the introduction of the target nucleic acid into the cells, but the lipid concentration is usually 1 to 100 nmol / mL, and may be 0.1 to 10 μg / mL.

[0244] After adding the above suspension to the cells, the cells are cultured. The culture temperature, humidity, and CO 2 The concentration and other factors are appropriately set in consideration of the cell type. When the cells are mammalian cells, the temperature is usually about 37° C., the humidity is about 95%, and the CO 2 The concentration is about 5%. In addition, the culture time can be appropriately set in consideration of conditions such as the cell type used, and may be in the range of 0.1 to 76 hours, or in the range of 0.2 to 24 hours, or may be 0.5 to 12 hours. If the culture time is too short, the nucleic acid may not be sufficiently introduced into the cells, and if the culture time is too long, the cells may become weak.

[0245] The nucleic acid is introduced into the cells by the above-mentioned culturing. The medium may be replaced with fresh medium, or new medium is added to the medium, and the culturing is continued. If the cells are mammalian cells, the fresh medium may contain serum or nutrient factors.

[0246] The lipid membrane structure of the present disclosure may further contain other components in addition to the ionic lipid of the present disclosure. Examples of other components include lipids (phospholipids (e.g., phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylcholine), glycolipids, peptide lipids, cholesterol, ionic lipids other than cationic lipids, PEG lipids, etc.), surfactants (e.g., 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, cholic acid sodium salt, octyl glycoside, ND-gluco-N-methylalkanamide), polyethylene glycol, proteins, etc.

[0247] The lipid membrane structure of the present disclosure is prepared by dispersing the ionic lipid of the present disclosure and other components (such as lipids) and, if necessary, a tissue in a suitable solvent or dispersion medium, for example, an aqueous solvent or an alcoholic solvent. It can be prepared by carrying out a procedure that induces crystallization.

[0248] Examples of the "operation for inducing organization" include an ethanol dilution method using a microchannel or vortex, a simple hydration method, such as ultrasonic treatment, heating, vortexing, ether injection, French press method, and cholic acid method. Examples of the operation include, but are not limited to, methods known per se, such as Ca2+ fusion method, freeze-thaw method, and reverse phase evaporation method.

[0249] Nucleic acids can be introduced into cells in vivo and / or in vitro by encapsulating the nucleic acids in a lipid membrane structure containing the ionic lipid of the present disclosure and contacting the structure with cells. Thus, the present disclosure provides a nucleic acid introduction agent comprising the ionic lipid or lipid membrane structure of the present disclosure.

[0250] The nucleic acid transfer agent of the present disclosure may transfer any nucleic acid into a cell. Examples of nucleic acids include, but are not limited to, DNA, RNA, RNA chimeric nucleic acid, DNA / RNA hybrid, etc. The nucleic acid may be any one of 1 to 3 strands, but may be single-stranded or double-stranded. The nucleic acid may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers with non-nucleotide backbones (e.g., commercially available peptide nucleic acids (PNAs), etc.) or other oligomers with special bonds. The oligomers may contain nucleotides with configurations that allow base pairing or base bonding as found in DNA or RNA.

[0251] The type of RNA that can be used in the present disclosure is not particularly limited and can be appropriately selected according to the purpose of use. For example, siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc., or siRNA, miRNA, shRNA, mRNA, antisense RNA, or RNA replicon.

[0252] The nucleic acids used in the present disclosure may be purified by methods commonly used by those of skill in the art.

[0253] The nucleic acid transfer agent of the present disclosure encapsulating the nucleic acid can be administered in vivo, for example, for the purpose of preventing and / or treating a disease. Therefore, the nucleic acid used in the present disclosure may be a nucleic acid having preventive and / or therapeutic activity against a certain disease (prophylactic / therapeutic nucleic acid). Examples of such nucleic acids include nucleic acids used in so-called gene therapy.

[0254] IV. Treatment Method The method of treatment described herein refers to the treatment of fibrotic disease and / or pulmonary disease and / or pulmonary fibrosis in a subject in need thereof by administering a composition comprising one or more TERT mRNA sequences. The compositions and methods of the present disclosure may be used to treat fibrotic conditions, including fibrosis. In some embodiments, the compositions and / or methods of use of the compositions of the present disclosure for treating fibrotic conditions, including fibrosis, induce TERT expression or increase TERT activity in lung cells. In some embodiments, the compositions and / or methods of use of the compositions of the present disclosure for treating fibrotic conditions, including fibrosis, do not induce cytotoxicity, tissue toxicity, or systemic toxicity. The compositions may be administered systemically, for example, intravenously.

[0255] Dosage and timing of telomerase reverse transcriptase (TERT) mRNA In the compositions and methods described herein, in some embodiments, TERT mRNA is administered to a subject in need thereof at a dose of about 0.001 mg / kg of the subject's body weight to about 2.0 mg / kg of the subject's body weight. In some embodiments, TERT mRNA is administered to a subject in need thereof at a dose of about 0.01 mg / kg; in some embodiments, at a dose of about 0.025 mg / kg; in some embodiments, at a dose of about 0.05 mg / kg; in some embodiments, at a dose of about 0.075 mg / kg; in some embodiments, at a dose of about 0.1 mg / kg; in some embodiments, at a dose of about 0.125 mg / kg; in some embodiments, at a dose of about 0.150 mg / kg; in some embodiments, at a dose of about 0.175 mg / kg; in some embodiments, at a dose of about 0.2 mg / kg; in some embodiments, at a dose of about 0.5 mg / kg; in some embodiments, at a dose of about 0.75 mg / kg; in some embodiments, at a dose of about 1.0 mg / kg; in some embodiments, at a dose of about 1.25 mg / kg; in some embodiments, at a dose of about 1.5 mg / kg; or in some embodiments, at a dose of about 2.0 mg / kg. In some embodiments, the TERT mRNA is administered to a subject in need thereof at a dose of 0.1 mg / kg. In some embodiments, the TERT mRNA is administered to a subject in need thereof at a dose of 0.125 mg / kg.

[0256] In some embodiments, TERT mRNA is administered once to a subject in need thereof. In some embodiments, TERT mRNA is administered twice, three times, four times, or five times or more to a subject in need thereof. In some embodiments, TERT mRNA is administered twice a week, every week, every two weeks, every four weeks, every six weeks, every twelve weeks, or every fifteen weeks. In some embodiments, TERT mRNA is administered monthly, every two months, every three months, every six months, once a year, continuously, or as determined by a physician.

[0257] TERT mRNA combination therapy In some embodiments, co-administration of TERT mRNA can be combined with other anti-fibrotic drugs used to treat fibrotic and / or pulmonary diseases. Drugs that can be used include, but are not limited to, nintedanib, pirfenidone, prednisone, azathioprine, cyclophosphamide, mycophenolate mofetil, pamrevlumab, and N-acetylcysteine.

[0258] Route of administration In some embodiments, TERT mRNA can be delivered orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intravaginally, intrarectally, or intraocularly. In exemplary embodiments, TERT mRNA can be administered intravenously or by inhalation.

[0259] Subjects and Treatments The treatment methods described herein are useful for treating pulmonary disease and / or pulmonary fibrosis in subjects in need thereof.Lung and pulmonary fibrotic diseases may include, but are not limited to, pulmonary fibrosis, lung cancer, familial pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary fibrosis with congenital dyskeratosis, interstitial lung disease (ILD), pneumonia, interstitial pneumonia, tuberculosis, bronchitis, emphysema, lung cancer, chronic obstructive pulmonary disease (COPD), aging-related fibrosis, pulmonary hypertension, asthma, and cystic fibrosis.

[0260] In some embodiments, the subject in need of the combination treatment described herein is a subject with a genetic disorder or a mutation in telomerase reverse transcriptase (TERT). In some embodiments, the subject does not have symptoms of fibrosis. In other embodiments, the subject has symptoms and the treatment completely or partially ameliorates the symptoms. In other embodiments, the treatment slows the progression of the symptoms.

[0261] In some embodiments, the subject in need of the treatment described herein is a subject with a genetic disorder or a mutation in telomerase reverse transcriptase (TERT). In some embodiments, the subject does not have symptoms of lung disease and / or pulmonary fibrosis. In other embodiments, the subject has symptoms and the treatment completely or partially ameliorates the symptoms. In other embodiments, the treatment slows the progression of the symptoms.

[0262] In some embodiments, the subject is a human.

[0263] In some embodiments, the efficacy of treatment may be measured in the lungs or pulmonary function may be performed by methods including, but not limited to, spirometry, plethysmography, methacholine inhalation challenge, 6-minute walk test, exhaled nitric oxide test, arterial blood gas test, lung volume test, pulmonary diffusion capacity, cardiopulmonary exercise test, ambulatory oximetry, respiratory muscle test, altitude simulation test, exercise challenge (with pre and post spirometry), shunt test (with 100% O 2 ), forced expiratory volume (FEV1), forced vital capacity (FVC), and maximum ventilation (MVV).

[0264] In some embodiments, administration of TERT mRNA reduces fibrotic tissue in a subject by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 100% during and / or after the treatment period.

[0265] In some embodiments, administration of TERT mRNA stops or slows the increase in fibrotic tissue over time compared to a subject not receiving treatment, in some embodiments, administration of TERT mRNA slows the increase in the amount of fibrotic tissue in a subject by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or at least 100% during and / or after a treatment period.

[0266] In some embodiments, administration of TERT mRNA increases lung function compared to a subject not receiving treatment, in some embodiments, administration of TERT mRNA increases the subject's lung function by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% during and / or after the treatment period.

[0267] In some embodiments, administration of TERT mRNA extends survival compared to subjects not receiving treatment. In some embodiments, administration of TERT mRNA extends lung transplant-free survival compared to subjects not receiving treatment. In some embodiments, administration of TERT mRNA extends survival of a subject by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 1000% during and / or after a treatment period. In some embodiments, administration of TERT mRNA reduces hospitalization time and / or number of hospitalizations for treating lung disease and / or pulmonary fibrosis. In some embodiments, administration of TERT mRNA extends time to lung transplant.

[0268] V. Drug Combinations In some embodiments, the composition comprising TERT mRNA includes an excipient or carrier, for example, an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline. The composition may contain pharma- ceutically acceptable auxiliary substances and buffers as required for appropriate physiological conditions, such as pH, and toxicity counteracting agents, for example, sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride, and sodium lactate. In some embodiments, the pharmaceutical composition includes 10 mM sodium citrate buffered to pH 6.4. The composition may contain a cryoprotectant, for example, glycerol, ethylene glycol, sucrose, propylene glycol, or dimethyl sulfoxide (DMSO). The concentration of active agent in these formulations can vary and is selected based on fluid volume, viscosity, and body weight according to the particular mode of administration selected and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0269] VI. Methods for Lengthening Telomeres In another aspect, the disclosure provides a method of lengthening telomeres, the method comprising administering any of the compounds or compositions described above to a cell having shortened telomeres, whereby telomeres are lengthened within the cell. The disclosure also provides a method of treatment, the method comprising administering any of the compounds or compositions described above to an animal subject in need of or that may benefit from telomere lengthening.

[0270] In some embodiments, the compound or composition is administered to a cell, which is an isolated cell or is part of a cell culture, an isolated tissue culture, an isolated organ, etc. (i.e., the administration is in vitro).

[0271] In other embodiments, the compound or composition is administered without isolating a cell(s), tissue, or organ from the subject (i.e., administration is in vivo). In some of these embodiments, the compound or composition is delivered to all or nearly all cells in the subject's body. In some embodiments, the compound or composition is delivered to a specific cell, cell type, tissue, or organ in the subject's body.

[0272] Administration of the disclosed compounds or compositions may result in the transient expression of telomerase activity in cells. The increase in activity may be measured by various assays, such as, for example, the telomerase repeat amplification protocol (TRAP) assay. Commercial versions of the TRAP assay are available, such as the Trapeze® Telomerase Detection Kit (Millipore), which provides highly sensitive detection and quantification of telomerase activity, although other measurement techniques are possible.

[0273] As previously noted, one advantage of the present approach is that the expression of telomerase activity in the treated cells is transient, in particular in contrast to previous techniques in which the telomerase reverse transcriptase gene persists in an episomal DNA segment or is inserted into the genomic sequence of the cell, or otherwise permanently alters the genetic makeup of the target cell and results in constitutive activity of the nucleic acid sequence.

[0274] Figure 1 graphically illustrates some of the advantages of the compounds, compositions, and methods disclosed herein. In particular, the rate of telomere elongation enabled by these compounds, compositions, and methods allows for the maintenance of telomeres with very infrequent delivery of TERT mRNA. The expressed telomerase activity rapidly extends telomeres for a short period of time before being turned over, allowing the protective anti-cancer mechanism of telomere shortening to function most of the time. During treatment, normal telomerase activity and telomere shortening are present, and therefore the anti-cancer safety mechanism of telomere shortening that prevents uncontrolled proliferation is maintained, while the risk of short telomere-related diseases remains low. In contrast, small molecule treatments to extend telomeres require long-term delivery, which may present the risk of chronic cancer and have minimal therapeutic effects.

[0275] In some embodiments of the methods of the present invention, the transient expression is cell cycle independent.

[0276] As described above, the transient expression of telomerase reverse transcriptase elongates shortened telomeres in treated cells. Telomere length can be measured using techniques such as terminal restriction fragment (TRF) length analysis, qPCR, MMqPCR, TeSLA, flow FISH, and Q-FISH, as will be understood by those skilled in the art. In some embodiments, the method increases the average telomere length of treated cells by at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, or more. In some embodiments, the method reduces the proportion of telomeres that have a length less than a certain length, for example, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, or more.

[0277] One advantage of the compounds, compositions, and methods of the present invention is the rapidity of telomere lengthening achieved by these techniques. The techniques allow for short treatment times, thereby allowing longer intervals between treatments, which may be performed safely, since the normal protective telomere shortening mechanisms remain intact in most cases, i.e., between treatments.

[0278] Transient expression of telomerase reverse transcriptase also results in an increase in the replicative capacity of treated cells. This increase in replicative capacity is easily monitored in cells approaching replicative senescence by measuring additional population doublings in such cells. Senescent cells do not divide in response to many conditions that cause normal cells to divide, such as passage in culture or treatment with serum. In addition, senescent cells are often characterized by expression of pH-dependent β-galactosidase activity, expression of the cell cycle inhibitors p53 and p19, as well as other changes in gene expression patterns, and an increase in cell size. It is known in the art that, without treatment with TERT mRNA, certain cell types (e.g., human lung fibroblasts) typically double 50-60 times before undergoing postnatal senescence. However, upon TERT mRNA treatment, these cells achieve an additional 16-28 population doublings. If treated again several weeks later, additional proliferative capacity is again conferred. This process of intermittent treatments to periodically re-lengthen telomeres can be applied many more times, with the interval between treatments depending on factors such as the rate of telomere shortening, the rate of cell division, and the amount of telomere elongation brought about by the treatment. Similarly, human microvascular dermal endothelial cells from elderly subjects that have not been treated with the composition may only achieve 1-2 population doublings, whereas treated cells may achieve 3, 4, or more population doublings.

[0279] Thus, in some embodiments, the treatment methods increase the population doubling number of the treated cells.

[0280] The compositions of the present disclosure may treat genetic diseases that are not directly involved in telomere maintenance but are caused by mutations in genes that result in telomere shortening, including, for example, dyskeratosis congenita (DC) and forms of pulmonary fibrosis, pulmonary disease, bone marrow failure, and aplastic anemia.

[0281] Furthermore, various types of cancer may be prevented or delayed by treatment with the compounds of the present disclosure; indeed, interchromosomal fusions caused by extremely short telomeres are believed to be a cause of cancer.

[0282] VII. Medical Kit Also contemplated herein are therapeutic kits that include a pharmaceutical composition of TERT mRNA or its sequence (including complementary sequences) and instructions for use. In some embodiments, the therapeutic kits include devices for administration, including, but not limited to, syringes, inhalers, nebulizers, and vials or containers.

[0283] In another aspect, the present disclosure provides a ready-to-use kit for use in elongating telomeres in mammalian cells. The kit includes any of the above compounds or compositions, together with instructions for use. In some embodiments, the kit further includes packaging materials. In some embodiments, the packaging materials are airtight. In these embodiments, the packaging materials may be optionally filled with an inert gas, such as nitrogen, argon, and the like. In some embodiments, the packaging materials include metal foil containers, such as sealed aluminum pouches. Such packaging materials are well known to those skilled in the art. The kit may also include a delivery vehicle, such as lipids, as described herein. In some embodiments, one or more components of the formulation are provided frozen or lyophilized with a cryoprotectant.

[0284] In some embodiments, the kit may further include a desiccant, a culture medium, an RNase inhibitor, or other such components. In some embodiments, the kit may further include a combination of two or more of these additional components. In some kit embodiments, the kit compositions are sterile.

[0285] Enumerated Embodiments The present disclosure may be defined by reference to the exemplary embodiments listed below.

[0286] Embodiment I Embodiment I-1. A composition comprising (i) a ribonucleic acid (RNA) encoding telomerase reverse transcriptase (TERT) and (ii) a delivery vehicle, wherein the RNA of (i) comprises one or more modified nucleotides, and the delivery vehicle of (ii) is operably linked to the RNA of (i).

[0287] Embodiment I-2. The composition of embodiment I-1, wherein the delivery vehicle comprises one or more of a nanoparticle, a liposome, a cationic lipid, an ionic lipid, an exosome, a lipid nanoparticle, a natural lipoprotein particle, and an artificial lipoprotein particle.

[0288] Embodiment I-3. The composition of embodiment I-1, wherein the delivery vehicle comprises a lipid nanoparticle.

[0289] Embodiment I-4. The composition of embodiment I-1, wherein the delivery vehicle comprises an ionic lipid nanoparticle.

[0290] Embodiment I-5. The composition of any one of embodiments I-1 to I-4, wherein the delivery vehicle comprises a targeting lipid.

[0291] Embodiment I-6. The composition of embodiment I-5, wherein said targeting lipid specifically or selectively interacts with liver cells.

[0292] Embodiment I-7. The composition of embodiment I-6, wherein the targeting lipid comprises cholesterol.

[0293] Embodiment I-8. The composition of embodiment I-5, wherein said targeting lipid specifically or selectively interacts with lung cells.

[0294] Embodiment I-9. The composition of embodiment I-8, wherein the targeting lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylammonium bromide (DABB), or 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

[0295] Embodiment I-10. The composition of any one of embodiments I-1 to I-9, wherein the delivery vehicle comprises a compound of formula I: [ka] (In the formula, R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms; X a and X b are each independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups,

[0296] R 2a and R 2b each independently represents an alkylene group or an oxydialkylene group having 8 or fewer carbon atoms;

[0297] Y a and Y b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond;

[0298] Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom;

[0299] R 3a and R 3b each independently represents a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride, or a residue derived from a reaction product with glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms).

[0300] Embodiment I-11. The composition of embodiment I-10, wherein the compound of formula I is: [ka]

[0301] Embodiment I-12. The composition of embodiment I-10, wherein the compound of formula I is: [ka]

[0302] Embodiment I-13. The composition of embodiment I-10, wherein the compound of formula I is: [ka]

[0303] Embodiment I-14. The composition of embodiment I-10, wherein the compound of formula I is: [ka]

[0304] Embodiment I-15. The composition of embodiment I-10, wherein the compound of formula I is: [ka]

[0305] Embodiment I-16. The composition of embodiment I-10, wherein the compound of formula I is: [ka]

[0306] Embodiment I-17. The composition of any one of embodiments I-1 to I-16, wherein the RNA comprises the human sequence of SEQ ID NO: 3 or 4, or a sequence at least 70% identical to the sequence of SEQ ID NO: 3 or 4.

[0307] Embodiment I-18. The composition of embodiment I-17, wherein the RNA comprises a 5' cap.

[0308] Embodiment I-19. The composition of embodiment I-18, wherein the 5' cap comprises an anti-reverse cap analog (ARCA).

[0309] Embodiment I-20. The composition of embodiment I-19, wherein said ARCA comprises the structure 3'-O-Me-m7G(5')ppp(5')G.

[0310] Embodiment I-21. The composition of embodiment I-18, wherein the 5' cap comprises m7G(5')ppp(5')(2'OMeA)pG.

[0311] Embodiment I-22. The composition of any one of embodiments I-1 to I-21, wherein said RNA further comprises at least one untranslated region (UTR).

[0312] Embodiment I-23. The composition of embodiment I-22, wherein said at least one UTR is located 5' to the RNA of (i).

[0313] Embodiment I-24. The composition of embodiment I-22, wherein said at least one UTR is located 3' to the RNA of (i).

[0314] Embodiment I-25. The composition of any one of embodiments I-22 to I-24, wherein the UTR comprises a human sequence.

[0315] Embodiment I-26. The composition of any one of embodiments I-22 to I-24, wherein the UTR comprises a non-human sequence.

[0316] Embodiment I-27. The composition of any one of embodiments I-22 to I-26, wherein the UTR comprises a chimeric sequence.

[0317] Embodiment I-28. The composition of embodiment I-27, wherein said chimeric sequence increases the stability of the RNA of (i), increases the transcription rate, or decreases the time to transcription initiation.

[0318] Embodiment I-29. The composition of any one of embodiments I-22 to I-28, wherein the UTR comprises a sequence having at least 70% identity to a UTR sequence isolated or derived from one or more of alpha-globin, beta-globin, c-fos, and tobacco etch virus.

[0319] Embodiment I-30. The composition of any one of embodiments I-1 to I-29, wherein the one or more modified nucleotides of the RNA of (i) include one or more of a modified adenine or analog thereof, a modified cytidine or analog thereof, a modified guanosine or analog thereof, and a modified uridine or analog thereof.

[0320] Embodiment I-31. The composition of any one of embodiments I-1 to I-30, wherein the one or more modified nucleotides of the RNA of (i) include one or more of 1-methylpseudouridine, pseudouridine, 2-thiouridine, and 5-methylcytidine.

[0321] Embodiment I-32. The composition of any one of embodiments I-1 to I-31, wherein the one or more modified nucleotides of the RNA of (i) comprises 5-methoxyuridine (5-moU).

[0322] Embodiment I-33. The one or more modified nucleotides of the RNA of (i) are selected from the group consisting of m1A 1-methyladenosine, m6A N6-methyladenosine, Am 2'-O-methyladenosine, i6A N6-isopentenyl adenosine, io6A N6-(cis-hydroxyisopentenyl) adenosine, ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, g6A N6-glycinylcarbamoyl adenosine, t6A N6-threonylcarbamoyl adenosine, ms2t6A 2-methylthio-N6-threonylcarbamoyl adenosine, Ar(p)2'-O-ribosyladenosine (phosphate), m6 2A N6,N6-dimethyladenosine, m6Am N6,2'-O-dimethyladenosine, m6 2Am N6,N6,2'-O-trimethyladenosine, m1Am 1,2'-O-dimethyladenosine, m3C 3-methylcytidine, m5C 5-methylcytidine, Cm 2'-O-methylcytidine, ac4C N4-acetylcytidine, f5C 5-formylcytidine, m4C N4-methylcytidine, hm5C 5-hydroxymethylcytidine, f5Cm 5-formyl-2'-O-methylcytidine, m1G 1-methylguanosine, m2G N2-methylguanosine, m7G 7-methylguanosine, Gm 2'-O-methylguanosine, m2 2G N2,N2-Dimethylguanosine, Gr(p)2'-O-ribosylguanosine (phosphate), yWwybutosine, o2yWperoxywybutosine, OHyWhydroxywybutosine, OHyW*hypomodifiedhydroxywybutosine, imGwybutosine, m2,7G N2,7-Dimethylguanosine, m2,2,7G N2,N2,7-Trimethylguanosine, Iinosine, m1I1-Methylinosine, Im2'-O-Methylinosine, Qqueuosine, galQgalactosyl-queuosine, manQmannosyl-queuosine, Ψpseudouridine, Ddihydrouridine, m5U5-Methyluridine, Um2'-O-Methyluridine, m5Um5,2'-O-Dimethyluridine, m1Ψ 1-methylpseudouridine, Ψm2'-O-methylpseudouridine, s2U 2-thiouridine, ho5U 5-hydroxyuridine, chm5U 5-(carboxyhydroxymethyl)uridine,mchm5U 5-(carboxyhydroxymethyl)uridine, methyl ester mcm5U 5-methoxycarbonylmethyluridine, mcm5Um 5-methoxycarbonylmethyl-2'-O-methyluridine, mcm5s2U 5-methoxycarbonylmethyl-2-thiouridine, ncm5U 5-carbamoylmethyluridine, ncm5Um 5-carbamoylmethyl-2'-O-methyluridine, cmnm5U 5-carboxymethylaminomethyluridine, m3U 3-methyluridine, m1acp3Ψ 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, cm5U 5-carboxymethyluridine, m3Um 3,2'-O-dimethyluridine, m5D 5-methyldihydrouridine, τm5U 5-taurinomethyluridine, τm5s2U 5-Taurinomethyl-2-thiouridine, 2-Aminoadenosine, 2-Amino-6-chloropurine riboside, 8-Azaadenosine, 6-Chloropurine riboside, 5-Iodocytidine, 5-Iodouridine, Inosine, 2'-O-Methylinosine, Xanthosine, 4-Thiouridine, O6-Methylguanosine, 5,6-Dihydrouridine, 2-Thiocytidine, 6-Azacytidine, 6-Azauridine, 2'-O-Methyl-2-aminoadenosine, 2'-O-Methylpseudouridine, N1-Methyladenosine, 2'-O-Methyl-5-methyluridine, 7-Deazaguanosine, 8-Azidoadenosine, 5-Bromocytidine, 5-Bromouridine, 7-Deazaadenosine, 5-Aminoallyluridine, 5-Aminoallyluridine, lysylcytidine, 8-oxoguanosine, 2-aminopurine-riboside, pseudoisocytidine, N1-methylpseudouridine, 5,6-dihydro-5-methyluridine, N6-methyl-2-aminoadenosine, 5-carboxycytidine, 5-hydroxymethyluridine, thienoguanosine, 5-hydroxycytidine, 5-formyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-methoxycytidine, thienouridine, 5-carboxymethylesteruridine, thienocytidine, 8-oxoadenosine, isoguanosine, N1-ethylpseudouridine, N1-methyl-2'-O-methylpseudouridine, N1-methoxymethylpseudouridine, N1-propylpseudouridine,2'-O-methyl-N6-methyladenosine, 2-amino-6-Cl-purine-2'-deoxyriboside, 2-amino-2'-deoxyadenosine, 2-aminopurine-2'-deoxyriboside, 5-bromo-2'-deoxycytidine, 5-bromo-2'-deoxyuridine, 6-chloropurine-2'-deoxyriboside, 7-deaza-2'-deoxyadenosine, 7-deaza-2'-deoxyguanosine, 2'-deoxyinosine, 5-propynyl-2'-deoxycytidine, 5-propynyl-2'-deoxyuridine, 5-fluoro-2'-deoxy Xyuridine, 5-iodo-2'-deoxycytidine, 5-iodo-2'-deoxyuridine, N6-methyl-2'-deoxyadenosine, 5-methyl-2'-deoxycytidine, O6-methyl-2'-deoxyguanosine, N2-methyl-2'-deoxyguanosine, 8-oxo-2'-deoxyadenosine, 8-oxo-2'-deoxyguanosine, 2-thiothymidine, 2'-deoxy-P-nucleoside, 5-hydroxy-2'-deoxycytidine, 4-thiothymidine, 2-thio-2'-deoxycytidine, 6-aza-2'-deoxyuridine , 6-thio-2'-deoxyguanosine, 8-chloro-2'-deoxyadenosine, 5-aminoallyl-2'-deoxycytidine, 5-aminoallyl-2'-deoxyuridine, N4-methyl-2'-deoxycytidine, 2'-deoxyzebularine, 5-hydroxymethyl-2'-deoxyuridine, 5-hydroxymethyl-2'-deoxycytidine, 5-propargylamino-2'-deoxycytidine, 5-propargylamino-2'-deoxyuridine, 5-carboxy-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5- [(3-indolyl)propionamido-N-allyl]-2'-deoxyuridine, 5-carboxy-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, 7-deaza-7-propargylamino-2'-deoxyadenosine, 7-deaza-7-propargylamino-2'-deoxyguanosine, biotin-16-aminoallyl-2'-dUTP, biotin-16-aminoallyl-2'-dCTP, biotin-16-aminoallylcytidine, N4-biotin-OBEA-2'-deoxycytidine, biotin-16-aminoallyluridine,Dabsyl-5-3-aminoallyl-2'-dUTP, desthiobiotin-6-aminoallyl-2'-deoxycytidine, desthiobiotin-16-aminoallyl-uridine, biotin-16-7-deaza-7-propargylamino-2'-deoxyguanosine, cyanine 3-5-propargylamino-2'-deoxycytidine, cyanine 3-6-propargylamino-2'-deoxyuridine, cyanine 5-6-propargylamino-2'-deoxycytidine, cyanine 5-6-propargylamino-2'-deoxyuridine, cyanine 3-aminoallyl allyl cytidine, cyanine 3-aminoallyl uridine, cyanine 5-aminoallyl cytidine, cyanine 5-aminoallyl uridine, cyanine 7-aminoallyl uridine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, 2'-O-methyl uridine, puromycin, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxy Oxyuridine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine, aracytidine, arauridine, 2'-azido-2'-deoxyadenosine, 2'-amino-2'-deoxyadenosine, araadenosine, 2'-fluoro-thymidine, 3'-O-methyladenosine, 3'-O-methylcytidine, 3'-O-methylguanosine, 3'-O-methyluridine, 2'-azido-2'-deoxyguanosine, araguanosine, 2'-deoxyuridine, 3'-O-(2-nitrobenzyl)-2'-deoxyadenosine, 3' -O-(2-nitrobenzyl)-2'-deoxyinosine, 3'-deoxyadenosine, 3'-deoxyguanosine, 3'-deoxycytidine, 3'-deoxy-5-methyluridine, 3'-deoxyuridine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'-dideoxyuridine, 2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 3'-azido-2',3'-dideoxyadenosine, 3'-azido-2',3'-dideoxythymidine, 3'-amino-2',3'-dideoxyadenosine,3'-amino-2',3'-dideoxycytidine, 3'-amino-2',3'-dideoxyguanosine, 3'-amino-2',3'-dideoxythymidine, 3'-azido-2',3'-dideoxycytidine, 3'-azido-2',3'-dideoxyuridine, 5-bromo-2',3'-dideoxyuridine, 2',3'-dideoxyinosine, 2'-deoxyadenosine-5'-O-(1-thio 5'-O-(1-thiotriphosphate), 2'-deoxycytidine-5'-O-(1-thiotriphosphate), 2'-deoxyguanosine-5'-O-(1-thiotriphosphate), 2'-deoxythymidine-5'-O-(1-thiotriphosphate), adenosine-5'-O-(1-thiotriphosphate), cytidine-5'-O-(1-thiotriphosphate), guanosine-5'-O-(1-thiotriphosphate), uridine-5'-O-(1-thiotriphosphate) , 2',3'-dideoxyadenosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxycytidine-5'-O-(1-thiotriphosphate), 2',3'-dideoxyguanosine-5'-O-(1-thiotriphosphate), 3'-deoxythymidine-5'-O-(1-thiotriphosphate), 3'-azido-2',3'-dideoxythymidine-5'-O-(1-thiotriphosphate), 2',3'-dideoxy The composition of any one of embodiments I-1 to I-32, comprising one or more of thiouridine-5'-O-(1-thiotriphosphate), 2'-deoxyadenosine-5'-O-(1-boranotriphosphate), 2'-deoxycytidine-5'-O-(1-boranotriphosphate), 2'-deoxyguanosine-5'-O-(1-boranotriphosphate), and 2'-deoxythymidine-5'-O-(1-boranotriphosphate).

[0323] Embodiment I-34. The composition of any one of embodiments I-1 to I-33, wherein the composition further comprises a ribonucleic acid (RNA) encoding a telomerase RNA component (TERC).

[0324] Embodiment I-35. The composition of any one of embodiments I-1 to I-34, wherein the delivery vehicle comprises RNA encoding the TERT.

[0325] Embodiment I-36. The composition of embodiment I-35, wherein one or more of the surfaces, layers, or volumes of said delivery vehicle comprises RNA encoding said TERT.

[0326] Embodiment I-37. The composition of embodiment I-36, wherein the surface comprises an exterior surface or an interior surface.

[0327] Embodiment I-38. The composition of embodiment I-36, wherein the layer comprises a lipid monolayer or a lipid bilayer.

[0328] Embodiment I-39. The composition of embodiment I-36, wherein said volume comprises an interior volume.

[0329] Embodiment I-40. The composition of any one of embodiments I-1 to I-39, wherein the delivery vehicle is operably linked to a ribonucleic acid (RNA) encoding a telomerase RNA component (TERC).

[0330] Embodiment I-41. The composition of embodiment I-40, wherein said delivery vehicle comprises RNA encoding TERC.

[0331] Embodiment I-42. The composition of embodiment I-35, wherein one or more of the surfaces, layers, or volumes of said delivery vehicle comprises RNA encoding TERC.

[0332] Embodiment I-43. The composition of embodiment I-42, wherein the surface comprises an exterior surface or an interior surface.

[0333] Embodiment I-44. The composition of embodiment I-42, wherein the layer comprises a lipid monolayer or a lipid bilayer.

[0334] Embodiment I-45. The composition of embodiment I-42, wherein said volume comprises an interior volume.

[0335] Embodiment I-46. A method of increasing telomerase activity in a cell comprising contacting the cell with a composition of any one of embodiments I-1 to I-45.

[0336] Embodiment I-47. A method of extending telomeres in a cell comprising contacting the cell with a composition of any one of embodiments I-1 to I-45.

[0337] Embodiment I-48. The method of embodiment I-46 or I-47, wherein the cell is in vivo, ex vivo, or in vitro.

[0338] Embodiment I-49. A cell comprising the composition of any one of embodiments I-1 to I-45.

[0339] Embodiment I-50. A formulation comprising the cells of embodiment I-49.

[0340] Embodiment I-51. The formulation of embodiment I-50, wherein the plurality of cells comprises the cells of embodiment I-49.

[0341] Embodiment I-52. The formulation of embodiment I-51, wherein each of the plurality of cells is a cell of embodiment I-49.

[0342] Embodiment I-53. A method of treating a disease or disorder comprising administering to a subject an effective amount of the composition of any one of embodiments I-1 to I-45.

[0343] Embodiment I-54. A method of treating a disease or disorder comprising administering to a subject an effective amount of the cell of embodiment I-49.

[0344] Embodiment I-55. A method of treating a disease or disorder comprising administering to a subject an effective amount of the formulation of any one of embodiments I-50 to I-52.

[0345] Embodiment I-56. A method of delaying the onset of a disease comprising administering to a subject an effective amount of the composition of any one of embodiments I-1 to I-45.

[0346] Embodiment I-57. A method of delaying the onset of a disease comprising administering to a subject an effective amount of a cell of embodiment I-49.

[0347] Embodiment I-58. A method of delaying the onset of a disease comprising administering to a subject an effective amount of the formulation of any one of embodiments I-50 to I-52.

[0348] Embodiment I-59. The composition of any one of embodiments I-1 to I-45, wherein the composition is capable of transfecting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the lung cell population.

[0349] Embodiment I-60. A composition comprising a lipid nanoparticle (LNP) capable of transfecting at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of a lung cell population.

[0350] Embodiment I-61. The composition of embodiment I-60, comprising a polynucleotide.

[0351] Embodiment I-62. The composition of embodiment I-61, wherein the polynucleotide is RNA.

[0352] Embodiment I-63. The composition of embodiment I-62, wherein the RNA is mRNA.

[0353] Embodiment I-64. The composition of any one of embodiments I-60 to I-63, wherein the LNP is capable of transfecting about 50% to 99%, about 60% to 99%, about 70% to 99%, about 80% to 99%, or about 90% to 99% of the lung cell population.

[0354] Embodiment I-65. The composition of any one of embodiments I-60 to I-63, wherein the LNP is capable of transfecting between 50% and 95%, between about 60% and 95%, between about 70% and 95%, between about 80% and 95%, or between about 90% and 95% of the lung cell population.

[0355] Embodiment I-66. The composition of any one of embodiments I-60 to I-65, wherein the pulmonary cell population comprises pulmonary endothelial cells.

[0356] Embodiment I-67. The composition of embodiment I-66, wherein the pulmonary endothelial cells comprise vascular endothelial cells.

[0357] Embodiment I-68. The composition of embodiment I-66, wherein said pulmonary endothelial cells comprise alveolar endothelial cells.

[0358] Embodiment I-69. The composition of any one of embodiments I-60 to I-68, wherein said pulmonary cell population comprises pulmonary epithelial cells.

[0359] Embodiment I-70. The composition of embodiment I-69, wherein the pulmonary epithelial cells comprise alveolar epithelial cells.

[0360] Embodiment I-71. The composition of embodiment I-70, wherein said alveolar epithelial cells comprise alveolar type 1 (AT1) cells.

[0361] Embodiment I-72. The composition of embodiment I-70, wherein the alveolar epithelial cells comprise alveolar type 2 (AT2) cells.

[0362] Embodiment I-73. The composition of any one of embodiments I-60 to I-72, wherein the lung cells comprise any one or more of macrophages, mast cells, club cells, brush cells, neuroepithelial cells, and goblet cells.

[0363] Embodiment I-74. The composition of any one of embodiments I-60 to I-73, wherein the lung cells comprise one or more of fibroblasts, myofibroblasts, lipofibroblasts, and fibromyocells.

[0364] Embodiment I-75. The composition of any one of embodiments I-60 to I-74, wherein said pulmonary cell population comprises bronchial cells.

[0365] Embodiment I-76. The composition of any one of embodiments I-60 to I-75, wherein said lung cell population comprises bronchoalveolar stem cells.

[0366] Embodiment I-77. The composition of any one of embodiments I-60 to I-76, wherein said pulmonary cell population comprises pulmonary immune cells.

[0367] Embodiment I-78. The composition of any one of embodiments I-60 to I-77, wherein the lung cell population comprises lung fibroblasts.

[0368] Embodiment I-79. The composition of any one of embodiments I-60 to I-78, wherein the lung cell population comprises one or more of lung precancerous cells and cancer cells.

[0369] Embodiment I-80. The composition of any one of embodiments I-60 to I-79, wherein the pulmonary cell population comprises alveolar endothelial cells and alveolar epithelial cells, and at least 60%, at least 70%, at least 80%, or at least 90% of the pulmonary endothelial cells are transfected, and at least 60%, at least 70%, at least 80%, or at least 90% of the pulmonary epithelial cells are transfected.

[0370] Embodiment I-81. The composition of any one of embodiments I-60 to I-80, wherein said lung cells are transfected when administered to a subject by intravenous injection.

[0371] Embodiment I-82. The composition of any one of embodiments I-60 to I-80, wherein said lung cells are transfected when administered to a subject by one or more of aerosol injection, aerosolization, inhalation, nebulization, or instillation.

[0372] Embodiment I-83. The composition of any one of embodiments I-60 to I-82, wherein the composition comprises an ionic lipid.

[0373] Embodiment I-84. The composition of embodiment I-83, wherein the ionizable lipid is about 40-60% of the molar percentage of the LNP.

[0374] Embodiment I-85. The composition of any one of embodiments I-60 to I-84, wherein the composition comprises a compound of formula I: [ka]

[0375] (In the formula, R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms; X a and X b are each independently a non-cyclic alkyl tertiary amino group having 1 to 6 carbon atoms and one tertiary amino group, or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and one or two tertiary amino groups,

[0376] R 2a and R 2b each independently represents an alkylene group or an oxydialkylene group having 8 or fewer carbon atoms;

[0377] Y a and Y b each independently represents an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond;

[0378] Z a and Z b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, at least one aromatic ring, and optionally a heteroatom;

[0379] R 3a and R 3b each independently represents a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride, or a residue derived from a reaction product of a sterol derivative having a hydroxyl group with succinic anhydride, or a residue derived from a reaction product with glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms).

[0380] Embodiment I-86. The composition of embodiment I-84, wherein the compound of formula I is: [ka]

[0381] Embodiment I-87. The composition of embodiment I-84, wherein the compound of formula I is: [ka]

[0382] Embodiment I-88. The composition of embodiment I-84, wherein the compound of formula I is: [ka]

[0383] Embodiment I-89. The composition of embodiment I-84, wherein the compound of formula I is: [ka]

[0384] Embodiment I-90. The composition of embodiment I-84, wherein the compound of formula I is: [ka]

[0385] Embodiment I-91. The composition of embodiment I-84, wherein the compound of formula I is: [ka]

[0386] Embodiment I-92. The composition of any one of Embodiments I-60 to I-91, wherein the compound of formula I comprises a carbon chain having from 8 to 24 carbon atoms.

[0387] Embodiment I-93. The composition of any one of embodiments I-60 to I-92, wherein the LNP comprises a mixture of two or more ionizable lipids.

[0388] Embodiment I-94. The composition of any one of embodiments I-82 to I-92, wherein the cationic lipid is an ionic lipid and a targeting lipid.

[0389] Embodiment I-95. The composition of any one of embodiments I-60 to I-93, wherein the LNP comprises a sterol.

[0390] Embodiment I-96. The composition of embodiment I-94, wherein the sterol is cholesterol.

[0391] Embodiment I-97. The composition of any one of embodiments I-94 to I-96, wherein the sterol is about 10-30% of the molar percentage of the LNP.

[0392] Embodiment I-98. The composition of any one of embodiments I-1 to I-97, wherein the LNP comprises an insulator lipid.

[0393] Embodiment I-99. The composition of embodiment I-98, wherein the insulator lipid is a PEGylated lipid.

[0394] Embodiment I-100. The composition of any one of embodiments I-98 to I-99, wherein the PEGylated lipid is linear.

[0395] Embodiment I-101. The composition of any one of embodiments I-98 to I-99, wherein the PEGylated lipid is branched.

[0396] Embodiment I-102. The composition of any one of embodiments I-98 to I-101, wherein the PEGylated lipid comprises a carbon chain having from 8 to 24 carbon atoms.

[0397] Embodiment I-103. The composition of any one of embodiments I-98 to I-102, wherein the insulator lipid is conjugated to a blood protein or a peptide sequence of a blood protein, and the blood protein is an albumin or a globulin.

[0398] Embodiment I-104. The composition of any one of embodiments I-98 to I-101, wherein said insulator lipid is at least 0.25-5% of the molar percentage of said LNP.

[0399] Embodiment I-105. The composition of any one of embodiments I-1 to I-104, wherein the LNP comprises a cationic lipid.

[0400] Embodiment I-106. The composition of embodiment I-105, wherein the cationic lipid is any one or more of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP analogs.

[0401] Embodiment I-107. The cationic lipid is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). , 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecylammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium Dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-1-(cis,cis-9',1-2'-octadecadienoxy)propane (C pLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,The composition of embodiment I-105, wherein the aryl ester is any one or more of DLin-K-DMA, 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), or 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

[0402] Embodiment I-108. The composition of any one of embodiments I-1 to I-107, wherein the LNP comprises a structured lipid.

[0403] Embodiment I-109. The structured lipid is 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol-monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPG), dioleoyl ... The composition of embodiment I-108, wherein the sterol is any one or more of 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or another sterol, and mixtures thereof.

[0404] Embodiment I-110. The LNP comprises at least a first component, a second component, and a third component;

[0405] the first component is an ionic lipid;

[0406] the second component is a cationic lipid;

[0407] The third component is a structured lipid; The composition of any one of embodiments I-1 to I-109.

[0408] Embodiment I-111. The composition of embodiment I-110, further comprising a sterol.

[0409] The composition of any one of embodiments I-110 to I-111, comprising PEG2000 PE and DMG-PEGylated lipid of embodiment I-112.14:0, and an insulator lipid selected from a lipid conjugated to a blood protein or a peptide sequence of a blood protein, wherein the blood protein is albumin or globulin.

[0410] Embodiment I-113. The composition of any one of embodiments I-1 to I-112, wherein the LNP comprises SS-OP or a SS-OP analog, DOPC, cholesterol, DMG-PEG2000, and DOTAP.

[0411] Embodiment I-114. The composition of embodiment I-113, wherein the LNP comprises 45-55% cationic lipid and 20-40% SS-OP or an analog thereof.

[0412] Embodiment I-115. The composition of embodiment I-114, wherein the LNPs comprise 45-55% DOTAP.

[0413] Embodiment I-116 The LNP of embodiment I-113, wherein the LNP comprises SS-OP, DOPC, cholesterol, DMG-PEG2000, and DOTAP.

[0414] Embodiment I-117. The composition of embodiment I-114, wherein said LNP comprises 25-29% SS-OP; 1-3% DOPC; 15-35% cholesterol; 0.8-1.6% DMG-PEG2000; and 45-55% DOTAP.

[0415] Embodiment I-118. The composition of embodiment I-114, wherein said LNP comprises 27% SS-OP; 2.5% DOPC; 20% cholesterol; 1.2% DMG-PEG2000; and 50% DOTAP.

[0416] Embodiment I-119. The composition of any one of embodiments I-60 to I-118, wherein said LNP comprises cKK-E12 or a cKK-E12 analog, DOPE, cholesterol, PEG2000PE, and DOTAP.

[0417] Embodiment I-120. A method of delivering a cargo to a lung cell population in a subject in need thereof, comprising administering to the subject a composition of any one of embodiments I-1 to I-45 or embodiments I-1 to I-119.

[0418] Embodiment I-121. A method of treating a pulmonary disease or disorder in a subject in need thereof comprising administering to the subject a composition of any one of embodiments I-1 to I-45 or embodiments I-60 to I-119.

[0419] Embodiment I-122. The method of any one of embodiments I-120 to I-121, comprising administering said composition by intravenous injection.

[0420] Embodiment I-123. The method of any one of embodiments I-120 to I-121 comprising administering said LNP by one or more of aerosol injection, aerosolization, inhalation, nebulization, or instillation.

[0421] Embodiment I-124. The method of any one of embodiments I-120 to I-123, wherein the cargo comprises a polynucleotide.

[0422] Embodiment I-125. The method of embodiment I-124, wherein the polynucleotide is RNA.

[0423] Embodiment I-126. The method of embodiment I-125, wherein said RNA is mRNA.

[0424] Embodiment I-127. The method of embodiment I-126, wherein the mRNA encodes one or more of TERT, a cystic fibrosis transmembrane conductance regulator (CFTR) protein, a growth factor, a transcription factor, and a gene-editing protein.

[0425] Embodiment I-128. The method of any one of embodiments I-120 to I-127, wherein at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subject's lung cell population is transfected.

[0426] Embodiment I-129. The method of any one of embodiments I-120 to I-128, wherein about 50% to 99%, about 60% to 99%, about 70% to 99%, about 80% to 99%, or about 90% to 99% of the subject's lung cell population is transfected.

[0427] Embodiment I-130. The method of any one of embodiments I-120 to I-129, wherein about 50% to 95%, about 60% to 95%, about 70% to 95%, about 80% to 95%, or about 90% to 95% of the subject's lung cell population is transfected.

[0428] Embodiment I-131. The method of any one of embodiments I-120 to I-130, wherein the pulmonary disease or disorder is selected from pulmonary fibrosis, idiopathic pulmonary fibrosis, emphysema, interstitial lung disease, chronic obstructive pulmonary disease (COPD), pulmonary infection, pneumonia, tuberculosis, gastric reflux, lung cancer, cystic fibrosis, dyskeratosis congenita, alpha-1 antitrypsin deficiency, and other inherited pulmonary diseases.

[0429] Embodiment I-132. The method of any one of embodiments I-120 to I-131, wherein the LNP is administered to the subject as a single dose.

[0430] Embodiment I-133. The method of any one of embodiments I-120 to I-131, wherein said LNP is administered to said subject as multiple doses, said multiple doses comprising at least two doses.

[0431] Embodiment I-134. The method of any one of embodiments I-120 to I-133, wherein the dose of LNP administered to the subject is at least 0.01, 0.1, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, or 80 mg / kg of the subject's weight.

[0432] Embodiment I-135. The method of any one of embodiments I-120 to I-134, wherein the cargo is mRNA and the mRNA is administered to the subject at a dose of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, or about 2.5 mg / kg of weight of the subject.

[0433] Embodiment I-136. The method of any one of embodiments I-120 to I-135, wherein the subject is a human.

[0434] Embodiment II Embodiment II-1. The method comprises administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) by intravenous injection,

[0435] (i) a molar percentage of a cationic lipid of about 20% to about 50%;

[0436] (ii) about 20% to about 60% molar percentage of SS-OP or SS-OP analogue 23. A method for delivering a polynucleotide to the lung of a subject, comprising:

[0437] Embodiment II-2. The method of embodiment II-1, wherein the SS-OP analog is any one or more of SS-M, SS-E, SS-EC, SS-LC, and SS-OC.

[0438] Embodiment II-3. The cationic lipid is 2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), imidazole cholesterol ester (ICE), 25-hydroxycholesterol (25OH Chol), 20α-hydroxycholesterol 5-cholestene-3α, 20α-diol (20α Chol), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearyl ... Dimethyldioctadecylammonium chloride (DOD), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecylammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyloxy-N,N-dimethylammonium chloride (DOD AC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene -3-beta-oxy)-3'-oxapentoxy)-3-dimethyl 1-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,The method of embodiment II-1 or II-2, wherein the aryl group is any one or more of N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

[0439] The method of embodiment II-4, wherein the cationic lipid is DOTAP.

[0440] Embodiment II-5. The method of any one of embodiments II-1 to II-4, wherein the LNP comprises a molar percentage of the cationic lipid of about 25% to about 35%.

[0441] Embodiment II-6. The method of any one of embodiments II-1 to II-4, wherein the LNP comprises a molar percentage of the cationic lipid of about 30%.

[0442] Embodiment II-7. The method of any one of embodiments II-1 to II-6, wherein the LNP comprises structured lipids.

[0443] Embodiment II-8. The structured lipid is 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPG), dioleoyl ... The method of embodiment II-7, wherein the phosphatidylethanolamine is any one or more of 1-(N-maleimidomethyl)-2-oleoyl-2-oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or another sterol, and mixtures thereof.

[0444] Embodiment II-9. The method of embodiment II-7, wherein the structured lipid is DOPC.

[0445] The method of embodiment II-9, wherein the LNPs comprise about 1% to about 5% DOPC.

[0446] Embodiment II-11. The method of any one of embodiments II-1 to II-6, wherein said LNPs are substantially free of structured lipids and / or contain up to 1% structured lipids.

[0447] The method of any one of embodiments II-1 to II-11, wherein said LNPs comprise about 20% to about 40% cholesterol.

[0448] Embodiment II-13. The method of any one of embodiments II-1 to II-11, wherein said LNPs are substantially free of cholesterol.

[0449] The method of any one of embodiments II-1 to II-13, wherein the LNP comprises an insulator lipid.

[0450] The method of any one of embodiments II-1 to II-13, wherein the LNP is substantially free of insulator lipids.

[0451] Embodiment II-16. The method of any one of embodiments II-1 to II-15, wherein the LNP is preferentially delivered to and / or transfected in the lung compared to the liver.

[0452] Embodiment II-17. The method of any one of embodiments II-1 to II-16, wherein said polynucleotide is a synthetic ribonucleic acid (RNA).

[0453] Embodiment II-18. The method of embodiment II-17, wherein said synthetic ribonucleic acid (RNA) encodes telomerase reverse transcriptase (TERT).

[0454] Embodiment II-19. A method of treating pulmonary fibrosis in a subject in need thereof comprising administering an effective amount of a composition comprising a delivery vehicle comprising synthetic ribonucleic acid (RNA) encoding telomerase reverse transcriptase (TERT).

[0455] Embodiment II-20. The method of embodiment II-19, wherein said delivery vehicle is a lipid nanoparticle (LNP).

[0456] The method of embodiment II-20, wherein the LNP comprises about 20% to about 50% molar percentage of cationic lipid, about 20% to about 60% molar percentage of SS-OP or SS-OP analog, and optionally one or more of a structural lipid, an insulator lipid, and cholesterol.

[0457] Embodiment II-22. The method of any one of embodiments II-19 to II-21, wherein said TERT synthetic mRNA comprises at least one modified nucleoside from the list in Table 2.

[0458] Embodiment II-23. The method of embodiment II-22, wherein said modified nucleoside is pseudouridine or a pseudouridine analog.

[0459] Embodiment II-24. The method of embodiment II-22, wherein said pseudouridine analog is N-1-methylpseudouridine.

[0460] Embodiment II-25. The method of any one of embodiments II-19 to II-24, wherein the TERT synthetic mRNA comprises an untranslated region (UTR).

[0461] Embodiment II-26. The method of any one of embodiments II-19 to II-25, wherein the TERT synthetic mRNA comprises a 5' cap structure, and the 5' cap structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, CleanCapTM, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0462] Embodiment II-27. The method of any one of embodiments II-19 to II-26, wherein the TERT synthetic mRNA comprises a polyadenosine (polyA) nucleotide sequence 3' to the coding region.

[0463] Embodiment II-28. The method of any one of embodiments II-19 to II-27, wherein the TERT synthetic mRNA comprises a chain-terminating nucleotide, and the nucleotide is 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, a 2',3'-dideoxynucleoside, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or an -O-methylnucleoside.

[0464] Embodiment II-29. The method of any one of embodiments II-19 to II-28, wherein said TERT synthetic mRNA is codon-optimized.

[0465] Embodiment II-30. The method of any one of embodiments II-19 to II-28, wherein said pulmonary fibrosis is associated with a lung disease.

[0466] Embodiment II-31. The method of embodiment II-30, wherein said pulmonary disease is pulmonary fibrosis, familial pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary fibrosis with dyskeratosis congenita, interstitial lung disease, pneumonia, interstitial pneumonia, emphysema, or lung cancer.

[0467] Embodiment II-32. The method of any one of embodiments II-19 to II-31, wherein said pulmonary fibrosis is associated with a TERT mutation.

[0468] Embodiment II-33. The method of any one of embodiments II-19 to II-32, wherein the subject is a human.

[0469] Embodiment II-34. The method of any one of embodiments II-19 to II-33, wherein the composition is administered to the subject via intravenous injection.

[0470] Embodiment II-35. The method of any one of embodiments II-19 to II-33, wherein the composition is administered to the subject by inhalation.

[0471] Embodiment II-36.

[0472] (i) a molar percentage of a cationic lipid of about 20% to about 50%;

[0473] (ii) about 20% to about 60% molar percentage of SS-OP or SS-OP analogue A composition comprising a polynucleotide encapsulated in a lipid nanoparticle (LNP), comprising:

[0474] Embodiment II-37. The composition of embodiment II-36, wherein said SS-OP analog is any one or more of SS-M, SS-E, SS-EC, SS-LC, and SS-OC.

[0475] Embodiment II-38. The cationic lipid is 2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), imidazole cholesterol ester (ICE), 25-hydroxycholesterol (25OH Chol), 20α-hydroxycholesterol 5-cholestene-3α, 20α-diol (20α Chol), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearyl ... Dimethyldioctadecylammonium chloride (DOD), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecylammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyloxy-N,N-dimethylammonium chloride (DOD AC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene -3-beta-oxy)-3'-oxapentoxy)-3-dimethyl 1-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,The composition of embodiment II-36, which is any one or more of N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

[0476] Embodiment II-39. The composition of embodiment II-38, wherein the cationic lipid is DOTAP.

[0477] Embodiment II-40. The composition of any one of embodiments II-36 to II-39, wherein the LNP comprises a molar percentage of the cationic lipid of about 25% to about 35%.

[0478] Embodiment II-41. The composition of any one of embodiments II-36 to II-39, wherein said LNP comprises a molar percentage of said cationic lipid of about 30%.

[0479] Embodiment II-42. The composition of any one of embodiments II-36 to II-41, wherein the LNP comprises a structured lipid.

[0480] Embodiment II-43. The structured lipid is 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPG), dioleoyl ... The composition of embodiment II-42, wherein the sterol is any one or more of 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or another sterol, and mixtures thereof.

[0481] Embodiment II-44. The composition of embodiment II-43, wherein the structured lipid is DOPC.

[0482] Embodiment II-45. The composition of embodiment II-44, wherein the LNP comprises about 1% to about 5% DOPC.

[0483] Embodiment II-46. The composition of any one of embodiments II-36 to II-45, wherein the LNP is substantially free of structured lipids and / or comprises up to 1% structured lipids.

[0484] Embodiment II-47. The composition of any one of embodiments II-36 to II-46, wherein said LNPs comprise about 20% to about 40% cholesterol.

[0485] Embodiment II-48. The composition of any one of embodiments II-36 to II-46, wherein said LNPs are substantially free of cholesterol.

[0486] Embodiment II-49. The composition of any one of embodiments II-36 to II-48, wherein the LNP comprises an insulator lipid.

[0487] Embodiment II-50. The composition of any one of embodiments II-36 to II-48, wherein the LNP is substantially free of insulator lipids.

[0488] Embodiment II-51. The composition of any one of embodiments II-36 to II-50, wherein the LNP is preferentially delivered to and / or transfected in the lung compared to the liver.

[0489] Embodiment II-52. The composition of any one of embodiments II-36 to II-51, wherein the polynucleotide is a synthetic ribonucleic acid (RNA).

[0490] Embodiment II-53. The composition of embodiment II-52, wherein said synthetic ribonucleic acid (RNA) encodes telomerase reverse transcriptase (TERT).

[0491] Embodiment II-54. The composition of embodiment II-53, wherein said TERT synthetic RNA comprises at least one modified nucleoside listed in Table 2.

[0492] Embodiment II-55. The composition of embodiment II-54, wherein said modified nucleoside is pseudouridine or a pseudouridine analog.

[0493] Embodiment II-56. The composition of embodiment II-54, wherein said pseudouridine analog is N-1-methylpseudouridine.

[0494] Embodiment II-57. The composition of embodiments II-53 to II-56, wherein the TERT synthetic RNA comprises an untranslated region (UTR).

[0495] Embodiment II-58. The composition of any one of embodiments II-53 to II-57, wherein the TERT synthetic RNA comprises a 5' cap structure, and the 5' cap structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, CleanCapTM, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0496] Embodiment II-59. The composition of any one of embodiments II-53 to II-58, wherein the TERT synthetic RNA comprises a poly-adenosine (polyA) nucleotide sequence 3' to the coding region.

[0497] Embodiment II-60. The composition of any one of embodiments II-53 to II-59, wherein the TERT synthetic RNA comprises a chain-terminating nucleotide, and the nucleotide is 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, a 2',3'-dideoxynucleoside, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or an -O-methylnucleoside.

[0498] Embodiment II-61. The composition of any one of embodiments II-53 to II-60, wherein the TERT synthetic RNA is codon-optimized.

[0499] Embodiment II-62. Use of the composition of any one of embodiments II-36 to II-61 for treating pulmonary fibrosis in a subject in need of such treatment.

[0500] Embodiment II-63. The use of a composition according to embodiment II-62, wherein said pulmonary fibrosis is associated with a lung disease, and said lung disease is pulmonary fibrosis, familial pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary fibrosis with dyskeratosis congenita, interstitial lung disease, pneumonia, interstitial pneumonia, emphysema, or lung cancer.

[0501] Embodiment II-64. The use of the composition according to embodiment II-62 or II-63, wherein said pulmonary fibrosis is associated with a TERT mutation in said subject.

[0502] Embodiment II-65. The use of a composition according to any of embodiments II-62 to II-64, wherein said composition is administered to said subject by intravenous injection.

[0503] Embodiment II-66.

[0504] (i) a delivery vehicle comprising ribonucleic acid (RNA) encoding telomerase reverse transcriptase (TERT); and

[0505] (ii) a pharma- ceutically acceptable solvent or excipient; Including,

[0506] A pharmaceutical composition, wherein said delivery vehicle is capable of preferentially delivering to and / or transfecting pulmonary cells.

[0507] Embodiment II-67. The pharmaceutical composition of embodiment II-66, wherein said delivery vehicle is an LNP.

[0508] Embodiment II-68. The LNP comprises:

[0509] (i) a molar percentage of a cationic lipid of about 20% to about 50%; and

[0510] (ii) about 20% to about 60% molar percentage of SS-OP or SS-OP analogue The pharmaceutical composition of embodiment II-67, comprising:

[0511] Embodiment II-69. The pharmaceutical composition of embodiment II-68, wherein said SS-OP analog is any one or more of SS-M, SS-E, SS-EC, SS-LC, and SS-OC.

[0512] Embodiment II-70. The cationic lipid is 2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), imidazole cholesterol ester (ICE), 25-hydroxycholesterol (25OH Chol), 20α-hydroxycholesterol 5-cholestene-3α, 20α-diol (20α Chol), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearyl Oxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecylammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DO DAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene -3-beta-oxy)-3'-oxapentoxy)-3-dimethyl 1-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,The pharmaceutical composition of embodiment II-68 or II-69, which is any one or more of N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

[0513] Embodiment II-71. The pharmaceutical composition of embodiment II-70, wherein the cationic lipid is DOTAP.

[0514] Embodiment II-72. The pharmaceutical composition of any one of embodiments II-68 to II-71, wherein the LNP comprises a molar percentage of the cationic lipid of about 25% to about 35%.

[0515] Embodiment II-73. The pharmaceutical composition of any one of embodiments II-68 to II-71, wherein said LNP comprises a molar percentage of said cationic lipid of about 30%.

[0516] Embodiment II-74. The pharmaceutical composition of any one of embodiments II-68 to II-73, wherein the LNP comprises a structured lipid.

[0517] Embodiment II-75. The structured lipid is 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or another sterol, and mixtures thereof.

[0518] Embodiment II-76. The pharmaceutical composition of embodiment II-75, wherein the structured lipid is DOPC.

[0519] Embodiment II-77. The pharmaceutical composition of embodiment II-76, wherein the LNP comprises about 1% to about 5% DOPC.

[0520] Embodiment II-78. The pharmaceutical composition of any one of embodiments II-68 to II-77, wherein said LNPs are substantially free of structured lipids and / or contain up to 1% structured lipids.

[0521] Embodiment II-79. The pharmaceutical composition of any one of embodiments II-68 to II-78, wherein said LNP comprises about 20% to about 40% cholesterol.

[0522] Embodiment II-80. The pharmaceutical composition of any one of embodiments II-68 to II-78, wherein said LNPs are substantially free of cholesterol.

[0523] Embodiment II-81. The pharmaceutical composition of any one of embodiments II-68 to II-80, wherein the LNP comprises an insulator lipid.

[0524] Embodiment II-82. The pharmaceutical composition of any one of embodiments II-68 to II-80, wherein said LNP is substantially free of insulator lipids.

[0525] Embodiment II-83. The pharmaceutical composition of any one of embodiments II-68 to II-82, wherein the LNP is preferentially delivered to and / or transfected in the lung compared to the liver.

[0526] Embodiment II-84. The pharmaceutical composition of any one of embodiments II-66 to II-83, wherein said TERT synthetic RNA comprises at least one modified nucleoside listed in Table 2.

[0527] Embodiment II-85. The pharmaceutical composition of embodiment II-84, wherein said modified nucleoside is pseudouridine or a pseudouridine analog.

[0528] Embodiment II-86. The pharmaceutical composition of embodiment II-85, wherein said pseudouridine analog is N-1-methylpseudouridine.

[0529] Embodiment II-87. The pharmaceutical composition of any one of embodiments II-66 to II-86, wherein the TERT synthetic RNA comprises an untranslated region (UTR).

[0530] Embodiment II-88. The pharmaceutical composition of any one of embodiments II-66 to II-87, wherein the TERT synthetic RNA comprises a 5' cap structure, and the 5' cap structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, CleanCapTM, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0531] Embodiment II-89. The pharmaceutical composition of any one of embodiments II-66 to II-88, wherein said TERT synthetic RNA comprises a poly-adenosine (polyA) nucleotide sequence 3' to said coding region.

[0532] Embodiment II-90. The pharmaceutical composition of any one of embodiments II-66 to II-89, wherein the TERT synthetic RNA comprises a chain-terminating nucleotide, and the nucleotide is 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, a 2',3'-dideoxynucleoside, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, or an -O-methylnucleoside.

[0533] Embodiment II-91. The pharmaceutical composition of any one of embodiments II-66 to II-90, wherein said TERT synthetic RNA is codon-optimized.

[0534] Embodiment II-92. Use of the pharmaceutical composition of any one of embodiments II-66 to II-91 for treating pulmonary fibrosis in a subject in need of such treatment.

[0535] Embodiment II-93. The use of a pharmaceutical composition according to embodiment II-92, wherein said pulmonary fibrosis is associated with a lung disease, and said lung disease is pulmonary fibrosis, familial pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary fibrosis with dyskeratosis congenita, interstitial lung disease, pneumonia, interstitial pneumonia, emphysema, or lung cancer.

[0536] Embodiment II-94. Use of a pharmaceutical composition according to embodiment II-92 or II-93, wherein said pulmonary fibrosis is associated with a TERT mutation in said subject.

[0537] Embodiment II-95. The use of a pharmaceutical composition according to any of embodiments II-92 to II-94, wherein said composition is administered to said subject by intravenous injection. EXAMPLES

[0538] The following examples are included for illustrative purposes and are not intended to limit the scope of the disclosure.

[0539] Example 1: Lipid nanoparticle formulation The compositions and methods of the present disclosure can be used to deliver cargo such as polynucleotides to lung cells by a delivery vehicle. In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP) as disclosed herein. In some embodiments, the LNP as disclosed herein is used to deliver mRNA to lung cell populations.

[0540] Tables 6A-6B below provide exemplary formulations of LNPs that can be used as delivery vehicles, and Table 6C below provides examples of molar percentage ranges of LNPs containing lipid classes that can be used as delivery vehicles.

[0541] FIG. 2 shows a representative dynamic light scattering (DLS) plot of mRNA-LNPs made using the exemplary lipid components shown in Table 6A. [Table 6] [Table 7] [Table 8]

[0542] Example 2: Intravenous injection of mRNA-LNPs into mice Figure 3 shows bioluminescence imaging of whole organs of mice injected with mRNA-LNPs. The mRNA-LNPs were prepared by formulation using microfluidic mixing, then processed by concentration and buffer exchange before injection. Mice were intravenously administered luciferase mRNA-LNPs at 0.6 mg / kg total mRNA by body weight, 26 hours after which organs were harvested and imaged. Bioluminescence imaging shows that the LNP delivery vehicle is capable of delivering mRNA cargo to the lungs.

[0543] Example 3: mRNA translation by intravenous injection in mice Figures 4A-4B show immunohistochemical staining of tdTomato in lung cells from mice treated with the mRNA reporter and untreated control mice, respectively. tdTomato fl / fl mice were intravenously administered 0.6 mg / kg Cre mRNA LNP or saline. Four days later, lungs were harvested. These figures show that in cells successfully targeted by Cre mRNA-LNP, Cre mRNA was translated, and Cre recombinase excised the STOP codon, allowing tdTomato expression under the CAG promoter of the Rosa26 locus. In Figures 4A-4B, the scale bar is 50 μM.

[0544] Example 4: Measurement of telomerase activity in mouse lungs after TERT mRNA-LNA delivery Figures 5A-5B show the measurement of telomerase activity in mouse lungs after TERT mRNA-LNP delivery and in untreated control mouse lungs, respectively. TERT knockout (KO) mice were administered TERT mRNA LNPs intravenously at 2.0 mg / kg, and lungs were harvested 19 hours later. As a control, lungs were also harvested from untreated TERT KO mice. Telomere repeat amplification protocol (TRAP) was performed on lung lysates. The banding patterns observed in lung lysates from TERT mRNA LNP-treated mice indicate that telomerase activity was present.

[0545] The TRAP assay was performed as follows: Protein lysates from cells or tissues were incubated with artificial telomeres (DNA oligonucleotides). If active telomerase is present, the artificial telomeres are extended 6 bp at a time, generating a ladder pattern. This extension reaction is then amplified by PCR and run on a gel (using an Agilent bioanalyzer, microfluidic agarose gel). The presence of a ladder of 6 bp increments indicates telomerase activity.

[0546] Example 5: Transfection Efficiency of Pulmonary Delivery Vehicle Formulations Figure 6A is a bar graph showing the transfection efficiency of an exemplary pulmonary delivery vehicle formulation as a function of Cre dose, dose number, and genotype in mice. Cre mRNA complexed with the LNP pulmonary vehicles of Table 6A was delivered at the indicated doses via tail vein injection to mice carrying the tdTomato reporter gene required to turn on Cre. Lungs were harvested for histological analysis by immunostaining of the reporter gene. Figure 6A shows that the Cre mRNA LNP formulation transfected cells in the lung and turned on the reporter gene in a dose-dependent manner.

[0547] Figures 6B-6F show representative images of lung sections taken from the above mice, with the reporter protein shown as a darkened stain. Lung cells were harvested after treatment with saline control (Figure 6B), 0.1 mg / kg (Figure 6C), 0.3 mg / kg (Figure 6D), 0.6 mg / kg (Figure 6E), or 2.0 mg / kg (Figure 6F) of body weight dose of mRNA.

[0548] Example 6: Treatment of pulmonary fibrosis in mice with TERT mRNA LNPs Figures 7A-7C show computed tomography (CT) x-ray scans of mouse lungs examined for pulmonary fibrosis. Mice were instilled intratracheally with 1 U / kg bleomycin on day 0 and intravenously injected with 0.6 mg / kg TERT mRNA LNPs or saline on days 4, 7, and 11. CT images were acquired two weeks later. Figure 7A shows a control without bleomycin treatment, and Figures 7B-7C show the use of 1 U / kg bleomycin to induce pulmonary fibrosis. Figures 7B-7C were treated with saline and TERT mRNA LNPs, respectively. Healthy lung tissue appears darker and fibrotic lung tissue appears lighter. Less fibrosis is shown in TERT mRNA LNP-treated mice than in saline-treated controls. These figures show the effect of treatment with TERT mRNA LNPs on reducing fibrosis in a bleomycin mouse model of pulmonary fibrosis.

[0549] Example 7: Reduction of toxicity by use of SS-OP DOTAP in mice FIG. 8 shows a graph depicting the mortality of mice administered various formulations of lung-targeted LNP. C57Bl / 6 mice were administered formulations of lung-targeted LNP containing SS-OP and DOTAP according to the exemplary formulations in Table 6A (N=2-12 per time point), or formulations of lung-targeted LNP containing cKK DOTAP according to the exemplary formulations in Table 6B (N=2-4 per time point). Mortality is shown as the percentage of mice surviving acute treatment. Mice were dosed based on mg / kg of total reporter mRNA. These results demonstrate improved tolerability and reduced toxicity rates of the exemplary formulations in Table 6A using SS-OP and DOTAP.

[0550] Example 8: Delivery of mRNA to alveolar cells in a mouse fibrosis model Figures 9A-9D show various lung samples from mice treated with bleomycin to induce pulmonary fibrosis and treated with CRE mRNA or saline to demonstrate delivery of mRNA to alveolar cells. In summary, Ai14 (ROSA26 Lox-stop-lox tdTomato) mice were given 1 U / kg bleomycin by oral aspiration (OA) to induce fibrosis. On day 21 after bleomycin administration, mice were administered saline or 2 mg / kg Cre mRNA, whose translation allows expression of tdTomato. On day 3 after administration (day 24), mice were killed and lungs were inflated and fixed with PFA. Lung sections were stained with anti-tdTomato antibody and trichrome. These results indicate that Cre mRNA can be successfully delivered to alveolar cells even in pulmonary fibrosis mice, which may address the question of whether shunting of LNPs in disease settings affects mRNA delivery.

[0551] Example 9: Pulmonary delivery of mRNA via SS-OP DOTAP IV and OA routes To compare intravenous and oral administration of SS-OP DOTAP LNP formulations, lipid nanoparticle (LNP) compositions encapsulating firefly luciferase (Luc) mRNA were formulated according to Tables 2A and 2B. The lipid to mRNA ratios (wt / wt) used in these experiments were 75:1 and 40:1. C57Bl / 6 mice were administered 0.5 mg / kg LNP-mRNA by intravenous injection (IV) or 0.2 mg / kg LNP-mRNA by oropharyngeal aspiration (OA). Lungs were imaged ex vivo 24 hours later. Figure 10 shows the mean radiance (photons / sec / cm2 / sr) of luciferase in the lungs. Intravenous administration of SS-OP DOTAP formulations resulted in significantly higher lung transfection than oral administration (Figures 10 and 11). Furthermore, SS-OP DOTAP LNPs with lipid:RNA ratios of either 75:1 or 40:1 showed similar transfection efficiency when administered intravenously.

[0552] Example 10: Positively charged lipids targeting mRNA to the lung To compare the SS-OP LNP formulation with other cationic lipids, Tomato fl / fl mice were administered various LNP formulations encapsulating Cre mRNA, delivered intravenously at a range of 0.1-0.5 mg / kg, as shown in FIG. 12. The lipid to mRNA ratio for the SS-OP DOTAP, SS-OP 20α Chol, SS-OP DDAB, and 25OH Chol formulations was 80 μmol lipid:1 mg mRNA. The lipid to mRNA ratio for the SS-OP ICE was 50:1. The lipid to mRNA ratio for the SS-OP DOTAP ICE formulation was 70:1. After 3 days, lungs were harvested and processed by formalin fixation and paraffin embedding. Positive cells were labeled with anti-tdTomato antibody. FIG. 12 shows the percentage of positive Tomato cells in the lung parenchyma.

[0553] The transfection results are shown in Figure 12. Of note, other cationic lipids with a net positive charge at physiological pH (7.4) can be substituted for DOTAP, including the positively charged form of cholesterol (ICE) in the SS-OP LNP formulation. The LNP formulations tested are shown in Tables 7-12 below. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0554] Example 11: Different flow rates of mRNA LNP formulations for pulmonary delivery To compare the flow rates of the formulations of SS-OP DOTAP LNP (Table 6A) encapsulation of mRNA, SS-OP DOTAP LNP encapsulating luciferase mRNA was formulated at the ratios in Table 6A. The lipid:mRNA wt / wt ratio was 50:1. The aqueous (mRNA) to ethanol (lipid) flow rate ratio was 3:1. The overall flow rate was varied as shown in Figure 13. C57Bl / 6 mice were administered 0.1 mg / kg by intravenous injection and the lungs were imaged ex vivo 20 hours later. The mean radiance (photons / sec / cm2 / sr) of the lungs is shown. The highest signal for the SS OP DOTAP formulation was found at a flow rate of 8 ml / min for LNP mRNA encapsulation (Figure 13). Table 13 compares the flow rates of the SS OP DOTAP formulations of Table 6A with the particle size, zeta potential, and encapsulation efficiency. [Table 15]

[0555] Example 12: DOPC and cholesterol were not required for SS-OP DOTAP LNP mRNA delivery. To determine the components of SS-OP DOTAP LNP required to transfect the lungs, the SS-OP DOTAP LNP formulations of Table 6A were modified according to the formulations presented in Table 14 below. C57Bl / 6 mice were administered the LNP formulations of Table 14 containing luciferase mRNA by intravenous injection and the lungs were imaged ex vivo 16 hours later. Figure 14 shows the mean radiance (photons / sec / cm) of luciferase activity in the lungs per formulation. 2 / sr). Notably, neither neutral lipids, e.g., DOPC nor cholesterol, were required for pulmonary transfection with SS-OP DOTAP LNP formulations. Table 14 shows the SS-OP DOTAP formulations that were tested both with and without DOPC and / or cholesterol. [Table 16]

[0556] Example 13: Varying mRNA:lipid ratios showed different activities To determine the association of mRNA delivery with mRNA to lipid ratio, SS-OP DOTAP LNPs containing luciferase mRNA according to the formulations in Table 6A and at mRNA:lipid ratios (wt / wt) of 1:62, 1:45, and 1:30 were administered intravenously to C57Bl / 6 mice. Mice were dosed at 1.5 mg / kg by intravenous injection and lungs were imaged ex vivo 26 hours later. Figure 15 shows that mRNA:lipid ratios of 1:30 to 1:62 were the best performing mRNA:lipid ratios, with 1:30 showing the highest bioluminescence.

[0557] SS-OP DOTAP LNPs without DOPC and containing various ratios of firefly luciferase (Luc) mRNA were intravenously injected into C57Bl / 6 mice at a dose of 1.5 mg / kg. After 26 hours, lungs were imaged ex vivo. The mean radiance (photons / sec / cm2 / sr) of the lungs is shown.

[0558] No LNPs were formed at an mRNA:lipid ratio of 1:5 (ND means not administered). In these SS-OP DOTAP LNP formulations without DOPC, an mRNA:lipid ratio of 1:62 showed the highest luciferase signal (Figure 16).

[0559] Example 14: Varying the percentage of PEGylated lipids showed different activities To determine the effect of PEGylated lipid percentage on mRNA delivery by SS-OP DOTAP LNPs, LNPs were formulated with luciferase mRNA per the ratios in Table 15 below and administered intravenously to C57Bl / 6 mice at a dose of 1.5 mg / kg. After 19 hours, lungs were imaged ex vivo. Figures 17A and 17B show the mean radiance (photons / sec / cm) of the lungs. 2 / sr) It was observed that as the PEG signal increased, the bioluminescence signal decreased.

[0560] The optimal PEGylated lipid range for SS-OP DOTAP LNPw was found to be 0-3%, with 0.5% providing the best LNP delivery of mRNA (Figures 17A and 17B). Table 15 shows SS-OP DOTAP formulations containing various percentages of PEGylated lipid. [Table 17] Example 16: PEGylated lipids are not required for lung targeting To determine whether PEGylated lipids are even necessary for SS-OP DOTAP LNP mRNA delivery, LNPs were formulated with luciferase mRNA at the ratios in Table 16 below. C57Bl / 6 mice were administered intravenous injection and lungs were imaged ex vivo 21 hours later. LNP-mRNA dose was 1.5 mg mRNA per kg animal weight for the 1.2% PEG and 0.1% PEG conditions. Lipid to mRNA ratio was 63. For the 0% PEG condition, mice were administered 0.2 mg / kg. Figures 18A and 18B show the mean lung radiance (photons / sec / cm2 / sr). These results indicate that SS-OP DOTAP LNP formulations containing 0.1-1.2% PEGylated may be optimal. However, LNP formulations without PEGylated lipid successfully delivered mRNA to the lung. Table 16 shows low PEGylated and non-PEGylated lipid SS-OP DOTAP formulations. [Table 18]

[0561] Example 15: Varying DOTAP ratios show differences in lung activity To determine the effect of different DOTAP percentages on SS-OP DOTAP LNPs, SS-OP DOTAP LNPs were formulated with firefly luciferase (Luc) mRNA per the ratios in Table 17. C57Bl / 6 mice were dosed by intravenous injection at 1.5 mg / kg and lungs were imaged ex vivo 19 hours later. Figure 19 shows the relationship between the mean lung radiance (photons / sec / cm) versus the percentage of DOTAP in the LNPs. 2 / sr) are shown. Among the formulations in Table 17, the most effective delivery of SS-OP DOTAP LNP to the lung was with 30% DOTAP. Minimal liver targeting was observed with 20% or more DOTAP LNP, while a strong liver signal was seen with 10% DOTAP (Figures 20A and 20B). Table 17 shows SS-OP DOTAP formulations with various DOTAP percentages. [Table 19]

[0562] Example 17: Delivery of LNP-mRNA to the lungs transfects alveolar epithelial cells To observe the cell types transfected with SS-OP DOTAP LNPs according to the formula in Table 6A, LNPs were formulated with Cre mRNA and administered at 2.0 mg / kg to tomato fl / fl mice (Figure 21A). Figure 21B shows the PBS negative control. After 3 days, lungs were harvested and processed by formalin fixation and paraffin embedding. 4 μm sections were stained with:

[0563] Anti-IGFBP2, a marker for AT1 alveolar epithelial lung cells, anti-Prosurfactant Protein C (SPC), a marker for AT2 alveolar epithelial lung stem cells, anti-tdTomato, and DAPI as a DNA marker to identify the cells. Cells positive for both SPC and tdTomato indicated that the LNP formulation successfully transfected pulmonary AT2 alveolar epithelial stem cells. Cells positive for both IGFBP2 and tdTomato indicated that the LNP formulation successfully transfected pulmonary AT1 alveolar epithelial cells. The absence of tdTomato staining in the control indicated that the anti-tdTomato staining was specific.

[0564] Example 18: TERT mRNA delivery in KO mice using SS-OP DOTAP To determine SS-OP DOTAP delivery of therapeutic mRNA as disease treatment, SS-OP DOTAP LNPs encapsulating TERT mRNA were administered to pulmonary fibrosis model mice. The mouse pulmonary fibrosis model lungs and treatment timeline are shown in FIG. 22. Pulmonary fibrosis was induced in third generation (G3) TERT knockout (KO) mice with human-like telomere length. Bleomycin was administered continuously via subcutaneously implanted osmotic minipumps. The total dose delivered was 100 U / kg. TERT mRNA encapsulated in the SS-OP DOTAP LNP formulations of Table 6A was administered intravenously four times as indicated, starting on day 10. Compared to the firefly luciferase mRNA control, delivery of TERT mRNA prolonged mouse survival by 210% at the endpoint.

[0565] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure herein may be used in implementing the present disclosure. The following claims define the scope of the present disclosure, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. 1. A lipid nanoparticle (LNP) composition, comprising a lipid nanoparticle (LNP) and a ribonucleic acid (RNA), formulated for use in intravenous delivery to the lungs of a subject, The LNP is (a) structured lipids at a molar percentage of 0% to 15% of the total lipids in the LNP; (b) an insulator lipid at a molar percentage of 0.2% to 6% of the total lipid in the LNP; (c) cholesterol at a molar percentage of 0% to about 40% of the total lipids in the LNP; (d) an ionizable lipid at a molar percentage of 10% to about 50% of the total lipid in the LNP; and (e) a molar percentage of cationic lipid of about 15% to 80% of the total lipid in the LNP; Including, The (d) ionic lipid comprises SS-OP or an SS-OP analog; The liposome pKa of the LNP is 5.5 to 7.2; An LNP composition, wherein the RNA is encapsulated in the LNP and encodes a telomerase reverse transcriptase (TERT) mRNA having one or more modified nucleotides.

2. (a) the structured lipid comprises DOPC; (b) the insulator lipid comprises a PEGylated lipid; (d) the ionic lipid comprises a molar percentage of SS-OP of about 20% to 60%; (e) the LNP composition of claim 1, wherein the cationic lipid comprises DOTAP.

3. (e) Cationic lipids include 2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), imidazole cholesterol ester (ICE), 25-hydroxycholesterol (25OH Chol), 20α-hydroxycholesterol 5-cholestene-3α, 20α-diol (20α Chol), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermylglycine dioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearyl Oxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecylammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DO DAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,The LNP composition according to claim 1 or 2, which is any one or more of N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

4. (a) the structured lipid comprises a molar percentage of DOPC of about 1% to about 5%; (b) the insulator lipid comprises a molar percentage of PEGylated lipid of about 0.1% to about 2%; (c) cholesterol is contained in a molar percentage of 15% to 35%; (d) the ionizable lipid comprises a molar percentage of SS-OP of about 20% to about 40%; (e) the LNP composition of any one of claims 1 to 3, wherein the cationic lipid comprises DOTAP at a molar percentage of about 25% to about 35%.

5. (a) Structured lipids are 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE ...ethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylethanolamine (DOPE), palmitoyl 5. The LNP composition of any one of claims 1-4, wherein the sterol is any one or more of leoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or another sterol, and mixtures thereof.

6. The LNP composition of any one of claims 1 to 5, wherein the LNPs are preferentially delivered to and / or transfected in the lungs compared to the liver.

7. The LNP composition of any one of claims 1 to 6, wherein the TERT mRNA comprises a nucleic acid sequence that is at least 50% identical to any one of SEQ ID NOs: 38 to 40.

8. 1. A lipid nanoparticle (LNP) composition comprising a lipid nanoparticle (LNP) and a synthetic ribonucleic acid (RNA) formulated for use in treating a pulmonary disease and / or pulmonary fibrosis in a subject in need thereof, The LNP is (a) structured lipids at a molar percentage of 0% to 15% of the total lipids in the LNP; (b) an insulator lipid at a molar percentage of 0.2% to 6% of the total lipid in the LNP; (c) cholesterol at a molar percentage of 0% to about 40% of the total lipids in the LNP; (d) an ionizable lipid at a molar percentage of 10% to about 50% of the total lipid in the LNP; and (e) a molar percentage of cationic lipid of about 20% to 80% of the total lipid in the LNP; Including, The (d) ionic lipid comprises SS-OP or an SS-OP analog; The liposome pKa of the LNP is 5.5 to 7.2; The LNPs encapsulate synthetic ribonucleic acid (RNA), The LNP composition, wherein the synthetic RNA encodes a TERT mRNA comprising a nucleic acid sequence at least 50% identical to any one of SEQ ID NOs: 38-40.

9. The TERT synthetic mRNA is selected from the group consisting of the following list: 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 The LNP composition of claim 8, comprising at least one modified nucleoside of:

10. The LNP composition of claim 8 or 9, wherein the lung disease and / or pulmonary fibrosis is familial pulmonary fibrosis, idiopathic pulmonary fibrosis, pulmonary fibrosis associated with dyskeratosis congenita, interstitial lung disease, pneumonia, interstitial pneumonia, emphysema, chronic obstructive pulmonary disease, cystic fibrosis, infectious disease, coronavirus disease, or lung cancer.

11. The LNP composition of any one of claims 8 to 10, wherein the pulmonary fibrosis is associated with a TERT mutation.

12. The LNP composition according to any one of claims 8 to 11, wherein the LNP composition is administered to the subject via intravenous injection.

13. The LNP composition according to any one of claims 8 to 11, wherein the LNP composition is administered to the subject by inhalation.

14. The LNP composition of any one of claims 1 to 13, wherein the LNP further comprises ribonucleic acid (RNA) encoding a telomerase RNA component (TERC).

15. The LNP composition of any one of claims 1 to 14, wherein the RNA or synthetic RNA comprises self-replicating RNA or circular RNA.

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