Compositions and methods for delivery of RNA

Abstract

The present invention relates to lipid nanoparticle (LNP) compositions and therapeutic polynucleotides, such as TERT mRNA, which may be delivered in formulation with the LNP compositions for use in the prevention or treatment of pulmonary disease and / or fibrosis.

Description

[0001] Many lung diseases have been identified for which there are currently no treatments. Lung diseases such as pulmonary fibrosis, interstitial lung disease, and lung cancer often induce fibrosis as part of disease progression, which further limits the extent to which patient recovery may occur. Treatment options for lung diseases remain limited. Delivery of polynucleotides to the lungs may provide a much-needed treatment option for patients with lung diseases; however, compositions and methods for delivering polynucleotides need to be improved.

[0002] Therefore, there is still a need in the art for a delivery formulation capable of delivering polynucleotides such as mRNA to the lungs for preventing or treating lung diseases and / or pulmonary fibrosis.

[0003] The present invention addresses this need as well as other related needs in the art. Summary of the invention

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

[0005] According to related embodiments, the cationic lipid is (a) between about 25% and about 60% SS-OP or an SS-OP analog at a molar percentage, wherein the LNP has a lipid nitrogen:polynucleotide phosphate ratio (N / P ratio) between 10 and 30, or (b) between about 30% and about 50% DLin-MC3-DMA or a DLin-MC3-DMA analog at a molar percentage, wherein the LNP has an N / P ratio between 3.5 and 10. The term "MC3" used alone herein means "DLin-MC3-DMA."

[0006] According to one embodiment, the lipid DOTAP has an in vivo half-life in the lung of a subject of at least 35 hours; an in vivo half-life in the liver of a subject of no more than 15 hours; and / or an in vivo half-life in the spleen of a subject of no more than 9 hours.

[0007] According to related embodiments, the in vivo half-life of the lipid DOTAP in the lung of a subject is at least 5, 7, 9, 10, 15, 20, 25, 30, 35, 37, 40, 45 or more hours. In addition, according to related embodiments, the in vivo half-life of the lipid DOTAP in the liver of a subject does not exceed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 15 hours. In addition, according to related embodiments, the in vivo half-life of the lipid DOTAP in the spleen of a subject does not exceed 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 15 hours.

[0008] Also according to embodiments provided herein is a method of delivering a polynucleotide to a subject, comprising administering by intravenous injection a polynucleotide encapsulated in a lipid nanoparticle (LNP), wherein the lipid nanoparticle comprises: (i) a molar percentage of between about 20% and about 50% cationic lipid, (ii) a molar percentage of between about 20% and about 60% SS-OP or an SS-OP analog, wherein the polynucleotide comprises a synthetic RNA, which is translated into a corresponding protein encoded by the synthetic RNA in the subject upon or after administration of the LNP, and wherein at or about 6 hours after intravenous infusion of the LNP, the lung:liver ratio of the amount of protein per mg of tissue is at least 3:1, 4:1 or 5:1. Typically, according to related embodiments, the ratio of the amount of lipid DOTAP per mg of tissue in lung: liver 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 typically, according to related embodiments, the ratio is at or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 48, 72, or 168 hours after intravenous infusion of the mRNA-encapsulated LNPs.

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

[0010] The synthetic RNA may comprise a sequence encoding a telomerase reverse transcriptase (TERT) protein or a portion thereof, the telomerase reverse transcriptase being selected from human TERT (hTERT), mouse TERT (mTERT) or TERT of another mammalian species. According to the presently contemplated embodiments, the mRNA generally comprises SEQ ID NO: 1 or a fragment thereof. In addition, according to the presently contemplated embodiments, the synthetic ribonucleic acid (RNA) encodes a telomerase reverse transcriptase (TERT), wherein optionally the TERT mRNA comprises a nucleic acid sequence having 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% identity to any one of SEQ ID NO: 1-5 or SEQ ID NO: 38-40 of PCT / US22 / 22642.

[0011] Typically, the mRNA synthesized by TERT comprises a 5' cap structure, wherein the 5' cap structure is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, IRES, Cap0, Capl, ARCA, inosine, Nl-methylguanosine, 2'fluoro-guanosine, 7-deaza-guanosine, CleanCapTM, 8-oxo-guanosine, 2-aminoguanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003 or CAP-225.

[0012] Typically, according to the methods of the present invention, the delivery of polynucleotides is used to prevent and / or treat a subject's lung disease and / or pulmonary fibrosis. Additionally typically, according to the methods of the present invention, the method of delivering polynucleotides is used to regulate a subject's lung disease and / or pulmonary fibrosis.

[0013] According to embodiments generally included, the LNP has a lipid nitrogen:polynucleotide phosphate (N / P) ratio between 28 and 31. In certain embodiments, the N / P ratio is 29.1 or about 29.1. Typically in such embodiments, the ionizable lipid is SS-OP, and the ratio of the cationic lipid of the LNP to the ionizable lipid is 1.8 or about 1.8 and is composed of 5 lipids. In related embodiments, the ionizable lipid is SS-OP, and the ratio of the cationic lipid of the LNP to the ionizable lipid is 1.8 or about 1.8 and is composed of 5 lipids, including DOTAP and DOPC. In related embodiments, the ionizable lipid is SS-OP, and the ratio of the cationic lipid of the LNP to the ionizable lipid is 1.8 or about 1.8 and is composed of 5 lipids, including DMG-PEG2000, DOPC and DOTAP.

[0014] According to embodiments generally contemplated, the LNP has an N / P ratio between 11 and 13. In certain embodiments, the N / P ratio is 12 or about 12. Typically in such embodiments, the ionizable lipid is SS-OP, and the LNP has a cationic lipid to ionizable lipid ratio of 0.8 or about 0.8 and consists of 3 or 5 lipids. In related embodiments, the ionizable lipid is SS-OP, and the LNP has a cationic lipid to ionizable lipid ratio of 0.8 or about 0.8 and consists of 3 or 5 lipids, including DMG-PEG2000 and DOTAP.

[0015] In some embodiments, the N / P ratio of LNP is between 4 and 5. In some embodiments, the N / P ratio is 4.5 or about 4. ...

[0016] In addition, according to the methods provided herein, the ionizable lipid is DLin-MC3-DMA, and the ratio of cationic lipid to ionizable lipid of the LNP is 1.5 or about 1.5 and is composed of 3 lipids, including DMG-PEG2000 and DOTAP, and the N / P ratio is 4.5 or about 4.5.

[0017] Furthermore, according to generally contemplated embodiments, the LNP is selected from the LNPs of Tables 8-10.

[0018] According to the implementation plan ( Fig.22A ), LNPs have only a single cationic lipid, such as DOTAP.

[0019] Also provided herein is a method for making LNP. In a specific example, the manufacturing method of LNP is to form LNP before adding nucleic acid. In a specific embodiment, this type of LNP is 2,3 or 5 lipid LNP as described herein in Table 8, and the method adopts vortex mixing during adding nucleic acid. In certain embodiments, LNP is composed of 2 or 3 lipid LNP as described herein.

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

[0021] Those skilled in the art will understand that the drawings, described below, are for illustration purposes only.

[0022] Figures 1A-1D The pharmacokinetics of specific lipid nanoparticles (LNPs) are depicted. The serum ( Figure 1A ),lung( Figure 1B ) and liver( Figure 1C )Horizontal timeline. Figure 1D Provide with Figures 1A-1C Related tabular data.

[0023] Figure 2 Depicted are the ratios of bioluminescent signals from the indicated organs generated by luciferase protein translated from mRNA preparations in LNPs ("mRNALNPs") injected intravenously at the indicated time points after intravenous injection.

[0024] Figure 3 LNPs formulated with TERT mRNA ("TERT mRNA LNPs") are shown to extend telomeres in lung cells in vivo, where telomere length was measured in lung tissue sections using Q-FISH and telomere signal intensity (green / lighter) is proportional to signal length.

[0025] Figure 4 TERT mRNA LNPs were shown to extend telomeres in lung cells in vivo, with TERT mRNA LNPs extending median and mean telomere length in AT2 cells. At least 50 pro-SPC-positive nuclei were quantified per mouse. Error bars are standard error of the mean (SEM) of telomere length between mice.

[0026] Figure 5 TERT mRNA LNPs were shown to extend telomeres in lung cells in vivo, where TERT mRNA LNPs extended median and mean telomere length in alveolar cells. Telomeres of at least 100 alveolar cells were quantified per mouse. Error bars are standard error of the mean (SEM) of telomere length between mice.

[0027] Fig. 6A Shows Figure 3-12 and Figure 20-21 Experimental design and dosing timeline for the data presented in the present disclosure.

[0028] Figure 6B Shows Figure 13-16 Experimental design and dosing timeline for the data presented in the present disclosure.

[0029] Figure 7 TERT mRNA LNPs were shown to reduce lung fibrosis in vivo, where Sirius red staining was used to map high-density fibrotic foci (yellow / darker) in the lungs of mice treated with bleomycin (treated with control mRNA LNPs (carrying luciferase mRNA) or TERT mRNA LNPs). "No bleomycin" represents lung sections from normal healthy mice that were not treated with bleomycin.

[0030] Figure 8 TERT mRNA LNPs were shown to reduce lung fibrosis in vivo, as measured by quantification of high-density fibrotic foci in Sirius red-stained tissue sections.

[0031] Fig. 9 TERT mRNA LNPs were shown to increase useful lung capacity in vivo as determined by quantifying changes from baseline in the ratio of normally ventilated lung using 3D quantification of fibrosis detected by signal intensity in X-ray computed tomography data.

[0032] Fig.10 TERT mRNA LNPs were shown to improve tissue elasticity in vivo as determined by quantification of measurements of mouse tissue elasticity using the Flexivent system at the end of the study.

[0033] Fig.11TERT mRNA LNPs were shown to improve lung function in vivo, as determined by measuring the forced expiratory volume of mice using the Flexivent system at the end of the study.

[0034] Fig.12 TERT mRNA LNPs were shown to improve lung function in vivo as determined by measuring the forced vital capacity of mice using the Flexivent system at the end of the study.

[0035] Fig.13 TERT mRNA LNPs were shown to improve lung architecture in vivo, where digitized images of tissue sections were analyzed using Morpho-Quant Lung software to quantify the number of alveoli per unit area.

[0036] Fig.14 TERT mRNA LNPs were shown to improve lung structure in vivo, where digitized images of tissue sections were analyzed using Morpho-Quant Lung software to quantify alveolar diameter.

[0037] Fig.15 TERT mRNA LNPs were shown to improve lung architecture in vivo, where digitized images of tissue sections were analyzed using Morpho-Quant Lung software to quantify alveolar circularity.

[0038] Fig.16 TERT mRNA LNPs were shown to improve lung structure in vivo, where representative images of alveoli were falsely colored by Morpho-Quant Lung software during digital analysis of alveolar structure were shown.

[0039] Fig.17 Shown are pharmacodynamic data for TERT mRNA in vivo following intravenous infusion in a TERT mRNA LNP formulation, where telomerase activity levels in lung tissue were quantified at indicated time points following infusion.

[0040] Fig.18 Shown is the experimental scheme used to quantify the effect of TERT mRNA LNPs on the colony-forming capacity of human primary epithelial cells.

[0041] Fig.19 It was shown that TERT mRNA LNP Fig.18 The colony-forming capacity of human primary epithelial cells was increased in the experimental model shown.

[0042] Fig. 20 according to Fig. 6A The experimental model shown depicts the level of P21+ senescent alveolar cells exposed to LNPs in Table 3

[0043] Fig.21 Representative immunohistochemical images of lung sections stained with anti-P21 are shown in Fig. 20 Quantity.

[0044] Fig.22A , 22B , 22C and 22D provide lung delivery data obtained using 2 lipid LNP compositions.

[0045] Fig.23 Described are the results of titrating the lipid:mRNA ratio in 2-lipid LNPs.

[0046] Fig.24 Depicted is the time course of protein expression following delivery of lung-targeted mRNA-LNPs encoding firefly luciferase using the LNPs of Table 3.

[0047] Fig.25A , 25B and 25C provide the use of Fig.25D Lung radiance, encapsulation efficiency, and body weight changes when SS-OP lipids were titrated into 'DOTAP+PEG' LNPs.

[0048] Fig.26A and 26B Lung and liver delivery data of the LNPs used in Table 4.

[0049] Fig.27A and 27B Exemplary LNPs and their encapsulation efficiency are provided.

[0050] Fig.27C and 27D Provides the use of Fig.27A Lung delivery data and body weight change data when SS-OP lipids were titrated into 'DOTAP+PEG' LNPs.

[0051] Fig.28A and 28B Comparison of transfection and weight data for fresh lungs versus long-term cryopreserved lungs for the LNPs in Table 3 is provided.

[0052] Fig.29A and 29B A comparison of transfection and particle size data for fresh lung versus short-term cryopreserved lung is provided for the LNPs in Table 3 .

[0053] Fig. 30A Exemplary 2-, 3-, and 5-lipid LNPs are provided.

[0054] Fig. 30B and 30C Provides the ability to titrate N / P ratios and compare Fig. 30ALung delivery data and body weight change data for 2, 3 and 5 lipid formulations.

[0055] Fig.31A and 31B Exemplary 3- and 5-lipid LNPs and their encapsulation efficiencies are provided.

[0056] Fig. 31C Provides the use of 3 and 5 lipid formulations Fig. 30A Lung delivery data upon titration of the N / ratio (mRNA to lipid ratio).

[0057] Fig.32A and 32B Lung transfection data for LNPs with lipid ratios of Table 3 prepared using vortexing or microfluidic mixing are provided as well as LNP size and encapsulation efficiency data.

[0058] Fig.33A Exemplary 3- and 5-lipid LNPs are provided.

[0059] Fig.33B Provides the use of Fig.33A Lung transfection data of LNPs.

[0060] Fig.34A Exemplary 2-, 3-, and 5-lipid LNPs are provided.

[0061] Fig.34B and 34C Provides the use of Fig.34A Lung transfection data of LNPs as well as Fig.34A Particle size and encapsulation efficiency of LNPs.

[0062] Fig.35A Exemplary 5-lipid LNPs prepared using different flow rates are provided.

[0063] Fig.35B and 35C Provides the use of Fig.35A LNP encapsulation percentage and lung transfection data.

[0064] Fig.36A Exemplary 3-lipid LNPs prepared using different flow rates are provided.

[0065] Fig.36B and 36C Provide use Fig.36A Lung transfection of LNPs and body weight change data.

[0066] Fig.37A and 37B Exemplary LNPs prepared by replacing SS-OP with different ionizable lipids (DLin-MC3-DMA) are provided, including the encapsulation percentage.

[0067] Fig.37C and 37D Provides the use of Fig.37A Lung transfection of LNPs and body weight change data.

[0068] Fig.38A Exemplary LNPs prepared by replacing SS-OP with DLin-MC3-DMA in 3-lipid LNPs are provided.

[0069] Fig.38B and 38C Provides the use of Fig.38A LNP encapsulation percentage and lung transfection data.

[0070] Fig.39A Exemplary LNPs prepared by replacing SS-OP with DLin-MC3-DMA in 5-lipid LNPs are provided.

[0071] Fig.39B and 39C Provides the use of Fig.39A LNP encapsulation percentage and lung transfection data.

[0072] Fig.40A A description of the titration time over which mRNA is adsorbed to form LNPs in a 2-lipid formulation is provided.

[0073] Fig.40B Provides the use of Fig.40A Weight change data of LNP.

[0074] Fig.41A and 41B Data for lung transfection and percent encapsulation of 2-lipid LNPs made at titrated mRNA:lipid ratios are presented.

[0075] Fig.42A and 42B Data for lung transfection and percent encapsulation of 3-lipid LNPs made at titrated N / P ratios are presented.

[0076] Fig.43A and 43B Lung transfection and body weight change data are provided using the listed 2 lipid LNPs titrated with DMG-PEG2000.

[0077] Fig.44 Comparison of lung mean radiances from microfluidics versus manual mixing of lipids in ethanol and malate buffer is provided.

[0078] Figures 45A-45D Formulation, transfection, and IHC data comparing MC3 formulations to the 5-lipid LNP compositions of the present description are provided.

[0079] Fig.46A Exemplary 2-, 3-, and 5-lipid LNPs are provided.

[0080] Fig.46B , 46C and 46D provide the use of Fig.46A Lung, liver and spleen cell transfection data of LNPs.

[0081] Fig.46E IHC data related to the use of exemplary 2, 3 and 5 lipid LNPs in lung, liver and spleen tissues are provided.

[0082] Fig.47A , 47B and 47C characterized the pharmacokinetics of specific 2 lipid LNPs in plasma, lung, and liver.

[0083] Fig.48 The pharmacokinetics of specific 5 lipid LNPs from Table 1 are depicted.

[0084] Fig.49A and 49B Depicted are the biodistribution of exemplary 5 lipid LNPs from Table 1 when administered to a mammal.

[0085] Fig.50A , 50B and 50C depicts Fig.46A Biodistribution of the 3 lipid LNPs described in when administered to mammals.

[0086] Fig.50D , 50E and 50F depict organ-level bioluminescence associated with the use of 3 lipid LNPs in mammals, as described in Example 33.

[0087] Figure 50G Describes Fig.46A Biodistribution of the exemplary 3 lipid LNPs described in when administered to a mammal.

[0088] Fig.51A and 51B Data related to telomere elongation and telomerase activity in lung epithelial cells using the LNPs of Table 3 are provided.

[0089] Fig.52 Data correlating telomerase activity in lung fibroblasts using the LNPs of Table 3 are provided.

[0090] Fig.53A and 53B Lung transfection and encapsulation efficiency data using the LNPs of Example 38 are provided.

[0091] Fig.54A and54B Lung transfection and encapsulation efficiency data using the LNPs of Example 39 are provided.

[0092] Fig.55 Shown is the lung mean radiance of freeze-dried (lyophilized) LNPs of Example 36.

[0093] Fig.56 Modeling is carried out for the best cationic lipid and ionizable lipid ratio (" C / I ratio ") and the best lipid nitrogen of the LNP containing the ionizable lipid of SS-OP family or DLin-MC3-DMA family: polynucleotide phosphoric acid ratio (" N / P ratio ").Gray box indicates the optimal range of C / I ratio and N / P ratio.The threshold value of the relative lung radiance selected by horizontal dotted line indication makes the convex peak of relative lung radiance in each figure basically above the dotted line.The virtual horizontal line is the threshold value of relative yield, makes the convex curve of relative yield basically higher than dotted line.The limit (grey box) of optimal range is determined by the intersection of the curve of relative lung radiance and relative yield and horizontal dotted line or dotted line threshold line respectively. DETAILED DESCRIPTION

[0094] For clarity of disclosure, and not by way of limitation, the detailed description of the present invention is divided into the following subsections.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. All patents, applications, published applications, and other publications referenced herein are incorporated herein by reference in their entirety. If the definitions set forth in this section are contrary to or inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this section take precedence over the definitions incorporated herein by reference.

[0096] As used herein, "a / an" means "at least one" or "one or more"

[0097] As used herein, the term "about" or "approximately" as applied to one or more values ​​of interest refers to a value similar in size and / or in a similar range to the reference value. In certain embodiments, unless otherwise specified or apparent from the context (except for the case where such numbers will exceed 100% of the possible value), the term "about" or "approximately" may refer to a value range falling 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 (greater than or less than) of the reference value. In the case of providing a value range, it should be understood that unless the context clearly indicates otherwise, each intermediate value to one-tenth of the lower limit unit between the upper and lower limits of the range and any other described value or intermediate value in the described range are included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed in the present disclosure, subject to any specifically excluded limit in the stated range. When the stated range includes one or two limits, ranges excluding any one or both of those included limits are also included in the present disclosure.

[0098] As used herein, the term "and / or" may mean "and", it may mean "or", it may mean "exclusive or", it may mean "one", it may mean "some, but not all", it may mean "neither", and / or it may mean "both".

[0099] The terms "individual", "subject" and "patient" are used interchangeably herein and refer to any subject in need of treatment or therapy. The subject can 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., cattle, sheep, pigs, horses, goats, etc.), etc. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion or service animal (e.g., a cat or dog).

[0100] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, consisting of monomers (nucleotides) containing sugars, phosphates and purine or pyrimidine bases. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acids 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 explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating a nucleic acid sequence that, when translated (if RNA) or transcribed and translated (if DNA), produces a sequence of amino acid residues identical to the original nucleic acid sequence; or, wherein the third position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0101] The term "nucleotide sequence" refers to a DNA or RNA polymer that may be single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.

[0102] As used herein, "ribonucleic acid" means a polymer of ribonucleotides.

[0103] As used herein, "mRNA species" means mRNA molecules having the same ribonucleotide length and sequence.

[0104] Telomeres are repetitive DNA sequences bound by proteins at the end of each chromosome arm that protect the chromosome tip by forming a loop. Telomeres shorten as cells divide, and if telomeres become too short to form a loop, the DNA tip becomes exposed, activating the DNA damage signaling P53 / P21, leading to cell senescence and apoptosis. The enzyme telomerase reverse transcriptase (TERT) lengthens telomeres and is transiently activated periodically in normal stem and progenitor cells throughout life.

[0105] Alveolar type 2 (AT2) cells are stem cells that can renew and repair the alveolar epithelium. Increasing evidence suggests that AT2 cell telomeres are involved in the etiology and pathogenesis of idiopathic pulmonary fibrosis (IPF). In the telomere model of IPF, alveolar cell damage from different sources prompts AT2 cells to divide to repair and regenerate alveoli, and because telomeres shorten during cell division, AT2 cell telomeres eventually become "extremely short", which means that protective telomere loops can no longer form, P53 and P21 are chronically activated, and harmful consequences ensue. This model suggests that individuals who are born with short telomeres or are prone to short telomeres are susceptible to IPF. In fact, the average telomere length of leukocytes in IPF patients is lower than the 10th percentile of the general population. 15% of familial IPF patients carry loss-of-function mutations in telomere maintenance genes, the most common of which is telomerase reverse transcriptase (TERT), an enzyme that lengthens telomeres and is expressed by alveolar epithelial AT2 cells. Regardless of TERT mutation status, TERT mRNA and TERT protein levels are reduced in lung tissues of IPF patients. The model also showed that AT2 cells within an individual have relatively short telomeres compared to other cell types. In fact, in IPF patients, telomeres in AT2 cells were shorter than in other lung cells, including myofibroblasts. Moreover, AT2 cell telomeres were shorter in fibrotic areas than in non-fibrotic areas.

[0106] Short telomeres lead to cellular senescence, and the lungs of IPF patients show an increase in senescence markers including P21. Short telomeres lead to AT2 cell apoptosis, and the number of AT2 cells in the lungs of IPF patients decreases. Consistent with the insufficient repair of alveoli by AT2 cells caused by telomere-induced senescence and apoptosis, serum markers of epithelial damage in IPF patients (such as CA-125 and SP-D) increase.

[0107] Consistent with the role of short telomere-induced senescence in altering intercellular signaling, IPF patients also have elevated levels of inflammatory molecules (SASP) such as TGFβ, which are associated with activation and transformation of lung fibroblasts into myofibroblasts. Consistent with the fact that short telomere-induced senescence impairs successful AT2 differentiation, IPF patients' lungs also accumulate a transitional cell phenotype with features of senescence.

[0108] Consistent with these consequences of short telomeres, longer telomere length in patients with IPF was associated with graft-free survival, independent of age, sex, forced vital capacity, or carbon monoxide diffusing capacity. Thus, in the telomere model of IPF, alveolar epithelial cell telomeres serve as etiological links, first integrating alveolar damage over time until they become critically short, subsequently causing multiple pathogenic consequences and ultimately leading to death.

[0109] The telomere model of IPF has inspired the quest for telomere lengthening. To avoid the risks of constitutive telomerase activity associated with cancer and AAV delivery of TERT DNA, we focused here on an approach that only transiently increases telomerase activity, similar to how stem cells periodically increase telomerase activity throughout their lifespan.

[0110] The data provided herein demonstrate or support the pharmacokinetics of, for example, the exemplified LNPs in vivo, particularly information and data related to the time course of concentration levels of lipids in the LNPs from the lungs, 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 lungs, lung tissue, and / or lung cells. With respect to rapid clearance, LNP compositions are provided herein that provide a ribonucleic acid payload that does not reside in the lungs, lung tissue, and / or lung cells for approximately 9 to 11 hours after administration to a subject. Fig.50A In the tissues listed above (except lung) (90% to 100% have been cleared). In contrast, with respect to rapid clearance, the LNP compositions provided herein provide a ribonucleic acid payload that resides in the lungs about 9 to 11 hours after administration, but is not present at all, or not present at any significant level, in one or more (including all) of the spleen, liver, adrenal glands, kidneys, bone marrow, gallbladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenum tissues.

[0111] The data provided herein also demonstrate or support, for example, the use of LNPs in which mRNA encodes telomerase. The present invention specifically provides the demonstration of telomere extension in alveolar cells in mouse lungs and in AT2 cells in mouse alveoli. The demonstration of improving the colony forming ability of primary alveolar epithelial cells of humans by treatment with TERT mRNA LNP is also provided.

[0112] Demonstration of the pharmacodynamics of TERT mRNA LNPs, specifically the time course of telomerase activity in lung tissue following intravenous infusion of TERT mRNA LNPs in mice, is also provided.

[0113] Also provided are efficacy data for TERT mRNA LNPs in vivo for the treatment of pulmonary fibrosis, specifically improvements in lung function (FEV0.1 and FVC), reductions in lung fibrosis, increases in the normal ventilated lung fraction, and improvements in lung structure (alveolar density or number, roundness, and diameter).

[0114] Also provided herein is a method for making LNP. In a specific example, the manufacturing mode of LNP is to form LNP before adding nucleic acid. In its specific embodiment, this type of LNP is 2,3 or 5 lipid LNP as described herein, and the method adopts vortex mixing during adding nucleic acid.

[0115] The inventor finds that in the LNP suitable for working according to the method and purposes currently considered, the ratio of cationic lipid to ionizable lipid (such as SSOP:DOTAP or MC3:DOTAP) is unpredictable according to the specific composition of LNP. Specifically, although it is not intended to be bound by any particular theory, experiments show that the specific ionizable lipid in LNP can affect the ratio of cationic lipid to ionizable lipid in a manner that requires experimental confirmation. The experimental evidence provided herein is based on the ionizable lipid identified in LNP, and is the ratio of specific cationic lipid to ionizable lipid or the specific ratio range to provide support. The table and accompanying drawing (such as Table 8) provided herein provide the additional examples of the specific ratio of cationic lipid to ionizable lipid of 3 and 5 lipid LNP considered herein. In certain embodiments, the ratio of cationic lipid to ionizable lipid is 0.8 or about 0.8, and ionizable lipid is SS-OP. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of LNP is 0.8 or about 0.8, and is composed of 3 or 5 lipids. In addition in certain related embodiments, the ratio of the cationic lipid of LNP to ionizable lipid is 0.8 or about 0.8 and is composed of 3 or 5 lipids, including DOTAP. In addition in certain related embodiments, the ratio of the cationic lipid of LNP to ionizable lipid is 0.8 or about 0.8 and is composed of 3 or 5 lipids, including DOTAP.

[0116] In some embodiments, the ratio of cationic lipid to ionizable lipid is 1.8 or about 1.8, and ionizable lipid is SS-OP. In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.8 or about 1.8, and is composed of 5 kinds of lipids. In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.8 or about 1.8, and is composed of 5 kinds of lipids. In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.8 or about 1.8, and is composed of 5 kinds of lipids, including DOTAP and DOPC. In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.8 or about 1.8, and is composed of 5 kinds of lipids, including pegylated lipid (e.g., DMG-PEG2000), DOPC and DOTAP.

[0117] In some embodiments, the ratio of cationic lipid to ionizable lipid is 1.5 or about 1.5, and ionizable lipid is DLin-MC3-DMA.In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.5 or about 1.5, and is made up of 3 kinds of lipids.In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.5 or about 1.5, and is made up of 3 kinds of lipids, including DOTAP.In some related embodiments, the ratio of cationic lipid to ionizable lipid is 1.5 or about 1.5, and is made up of 3 kinds of lipids, including Pegylated lipid (for example, DMG-PEG2000) and DOTAP.

[0118] The LNP compositions of the present disclosure include and encompass a variety of lipid ratios and lipid:mRNA ratios (lipid:mRNA ratios are also referred to herein as "N / P ratios"). When referred to herein, LNP compositions refer to the final LNP composition or the formulation used to produce the LNP, or both, which will be apparent from the context in which the phrase is used. Specific examples of the contemplated compositions are provided in the Examples, including supporting figures. For example, particularly preferred LNP compositions of the present disclosure are provided in Table 8 and other tables, figures, and descriptions provided herein.

[0119] According to preferred embodiments herein, there is provided a LNP composition containing nucleic acid composed of 5 different lipids, including ionizable lipids and cationic lipids, and having an N / P ratio between 10 and 30. Table 8 provides some such exemplary compositions. Generally according to such embodiments, such LNP compositions are further characterized in having a ratio of cationic lipids to ionizable lipids of 0.8 to 1.8. In addition according to such embodiments, such LNP compositions are further characterized in having a ratio of cationic lipids to ionizable lipids of 0.8 or about 0.8. In addition according to such embodiments, such LNP compositions comprise SS-OP as an ionizable lipid in the composition. In addition according to such embodiments, such LNP compositions are used in mammalian subjects and target lung tissue and / or lung cells in a manner that provides transfection of nucleic acid payloads in lung tissue and / or lung cells. Therefore, it is contemplated herein that such compositions are adapted to target lung tissue and / or lung cells. Further according to such embodiments, such LNP compositions preferentially target lung tissue and / or lung cells of a subject relative to other tissues, including, for example, spleen, liver, adrenal glands, kidney, bone marrow, gall bladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenal tissue. Further according to such embodiments, such LNP compositions have an increased half-life in lung tissue and / or lung cells of a subject relative to the half-life in other tissues, including, for example, spleen, liver, adrenal glands, kidney, bone marrow, gall bladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenal tissue. According to such embodiments, the half-life of such LNP compositions in lung tissue is greater than 30 hours, or between 30 hours and 40 hours, or about 37 hours.

[0120] In addition, according to preferred embodiments of the present invention, there is provided an LNP composition containing nucleic acid composed of 3 different lipids, including ionizable lipids and cationic lipids, and having an N / P ratio between 4 and 24, or an N / P ratio between 4.5 and 12. Table 8 provides some such exemplary compositions. Generally, according to such embodiments, such LNP compositions are further characterized in that there is a ratio of cationic lipids to ionizable lipids of 0.8 to 1.5. In addition, according to such embodiments, such LNP compositions are further characterized in that there is a ratio of cationic lipids to ionizable lipids of 0.8 or about 0.8. In addition, according to such embodiments, such LNP compositions include SS-OP as the ionizable lipid in the composition, and the ratio of cationic lipids to ionizable lipids is 0.8 or about 0.8. In addition, according to such embodiments, such LNP compositions include DLin-MC3-DMA as the ionizable lipid in the composition, and the ratio of cationic lipids to ionizable lipids is 1.5 or about 1.5, and optionally the N / P ratio is 4.5 or about 4.5. In addition, according to such embodiments, such LNP compositions are used in mammalian subjects and target lung tissue and / or lung cells in a manner that provides transfection of nucleic acid payloads in lung tissue and / or lung cells. Therefore, it is contemplated herein that such compositions are adapted to target lung tissue and / or lung cells. In addition, according to such embodiments, relative to other tissues, such LNP compositions preferentially target lung tissue and / or lung cells of subjects, and other tissues include, for example, spleen, liver, adrenal glands, kidney, bone marrow, gall bladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenal tissues. In addition, according to such embodiments, compared to the half-life in other tissues, such LNP compositions have an increased half-life in lung tissue and / or lung cells of subjects, and other tissues include, for example, spleen, liver, adrenal glands, kidney, bone marrow, gall bladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenal tissues. According to such embodiments, the half-life of such LNP compositions in lung tissue is greater than 5 hours, greater than 30 hours, or between 30 hours and 40 hours, or about 37 hours.

[0121] In addition, according to a preferred embodiment of the present invention, there is provided a nucleic acid-containing LNP composition composed of 3 or 5 different lipids, including DOTAP, including ionizable lipids and cationic lipids, and having an N / P ratio between 4 and 24, or an N / P ratio of 12 or about 12. In addition, according to a preferred embodiment of the present invention, there is provided a nucleic acid-containing LNP composition composed of 3 different lipids, including DLin-MC3-DMA, including ionizable lipids and cationic lipids, and having an N / P ratio between 4 and 24, or an N / P ratio of 4.5 or about 4.5.

[0122] In addition, according to embodiments often included herein, there is provided a LNP composition containing nucleic acids composed of 2 different lipids having an N / P ratio of 4 or about 4. Preferably, in such embodiments, one of the two lipids is DOTAP or comprises DOTAP. In addition, according to such embodiments, such LNP compositions are used in mammalian subjects and target lung tissue and / or lung cells in a manner that provides transfection of nucleic acid payloads in lung tissue and / or lung cells. Therefore, it is contemplated herein that such compositions are adapted to target lung tissue and / or lung cells. In addition, according to such embodiments, such LNP compositions preferentially target lung tissue and / or lung cells of subjects relative to other tissues, and other tissues include, for example, spleen, liver, adrenal glands, kidneys, bone marrow, gall bladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenal tissues. Further according to such embodiments, such LNP compositions have an increased half-life in lung tissue and / or lung cells of a subject compared to the half-life in other tissues, including, for example, spleen, liver, adrenal glands, kidney, bone marrow, gall bladder, gonads, heart, brain, pancreas, fat, thymus, stomach, colon and / or duodenum tissue. According to such embodiments, such LNP compositions have a half-life in lung tissue of more than 30 hours, or between 30 hours and 40 hours, or about 37 hours.

[0123] 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 a range of 5% to 25% included in the final composition provides a preferred level of cryoprotection. In a specific embodiment, a cryoprotectant (e.g., sucrose) is included at or about 15% of the final composition.

[0124] Delivery of synthetic nucleoside-modified mRNA encoding TERT (TERT mRNA) was able to temporarily increase telomerase activity, which lengthened telomeres within hours, sufficient to reverse years of telomere shortening. Importantly for safety, the mRNA and the resulting TERT protein degraded within hours and days, respectively, while the resulting telomerase activity persisted for several days ( Fig.17 ). Thus, delivery of synthetic TERT mRNA recapitulates normal telomere lengthening of endogenous TERT mRNA, as occurs periodically throughout life in stem cells. Immediately after treatment ends, the lengthened telomeres resume shortening at their normal rate. Recent advances in lipid nanoparticle (LNP) vehicles enable delivery of mRNA to up to 90% of lung epithelial cells with high tolerability via intravenous (IV) infusion.

[0125] Synthetic mRNA

[0126] As used herein, synthetic ribonucleic acid (RNA) may refer to any RNA sequence comprising a mutation (point mutation or deletion) or additional nucleotides that are not present in the wild-type sequence. For example, messenger RNA (mRNA) may refer to a wild-type sequence encoding a human sequence, 1, 2, 3, 10, 100 or more nucleotides of which are flanked by additions. Similarly, the nucleotides themselves may encode amino acids different from the wild type, or may be modified to reduce immunogenicity in cells or tissues. In some embodiments, the mRNA sequence may include any of the following modifications, including but not limited to untranslated regions (UTRs), 5' caps, and polyadenosine tails. In some embodiments, RNA may be circular and / or self-replicating.

[0127] Illustrative methods for making circular mRNA are provided in Chen et al. Science. 1995 Apr 21;268(5209):415-7; Perriman R. (2002) Circular mRNA Encoding for Monomeric and Polymeric Green Fluorescent Protein. Hicks BW (ed.) 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 making self-replicating mRNA are provided in Tews BA, Meyers G. (2017) Self-Replicating RNA. 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.

[0128] In some embodiments, the mRNA may comprise a codon-optimized sequence. In some embodiments, the mRNA may comprise a uridine-depleted sequence.

[0129] In some embodiments, the 5' cap of the RNA is a non-immunogenic cap. In some embodiments, the 5' cap can increase the translation of the RNA. In some embodiments, the 5' cap can be treated with a phosphatase to modulate the innate immunogenicity of the RNA. 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 structural analog. In some embodiments, the 5' cap is m7G(5')ppp(5')(2'OmeA)pG (also known as AG). In some embodiments, the 5' cap is m7(3'Ome G)(5')ppp(5')(2'OmeA)pG (also known as AG(3'OMe)).

[0130] The above-mentioned characteristics or other characteristics can increase the translation of the protein encoded by the ribonucleic acid, can increase or reduce the stability of the ribonucleic acid itself in a cell type-specific or non-cell type-dependent manner, or can have both. In some embodiments, 5'UTR and / or 3'UTR are from genes with very stable mRNA and / or rapidly translated mRNA, such as alpha-globulin or beta-globulin, c-fos or tobacco etch virus. In some embodiments, 5'UTR and 3'UTR are from different genes or from species different from the species of the delivery composition. UTR can also be an assembly of the UTR part of the mRNA from different genes, wherein the part is selected to realize a certain combination of stability and translation efficiency. UTR can also include a design sequence that gives RNA characteristics such as cell type-specific stability or non-cell type-dependent stability.

[0131] The ribonucleic acid of the present disclosure may include one or more modified nucleosides, and / or include a primary sequence of nucleosides, which regulates the translation, stability or immunogenicity of RNA. Most mature RNA molecules in eukaryotic cells contain nucleosides that are modified forms of unmodified RNA nucleosides (i.e., adenine, cytidine, guanosine and uridine) as standard. For example, the 5' cap of mature RNA includes modified nucleosides, and other modified nucleosides generally appear in other positions in RNA. These modifications can prevent RNA from being identified as exogenous RNA. The immunogenicity of synthetic RNA molecules prepared using certain nucleosides is much lower than that of unmodified RNA. By purifying the synthesized mRNA, for example, by using high performance liquid chromatography (HPLC), immunogenicity can even be further reduced. Modified nucleosides can, for example, be selected from the nucleosides listed below. In some embodiments, nucleosides are pseudouridine, 1-methyl pseudouridine, 2-thiouridine, 5-methoxyuridine or 5-methylcytidine. The primary sequence can be modified in a manner that increases or decreases immunogenicity. In some cases, it may be desirable for the modified RNA to retain some immunogenicity.

[0132] Thus, in some embodiments, the ribonucleic acid of the compositions of the invention comprises 1-methylpseudouridine, pseudouridine, 5-methoxyuridine (5-moU), 2-thiouridine, 5-methylcytidine, or another modified nucleoside. Modified nucleosides present in eukaryotic cells include m1A 1-methyladenosine, m6A N6-methyladenosine, Am 2'-O-methyladenosine, i6A N6-isopentenyladenosine, io6A N6-(cis-hydroxyisopentenyl)adenosine, ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, g6A N6-glycinecarbamoyladenosine, t6A N6-threonylcarbamoyladenosine, ms2t6A2-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, hm5C5-hydroxymethylcytidine, f5Cm 5-formyl-2'-O-methylcytidine, m1G1-methylguanosine, m2GN2-methylguanosine, m7G 7-methylguanosine, Gm 2'-O-methylguanosine, m2 2G N2,N2-dimethylguanosine, Gr(p)2'-O-ribosylguanosine (phosphate), yW wyoside, o2yW peroxy wyoside, OhyW hydroxy wyoside, OhyW* undermodified hydroxy wyoside, imG wyoside, m2,7G N2,7-dimethylguanosine, m2,2,7G N2,N2,7-trimethylguanosine I inosine, m1I 1-methylinosine, Im 2'-O-methylinosine, Q braid, galQ galactosyl-braid, manQ mannosyl-braid, Ψ pseudouridine, D dihydrouridine, m5U 5-methyluridine, Um 2'-O-methyluridine, m5Um 5,2'-O-dimethyluridine, m1Ψ1-methyl pseudouridine, Ψm 2'-O-methyl pseudouridine, 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,m1acp3Ψ1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, cm5U 5-carboxymethyluridine, m3Um 3,2'-O-dimethyluridine, m5D 5-methyldihydrouridine, τm5U 5-taurine methyluridine, τm5s2U 5-taurine methyl-2-thiouridine, 2-aminoadenosine, 2-amino-6-chloropurine nucleoside, 8-azaadenosine, 6-chloropurine nucleoside, 5-iodocytidine, 5-iodouridine, inosine, 2'-O-methylinosine, xanthosine, 4-thiouridine, O6-methylguanosine, 5,6-dihydrouridine, 2-thiocytidine, 6-azacytidine, 6-azauridine, 2'-O-methyl-2-aminoadenosine, 2'-O-methylpseudouridine, N1-methyladenosine, 2'-O-methyl-5-methyluridine, 7-deazaguanosine, 8-azidoadenosine, 5-bromocytidine, 5-bromouridine, 7-deazaadenosine, 5-aminoallyl uridine, 5-aminoallyl Cytidine, 8-oxoguanosine, 2-aminopurine nucleoside, 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-carboxymethyluridine, 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'-deoxynucleoside, 2-amino-2'-deoxyadenosine, 2-aminopurine-2'-deoxynucleoside, 5-bromo-2'-deoxycytidine, 5-bromo-2'-deoxyuridine, 6-chloropurine-2'-deoxynucleoside, 7-deaza-2'-deoxyadenosine, 7-deaza-2'-deoxyguanosine, 2'-deoxyinosine, 5-propynyl-2'-deoxycytidine, 5-propynyl-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-iodo-2'-deoxycytidine, 5-iodo-2'-deoxyuridine, N6-methyl-2'-deoxyadenosine, 5-methyl-2'-deoxycytidine, O6-methyl-2'-deoxyguanosine, N2-methyl- 2'-deoxyguanosine, 8-oxo-2'-deoxyadenosine, 8-oxo-2'-deoxyguanosine, 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)propionamide-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-aminoallyl cytidine, N4-biotin-OBEA-2'-deoxycytidine, biotin-16-aminoallyl uridine, Dabcyl-5-3-aminoene Propyl-2'-dUTP, desthiobiotin-6-aminoallyl-2'-deoxycytidine, desthiobiotin-16-aminoallyl-uridine, biotin-16-7-deaza-7-propargylamino-2'-deoxyguanosine, anthocyanidin 3-5-propargylamino-2'-deoxycytidine, anthocyanidin 3-6-propargylamino-2'-deoxyuridine, anthocyanidin 5-6-propargylamino-2'-deoxycytidine, anthocyanidin 5-6-propargylamino-2'-deoxyuridine, anthocyanidin 3-aminoallyl cytidine, anthocyanidin 3-aminoallyl uridine, anthocyanidin 5-aminoallyl cytidine, anthocyanidin 5-aminoallyl uridine, anthocyanidin 7-aminoallyl uridine, 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, cytosine arabinoside, arabinouridine, 2'-azido-2'-deoxyadenosine, 2'-amino-2'-deoxyadenosine, adenosine, 2'-fluoro-thymidine, 3'-O-methyladenosine, 3'-O-methylcytidine, 3'-O-methylguanosine, 3'-O-methyluridine, 2'-azido-2'-deoxyguanosine, 2'-arabinouridine, '-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-phosphorothioate), 2'-deoxycytidine-5'-O-(1-phosphorothioate), 2'-deoxyguanosine-5'-O-(1-phosphorothioate), 2'-deoxythymidine-5'-O-(1-phosphorothioate), adenosine-5'-O-(1-phosphorothioate), cytidine-5'-O-(1-phosphorothioate), guanosine-5'-O-(1-phosphorothioate), uridine-5'-O-(1-phosphorothioate), 2',3 '-dideoxyadenosine-5'-O-(1-phosphorothioate), 2',3'-dideoxycytidine-5'-O-(1-phosphorothioate), 2',3'-dideoxyguanosine-5'-O-(1-phosphorothioate), 3'-deoxythymidine-5'-O-(1-phosphorothioate), 3'-azido-2',3'-dideoxythymidine-5'-O-(1-phosphorothioate), 2',3'-dideoxyuridine-5'-O-(1-phosphorothioate), 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).

[0133] Without wishing to be bound by theory, the presence of modified nucleosides and / or nucleoside sequences that change the secondary structure of RNA and / or the binding of RNA to RNA binding proteins or microRNAs can enable mRNA to avoid activation of immune responses mediated by various receptors, including Toll-like receptors and RIG-1. Non-immunogenic mRNA has been used as a therapeutic agent for mice by local delivery. Kormann et al. (2011) Nature Biotechnology 29:154-157. In some embodiments, ribonucleic acid comprises more than one of the above-mentioned nucleosides or a combination of the above-mentioned nucleosides. In some embodiments, ribonucleic acid comprises 1-methyl pseudouridine, 5-methoxyuridine, or pseudouridine and 5-methylcytidine.

[0134] In some embodiments, an immune response to the mRNA may be desired, and the RNA may be modified to induce optimal levels of innate immunity. In other embodiments, an immune response to the mRNA may not be desired, and the RNA may be modified to minimize such a response. The RNA may be modified for either situation.

[0135] The ribonucleic acid molecule can be a synthetic ribonucleic acid. As used herein, the term "synthetic" may mean that in some embodiments, molecular biology tools are used to prepare ribonucleic acids under the guidance of people, such as as described below. Synthetic ribonucleic acids can be prepared by in vitro synthesis, for example, using cell extracts or purified enzymes and nucleic acid templates. In some embodiments, synthetic ribonucleic acids can be prepared partially or completely by chemical synthesis. Alternatively or in addition to this, in some embodiments, synthetic ribonucleic acids can be expressed by engineering in cells, then destroy cells and at least partially purify ribonucleic acids to prepare.

[0136] The ribonucleic acid of the present disclosure can be prepared using a variety of techniques, as will be understood by those of ordinary skill in the art. In some embodiments, the ribonucleic acid can be prepared by in vitro synthesis. In some embodiments, the ribonucleic acid can be prepared by chemical synthesis. In some embodiments, the ribonucleic acid can be prepared by a combination of in vitro synthesis and chemical synthesis. As mentioned above, the term "synthetic" should be construed as including ribonucleic acids prepared by chemical synthesis, by in vitro synthesis, by in vivo expression and at least partial purification, or by a combination of these or other chemical or molecular biological methods.

[0137] In some embodiments, ribonucleic acid can be purified. As mentioned above, purification can reduce the immunogenicity of ribonucleic acid, and may be advantageous in some cases. In some embodiments, ribonucleic acid is purified by one or more of HPLC, DNA enzyme treatment, protease treatment or by affinity capture and elution.

[0138] In some embodiments, the mRNA sequence can be synthesized as unmodified or modified mRNA. mRNA can be modified to enhance stability and / or escape immune detection and degradation. Modified mRNA may include one or more of, for example, nucleotide modification, nucleoside modification, backbone modification, sugar modification and / or base modification. In some embodiments, modified nucleoside is pseudouridine or pseudouridine analog. In some embodiments, pseudouridine analog is N-1-methyl pseudouridine. In some embodiments, modified nucleoside is 5-methoxyuridine. In some embodiments, modified nucleoside as used herein may include any part listed in Table 2.

[0139] Table 2

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150] In some embodiments, RNA, such as mRNA, can be synthesized from naturally occurring bases and / or base analogs (modified bases), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and their analogs and derivatives, 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, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, quercetin, β-D-mannosyl-quercetin, whibutoxin, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.

[0151] In some embodiments, RNA, such as mRNA, can be synthesized from naturally occurring nucleosides and / or nucleoside analogs (modified nucleosides), including but not limited to nucleosides comprising adenosine (A), guanosine (G), or pyrimidine (thymine (T), cytidine (C), uridine (U)), and nucleosides including analogs and derivatives thereof, such as 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytidine, 3'-deoxyguanosine, 3'-deoxythymine, 2',3'-dideoxynucleosides, 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytidine, 2',3'-dideoxyguanosine, 2',3'-dideoxythymine, a 2'-deoxynucleoside, -O-methylnucleoside, 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-carboxymethyl aminomethyl-2-thio-uridine, 5-(carboxyhydroxymethyl)-uridine, 5-fluoro-uridine, 5-bromo-uridine, 5-carboxymethylaminomethyl-uridine, 5-methyl-2-thio-uridine, 5-methyl-uridine, N-uridine-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uridine, 5-methoxyaminomethyl-2-thio-uridine, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouridine, braided uridine, β-D-mannosyl-braided uridine, whibutoxin, 7-deazaguanosine, 5-methylcytosine and inosine.

[0152] The preparation of such bases, nucleosides, nucleotides and backbone analogs, modifications and derivatives are known to those skilled 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. Nos. 5,262,530 and 5,700,642, all of which are incorporated by reference in their entirety.

[0153] In some embodiments, the mRNA is about 80%, about 90%, 95%, 99%, or 100% depleted of uridine and replaced with a uridine analog, such as pseudouridine, 5-methoxyuridine, or N-1-methyl-pseudouridine.

[0154] In some embodiments, RNA may include RNA backbone modifications. Generally, backbone modifications are modifications in which the phosphates of the nucleotide backbones included in the RNA are chemically modified. Exemplary backbone modifications may include, but are not limited to, modifications in which the phosphodiester bond is selected from a group consisting of a peptide, methylphosphonate, methylphosphoramidate, phosphoramidate, thiophosphate (e.g., cytidine 5'-0-(l-phosphorothioate)), borane phosphate and / or a positively charged guanidine group, or other ways of replacing the phosphodiester bond.

[0155] In some embodiments, the RNA may include sugar modifications. Sugar modifications may include, but are not limited to, 2'O-methyl sugar modifications, 2'fluoro sugar modifications (e.g., 2'-fluoro ribose), 3'amino sugar modifications, 2'thio sugar modifications, 2'-O-alkyl sugar modifications, 5-methylthio ribose, and 2'-deoxy-2'-fluoro-ribonucleotides (2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine), 2'-deoxy-2'-deamine-ribonucleotides (2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine), 2'-deoxy-2'-deamine-ribonucleotides (2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine, 2'-deoxy-2'-deoxyuridine, 2'-deoxy-2'-deoxyuridine, 2'-deoxy-2'-deamine-ribonucleotides (2'-amino-2'-deoxycytidine, 2'-deoxy-2'-deoxyur ... Sugar modifications of 2'-O-alkyl ribonucleotides, 2'-deoxy-2'-C-alkyl ribonucleotides (2'-O-methyl cytidine, 2'-methyl uridine), 2'-C-alkyl ribonucleotides, and their isomers (2'-arabinocytidine, 2'-arabinouridine), or azidophosphates (2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine).

[0156] In some embodiments, the RNA can be synthesized from one or more nucleotide triphosphates, comprising any of the nucleosides and nucleotides disclosed herein, or 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'-dideoxyadenosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxyadenosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxycytidine-5'-O-(1-thiotriphosphate), 2',3'-dideoxyguanosine-5'-O-(1-thiotriphosphate), 2',3'-dideoxythymidine-5'-O-(1-thiotriphosphate), -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-boranetriphosphate), 2'-deoxycytidine-5'-O-(1-boranetriphosphate), 2'-deoxyguanosine-5'-O-(1-boranetriphosphate), and 2'-deoxythymidine-5'-O-(1-boranetriphosphate).

[0157] In some embodiments, the mRNA may include a "cap" added at the N-terminal (5') end, and a "tail" added at the C-terminal (3') end. The presence of the cap can provide resistance to nucleases present in eukaryotic cells. The presence of the "tail" can protect the mRNA from exonuclease degradation.

[0158] Cap structure

[0159] In some embodiments, the mRNA may include a 5' cap structure. The 5' cap may include, for example, a triphosphate bond and a guanine nucleotide, wherein 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. The naturally occurring cap structure includes 7-methylguanosine, which is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge to give a dinucleotide cap of m7G(5')ppp(5')N, wherein N is any nucleoside. In vivo, the cap is added to the nucleus by the enzyme guanylyltransferase immediately after transcription initiation.

[0160] In some embodiments, the 5' cap may comprise m7(3'OmeG)(5')ppp(5')(2'OmeA)pG or (CleanCap TMIn some embodiments, the 5' cap may comprise m7G(5')ppp(5')G. In some embodiments, the anti-reverse cap analog ("ARCA") or modified ARCA is a 5' cap in which the 2' or 3' OH group is replaced by -OCH3. In some embodiments, 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, m7,3dGpppG, and tetraphosphate derivatives thereof) (see, e.g., Jemielity, J. et al., 'Wove anti-reverse cap analogs with superior translational properties", RNA, 9: 1108-1122 (2003)).

[0161] In some embodiments, a suitable cap is 7-methylguanylate ("m7G"), which is linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge to give m7G(5')ppp(5')N, where N is any nucleoside. An embodiment of the m7G cap used in the embodiments of the present disclosure is m7G(5')ppp(5')G. In some embodiments, the cap is a Cap0 structure. The Cap0 structure lacks the 2'-0-methyl residue of the ribose linked to bases 1 and 2. In some embodiments, the cap is a Cap1 structure. The Cap1 structure has a 2'-0-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. The Cap2 structure has a 2'-0-methyl residue linked to bases 2 and 3.

[0162] A variety of m7G cap analogs are known in the art, many of which are commercially available. These include the m7GpppG described above, as well as 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. Pat. Nos. 8,093,367 and 8,304,529 (including biotinylated cap analogs), which are incorporated herein by reference.

[0163] In some embodiments, the 5' cap is inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, CleanCap TM , m7(3'OmeG)(5')ppp(5')(2'OmeA)pG, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003 or CAP-225.

[0164] In some embodiments, the 5' cap comprises or consists of an internal ribosome entry site (IRES). In some embodiments, the IRES is located within the 5'UTR. In some embodiments, the 5' cap comprises or consists of a 2A self-cleaving peptide, such as one or more of P2A, T2A, E2A, and F2A.

[0165] Tail Structure

[0166] The presence of the "tail" can be used to protect mRNA from exonuclease degradation. It is believed that the poly-A tail stabilizes natural messengers and synthetic sense RNA. Therefore, in certain embodiments, a long poly-A tail can be added to the mRNA molecule to make the RNA more stable. The poly-A tail can be added using a variety of techniques recognized in the art. For example, a long poly-A tail can be added to a synthetic or in vitro transcribed RNA using a poly-A polymerase (Yokoe et al., Nature Biotechnology. 1996; 14: 1252-1256). The transcription vector can also encode a long poly-A tail. In addition, the poly-A tail can be added by directly transcribing from the PCR product. It is also possible to connect poly-A to the 3' end of the sense RNA with RNA ligase (see, for example, Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

[0167] 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 on the 3' end of the mRNA may include about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides or about 20 to 60 adenosine nucleotides). In some embodiments, the poly A tail is 120 adenosine nucleotides.

[0168] In some embodiments, mRNA may include a 3' poly-C tail structure. The poly-C tail on 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 the poly-A tail or may 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 is therefore related to the transcription of the protein. For example, because the length of the poly-A tail may affect the half-life of the sense mRNA molecule, the length of the poly-A tail can be adjusted to change the resistance level of the mRNA to nucleases, thereby providing more control over the time course of polynucleotide expression and / or polypeptide production.

[0169] 5' and 3' untranslated regions (UTR)

[0170] In some embodiments, mRNA may include a 5' untranslated region (UTR) and / or a 3'UTR. In some embodiments, the 5'UTR may include one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5'UTR may, for example, include an iron response element. In some embodiments, the length of the 5'UTR may be between about 50 to about 100 or about 50 to about 500 nucleotides. In some embodiments, the 3'UTR includes one or more of a poly-A signal, a protein binding site that may affect the stability or localization of the mRNA, or a binding site for one or more miRNAs. In some embodiments, the length of the 3'UTR may be between about 0 and about 50 nucleotides, or between about 50 to about 100 nucleotides.

[0171] Exemplary 3' and 5'UTR sequences can be derived from mRNA with a relatively long half-life (e.g., globulin, actin, GAPDH, tubulin, histone or citric acid cycle enzyme) to increase the stability of sense mRNA molecules. For example, the 5'UTR sequence can include a partial sequence of a cytomegalovirus (CMV) immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or extend the half-life of a polynucleotide. In another example, the 5'UTR may include a sequence of a tobacco etch virus (TEV). Generally speaking, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of polynucleotides relative to their unmodified counterparts, and include modifications such as those performed to improve the resistance of such polynucleotides to nuclease digestion in vivo.

[0172] In some embodiments, the UTR can improve tissue-specific expression in, for example, the lung. In some embodiments, the 3'UTR is a mouse alpha-globin 3'UTR. In another example, the 3'UTR can include the sequence of Xenopus globin. In some embodiments, the UTR can be any one of those described in any one of PCT Application No. WO2017053297A1 and Patent No. US10519189B2, or a functional variant thereof, all of which are incorporated herein in their entirety.

[0173] Ionizable lipids

[0174] In some embodiments of the present disclosure, LNP may include ionizable lipids, such as SS-OP or its analogs, or MC3 or its analogs. The charge of the lipid may depend on the pH of the surrounding solution, making it an ionizable lipid. The ionizable lipid may also be cleavable. The ionizable lipid may be cationic within the pH range in the endosome or lysosome of a mammalian cell.

[0175] An ionizable lipid may refer to any of a variety of lipid species having a net positive charge at a selected pH, such as physiological pH. In some embodiments, the LNP may comprise an ionizable lipid as disclosed in any one of WO 2010 / 053572 or WO 2012 / 170930, or a variation thereof, which are incorporated herein by reference in their entirety.

[0176] In some embodiments, the LNP may include one or more of the following: MC3 (((6Z,9Z,28Z,31Z)-triacontriacontane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate), DLin-MC3-DMA (4-(dimethylamino)-butyric acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecanadien-1-yl ester), or Its analogs, including but not limited to LenMC3, γ-LenMC3, MC3MC, MC2C, MC2MC, 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, lipid 5, SM-102, ALC-0315 and combinations thereof.

[0177] In some embodiments, the LNP may include one or more of cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), DLinDAP (1,2-dilinoleoyl-3-dimethylammonium-propane), DLin-DMA, DLin-D-DMA, DLin-KC2-DMA (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), and DODMA.

[0178] 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):

[0179]

[0180] In formula (1): R 1a and R 1b Each independently represents an alkylene group having 1 to 6 carbon atoms, and may be linear or branched. The alkylene group may have 1 to 4 carbon atoms, or may have 1 to 2 carbon atoms. Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylidene group, a tetramethylene group, an isobutylene group, a pentamethylene group, and a neopentylene group. R 1a and R 1b Each may independently be a methylene group, an ethylene group, a trimethylene group, an isopropylene group or a tetramethylene group, and may be an ethylene group.

[0181] R 1a Can be used with R 1b Different or the same.

[0182] X a and X b Each independently represents an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, or 2 to 5 carbon atoms, and a cyclic alkylene tertiary amino group having 1 to 2 tertiary amino groups, and / or each independently represents a cyclic alkylene group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups and an alkylene tertiary amino group.

[0183] In the acyclic alkyl tertiary amino group with 1 to 6 carbon atoms and 1 tertiary amino group, the alkyl group with 1 to 6 carbon atoms is branched, although it is straight chain. The alkyl group can be annular. The alkyl group can have 1 to 3 carbon atoms. The specific example of the alkyl group with 1 to 6 carbon atoms includes methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, isobutyl group, tertiary butyl group, pentyl group and isopentyl group. Neopentyl group, tertiary pentyl group, 1,2-dimethylpropyl group, 2-methylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 2,2-dimethylbutyl group, 2,3-dimethylbutyl group, cyclohexyl group etc. can be mentioned.

[0184] The specific structure of the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group is represented by X 1 express.

[0185]

[0186] X 1 R 5 Represents an alkyl group with 1 to 6 carbon atoms, and can be linear, branched or cyclic. The alkyl group can have 1 to 3 carbon atoms. Specific examples of alkyl groups with 1 to 6 carbon atoms include methyl groups, ethyl groups, propyl groups, isopropyl groups, n-butyl groups, sec-butyl groups, isobutyl groups, tert-butyl groups, pentyl groups and isopentyl groups. Neopentyl groups, tert-pentyl groups, 1,2-dimethylpropyl groups, 2-methylbutyl groups, 2-methylpentyl groups, 3-methylpentyl groups, 2,2-dimethylbutyl groups, 2,3-dimethylbutyl groups, cyclohexyl groups, etc. can be mentioned.

[0187] 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 aziridinyl group, an azetidinyl group, a pyrrolidinyl group, a piperidinyl group, an imidazolidinyl group, a piperazinyl group, an optional pyrrolidinyl group, a piperidinyl group or a piperazinyl group.

[0188] The specific structure of the alkylene tertiary amino group having 2 to 5 carbon atoms and containing one cyclic tertiary amino group is represented by X 2 express.

[0189]

[0190] X 2 p is 1 or 2. When p is 1, X 2is a pyrrolidinylene group, and when p is 2, X 2 It is a piperidinylene group.

[0191] The specific structure of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups is represented by X 3 express.

[0192]

[0193] X 3 w is 1 or 2. When w is 1, X 3 is an imidazolidinyl group, and when w is 2, X 3 It is a piperazinylene group.

[0194] X a Can be different from X b .

[0195] R 2a and R 2b Each independently represents an alkylene group or an oxydialkylene group having 8 or less carbon atoms, and optionally each independently represents an alkylene group having 8 or less carbon atoms.

[0196] The alkylene group with 8 or less carbon atoms can be straight or branched, but is optionally straight. The number of carbon atoms contained in the alkylene group is optionally 6 or less, and is optionally 4 or less. The specific example of the alkylene group with 8 or less carbon atoms includes a methylene group, an ethylene group, a propylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, etc. In some embodiments, a methylene group, an ethylene group, a propylene group and a tetramethylene group are included.

[0197] The oxydialkylene group with 8 or less carbon atoms refers to an alkylene group (alkylene-O-alkylene) via an ether bond, and the total number of carbon atoms of the two alkylene groups is 8 or less. Here, the two alkylene groups may be the same or different, but are optionally the same. Specific examples of the oxydialkylene group with 8 or less carbon atoms include oxydimethylene groups, oxydiethylene groups, oxydipropylene groups, and oxydibutylene groups.

[0198] R 2a Can be used with R 2b Same or different.

[0199] Y a and Y bEach is independently an ester bond, an amide bond, a carbamate bond, an ether bond or a urea bond, and optionally each is independently an ester bond, an amide bond or a carbamate bond. a and Y b The Y binding orientation is not restricted, but if Y a and Y b is an ester bond, optionally -Z a -CO-R 2a -and-Z b -CO-OR 2b -structure.

[0200] Y a Can be used with Y b Different or the same.

[0201] Z a and Z b Each independently is a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring, and optionally having a heteroatom. The number of carbon atoms contained in the aromatic compound is optionally 6 to 12, or 6 to 7. In addition, the number of aromatic rings contained in the aromatic compound is optionally one.

[0202] As the type of aromatic ring contained in the aromatic compound having 3 to 16 carbon atoms, for aromatic hydrocarbon rings such as benzene ring, naphthalene ring, anthracene ring, and aromatic heterocyclic rings such as imidazole ring, pyrazole ring, oxazole ring, isoxazole ring, thiazole ring, isothiazole ring, triazine ring, pyrrole ring, furothiphene ring, pyrimidine ring, pyridazine ring, pyrazine ring, pyridine ring, purine ring, pteridine ring, benzimidazole ring, indole ring, benzofuran ring, quinazoline ring, phthalazine ring, quinoline ring, isoquinoline ring, coumarin ring, chromone ring, benzodiazepine 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, and a 2 to 2 carbon atom group. The invention also includes but is not limited to an acyloxy group having 1 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 alkylthio group having 1 to 4 carbon atoms, a 1-carbon atom alkylsulfonyl group having 4 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, an aryloxy group having 6 to 10 carbon atoms, and the like. Some examples include an acetyl group, a methoxycarbonyl group, a methyl carbonate group and the like, a moyl group, an acetoxy group, an acetamide group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylthio group, a benzenesulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group, and a phenoxy group.

[0203] Z a and Z b The specific structure includes Z 1 .

[0204]

[0205] Here, s represents an integer from 0 to 3, t represents an integer from 0 to 3, and u represents an integer from 0 to 4.

[0206] Z 1 The S in is optionally an integer from 0 to 1.

[0207] Z 1 T in is optionally an integer from 0 to 2.

[0208] Z 1 U in is optionally an integer from 0 to 2.

[0209] Z 1R4 in is a substituent of an aromatic ring (benzene ring) contained in an aromatic compound having 3 to 16 carbon atoms, which does not inhibit the reaction in the process of synthesizing an ionizable lipid. Examples of substituents include acyl groups having 2 to 4 carbon atoms, alkoxycarbonyl groups having 2 to 4 carbon atoms, carbamoyl groups having 2 to 4 carbon atoms, acyloxy groups having 2 to 4 carbon atoms, amido groups having 2 to 4 carbon atoms, alkoxycarbonylamino groups having 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylthio groups having 1 to 4 carbon atoms, alkylsulfonyl groups having 1 to 4 carbon atoms, arylsulfonyl groups having 6 to 10 carbon atoms, nitro groups, trifluoromethyl groups, cyano groups, alkyl groups having 1 to 4 carbon atoms, The ureido group, the alkoxy group having 1 to 4 carbon atoms, the aryl group having 6 to 10 carbon atoms and the aryloxy group having 6 to 10 carbon atoms, and examples include an acetyl group, a methoxycarbonyl group, a methylcarbamoyl group, an acetoxy group, a Mido group, a methoxycarbonylamino group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a methylthio group, a benzenesulfonyl group, a nitro group, a trifluoromethyl group, a cyano group, a methyl group, an ethyl group, a propyl group, an isopropyl group, a tert-butyl group, a ureido group, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a tert-butoxy group, a phenyl group and a phenoxy group. When a plurality of R 4 When each R 4 Can be the same or different.

[0210] Z a Can be used with Z b Different or even the same.

[0211] R 3a and R 3b Each independently is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride, or a sterol derivative having a hydroxyl group and succinic anhydride or glutaric acid. represents a residue derived from a reaction product with an acid anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms, and optionally each independently is a residue of a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride. or a C12-22 aliphatic hydrocarbon group, and optionally each independently is an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

[0212] Examples of fat-soluble vitamins having a hydroxyl group include retinol, ergosterol, 7-dehydrocholesterol, calciferol, corcalciferol, dihydroergocalciferol, dihydrotaxol, tocopherol and tocotrienol. The fat-soluble vitamin having a hydroxyl group is optionally tocopherol.

[0213] Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, and the like, optionally cholesterol or cholestanol.

[0214] The aliphatic hydrocarbon group with 12 to 22 carbon atoms can be straight or branched. The aliphatic hydrocarbon group can be saturated or unsaturated. For unsaturated aliphatic hydrocarbon groups, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is generally 1 to 6, optionally 1 to 3 or 1 to 2. 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. The aliphatic hydrocarbon group includes an alkyl group, an alkenyl group, an alkynyl group, etc., and optionally includes an alkyl group or an alkenyl group. Specific examples of the aliphatic hydrocarbon group having 12 to 22 carbon atoms include dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, a dodecenyl group, a tridecenyl group, a tetradecenyl group, a pentadecenyl group, a hexadecenyl group, a heptadecenyl group, an octadecenyl group, a nonadecenyl group, an eicosenyl group, a heneicosenyl group, a docosenyl group, a dodecadienyl group, a tridecadienyl group The aliphatic hydrocarbon group having 12 to 22 carbon atoms is optionally a tridecyl group, a pentadecyl group, a heptadecadienyl group, a nonadecadienyl group, an eicosadienyl group, a hexadienyl group, a docosadienyl group, an octadecadienyl group, an eicosadienyl group, a hexadienyl group, a docosadienyl group, an octadecatrienyl group, an eicosatrienyl group, an eicosatetraenyl group, an eicosapentaenyl group, a docosahexenyl group, an isostearyl group, a 1-hexylheptyl group, a 1-hexylnonyl group, a 1-octylnonyl group, a 1-octylundecyl group, a 1-decylundecyl group, etc. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is optionally a tridecyl group, a pentadecyl group, a heptadecadecyl group, a nonadecadecyl group, a heptadecaenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, or a tridecyl group, a heptadecadecyl group, a heptadecaenyl group, and a heptadecadienyl group.

[0215] In one embodiment of the present disclosure, R 3a and R 3b The aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by 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.

[0216] R 3a Can be used with R 3b Different or the same.

[0217] In one embodiment of the present disclosure, R 1a With R 1b Same, X a With X b Same, R 2a With R 2b Same, Y a With Y b The same, and Z a With Z b Same, R 3a With R 3b same.

[0218] Preferred examples of the ionizable lipid represented by formula (1) include the following ionizable lipids: ionizable 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 alkylene tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (e.g., -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (e.g., a piperidinyl group); R 2a and R 2b are each independently an alkylene group having 8 or less carbon atoms (eg, a methylene group, an ethylene group, a propylene group); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each is independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms, having at least one aromatic ring and optionally having a heteroatom. (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); R 3a and R 3b Each is independently a residue derived from the reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., tocopherol) and succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group);

[0219] Ionizable lipids (1-2); R 1a and R 1b Each is independently an alkylene group having 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 having 1 to 3 carbon atoms and 1 tertiary amino group (e.g., -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 tertiary amino group (e.g., a piperidinyl group); R 2a and R 2bare each independently an alkylene group having 6 or less carbon atoms (eg, a methylene group, an ethylene group, a propylene group); Y a and Y b Each is 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 having a heteroatom (e.g., -C6H4-CH2-, -CH2-C6H4-CH2-); R 3a and R 3b Each is independently a residue derived from a 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., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group).

[0220] Ionizable lipids (1-3); R 1a and R 1b are each independently an alkylene group having 1 to 2 carbon atoms (eg, a methylene group, an ethylene group); X a and X b Each independently is X 1 :

[0221]

[0222] Where R 5 is an alkyl group having 1 to 3 carbon atoms (eg, a methyl group), or X 2 :

[0223]

[0224] Where p is 1 or 2), R 2a and R 2b are each independently an alkylene group having 4 or less carbon atoms (eg, a methylene group, an ethylene group, a propylene group); Y a and Y b Each is independently an ester bond or an amide bond; Z a and Z b Each independently is Z 1 :

[0225]

[0226] wherein s is an integer from 0 to 1, t is an integer from 0 to 2, u is an integer from 0 to 2 (optionally 0), and (R 4 )u each independently represents a substituent. 3a and R 3bEach is 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 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group);

[0227] Specific examples of ionizable 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 following table.

[0228] Ionizable lipids

[0229]

[0230]

[0231] Ionizable lipids

[0232]

[0233] The lipids having the structure of Formula I are shown in the table below. For example, SS-OP is also known as O-Ph-P4C2. As used herein, the term "SS-OP analog" refers to a compound of Formula I.

[0234] Lipid nomenclature

[0235]

[0236] Cationic lipids

[0237] In some embodiments, LNP of the present disclosure comprises cationic lipid, such as DOTAP or its variant. Cationic lipid can be "permanent cationic lipid". The term cationic lipid can be cationic in the pH range present in mammalian physiological environment such as blood or interstitial fluid. Cationic lipid can be composed of cationic amine part and lipid part, and cationic amine part and polyanionic nucleic acid can interact to form positively charged liposome or lipid membrane structure. Therefore, it is possible to promote uptake into cells and deliver nucleic acid into cells.

[0238] In some embodiments, cationic lipids can be selected from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylammonium bromide (DABB) or 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) One or more. In some embodiments, LNPs contain ionizable lipids, wherein the ionizable lipids are one or more of the following: N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxysperminylglycine dioctadecylamide (DOGS), 2,3-dioleoyloxy-N-[2 (spermine-formylamino)ethyl]-N,N-dimethyl-1-propylammonium (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 11,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA) 、1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyldioctadecyl ammonium (DDA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DM RIE), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5′-(cholest-5-ene-3-β-oxy)-3′-oxapentyloxy)-3-dimethyl1-1-(cis, cis-9′,1-2′-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbD AP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane or (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA) and mixtures thereof.

[0239] In some embodiments, cationic lipid refers to cationic cholesterol lipid. In some embodiments of the present disclosure, LNP comprises imidazole cholesterol ester (ICE). In some embodiments, ICE structure is substantially similar to:

[0240]

[0241] In some embodiments of the present disclosure, LNP comprises 25-hydroxycholesterol (25OH Chol). In some embodiments, the 25OH Chol structure is substantially similar to:

[0242]

[0243] In some embodiments of the present disclosure, the LNP comprises 20α-hydroxycholesterol 5-cholestene-3α.

[0244] In some embodiments, 20α-hydroxycholesterol 5-cholestene-3α (also known as 20α-diol or 20αchol structure) is substantially similar to:

[0245]

[0246] In some embodiments, the cationic lipid refers to dimethyldioctadecyl ammonium bromide (DDAB). In some embodiments of the present disclosure, LNP comprises dimethyldioctadecyl ammonium bromide (DDAB). In some embodiments, the structure of dimethyldioctadecyl ammonium bromide (DDAB) is substantially similar to:

[0247]

[0248] Structured lipids

[0249] In some embodiments, LNP comprises structural lipid.As used herein, structural lipid is as the supplement of electric charge or is independent of the lipid that contributes physical or chemical properties to LNP.For example, structural lipid can tend to have the shape, size, rigidity, hydrophobicity or other characteristics that increase the therapeutic utility of LNP, such as by increasing its stability, half-life, deformability, transfection efficiency, tropism, thermostability, anti-aggregation, membrane fluidity or other parameters.In some embodiments, structural lipid is charge neutral, this is due to lack of charged part, or due to being zwitterionic, has the balance charge that amounts to zero net charge.

[0250] In some embodiments, the LNP may include one or more structural lipids 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 (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE) or variants thereof.

[0251] In some embodiments, LNP may include one or more phosphatidyl lipids, such as phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine). In some embodiments, LNP may include sphingolipids, such as but not limited to sphingosine, ceramide, sphingomyelin, cerebrosides and gangliosides. In some embodiments, the aforementioned "structural" lipid contributes to the stability and / or specificity of the LNP composition.

[0252] Cholesterol-based lipids

[0253] In some embodiments, LNP may include one or more cholesterol-based lipids. Cholesterol-based lipids may include, but are not limited to, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylformylamino cholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine.

[0254] PEGylated lipids

[0255] In some embodiments of the present disclosure, LNP may include one or more PEGylated lipids. For example, phospholipids and derived lipids such as derived ceramides (PEG-CER) modified with polyethylene glycol (PEG), including the use of N-octanoyl-sphingosine-l-[succinyl (methoxy polyethylene glycol)-2000] (C8 PEG-2000 ceramide) are considered by the present disclosure to be combined with one or more ionizable lipids and / or other lipids. In some embodiments, the PEGylated lipid includes a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18). In some embodiments, the PEGylated lipid DSPE-PEG-maleimide-lectin can be used. Other PEGylated lipids considered include, but are not limited to, polyethylene glycol chains up to 5kDa in length covalently attached to lipids with an alkyl chain with a length of C6-C20. Without wishing to be bound by a particular theory, it is contemplated that the addition of PEGylated lipids prevents aggregation of the complexes and increases circulation lifetime to facilitate delivery of liposome-encapsulated mRNA to target cells.

[0256] Treatment

[0257] Methods of treatment as described herein refer to extending telomeres in the lungs of subjects in need 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 diseases in lungs or lung cells or diseases involving lungs or lung cells. In some embodiments, the compositions of the present disclosure and / or the methods of use of the compositions are intended to be used to treat fibrotic diseases (including fibrosis). In some embodiments, the compositions of the present disclosure and / or the methods of use of the compositions intended to be used to treat fibrotic diseases (including fibrosis) induce the expression of encapsulated mRNA in lung tissue. In some embodiments, the compositions of the present disclosure and / or the methods of use of the compositions intended to be used for treatment do not induce cell, tissue or systemic toxicity. The composition can be administered systemically, such as intravenously. In some embodiments, the compositions of the present disclosure and / or the methods of use of the compositions intended to be used to treat diseases in lung tissue cells or diseases involving lung tissue cells do not induce cell or systemic toxicity. The composition can be administered systemically, such as intravenously.

[0258] 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 to about 2.0 mg / kg of subject body weight. In some embodiments, targeted LNP is administered to a subject in need at a dosage of about 0.01 mg / kg; in some embodiments at a dosage of about 0.025 mg / kg; in some embodiments at a dosage of about 0.05 mg / kg; in some embodiments at a dosage of about 0.075 mg / kg; in some embodiments at a dosage of about 0.1 mg / kg; in some embodiments at a dosage of about 0.125 mg / kg; in some embodiments at a dosage of about 0.150 mg / kg; in some embodiments at a dosage of about 0.175 mg / kg; in some embodiments at a dosage of about 0.2 mg / kg; in some embodiments at a dosage of about 0.5 mg / kg; in some embodiments at a dosage of about 0.75 mg / kg; in some embodiments at a dosage of about 1.0 mg / kg; in some embodiments at a dosage of about 1.25 mg / kg; in some embodiments at a dosage of about 1.5 mg / kg; or in some embodiments at a dosage of about 2.0 mg / kg. In some embodiments, LNP is administered to a subject in need at a dosage of 0.1 mg / kg. In some embodiments, LNP is administered to a subject in need thereof at a dose of 0.125 mg / kg.

[0259] In some embodiments, the LNP pharmaceutical compositions contemplated herein are administered to a subject in need in a single dose. In some embodiments, the LNPs are administered to a subject in need two, three, four, or five times or more. In some embodiments, the LNPs are administered twice a week, once a week, once every two weeks, once every four weeks, once every six weeks, once every twelve weeks, or once every fifteen weeks. In some embodiments, the LNPs are administered once a month, once every two months, once every three months, once every six months, once a year, continuously or as determined by their physician.

[0260] In some embodiments, the contemplated LNP pharmaceutical compositions are delivered in the form of aerosol inhalation, orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In an exemplary embodiment, the targeted LNP is administered intravenously.

[0261] In some embodiments, exemplary LNP pharmaceutical compositions include excipients or carriers, such as aqueous carriers. A variety of aqueous carriers can be used, such as buffered saline. The composition may contain pharmaceutically acceptable auxiliary substances, such as those required for close physiological conditions, such as pH and buffers, toxic antagonists, such as sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride and sodium lactate. In some embodiments, the pharmaceutical composition includes 10mM sodium citrate buffered to pH 6.4. The composition may contain cryoprotectants, such as glycerol, ethylene glycol, sucrose, propylene glycol or dimethyl sulfoxide (DMSO). The concentration of the active agent in these preparations can vary, and is selected based on fluid volume, viscosity and body weight according to the selected specific mode of administration and the needs of the patient (e.g., Remington's Pharmaceutical Science (15th edition, 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., 1996)).

[0262] In other embodiments, the compound or composition is administered without separating one or more cells, tissues or organs from the subject (i.e., administration is in vivo). In some of these embodiments, the compound or composition is delivered to all or nearly all cells in the subject. In some embodiments, the compound or composition is delivered to specific cells, cell types, tissues or organs in the subject.

[0263] Administration of a compound or composition of the present disclosure can result in transient expression of mRNA within the LNP in the cell. The increased activity can be measured by various assays, such as, for example, the telomerase repeat amplification protocol (TRAP) assay. Commercial versions of the TRAP assay are available, for example Telomerase Detection Kit (Millipore), which provides sensitive detection and quantification of telomerase activity, but other measurement techniques are possible.

[0264] As previously described, one advantage of the technology of the present invention is that the expression of mRNA in LNP is transient in transfected cells. Specifically, this transient expression is in contrast to previous technologies, in which, for example, the telomerase reverse transcriptase gene persists in the free DNA fraction, or is inserted into the genomic sequence of the cell, or otherwise permanently modifies the genetic composition of the target cell and results in the intrinsic activity of the nucleic acid sequence.

[0265] In some embodiments of the methods of the invention, transient expression is independent of the cell cycle.

[0266] Treatment kit

[0267] Also contemplated herein are therapeutic kits comprising pharmaceutical compositions of the LNPs contemplated herein, or lipid components thereof provided in separate containers, and instructions for manufacture and / or use. In some embodiments, the therapeutic kit includes a device for administration, including but not limited to a syringe, an inhaler, a nebulizer, and a vial or container. In some embodiments, components of exemplary therapeutic kits are provided so that they can be mixed with commercially available microfluids or vortex mixers. Such kits are typically with or without nucleic acids (e.g., mRNA), which will be encapsulated in the LNPs made with the components of the therapeutic kit.

[0268] On the other hand, the present disclosure provides a ready-to-use kit for extending telomeres in mammalian cells. The kit includes any of the above compounds or compositions, and their instructions for use. In some embodiments, the kit also includes packaging material. In some embodiments, the packaging material is airtight. In these embodiments, the packaging material can be optionally filled with an inert gas, such as, for example, nitrogen, argon, etc. In some embodiments, the packaging material includes a metal foil container, such as, for example, a sealed aluminum bag, etc. This packaging material is well known to those of ordinary skill 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 preparation are configured to be frozen or lyophilized together with a cryoprotectant.

[0269] In some embodiments, the kit may also include a desiccant, culture medium, RNase inhibitor or other such components. In some embodiments, the kit may also include a combination of more than one of these additional components. In some kit embodiments, the composition of the kit is sterile.

[0270] Implementation

[0271] In embodiment 1, a method for delivering a polynucleotide to a subject is provided, comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) by intravenous injection, wherein the lipid nanoparticle comprises: a cationic lipid at a molar percentage between about 25% and about 60%, a ratio of ionizable lipid to cationic lipid between 0.7 and 1.8, and the ionizable lipid comprises any of the following: (a) SS-OP or an SS-OP analog at a molar percentage between about 25% and about 60%, wherein the LNP has a lipid nitrogen:polynucleotide phosphate ratio (N / P ratio) between 10 and 30, or (b) DLin-MC3-DMA or a DLin-MC3-DMA analog at a molar percentage between 30% or about 30% and 50% or about 50%, wherein the LNP has an N / P ratio between 3.5 and 10.

[0272] In embodiment 2, a method of embodiment 1 is provided, wherein the polynucleotide comprises a synthetic RNA, the mRNA is translated into a corresponding protein encoded by the mRNA in the subject upon or after administration of the LNP, and the half-life of the lipid DOTAP in the subject is: a) at least 30 hours in the lungs of the subject; b) no more than 11 hours in the liver of the subject; and / or c) no more than 9 hours in the spleen of the subject.

[0273] In embodiment 3, the method of embodiment 2 is provided, wherein the protein has an in vivo half-life in the lungs of a subject of at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours or 35 hours.

[0274] In embodiment 4, a method of any one of embodiments 1 to 3 is provided, wherein the polynucleotide comprises a synthetic RNA, the mRNA is translated into a corresponding protein encoded by the mRNA in the subject upon or after administration of the LNP, and wherein the lung:liver ratio of the amount of protein per mg of tissue is at least 2:1, 3:1 or 4:1 6 hours or about 6 hours after intravenous infusion of the LNP.

[0275] In embodiment 5, the method of any one of embodiments 1 to 4 is provided, wherein the lung:liver ratio of the amount 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.

[0276] In embodiment 6, the method of any one of embodiments 1 to 5 is provided, wherein the polynucleotide comprises a synthetic RNA encoding a telomerase reverse transcriptase (TERT), and upon or after administration of the LNP, the mRNA is translated into a corresponding TERT protein in the subject.

[0277] In embodiment 7, the method of any one of embodiments 1 to 6 is provided, wherein the median telomere length of the alveolar cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%.

[0278] In embodiment 8, the method of any one of embodiments 1 to 7 is provided, wherein the median telomere length of lung AT2 cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%.

[0279] In embodiment 9, the method of any one of embodiments 1 to 8 is provided, wherein the increase in telomerase activity in the lung is detectable by a TRAP assay for at least 24 or 48 hours.

[0280] In embodiment 10, the method of any one of embodiments 1 to 9 is provided, wherein the LNP has an N / P ratio between 28 and 31.

[0281] In embodiment 11, the method of embodiment 10 is provided, wherein the LNP has an N / P ratio of at or about 29.1.

[0282] In embodiment 12, the method of any one of embodiments 1 to 9 is provided, wherein the LNP has an N / P ratio between 11 and 13.

[0283] In embodiment 13, the method of embodiment 12 is provided, wherein the LNP has an N / P ratio of 12 or about 12 or at or about 12.1.

[0284] In embodiment 14, the method of any one of embodiments 1 to 9 is provided, wherein the LNP has an N / P ratio between 4 and 5.

[0285] In embodiment 15, the method of embodiment 14 is provided, wherein the LNP has an N / P ratio of at or about 4.5.

[0286] In embodiment 16, a method according to any one of embodiments 1 to 11 is provided, wherein the ionizable lipid is SS-OP and the LNP has a ratio of cationic lipid to ionizable lipid of at or about 1.8 and consists of 5 lipids.

[0287] In embodiment 17, a method according to any one of embodiments 1 to 11 is provided, wherein the ionizable lipid is SS-OP and the LNP has a ratio of cationic lipid to ionizable lipid of at or about 1.8 and is composed of 5 lipids, including DOTAP and DOPC.

[0288] In embodiment 18, a method according to any one of embodiments 1 to 11 is provided, wherein the ionizable lipid is SS-OP and the LNP has a ratio of cationic lipid to ionizable lipid of 1.8 or about 1.8 and is composed of 5 lipids, including DMG-PEG2000, DOPC and DOTAP.

[0289] In embodiment 19, a method according to any one of embodiments 1 to 9 or 12-13 is provided, wherein the ionizable lipid is SS-OP and the LNP has a ratio of cationic lipid to ionizable lipid of 0.8 or about 0.8 and consists of 3 or 5 lipids.

[0290] In embodiment 20, a method according to any one of embodiments 1 to 9 or 12-13 is provided, wherein the ionizable lipid is SS-OP and the LNP has a ratio of cationic lipid to ionizable lipid of 0.8 or about 0.8 and consists of 3 or 5 lipids, including DOTAP.

[0291] In embodiment 21, a method of any one of embodiments 1 to 9 or 12-13 is provided, wherein the ionizable lipid is SS-OP and the LNP has a ratio of cationic lipid to ionizable lipid of 0.8 or about 0.8 and is composed of 3 or 5 lipids, including DMG-PEG2000 and DOTAP.

[0292] In embodiment 22, a method according to any one of embodiments 1 to 9 or 14-15 is provided, wherein the ionizable lipid is DLin-MC3-DMA and the LNP has a ratio of cationic lipid to ionizable lipid of at or about 1.5 and is composed of 3 lipids.

[0293] In embodiment 23, a method according to any one of embodiments 1 to 9 or 14-15 is provided, wherein the ionizable lipid is DLin-MC3-DMA and the LNP has a ratio of cationic lipid to ionizable lipid of 1.5 or about 1.5 and is composed of 3 lipids, including DOTAP.

[0294] In embodiment 24, a method according to any one of embodiments 1 to 9 or 14-15 is provided, wherein the ionizable lipid is DLin-MC3-DMA, and the LNP has a ratio of cationic lipid to ionizable lipid of 1.5 or about 1.5 and is composed of 3 lipids, including DMG-PEG2000 and DOTAP.

[0295] In embodiment 25, a method according to any one of embodiments 1 to 9 or 14-15 is provided, wherein the ionizable lipid is DLin-MC3-DMA, and the LNP has a ratio of cationic lipid to ionizable lipid of 1.5 or about 1.5 and is composed of 3 lipids, including DMG-PEG2000 and DOTAP, and the N / P ratio is about 4.5.

[0296] In embodiment 26, the method of any one of claims 1 to 15 is provided, wherein the LNP is selected from the LNPs of Table 8.

[0297] In embodiment 27, the LNP of any one of embodiments 1 to 26 comprises a nucleic acid, such as ribonucleic acid or deoxyribonucleic acid.

[0298] In embodiment 28, the ribonucleic acid of embodiment 27 comprises TERT mRNA.

[0299] In embodiment 29, the ribonucleic acid of any one of embodiments 27 to 28 is transiently expressed in a cell.

[0300] In embodiment 30, the cells of embodiment 29 include lung tissue cells.

[0301] In embodiment 31, the lung tissue cells of embodiment 30 include alveolar cells, such as ST2 cells.

[0302] In embodiment 32, the LNP used in any one of embodiments 1 to 31 is provided.

[0303] In embodiment 33, 2-lipid LNPs comprising DOTAP and DMG-PEG2000 and having an N / P ratio of 4 or about 4 are provided for use in any one of embodiments 1 to 31.

[0304] In embodiment 34, a cell is provided, comprising the LNP of any one of embodiments 1 to 33.

[0305] In embodiment 35, a kit comprising the components of the LNP of any one of embodiments 1 to 34 is provided.

[0306] In embodiment 36, a kit comprising the LNP of any one of embodiments 1 to 34 is provided.

[0307] In embodiment 37, a kit of embodiment 35 or 36 is provided, wherein the kit further comprises a nucleic acid, such as ribonucleic acid or deoxyribonucleic acid.

[0308] In embodiment 38, a method of making the LNP of any one of embodiments 1 to 37, the LNP used in any one of embodiments 1 to 37, or the LNP formed from the components of any one of embodiments 1 to 37 is provided.

[0309] In embodiment 39, the manufacturing method of embodiment 38 is provided, wherein the LNP is formed before the ribonucleic acid or ribonucleic acid is introduced into the solution containing the LNP.

[0310] Example

[0311] Example 1

[0312] Figures 1 and 48 depict exemplary pharmacokinetics of the 5 lipid LNPs of Table 1. A timeline of plasma, lung and liver levels of the 5 lipid LNPs as determined by measuring one of the lipids DOTAP is presented. In the process of processing these data, LNPs were formulated with TERT mRNA (human TERT opti-SEQ ID NO: 1) and lipids according to the ratios of Table 1. Male C57Bl / 6 mice were dosed at 1 mg / kg, and plasma and tissues were collected at the above time points. DOTAP in plasma, lungs and liver was measured using LC-MS / MS. The nitrogen: phosphorus N / P charge ratio of the composition of Table 1 is 12.

[0313] Table 1

[0314] Compound Molar ratio SS-OP 55 DOPC 5 cholesterol 40 DMG-PEG2000 2.5 DOTAP 44

[0315] N / P ratio (nitrogen:phosphorus charge ratio): 12

[0316] Example 2

[0317] Figure 2 The ratio of the bioluminescent signal generated by the luciferase protein translated from the mRNA injected intravenously at the indicated time points after intravenous injection of the 5 lipid LNPs in Table 2 is depicted. In this process, LNPs were formulated with firefly luciferase mRNA and lipids at the ratios in Table 2. Male C57B16 mice were dosed at 1.3 mg / kg and tissues were collected and imaged in vitro at the above time points. The average radiance values ​​were baseline normalized by subtracting the average radiance of animals treated with negative controls (no LNPs). The relative radiance between the lungs, liver, and spleen is plotted on Figure 2 The tissue was also homogenized and bioluminescence was measured from equal weights of the homogenate. The composition of Table 2 had a nitrogen:phosphorus N / P charge ratio of 12.

[0318] Table 2

[0319] Compound Molar ratio SS-OP 55 DOPC 5 cholesterol 40 DMG-PEG2000 2.5 DOTAP 44

[0320] N / P ratio (nitrogen:phosphorus charge ratio): 12

[0321] Example 3

[0322] Figure 3-17 and Figure 20-21 Experiments involving the use of different 5-lipid LNP compositions are described in which pulmonary fibrosis was induced in third generation (G3) TERT knockout (KO) mice, which have telomere lengths similar to humans. Bleomycin was administered at 2.0 U / kg by oropharyngeal inhalation. Figure 13-16, one week before the 2.0U / kg dose, an initial dose of bleomycin was administered by oropharyngeal inhalation at 0.5U / kg. LNPs were formulated with firefly luciferase mRNA (control) or TERT mRNA (mouse SEQ ID NO: 2, including synthetic 5'UTR (SEQ ID NO: 3), wt mTert coding sequence (SEQ ID NO: 4) and mouse α-globin 3'UTR (SEQ ID NO: 5)) (referred to herein as "TERT mRNA LNP" or in the figures as "TERT" or "TERT mRNA") and lipids according to the ratio of Table 3. The DOTAP molar ratio is 2.27 times higher in the 5 lipid LNPs of Table 3. Starting from the 7th day after bleomycin, mice were administered with a dosage of 1.5mg / kg twice a week for two weeks. Some mice received bleomycin, but did not receive LNP treatment (without LNP), and control mice received saline instead of bleomycin and LNP treatment (without bleomycin). Tissue was harvested on the 24th day after bleomycin. The lungs were fixed with paraffin and embedded in formalin. To measure telomere length, sections were stained using the Q-FISH method with a telomere probe and anti-surfactant precursor protein C (SPC) antibody to mark alveolar type II (AT2) cells. Telomerase activity in lung tissue lysates was measured using the telomeric repeat amplification protocol (TRAP) assay.

[0323] Table 3

[0324] Compound Molar ratio SS-OP 55 DOPC 5 cholesterol 40 DMG-PEG2000 2.5 DOTAP 100

[0325] N / P ratio (nitrogen:phosphorus charge ratio): 26.5

[0326] Figure 3 Depicted are representative immunofluorescence micrographs of lung sections used to measure telomere length by the Q-FISH method known in the art, in which the intensity of the telomere probe is proportional to telomere length and cells are co-stained with an antibody to the AT2 cell marker precursor SPC.

[0327] Figure 4 Depicted is the measurement of telomere length by Q-FISH in mouse AT2 cells, where mice had been induced to fibrosis using bleomycin ("Bleo") as described above. Treatment with TERT mRNA LNPs of Table 3 ("TERT" in the figure) increased the median telomere length and the first telomere length of AT2 cells (cells scored as precursor SPC+ by immunofluorescence staining) as measured by Q-FISH relative to mice receiving luciferase mRNA LNPs (referred to as "control" in the figure). 10 The percentile telomere length almost reached the length found in normal mice that did not receive bleomycin ("No Bleomycin" in the figure).

[0328] Figure 5 Describe the measurement of telomere length in mouse AT2 cells by Q-FISH, wherein mice have been induced to fibrosis using bleomycin (" Bleo ") as described above. Compared with mice receiving luciferase mRNA LNP (referred to as " control " in the figure), treatment with TERT mRNA LNP (" TERT " in the figure) in Table 3 increased the median telomere length and the 10th percentile telomere length of alveolar cells as measured by Q-FISH, almost reaching the length found in normal mice that do not receive bleomycin (" without bleomycin " in the figure). The visual field that is mainly filled with alveoli is used for quantitative, and includes the telomere length of all cell types present. Typically, alveolar tissue includes epithelial cells, endothelial cells, fibroblasts, immune cells and other cell types.

[0329] Fig. 6A Depicted is a timeline of the bleomycin mouse experiment described above. "CT" indicates that the mice were subjected to X-ray computed tomography to quantify normal ventilated lung volumes.

[0330] Figure 6B Depicted Figure 13-16 Timeline of the bleomycin mouse experiments.

[0331] Figure 7 Depicted are false color images of lung sections stained with Sirius red for quantification of fibrotic lesions and fibrosis by tissue area covered by collagen deposition. IV infusion of TERT mRNA LNPs of Table 3 reduced fibrosis by 62%.

[0332] Figure 8 Depicted is the reduction of fibrotic lesions caused by TERT mRNA LNP treatment of Table 3 in bleomycin-treated mice as measured by Sirius red staining.

[0333] Fig. 9 Depicted are the increase in normal ventilated lung ratio or amount of useful lung capacity caused by TERT mRNA LNP treatment of Table 3 in bleomycin-treated mice as measured by x-ray computed tomography imaging followed by quantification of fibrotic tissue based on voxel-based x-ray intensity.

[0334] Fig.10 Depicted are improvements in lung elasticity resulting from TERT mRNA LNP treatment of Table 3 in bleomycin-treated mice as measured by the Flexivent system.

[0335] Fig.11 and 12Depicted are improvements in forced expiratory volume (FEV0.1) and forced vital capacity (FVC) caused by TERT mRNA LNPs of Table 3 in bleomycin-treated mice, as measured by the Flexivent system, respectively.

[0336] Fig.13 , 14 and 15 depict the improvements in alveolar density, alveolar diameter, and alveolar circularity, respectively, caused by TERT mRNA LNP treatment of Table 3 in bleomycin-treated mice as measured by machine vision analysis of lung section micrographs by Biocellvia.

[0337] Fig.16 Depicted by Biocellvia in the analysis Figure 13-15 Example of a false color image of a lung tissue section generated during quantification of alveolar architecture.

[0338] Fig.17 Depicted are the results of measurement of telomerase activity in isolated primary human alveolar epithelial cells from a 50 year old donor at the indicated time points following treatment with TERT mRNA LNPs of Table 3 at a concentration of 500 ng / ml as measured using the Telomere Repeat Amplification Protocol (TRAP) assay.

[0339] Fig.18 Depicted is a timeline of an experiment measuring the effects of TERT mRNA LNP treatment of Table 3 on the colony forming capacity of isolated primary human alveolar epithelial cells from a 50-year-old donor. Cells were seeded in a 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 as Fig.19 Report.

[0340] Fig.19 Depicted is the increase in colony-forming capacity of human alveolar epithelial cells from a 50-year-old donor caused by treatment with TERT mRNA LNPs of Table 3.

[0341] Example 4 - Reduced senescence after telomerase mRNA LNP treatment in mice

[0342] Method: As about Figure 3-12 The experiments were performed as described.

[0343] Results: Figure 20-21 As shown, the senescence marker P21 was reduced by 40% in alveolar cells of mice treated with telomerase mRNA LNPs compared to mice treated with luciferase mRNA LNPs (control). Fig.21 Dark cells are positive. Senescence increases after bleomycin treatment.

[0344] Example 5 - Efficient lung delivery using 2 lipid LNPs

[0345] Method: According to Fig. 22C 2 lipid LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1.25 mg / kg via intravenous injection (IV) to C57B1 / 6 male mice, and organs were imaged ex vivo 21 hours later. Shown in the figure are the average radiance (photons / s / cm 2 / sr). Fig.22A -B, D.

[0346] Results: Bioluminescent signals were visible in the lungs with all formulations, indicating that: 1. Toxicity at low PEG concentrations - one mouse died at 0.1% PEG, suggesting the optimal amount may be between 0.2-2% PEG. 2. The signal peaked at 0.5% PEG.

[0347] Example 6 - Titration of lipid:mRNA ratio in 2-lipid LNPs

[0348] 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; the ratio of nitrogen to phosphate molecules. Each DOTAP molecule has a single nitrogen, while each nucleoside on the mRNA has one phosphate. C57Bl / 6 male mice were dosed at 1.5 mg / kg via intravenous injection (IV) and lungs were removed 23 hours later for ex vivo imaging. Fig.23 The average radiance (photons / s / cm 2 / sr).

[0349] Results: Bioluminescent signals were seen in the lungs at all N / P ratios. Increased lung signal corresponded to increased weight loss - the more signal, the greater the toxicity. 20 Nitrogen:Phosphorus ratio (N / P) was a local minimum for signal and weight loss

[0350] Example 7 - Time course of protein expression following delivery of lung-targeted mRNA-LNPs encoding firefly luciferase

[0351] Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Provisional Table 3. C57Bl / 6 male mice were dosed at 1.5 mg / kg via intravenous injection (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"). Fig.24 The average radiance (photons / s / cm 2 / sr).

[0352] Results: Given the known half-life of the firefly luciferase protein, the bioluminescent signal declined over time, suggesting that the mRNA half-life is not long and that the mRNA-LNPs do not circulate in the lungs and re-transfect cells over time.

[0353] Example 8 - Titration of SS-OP lipids into "DOTAP+PEG" LNPs

[0354] Method: According to Fig.25D LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1.5. C57B1 / 6 male mice were dosed via intravenous injection (IV) at 1.5 mg / kg and organs were imaged ex vivo 22 hours later. Fig.25A The average radiance (photons / s / cm 2 / sr).

[0355] result:

[0356] 1. Encapsulation efficiency is 98% or higher as measured by Ribo-Green microplate reader assay. Fig.25B

[0357] 2. Stable body weight indicates low or no toxicity. Fig.25C

[0358] 3. Lung radioactivity peaks at 50% SS-OP lipids

[0359] Local maximum at 4.5% SS-OP

[0360] Example 9 - Delivery to the lung and liver using 2 LNPs

[0361] 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 dosed at 1.0 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 19 hours later. Conditions:

[0362] · Lung-targeted LNPs are administered only “lung only”

[0363] Liver-targeted LNPs "Liver Only"

[0364] Lung-targeted LNPs were administered first, followed by liver-targeted LNPs 1 hour later (1) Lung (2) Liver

[0365] ·First, administer liver-targeted LNP, and 1 hour later administer lung-targeted LNP "(1) liver (2) lung"

[0366] Mix lung-targeted LNP and liver-targeted LNP and administer twice, 1 hour apart "liver / lung mixture"

[0367] · Fig.26A and 26B Provides the average radiance (photons / s / cm 2 / sr).

[0368] Table 4

[0369] Compound Mole % SS-OP 54.2％ DOPC 4.9％ cholesterol 39.4％ DMG-PEG2000 1.5％

[0370] result:

[0371] 1. In the case of lung-targeted LNP administration, the bioluminescent signal in the lung was consistent. The highest was in the liver / lung mixture. Fig.26A

[0372] 2. Liver BLI signal was variable, but very low when lung LNPs were administered first. Fig.26B .

[0373] 3. The best order for these two organs is lung first, then liver. The second best option is to mix and deliver.

[0374] Example 10 - Titration of SS-OP lipids into "DOTAP+PEG" LNPs

[0375] Method: According to Fig.27A LNPs were formulated with firefly luciferase mRNA (Luc) at the ratios shown in the table. C57B1 / 6 male mice were dosed at 1.5 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 23 hours later. Fig.27C The average radiance (photons / s / cm 2 / sr).

[0376] Body weight was measured and clinical signs were assessed using a scoring system that included a visual assessment of the appearance and behavior of the mice, with particular attention to locomotion, posture, and eyes (abbreviated “LPE”) (Nunamaker 2013, PMID: 24209966). Fig.27D .

[0377] result:

[0378] 1. When the ratio of SS-OP lipids increased to 75%, tolerance decreased and both mice needed to be euthanized.

[0379] 2. Combined with the data of Example 7, this study shows that 35-65% SS-OP provides relatively lung-specific transfection (as measured by bioluminescent signal) and relatively high tolerability (<10% body weight, no clinical signs (0LPE), no death) compared to other percentages of SS-OP. 55% SS-OP scores particularly well and can be considered optimal in this regard.

[0380] Example 11 - Stability under frozen storage - long term

[0381] Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. They were formulated and administered "fresh" on the same day, or stored frozen at -20C, -80C 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 via intravenous injection (IV) and organs were imaged ex vivo 23 hours later. Fig.28A The average radiance (photons / s / cm 2 / sr)+ / -SEM.

[0382] Results: No clinical signs and slight weight loss were observed. Fig.28B In all cases, the lung signal was present in similar amounts, indicating that cryopreservation was effective.

[0383] Example 12 - Stability under cryopreservation - short term

[0384] Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. They were formulated and administered "fresh" on the same day, or stored frozen at 4C, -20C, -80C or in liquid nitrogen and thawed after 3 days. Sucrose was added to 15% as a cryoprotectant before freezing, except for the "4C no sucrose" condition. CD1 female mice were dosed at 0.4 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 23 hours later. Fig.29A The average radiance (photons / s / cm 2 / sr).

[0385] Results: In stored conditions, the lung signal was lower than the "fresh" signal. Based on particle size measured with dynamic light scattering, 4C had sucrose and -20 was out of range (see next slide). Fig.29B .

[0386] Example 13 - Titration of N / P ratio and comparison of 2, 3 and 5 lipid formulations

[0387] Method: According to Fig. 30ALNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1.5 mg / kg to C57B1 / 6 male mice via intravenous injection (IV), and organs were imaged ex vivo 23 hours later. Fig. 30B The average radiance (photons / s / cm 2 / sr).

[0388] result:

[0389] 1. The average signal of the 3 and 5 lipid (SS and "5 lipid") formulations is higher than that of the 2 lipid (1% PEG) formulation

[0390] 2. Among the lipid preparations 3 and 5, 10N / P is the best

[0391] 3. For 2 lipids, 15NP is better than 10NP

[0392] 4. Given the high radiative yield and less than 10% weight loss, all formulations showed promise as lung-targeted LNPs, with the exception of the low activity of 2-lipid 10N / P. Fig. 30C .

[0393] Example 14 - Titration of N / P Ratio (mRNA to Lipid Ratio) Using 3 and 5 Lipid Formulations

[0394] Method: According to Fig.31A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1.5. Bl / 6 male mice were dosed via intravenous injection (IV) at 1.5 mg / kg and organs were imaged ex vivo 21 hours later. Fig. 31C The average radiance (photons / s / cm 2 / sr).

[0395] result:

[0396] 1. All three lipid preparations at 3 and 6 N / P conditions were significantly toxic

[0397] 2. For 5-lipid lung-targeted LNPs, the encapsulation efficiency began to drop below 8N / P. Fig.31B

[0398] 3. In this study, considering the above considerations, lipids 3 and 5 with 10N / P are generally superior

[0399] Example 15 - Testing vortexing and microfluidic mixing for mRNA-LNP formulations

[0400] Methods: LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. Balb c / J male mice were dosed at 0.1 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 21 hours later. Fig.32AThe average radiance (photons / s / cm 2 / sr).

[0401] result:

[0402] 1. LNPs produced by vortex-mixed mRNA-LNPs produced high BLI signals in the lungs and had slightly larger particle sizes (113 nm vs. 86 nm). Fig.32B .

[0403] 2. The encapsulation efficiency of LNPs produced by vortex-mixed mRNA-LNPs was 63%, much lower than the 94% of the microfluidic-mixed counterparts. Fig.32B .

[0404] Example 16 - Titration of NP ratios of 5 lipid mixtures, comparison of input concentrations

[0405] Method: According to Fig.33A LNPs were formulated with firefly luciferase mRNA (Luc) at the ratios shown in the table. C57B1 / 6 male mice were dosed at 1.5 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 21 hours later. Fig.33B The average radiance (photons / s / cm 2 / sr).

[0406] For the conditions labeled "dil", the mRNA and lipids were diluted to 30% of their original values ​​in buffer before mixing, but the ratios of the components remained the same. The input mRNA concentration was changed from 0.147 mg / ml to 0.44 mg / ml.

[0407] result:

[0408] 1. Reducing the DOTAP molar percentage from 49% to 30% increased the signal in the 5 lipid preparation by 30%

[0409] 2. Encapsulation efficiency of all formulations exceeded 90%, and all mice showed no movement, posture, or eye abnormalities (LPE score)

[0410] Example 17 - Titration of NP ratios of 5-lipid mixtures; comparison of 5, 3, 2 lipid LNP formulations

[0411] Method: According to Fig.34A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1.5 mg / kg to BALB / cJ male mice via intravenous injection (IV), and organs were imaged ex vivo 20 hours later. Fig.34B The average radiance (photons / s / cm 2 / sr). This formulation used 5 lipids, except for "3 lipid 12NP" and "1% PEG 15NP".

[0412] Results: The encapsulation efficiency of all formulations was ≥90%. Fig.34C For both 3 and 5 lipid lung-targeted LNPs, the highest signal was observed in the case of 12NP.

[0413] Example 18 - Testing 5 Different Total Flow Rates of Lipid Formulations

[0414] Method: According to Fig.35A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1. C57B1 / 6 male mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 17 hours later. Fig.35C The average radiance (photons / s / cm 2 The formulation was the same, the variable changed was the flow rate through the PrecisionNanosystems Nanoassemblyr microfluidic mixing platform.

[0415] Results: All three flow rates had high bioluminescent signals, encapsulation efficiency >95%, and no clinical signs (LPE=0, no mortality). Fig.35B The optimal flow rate is 12 ml / min.

[0416] Example 19 - Test 3 Different Total Flow Rates of Lipid Formulations ("PDS")

[0417] Method: According to Fig.36A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1.5 mg / kg to BALB / cJ male mice via intravenous injection (IV), and organs were imaged ex vivo 26 hours later. Fig.36B The average radiance (photons / s / cm 2 / sr). The total flow rate was varied, but the ratio of mRNA (aqueous) to lipid (organic) flow was kept constant at 3:1. Therefore, the flow rate of the mRNA flow was the total flow rate multiplied by 0.75, and the flow rate of the lipid flow was the total flow rate multiplied by 0.25.

[0418] result:

[0419] 1. Bioluminescent signal was high for all 3 flow rates, encapsulation efficiency was greater than 98%, and weight loss was less than 5% ( Fig.36B and 36C ).

[0420] Example 20—Replacing SS-OP in 3-lipid LNPs with a different ionizable lipid, DLin-MC3-DMA ("MC3"). Titrate the MC3 to DOTAP ratio from 9% to 50% MC3.

[0421] Method: According to Fig.37A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of . Fig.37B The encapsulation percentage of LNPs is provided in. BALB / cJ female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later. Fig.37C The average radiance (photons / s / cm 2 / sr).

[0422] result:

[0423] 1. Comparable bioluminescent signals were observed in the lungs for all conditions, and the signals showed a decreasing trend as the MC3 concentration decreased. Fig.37C .

[0424] 2. The lowest MC3 concentration (3-MC3 0.1) and total MC3 of 9% resulted in death. Fig.37B .

[0425] 3. As the concentration of MC3 increased, the weight loss showed an upward trend, with the highest average weight loss at 50% MC3. Fig.37D .

[0426] Example 21 - Replacement of the ionizable lipid SS-OP with DLin-MC3-DMA in 3-lipid LNPs.

[0427] Method: According to Fig.38A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1. MC3 is the abbreviation for DLin-MC3-DMA. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 26 hours later. Fig.38C The average radiance (photons / s / cm 2 / sr).

[0428] result:

[0429] 1. Deaths occurred in LNPs containing 70.5% 3-MC, indicating poor tolerability.

[0430] 2. Encapsulation efficiency was >95% under all conditions. Fig.38B

[0431] 3. Mean lung radiances were comparable, with the LNP formulation containing 33 mol% MC3 showing the highest signal on average.

[0432] Example 22—Replacing the ionizable lipid SS-OP with DLin-MC3-DMA in 5-lipid LNPs.

[0433] Method: According to Fig.39ALNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1. MC3 is the abbreviation for DLin-MC3-DMA. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 26 hours later. Fig.39C The average radiance (photons / s / cm 2 / sr).

[0434] result:

[0435] 1. Encapsulation efficiency was >95% under all conditions. Fig.39B .

[0436] 2. Under the 5-lipid condition containing SS-OP, the mean lung radiance was at least 7.2 times higher than that of any LNP containing DLin-MC3-DMA. Fig.39C .

[0437] Example 23 - Titration 2 Time for mRNA to be adsorbed to the formed LNPs in lipid formulation

[0438] Methods: DOTAP and DMG-PEG2000 were mixed with malic acid at a molar ratio of 100:1 by microfluidics, and then the buffer was exchanged to 0.1 M sodium acetate and concentrated. The lipid concentration was measured by HPLC with UV detection, and then firefly luciferase mRNA (Luc) was added to the lipids to give a final N / P ratio of 15. mRNA was adsorbed before adding PBS. Fig.40A CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 24 hours later. Fig.40A The average radiance (photons / s / cm 2 / sr).

[0439] result:

[0440] 1. The strongest lung signal was observed under 30-minute incubation conditions

[0441] 2. Body weight loss did not exceed 5% in any animal and no mortality was observed, indicating tolerance. Fig.40B

[0442] For all conditions, the encapsulation efficiency was greater than 98%.

[0443] Example 24 - Titration of mRNA:lipid ratio of 2 lipid LNPs

[0444] Methods: DOTAP and DMG-PEG2000 were mixed with malic acid at a molar ratio of 100:1 by microfluidics, and then the buffer was exchanged to 0.1 M sodium acetate and concentrated. The lipid concentration was measured by HPLC, and then firefly luciferase mRNA (Luc) was added to the lipids to achieve Fig.41A N / P ratio shown. Fig.41B The encapsulation percentage for each composition is provided in. mRNA was allowed to adsorb for 30 minutes before adding PBS. C57BL / 6 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 27 hours later. Fig.41A The average radiance (photons / s / cm 2 / sr).

[0445] result:

[0446] 1. LNPs with NP 2, not shown, did not produce enough mRNA for dosing.

[0447] 2. Lung bioluminescence signals are comparable between different conditions

[0448] Example 25 - Titration of the N / P ratio of 3 lipid LNPs

[0449] Methods: LNP "PDS" was formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 6. Fig.42B The encapsulation percentage of each composition is provided in the Figure. 3-MC3 LNPs were formulated with 39.6% MC3, 59.4% DOTAP and 1% DMG-PEG2000 molar percentages at the N / P ratios listed in the Figure. C57BL / 6 male mice were dosed at 2.0 mg / kg by intravenous injection (IV), and organs were imaged ex vivo 20 hours later. Fig.42A The average radiance (photons / s / cm 2 / sr).

[0450] Table 6

[0451]

[0452] result:

[0453] 1. Deaths occurred in LNPs containing MC3 at an N / P ratio of 20, and clinical signs (slowed movements and hunched posture) were seen in MC3 LNPs at an N / P ratio of 15, indicating that these drugs were poorly tolerated.

[0454] 2. Encapsulation efficiency >98% under all conditions

[0455] 3. The average lung radiation rate decreases as the N / P ratio increases

[0456] 4. Very little mRNA-LNP was recovered in the NP 1.5 and NP 2 conditions, indicating that these mRNA:lipid ratios were not suitable for LNP formation under these conditions. NP 2.5 and NP 3 had lower yields and were therefore dosed to a single animal.

[0457] 5. In this study, LNPs containing DLin-MC3-DMA with N / P ratios of 4 and 5 were the most promising.

[0458] Example 26 - Titration of DMG-PEG 2000 in 2 lipid LNPs

[0459] Method: DOTAP and DMG-PEG2000 were prepared as follows Fig.43A and 43B The molar percentage of PEGylated lipids shown in Figure 1 is mixed with malic acid through microfluidics, and then buffer exchange is 0.1M sodium acetate and concentrated. Lipid concentration is measured by HPLC, and then firefly luciferase mRNA (Luc) is added to N / P ratio of 5.0. Before adding PBS, mRNA is adsorbed for 30 minutes. Via intravenous injection (IV), C57BL / 6 female mice are administered with 2.0mg / kg, and organs are imaged in vitro after 20 hours. Fig.43A The average radiance (photons / s / cm 2 / sr).

[0460] result:

[0461] 1. A single cationic lipid—DOTAP—is sufficient to achieve tolerable lung targeting.

[0462] 2. With the increase of DMG-PEG2000, the mean lung radioactivity decreased.

[0463] Example 27 - Microfluidics versus Manual Mixing of Lipids in Ethanol and Malic Acid Buffer

[0464] Methods: DOTAP and DMG-PEG2000 were mixed with malic acid at a molar ratio of 100:1 and subjected to microfluidic mixing or manual vortex mixing, and then the buffer was exchanged to 0.1M sodium acetate and concentrated. Lipid concentration was measured by HPLC, and then firefly luciferase mRNA (Luc) was added to an N / P ratio of 5.0. Before adding PBS, mRNA was adsorbed for 30 minutes. C57BL / 6 female mice were administered with 0.5mg / kg via intravenous injection (IV), and organs were imaged in vitro 20 hours later. Fig.44 The average radiance (photons / s / cm 2 / sr).

[0465] result:

[0466] 1. Microfluidic mixing increases average lung radiation rate by 2 times

[0467] 2. The mRNA encapsulation efficiency under both conditions was greater than 98%

[0468] Example 28 - Comparison of published MC3 formulations with 5-lipid LNPs containing SS-OP

[0469] Method 1: Follow Fig.45A LNPs were formulated with firefly luciferase mRNA (Luc) at a ratio of 1:1. C57BL / 6J female mice were dosed via intravenous injection (IV) at the indicated mg / kg, and organs were imaged ex vivo 20 hours later. Fig.45B The average radiance (photons / s / cm 2 / sr). MC3 formulation is based on Dilliard et al. 2021 PNAS (https: / / doi.org / 10.1073 / pnas.2109256118).

[0470] Result 1:

[0471] 1. Compared with the published lung-targeted LNPs containing DLin-MC3-DMA, the LNPs containing 5-lipid SS-OP showed 6.3- and 5.2-fold higher average lung irradiation rates in the 0.5 and 2.0 mg / kg dose groups, respectively. Fig.45B

[0472] 2. Encapsulation efficiency of both LNPs was >98% and overall yield was comparable. Modal particle size measured by dynamic light scattering was between 35-55 nm.

[0473] Method 2: Follow Fig.45A LNPs were also formulated with Cre mRNA at a ratio of 1:1. The MC3 formulation was based on Dilliard et al. 2021 PNAS (https: / / doi.org / 10.1073 / pnas.2109256118). Ai14 Tomatofl / fl female mice were intravenously administered Cre mRNA at 2.0 mg / kg. Lungs were harvested 3 days later and processed via formalin fixation and paraffin embedding. Positive cells were labeled with anti-tdTomato antibodies. Fig.45C Shown in are the percentages of positive cells in alveolar cells.

[0474] Result 2:

[0475] 1. The transfection efficiency of LNPs containing 5-lipid SS-OP on alveolar cells was 4.9 times higher.

[0476] 2. Shown are representative images of the alveolar region

[0477] Example 29 - Cell transfection efficiency in three organs

[0478] Method: Use Fig.46A LNPs were prepared with lipid molar ratios and lipid:mRNA ratios as shown in . PD (*) formulations were prepared as described above (Example 26). CremRNA was administered intravenously to Ai14 Tomato fl / fl female mice at 1.5 mg / kg. Lungs were harvested 3 days later and processed via formalin fixation and paraffin embedding. Positive cells were labeled with anti-tdTomato antibodies. Shown are alveolar cavities ( Fig.46B ), hepatocytes (non-hepatocytes were excluded using nuclear size and circularity) ( Fig.46C ) and spleen cells ( Fig.46D ) The percentage of positive cells in . Fig.46E The relevant IHC data are provided in . Dark cells are tdTomato-positive cells transfected with Cre mRNA.

[0479] result:

[0480] The transfection efficiency of 1.5 lipid LNP (5Lip) and 3 lipid LNP (PDS) in lung and spleen was comparable, but the transfection efficiency of 5 lipid LNP in liver hepatocytes was 8 times higher.

[0481] 2. Fig.45D Shown are representative images of alveolar regions. Dark cells are transfected (tdTomato positive).

[0482] Example 30 - Pharmacokinetics using 2 lipid LNPs

[0483] Methods: PD preparations were prepared as described above (Example 26) using mRNA encoding human telomerase. C57Bl / 6 male mice were dosed at 1.0 mg / kg and plasma and tissues were collected at designated time points as shown in Figure 1. DOTAP was measured using LC-MS / MS.

[0484] DMG-PEG 2000% DOTAP% Lipid:mRNA weight / weight N / P 1.00％ 99.00％ 8.9 4.0

[0485] result:

[0486] organize Half-life (h) Decay constant plasma 3.57 0.194 lung 35.7 0.0194 liver 13.0 0.0534

[0487] Example 31 - Pharmacokinetics using 5-lipid LNP

[0488] Methods: LNPs were formulated with mRNA encoding human telomerase using the lipid molar ratios in Table 1. CD1 male mice were dosed at 1.0 mg mRNA / kg body weight and plasma and tissues were collected at time points 0 (<5 min) and 24 hours. DOTAP was measured using LC-MS / MS. mRNA was measured by qRT-PCR. "Liver remnant" is a liver sample excluding the left lobe.

[0489] result:

[0490] molecular Matrix Half-life hours Decay constant DOTAP Left lobe of liver 10.52 0.066 DOTAP Liver remnant 13.79 0.050 DOTAP Left lobe of lung 7.37 0.094 DOTAP Right lobe 9.51 0.073 DOTAP spleen 5.09 0.136 DOTAP plasma 3.63 0.191 mRNA plasma 3.26 0.212

[0491] Example 32 - Biodistribution using 5-lipid LNP

[0492] Methods: LNPs were formulated with mRNA encoding firefly luciferase using the lipid molar ratios in Table 1. New Zealand white rabbits were dosed intravenously (IV) at the mg mRNA / kg body weight levels indicated in the figure. Organs were imaged ex vivo 6 hours later. Shown are average radiances (photons / s / cm 2 / sr).

[0493] The results are Fig.49A and 49B Available in.

[0494] Example 33 - Biodistribution of LNPs using 3 lipids

[0495] Methods: LNPs were formulated with mRNA encoding firefly luciferase using the lipid molar ratios in Table 7. Rhesus monkeys were dosed intravenously (IV) at mg mRNA / kg body weight levels indicated in the figures. Organs were imaged ex vivo 6 hours after luciferin addition. Fig.50A is the average radiance (photons / s / cm 2 / sr). Fig.50B and 50C The relative radiation rates between low-dose and high-dose organs are shown. Figures 50D-50F Representative bioluminescent images of organs from high-dose animals are shown.

[0496] Table 7

[0497] Compound Mole Percent SS-OP 54.9％ DMG-PEG2000 1.0％ DOTAP 44.1％

[0498] Figure 50G Biodistribution results of the bioluminescence of the organs are provided, depicted as fold change in radiance.

[0499] Example 34 - Telomerase activity and telomere elongation in human small airway epithelial cells in the presence of SOC

[0500] Methods: LNPs were prepared with mRNA encoding human telomerase or mCherry as a control using the lipid molar ratios in Table 3. LNPs were added to human small airway epithelial cells (SAEC) obtained from Lonza in culture at 0.5 mg / ml. SAECs were grown in Lonza SAGM until 70% confluence. The day before, pirfenidone (1uM) and nintedanib (0.5uM), drugs representing the standard of care (SOC) for patients with pulmonary fibrosis, were added to the culture and replaced each time the culture medium was changed. To measure telomerase activity, cells were harvested 24 hours after treatment with LNPs and lysed in CHAPS buffer for TRAP assay. The lysate was exposed to an artificial telomerase single-stranded DNA template, and telomerase activity was then detected using PCR amplification. Fig.51B .

[0501] To measure telomere length, cells were harvested and fixed five days after the addition of telomerase mRNA LNPs. Fig.51A The Q-FISH protocol was performed as follows: cells were spun down onto glass slides, then permeabilized and telomeres labeled with a fluorescent probe. Fig.51B Microscopy and quantitative image analysis were performed to determine the length of individual telomeres. All hTERTLNP samples (+ / - SOC) were pooled for analysis to test for differences in telomere length.

[0502] result:

[0503] 1. Telomerase activity was detected in human small airway epithelial cells after adding hTERT LNP

[0504] 2. Similar levels of telomerase activity were detected in cells when standard-of-care drugs were added

[0505] 3. After adding a single dose of hTERT LNP, the median and 20th percentile telomere lengths increased significantly by 492 and 435 base pairs, respectively.

[0506] Example 35 - Telomerase activity and telomere elongation in human lung fibroblasts in the presence of SOC

[0507] Methods: LNPs were formulated with mRNA encoding human telomerase or mCherry as a control using the lipid molar ratios in Table 3. LNPs were added to fetal human lung fibroblast MRC-5 cells (passage 5) and grown in DMEM + 10% FBS until 70-80% confluence. MRC-5 cells were pretreated with standard of care drugs pirfenidone (1uM) and nintedanib (1uM) 24 hours before LNP treatment and then treated with 500ng / ml hTERT LNPs or 500ng / ml mCherry LNPs (control). Fresh standard of care drugs were added at each media change.

[0508] To measure telomerase activity, cells were harvested 24 hours after LNP treatment and lysed in CHAPS buffer for TRAP assay. The lysates were exposed to an artificial telomerase single-stranded DNA template and then telomerase activity was detected using PCR amplification. Fig.52 .

[0509] result:

[0510] 1. Telomerase activity was detected in human lung fibroblasts after addition of hTERT LNP.

[0511] 2. Similar levels of telomerase activity were detected in the cells when standard of care drugs were added.

[0512] Example 36 - Lyophilization of 3 and 5 lipid LNPs

[0513] Methods: LNPs were formulated with mRNA encoding firefly luciferase using the lipid molar ratios described herein. The LNP ratios used are described in Table 8. The LNPs were mixed with 15% sucrose and frozen at -80°C. Next, they were placed in the pre-freeze rack of an L-200Pro 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. The LNPs were stored at 4°C for 2 days and then resuspended 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 dosed with 0.3 mg mRNA / kg body weight by intravenous injection (IV), and organs were imaged ex vivo 18 hours later. Fig.55 .

[0514] Results: The in vivo activities of 5-lipid and PDS were comparable to each other and to frozen PDSLNPs that were not lyophilized (freeze-dried). Freeze-dried and reconstituted PD LNPs exhibited at least 8-fold higher activity compared to the other LNPs.

[0515] Example 37 - Summary of Exemplary LNP Formulations of the Disclosure

[0516] Table 8 provides the mole percent composition, N / P ratio, molar ratio of cationic lipid:ionizable lipid, and lipid:mRNA ratio (weight / weight) of exemplary LNPs of the present disclosure (named "SSOP-DOTAP", "5 lipid", "PDS", "3-MC3", "PD", respectively).

[0517] Table 8

[0518]

[0519] Fig.56 The optimal cationic lipid to ionizable lipid ratio ("C / I ratio") and N / P ratio determined by the inventors for LNPs containing ionizable lipids from the SS-OP family or the DLin-MC3-DMA family were modeled.

[0520] Example 38 - Addition of mRNA after LNP formation

[0521] LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 8. For LNPs with asterisks (*), only buffer was used for microfluidic mixing without mRNA. The buffer was then exchanged for 0.1M sodium acetate and concentrated. Firefly luciferase mRNA (Luc) was added to lipids to achieve the N / P ratio shown in the figure. mRNA was adsorbed for 2 hours before adding PBS, 0.2μM filtration and administration. mRNA LNPs were delivered intravenously to C57BL / 6 male mice, and organs were imaged in vitro 17 hours later.

[0522] result:

[0523] 1. Addition of mRNA at the end of the study resulted in the following changes in mean lung radiance ( Fig.53A ):

[0524] PD LNP increased by 85%

[0525] PDS LNP fell 68%

[0526] 5. Reduce lipids by 16%

[0527] 2. In the case where mRNA was added at the end of the purification process, encapsulation efficiency was >95% only in the PD (*) condition, but not in the PDS (*) and 5 lipid (*) conditions. Fig.53B .

[0528] Example 39 - Addition of mRNA after LNP formation

[0529] LNPs were formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 8. For PD (*), it means that only buffer was used for microfluidic mixing without mRNA. The buffer was then exchanged for 0.1M sodium acetate and concentrated. Firefly luciferase mRNA (Luc) was added to lipids to achieve the N / P ratio shown in the figure. mRNA was adsorbed for 2 hours before adding PBS, 0.2μM filtration and administration. mRNA LNPs were delivered intravenously to BALB / cJ male mice at 1.5mg / kg, and organs were imaged in vitro 26 hours later.

[0530] result:

[0531] 1. Addition of mRNA at the end of PD formulation resulted in a 31.8-fold increase in bioluminescent signal. Fig.54A .

[0532] 2. The encapsulation efficiency of all formulations was >95%. Fig.54B .

[0533] A list of exemplary nucleic acid sequences used in the presently described embodiments 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.

[0534] SEQ ID NO:1

[0535]

[0536] SEQ ID NO:2

[0537]

[0538] SEQ ID NO:3

[0539]

[0540] SEQ ID NO:4

[0541]

[0542]

[0543] SEQ ID NO:5

[0544]

[0545] The above embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Many variations of the above are possible. Since modifications and variations to the above-described embodiments will be apparent to those skilled in the art, the present invention is intended to be limited only by the scope of the appended claims.

[0546] Citation of the above publications or documents is not intended to be an admission that any of the above is relevant prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

Claims

1. A method of delivering a polynucleotide to a subject, comprising administering by intravenous injection a polynucleotide encapsulated in a lipid nanoparticle (LNP), wherein the LNP comprises a cationic lipid in a molar percentage between about 25% and about 60%, and any of the following: (a) between about 25% and about 65% ionizable lipid by mole percentage, comprising SS-OP or an SS-OP analog, wherein the LNP has a cationic lipid:ionizable lipid molar (C / I) ratio between 0.5 and 1.2 and a lipid nitrogen:polynucleotide phosphate (N / P) ratio between 10.5 and 31; or, (b) a mole percentage of between 30% or about 30% and 50% or about 50% ionizable lipids, which includes DLin-MC3-DMA or a DLin-MC3-DMA analog, wherein the LNP has a C / I ratio between 0.5 and 2.5, and an N / P ratio between 2.5 and 8.

2. The method of claim 1, wherein the polynucleotide comprises a synthetic RNA, the mRNA is translated into a corresponding protein encoded by the mRNA in the subject upon or after administration of the LNP, and the lipid DOTAP has an in vivo half-life of: a) in the lungs of the subject for at least 30 hours; b) in the subject's liver for no longer than 11 hours; and / or c) in the spleen of said subject for no longer than 9 hours.

3. The method of claim 2, wherein the protein has an in vivo half-life in the lungs of the subject of at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours or 35 hours.

4. The method of claim 1, wherein the polynucleotide comprises a synthetic RNA that is translated into a corresponding protein encoded by the mRNA in the subject upon or after administration of the LNP, and wherein the amount of the protein per mg of tissue has a lung:liver ratio of at least 2:1, 3:1, or 4:1 at or about 6 hours after intravenous infusion of the LNP.

5. The method of claim 4, wherein the amount of the protein per mg of tissue is in a lung:liver ratio of at least 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, or 50:

1.

6. The method of claim 1, wherein the polynucleotide comprises a synthetic RNA encoding a telomerase reverse transcriptase (TERT), and upon or after administration of the LNP, the mRNA is translated into a corresponding TERT protein in the subject.

7. The method of claim 6, wherein the median telomere length of the alveolar cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%.

8. The method of claim 6, wherein the median telomere length of lung AT2 cells is extended by at least 5%, at least 10%, at least 20%, or at least 50%.

9. The method of claim 6, wherein the increase in telomerase activity in the lung is detectable by the TRAP assay for at least 24 or 48 hours.

10. The method of claim 1, wherein the ionizable lipid comprises SS-OP or an SS-OP analog and the LNP has an N / P ratio between 28 and 31.

11. The method of claim 10, wherein the LNPs have an N / P ratio of 29 or about 29.

12. The method of claim 1, wherein the ionizable lipid comprises SS-OP or an SS-OP analog and the LNP has an N / P ratio between 10.5 and 15.

13. The method of claim 12, wherein the LNPs have an N / P ratio of at or about 12 or at or about 12.

1.

14. The method of claim 1, wherein the ionizable lipid comprises DLin-MC3-DMA or a DLin-MC3-DMA analog, and the LNP has an N / P ratio between 3 and 5.

15. The method of claim 14, wherein the LNPs have an N / P ratio of 4.5 or about 4.

5.

16. The method of any one of claims 1 to 11, wherein the ionizable lipid is SS-OP and the LNP has a C / I ratio of at or about 1.8 and consists of 5 lipids.

17. The method of any one of claims 1 to 11, wherein the ionizable lipid is SS-OP and the LNP has a C / I ratio of at or about 1.8 and is composed of 5 lipids, including DOTAP and DOPC.

18. The method of any one of claims 1 to 11, wherein the ionizable lipid is SS-OP and the LNP has a C / I ratio of at or about 1.8 and is composed of 5 lipids, including DMG-PEG2000, DOPC, and DOTAP.

19. The method of any one of claims 1 to 9 or 12-13, wherein the ionizable lipid is SS-OP and the LNP has a C / I ratio of at or about 0.8 and is composed of 3 or 5 lipids.

20. The method of any one of claims 1 to 9 or 12-13, wherein the ionizable lipid is SS-OP and the LNP has a C / I ratio of at or about 0.8 and consists of 3 or 5 lipids, including DOTAP.

21. The method of any one of claims 1 to 9 or 12-13, wherein the ionizable lipid is SS-OP and the LNP has a C / I ratio of 0.8 or about 0.8 and consists of 3 or 5 lipids, including DMG-PEG2000 and DOTAP.

22. as described in any one in claim 1 to 9 or 14-15 method, wherein said ionizable lipid is DLin-MC3-DMA, and described LNP has the C / I ratio that is 1.5 or about 1.5 and is comprised of 3 kinds of lipids.

23. as described in any one in claim 1 to 9 or 14-15 method, wherein said ionizable lipid is DLin-MC3-DMA, and described LNP has the C / I ratio that is 1.5 or about 1.5 and is made up of 3 kinds of lipids, comprises DOTAP.

24. as described in any one in claim 1 to 9 or 14-15 method, wherein said ionizable lipid is DLin-MC3-DMA, and described LNP has the C / I ratio that is 1.5 or about 1.5 and is made up of 3 kinds of lipids, comprise DMG-PEG2000 and DOTAP.

25. as described in any one in claim 1 to 9 or 14-15 method, wherein said ionizable lipid is DLin-MC3-DMA, and described LNP has the C / I ratio that is 1.5 or about 1.5 and is made up of 3 kinds of lipids, comprises DMG-PEG2000 and DOTAP, and the N / P ratio is about 4.

5.

26. The method of any one of claims 1 to 15, wherein the LNP is selected from the LNPs of Tables 8-10.

Images