Compositions and methods for RNA delivery

Abstract

The present invention relates to lipid nanoparticle (LNP) compositions and therapeutic polynucleotides (e.g., TERT mRNA) that can be delivered with the LNP compositions in a formulation for the prevention or treatment of lung disease and / or fibrosis.

Description

[Technical Field]

[0001] Numerous pulmonary diseases have been identified for which there is currently no treatment. Pulmonary diseases (e.g., pulmonary fibrosis, interstitial lung disease, and lung cancer) often induce fibrosis as part of the disease progression, which further limits the extent to which patient recovery can occur. Treatment options for pulmonary diseases remain limited. Delivery of polynucleotides to the lungs could provide a much-needed treatment option for pulmonary disease patients, but compositions and methods for delivery of polynucleotides require improvement.

[0002] Therefore, there is still a need to prevent or treat lung disease and / or pulmonary fibrosis. There is a need in the art for delivery formulations capable of delivering polynucleotides (eg, mRNA) to the lung.

[0003] The present invention addresses this and other related needs in the art. Summary of the Invention

[0004] According to embodiments, provided herein is a method of delivering a polynucleotide to a subject, the method comprising administering by intravenous injection a polynucleotide encapsulated in a lipid nanoparticle (LNP), wherein the lipid nanoparticle comprises (i) about 20% to about 50% molar percentage of a cationic lipid, and ii) about 20% to about 60% molar percentage of an SS-OP or an SS-OP analog, and wherein the polynucleotide comprises a synthetic RNA, and wherein upon or after administration of the LNP, the synthetic RNA is translated in vivo in the subject into a corresponding protein encoded by the synthetic RNA.

[0005] According to related embodiments, the cationic lipid is (a) about 25% to about 60% molar percentage of SS-OP or SS-OP analog (LNPs having a lipid nitrogen:polynucleotide phosphate ratio (N / P ratio) of 10 to 30), or (b) about 30% to about 50% molar percentage of DLin-MC3-DMA or DLin-MC3-DMA analog (LNPs having an N / P ratio of 3.5 to 10). As used herein, the term "MC3" refers to "DLin-MC3-DMA."

[0006] According to one embodiment, the lipid DOTAP has an in vivo half-life of at least 35 hours in the lungs of a subject, not more than 15 hours in the liver of a subject, and / or not more than 9 hours in the spleen of a subject.

[0007] According to related embodiments, the in vivo half-life of lipid DOTAP in the lungs of a subject is at least 5, 7, 9, 10, 15, 20, 25, 30, 35, 37, 40, 45 hours or more. Also according to related embodiments, the in vivo half-life of lipid DOTAP in the liver of a subject is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 15 hours or less. Also according to related embodiments, the in vivo half-life of lipid DOTAP in the spleen of a subject is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 15 hours or less.

[0008] Also provided according to embodiments herein is a method of delivering a polynucleotide to a subject, the method comprising administering intravenously a polynucleotide encapsulated in lipid nanoparticles (LNPs), wherein the lipid nanoparticles comprise (i) about 20% to about 50% molar percentage of cationic lipids, and (ii) about 20% to about 60% molar percentage of SS-OP or SS-OP analogs, and the polynucleotide comprises synthetic RNA, wherein upon or after administration of the LNPs, the synthetic RNA is translated in vivo in the subject into a corresponding protein encoded by the synthetic RNA, and approximately 6 hours after intravenous infusion of the LNPs, a lung:liver ratio of protein per mg of tissue is at least 3:1, 4:1, or 5:1. Often, according to related embodiments, the lung:liver ratio of the amount of lipid DOTAP per mg of tissue is at least 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. Also, often, according to related embodiments, the recited ratios are approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, or 168 hours after intravenous infusion of LNPs encapsulating mRNA.

[0009] Also provided herein, according to embodiments, is a method of delivering a polynucleotide to a subject, the method comprising administering via intravenous injection a polynucleotide encapsulated in lipid nanoparticles (LNPs), wherein the lipid nanoparticles comprise (i) about 20% to about 50% molar percentage of a cationic lipid, and ii) about 20% to about 60% molar percentage of a SS-OP or SS-OP analog, and the polynucleotide comprises a synthetic RNA, wherein upon or after administration of the LNPs, the synthetic RNA is translated in vivo in the subject into a corresponding protein encoded by the synthetic RNA, and the median telomere length of alveolar cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%. In many cases, according to related embodiments, the median telomere length of alveolar cells is increased by at least 6%, 7%, 8%, 9%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 25%, 30%, 35%, 40%, 45%, or more. In many cases, according to related embodiments, the median telomere length of alveolar cells is increased by at least 50, 100, 200, 300, 400, 500, 1000 base pairs or more.

[0010] The synthetic RNA may include a sequence encoding a telomerase reverse transcriptase (TERT) protein or portion thereof selected from human TERT (hTERT), mouse TERT (mTERT), or TERT of another mammalian species. According to currently contemplated embodiments, the mRNA often includes SEQ ID NO: 1 or a fragment thereof. Also according to currently contemplated embodiments, the synthetic ribonucleic acid (RNA) encodes telomerase reverse transcriptase (TERT), and optionally, the TERT mRNA includes a nucleic acid sequence 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 or 38-40 of PCT / US22 / 22642.

[0011] In many cases, the TERT synthetic mRNA contains a 5' cap structure, which is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, Cap0, Cap1, ARCA, inosine, Nl-methylguanosine, 2'fluoroguanosine, 7-deazaguanosine, CleanCap™, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0012] In many cases, according to the methods of the invention, delivery of a polynucleotide is for the prevention and / or treatment of a lung disease and / or pulmonary fibrosis in a subject. Also, in many cases, according to the methods of the invention, delivery of a polynucleotide is for the modulation of a lung disease and / or pulmonary fibrosis in a subject.

[0013] According to commonly included embodiments, the LNPs have a lipid nitrogen:polynucleotide phosphate (N / P) ratio of 28-31. In particular embodiments, the N / P ratio is approximately 29.1. Often, in such embodiments, the ionic lipid is SS-OP, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 1.8 and are comprised of five lipids. In related embodiments, the ionic lipid is SS-OP, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 1.8 and are comprised of five lipids, including DOTAP and DOPC. In related embodiments, the ionic lipid is SS-OP, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 1.8 and are comprised of five lipids, including DMG-PEG2000, DOPC, and DOTAP.

[0014] According to common embodiments, the LNPs have an N / P ratio of 11 to 13. In particular embodiments, the N / P ratio is approximately 12. Often, in such embodiments, the ionic lipid is SS-OP, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 0.8 and are comprised of three or five lipids. In related embodiments, the ionic lipid is SS-OP, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 0.8 and are comprised of three or five lipids, including DMG-PEG2000 and DOTAP.

[0015] According to common embodiments, the LNPs have an N / P ratio of 4-5. In particular embodiments, the N / P ratio is approximately 4.5. Often, in such embodiments, the ionic lipid is DLin-MC3-DMA, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 1.5 and are comprised of three lipids, including DLin-MC3-DMA, and a cationic lipid to ionic lipid ratio of approximately 1.5, in related embodiments. In related embodiments, the ionic lipid is DLin-MC3-DMA, and the LNPs have a cationic lipid to ionic lipid ratio of approximately 1.5 and are comprised of three lipids, including DMG-PEG2000 and DOTAP.

[0016] Also, according to the methods provided herein, the ionic lipid is DLin-MC3-DMA, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5, and is composed of three lipids including DMG-PEG2000 and DOTAP, with an N / P ratio of approximately 4.5.

[0017] Also, according to preferred embodiments, the LNP is selected from the LNPs in Tables 8-10.

[0018] According to an embodiment (Figure 22A), the LNP has only a single cationic lipid (eg, DOTAP).

[0019] Also provided herein is a method for producing LNPs. In a specific example, LNPs are produced by a method in which the LNPs are formed before adding nucleic acid. In certain embodiments thereof, such LNPs are two, three, or five lipid LNPs as described in Table 8 herein, and the method uses vortex mixing during the addition of nucleic acid. In certain embodiments, the LNPs are composed of two or three lipid LNPs as described herein.

[0020] These and other embodiments, features, and advantages will become apparent to those skilled in the art upon review of the following more detailed description of various exemplary embodiments of the present disclosure in conjunction with the accompanying drawings.

[0021] Those skilled in the art will appreciate that the drawings, described below, are for illustrative purposes only. [Brief explanation of the drawings]

[0022] [Figure 1] Panels A–D show the pharmacokinetics of specific lipid nanoparticles (LNPs). Timelines of serum (Figure 1A), lung (Figure 1B), and liver (Figure 1C) levels of LNPs determined by measuring one of the lipids, DOTAP. Panel D presents tabular data related to panels A–C. [Figure 2] Shown are ratios of bioluminescence signals from the indicated organs generated from luciferase protein translated from a formulation of mRNA in intravenously injected LNPs ("mRNA LNPs") at the indicated time points after intravenous injection. [Figure 3] We demonstrate that LNPs formulated with TERT mRNA ("TERT mRNA LNPs") lengthen telomeres in lung cells in vivo, and use Q-FISH to measure telomere length in lung tissue sections, with telomere signal intensity (green / light color) being proportional to signal length. [Figure 4]We demonstrate that TERT mRNA LNPs extend telomeres in lung cells in vivo. TERT mRNA LNPs extend the median and mean telomere length of AT2 cells. At least 50 pro-SPC-positive nuclei were quantified per mouse. Error bars represent the standard error of the mean (SEM) of telomere length across mice. [Figure 5] We demonstrate that TERT mRNA LNPs extend lung cell telomeres in vivo, and that TERT mRNA LNPs extend the median and mean telomere length of alveolar cells. Telomeres were quantified in at least 100 alveolar cells per mouse. Error bars represent the standard error of the mean (SEM) of telomere length across mice. [Figure 6] Figure 6A shows the experimental design and administration timeline for the data of the present disclosure presented in Figures 3-12 and 20-21. Figure 6B shows the experimental design and administration timeline for the data of the present disclosure presented in Figures 13-16. [Figure 7] TERT mRNA LNPs reduce lung fibrosis in vivo. The figure shows picrosirius red staining to map dense fibrotic foci (yellow / dark color) in the lungs of bleomycin-treated mice treated with either control mRNA LNPs (carrying luciferase mRNA) or TERT mRNA LNPs. "No bleo" indicates a lung section from a normal, healthy mouse not treated with bleomycin. [Figure 8] We show that TERT mRNA LNPs reduce lung fibrosis in vivo, as measured by quantification of dense fibrotic foci in picrosirius red-stained tissue sections. [Figure 9] We show that TERT mRNA LNPs increase the amount of useful lung volume in vivo as determined by quantifying the change in normal aerated lung ratio from baseline using 3D quantification of fibrosis detected by signal intensity in X-ray computed tomography scan data. [Figure 10]1 shows that TERT mRNA LNPs improve tissue elasticity in vivo as determined by quantitation of tissue elasticity measurements in mice at the end of the study using the Flexivent system. [Figure 11] 1 shows that TERT mRNA LNPs improve lung function in vivo as determined by determining the forced expiratory volume of mice at the end of the study using the Flexivent system. [Figure 12] 1 shows that TERT mRNA LNPs improve lung function in vivo as determined by measuring the forced vital capacity of mice at the end of the study using the Flexivent system. [Figure 13] We demonstrated that TERT mRNA LNPs improved lung structure in vivo by analyzing digital images of tissue sections and quantifying the number of alveoli per unit area using Morpho-Quant lung software. [Figure 14] We demonstrated that TERT mRNA LNPs improved lung structure in vivo by analyzing digital images of tissue sections and quantifying alveolar diameters using Morpho-Quant Lung software. [Figure 15] We demonstrated that TERT mRNA LNPs improved lung structure in vivo by analyzing digital images of tissue sections and quantifying alveolar circularity using Morpho-Quant Lung software. [Figure 16] Representative images of alveoli false-colored by Morpho-Quant Lung software during digital analysis of alveolar structure are shown, demonstrating that TERT mRNA LNPs improve lung structure in vivo. [Figure 17] Pharmacodynamic data for in vivo TERT mRNA are shown following intravenous infusion of a formulation of TERT mRNA LNPs, quantifying the levels of telomerase activity in lung tissue at the indicated time points after infusion. [Figure 18] 1 shows an experimental scheme for quantifying the effect of TERT mRNA LNPs on human primary epithelial cell colony-forming ability. [Figure 19] TERT mRNA LNPs demonstrate increased colony-forming capacity of human primary epithelial cells in the experimental model shown in FIG. [Figure 20] Based on the experimental model shown in Figure 6A, the levels of P21+ senescent alveolar cells containing LNPs in Table 3 are shown. [Figure 21] Representative immunohistochemical images of anti-P21 staining of lung sections quantified in FIG. 20. [Figure 22A] Pulmonary delivery data obtained using two lipid LNP compositions are presented. [Figure 22B] Pulmonary delivery data obtained using two lipid LNP compositions are presented. [Figure 22C] Pulmonary delivery data obtained using two lipid LNP compositions are presented. [Figure 22D] Pulmonary delivery data obtained using two lipid LNP compositions are presented. [Figure 23] 2 shows the results of titration of lipid:mRNA ratio in lipid LNPs. [Figure 24] Table 3 shows the time course of protein expression after delivery of lung-targeted mRNA-LNPs encoding firefly luciferase using the LNPs. [Figure 25A] The LNPs in Figure 25D are used to show the variation in lung radiance, encapsulation efficiency, and body weight upon titration of SS-OP lipids into "DOTAP+PEG" LNPs. [Figure 25B] The LNPs in Figure 25D are used to show the variation in lung radiance, encapsulation efficiency, and body weight upon titration of SS-OP lipids into "DOTAP+PEG" LNPs. [Figure 25C] The LNPs in Figure 25D are used to show the variation in lung radiance, encapsulation efficiency, and body weight upon titration of SS-OP lipids into "DOTAP+PEG" LNPs. [Figure 25D] Present LNP. [Figure 26A] Table 4 Lung and liver delivery data using LNPs. [Figure 26B] Table 4 Lung and liver delivery data using LNPs. [Figure 27A] Exemplary LNPs and their encapsulation efficiencies are presented. [Figure 27B] Exemplary LNPs and their encapsulation efficiencies are presented. [Figure 27C] Pulmonary delivery and weight change data are presented using the LNPs of Figure 27A when titrating SS-OP lipids into "DOTAP+PEG" LNPs. [Figure 27D] Pulmonary delivery and weight change data are presented using the LNPs of Figure 27A when titrating SS-OP lipids into "DOTAP+PEG" LNPs. [Figure 28A] Table 3 presents a comparison of transfection and weight data for LNP fresh lung and long-term cryopreserved lung. [Figure 28B] Table 3 presents a comparison of transfection and weight data for LNP fresh lung and long-term cryopreserved lung. [Figure 29A] Table 3 presents a comparison of transfection and particle size data for LNP fresh lung and short-term cryopreserved lung. [Figure 29B] Table 3 presents a comparison of transfection and particle size data for LNP fresh lung and short-term cryopreserved lung. [Figure 30A] Exemplary 2-, 3-, and 5-lipid LNPs are presented. [Figure 30B] The N / P ratio was titrated and pulmonary delivery and body weight change data are presented comparing 2, 3, and 5 lipid formulations in Figure 30A. [Figure 30C] The N / P ratio was titrated and pulmonary delivery and body weight change data are presented comparing 2, 3, and 5 lipid formulations in Figure 30A. [Figure 31A] Exemplary 3- and 5-lipid LNPs and their encapsulation efficiencies are presented. [Figure 31B] Exemplary 3- and 5-lipid LNPs and their encapsulation efficiencies are presented. [Figure 31C] FIG. 30A shows pulmonary delivery data upon titration of N / P ratio (mRNA to lipid ratio) using 3 and 5 lipid formulations. [Figure 32A]We present lung transfection data and LNP size and encapsulation efficiency data for LNPS with the lipid ratios in Table 3 prepared using vortex or microfluidic mixing. [Figure 32B] We present lung transfection data and LNP size and encapsulation efficiency data for LNPS with the lipid ratios in Table 3 prepared using vortex or microfluidic mixing. [Figure 33A] Exemplary 3- and 5-lipid LNPs are presented. [Figure 33B] Lung transfection data using LNPs is presented in Figure 33A. [Figure 34A] Exemplary 2-, 3-, and 5-lipid LNPs are presented. [Figure 34B] Pulmonary transfection data using LNPs and particle size and encapsulation efficiency are presented in Figure 34A. [Figure 34C] Pulmonary transfection data using LNPs and particle size and encapsulation efficiency are presented in Figure 34A. [Figure 35A] An exemplary five-lipid LNP prepared using different flow rates is presented. [Figure 35B] Percent encapsulation and lung transfection data using LNPs in Figure 35A are presented. [Figure 35C] Percent encapsulation and lung transfection data using LNPs in Figure 35A are presented. [Figure 36A] An exemplary three-lipid LNP prepared using different flow rates is presented. [Figure 36B] Figure 36A presents data on lung transfection and body weight change using LNPs. [Figure 36C] Figure 36A presents data on lung transfection and body weight change using LNPs. [Figure 37A] Exemplary LNPs (including encapsulation percentages) prepared by substituting SS-OP with different ionic lipids (DLin-MC3-DMA) are presented. [Figure 37B]Exemplary LNPs (including encapsulation percentages) prepared by substituting SS-OP with different ionic lipids (DLin-MC3-DMA) are presented. [Figure 37C] Figure 37A presents data on lung transfection and body weight change using LNPs. [Figure 37D] Figure 37A presents data on lung transfection and body weight change using LNPs. [Figure 38A] Exemplary LNPs prepared by substituting DLin-MC3-DMA for SS-OP in three-lipid LNPs are presented. [Figure 38B] Percent encapsulation and lung transfection data using LNPs are presented in Figure 38A. [Figure 38C] Percent encapsulation and lung transfection data using LNPs are presented in Figure 38A. [Figure 39A] Exemplary LNPs prepared by substituting DLin-MC3-DMA for SS-OP in five-lipid LNPs are presented. [Figure 39B] Percent encapsulation and lung transfection data using LNPs are presented in Figure 39A. [Figure 39C] Percent encapsulation and lung transfection data using LNPs are presented in Figure 39A. [Figure 40A] The titration time for mRNA adsorption to form LNPs in the two lipid formulations is presented. [Figure 40B] Figure 40A presents weight change data using LNP. [Figure 41A] Lung transfection and encapsulation percentage data for two-lipid LNPs made with titrating mRNA:lipid ratios are presented. [Figure 41B] Lung transfection and encapsulation percentage data for two-lipid LNPs made with titrating mRNA:lipid ratios are presented. [Figure 42A] Lung transfection and encapsulation percentage data for three lipid LNPs made with titrating N / P ratios are presented. [Figure 42B] Lung transfection and encapsulation percentage data for three lipid LNPs made with titrating N / P ratios are presented. [Figure 43A] Lung transfection and weight change data using the described two-lipid LNPS titrated in DMG-PEG2000 are presented. [Figure 43B] Lung transfection and weight change data using the described two-lipid LNPS titrated in DMG-PEG2000 are presented. [Figure 44] Average lung radiance comparing microfluidic mixing of lipids in ethanol with malate buffer versus manual mixing is presented. [Figure 45A] Formulation, transfection, and IHC data comparing the MC3 formulation with five lipid LNP compositions herein are presented. [Figure 45B] Formulation, transfection, and IHC data comparing the MC3 formulation with five lipid LNP compositions herein are presented. [Figure 45C] Formulation, transfection, and IHC data comparing the MC3 formulation with five lipid LNP compositions herein are presented. [Figure 45D] Formulation, transfection, and IHC data comparing the MC3 formulation with five lipid LNP compositions herein are presented. [Figure 46A] Exemplary 2-, 3-, and 5-lipid LNPs are presented. [Figure 46B] Transfection data for lung, liver, and spleen cells using LNPs are presented in Figure 46A. [Figure 46C] Transfection data for lung, liver, and spleen cells using LNPs are presented in Figure 46A. [Figure 46D] Transfection data for lung, liver, and spleen cells using LNPs are presented in Figure 46A. [Figure 46E] IHC data relating to the use of exemplary 2-, 3-, and 5-lipid LNPs in lung, liver, and spleen tissues is presented. [Figure 47A]Pharmacokinetics of two specific lipid LNPs in plasma, lung, and liver. [Figure 47B] Pharmacokinetics of two specific lipid LNPs in plasma, lung, and liver. [Figure 47C] Pharmacokinetics of two specific lipid LNPs in plasma, lung, and liver. [Figure 48] Table 1 shows the pharmacokinetics of five specific lipid LNPs. [Figure 49A] 1 shows the biodistribution of exemplary five-lipid LNPs of Table 1 upon administration to mammals. [Figure 49B] 1 shows the biodistribution of exemplary five-lipid LNPs of Table 1 upon administration to mammals. [Figure 50A] 46B shows the biodistribution of the three lipid LNPs depicted in FIG. 46A upon administration to mammals. [Figure 50B] 46B shows the biodistribution of the three lipid LNPs depicted in FIG. 46A upon administration to mammals. [Figure 50C] 46B shows the biodistribution of the three lipid LNPs described in FIG. 46A upon administration to mammals. [Figure 50D] 1 shows organ-level bioluminescence associated with the use of three-lipid LNPs in mammals, as described in Example 33. [Figure 50E] 1 shows organ-level bioluminescence associated with the use of three-lipid LNPs in mammals, as described in Example 33. [Figure 50F] 1 shows organ-level bioluminescence associated with the use of three-lipid LNPs in mammals, as described in Example 33. [Figure 50G] 46B shows the biodistribution of the exemplary three-lipid LNP depicted in FIG. 46A upon administration to a mammal. [Figure 51A] Table 3 presents data on telomere elongation and telomerase activity in lung epithelial cells using LNPs. [Figure 51B] Table 3 presents data on telomere elongation and telomerase activity in lung epithelial cells using LNPs. [Figure 52] We present data on telomerase activity in lung fibroblasts using LNPs in Table 3. [Figure 53A] Pulmonary transfection and encapsulation efficiency data using the LNPs of Example 38 are presented. [Figure 53B] Pulmonary transfection and encapsulation efficiency data using the LNPs of Example 38 are presented. [Figure 54A] 1 presents pulmonary transfection and encapsulation efficiency data using the LNPs of Example 39. [Figure 54B] 1 presents pulmonary transfection and encapsulation efficiency data using the LNPs of Example 39. [Figure 55] 1 shows the mean lung radiance of freeze-dried (lyophilized) LNPs of Example 36. [Figure 56] The optimal cationic lipid to ionic lipid ratio ("C / I ratio") and optimal lipid nitrogen:polynucleotide phosphate ratio ("N / P ratio") for LNPs containing either the SS-OP or DLin-MC3-DMA family of ionic lipids are modeled. The gray boxes indicate the optimal ranges for C / I and N / P ratios. The horizontal dashed lines indicate the threshold for relative lung radiance, selected so that the convex peak(s) of relative lung radiance in each graph significantly exceed the dashed line. The horizontal dashed lines indicate the threshold for relative yield, so that the convex curve of relative yield significantly exceeds the dashed line. The limits of the optimal range (gray boxes) are determined where the relative lung radiance curve and relative yield intersect with the horizontal dashed or dotted threshold lines, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0023] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications, and other publications referenced herein are incorporated by reference in their entirety. To the extent that a definition set forth in this section contradicts or is inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth in this section shall take precedence over the definition incorporated herein by reference.

[0025] As used herein, "a" or "an" means "at least one" or "one or more."

[0026] As used herein, the term "approximately" or "about," when applied to one or more subject values, refers to a value that is similar in magnitude to and / or within a similar range of a stated reference value. In certain embodiments, the term "approximately" or "about" can refer to a range of values ​​that falls within 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 the stated reference value, unless otherwise stated or otherwise apparent from the context (unless such number exceeds 100% of possible values). When a range of values ​​is provided, 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 within the present disclosure unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0027] As used in this document, the term "and / or" can mean "and," can mean "or," can mean "exclusive or," can mean "one," can mean "some but not all," can mean "neither," and / or can mean "both."

[0028] 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.), etc. 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).

[0029] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides, and polymers thereof, in single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, a phosphate, and either a purine or a pyrimidine base. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions, when translated (in the case of RNA) or transcribed and translated (in the case of DNA), produce the same sequence of amino acid residues as the original nucleic acid sequence, or can be achieved by generating nucleic acid sequences in which the third position of one or more (or all) selected codons is substituted with mixed-base and / or deoxyinosine residues.

[0030] The term "nucleotide sequence" refers to a polymer of DNA or RNA, which may be single- or double-stranded, optionally containing synthetic, non-natural, or modified nucleotide bases that can be incorporated into a DNA or RNA polymer.

[0031] As used herein, "ribonucleic acid" means a polymer of ribonucleotides.

[0032] As used herein, "mRNA species" means mRNA molecules that have the same length and sequence of ribonucleotides.

[0033] Telomeres are repetitive DNA sequences bound by proteins at the ends of each chromosome arm, which protect the ends of chromosomes by forming loops. Telomeres shorten with cell division, and when they become too short to form loops, the ends of the DNA become exposed, activating DNA damage signaling (P53 / P21), leading to cellular senescence and apoptosis. Telomerase reverse transcriptase (TERT) lengthens telomeres and is transiently activated periodically throughout life in normal stem and progenitor cells.

[0034] Alveolar type 2 (AT2) cells are stem cells capable of regenerating and repairing the alveolar epithelium. Accumulating evidence implicates AT2 cell telomeres in the pathogenesis and development of idiopathic pulmonary fibrosis (IPF). In the telomere model of IPF, AT2 cells divide to repair and regenerate the alveoli following alveolar cell damage due to various causes. However, telomeres shorten during cell division, ultimately leading to "critically short" telomeres in AT2 cells (meaning that protective telomere loops cannot be formed), resulting in chronic activation of p53 and p21, leading to deleterious consequences. The model suggests that individuals born with short telomeres or individuals predisposed to have short telomeres are more susceptible to IPF. Indeed, the average telomere length of leukocytes in IPF patients is below the 10th percentile of the general population, implying that 15% of patients with familial IPF have loss-of-function mutations in telomere maintenance genes, most commonly telomerase reverse transcriptase (TERT), an enzyme that lengthens telomeres and is expressed in alveolar epithelial AT2 cells. Regardless of TERT mutation status, lung tissue from IPF patients has reduced levels of TERT mRNA and TERT protein. The model also suggests that telomeres in AT2 cells are relatively short within individuals compared with other cell types. Indeed, in IPF patients, telomeres in AT2 cells are shorter than those in other lung cells, including myofibroblasts. Furthermore, telomeres in AT2 cells are shorter in fibrotic than non-fibrotic regions.

[0035] Short telomeres induce cellular senescence, and lungs of IPF patients exhibit increased senescence markers, including P21. Short telomeres induce apoptosis of AT2 cells, and lungs of IPF patients exhibit reduced numbers of AT2 cells. Consistent with impaired alveolar repair by AT2 cells due to telomere-induced senescence and apoptosis, IPF patients also exhibit elevated serum markers of epithelial damage, such as CA-125 and SP-D.

[0036] Consistent with a role for short telomere-induced senescence in altering cell-cell signaling, IPF patients also have elevated levels of proinflammatory molecules (SASPs) (e.g., TGFβ) involved in the activation and transformation of lung fibroblasts into myofibroblasts. Consistent with short telomere-induced senescence inhibiting normal differentiation of AT2, the lungs of IPF patients also accumulate a transitional cellular phenotype with a senescent profile.

[0037] Consistent with the consequences of short telomeres, long telomere length in patients with IPF is associated with transplant-free survival independent of age, sex, forced vital capacity, and carbon monoxide diffusing capacity. Thus, in the telomere model of IPF, alveolar epithelial cell telomeres function as a pathogenetic link, first orchestrating alveolar damage over time until critical shortening, then triggering multiple pathogenic consequences and ultimately death.

[0038] The telomere model of IPF facilitates exploration of telomere lengthening. To avoid the risks of constitutive telomerase activity and AAV delivery of TERT DNA associated with cancer, we focus here on methods to only transiently increase telomerase activity, as stem cells do periodically throughout life.

[0039] The data provided herein substantiate or support, for example, information and data relating to the in vivo pharmacokinetics of exemplary LNPs, particularly the time course of lipid concentration levels from the LNPs in the lung, liver, and / or serum. In embodiments described herein, LNP compositions and formulations are provided that are rapidly cleared from a subject but have a long half-life in the lung, lung tissue, and / or lung cells. In terms of rapid clearance, provided herein are LNP compositions that provide a ribonucleic acid payload that does not persist (90%-100% cleared) in the tissues listed in Figure 50A , excluding the lung, approximately 9-11 hours after administration to a subject. Conversely, in terms of rapid clearance, provided herein are LNP compositions that provide a ribonucleic acid payload that is present in the lungs about 9-11 hours after administration, but is completely absent, or absent at any significant level, in one or more (including all) of the following tissues: spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose, thymus, stomach, colon, and / or duodenal tissues.

[0040] The data provided herein demonstrate or support the use of LNPs whose mRNA encodes telomerase, for example. Specifically provided herein is a demonstration of telomere lengthening in mouse lung alveolar cells and mouse lung alveolar AT2 cells. Also provided is a demonstration of the increased colony-forming ability of human primary alveolar epithelial cells by treatment with TERT mRNA LNPs.

[0041] Also provided is a demonstration of the pharmacodynamics of TERT mRNA LNPs, specifically the time course of telomerase activity in lung tissue after intravenous infusion of TERT mRNA LNPs in mice.

[0042] Also provided is efficacy data for TERT mRNA LNPs in treating pulmonary fibrosis in vivo, specifically improving lung function (FEV0.1 and FVC), reducing pulmonary fibrosis, increasing the normal aerated lung ratio, and improving lung structure (alveolar density or number, circularity, and diameter).

[0043] Also provided herein is a method for producing LNP.In a specific example, LNP is produced by a method that forms LNP before adding nucleic acid.In a specific embodiment thereof, such LNP is a two-, three-, or five-lipid LNP as described herein, and the method uses vortex mixing during adding nucleic acid.

[0044] The inventors have found that the ratio of cationic lipid to ionic lipid (e.g., SSOP:DOTAP or MC3:DOTAP) in LNPs adapted to function according to the presently contemplated methods and uses cannot be predicted based on the specific components of the LNP. Specifically, while not intending to be bound by any particular theory, it is apparent experimentally that the specific ionic lipids in an LNP influence the ratio of cationic lipid to ionic lipid in a manner that requires experimentation to confirm. The experimental evidence provided herein provides support for a specific ratio of cationic lipid to ionic lipid, or a specific ratio range, based on the ionic lipids identified in the LNP. Tables and figures presented herein (e.g., Table 8) provide additional examples of specific ratios of cationic lipid to ionic lipid for the three-lipid and five-lipid LNPs contemplated herein. In certain embodiments, the ratio of cationic lipid to ionic lipid is approximately 0.8, and the ionic lipid is SS-OP. In certain related embodiments, the LNPs have a cationic lipid to ionic lipid ratio of approximately 0.8 and are comprised of three or five lipids. Also, in certain related embodiments, the LNPs have a cationic lipid to ionic lipid ratio of approximately 0.8 and are comprised of three or five lipids including DOTAP. Also, in certain related embodiments, the LNPs have a cationic lipid to ionic lipid ratio of approximately 0.8 and are comprised of three or five lipids including a pegylated lipid (e.g., DMG-PEG2000) and DOTAP.

[0045] Also, in certain embodiments, the ratio of cationic lipid to ionic lipid is approximately 1.8, and the ionic lipid is SS-OP. In certain related embodiments, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.8 and is composed of five lipids. Also, in certain related embodiments, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.8 and is composed of five lipids, including DOTAP and DOPC. Also, in certain related embodiments, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.8 and is composed of five lipids, including a pegylated lipid (e.g., DMG-PEG2000), DOPC, and DOTAP.

[0046] In certain embodiments, the ratio of cationic lipid to ionic lipid is approximately 1.5, and the ionic lipid is DLin-MC3-DMA. In certain related embodiments, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5 and is composed of three lipids, including DOTAP. In certain related embodiments, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5 and is composed of three lipids, including DOTAP. In certain related embodiments, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5 and is composed of three lipids, including a PEGylated lipid (e.g., DMG-PEG2000) and DOTAP.

[0047] LNP compositions of the present disclosure include and encompass a variety of lipid and lipid:mRNA ratios (lipid:mRNA ratios are also referred to herein as "N / P ratios"). As referred to herein, LNP composition refers to either the final LNP composition or the formulation used to produce the LNPs, or both, as will be clear based on the context in which the term is used. Specific examples of contemplated compositions are provided in the Examples, including the Supplementary Figures. Particularly preferred LNP compositions of the present disclosure are presented, for example, in Table 8 and other tables, figures, and specification presented herein.

[0048] According to preferred embodiments of the present invention, nucleic acid-containing LNP compositions are provided that are composed of five different lipids, including ionizable lipids and cationic lipids, and have an N / P ratio of 10 to 30. Table 8 provides some exemplary configurations. Often, according to such embodiments, the LNP compositions are further characterized by a cationic lipid to ionic lipid ratio of 0.8 to 1.8. According to such embodiments, the LNP compositions are characterized by a cationic lipid to ionic lipid ratio of approximately 0.8. According to such embodiments, the LNP compositions include SS-OP as the ionic lipid in the composition. According to such embodiments, the LNP compositions are used in vivo in a mammalian subject to target lung tissue and / or lung cells in a manner that provides transfection of a nucleic acid payload into the lung tissue and / or lung cells. Accordingly, such compositions adapted to target lung tissue and / or lung cells are contemplated herein. Also, according to such embodiments, such LNP compositions preferentially target lung tissue and / or lung cells over other tissues of a subject (e.g., spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose tissue, thymus, stomach, colon, and / or duodenal tissue). Also, according to such embodiments, such LNP compositions have a half-life in lung tissue and / or lung cells that is increased relative to the half-life in other tissues of a subject (e.g., spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose tissue, thymus, stomach, colon, and / or duodenal tissue). According to such embodiments, such LNP compositions have a half-life in lung tissue of greater than 30 hours, or 30 to 40 hours, or about 37 hours.

[0049] Also provided according to preferred embodiments of the present specification are nucleic acid-containing LNP compositions composed of three different lipids, including an ionizable lipid and a cationic lipid, and having an N / P ratio of 4 to 24 or an N / P ratio of 4.5 to 12. Table 8 provides some exemplary configurations. Often, according to such embodiments, the LNP composition is further characterized by a cationic lipid to ionic lipid ratio of 0.8 to 1.5. Also, according to such embodiments, the LNP composition is characterized by a cationic lipid to ionic lipid ratio of approximately 0.8. Also, according to such embodiments, the LNP composition includes SS-OP as the ionic lipid, and the cationic lipid to ionic lipid ratio is approximately 0.8. Also, according to such embodiments, the LNP composition includes DLin-MC3-DMA as the ionic lipid, and the cationic lipid to ionic lipid ratio is approximately 1.5, and optionally, the N / P ratio is 4.5 or greater. Also, according to such embodiments, such LNP compositions are used in vivo in a mammalian subject to target lung tissue and / or lung cells in a manner that provides transfection of a nucleic acid payload into the lung tissue and / or lung cells. Accordingly, such compositions adapted to target lung tissue and / or lung cells are contemplated herein. Also, according to such embodiments, such LNP compositions preferentially target lung tissue and / or lung cells over other tissues of the subject (e.g., spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose tissue, thymus, stomach, colon, and / or duodenal tissue). Also, according to such embodiments, such LNP compositions have a half-life in lung tissue and / or lung cells that is increased relative to its half-life in other tissues of the subject (e.g., spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose tissue, thymus, stomach, colon, and / or duodenal tissue). According to such embodiments, such LNP compositions have a half-life in lung tissue of greater than 5 hours, greater than 30 hours, or 30-40 hours, or about 37 hours.

[0050] Furthermore, according to a preferred embodiment of the present specification, there is provided a nucleic acid-containing LNP composition that is composed of three or five different lipids including DOTAP, that includes an ionic lipid and a cationic lipid, and that has an N / P ratio of 4 to 24, or an N / P ratio of approximately 12. Furthermore, according to a preferred embodiment of the present specification, there is provided a nucleic acid-containing LNP composition that is composed of three different lipids including DLin-MC3-DMA, that includes an ionic lipid and a cationic lipid, and that has an N / P ratio of 4 to 24, or an N / P ratio of approximately 4.5.

[0051] Also contemplated herein, according to preferred embodiments, are nucleic acid-containing LNP compositions composed of two different lipids and having an N / P ratio of approximately 4. Preferably, in such embodiments, one of the two lipids is or comprises DOTAP. Also contemplated herein are such LNP compositions for use in vivo in a mammalian subject to target lung tissue and / or lung cells in a manner that provides transfection of a nucleic acid payload into the lung tissue and / or lung cells. Thus, such compositions adapted to target lung tissue and / or lung cells are contemplated herein. Also contemplated herein are such LNP compositions that preferentially target lung tissue and / or lung cells over other tissues of the subject (e.g., spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose tissue, thymus, stomach, colon, and / or duodenal tissue). Also, according to such embodiments, such LNP compositions have a half-life in lung tissue and / or lung cells that is increased relative to the half-life in other tissues of a subject (e.g., spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, adipose, thymus, stomach, colon, and / or duodenal tissue). According to such embodiments, such LNP compositions have a half-life in lung tissue of greater than 30 hours, or 30 to 40 hours, or about 37 hours.

[0052] Also provided herein are methods for producing and / or storing LNP compositions. The inventors have found that sucrose is a surprisingly good cryoprotectant for the LNP compositions currently under consideration. Specifically, it has been found that sucrose in the range of 5% to 25% of the final composition provides a desirable level of cryoprotection. In certain embodiments, the cryoprotectant (e.g., sucrose) is present at approximately 15% of the final composition.

[0053] Delivery of synthetic nucleoside-modified mRNA encoding TERT (TERT mRNA) transiently elevates telomerase activity, sufficiently lengthening telomeres within a few hours and reversing years of telomere shortening. Importantly for safety, the mRNA and resulting TERT protein are degraded within hours and days, respectively, and the resulting telomerase activity persists for several days (Figure 17). Thus, delivery of synthetic TERT mRNA recapitulates the normal telomere lengthening induced by endogenous TERT mRNA, which occurs periodically in stem cells throughout life. Shortly after treatment ends, lengthened telomeres resume shortening at their normal rate. Recent advances in lipid nanoparticle (LNP) vehicles enable the delivery of mRNA to up to 90% or more of lung epithelial cells via intravenous (IV) infusion, with good tolerability. synthetic mRNA

[0054] As used herein, synthetic ribonucleic acid (RNA) can refer to any RNA sequence that contains mutations (points or deletions) or additional nucleotides not found in the wild-type sequence. For example, messenger RNA (mRNA) can refer to a wild-type sequence encoding a human sequence flanked by 1, 2, 3, 10, 100, or more nucleotide additions. Similarly, the nucleotides themselves can be modified to encode amino acids different from the wild-type or to reduce immunogenicity in cells or tissues. In some embodiments, the mRNA sequence can include any of the following modifications, including, but not limited to, an untranslated region (UTR), a 5' cap, and a polyadenosine tail. In some embodiments, the RNA can be circular and / or self-replicating.

[0055] Exemplary methods for making circular mRNA are presented 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. Exemplary methods for generating self-replicating mRNA are presented 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.

[0056] In some embodiments, the mRNA may comprise a codon-optimized sequence. In some embodiments, the mRNA may comprise a uridine-deficient sequence.

[0057] In some embodiments, the 5' cap of the ribonucleic acid is a non-immunogenic cap. In some embodiments, the 5' cap can increase translation of the ribonucleic acid. In some embodiments, the 5' cap can 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)).

[0058] These or other features may increase translation of the protein encoded by the ribonucleic acid, 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 derived from genes with highly stable and / or rapidly translated mRNAs (e.g., α-globin or β-globin, c-fos, or tobacco etch virus). In some embodiments, the 5' UTR and 3' UTR are derived from different genes or from a species different from the species to which the composition is delivered. UTRs may also be a collection of portions of UTRs from mRNAs of different genes, selected to achieve a particular combination of translational stability and efficiency. UTRs may also contain engineered sequences that confer properties to the RNA, such as cell-type-specific or cell-type-independent stability.

[0059] The ribonucleic acids of the present disclosure may contain one or more modified nucleosides and / or primary sequences of nucleosides that modulate RNA translation, stability, or immunogenicity. 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 a mature RNA contains a modified nucleoside, and other modified nucleosides are often present elsewhere in the RNA. These modifications may prevent the RNA from being recognized as foreign RNA. Synthetic RNA molecules made using certain nucleosides are much less immunogenic than unmodified RNA. Immunogenicity can be further reduced by purifying the synthetic mRNA, for example, by using high-performance liquid chromatography (HPLC). Modified nucleosides may be selected, for example, from the nucleosides listed below. In some embodiments, the nucleoside is pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methoxyuridine, or 5-methylcytidine. The primary sequence can be modified in a way that increases or decreases immunogenicity. In some situations, it may be desirable for the modified RNA to retain some immunogenicity.

[0060] 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), yW wibutosine, o2yW peroxywibutosine, OhyW hydroxywibutosine, OhyW* hypomodified hydroxywibutosine, imG wibutosine, m2,7G N2,7-dimethylguanosine, m2,2,7G N2,N2,7-trimethylguanosine, I inosine, m1I 1-methylinosine, Im 2'-O-methylinosine, Q queusine, galQ galactosyl-queusine, manQ mannosyl-queusine, Ψ pseudouridine, D dihydrouridine, m5U 5-methyluridine, Um 2'-O-methyluridine, m5Um 5,2'-O-dimethyluridinem1Ψ 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'-de 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, arauidine, 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-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).

[0061] 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 enable 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 therapeutic agents in mice via local 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.

[0062] In some embodiments, an immune response to 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 mRNA may be undesirable, and the RNA may be modified to minimize such a response. The RNA may be modified in either situation.

[0063] A 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 the tools of molecular biology 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 partially or completely by 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.

[0064] The ribonucleic acids of the present disclosure may be prepared using a variety of techniques, as will be understood 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.

[0065] In some embodiments, the ribonucleic acid can be purified. As noted above, purification can reduce the immunogenicity of the ribonucleic acid, which can 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.

[0066] In some embodiments, the mRNA sequence may be synthesized as unmodified or modified mRNA. The mRNA may be modified to increase stability and / or to avoid immune detection and degradation. The modified mRNA may include, for example, one or more of a nucleotide modification, a nucleoside modification, a backbone modification, a sugar modification, and / or a base modification. 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 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

[0067] 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, such as 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, 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-mannosylqueuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0068] 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 pyrimidine (thymine (T), cytidine (C), uridine (U)), as well as nucleosides containing analogs and derivatives thereof, e.g., 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymine, 2'-deoxythiazol-1-yl (2'-thiazol-1-yl)-2'-pyrimidine ... ',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, 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-thio These include, but are not limited to, uridine, 5-methyl-uridine, N-uridine-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine, 5-methoxy-aminomethyl-2-thiouridine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 1-methyl-pseudouridine, queusine, beta-D-mannosyl-queusine, wybutoxosine, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0069] The preparation of such base, nucleoside, nucleotide, and backbone analogs, modifications, and derivatives is known to those of skill in the art, for example, from 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.

[0070] In some embodiments, the mRNA is about 80%, about 90%, 95%, 99%, or 100% depleted of uracil nucleosides and substituted with uracil nucleoside analogs, such as pseudouridine, 5-methoxyuridine, or N-1-methyl-pseudouridine.

[0071] In some embodiments, RNA may contain RNA backbone modification.Usually, backbone modification is the modification that 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 that phosphodiester bond is replaced with a member of the group consisting of peptide, methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate (for example, cytidine 5'-O-(1-thiophosphate)), boranophosphate, and / or positively charged guanidinium group, or other means of replacing phosphodiester bond.

[0072] 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'-arauidine), or azidophosphates (2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine).

[0073] In some embodiments, the RNA can 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).

[0074] In some embodiments, 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.

[0075] Cap Structure In some embodiments, an 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 a 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.

[0076] In some embodiments, the 5' cap may comprise an 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 may 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, m7,3 dGpppG 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)).

[0077] In some embodiments, a suitable cap is 7-methylguanylate ("m7G") attached to the 5' end of the initially 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 2'-O-methyl residues 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 2'-O-methyl residues attached to both bases 2 and 3.

[0078] Various m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG 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 for use 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 (incorporated herein by reference), including biotinylated cap analogs.

[0079] 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-aminoguanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0080] In some embodiments, the 5' cap comprises or consists of an internal ribosome entry site (IRES). In some embodiments, the IRES is within 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.

[0081] Tail structure The presence of a "tail" can help protect mRNA from exonuclease degradation. Poly-A tails are thought to stabilize natural messenger and synthetic sense RNA. Thus, in certain embodiments, a long poly-A tail can be added to an mRNA molecule, thereby making the RNA more stable. Poly-A tails can be added using a variety of techniques recognized in the art. For example, poly-A polymerase can be used to add long poly-A tails to synthetic or in vitro transcribed RNA (Yokoe, et al. Nature Biotechnology. 1996;14:1252-1256). Transcription vectors can also encode long poly-A tails. Additionally, poly-A tails can be added by direct transcription from PCR products. Poly-A can also be ligated to the 3' end of sense RNA using RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2002). nd Ed., ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0082] In some embodiments, the mRNA may include a 3' poly(A) tail structure. The length of the poly(A) tail may be at least about 10, 50, 100, 200, 300, 400, or at least about 500 nucleotides. In some embodiments, the poly(A) 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 poly(A) tail is 120 adenosine nucleotides.

[0083] 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 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 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 or poly-C tail is related to the stability of the modified sense mRNA and, therefore, to protein transcription. For example, because the length of the poly-A tail can affect the half-life of the sense mRNA molecule, the length of the poly-A tail can be adjusted to alter the level of resistance of the mRNA to nucleases, thereby providing greater control over the time course of polynucleotide expression and / or polypeptide production.

[0084] 5' and 3' Untranslated Regions (UTRs) In some embodiments, an mRNA may comprise a 5' untranslated region (UTR) and / or a 3' UTR. In some embodiments, the 5' UTR may comprise one or more elements that affect mRNA stability or translation. In some embodiments, for example, the 5' UTR may comprise 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 comprises one or more of a poly(A) signal, a binding site for a protein that can affect mRNA stability or localization, or one or more binding sites for miRNA. In some embodiments, the 3' UTR may be about 0 to about 50, or about 50 to about 100 nucleotides in length.

[0085] 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 subsequence 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 another example, the 5' UTR may include a tobacco etch virus (TEV) sequence. Generally, these modifications 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 in vivo nuclease digestion.

[0086] 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 another example, the 3'UTR may include a Xenopus globin sequence. In some embodiments, the UTR may be any of those described in PCT Application No. WO2017053297A1 and Patent No. US10519189B2, or a functional variant thereof, both of which are incorporated herein in their entirety.

[0087] ionic lipids In some embodiments of the present disclosure, the LNP may include an ionizable lipid (e.g., SS-OP or an analog thereof, or MC3 or an analog thereof). The charge of the lipid may depend on the pH of the surrounding solution, making the lipid 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.

[0088] Ionizable lipids can refer to any of a number of lipid species that have a net positive charge at a selected pH (e.g., physiological pH). In some embodiments, LNPs may comprise ionizable lipids disclosed in either WO2010 / 053572 or WO2012 / 170930 (both of which are incorporated by reference in their entireties), or variations thereof.

[0089] In some embodiments, the LNP is MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), DLin-MC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester), or analogs thereof (including but not limited to). The MC3 thioester, MC3 ether, MC4 ether, MC3 alkyne, MC3 amide, Pan-MC3, Pan-MC4, Pan-MC5, CP-LenMC3, CP-γ-LenMC3, CP-MC3, D-Lin-MC2-DMA, Lipid5, SM-102, ALC-0315, and combinations thereof.

[0090] In some embodiments, LNPs may comprise one or more of cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), DLinDAP (1,2-dilinoleyl-3-dimethylammonium-propane), DLin-DMA, DLin-D-DMA, DLin-KC2-DMA (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), and DODMA.

[0091] 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]

[0092] In equation (1), R 1a and R 1bR 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 1 to 2 carbon atoms. Specific examples of alkylene groups having 1 to 6 carbon atoms include methylene, ethylene, trimethylene, isopropylene, tetramethylene, isobutylene, pentamethylene, and neopentylene. 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.

[0093] R 1a is R 1b It may be different from or the same as

[0094] 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.

[0095] 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. Specific 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.

[0096] Specific structures of acyclic alkyl tertiary amino groups having 1 to 6 carbon atoms and one tertiary amino group are: X 1 It is expressed as: [ka]

[0097] 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 alkyl groups having 1 to 6 carbon atoms include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, and isopentyl groups. Specific examples of alkyl groups having 1 to 6 carbon atoms include neopentyl, t-pentyl, 1,2-dimethylpropyl, 2-methylbutyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, and cyclohexyl groups.

[0098] 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.

[0099] 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 formula: [ka]

[0100] X 2 In the formula, p is 1 or 2. When p is 1, X 2is a pyrrolidine group, and when p is 2, X 2 is a piperidylene group.

[0101] The specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and two tertiary amino groups is X 3 It is expressed as: [ka]

[0102] X 3 The value of w is either 1 or 2. If w is 1, then X 3 is an imidazolidylene group, and when w is 2, X 3 is a piperazylene group.

[0103] X a is X b It may be different from or the same as.

[0104] R 2a and R 2b each independently represent an alkylene group or an oxydialkylene group having up to 8 carbon atoms, optionally each independently represent an alkylene group having up to 8 carbon atoms.

[0105] Alkylene groups having 8 or fewer carbon atoms can 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.

[0106] An oxydialkylene group having 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) connected via an ether bond, where the total number of carbon atoms in 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.

[0107] R 2a and R 2b may be the same or different.

[0108] 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 Y a and Y b is an ester bond, optionally -Z a -CO---R 2a - and -Z b -CO-OR 2b -Structure.

[0109] Y a is Y b It may be different or the same as

[0110] 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. The aromatic compound optionally contains 6 to 12 or 6 to 7 carbon atoms. Furthermore, the aromatic compound optionally contains one aromatic ring.

[0111] Examples of the aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms include aromatic hydrocarbon rings such as a benzene ring, a naphthalene ring, and an anthracene ring; aromatic heterocycles such as an imidazole ring, a pyrazole ring, an oxazole ring, an isoxazole ring, a thiazole ring, an isothiazole ring, a triazine ring, a pyrrole ring, a furanthiophene ring, a pyrimidine ring, a pyridazine ring, a pyrazine ring, a pyridine ring, a purine ring, a pteridine ring, a benzimidazole ring, an indole ring, a benzofuran ring, a quinazoline ring, a phthalazine ring, a quinoline ring, an isoquinoline ring, a coumarin ring, a chromone ring, a benzodiazepine ring, a phenoxazine ring, a phenothiazine ring, and an acridine ring; and optionally, a benzene ring, a naphthalene ring, or an anthracene ring. The aromatic ring 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.

[0112] Z a and Z b The specific structure of Z 1 Examples include: [ka]

[0113] s represents an integer of 0 to 3, t represents an integer of 0 to 3, and u represents an integer of 0 to 4.

[0114] Z 1 In the formula, S is optionally an integer from 0 to 1.

[0115] Z 1 In the formula, T is optionally an integer from 0 to 2.

[0116] Z 1 In the formula, U is optionally an integer of 0 to 2.

[0117] 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, and mido groups. group), methoxycarbonylamino group, fluorine atom, chlorine atom, bromine atom, iodine atom, methylsulfanyl group, phenylsulfonyl group, nitro group, trifluoromethyl group, cyano group, methyl group, ethyl group, propyl group, isopropyl group, t-butyl group, ureido group, methoxy group, ethoxy group, propoxy group, isopropoxy group, t-butoxy group, phenyl group, and phenoxy group. 4 If there is, then each R 4 may be the same or different.

[0118] Z a is Z b It may be different from or the same as.

[0119] 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, an 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.

[0120] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, colcalciferol, dihydroergocalciferol, dihydrotaxolol, tocopherol, and tocotrienol. The fat-soluble vitamin having a hydroxyl group is optionally tocopherol.

[0121] Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, and the like, optionally cholesterol or cholestanol.

[0122] 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 alkyl groups, alkenyl groups, and alkynyl groups, and optionally include alkyl groups or alkenyl groups. 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, tetradecen ...icosenyl, hexadecenyl, hexadecenyl, octadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, octadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl, hexadecenyl Examples thereof 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.

[0123] 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.

[0124] R 3a is R 3b It may be different from or the same as.

[0125] In one embodiment of the present disclosure, R 1a is R 1b is the same as X a is 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 identical to R 3a is R 3b is the same as

[0126] 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 an acyclic 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, or a propylene group); Y a and Y b are each independently 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 an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0127] Ionic lipid (1-2); R 1a and R 1b are each independently an alkylene group of 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); Xa and Xb are each independently an acyclic alkyl tertiary amino group of 1 to 3 carbon atoms and one tertiary amino group (e.g., —N(CH3)—), or a cyclic alkylene tertiary amino group of 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, or a propylene group); Y a and Y b are each independently an ester bond or an amide bond; Za and Zb are each independently 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) and 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 R1b are each independently an alkylene group having 1 to 2 carbon atoms (e.g., a methylene group, an ethylene group); X a and X b are each independently, X 1 and: [ka] R 5 is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X 2 [ka] (p is 1 or 2), and 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, or a propylene group); Y a and Y b are each independently an ester bond or an amide bond, and Z a and Z b are each independently Z 1 and: [ka] 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), (R 4 )u each independently represents a substituent. 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 17 carbon atoms (e.g., heptadecenyl, heptadecadienyl, 1-hexylnonyl).

[0128] 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 the diagram below. [Table 2-1] [Table 2-2] [Table 3]

[0129] The lipid having the structure of Formula I is shown in the diagram below. For example, SS-OP is also called 0-Ph-P4C2. As used herein, the term "SS-OP analog" refers to the compound of Formula I.

[0130] [Table 4]

[0131] cationic lipids In some embodiments, the LNPs of the present disclosure comprise a cationic lipid (e.g., DOTAP or a variation thereof). The cationic lipid may be a "permanent cationic lipid." The term cationic lipid refers to a lipid that is cationic in the pH range found in mammalian physiological environments, such as blood or interstitial fluid. The cationic lipid may be composed of a cationic amine moiety and a lipid moiety, and the cationic amine moiety and the polyanionic nucleic acid may interact to form a positively charged liposome or lipid membrane structure. Thus, cellular uptake may be facilitated and the nucleic acid may be delivered into the cell.

[0132] 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 LNP comprises an ionic lipid, and the ionic lipid is 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 ...5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide (DOGS), 5-carboxyspermylglycine dioctadecylamide Pan (DODAP), 11,2-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-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane] St-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,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), or mixtures thereof.

[0133] In some embodiments, cationic lipid refers to cationic cholesterol lipid. In some embodiments of the present disclosure, the LNP comprises imidazole cholesterol ester (ICE). In some embodiments, the ICE structure is substantially similar to: [ka]

[0134] 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]

[0135] In some embodiments of the present disclosure, the LNP comprises 20α-hydroxycholesterol 5-cholesterol-3α.

[0136] In some embodiments, the 20α-hydroxycholesterol 5-cholestene-3α (also known as 20α-diol or 20α chol structure) is substantially similar to: [ka]

[0137] 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: [ka]

[0138] structural lipids In some embodiments, the LNP comprises a structured lipid. As used herein, a structured lipid is a lipid that imparts physical or chemical properties to the LNP in addition to or independent of charge. By way of example, structured lipids may tend to have shape, size, rigidity, hydrophobicity, or other properties that increase the therapeutic utility 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 lipids are charge-neutral because they lack charged moieties or are zwitterions whose balanced charges sum to a net charge of zero.

[0139] 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-phosphatidyl The lipids may comprise a structured lipid selected from one or more of 16-O-oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or variants thereof.

[0140] In some embodiments, LNPs may comprise one or more phosphatidyl lipids, such as phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine). In some embodiments, 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.

[0141] Cholesterol-based lipids In some embodiments, the LNPs may comprise one or more cholesterol-based lipids, which may include, but are not limited to, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), and 1,4-bis(3-N-oleylamino-propyl)piperazine.

[0142] PEGylated lipids In some embodiments of the present disclosure, LNPs may comprise one or more PEGylated lipids. For example, polyethylene glycol (PEG)-modified phospholipids and derivatized lipids, including derivatized ceramides (PEG-CER) (including N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8PEG-2000 ceramide)), in combination with one or more ionizable lipids and / or other lipids, are contemplated by the present disclosure. In some embodiments, the PEGylated lipid comprises 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 by any particular theory, it is believed that the addition of PEGylated lipids may prevent aggregation of the complex, increase circulation lifetime, and facilitate delivery of liposome-encapsulated mRNA to target cells.

[0143] Treatment method The treatment methods described herein refer to lengthening telomeres in the lungs of a subject in need thereof by administering LNPs of the present disclosure comprising one or more mRNA sequences. The compositions and methods of the present disclosure can be used to treat conditions in or involving the lungs or lung cells. In some embodiments, the compositions and / or methods of using the compositions of the present disclosure are intended to treat fibrotic conditions, including fibrosis. In some embodiments, the compositions and / or methods of using the compositions of the present disclosure intended to treat fibrotic conditions, including fibrosis, induce expression of encapsulated mRNA in lung tissue. In some embodiments, the compositions and / or methods of using the compositions of the present disclosure intended to treat fibrotic conditions, including fibrosis, do not induce cytotoxicity, tissue toxicity, or systemic toxicity. The compositions can be administered systemically (e.g., intravenously). In some embodiments, the compositions and / or methods of using the compositions of the present disclosure intended to treat conditions in or involving lung tissue cells do not induce cytotoxicity or systemic toxicity. The compositions can be administered systemically (e.g., intravenously).

[0144] In the compositions and methods described herein, in some embodiments, the LNP pharmaceutical compositions contemplated herein are administered to a subject in need thereof at a dose of about 0.001 mg / kg of subject body weight to about 2.0 mg / kg of subject body weight. In some embodiments, the targeted LNP 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 LNPs are administered to a subject in need thereof at a dose of 0.1 mg / kg. In some embodiments, the LNPs are administered to a subject in need thereof at a dose of 0.125 mg / kg.

[0145] In some embodiments, the LNP pharmaceutical compositions contemplated herein are administered to a subject in need thereof once. In some embodiments, the LNP is administered to a subject in need thereof two, three, four, or five or more times. In some embodiments, the LNP is administered twice a week, weekly, every two weeks, every four weeks, every six weeks, every 12 weeks, or every 15 weeks. In some embodiments, the LNP is administered monthly, every two months, every three months, every six months, once a year, continuously, or as determined by a physician.

[0146] In some embodiments, contemplated LNP pharmaceutical compositions are delivered orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, intravaginally, intrarectally, or intraocularly in an aerosolized inhaled form. In an exemplary embodiment, targeted LNPs are administered intravenously.

[0147] In some embodiments, exemplary LNP pharmaceutical compositions include an excipient or carrier (e.g., an aqueous carrier). A variety of aqueous carriers (e.g., buffered saline) can be used. The composition may contain pharmaceutically acceptable auxiliary substances and buffers as required for appropriate physiological conditions, such as pH, and toxicity counteracting agents, e.g., sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride, and sodium lactate. In some embodiments, the pharmaceutical composition comprises 10 mM sodium citrate buffered to pH 6.4. The composition may also contain a cryoprotectant, e.g., 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, depending on the selected mode of administration and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0148] In other embodiments, the compound or composition is administered without isolating cells, tissues, or organs from the subject (i.e., administration occurs 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 specific cells, cell types, tissues, or organs in the subject's body.

[0149] Administration of a compound or composition of the present disclosure can result in transient expression of mRNA within the LNP within the cell. Increased activity can be measured by various assays, such as the telomerase repeat amplification protocol (TRAP) assay. Commercial versions of the TRAP assay (e.g., Trapeze® Telomerase Detection Kit (Millipore)) are also available, which allow for sensitive detection and quantification of telomerase activity, although other measurement techniques are also possible.

[0150] As noted above, one advantage of this approach is that expression of the mRNA in the LNP is transient in the transfected cells, particularly in contrast to previous approaches in which, for example, the telomerase reverse transcriptase gene remains in an episomal DNA segment or is inserted into the cellular genomic sequence, or otherwise permanently modifies the genetic makeup of the target cell to result in constitutive activity of the nucleic acid sequence.

[0151] In some embodiments of the methods of the present invention, the transient expression is cell cycle independent.

[0152] medical kit A pharmaceutical composition of the LNP contemplated herein, or a treatment comprising the lipid component thereof, provided in a separate container. Kits and instructions for manufacture and / or use are also contemplated herein. In some embodiments, the therapeutic kits include administration devices (including, but not limited to, syringes, inhalers, nebulizers, and vials or containers). In some embodiments, the components of exemplary therapeutic kits are provided so that they can be mixed in a commercially available microfluidic mixer or vortex mixer. Often, such kits are provided with or without nucleic acids (e.g., mRNA) to be encapsulated in LNPs produced using the components of the therapeutic kit.

[0153] In another aspect, the present disclosure provides a ready-to-use kit for use in telomere lengthening in mammalian cells. The kit includes any of the compounds or compositions described above, along 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 optionally be filled with an inert gas, such as nitrogen, argon, or the like. In some embodiments, the packaging materials include, for example, 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 a lipid, as described herein. In some embodiments, one or more components of the formulation are provided frozen or lyophilized with a cryoprotectant.

[0154] 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.

[0155] Embodiment In embodiment 1, a method of delivering a polynucleotide to a subject is provided, the method comprising administering via intravenous injection a polynucleotide encapsulated in lipid nanoparticles (LNPs), the lipid nanoparticles comprising cationic lipids at a molar percentage of about 25% to about 60%, in an ionic lipid to cationic lipid ratio of 0.7 to 1.8, and an ionic lipid comprising either: (a) SS-OP or an SS-OP analog at a molar percentage of about 25% to about 60% (the LNPs having a lipid nitrogen:polynucleotide phosphate ratio (N / P ratio) of 10 to 30), or (b) DLin-MC3-DMA or a DLin-MC3-DMA analog at a molar percentage of about 30% to about 50% (the LNPs having an N / P ratio of 3.5 to 10).

[0156] In embodiment 2, the method of embodiment 1 is provided, wherein the polynucleotide comprises synthetic RNA, and upon or after administration of the LNP, the mRNA is translated in vivo in the subject into a corresponding protein encoded by the mRNA, and the in vivo half-life of the lipid DOTAP is a) at least 30 hours in the subject's lung, b) not more than 11 hours in the subject's liver, and / or c) not more than 9 hours in the subject's spleen.

[0157] In embodiment 3, there is provided a method according to embodiment 2, wherein the in vivo half-life of the protein in the lungs of the subject is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, or 35 hours.

[0158] In embodiment 4, there is provided a method of any of embodiments 1-3, wherein the polynucleotide comprises synthetic RNA, and upon or after administration of the LNP, the mRNA is translated in vivo in the subject into the corresponding protein encoded by the mRNA, and approximately 6 hours after intravenous infusion of the LNP, the lung:liver ratio of protein per mg of tissue is at least 2:1, 3:1, or 4:1.

[0159] In embodiment 5, there is provided a method according to any one of embodiments 1 to 4, wherein the lung:liver ratio of protein per mg of tissue is at least 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, or 50:1.

[0160] In embodiment 6, there is provided a method of any of embodiments 1-5, wherein the polynucleotide comprises a synthetic RNA encoding telomerase reverse transcriptase (TERT), and the mRNA is translated in vivo into the corresponding TERT protein in the subject upon or after administration of the LNP.

[0161] In embodiment 7, there is provided a method according to any of embodiments 1 to 6, wherein the median telomere length of the alveolar cells is increased by at least 5%, at least 10%, at least 20%, or at least 50%.

[0162] In embodiment 8, there is provided a method according to any one of embodiments 1 to 7, wherein the median telomere length of the lung AT2 cells is increased by at least 5%, at least 10%, at least 20%, or at least 50%.

[0163] In embodiment 9, there is provided a method according to any of embodiments 1 to 8, wherein the increase in telomerase activity in the lung is detectable by a TRAP assay for at least 24 or 48 hours.

[0164] In embodiment 10, there is provided a method according to any one of embodiments 1 to 9, wherein the LNP has an N / P ratio of 28 to 31.

[0165] In embodiment 11, there is provided the method of embodiment 10, wherein the LNP has an N / P ratio of approximately 29.1.

[0166] In embodiment 12, there is provided a method according to any one of embodiments 1 to 9, wherein the LNP has an N / P ratio of 11 to 13.

[0167] In embodiment 13, there is provided a method according to embodiment 12, wherein the LNP has an N / P ratio of greater than or equal to about 12, or about 12.1.

[0168] In embodiment 14, there is provided a method according to any one of embodiments 1 to 9, wherein the LNP has an N / P ratio of 4 to 5.

[0169] In embodiment 15, there is provided the method of embodiment 14, wherein the LNP has an N / P ratio of approximately 4.5.

[0170] In embodiment 16, there is provided a method according to any one of embodiments 1 to 11, wherein the ionic lipid is SS-OP and the LNP has a cationic lipid to ionic lipid ratio of approximately 1.8 and is composed of five lipids.

[0171] In embodiment 17, there is provided a method according to any one of embodiments 1 to 11, wherein the ionic lipid is SS-OP and the LNP has a cationic lipid to ionic lipid ratio of approximately 1.8 and is composed of five lipids, including DOTAP and DOPC.

[0172] In embodiment 18, there is provided a method according to any one of embodiments 1 to 11, wherein the ionic lipid is SS-OP, and the LNP has a cationic lipid to ionic lipid ratio of approximately 1.8 and is composed of five lipids including DMG-PEG2000, DOPC, and DOTAP.

[0173] In embodiment 19, there is provided a method according to any of embodiments 1-9 or 12-13, wherein the ionic lipid is SS-OP and the LNP has a cationic lipid to ionic lipid ratio of approximately 0.8 and is composed of three or five lipids.

[0174] In embodiment 20, there is provided a method according to any one of embodiments 1-9 or 12-13, wherein the ionic lipid is SS-OP and the LNP has a cationic lipid to ionic lipid ratio of approximately 0.8 and is composed of three or five lipids, including DOTAP.

[0175] In embodiment 21, there is provided a method according to any one of embodiments 1 to 9 or 12 to 13, wherein the ionic lipid is SS-OP and the LNP has a cationic lipid to ionic lipid ratio of approximately 0.8 and is composed of three or five lipids including DMG-PEG2000 and DOTAP.

[0176] In embodiment 22, there is provided a method according to any of embodiments 1-9 or 14-15, wherein the ionic lipid is DLin-MC3-DMA and the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5 and is composed of three lipids.

[0177] In embodiment 23, there is provided a method according to any one of embodiments 1-9 or 14-15, wherein the ionic lipid is DLin-MC3-DMA and the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5 and is composed of three lipids, including DOTAP.

[0178] In embodiment 24, there is provided a method according to any of embodiments 1-9 or 14-15, wherein the ionic lipid is DLin-MC3-DMA and the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5 and is composed of three lipids, including DMG-PEG2000 and DOTAP.

[0179] In embodiment 25, there is provided a method according to any one of embodiments 1 to 9 or 14 to 15, wherein the ionic lipid is DLin-MC3-DMA, the LNP has a cationic lipid to ionic lipid ratio of approximately 1.5, is composed of three lipids including DMG-PEG2000 and DOTAP, and the N / P is about 4.5.

[0180] In embodiment 26, there is provided a method according to any one of claims 1 to 15, wherein the LNP is selected from the LNPs in Table 8.

[0181] In embodiment 27, the LNP of any of embodiments 1-26 comprises a nucleic acid, such as a ribonucleic acid or a deoxyribonucleic acid.

[0182] In embodiment 28, the ribonucleic acid according to embodiment 27 comprises TERT mRNA.

[0183] In embodiment 29, the ribonucleic acid according to any of embodiments 27 to 28 is transiently expressed in a cell.

[0184] In embodiment 30, the cells of embodiment 29 comprise lung tissue cells.

[0185] In embodiment 31, the lung tissue cells of embodiment 30 include alveolar cells, such as ST2 cells.

[0186] In embodiment 32, an LNP for use in any of embodiments 1 to 31 is provided.

[0187] In embodiment 33, a two-lipid LNP comprising DOTAP and DMG-PEG2000 and having an N / P ratio of approximately 4 is provided, as used in any of embodiments 1 to 31.

[0188] In embodiment 34, a cell comprising an LNP according to any one of embodiments 1 to 33 is provided.

[0189] In embodiment 35, a kit is provided that includes components of an LNP described in any of embodiments 1-34.

[0190] In embodiment 36, a kit is provided comprising the LNP of any of embodiments 1-34.

[0191] In embodiment 37, there is provided a kit according to embodiment 35 or 36, wherein the kit further comprises a nucleic acid, such as a ribonucleic acid or a deoxyribonucleic acid.

[0192] In embodiment 38, a method for producing an LNP according to any one of embodiments 1 to 37, an LNP used in any one of embodiments 1 to 37, or an LNP formed by components according to any one of embodiments 1 to 37 is provided.

[0193] In embodiment 39, there is provided a method of manufacturing according to embodiment 38, wherein the ribonucleic acid is introduced after the LNPs have been formed, or the ribonucleic acid is introduced into a solution containing the LNPs. [Example]

[0194] Example 1 Figures 1 and 48 show exemplary pharmacokinetics of the five lipid LNPs in Table 1. Timelines of plasma, lung, and liver levels of the five lipid LNPs are presented, as determined by measuring one of the lipids, DOTAP. In the process of decomposing these data, LNPs were formulated with TERT mRNA (human TERT opti-SEQ ID NO: 1) and lipids at the ratios in Table 1. Male C57B1 / 6 mice were dosed at 1 mg / kg, and plasma and tissues were collected at the indicated time points. LC-MS / MS was used to measure DOTAP in plasma, lung, and liver. The nitrogen:phosphorus N / P charge ratio of the compositions in Table 1 is 12. [Table 5]

[0195] Example 2 Figure 2 shows the ratios of bioluminescence signals from the indicated organs generated from luciferase protein translated from intravenously injected mRNA at the indicated time points after intravenous injection of the five lipid LNPs in Table 2. In this process, LNPs were formulated with firefly luciferase mRNA and lipids at the ratios listed in Table 2. Male C57B16 mice were administered 1.3 mg / kg, and tissues were collected and imaged ex vivo at the indicated time points. Mean radiance values ​​were baseline normalized by subtracting the mean radiance of negative control (no LNP)-treated animals. The relative radiances between lung, liver, and spleen are plotted in Figure 2. Tissues were also homogenized, and bioluminescence was measured from equal amounts of homogenate by weight. The nitrogen:phosphorus N / P charge ratio for the compositions in Table 2 is 12. [Table 6]

[0196] Example 3 Figures 3-17 and Figures 20-21 show experiments involving the use of different five-lipid LNP compositions to induce pulmonary fibrosis in third-generation (G3) TERT knockout (KO) mice with telomere lengths similar to humans. Bleomycin was administered by oropharyngeal aspiration at 2.0 U / kg. In Figures 13-16, an initial dose of bleomycin was administered by oropharyngeal aspiration at 0.5 U / kg, one week prior to the 2.0 U / kg dose. LNPs were formulated with firefly luciferase mRNA (Ctrl) or TERT mRNA (mouse SEQ ID NO: 2, e.g., synthetic 5' UTR (SEQ ID NO: 3), wild-type mTert coding sequence (SEQ ID NO: 4), and mouse alpha globin 3' UTR (SEQ ID NO: 5)) (referred to herein as "TERT mRNA LNPs" and in the figures as "TERT" or "TERT mRNA") and lipid ratios in Table 3. The DOTAP molar ratio was 2.27-fold higher than the five-lipid LNPs in Table 3. Mice were treated with 1.5 mg / kg bleomycin twice weekly for 2 weeks, beginning on day 7 after bleomycin administration. Some mice received bleomycin but no LNP treatment (No LNP), and control mice received saline instead of bleomycin and LNP treatment (No Bleo). Tissues were harvested 24 days after bleomycin administration. Lungs were paraffin-fixed and formalin-embedded. To measure telomere length, sections were stained using Q-FISH with a telomere probe and an antibody against prosurfactant protein C (SPC) to label alveolar type II (AT2) cells. Telomerase activity in lung tissue lysates was measured using the telomeric repeat amplification protocol (TRAP) assay. [Table 7]

[0197] Figure 3 shows representative immunofluorescence micrographs of lung sections used to measure telomere length by the art-known Q-FISH method, in which the intensity of the telomere probe is proportional to telomere length, and cells were co-stained with an antibody against the AT2 cell marker pro-SPC.

[0198] Figure 4 shows telomere length measurements by Q-FISH in AT2 cells from mice in which fibrosis had been induced using bleomycin ("Bleo") as described above. Treatment with the TERT mRNA LNPs ("TERT" in the figure) from Table 3 increased the median and 10th percentile telomere lengths measured by Q-FISH in AT2 cells (cells scored as pro-SPC+ by immunofluorescence staining) compared with mice receiving luciferase mRNA LNPs (referred to as "Ctrl" in the figure), and was similar to the lengths seen in normal mice not receiving bleomycin ("No Bleo" in the figure).

[0199] Figure 5 shows telomere length measurements by Q-FISH in AT2 cells from mice in which fibrosis was induced using bleomycin ("Bleo") as described above. Treatment with the TERT mRNA LNPs (referred to as "TERT" in the figure) from Table 3 increased the median and 10th percentile telomere lengths of alveolar cells measured by Q-FISH compared with mice receiving luciferase mRNA LNPs (referred to as "Ctrl" in the figure), and these telomere lengths were approximately equivalent to those observed in normal mice not receiving bleomycin (referred to as "No Bleo" in the figure). Fields primarily filled with alveoli were used for quantification, and telomere lengths of all cell types present were included. Alveolar tissue typically contains epithelial cells, endothelial cells, fibroblasts, immune cells, and other cell types.

[0200] Figure 6A shows the timeline of the bleomycin mouse experiment described above. "CT" indicates X-ray computed tomography scanning of mice to quantify normal aerated lung volume.

[0201] Figure 6B shows the timeline of the bleomycin mouse experiments in Figures 13-16.

[0202] Figure 7 shows pseudocolor images of lung sections stained with Sirius Red for quantification of fibrotic foci and fibrosis by tissue area covered with collagen deposits. IV infusion of TERT mRNA LNPs from Table 3 reduced fibrosis by 62%.

[0203] FIG. 8 shows the reduction of fibrotic lesions in bleomycin-treated mice by treatment with the TERT mRNA LNPs of Table 3, as measured by Sirius Red staining.

[0204] FIG. 9 shows the increase in normal aerated lung ratio or useful lung volume in bleomycin-treated mice with treatment with the TERT mRNA LNPs of Table 3, as measured by X-ray computed tomography imaging followed by quantification of fibrotic tissue based on voxel X-ray intensity.

[0205] FIG. 10 shows improvement in lung elastance in bleomycin-treated mice by treatment with the TERT mRNA LNPs of Table 3, as measured with the Flexivent system.

[0206] 11 and 12 show improvements in pulmonary forced expiratory volume (FEV0.1) and forced vital capacity (FVC), respectively, in bleomycin-treated mice upon treatment with TERT mRNA LNPs from Table 3, as measured with the Flexivent system.

[0207] Figures 13, 14, and 15 show improvements in alveolar density, alveolar diameter, and alveolar circularity, respectively, in bleomycin-treated mice upon treatment with the TERT mRNA LNPs in Table 3, as measured by machine vision analysis of photomicrographs of lung sections by Biocellvia.

[0208] Figure 16 shows an example of a pseudocolor image generated by Biocellvia of a lung tissue section during analysis of the alveolar structures quantified in Figures 13-15.

[0209] FIG. 17 shows measurements of telomerase activity in primary human alveolar epithelial cells isolated from a 50-year-old donor at the indicated time points after treatment with the TERT mRNA LNPs of Table 3 at a concentration of 500 ng / ml, as measured using a telomeric repeat amplification protocol (TRAP) assay.

[0210] Figure 18 shows a timeline of an experiment measuring the effect of treatment with the TERT mRNA LNPs from Table 3 on the colony-forming ability of primary human alveolar epithelial cells isolated from a 50-year-old donor. As shown in Figure 19, cells were seeded in medium that supports colony formation, treated with TERT mRNA LNPs at a concentration of 500 ng / ml of TERT mRNA, and incubated for 7 days, after which colonies were counted.

[0211] FIG. 19 shows the increased colony-forming ability of human alveolar epithelial cells from a 50-year-old donor by treatment with the TERT mRNA LNPs of Table 3.

[0212] Example 4 - Reduction of senescence after telomerase mRNA LNP treatment in mice Methods: Experiments related to Figures 3-12 were carried out.

[0213] Results: As shown in Figures 20-21, a 40% reduction in the senescence marker P21 was observed in alveolar cells from mice treated with telomerase mRNA LNPs compared to mice treated with luciferase mRNA LNPs (control). In Figure 21, dark cells are positive. Increased senescence after bleomycin treatment.

[0214] Example 5-2 Efficient Pulmonary Delivery Using Lipid LNPs Methods: Two-lipid LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 22C. C57B1 / 6 male mice were administered 1.25 mg / kg intravenously (IV), and organs were imaged ex vivo 21 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in the graphs Figures 22A-B and 22D.

[0215] Results: All formulations showed bioluminescent signals in the lungs, demonstrating the following: 1. Low PEG concentrations were toxic (one mouse died at 0.1% PEG), suggesting that the optimal amount may be 0.2-2% PEG. 2. The signal peaked at 0.5% PEG.

[0216] Example 6-2 Titration of lipid:mRNA ratio in lipid LNPs Methods: DOTAP and PEG were formulated with firefly luciferase mRNA (Luc). The lipid ratio was 99% DOTAP and 1% PEG. The lipid:mRNA ratio is expressed as NP (nitrogen to phosphate molecule ratio). Each DOTAP molecule has one nitrogen, and each nucleoside on the mRNA has one phosphate. C57Bl / 6 male mice were administered 1.5 mg / kg intravenously (IV), and 23 hours later, lungs were removed for ex vivo imaging. Average radiance (photons / s / cm) was measured. 2 / sr) is shown in Figure 23.

[0217] Results: Bioluminescent signals were observed in the lungs at all N / P ratios. Increased lung signal corresponded to greater weight loss, with greater signal indicating greater toxicity. A nitrogen:phosphorus (N / P) ratio of 20 resulted in minimal signal and weight loss.

[0218] Example 7 - Time course of protein expression following delivery of lung-targeted mRNA-LNPs encoding firefly luciferase Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in provisional Table 3. C57Bl / 6 male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo at 16 hours ("Day 1"), 33 hours ("Day 2"), 73 hours ("Day 3"), and 91 hours ("Day 4"). Mean radiance (photons / s / cm) was measured. 2 / sr) is shown in Figure 24.

[0219] Results: Given the known half-life of the firefly luciferase protein, the bioluminescence signal decreased over time, as expected, suggesting that the half-life of the mRNA was not significant and that the mRNA-LNPs did not retransfect circulating cells in the lung over time.

[0220] Example 8 - Titration of SS-OP lipids into "DOTAP+PEG" LNPs Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 25D. C57B1 / 6 male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 22 hours later. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 25A.

[0221] result: 1. The encapsulation efficiency measured by Ribo-Green plate reader assay was over 98%. Figure 25B 2. Body weight remained stable, suggesting low or no toxicity. Figure 25C 3. Lung radiance peaked at 50% SS-OP lipid. Maximum value at 4.5% SS-OP

[0222] Example 9-2 Lung and Liver Delivery Using LNPs Methods: Liver-targeted LNPs were formulated according to the lipid ratios in Table 4, and lung-targeted LNPs were formulated according to the lipid ratios in Table 3. Both were formulated with firefly luciferase mRNA (Luc). CD1 male mice were administered 1.0 mg / kg intravenously (IV), and organs were imaged ex vivo 19 hours later. Conditions: ●Only lung-targeted LNP was administered "lung-only." ●Only liver-targeted LNP was administered "liver-only." Lung-targeted LNP was administered first, followed one hour later by liver-targeted LNP: (1) Lung (2) Liver. Liver-targeted LNP was administered first, followed one hour later by lung-targeted LNP: (1) Liver (2) Lung. Lung- and liver-targeted LNPs were mixed and administered twice, one hour apart: "liver / lung mixed" Figures 26A and 26B show the average radiance (photons / s / cm 2 / sr). [Table 8]

[0223] result: 1. Under conditions in which lung-targeted LNPs were administered, the bioluminescent signal in the lung was consistent. The highest was in the liver / lung mixture. Figure 26A 2. The liver BLI signal fluctuated but was very low when lung LNPs were administered first. Figure 26B. 3. The optimal order for both organs is lung first, then liver. The second optimal is to mix and deliver.

[0224] Example 10 - Titration of SS-OP lipids into "DOTAP+PEG" LNPs Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in the table in Figure 27A. C57B1 / 6 male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 23 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 27C.

[0225] Body weights were measured and clinical signs were assessed using a scoring system consisting of a visual assessment of mouse appearance and behavior, specifically gait, posture, and eyes (abbreviated as "LPE") (Nunamaker 2013, PMID:24209966). Figure 27D.

[0226] result: 1. When the proportion of SS-OP lipid was increased to 75%, tolerance decreased and both mice required euthanasia. 2. Combined with the data from Example 7, the study suggests that 35-65% SS-OP provides relatively lung-specific transfection (measured by bioluminescence signal) and relatively high tolerability (less than 10% of body weight, no clinical signs (LPE0), no deaths) compared to other SS-OP percentages. 55% SS-OP scores particularly well and may be considered optimal in these respects.

[0227] Example 11 - Stability under cryopreservation - Long term Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. They were either "fresh" (formulated and administered on the same day) or frozen at -20°C, -80°C, or in liquid nitrogen and thawed after 70 days. Sucrose was added to 7% as a cryoprotectant before freezing. CD1 male mice were administered 1.0 mg / kg intravenously (IV), and organs were imaged ex vivo 19 hours later. Mean radiance (photons / s / cm) 2 / sr) + / - SEM is shown in Figure 28A.

[0228] Results: No clinical symptoms and minimal weight loss were observed (Figure 28B). Lung signals were present in similar amounts in all conditions, suggesting that cryopreservation was effective.

[0229] Example 12 - Stability under cryopreservation - short term Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. They were either "fresh" (formulated and administered on the same day) or frozen at 4°C, -20°C, -80°C, or in liquid nitrogen and thawed after 3 days. Except for the "4°C w / o sucrose" condition, sucrose was added to 15% as a cryoprotectant before freezing. CD1 female mice were administered 0.4 mg / kg intravenously (IV), and organs were imaged ex vivo 23 hours later. Mean radiance (photons / s / cm) was measured. 2 / sr) is shown in Figure 29A.

[0230] Results: The signal from preserved lungs was lower than that from "fresh." Particle size measured by dynamic light scattering was out of range at 4C and -20 with sucrose (see next slide). Figure 29B.

[0231] Example 13 - Titration of N / P ratio and comparison of 2, 3, and 5 lipid formulations Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 30A. C57B1 / 6 male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 23 hours later. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 30B.

[0232] result: The 1.3 lipid and 5 lipid (SS and "5 lipid") formulations showed higher signals on average than the 2 lipid (1% PEG) formulation. For the 2, 3 and 5 lipid formulations, 10N / P was optimal. 3.2 For lipids, 15NP was superior to 10NP. 4. All formulations showed promise as lung-targeted LNPs, given their high radiance and less than 10% weight loss, except for the low activity of 2 lipid 10N / P. Figure 30C.

[0233] Example 14 - Titration of N / P ratio (mRNA to lipid ratio) using lipid formulations 3 and 5 Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 31A. Bl / 6 male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 21 hours later. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 31C.

[0234] result: There was significant toxicity in the three lipid formulations performed at 1.3 and 6N / P. For lung-targeted LNPs with 2.5 lipids, encapsulation efficiency began to fall below 8 N / P. 3. In this study, 10N / P 3 and 5 lipids were overall superior, taking into account the above considerations.

[0235] Example 15 - Testing vortex and microfluidic mixing of mRNA-LNP formulations Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. Male Balb c / J mice were administered 0.1 mg / kg intravenously (IV), and organs were imaged ex vivo 21 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 32A.

[0236] result: 1. Vortex-mixed mRNA-LNPs produced LNPs with a slightly larger particle size (113 nm vs. 86 nm) that produced a higher BLI signal in the lung. Figure 32B. 2. Vortex-mixed mRNA-LNPs produced LNPs with an encapsulation efficiency of 63%, far below the 94% of their microfluidically mixed counterparts. Figure 32B.

[0237] Example 16 - Titration of NP ratios of five lipid mixtures comparing input concentrations Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in the table in Figure 33A. C57B1 / 6 male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 21 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 33B.

[0238] In the condition labeled "dil," mRNA and lipids were diluted to 30% of their original concentrations in buffer before mixing, but the ratio of components remained the same. Input mRNA concentrations ranged from 0.147 mg / ml to 0.44 mg / ml.

[0239] result: 1. Decreasing the molar percentage of DOTAP from 49% to 30% improved the signal in the 5 lipid formulation by 30%. 2. Encapsulation efficiency was over 90% for all formulations, and no abnormalities in mouse movement, posture, or eyes were observed (LPE score).

[0240] Example 17 - Titration of NP ratios of five lipid mixtures; comparison of five-lipid, three-lipid, and two-lipid LNP formulations Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 34A. BALB / cJ male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 20 hours later. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 34B. The formulations use 5 lipids, except for "3 lipids 12NP" and "1% PEG 15NP."

[0241] Results: Encapsulation efficiency was greater than 90% for all formulations. Figure 34C. The highest signal was observed with 12NP for both 3-lipid lung-targeted LNP and 5-lipid lung-targeted LNP.

[0242] Example 18-5 Testing Different Total Fluxes of Lipid Formulations Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 35A. C57B1 / 6 male mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 17 hours later. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 35C. The formulation was the same, the only variable that was changed was the flow rate through the PrecisionNanosystems Nanoassemblr microfluidic mixing platform.

[0243] Results: All three flow rates had high bioluminescence signals, over 95% encapsulation efficiency, and no clinical symptoms (LPE=0, no deaths). Figure 35B. The preferred flow rate was 12 ml / min.

[0244] Example 19-3 Testing Different Total Fluxes of Lipid Formulations ("PDS") Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 36A. BALB / cJ male mice were administered 1.5 mg / kg intravenously (IV), and organs were imaged ex vivo 26 hours later. Mean radiance (photons / s / cm 2 The flow rates of the mRNA (aqueous) stream and the lipid (organic) stream are shown in Figure 36B. The total flow rate was varied, but the ratio of the mRNA (aqueous) stream to the lipid (organic) stream was maintained at 3:1. Thus, the flow rate of the mRNA stream is the total flow rate multiplied by 0.75, and the flow rate of the lipid stream is the total flow rate multiplied by 0.25.

[0245] result: 1. At all three flow rates, the bioluminescence signal was high, the encapsulation efficiency was over 98%, and the weight loss was less than 5% (Figures 36B and 36C).

[0246] Example 20-3 Replacing SS-OP in Lipid LNPs with Another Ionic Lipid, DLin-MC3-DMA ("MC3") Titrating the MC3 to DOTAP Ratio from 9 to 50% MC3 Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 37A. The encapsulation percentage of LNPS is presented in Figure 37B. BALB / cJ female mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 24 hours later. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 37C.

[0247] result: 1. In the lung, comparable bioluminescent signals were seen in all conditions, with a trend towards lower signal as MC3 concentration decreased. Figure 37C. 2. At 9% total MC3, the lowest MC3 concentration (3-MC3 0.1) resulted in a fatal event. Figure 37B. 3. Weight loss tended to increase with increasing MC3 concentration, with the highest mean weight loss at 50% MC3 (Figure 37D).

[0248] Example 21-3 Replacing the ionic lipid SS-OP with DLin-MC3-DMA in lipid LNPs Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 38A. MC3 stands for DLin-MC3-DMA. CD1 female mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 26 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 38C.

[0249] result: There were fatal events in LNP with 1.70.5% 3-MC, suggesting that it was not well tolerated. 2. Encapsulation efficiency was greater than 95% in all conditions. Figure 38B 3. The mean lung radiance was similar, with the LNP formulation containing 33% molar percentage of MC3 showing the highest signal on average.

[0250] Example 22-5 Replacing the ionic lipid SS-OP with DLin-MC3-DMA in lipid LNPs. Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 39A. MC3 stands for DLin-MC3-DMA. CD1 female mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 26 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 39C.

[0251] result: 1. Encapsulation efficiency was over 95% in all conditions. Figure 39B. 2. Mean lung radiance was at least 7.2-fold higher in the five lipid conditions containing SS-OP than in any of the LNPs containing DLin-MC3-DMA. Figure 39C.

[0252] Example 23 - Titration of the time that mRNA is adsorbed to formed LNPs in two lipid formulations Methods: DOTAP and DMG-PEG2000 at a 100:1 molar ratio were microfluidically mixed with malic acid, followed by buffer exchange into 0.1 M sodium acetate and concentration. Lipid concentration was measured by HPLC with UV detection, and then firefly luciferase mRNA (Luc) was added to the lipids to a final N / P ratio of 15. The mRNA was allowed to adsorb for the time indicated in Figure 40A before adding PBS. CD1 female mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 24 hours later. Mean radiance (photons / s / cm) was measured. 2 / sr) is shown in Figure 40A.

[0253] result: 1. The largest lung signal was observed at the 30 minute incubation condition. 2. In all animals, body weight loss was less than 5% and no deaths were observed, suggesting tolerability. (Figure 40B) Encapsulation efficiency was greater than 98% in all conditions.

[0254] Example 24-2 Titration of mRNA:lipid ratio of lipid LNPs Methods: DOTAP and DMG-PEG2000 at a 100:1 molar ratio underwent microfluidic mixing with malic acid, followed by buffer exchange into 0.1 M sodium acetate and concentration. Lipid concentrations were measured by HPLC, and then firefly luciferase mRNA (Luc) was added to the lipids to the N / P ratios shown in Figure 41A. The encapsulation percentage for each composition is presented in Figure 41B. The mRNA was allowed to adsorb for 30 minutes before adding PBS. C57BL / 6 female mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 27 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 41A.

[0255] result: 1. LNPs containing NP2, not shown, did not produce enough mRNA to be administered. 2. The lung bioluminescence signals were comparable among different conditions.

[0256] Example 25-3 Titration of N / P ratio of lipid LNP Methods: LNP "PDS" was formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 6. The encapsulation percentage of each composition is provided in Figure 42B. 3-MC3 LNP was formulated with the molar percentages of 39.6% MC3, 59.4% DOTAP, and 1% DMG-PEG2000 at the N / P ratios indicated in the graph. C57BL / 6 male mice were administered 2.0 mg / kg by intravenous injection (IV), and organs were imaged ex vivo 20 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown in Figure 42A. [Table 9]

[0257] result: MC3-containing LNP with an N / P ratio of 1.20 had a fatal event, and MC3 LNP with an N / P ratio of 15 had clinical signs (decreased gait and hunched posture), suggesting that they were not well tolerated. 2. Encapsulation efficiency was over 98% under all conditions. 3. The mean lung radiance decreased with increasing N / P ratio. 4. In the NP1.5 and NP2 conditions, little mRNA-LNP was recovered, suggesting that these mRNA:lipid ratios were not suitable for LNP formation in this setting. NP2.5 and NP3 were administered to only one animal due to low yields. 5. In this study, DLin-MC3-DMA-containing LNPs with N / P ratios of 4 and 5 were the most promising.

[0258] Example 26-2 Titration of DMG-PEG2000 in Lipid LNPs Methods: As shown in Figures 43A and 43B for PEGylated lipids, molar percentages of DOTAP and DMG-PEG2000 were subjected to microfluidic mixing with malic acid, followed by buffer exchange into 0.1 M sodium acetate and concentration. Lipid concentrations were measured by HPLC, and then firefly luciferase mRNA (Luc) was added to an N / P ratio of 5.0. The mRNA was allowed to adsorb for 30 minutes before adding PBS. C57BL / 6 female mice were administered 2.0 mg / kg intravenously (IV), and organs were imaged ex vivo 20 hours later. Mean radiance (photons / s / cm) was measured. 2 / sr) is shown in Figure 43A.

[0259] result: 1. A single cationic lipid (DOTAP) is sufficient for acceptable pulmonary targeting. 2. Mean lung radiance decreased with increasing DMG-PEG2000.

[0260] Example 27 - Microfluidic mixing versus manual mixing of lipids in ethanol with malate buffer Methods: DOTAP and DMG-PEG2000 at a molar ratio of 100:1 were mixed with malic acid by microfluidic mixing or manual vortexing, then buffer-exchanged into 0.1 M sodium acetate and concentrated. Lipid concentrations were measured by HPLC, and firefly luciferase mRNA (Luc) was then added to an N / P ratio of 5.0. The mRNA was allowed to adsorb for 30 minutes before adding PBS. C57BL / 6 female mice were administered 0.5 mg / kg intravenously (IV), and organs were imaged ex vivo 20 hours later. Mean radiance (photons / s / cm) was measured. 2 / sr) is shown in Figure 44.

[0261] result: 1. Microfluidic mixing resulted in a two-fold increase in mean lung radiance. 2. The encapsulation efficiency of mRNA was over 98% under both conditions.

[0262] Example 28 - Comparison of the published MC3 formulation of 5 lipid LNP with SS-OP Method 1: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Figure 45A. C57BL / 6J female mice were administered the indicated mg / kg via intravenous injection (IV), and 20 hours later, organs were imaged ex vivo. Mean radiance (photons / s / cm 2 / sr) is shown in Figure 45B. The MC3 formulation was based on: Dilliard et al. 2021 PNAS (https: / / doi.org / 10.1073 / pnas.2109256118).

[0263] Result 1: The 1.5 lipid SS-OP-containing LNPs had 6.3-fold and 5.2-fold higher mean lung radiance at the 0.5 mg / kg and 2.0 mg / kg dose groups, respectively, compared to published DLin-MC3-DMA-containing lung-targeted LNPs. 2. Encapsulation was greater than 98% for both LNPs, with similar overall yields. The modal particle size by dynamic light scattering was 35-55 nm.

[0264] Method 2: LNPs were also formulated with Cre mRNA according to the ratios in Figure 45A. The MC3 formulation was based on Dilliard et al. 2021 PNAS (https: / / doi.org / 10.1073 / pnas.2109256118). Ai14 Tomato fl / fl female mice were intravenously administered Cre mRNA at 2.0 mg / kg. Three days later, lungs were harvested and processed for formalin fixation and paraffin embedding. Positive cells were labeled with anti-tdTomato antibody. The percentage of positive cells among alveolar cells is shown in Figure 45C.

[0265] Result 2: The 1.5 lipid SS-OP-containing LNPs resulted in a 4.9-fold higher transfection rate of alveolar cells. 2. Representative images of the alveolar region are shown.

[0266] Example 29 - Cell transfection rates in three organs Methods: LNPs were formulated using the lipid molar ratio and lipid:mRNA ratio shown in Figure 46A. PD(*) formulations were prepared as described above (Example 26). Ai14 Tomato fl / fl female mice were intravenously administered Cre mRNA at 1.5 mg / kg. Three days later, lungs were harvested and processed by formalin fixation and paraffin embedding. Positive cells were labeled with anti-tdTomato antibody. The percentage of positive cells in the alveolar space (Figure 46B), liver hepatocytes (nuclear size and circularity were used to exclude non-hepatocytes) (Figure 46C), and spleen cells (Figure 46D) are shown. Related IHC data are presented in Figure 46E. Dark cells are tdTomato-positive cells transfected with Cre mRNA.

[0267] result: 1.5 lipid LNP (5Lip) and 3 lipid LNP (PDS) had comparable transfection in lung and spleen, while 5 lipid LNP had 8-fold greater transfection in hepatocytes in the liver. 2. A representative image of the alveolar region is shown in Figure 45D. Dark cells are transfected (tdTomato positive).

[0268] Example 30-2 Pharmacokinetics of Lipid LNPs Methods: PD formulations were prepared using mRNA encoding human telomerase as described above (Example 26). C57B1 / 6 male mice were administered 1.0 mg / kg, and plasma and tissues were collected at the indicated time points, as shown in Figure 1. DOTAP was measured using LC-MS / MS. [Table 10]

[0269] result: [Table 11]

[0270] Example 31-5 Pharmacokinetics of Lipid LNPs Methods: LNPs were formulated with mRNA encoding human telomerase using the lipid molar ratios in Table 1. CD1 male mice were administered 1.0 mg mRNA / kg body weight, and plasma and tissues were collected at 0 (<5 min) and 24 h. DOTAP was measured using LC-MS / MS. mRNA was measured by qRT-PCR. "Liver remnant" refers to liver samples excluding the left lobe.

[0271] result: [Table 12]

[0272] Example 32-5 Biodistribution of Lipid LNPs Methods: LNPs were formulated with mRNA encoding firefly luciferase using the lipid molar ratios in Table 1. New Zealand White rabbits were administered intravenously (IV) at the levels shown in mg mRNA / kg body weight. Organs were imaged ex vivo 6 hours later. Mean radiance (photons / s / cm) 2 / sr) is shown on the graph.

[0273] The results are presented in Figures 49A and 49B.

[0274] Example 33-3 Biodistribution of Lipid LNPs Methods: LNPs were formulated with mRNA encoding firefly luciferase using the lipid molar ratios in Table 7. Cynomolgus monkeys were administered intravenously (IV) at the levels shown in mg mRNA / kg body weight. Organs were imaged ex vivo 6 hours after addition of luciferin. Figure 50A shows the mean radiance (photons / s / cm) of each imaged organ. 2 / sr). Figures 50B and 50C show the relative radiance between organs at low and high doses. Figures 50D-50F show representative bioluminescence images of organs from high-dose animals, as shown below: [Table 13]

[0275] The results of bioluminescence biodistribution by organ, expressed in terms of fold change in radiance, are shown in Figure 50G.

[0276] Example 34 - Telomerase activity and telomere elongation in human small airway epithelial cells in the presence of SOC Methods: LNPs were formulated with mRNA encoding human telomerase or mCherry as a control, using the lipid molar ratios listed in Table 3. LNPs were added to human small airway epithelial cells (SAECs) obtained from Lonza at 0.5 mg / ml in culture. SAECs were grown to 70% confluence in Lonza SAGM. Pirfenidone (1 μM) and nintedanib (0.5 μM), drugs representing the standard of care (SOC) for patients with pulmonary fibrosis, were added to the cultures the day before and replaced with each media change. For telomerase activity measurements, cells were harvested and lysed in CHAPS buffer 24 hours after treatment with LNPs for the TRAP assay. Lysates were exposed to an artificial telomerase single-stranded DNA template, and telomerase activity was detected using PCR amplification (Figure 51B).

[0277] To measure telomere length, cells were harvested and fixed 5 days after addition of telomerase mRNA LNPs (Figure 51A). The Q-FISH protocol was performed as follows: cells were spun down onto glass slides, permeabilized, and telomeres were labeled with a fluorescent probe (Figure 51B). Microscopy and quantitative image analysis were performed to determine the length of individual telomeres. In the analysis, all hTERT LNP samples (+ / - SOC) were pooled and tested for differences in telomere length.

[0278] result: 1. Telomerase activity was detected in human small airway epithelial cells after the addition of hTERT LNP. 2. When standard treatments were added, telomerase activity was detected in the cells at similar levels. 3. After a single dose of hTERT LNP, the median and 20th percentile telomere lengths significantly increased by 492 base pairs and 435 base pairs, respectively.

[0279] Example 35 - Telomerase activity and telomere elongation in human lung fibroblasts in the presence of SOC Methods: LNPs were formulated using the lipid molar ratios listed in Table 3 with either human telomerase-encoding mRNA or mCherry as a control. LNPs were added to human fetal lung fibroblast MRC-5 cells (passage 5) and grown to 70-80% confluence in DMEM + 10% FBS. Twenty-four hours prior to LNP treatment, MRC-5 cells were pretreated with standard therapies pirfenidone (1 μM) and nintedanib (1 μM), followed by treatment with 500 ng / ml hTERT LNPs or 500 ng / ml mCherry LNPs (control). Standard therapies were added freshly after each medium change.

[0280] For telomerase activity measurements, cells were harvested and lysed in CHAPS buffer 24 hours after treatment with LNP for the TRAP assay. Lysates were exposed to an artificial telomerase single-stranded DNA template, and telomerase activity was detected using PCR amplification. (Figure 52)

[0281] result: 1. Telomerase activity was detected in human lung fibroblasts after the addition of hTERT LNPs. 2. When standard treatments were added, telomerase activity was detected in the cells at similar levels.

[0282] Example 36 - Lyophilization of 3-lipid and 5-lipid LNPs Methods: LNPs were formulated with mRNA encoding firefly luciferase using the lipid molar ratios described herein. The LNP ratios used are listed in Table 8. LNPs were mixed with 15% sucrose and frozen at -80°C. They were then placed on the pre-frozen shelf of an L-200 Pro Lyovapor freeze dryer. The vacuum was set to 1 mbar, the condenser was set to -55°C, and the total run time was 22 hours. LNPs were stored at 4°C for 2 days before resuspension in water. As a control, "PDS" LNPs were frozen with 15% sucrose and thawed without lyophilization. Second-generation Tert- / - female mice on a C57Bl / 6 background were administered 0.3 mg mRNA / kg body weight via intravenous injection (IV), and organs were imaged ex vivo 18 hours later. Figure 55.

[0283] Results: The in vivo activity of the five lipids and PDS was comparable to each other and to that of frozen, non-lyophilized PDS LNPs. Lyophilized and reconstituted PD LNPs showed at least 8-fold higher activity than other LNPs.

[0284] Example 37 - Overview of exemplary LNP formulations of the present disclosure Table 8 presents the molar percentage composition, N / P ratio, cationic lipid:ionic lipid molar ratio, and lipid:mRNA ratio (wt / wt) of exemplary LNPs of the present disclosure designated "SSOP-DOTAP," "5 lipid," "PDS," "3-MC3," and "PD," respectively. [Table 14]

[0285] The optimal cationic lipid to ionic lipid ratios ("C / I ratios") and N / P ratios that we identify for LNPs containing either the SS-OP family or the DLin-MC3-DMA family of ionic lipids are modeled in Figure 56.

[0286] Example 38 - Addition of mRNA after LNPs are formed LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 8. For LNPs marked with an asterisk (*), microfluidic mixing was performed using buffer only, without mRNA. This was followed by buffer exchange into 0.1 M sodium acetate and concentration. Firefly luciferase mRNA (Luc) was added to lipids at the N / P ratios indicated on the graph. The mRNA was added to PBS, filtered to 0.2 μM, and allowed to absorb for 2 hours before administration. The mRNA LNPs were administered intravenously to C57BL / 6 male mice, and organs were imaged ex vivo 17 hours later.

[0287] result: 1. Finally, addition of mRNA resulted in the following changes in mean lung radiance (Fig. 53A): ● PD LNP increased by 85% ●For PDS LNP, the decrease was 68% ●For 5 lipids, 16% reduction 2. Encapsulation efficiency was above 95% only under the PD(*) condition, but not under PDS(*) or 5Lipid(*), in which mRNA was added at the end of the purification process. Figure 53B.

[0288] Example 39 - Addition of mRNA after LNPs are formed LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 8. For PD(*), microfluidic mixing was performed using only buffer, without mRNA. This was followed by buffer exchange into 0.1 M sodium acetate and concentration. Firefly luciferase mRNA (Luc) was added to lipids at the N / P ratios shown on the graph. The mRNA was added to PBS, filtered to 0.2 μM, and allowed to adsorb for 2 hours before administration. mRNA LNPs were administered intravenously at 1.5 mg / kg to BALB / cJ male mice, and organs were imaged ex vivo 26 hours later.

[0289] result: 1. Adding mRNA at the end of the PD formulation increased the bioluminescence signal by 31.8-fold. Figure 54A. 2. Encapsulation efficiency was greater than 95% for all formulations. Figure 54B.

[0290] A list of exemplary nucleic acid sequences used in the examples described herein is provided below. Additional sequences, such as those described in PCT / US22 / 22642, filed March 30, 2022, U.S. Application No. 17 / 709,108, filed March 30, 2022, and U.S. Provisional Application No. 63 / 169,118, filed March 31, 2021, are incorporated herein by reference. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0291] The above examples are included for illustrative purposes only and do not limit the scope of the invention. Many variations on those described above are possible. Since modifications and variations to the above-described examples will be apparent to those skilled in the art, it is intended that this invention be limited only by the scope of the appended claims.

[0292] Citation of any of the above publications or documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or dates of these publications or documents.

Claims

1. Polynucleotides encapsulated in lipid nanoparticles (LNPs), The aforementioned LNP contains cationic lipids in a molar percentage of approximately 20% to approximately 60%, (a) An ionic lipid comprising SS-OP or an SS-OP analog in a molar percentage of about 25% to about 65%, wherein the LNP has a cationic lipid:ionic lipid molar (C / I) ratio of 0.5 to 1.8 and a lipid nitrogen:polynucleotide phosphate (N / P) ratio of 10.5 to 31, or (b) An ionic lipid comprising any of the above ionic lipids, wherein DLin-MC3-DMA or DLin-MC3-DMA analog is contained in a molar percentage of approximately 30% to approximately 50%, and the LNP has a C / I ratio of 0.5 to 2.5 and an N / P ratio of 2.5 to 8. This is intended for use in a method for delivering the aforementioned polynucleotides. The method comprises administering the polynucleotide encapsulated in lipid nanoparticles (LNPs) by intravenous injection.

2. The polynucleotide comprises synthetic RNA, and at the time of or after administration of the LNP, the mRNA is translated in vivo into the corresponding protein encoded by the mRNA in the subject. (a) The in vivo half-life of lipid DOTAP is (i) In the lung of the subject, for at least 30 hours, (ii) In the liver of the subject, within 11 hours, or (iii) In the spleen of the subject, within 9 hours and / or (b) The polynucleotide for use according to claim 1, wherein the in vivo half-life of the protein in the lung of the subject is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, or 35 hours.

3. The polynucleotide for use according to claim 1, wherein the polynucleotide comprises synthetic RNA, and at the time of or after administration of the LNP, the mRNA is translated in vivo into the corresponding protein encoded by the mRNA in the subject, and approximately 6 hours after intravenous infusion of the LNP, the lung:liver ratio of the amount of the protein per 1 mg of tissue is at least 2:1, 3:1, or 4:

1.

4. The polynucleotide for use according to claim 3, wherein the lung:liver ratio of the amount of protein per 1 mg of tissue is at least 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, or 50:

1.

5. The polynucleotide for use according to claim 1, wherein the polynucleotide comprises synthetic RNA encoding telomerase reverse transcriptase (TERT), and the mRNA is translated in vivo into the corresponding TERT protein in the subject at or after administration of the LNP.

6. The polynucleotide for use according to claim 5, wherein the median telomere length of alveolar cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%, or the median telomere length of lung AT2 cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%.

7. The polynucleotide for use according to claim 5, wherein an increase in telomerase activity in the lungs is detectable by a TRAP assay for at least 24 or 48 hours.

8. The polynucleotide for use according to claim 1, wherein the ionic lipid comprises SS-OP or an SS-OP analog, and the LNP has (a) an N / P ratio of 28 to 31, or (b) an N / P ratio of approximately 29.

9. The polynucleotide for use according to claim 1, wherein the ionic lipid comprises SS-OP or an SS-OP analog, and the LNP has (a) an N / P ratio of 10.5 to 15, (b) an N / P ratio of approximately 12, or (c) an N / P ratio of approximately 12.

1.

10. The polynucleotide for use according to claim 1, wherein the ionic lipid comprises DLin-MC3-DMA or a DLin-MC3-DMA analog, and the LNP has (a) an N / P ratio of 3 to 5, or (b) an N / P ratio of approximately 4.

5.

11. The polynucleotide for use according to any one of claims 1 to 8, wherein the ionic lipid is SS-OP, and the LNP has a C / I ratio of approximately 1.8 and is composed of five different lipids.

12. The polynucleotide for use according to any one of claims 1 to 8, wherein the ionic lipid is SS-OP, and the LNP has a C / I ratio of approximately 1.8 and is composed of five lipids including DOTAP and DOPC.

13. The polynucleotide for use according to any one of claims 1 to 8, wherein the ionic lipid is SS-OP, and the LNP has a C / I ratio of approximately 1.8 and is composed of five lipids including DMG-PEG2000, DOPC, and DOTAP.

14. The polynucleotide for use according to any one of claims 1 to 7 or 9, wherein the ionic lipid is SS-OP, and the LNP has a C / I ratio of approximately 0.8 and is composed of 3 or 5 different lipids.

15. The polynucleotide for use according to any one of claims 1 to 7 or 9, wherein the ionic lipid is SS-OP, and the LNP is composed of three or five lipids having a C / I ratio of approximately 0.8 and including DOTAP.

16. The polynucleotide for use according to any one of claims 1 to 7 or 9, wherein the ionic lipid is SS-OP, and the LNP is composed of three or five lipids having a C / I ratio of approximately 0.8, including DMG-PEG2000 and DOTAP.

17. The polynucleotide for use according to any one of claims 1 to 7 or 10, wherein the ionic lipid is DLin-MC3-DMA, and the LNP has a C / I ratio of approximately 1.5 and is composed of three lipids.

18. The polynucleotide for use according to any one of claims 1 to 7 or 10, wherein the ionic lipid is DLin-MC3-DMA, and the LNP is composed of three lipids having a C / I ratio of approximately 1.5 and including DOTAP.

19. The polynucleotide for use according to any one of claims 1 to 7 or 10, wherein the ionic lipid is DLin-MC3-DMA, and the LNP is composed of three lipids having a C / I ratio of approximately 1.5 and including DMG-PEG2000 and DOTAP.

20. The polynucleotide for use according to any one of claims 1 to 7 or 10, wherein the ionic lipid is DLin-MC3-DMA, and the LNP is composed of three lipids having a C / I ratio of approximately 1.5, including DMG-PEG2000 and DOTAP, and has an N / P ratio of approximately 4.

5.

21. (1) The LNP is as shown in the table below: Table 1 Selected from the LNPs, and / or (2) The polynucleotide for use according to any one of claims 1 to 10, wherein the delivery of the polynucleotide is performed for the prevention and / or treatment and / or regulation of the lung disease and / or pulmonary fibrosis of the subject.

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