Compositions and methods for extensive delivery of RNA to tissue

Abstract

The present invention relates to lipid nanoparticle (LNP) compositions, as well as diagnostic or therapeutic polynucleotides, such as TERT mRNA, that can be delivered in a formulation together with the LNP compositions to various tissue and cell types in the whole body of a mammal, such as, for example, TNP mRNA. Comprising stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenic cells, hepatocells, kidney cells and / or osteoblasts, for example, for use in the diagnosis, prevention and / or treatment of a condition or disease.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 579,345, filed August 29, 2023, the contents of which are incorporated by reference in their entirety.

[0002] Incorporation by Reference of the Electronic Sequence Listing The instant application contains a Sequence Listing which has been submitted electronically in XML format. The XML copy, created on August 28, 2023, is named “REJU11PRV_seq_listing.xml” and is 15.1 KB. The Sequence Listing contained in the XML file is part of the specification and is herein incorporated by reference in its entirety. BACKGROUND

[0003] A variety of diseases have been identified, but there are currently no practical diagnostic means, and / or there are currently no or limited treatment methods. Delivery of polynucleotides to cells can provide much-needed treatment options for patients with diseases; however, compositions and methods for delivering polynucleotides need improvement.

[0004] Developing delivery systems and methods remains the most important challenge to realize the tremendous potential of delivering nucleic acids for gene therapy. RNA- and DNA-based biologies have a wide range of abilities to modulate cellular activities, which can be used to treat genetic and acquired diseases. Among non-viral gene delivery vehicles, the recent clinical success of LNPs has drawn widespread attention. Most of the lipid-based nucleic acid delivery platforms that are currently in clinical investigation or have been marketed consist of four or five lipid components. Recent studies report that not only the selection of lipid components, but also the relative proportion of lipid components in the formulation greatly influences the in vivo transfection efficiency and tissue-specific delivery.

[0005] Although these formulations have proven the ability to encapsulate mRNA or siRNA and mediate cellular uptake and endosomal escape, there is currently a lack of in-depth analysis of the effects of lipid charge and the relative ratio of LNP components on the transfection efficiency of nucleic acid delivery. In addition, the large number of LNP systems used to screen candidates for effective in vivo delivery makes it difficult to reasonably determine the optimal formulation for a specific tissue or disease target.

[0006] Accordingly, there remains a need in the art for delivery formulations capable of delivering polynucleotides, such as mRNA, to cells that prevent or treat a wide variety of conditions or diseases.

[0007] The present disclosure satisfies this and other related needs in the art. SUMMARY

[0008] According to embodiments provided herein, are methods of delivering a polynucleotide to one or more of a broad range of different cell types of a subject, comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or SS-OP analog at a molar percentage between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between.6 and 1, wherein the polynucleotide comprises a synthetic RNA that is translated in vivo in one or more different cell types of the subject into a corresponding protein encoded by the synthetic RNA at or after administration of the LNP.

[0009] According to embodiments provided herein, are methods of delivering a polynucleotide to one or more of a broad range of different cell types of a subject, comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP) comprising: (i) a SS-OP or SS-OP analog at a molar percentage between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between.6 and 1, wherein the polynucleotide comprises a synthetic RNA that is translated in vivo in one or more different cell types of the subject into a corresponding protein encoded by the synthetic RNA at or after administration of the LNP.

[0010] In the above embodiments, the PEGylated lipid is DMG-PEG2000 according to embodiments described herein. Further in the above embodiments, the cationic lipid is DOTAP according to embodiments described herein. According to presently contemplated embodiments, the LNP of the above methods and methods described herein are composed of the lipids listed in Table 7.

[0011] According to embodiments described herein, the target cell type is located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 different organs of the subject. According to related embodiments described herein, the target cell type is located in 4 to 6 different organs of the subject. According to related embodiments described herein, the target cell type is located in 5 to 8 different organs of the subject. According to related embodiments described herein, the target cell type is located in 7 to 10 different organs of the subject. According to related embodiments described herein, the target cell type is located in 9 to 12 different organs of the subject. According to related embodiments described herein, the target cell type is located in 10 to 15 different organs of the subject.

[0012] According to embodiments provided herein, the delivery is to a target cell. Such target cells are typically in any of a variety of tissues and cell types throughout the mammal's body, including stem cells, progenitor cells, hematopoietic stem cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0013] According to embodiments described herein, the administration involves the use of the LNP described herein to introduce the agent into the subject, the administration is intravenous injection, transdermal injection, intradermal injection via a microneedle array, or local delivery involving a step of using a mechanical means such as a microneedle treatment and / or applying a skin permeabilizing agent to permeabilize the skin barrier. Exemplary skin permeabilizing agents include solutions and mixtures containing one or more proteases, one or more lipases, or other skin permeabilizing agents known in the art.

[0014] According to certain embodiments, the corresponding protein encoded by the synthetic RNA is expressed in two or more of bone marrow cells, spleen cells, liver cells, and / or kidney cells. According to certain embodiments, the corresponding protein encoded by the synthetic RNA is expressed in each of bone marrow cells, spleen cells, liver cells, and / or kidney cells. According to related embodiments, the bone marrow cells are bone marrow stem and / or progenitor cells.

[0015] According to certain embodiments, the corresponding protein encoded by the synthetic RNA is expressed in two or more of stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, and / or bone cells. According to certain embodiments, the corresponding protein encoded by the synthetic RNA is expressed in each of bone marrow cells, spleen cells, liver cells, and / or kidney cells. According to related embodiments, the bone marrow cells are bone marrow stem and / or progenitor cells and / or stem cells such as hematopoietic stem cells.

[0016] The polynucleotide in the LNP will typically comprise a sequence encoding any therapeutic protein or protein useful for diagnosis that can be expressed in a target cell, such as, for example, a telomerase reverse transcriptase (TERT) protein or a portion thereof, selected from a human TERT (hTERT), a mouse TERT (mTERT), or a TERT of other mammalian species. According to presently contemplated embodiments, the polynucleotide (e.g., mRNA) typically comprises SEQ ID NO: 1 or a fragment thereof. Further according to presently contemplated embodiments, the synthetic ribonucleic acid (RNA) encodes a telomerase reverse transcriptase (TERT), wherein optionally the TERT mRNA comprises a nucleic acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1-5 or SEQ ID NOs: 38-40 of PCT / US22 / 22642.

[0017] Typically the TERT synthetic mRNA 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, CleanCap®, 8-oxo-guanosine, 2-aminoguanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0018] According to the present embodiments, there is provided a method of delivering a polynucleotide to bone marrow cells, comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP), the lipid nanoparticle comprising: (i) a molar percentage between about 20% and about 60% of SS-OP or SS-OP analog, a molar percentage between about 0.5% and about 2% of a PEGylated lipid, and a molar percentage between about 40% and about 50% of a cationic lipid, wherein the cationic lipid:ionizable lipid (C / I) ratio is between.6 and 1; and / or (ii) a molar percentage between about 30% and about 50% of DLin-MC3-DMA, a molar percentage between about 0.5% and about 2% of DMG-PEG2000, and a molar percentage between about 50% and about 70% of a cationic lipid, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 1 and 2, wherein the polynucleotide comprises a polynucleotide that is translated in vivo in the bone marrow cells of the subject into the corresponding protein encoded by the polynucleotide at or after administration of the LNP. Typically in such embodiments, the PEGylated lipid is DMG-PEG2000. Also typically, the cationic lipid is DOTAP. Also typically, the PEGylated lipid is DMG-PEG2000, and wherein the cationic lipid is DOTAP.

[0019] According to the present embodiments, there is provided a method of delivering a polynucleotide to one or more of bone marrow cells, spleen cells, liver cells, and / or kidney cells, comprising administering a polynucleotide encapsulated in a lipid nanoparticle (LNP), the lipid nanoparticle comprising: (i) a molar percentage between about 20% and about 60% of SS-OP or SS-OP analog, a molar percentage between about 0.5% and about 2% of a PEGylated lipid, and a molar percentage between about 40% and about 50% of a cationic lipid, wherein the cationic lipid:ionizable lipid (C / I) ratio is between.6 and 1; and / or (ii) a molar percentage between about 30% and about 50% of DLin-MC3-DMA, a molar percentage between about 0.5% and about 2% of a PEGylated lipid, and a molar percentage between about 50% and about 70% of a cationic lipid, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 1 and 2, wherein the polynucleotide comprises a polynucleotide that is translated in vivo in one or more of the bone marrow cells, spleen cells, liver cells, and / or kidney cells of the subject into the corresponding protein encoded by the polynucleotide at or after administration of the LNP. Typically in such embodiments, the PEGylated lipid is DMG-PEG2000. Also typically, the cationic lipid is DOTAP. Also typically, the PEGylated lipid is DMG-PEG2000, and wherein the cationic lipid is DOTAP.

[0020] According to embodiments described herein, the respective protein encoded by the polynucleotide is expressed in two or more of bone marrow cells, spleen cells, liver cells, and / or kidney cells. Typically, the respective protein encoded by the polynucleotide is expressed in each of bone marrow cells, spleen cells, liver cells, and / or kidney cells.

[0021] According to embodiments described herein, the bone marrow cells are bone marrow stem and / or progenitor cells.

[0022] According to embodiments described herein, the polynucleotide typically comprises a sequence encoding a diagnostic or therapeutic protein expressible in the target cell.

[0023] Further according to embodiments described herein, the LNP further comprises a targeting ligand suitable for specific targeting of the cell. In such embodiments, suitable for specific targeting of the cell is typically binding or other interaction of the LNP with the cell.

[0024] Further according to embodiments described herein, the targeting ligand comprises a targeting group selected from the group consisting of a cell targeting agent, a tissue targeting agent, a lectin, a glycoprotein, a lipid, a protein, a peptide, an antibody suitable for binding to the target cell type, an aptamer, a small molecule, a carbohydrate, a lipid, a nanobody, and an aptamer-antibody conjugate.

[0025] Further according to embodiments described herein, the method is suitable for diagnosing, preventing, or treating a disorder or disease involving the cell.

[0026] Currently, polynucleotide delivery is typically used for the diagnosis, prevention, and / or treatment of various tissue and cell types throughout the mammalian body using existing methods. Examples of diseases or ailments include influenza, asthma, type 1 diabetes, type 2 diabetes, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer's disease, Parkinson's disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn's disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, and prostate cancer. Lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, stomach cancer, brain cancer, endometrial cancer, bone cancer, sarcoma, multiple myeloma, skin cancer, basal cell carcinoma, squamous cell carcinoma, tuberculosis, pneumonia, bronchitis, sinusitis, otitis media, urinary tract infection (UTI), hepatitis A, hepatitis B, hepatitis C, HIV / AIDS, syphilis, gonorrhea, chlamydia, herpes simplex virus (HSV), human papillomavirus (HPV), scabies, tinea pedis (athlete's) Foot, tinea, lice, measles, mumps, rubella, chickenpox, herpes zoster, pertussis, diphtheria, tetanus, poliomyelitis, rabies, malaria, dengue fever, yellow fever, Zika virus, Lyme disease, Rocky Mountain spotted fever, toxoplasmosis, giardiasis, amoebiasis, ascariasis, trichinosis, echinococcosis, leishmaniasis, anthrax, botulism, tetanus, plague, cholera, typhus, salmonellosis, campylobacteriosis, listeriosis, Clostridium difficile infection, norovirus infection, rotavirus infection, cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection, parvovirus infection, West Nile virus infection, hantavirus infection. Infections, Ebola virus disease, Marburg virus disease, SARS, MERS, COVID-19, sepsis, cellulitis, osteomyelitis, endocarditis, meningitis, encephalitis, Guillain-Barré syndrome, amyotrophic lateral sclerosis (ALS), Huntington's disease, cystic fibrosis, Duchenne muscular dystrophy, Becker's muscular dystrophy, fragile X syndrome, Down syndrome, Turner syndrome, Klinefelter syndrome, Marfan syndromesyndrome), Ehlers-Danlos syndrome, polycystic kidney disease, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson’s disease, Gaucher disease, Fabry disease, Niemann-Pick disease, Tay-Sachs disease, Huntington’s chorea, Rett syndrome, Prader-Willi syndrome, Angelman syndrome, Sjogren’s syndrome, Addison’s disease, Cushing’s syndrome, acromegaly, gigantism, pheochromocytoma, hyperparathyroidism, hypoparathyroidism, rickets, osteomalacia, osteoporosis, Paget’s disease of bone, gout, bursitis, tendonitis, tennis elbow, carpal tunnel syndrome, plantar fasciitis, fibromyalgia, chronic fatigue syndrome, migraine, tension headaches, cluster headaches, epilepsy, narcolepsy, restless leg syndrome, sleep apnea, insomnia, bipolar disorder, depression, anxiety, schizophrenia, obsessive compulsive disorder (OCD), post-traumatic stress disorder (PTSD), attention deficit / hyperactivity disorder (ADHD), autism spectrum disorder (ASD), anorexia nervosa, bulimia nervosa, binge eating disorder, alcohol use disorder, substance use disorder, personality disorder, somatic symptom disorder, dissociative identity disorder, hypochondriasis, Munchausen syndrome, conversion disorder, delirium, dementia, fetal alcohol syndrome, neonatal abstinence syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cirrhosis, liver fibrosis, nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), chronic hepatitis B-related fibrosis, chronic hepatitis C-related fibrosis, systemic sclerosis (scleroderma), cystic fibrosis, myocardial fibrosis, hypertrophic cardiomyopathy, restrictive cardiomyopathy, dilated cardiomyopathy, chronic kidney disease (CKD), glomerulonephritis, diabetic nephropathy, interstitial nephritis, retroperitoneal fibrosis, peritoneal fibrosis, Dupuytren’s contracture, Peyronie’s disease, myelofibrosis, bone marrow fibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myelofibrosis, myeloffibrosis), keloid formation, scar tissue formation, adhesive capsulitis (frozen shoulder), chronic pancreatitis, pancreatitis-associated fibrosis, Crohn's disease-associated fibrosis, ulcerative colitis-associated fibrosis, dermal fibrosis, radiation-induced fibrosis, radiation pneumonitis, post-surgical adhesions, asbestosis, silicosis, sarcoidosis-associated fibrosis, and eosinophilic fasciitis. Cell types can include, for example, stem cells (e.g., hematopoietic stem cells, progenitor cells), differentiated cells, terminally differentiated cells, endothelial cells, epithelial cells, splenocytes, hepatocytes, renal cells, cancer cells, and / or bone cells of the subject. Also generally, according to the current methods, the methods of delivering polynucleotides are used to modulate a condition or disease of various tissues and cell types of the mammal's body (including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenocytes, hepatocytes, renal cells, and / or bone cells of the subject). Also generally, according to the current methods, the methods of delivering polynucleotides are used to increase or initiate expression of a protein (e.g., a therapeutic or supplemental protein) in target cells in various tissues and cell types of the mammal's body (including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenocytes, hepatocytes, renal cells, and / or bone cells of the subject).

[0027] When it is desirable to prevent such a condition or disease, the presently described LNP compositions are administered at some time prior to the subject having or being suspected of having the condition or disease.

[0028] In each instance in which a polynucleotide is delivered for the diagnosis, prevention, and / or treatment of a condition or disease, the presently contemplated LNP is capable of targeting the cell types and tissues in which the condition or disease manifests. In this regard, the presently described LNP has been demonstrated to provide the ability to transfect cells throughout the body of a subject with an encapsulated polynucleotide, including cell types present throughout the body. More specific targeting of cell types and tissues for transfection can be provided using a targeting ligand or moiety, generally as desired according to the purpose of the diagnosis, prevention, and / or treatment and the particular type or types of conditions or diseases that are the subject of administration to the subject. While the polynucleotide encapsulated in the LNP will vary according to the condition or disease and the purpose of the delivery (diagnosis, prevention, or treatment), the presently described LNP is provided as an effective delivery vehicle for such polynucleotides so that it can be delivered to areas previously difficult or impossible to deliver polynucleotides in vivo in a subject (e.g., a mammal).

[0029] Further according to generally included embodiments, the LNP is selected from the LNP of Table 7.

[0030] According to certain embodiments, the LNP has only a single charged lipid, such as DOTAP.

[0031] Also provided herein are methods of manufacturing LNP (e.g., 3-MC3 and / or PDS) for introducing nucleic acids into a wide range of different cell populations. In one particular example, LNP are manufactured in a manner that forms the LNP prior to the addition of the nucleic acid. In particular embodiments thereof, such LNP are 2, 3, or 5 lipid LNP described herein in Table 7, and the method employs vortex mixing during the addition of the nucleic acid. In certain embodiments, the LNP consists of 2 or 3 lipid LNP described herein.

[0032] These and other embodiments, features, and advantages will become apparent to those of ordinary skill in the art in view of the following more detailed descriptions of various example embodiments of the disclosure in conjunction with the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0033] Those of skill in the art will understand that the accompanying drawings described below are for illustrative purposes and are not a limitation of the described embodiments.

[0034] FIGS. 1A-1D Pharmacokinetics of a particular lipid nanoparticle (LNP) are depicted. Time course of serum ( FIG. 1A ), lung ( FIG. 1B ), and liver ( FIG. 1C ) levels of LNP as determined by measuring one of the lipids, DOTAP. FIG. 1D Table data related to FIGS. 1A-1C is provided.

[0035] FIG. 2 Depiction of the ratio of bioluminescent signal produced by luciferase protein translated from mRNA formulation in LNP (“mRNA LNP”) intravenously injected at specified time points after intravenous injection from the specified organ.

[0036] FIG. 3 Depiction of LNP formulated with TERT mRNA (“TERT mRNA LNP”) extending 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.

[0037] FIG. 4 Depiction of TERT mRNA LNP extending telomeres in lung cells in vivo, where TERT mRNA LNP extended median and mean telomere length in AT2 cells. At least 50 precursor SPC positive nuclei were quantified per mouse. Error bars are standard error of the mean (SEM) of telomere length between mice.

[0038] FIG. 5The results showed that TERT mRNA LNPs elongated telomeres in lung cells in vivo, with TERT mRNA LNPs extending the median and mean telomere lengths in alveolar cells. Telomeres from at least 100 alveolar cells were quantified from each mouse. The error bar represents the standard error (SEM) of the mean telomere length across mice.

[0039] FIG. 6A Showing FIGS. 3-12 and FIGS. 20-21 The experimental design and dosing timeline of the data presented in this disclosure are described.

[0040] FIG. 6B Showing FIGS. 13-16 The experimental design and dosing timeline of the data presented in this disclosure are described.

[0041] FIG. 7 The results show that TERT mRNA LNP reduces pulmonary fibrosis in vivo, with high-density fibrotic lesions (yellow / dark) mapped in the lungs of mice treated with bleomycin (treated with either control mRNA LNP (carrying luciferase mRNA) or TERT mRNA LNP) using Sirius red staining. "Bleomycin-free" indicates lung sections from normal healthy mice that were not treated with bleomycin.

[0042] FIG. 8 The results showed that TERT mRNA LNP reduced pulmonary fibrosis in vivo, as measured by high-density fibrotic lesions in tissue sections stained with quantitative Sirius red.

[0043] FIG. 9 The TERT mRNA LNP showed that it increased useful lung volume in vivo, as determined by quantifying the change in normal ventilated lung ratio relative to baseline using 3D quantification of fibrosis detected by signal intensity in X-ray computed tomography data.

[0044] FIG. 10 The results showed that TERT mRNA LNP improved tissue elasticity in vivo, as determined by quantification using the Flexivent system to measure tissue elasticity in mice at the end of the study.

[0045] FIG. 11 The results showed that TERT mRNA LNP improved lung function in vivo, as determined by measuring forced expiratory volume in mice using the Flexivent system at the end of the study.

[0046] FIG. 12 The results showed that TERT mRNA LNP improved lung function in vivo, as determined by measuring forced vital capacity in mice using the Flexivent system at the end of the study.

[0047] FIG. 13 TERT mRNA LNPs were shown to improve lung structure in vivo, where digital images of tissue sections were analyzed using the Morpho-Quant Lung software to quantify the number of alveoli per unit area.

[0048] FIG. 14 TERT mRNA LNPs were shown to improve lung structure in vivo, where digital images of tissue sections were analyzed using the Morpho-Quant Lung software to quantify alveolar diameter.

[0049] FIG. 15 TERT mRNA LNPs were shown to improve lung structure in vivo, where digital images of tissue sections were analyzed using the Morpho-Quant Lung software to quantify alveolar roundness.

[0050] FIG. 16 TERT mRNA LNPs were shown to improve lung structure in vivo, where representative images of alveoli, as false-colored by the Morpho-Quant Lung software during the alveolar structure digitalization process, are shown.

[0051] FIG. 17 Pharmacodynamic data of TERT mRNA in vivo after intravenous infusion in TERT mRNA LNP formulations were shown, where the level of telomerase activity in lung tissue was quantified at specified time points post-infusion.

[0052] FIG. 18 Experimental protocol for quantifying the effect of TERT mRNA LNPs on the colony forming ability of human primary epithelial cells was shown.

[0053] FIG. 19 TERT mRNA LNPs were shown to increase the colony forming ability of human primary epithelial cells in FIG. 18 the experimental model shown.

[0054] FIG. 20 Depiction of the experimental model shown according to FIG. 6A the levels of P21+ senescent alveolar cells involved in the experimental model shown.

[0055] FIG. 21 are representative immunohistochemistry images of anti-P21 staining of lung sections, as FIG. 20 quantified.

[0056] FIG. 22A , 22B , 22C and 22D provide lung delivery data obtained using 2-lipid LNP compositions.

[0057] FIG. 23Results of titrating the lipid:mRNA ratio in 2 lipid LNPs are described.

[0058] FIG. 24 Time courses of protein expression following delivery of lung-targeted mRNA-LNPs encoding firefly luciferase using the LNPs of Table 3 are depicted.

[0059] FIG. 25A , 25B Lung radioactivity, encapsulation efficiency, and body weight change when SS-OP lipids are titrated into ‘DOTAP + PEG’ LNPs using the LNPs of FIG. 25D are provided.

[0060] FIG. 26A and 26B Lung and liver delivery data using the LNPs of Table 4.

[0061] FIG. 27A and 27B Exemplary LNPs and their encapsulation efficiencies are provided.

[0062] FIG. 27C and 27D Lung delivery data and body weight change data when SS-OP lipids are titrated into ‘DOTAP + PEG’ LNPs using the LNPs of FIG. 27A are provided.

[0063] FIG. 28A and 28B Comparison of transfection and weight data for fresh lungs versus long-term cryopreserved lungs for the LNPs of Table 3 are provided.

[0064] FIG. 29A and 29B Comparison of transfection and particle size data for fresh lungs versus short-term cryopreserved lungs for the LNPs of Table 3 are provided.

[0065] FIG. 30A Exemplary 2, 3, and 5 lipid LNPs are provided.

[0066] FIG. 30B and 30C Lung delivery data and body weight change data when N / P ratios are titrated and 2, 3, and 5 lipid formulations of FIG. 30A are compared are provided.

[0067] FIG. 31A and 31B Exemplary 3 and 5 lipid LNPs and their encapsulation efficiencies are provided.

[0068] FIG. 31C Lung delivery data when N / ratios (mRNA to lipid ratios) are titrated using the 3 and 5 lipid formulations 30A are provided.

[0069] FIG. 32A and 32B Lung transfection data for LNP prepared using vortex or microfluidic mixing with the lipid ratios of Table 3 are provided along with LNP size and encapsulation efficiency data.

[0070] FIG. 33A Exemplary 3 and 5 lipid LNP are provided.

[0071] FIG. 33B Lung transfection data for LNP prepared using FIG. 33A are provided.

[0072] FIG. 34A Exemplary 2, 3 and 5 lipid LNP are provided.

[0073] FIG. 34B and 34C Lung transfection data for LNP prepared using FIG. 34A are provided along with particle size and encapsulation efficiency for LNP FIG. 34A .

[0074] FIG. 35A Exemplary 5 lipid LNP prepared using different flow rates are provided.

[0075] FIG. 35B and 35C Encapsulation percentage and lung transfection data for LNP prepared using FIG. 35A are provided.

[0076] FIG. 36A Exemplary 3 lipid LNP prepared using different flow rates are provided.

[0077] FIG. 36B and 36C Lung transfection and body weight change data for LNP prepared using FIG. 36A are provided.

[0078] FIG. 37A and 37B Exemplary LNP prepared by replacing SS-OP with a different ionizable lipid (DLin-MC3-DMA) in 3 lipid LNP are provided, including encapsulation percentage.

[0079] FIG. 37C and 37D Lung transfection and body weight change data for LNP prepared using FIG. 37A are provided.

[0080] FIG. 38A Exemplary LNP prepared by replacing SS-OP with DLin-MC3-DMA in 3 lipid LNP are provided.

[0081] FIG. 38Band 38C Encapsulation percentage and lung transfection data for LNP using FIG. 38A are provided.

[0082] FIG. 39A Exemplary LNP prepared by replacing SS-OP with DLin-MC3-DMA in 5 lipid LNP are provided.

[0083] FIG. 39B and 39C Encapsulation percentage and lung transfection data for LNP using FIG. 39A are provided.

[0084] FIG. 40A A description of the titration time for mRNA to be adsorbed to form LNP in 2-lipid formulations is provided.

[0085] FIG. 40B Body weight change data for LNP using FIG. 40A are provided.

[0086] FIG. 41A and 41B Lung transfection and encapsulation percentage data for 2 lipid LNP made while titrating mRNA:lipid ratio are provided.

[0087] FIG. 42A and 42B Lung transfection and encapsulation percentage data for 3 lipid LNP made while titrating N / P ratio are provided.

[0088] FIG. 43A and 43B Lung transfection and body weight change data for 2 lipid LNP using the listed titration with DMG-PEG2000 are provided.

[0089] FIG. 44 Lung mean radiant rates comparing microfluidic versus manual mixing of lipids and malic acid buffer in ethanol are provided.

[0090] FIGS. 45A-45D Formulation, transfection, and IHC data comparing MC3 formulations to the 5 lipid LNP compositions of the present specification demonstrating transfection of endothelial and epithelial cells of the alveolar region are provided.

[0091] FIG. 46A Exemplary 2, 3, and 5 lipid LNP are provided.

[0092] FIG. 46B , 46C Lung, liver, and spleen cell transfection data for LNP using FIG. 46A are provided.

[0093] FIG. 46EIHC data related to use of exemplary 2, 3, and 5 lipid LNPs in lung, liver, and spleen tissue is provided.

[0094] FIG. 47A , 47B and 47C depict pharmacokinetics of a particular 2 lipid LNP in plasma, lung, and liver.

[0095] FIG. 48 Pharmacokinetics from a particular 5 lipid LNP in Table 1 is depicted.

[0096] FIG. 49A and 49B Depicts biodistribution from exemplary 5 lipid LNPs in Table 1 upon administration to a mammal.

[0097] FIG. 50A , 50B and 50C depict biodistribution of 3 lipid LNPs as described in FIG. 46A upon administration to a mammal.

[0098] FIG. 50D , 50E and 50F depict organ level bioluminescence related to use of 3 lipid LNPs in a mammal as described in Example 33.

[0099] FIG. 50G Depicts biodistribution of exemplary 3 lipid LNPs as described in FIG. 46A upon administration to a mammal.

[0100] FIG. 51A and 51B Data related to telomere elongation and telomerase activity in lung epithelial cells using LNPs of Table 3 is provided.

[0101] FIG. 52 Data related to telomerase activity in lung fibroblasts using LNPs of Table 3 is provided.

[0102] FIG. 53A and 53B Pulmonary transfection and encapsulation efficiency data using LNPs of Example 38 is provided.

[0103] FIG. 54A and 54B Pulmonary transfection and encapsulation efficiency data using LNPs of Example 39 is provided.

[0104] FIG. 55 Pulmonary mean radiant emittance of lyophilized (freeze-dried) LNPs of Example 36 is shown.

[0105] FIG. 56Optimal cationic lipid to ionizable lipid ratios (“C / I ratio”) and optimal lipid nitrogen:polynucleotide phosphate ratios (“N / P ratio”) were modeled for LNPs containing ionizable lipids of the SS-OP family or DLin-MC3-DMA family. Gray boxes indicate the optimal ranges for the C / I and N / P ratios. Horizontal dashed lines indicate the selected threshold for relative lung radiance, such that the peak of relative lung radiance in each plot lies substantially above the dashed line. Horizontal dashed lines represent the threshold for relative yield, such that the convex curve of relative yield lies substantially above the dashed line. The limits of the optimal ranges (gray boxes) are determined by the intersections of the relative lung radiance and relative yield curves with the horizontal dashed line or the dotted threshold line, respectively.

[0106] FIG. 57 An exemplary flow cytometry gating strategy is illustrated, including gating live bone marrow cells using DAPI.

[0107] FIG. 58A , 58B 58C provides flow cytometry results of bone marrow cells collected six days after administration of mRNA-LNP, which encodes Cre recombinase in exemplary PDS and 3-MC3 LNP and lipid formulation.

[0108] FIG. 58D Involving FIG. 58A , 58B The results for 58C are presented, along with exemplary bone marrow transfection efficiency results for PDS and 3-MC3 LNP.

[0109] FIG. 59A and 59B Fluorescence imaging data (compared to control) are shown in Ai14tdTomato flox / flox mice that received injections of exemplary 3-MC3 containing Cre mRNA and PDS LNP. FIG. 59A The test Cy3 channel is shown, and FIG. 59B The Cy7 channel is shown in the comparison.

[0110] FIG. 60A The dorsal fluorescence imaging data (whole-body gating) of Ai14tdTomato flox / flox mice that received injections of exemplary 3-MC3 containing Cre mRNA and PDS LNP are shown (compared to control).

[0111] FIG. 60B Showing FIG. 60A Quantitative analysis of Cy3 images.

[0112] FIG. 61AThe dorsal fluorescence imaging data (gated around the limbs, tail, and pelvis) of Ai14tdTomato flox / flox mice that received injections of exemplary 3-MC3 containing Cre mRNA and PDS LNP are shown (compared to control).

[0113] FIG. 61B Showing FIG. 61A Quantitative analysis of Cy3 images.

[0114] FIG. 62A The results show the assay results of bone marrow cells isolated from the mice in the previous slide 126 days after drug administration. Assume the phylum structure is as follows... FIG. 57 and 58A The results are shown at -58°C. The percentage of tdTomato+ cells in bone marrow is displayed for PBS control, 3-MC3, and PDS.

[0115] FIG. 62B represent FIG. 62A The same data were used, but the percentage of tdTomato+ cells in the PBS control was subtracted from all samples at each time point for direct comparison.

[0116] FIG. 63A The percentage of transfected cells is shown in hepatocytes, splenic white pulp cells, splenic red pulp cells, kidney cells, brain tissue cells, and cells obtained from foot and leg tissues of the same Ai14 mice discussed in conjunction with the data in Figures 58-62 obtained on day 126 post-delivery.

[0117] FIGS. 63B-63F The data shown in Figures 58-63 are discussed in conjunction with the liver data from the same Ai14 mice. FIG. 63B ),spleen( FIG. 63C ), kidney tissue ( FIG. 63D ),leg( FIG. 63E ) and tail( FIG. 63F The example 3-MC3 and PDS LNP-related IHC data are used in the example.

[0118] FIGS. 64A-64B Flow cytometry experiments were shown to demonstrate the targeting of hematopoietic stem cells in mammals (lineage-, Sca1+, cKit+ bone marrow cells) using lipid nanoparticles conjugated with PEGylated lipids.

[0119] FIG. 65 The transfection of hematopoietic stem cells (lineage-, Sca1+, cKit+ bone marrow cells) using multiple doses of PDS LNP is shown. Detailed Implementation

[0120] For the sake of clarity, the detailed description of the application has been divided into the following subsections.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents, applications, published applications and other publications referred to in this section are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition incorporated herein by reference.

[0122] As used herein, “a” or “an” means “at least one” or “one or more” As used herein, the term “about” or “approximately,” as applied to one or more stated reference value. In certain embodiments, unless otherwise stated, or as is otherwise apparent from the context, the term “about” or “approximately” can mean ranges 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 (i.e., greater than or less than) of the stated reference value unless otherwise stated or otherwise apparent from the context. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the application.

[0123] As used herein, the term “and / or” can mean “and,” it can mean “or,” it can mean “exclusive-or,” it can mean “one,” it can mean “some, but not all,” it can mean “neither,” and / or it can mean “both.” The terms“individual,”“subject,” and“patient” are used interchangeably herein and refer to any subject in need of diagnosis, prevention, or treatment. 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., cows, sheep, pigs, horses, goats, etc.), and the like. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate, such as a cynomolgus monkey. In some embodiments, the subject is a companion or service animal (e.g., a cat or dog).

[0124] The term“nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, consisting of monomers (nucleotides) containing a sugar, phosphate, and a purine or pyrimidine base. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also

[0125] The term“nucleotide sequence” refers to a DNA or RNA polymer that can be single- or double-stranded, optionally containing synthetic, non-natural, or altered nucleotide bases capable of being incorporated into a DNA or RNA polymer.

[0126] As used herein, “ribonucleic acid” means a polymer of ribonucleotides.

[0127] As used herein, “mRNA species” means mRNA molecules having the same length and sequence of ribonucleotides.

[0128] Provided herein are compositions that are capable of delivering polynucleotides to sites (tissues and cells) in a subject that are traditionally difficult to reach. This includes, for example, transfection of stem cells (e.g., hematopoietic stem cells, progenitor cells), tumor cells, or regions that receive low levels of circulation. The broad transfection capabilities exhibited by the present compositions enable them to deliver nucleic acid payloads to any of a variety of body tissues and cells, with or without the inclusion of specific targeting ligands (the inclusion of these ligands (e.g., 3-MC3 and / or PDS) in the LNP of the present invention is also contemplated), including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenic cells, hepatic cells, renal cells, and / or bone cells. Such LNP (e.g., 3-MC3 and / or PDS) have very diverse application opportunities as they have been shown to be capable of transfecting a wide variety of cell and tissue types. The present disclosure is not intended to be limited to the use of the presently described LNP (particularly those listed in Table 7 as 3-MC3 and PDS) in any particular tissue or cell type. While stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenic cells, hepatic cells, renal cells, and / or bone cells are specifically illustrated, these are merely examples.

[0129] The presently described LNP (e.g., 3-MC3 and / or PDS) can also include the attachment of ligands to the surface of the liposome. In some embodiments, the lipid particle comprises a targeting agent, such as a targeting lipid described herein. Targeting is typically a moiety specific for a cell type or tissue. The targeting of LNP (e.g., 3-MC3 and / or PDS) using various targeting moieties (such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin)) and monoclonal antibodies is a known concept. The targeting moiety can include an entire protein or a fragment thereof. The targeting mechanism generally requires positioning of the targeting agent on the surface of the lipid particle so that the targeting moiety is able to interact with the target (e.g., cell surface receptor). A variety of different targeting agents and methods are known in the art and can be used.

[0130] In one approach, a ligand (e.g., an antibody) that targets the lipid particle is conjugated to the polar head group of the lipid that forms the lipid particle. Standard methods for conjugating targeting agents can be used. Examples of targeting moieties can include other proteins specific for cellular components, including antigens associated with stem cells, progenitor cells, germ cells, differentiated cells and terminally differentiated cells, neoplasms and tumors. Proteins used as targeting moieties can be covalently attached to the liposome (see Heath, Covalent Attachment of Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)). Other targeting methods include the biotin-avidin system, the maleimide-thiol reaction, and the succinimidyl ester-amine reaction.

[0131] A wide variety of entities can be attached to the oligonucleotides and lipids of the present disclosure. Preferred moieties are ligands that are bound, preferably covalently bound, directly or indirectly through an intermediate tether.

[0132] In preferred embodiments, the ligand alters the distribution, targeting, or lifetime of the molecule to which it is bound. In preferred embodiments, the ligand is selected from a target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cellular compartment or an organ compartment), a tissue, an organ or a region of the body to which affinity is enhanced). Ligands that enhance affinity for a selected target are also referred to as targeting ligands. Preferred ligands for conjugation to the lipids of the present application are targeting ligands.

[0133] Some ligands can have endosome lysing properties. Endosome lysing ligands facilitate endosome lysis and / or transport of the compositions of the present application or components thereof from the endosome to the cytoplasm of the cell. Endosome lysing ligands can be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenic properties. In certain embodiments, the endosome lysing ligand assumes its active conformation at endosomal pH. An “active” conformation is a conformation in which the endosome lysing ligand facilitates endosome lysis and / or transport of the compositions of the present application or components thereof from the endosome to the cytoplasm of the cell. Exemplary endosome lysing ligands include GALA peptides and derivatives thereof. In certain embodiments, the endosome lysing component can comprise a chemical group (e.g., an amino acid) that changes charge or protonates in response to a change in pH. The endosome lysing component can be linear or branched.

[0134] Preferred ligands can improve transport properties, hybridization properties, and specificity properties, and the resulting natural or modified oligoribonucleotides, or monomers and / or natural ribonucleotides described herein. It can also increase the nuclease resistance of a polymer molecule containing any combination of nucleotides or modified ribonucleotides.

[0135] Ligands generally include, for example, therapeutic modifiers for enhancing uptake; diagnostic compounds or reporter groups for monitoring distribution; cross-linking agents; and moieties that confer nuclease resistance. It can. General examples include lipids, steroids, vitamins, saccharides, proteins, peptides, polyamines, and peptidomimetics.

[0136] Ligands include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can be recombinant molecules or synthetic molecules such as synthetic polymers (e.g., synthetic polyamino acids), oligonucleotides (e.g., aptamers). Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether maleic anhydride copolymer, polyamino acid that is N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphadine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine-based salt, or alpha helical peptide.

[0137] The ligand can also comprise a targeting group, such as a cell targeting agent or a tissue targeting agent (e.g., a lectin, a glycoprotein, a lipid, or a protein (e.g., an antibody that binds to a particular cell type such as a stem cell, a progenitor cell, a germ cell, a differentiated cell, a terminally differentiated cell, or a particular cell type, e.g., a kidney cell)). Types of targeting groups include antibodies, peptides, aptamers, small molecules, carbohydrates, lipids, nanobodies, and aptamer-antibody conjugates. Examples of targeting groups include a thyroid stimulating hormone, a melanocyte stimulating hormone, a lectin, a glycoprotein, a surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, a N-acetylgalactosamine, a N-acetylglucosamine, a multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, a transferrin, a bisphosphonate, a polyglutamic acid, a polyaspartic acid, a lipid, a cholesterol, a steroid, a bile acid, a folate, a vitamin B12, a biotin, an RSG peptide, an RSG peptide mimetic, or can be an aptamer. Other examples include antibodies, nanobodies, and aptamers that target a cluster of differentiation on the surface of a cell, e.g., CD1a, CD1b, CD1c, CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49d, CD49e, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62L, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117,CD118, CD119, CD120, CD121, CD122, CD123, CD124, CD125, CD126, CD127, CD128, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140, CD141, CD142, CD143, CD144, CD145, CD146, CD147, CD148, CD149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158, CD159, CD160, CD161, CD162, CD163, CD164, CD165, CD166, CD167, CD168, CD169, CD170, CD171, CD172, CD173, CD174, CD175, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD187, CD188, CD189, CD190, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CD198, CD199, CD200, CD201, CD202, CD203, CD204, CD205, CD206, CD207, CD208, CD209, CD210, CD211, CD212, CD213, CD214, CD215, CD216, CD217, CD218, CD219, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235, CD236, CD237, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, CD259, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282,CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD291, CD292, CD293, CD294, CD295, CD296, CD297, CD298, CD299, CD300, D301, CD302, CD303, CD304, CD305, CD306, CD307, CD308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD323, CD324, CD325, CD326, CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD341, CD342, CD343, CD344, CD345, CD346, CD347, CD348, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD356, CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, CD372, CD373, CD374, CD375, CD376, CD377, CD378, CD379, CD380, CD381, CD382, CD383, CD384, CD385, CD386, CD387, CD388, CD389, CD390, CD391, CD392, CD393, CD394, CD395, CD396, CD397, CD398, CD399, and CD400.

[0138] Other examples of ligands include dyes, intercalators (e.g., acridines), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, suffirin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)glycholic acid, 03-(oleoyl)lithocholic acid, dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, amines, thiols, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabels, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folate), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates), Gates, tetraazamacrocyclic Eu3+complexes), dinitrophenyl, HRP, or AP.

[0139] The ligand can be a protein (e.g., a glycoprotein), or a peptide (e.g., a molecule with specific affinity for a co-ligand) or an antibody (e.g., specific for a particular type of cell, e.g., a stem cell, progenitor cell, germ cell, differentiated cell or terminally differentiated cell, a cancer cell, an endothelial cell, or a bone cell). The ligand can also include hormones and hormone receptors. Ligands include non-peptides, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine, multivalent mannose, multivalent fucose, or aptamer species can also be included. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.

[0140] The ligand is a substance that increases uptake of the polynucleotide of the LNP into a cell, e.g., by increasing the affinity of the LNP for a molecule on the surface of the cell, or increasing the affinity of the LNP for a molecule that in turn binds to a molecule on the surface of the cell, or by disrupting the cytoskeleton of the cell (e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell, e.g., a drug). The drug can be, for example, taxane, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanosine, or myoserbin.

[0141] Ligands can increase uptake of the polynucleotide of the LNP into a cell, for example, by endocytosis, or by activating an inflammatory response. Exemplary ligands with such effects include tumor necrosis factor alpha (TNFa), interleukin-1 beta, or gamma interferon.

[0142] In one aspect, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow the conjugate to distribute to target tissues (e.g., target tissues other than the human kidney). For example, the target tissue can be bone marrow containing hematopoietic stem cells. Other molecules that can bind to target tissues can also be used as ligands.

[0143] Lipid-based ligands can be used, for example, to control binding of the conjugate to target tissues.

[0144] The LNP of the disclosure (e.g., 3-MC3, PDS, etc.) are provided in combination with one or more targeting ligands for use in the diagnosis, prevention, and / or treatment of suspected or actual underlying diseases or conditions in specifically targeted cell types and / or tissues, including those listed in the SUMMARY above. In each case in which a polynucleotide is delivered for the diagnosis, prevention, and / or treatment of a condition or disease, the presently contemplated LNP is capable of targeting the cell types and tissues in which the condition or disease manifests. In this regard, the presently described LNP have been demonstrated to provide the ability to transfect cells throughout the body of a subject with an encapsulated polynucleotide, including cell types present throughout the body. While the polynucleotide encapsulated in the LNP will vary depending on the condition or disease and the purpose of delivery (diagnosis, prevention, or treatment), the presently described LNP are provided as effective delivery vehicles for such polynucleotides so that they can be delivered to areas of the subject (e.g., mammal) where in vivo delivery of polynucleotides has previously been difficult or impossible.

[0145] While telomere elongation can provide potential benefits in alleviating some age-related changes, it is critical to consider the associated risks, such as cellular immortalization, which results from the delivery of telomerase DNA into cells. In contrast, as demonstrated by the present inventors, delivery of mRNA or circular RNA encoding telomerase reverse transcriptase (TERT) is able to achieve safe telomere elongation in vitro and in vivo in a wide range of different cell populations without the risk of immortalization. Specifically, in a wide range of different cell populations across various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells, the use of TERT mRNA or TERT circular RNA delivered in vivo with the delivery vehicles disclosed herein provides a means to provide a variety of benefits. One such benefit includes enhancing the replicative and regenerative capacity of a wide range of different cell populations, where the increase in telomerase activity can delay replicative senescence of the specifically targeted cell population, thereby facilitating tissue renewal and homeostasis in vivo. Another benefit is to help preserve or enhance tissue architecture in a variety of organs and tissues, where improved structural integrity through telomere lengthening can prevent or slow the rate of degeneration of the tissue. Yet another such benefit includes the ability to accelerate wound healing in tissues, where the promotion of cell proliferation and migration can enhance the regenerative capacity of the tissue. Further, another benefit includes reducing the risk of cancer in a wide range of different cell populations, whereby maintaining telomere length and preventing chromosomal instability, the telomere lengthening strategy can provide a potential approach to reduce the risk of a variety of cancers associated with aging.

[0146] To provide these benefits, the LNP described herein can be used to deliver diagnostic and / or therapeutic agents, such as mRNA (including TERT mRNA), to cells in methods of treating, ameliorating, or preventing a condition or disease that can be modulated by an engineered polynucleotide, such as a modified mRNA. In this regard, a variety of conditions or diseases are contemplated, where the condition or disease itself or its symptoms can be modulated (e.g., treated, ameliorated, or prevented) by the presently described and encompassed LNP compositions and formulations. These include preventing, treating, or ameliorating a condition or disease in a wide range of different cell populations across various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0147] The data provided herein demonstrate or support, for example, the in vivo pharmacokinetics of the exemplified LNP and, in particular, information and data relating to the time course of concentration levels of lipids from the LNP in various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. In the embodiments described herein, LNP compositions and formulations are provided that are rapidly cleared from the circulatory system of a subject but have a longer half-life in tissues of the body.

[0148] The data provided herein also demonstrate or support, for example, the use of LNP in which the mRNA encodes a telomerase.

[0149] Also provided is demonstration of the pharmacodynamics of the presently described 3-MC3 and PDS LNP, in particular, the time course of protein expression activity in various tissues following intravenous infusion of the LNP in mice.

[0150] Also provided herein are methods of manufacturing LNP for introducing a nucleic acid into a tissue of the body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. In one particular example, the LNP is manufactured in a manner that forms the LNP prior to addition of the nucleic acid. In particular embodiments thereof, such LNP is a 2, 3, or 5 lipid LNP as described herein and the method generally employs vortex mixing during addition of the nucleic acid.

[0151] The present inventors have discovered that the ratio of cationic lipid to ionizable lipid (e.g., SSOP:DOTAP or MC3:DOTAP) in LNP suitable for working according to the presently contemplated methods and uses is unpredictable depending on the specific composition of the LNP. In particular, while not intending to be bound by any particular theory, experiments have shown that the specific ionizable lipid in the LNP influences the ratio of cationic lipid to ionizable lipid in a manner that requires experimental demonstration. The experimental evidence provided herein supports a specific ratio of cationic lipid to ionizable lipid or a specific range of ratios for particular cationic lipids and ionizable lipids based on the ionizable lipids identified in the LNP. Tables and figures provided herein (e.g., Table 7) provide additional examples of specific ratios of cationic lipid to ionizable lipid for 3 and 5 lipid LNP contemplated herein. In certain embodiments, the ratio of cationic lipid to ionizable lipid is 0.8 or about 0.8, and the ionizable lipid is SS-OP. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 0.8 or about 0.8, and the LNP consists of 3 or 5 lipids. Further in certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 0.8 or about 0.8 and the LNP consists of 3 or 5 lipids, including DOTAP. Further in certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 0.8 or about 0.8 and the LNP consists of 3 or 5 lipids, including a pegylated lipid (e.g., DMG-PEG2000) and DOTAP. Such LNP are used in the presently described methods to introduce a gene payload (e.g., mRNA, including TERT mRNA) in an operable form such that the gene payload is expressed in various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0152] In certain embodiments, the ratio of cationic lipid to ionizable lipid is 1.8 or about 1.8, and the ionizable lipid is SS-OP. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 1.8 or about 1.8, and the LNP consists of 5 lipids. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 1.8 or about 1.8, and the LNP consists of 5 lipids, including DOTAP and DOPC. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 1.8 or about 1.8, and the LNP consists of 5 lipids, including a pegylated lipid (e.g., DMG-PEG2000), DOPC, and DOTAP. Such LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in an operable form such that the genetic payload is expressed in various tissues and cell types throughout the mammal, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0153] In certain embodiments, the ratio of cationic lipid to ionizable lipid is 1.5 or about 1.5, and the ionizable lipid is DLin-MC3-DMA. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 1.5 or about 1.5, and the LNP consists of 3 lipids. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 1.5 or about 1.5, and the LNP consists of 3 lipids, including DOTAP. In certain related embodiments, the ratio of cationic lipid to ionizable lipid of the LNP is 1.5 or about 1.5, and the LNP consists of 3 lipids, including a pegylated lipid (e.g., DMG-PEG2000) and DOTAP. Such LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in an operable form such that the genetic payload is expressed in various tissues and cell types throughout the mammal, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0154] 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 composition means either the final LNP composition or the formulation used to produce the LNP, or both, as will be apparent from the context in which the phrase is used. Specific examples of contemplated compositions are provided in the Examples, including supporting figures. For example, particularly preferred LNP compositions of the present disclosure are provided in Table 7, as well as other tables, figures, and descriptions provided herein.

[0155] According to preferred embodiments herein, there are provided nucleic acid- containing LNP compositions composed of five different lipids, including an ionizable lipid and a cationic lipid, and having an N / P ratio of between 10 and 30. Table 7 provides some such exemplary compositions. Typically according to such embodiments, such LNP compositions are further characterized as having a ratio of cationic lipid to ionizable lipid of 0.8 to 1.8. Further according to such embodiments, such LNP compositions are further characterized as having a ratio of cationic lipid to ionizable lipid of 0.8 or about 0.8. Further according to such embodiments, such LNP compositions include SS-OP as the ionizable lipid in the composition. Such LNPs are used in the presently described methods to introduce a gene payload (e.g., mRNA, including TERT mRNA) in operable form such that the gene payload is expressed in various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. According to such embodiments, such LNP compositions are used in vivo in a mammalian subject, and target various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells, in a manner that provides transfection of the nucleic acid payload in various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. Thus, such compositions are contemplated herein as suitable for various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. Further according to such embodiments, such LNP compositions preferentially target various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. Further according to such embodiments, such LNP compositions have increased half-life in various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.According to such embodiments, such LNP compositions have a half-life in various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells) of greater than 30 hours, or between 30 and 40 hours, or about 37 hours.

[0156] Further according to preferred embodiments herein, provided are nucleic acid- containing LNP compositions composed of 3 different lipids, including an ionizable lipid and a cationic lipid, and having an N / P ratio of between 4 and 24, or an N / P ratio of between 4.5 and 12. Table 7 provides some such exemplary compositions. Generally according to such embodiments, such LNP compositions are further characterized as having a ratio of cationic lipid to ionizable lipid of 0.8 to 1.5. Further according to such embodiments, such LNP compositions are further characterized as having a ratio of cationic lipid to ionizable lipid of 0.8 or about 0.8. Further according to such embodiments, such LNP compositions include SS-OP as the ionizable lipid in the composition, and the ratio of cationic lipid to ionizable lipid is 0.8 or about 0.8. Further according to such embodiments, such LNP compositions include DLin-MC3-DMA as the ionizable lipid in the composition, and the ratio of cationic lipid to ionizable lipid is 1.5 or about 1.5, and optionally the N / P ratio is 4.5 or about 4.5. Further according to such embodiments, such LNP compositions are for in vivo use in a mammalian subject, and target various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells) in a manner that provides transfection of the nucleic acid payload in various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells). Thus, such compositions are contemplated herein as suitable for targeting various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells). Further according to such embodiments, such LNP compositions preferentially target various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells).

[0157] Further according to preferred embodiments herein, nucleic acid-containing LNP compositions are provided that consist of 3 or 5 different lipids, including DOTAP, including an ionizable lipid and a cationic lipid, and have an N / P ratio of between 4 and 24, or an N / P ratio of 12 or about 12. Further according to preferred embodiments herein, nucleic acid-containing LNP compositions are provided that consist of 3 different lipids, including DLin-MC3-DMA, including an ionizable lipid and a cationic lipid, and have an N / P ratio of between 4 and 24, or an N / P ratio of 4.5 or about 4.5. Such LNPs are used in the presently described methods to introduce a genetic payload (e.g., mRNA, including TERT mRNA) in an operable form such that the genetic payload is expressed in various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0158] Further according to generally included embodiments contemplated herein, nucleic acid-containing LNP compositions are provided that consist 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. Further according to such embodiments, such LNP compositions are used in vivo in a mammalian subject, and target various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells, in a manner that provides transfection of the nucleic acid payload in various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. Accordingly, such compositions are contemplated herein as suitable for targeting various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0159] Also provided herein are methods of producing and / or storing LNP compositions. The present inventors have discovered that sucrose is a surprisingly good cryoprotectant for the presently contemplated LNP compositions. In particular, it has been discovered that the inclusion of between 5% and 25% sucrose in the final composition provides a preferred level of cryoprotection. In particular embodiments, the cryoprotectant (e.g., sucrose) is included at 15% or about 15% of the final composition. Such LNP are used in the presently described methods to introduce a gene payload (e.g., mRNA, including TERT mRNA) in an operable form such that the gene payload is expressed in various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells.

[0160] Delivery of synthetic, nucleoside-modified mRNA encoding TERT (TERT mRNA) results in a transient increase in telomerase activity in various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells, which extends telomeres over a period of hours sufficient to reverse years of telomere shortening.

[0161] The lipid nanoparticle (LNP) vehicles provided by the present inventors herein enable delivery of mRNA to various tissues and cell types of the mammal’s body, including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells, by intravenous (IV) infusion at high tolerability. Administration can also be provided by transdermally, intradermally by micro-needle array, locally (e.g., in combination with a skin barrier permeabilization step such as micro-needle treatment or application of a permeabilization agent such as a mixture containing proteases, lipases, or other such agents known in the art).

[0162] Synthetic mRNA Synthetic ribonucleic acid (RNA) as used herein can refer to any RNA sequence comprising a mutation (point mutation or deletion) or additional nucleotides not present in the wild-type sequence. For example, messenger RNA (mRNA) can refer to a wild-type sequence encoding a human sequence flanked by 1, 2, 3, 10, 100, or more nucleotides added. Similarly, a nucleotide can itself encode an amino acid different from the wild-type, or be modified to reduce immunogenicity in a cell or tissue. In some embodiments, an mRNA sequence can comprise any of the following modifications, including but not limited to untranslated regions (UTRs), 5’ cap, and poly-A tail. In some embodiments, the RNA can be circular and / or self-replicating.

[0163] Illustrative methods of 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 B.W. (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 of making self-replicating mRNA are provided in Tews B.A., 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.

[0164] In some embodiments, the mRNA can comprise a codon-optimized sequence. In some embodiments, the mRNA can comprise a uridine-depleted sequence.

[0165] In some embodiments, the 5’ cap of the ribonucleic acid is a non-immunogenic cap. In some embodiments, the 5’ cap can increase translation of the ribonucleic acid. In some embodiments, the 5’ cap can be treated with a phosphatase to modulate the innate immunogenicity of the ribonucleic acid. In some embodiments, the 5’ cap is an anti-reverse cap analog (“ARCA”), such as a 3’-O-Me-m7G(5’)ppp(5’)G RNA cap structure analog. In some embodiments, the 5’ cap is m7G(5’)ppp(5’)(2’OmeA)pG (also known as CleanCap® AG). In some embodiments, the 5’ cap is m7(3’OmeG)(5’)ppp(5’)(2’OmeA)pG (also known as CleanCap® AG (3’ OMe)).

[0166] The above-mentioned features or other features can increase translation of a protein encoded by the ribonucleic acid, can increase or decrease stability of the ribonucleic acid itself in a cell type-specific or non-cell type-dependent manner, or can do both. In some embodiments, the 5’ UTR and / or 3’ UTR is from a gene with very stable mRNA and / or rapid translation of the mRNA, such as alpha-globin or beta-globin, c-fos, or tobacco etch virus. In some embodiments, the 5’ UTR and 3’ UTR are from different genes or from a different species than the species in which the delivery composition is delivered. The UTRs can also be an assembly of UTR portions from different mRNAs, where the portions are selected to achieve some combination of stability and translation efficiency. The UTRs can also comprise designed sequences that impart properties to the RNA such as cell type-specific stability or non-cell type-dependent stability.

[0167] Ribonucleic acids of the present disclosure can comprise one or more modified nucleosides, and / or comprise a primary sequence of nucleosides that modulates translation, stability, or immunogenicity of the RNA. Most mature RNA molecules in eukaryotic cells contain nucleosides that are modified forms of canonical unmodified RNA nucleosides (i.e., adenine, cytidine, guanosine, and uridine). For example, the 5' cap of mature RNA comprises a modified nucleoside, and other modified nucleosides are commonly found elsewhere in the RNA. These modifications can prevent the RNA from being recognized as foreign. Synthetic RNA molecules made using certain nucleosides are much less immunogenic than unmodified RNA. The immunogenicity can be reduced even further by purifying the synthetic mRNA, for example, by using high performance liquid chromatography (HPLC). The modified nucleosides can be, for example, selected from the nucleosides listed below. In some embodiments, the nucleoside is pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methoxyuridine, or 5-methylcytidine. The primary sequence can be modified in a way that increases or decreases immunogenicity. In some cases, it can be desirable for the modified RNA to retain some immunogenicity.

[0168] Thus, in some embodiments, the ribonucleic acids of the present compositions comprise 1-methylpseudouridine, pseudouridine, 5-methoxyuridine (5-moU), 2-thiouridine, 5-methylcytidine, or another modified nucleoside. Modified nucleosides present in eukaryotic cells include mlA 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-glycinamidoadenosine, t6A N6-threonyl- adenosine, ms2t6A 2-methylthio-N6-threonylcarbamoyladenosine, Ar(p) 2'-O- ribosyladenosine (phosphate), m6 2A N6,N6-dimethyladenosine, m6Am N6,2'-O- dimethyladenosine, m6 2Am N6,N6,2'-O-trimethyladenosine, mlAm 1,2'-O- dimethyladenosine, m3C 3-methylcytidine, m5C 5-methylcytidine, Cm 2'-O- methylcytidine, ac4C N4-acetylcytidine, f5C 5-formylcytidine, m4C N4-methylcytidine, hm5C 5-hydroxymethylcytidine, f5Cm 5-formyl-2'-O-methylcytidine, mlG 1- methylguanosine, m2G N2-methylguanosine, m7G 7-methylguanosine, Gm 2'-O- methylguanosine, m2 2G N2,N2-dimethylguanosine, Gr(p) 2'-O-ribosylguanosine (phosphate), yW wyosine, o2yW peroxynucleoside, OhyW hydroxywyosine, OhyW* undermodified hydroxywyosine, imG inosine, m2,7G N2,7-dimethylguanosine, m2,2,7G N2,N2,7- trimethylguanosine I inosine, mlI 1-methylinosine, Im 2'-O-methylinosine, Q queuosine, galQ galactosyl-queuosine, manQ mannosyl-queuosine, Ψ pseudouridine, D dihydrouridine, m5U 5-methyluridine, Um 2'-O-methyluridine, m5Um 5,2'-O- dimethyluridine, mlΨ 1-methylpseudouridine, Ψm 2'-O-methylpseudouridine, s2U 2- thiouridine, ho5U 5-hydroxyuridine, chm5U 5-(carboxyhydroxymethyl)uridine, mchm5U 5-(carboxyhydroxymethyl)uridine, methyl ester mcm5U 5-methoxycarbonylmethyluridine, mcm5Um 5-methoxycarbonylmethyl-2'-O-methyluridine, mcm5s2U 5- methoxycarbonylmethyl-2-thiouridine, ncm5U 5-carbamoylmethyluridine, ncm5Um 5- carbamoylmethyl-2'-O-methyluridine, cmnm5U 5-carboxymethylaminomethyluridine, m3U 3-methyluridine,m1acp3Ψ 1 -methyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine, cm5U 5-carboxymethyluridine, m3Um 3,2'-O-dimethyluridine, m5D 5-methyl dihydrouridine, τm5U 5-taurinomethyluridine, τm5s2U 5-taurinomethyl-2-thiouridine, 2-aminoadenosine, 2-amino-6-chloropurine riboside, 8-az- adenosine, 6-chloropurine riboside, 5-iodocytidine, 5-iodouridine, inosine, 2'-O-methyl inosine, 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-deazoadenosine, 5-aminoallyluridine, 5-aminoallylcytidine, 8-oxoguanosine, 2-aminopurine riboside, pseudoisocytidine, N1-methylpseudouridine, 5,6-dihydro-5-methyluridine, N6-methyl-2-aminoadenosine, 5-carboxycytidine, 5-hydroxymethyluridine, thienoguanosine, 5-hydroxycytidine, 5-formyluridine, 5-carboxyuridine, 5-methoxyuridine, 5-methoxycytidine, thienouridine, 5-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'-deoxyriboside, 2-amino-2'-deoxyadenosine, 2-aminopurine-2'-deoxyriboside, 5-bromo-2'-deoxycytidine, 5-bromo-2'-deoxyuridine, 6-chloropurine-2'-deoxyriboside, 7-deaza-2'-deoxyadenosine, 7-deaza-2'-deoxyguanosine, 2'-deoxyinosine, 5-propynyl-2'-deoxycytidine, 5-propynyl-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-iodo-2'-deoxycytidine, 5-iodo-2'-deoxyuridine, N6-methyl-2'-deoxyadenosine, 5-methyl-2'-deoxycytidine, O6-methyl-2'-deoxyguanosine, N2-methyl-2'-deoxyguanosine, 8-oxo-2'-deoxyadenosine, 8-oxo-2'-deoxyguanosine, 2-thiothymidine, 2'-deoxy-P-riboside, 5-hydroxy-2'-deoxycytidine, 4-thiothymidine, 2-thio-2'-deoxycytidine, 6-aza-2'-deoxyuridine, 6-thio-2'-deoxyguanosine, 8-chloro-2'-deoxyadenosine, 5-aminoallyl-2'-deoxycytidine, 5-aminoallyl-2'-deoxyuridine, N4-methyl-2'-deoxycytidine, 2'-deoxyzebularine, 5-hydroxymethyl-2'-deoxyuridine, 5-hydroxymethyl-2'-deoxycytidine, 5-propargylamino-2'-deoxycytidine,5-propargylamino-2'-deoxyuridine, 5-carboxy-2'-deoxycytidine, 5-formyl-2'- deoxycytidine, 5-[(3-indolyl)propionamido-N-allyl]-2'-deoxyuridine, 5-carboxy-2'- deoxyuridine, 5-formyl-2'-deoxyuridine, 7-deaza-7-propargylamino-2'-deoxyadenosine, 7-deaza-7-propargylamino-2'-deoxyguanosine, biotin-16-aminoallyl-2'-dUTP, biotin- 16-aminoallyl-2'-dCTP, biotin-16-aminoallylcytidine, N4-biotin-OBEA-2'-deoxycytidine, biotin-16-aminoallyluridine, Dabcyl-5-3-aminoallyl-2'-dUTP, desulfo-biotin-6- aminoallyl-2'-deoxycytidine, desulfo-biotin-16-aminoallyl-uridine, biotin-16-7-deaza-7- propargylamino-2'-deoxyguanosine, cyanine 3-5-propargylamino-2'-deoxycytidine, cyanine 3-6-propargylamino-2'-deoxyuridine, cyanine 5-6-propargylamino-2'- deoxycytidine, cyanine 5-6-propargylamino-2'-deoxyuridine, cyanine 3-aminoallylcytidine, cyanine 3-aminoallyluridine, cyanine 5-aminoallylcytidine, cyanine 5-aminoallyluridine, cyanine 7-aminoallyluridine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'- deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methyluridine, puromycin, 2'-amino-2'- deoxycytidine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxycytidine, 2'-azido-2'- deoxyuridine, cytarabine, uridine arabinoside, 2'-azido-2'-deoxyadenosine, 2'-amino-2'- deoxyadenosine, arabinoadenosine, 2'-fluoro-thymidine, 3'-O-methyladenosine, 3'-O- methylcytidine, 3'-O-methylguanosine, 3'-O-methyluridine, 2'-azido-2'-deoxyguanosine, arabinoguanosine, 2'-deoxyuridine, 3'-O-(2-nitrobenzyl)-2'-deoxyadenosine, 3'-O-(2- nitrobenzyl)-2'-deoxyinosine, 3'-deoxyadenosine, 3'-deoxyguanosine, 3'-deoxycytidine, 3'- deoxy-5-methyluridine, 3'-deoxyuridine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine, 2',3'- dideoxyuridine, 2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 3'-azido-2',3'-dideoxyadenosine, 3'-azido-2',3'-dideoxythymidine, 3'-amino-2',3'-dideoxyadenosine, 3'-amino-2',3'- dideoxycytidine, 3'-amino-2',3'-dideoxyguanosine,3'-amino-2',3'-dideoxythymidine, 3'-azido-2',3'-dideoxycytidine, 3'-azido-2',3'- dideoxyuridine, 5-bromo-2',3'-dideoxyuridine, 2',3'-dideoxymosine, 2'-deoxyadenosine- 5'-0-(l-thiophosphoester), 2'-deoxycytidine-5'-0-(l-thiophosphoester), 2'-deoxyguanosine- 5'-0-(l-thiophosphoester), 2'-deoxythymidine-5'-0-(l-thiophosphoester), adenosine-5'-0- (l-thiophosphoester), cytidine-5'-0-(l-thiophosphoester), guanosine-5'-0-(l- thiophosphoester), uridine-5'-0-(l-thiophosphoester), 2',3'-dideoxyadenosine-5'-0-(l- thiophosphoester), 2',3'-dideoxycytidine-5'-0-(l-thiophosphoester), 2',3'-dideoxyguanosine- 5'-0-(l-thiophosphoester), 3'-deoxythymidine-5'-0-(l-thiophosphoester), 3'-azido-2',3'- dideoxythymidine-5'-0-(l-thiophosphoester), 2',3'-dideoxyuridine-5'-0-(l- thiophosphoester), 2'-deoxyadenosine-5'-0-(l-borano phosphoester), 2'-deoxycytidine- 5'-0-(l-borano phosphoester), 2'-deoxyguanosine-5'-0-(l-borano phosphoester), and 2'- deoxythymidine-5'-0-(l-borano phosphoester).

[0169] Without wishing to be bound by theory, the presence of modified nucleosides and / or nucleotide sequences that alter the secondary structure of the RNA and / or the binding of the RNA to RNA binding proteins or microRNAs can enable the mRNA to avoid activation of the immune response mediated by various receptors, including Toll-like receptors and RIG-1. Non-immunogenic mRNA has been used as a therapeutic agent for mice with local delivery. Kormann et al. (2011) Nature Biotechnology 29: 154-157. In some embodiments, the ribonucleic acid comprises more than one of the above-mentioned nucleosides or combinations of the above-mentioned nucleosides. In some embodiments, the ribonucleic acid comprises 1-methylpseudouridine, 5-methoxyuridine, or pseudouridine and 5-methylcytidine.

[0170] In some embodiments, an immune response to the mRNA can be desired, and the RNA can be modified to induce optimal levels of innate immunity. In other embodiments, an immune response to the mRNA can not be desired, and the RNA can be modified to minimize such a response. The RNA can be modified for either case.

[0171] The ribonucleic acid molecule can be a synthetic ribonucleic acid. As used herein, the term "synthetic" can mean that the ribonucleic acid is prepared under the direction of a human using molecular biology tools, in some embodiments, for example, as described below. Synthetic ribonucleic acids can be prepared, for example, by in vitro synthesis using cellular extracts or purified enzymes and nucleic acid templates. In some embodiments, synthetic ribonucleic acids can be prepared, in part or in whole, by chemical synthesis. Alternatively or additionally, in some embodiments, synthetic ribonucleic acids can be prepared by engineering expression in a cell, followed by destruction of the cell and at least partial purification of the ribonucleic acid.

[0172] Ribonucleic acids of the present disclosure can be prepared using a variety of techniques, as will be appreciated by one of ordinary skill in the art. In some embodiments, ribonucleic acids can be prepared by in vitro synthesis. In some embodiments, ribonucleic acids can be prepared by chemical synthesis. In some embodiments, ribonucleic acids can be prepared by a combination of in vitro synthesis and chemical synthesis. As noted above, the term "synthetic" should be understood to include ribonucleic acids prepared by chemical synthesis, by in vitro synthesis, by in vivo expression and at least partial purification, or by a combination of these or other chemical or molecular biology methods.

[0173] In some embodiments, ribonucleic acids can be purified. As noted above, purification can reduce the immunogenicity of ribonucleic acids, and can be advantageous in some cases. In some embodiments, ribonucleic acids are purified by one or more of HPLC, DNase treatment, protease treatment, or by affinity capture and elution.

[0174] In some embodiments, mRNA sequences can be synthesized as unmodified or modified mRNA. mRNA can be modified to enhance stability and / or to evade immune detection and degradation. Modified mRNA can include, for example, one or more of nucleotide modifications, nucleoside modifications, backbone modifications, sugar modifications, and / or base modifications. In some embodiments, the modified nucleoside is pseudouridine or a pseudouridine analog. In some embodiments, the pseudouridine analog is N-l-methylpseudouridine. In some embodiments, the modified nucleoside is 5-methoxyuridine. In some embodiments, modified nucleosides as used herein can include any of the moieties listed in Table A.

[0175] Table A

[0176] In some embodiments, RNA, e.g., 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 analogs and derivatives thereof, such as 1 -methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1 -methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methyl-inosine, pseudouracil (5-uracil), pseudouridine, N-1 -methyl-pseudouridine, dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxylmethyl)-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, queuosine, β-D-mannosyl-queuosine, wyosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0177] In some embodiments, RNA, e.g., 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 pyrimidines (thymine (T), cytidine (C), uridine), 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-methyl nucleoside, 1 -methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 5-methyl-cytidine, 2,6-diaminopurine, 1 -methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methyl-inosine, pseudouridine, N-1 -methyl-pseudouridine, dihydro-uracil, 2-thio-uracil, 4-thio-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-(carboxyhydroxylmethyl)-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, queuosine, beta-D-mannosyl-queuosine, wyosine, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0178] The preparation of such base, nucleoside, nucleotide, and backbone analogs, modifications, and derivatives are known to those skilled in the art, e.g., from U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and 5,700,642, all of which are incorporated by reference in their entirety.

[0179] In some embodiments, the uridine of the mRNA is depleted by about 80%, about 90%, 95%, 99%, or 100% and replaced with a uridine analog, such as pseudouridine, 5-methoxyuridine, or N-l-methyl-pseudouridine.

[0180] In some embodiments, the RNA can comprise a RNA backbone modification. Generally, a backbone modification is a modification in which the phosphates of the nucleotide backbone contained in the RNA are chemically modified. Exemplary backbone modifications can include, but are not limited to, modifications in which the phosphodiester bond is replaced by a member selected from the group consisting of a peptide, a methylphosphonate, a methylphosphoramidate, a phosphoramidate, a phosphorothioate (e.g., cytidine 5'-0-(l- thio phosphate)), a boranophosphonate, and / or a positively charged guanidinium group, or other ways of replacing the phosphodiester bond.

[0181] In some embodiments, the RNA can comprise a sugar modification. Sugar modifications can 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-methylthioribose, and 2'-deoxy-2'-fluoro- ribonucleotides (2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyuridine), 2'-deoxy-2'-deamine- ribonucleotides (2'-amino-2'-deoxycytidine, 2,-amino-2'-deoxyuridine), 2'-O-alkyl ribonucleotides, 2'-deoxy-2'-C-alkyl ribonucleotides (2'-O-methylcytidine, 2'-methyluridine), 2'-C-alkyl ribonucleotides, and their isomers (2'-arabinocytidine, 2'-arabinouridine), or azido phosphates (2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyuridine).

[0182] 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'-0-(1 -thio triphosphate), 2'-deoxycytidine-5'-0-(1 -thio triphosphate), 2'-deoxyguanosine-5'-0-(1 -thio triphosphate), 2'-deoxythymidine-5'-0-(1 -thio triphosphate), adenosine-5'-0-(1 -thio triphosphate), cytidine-5'-0-(1 -thio triphosphate), guanosine-5'-0-(1 -thio triphosphate), uridine-5'-0-(1 -thio triphosphate), 2',3'- dideoxyadenosine-5'-0-(1 -thio triphosphate), 2',3'-dideoxycytidine-5'-0-(1 -thio triphosphate), 2',3'-dideoxyguanosine-5'-0-(1 -thio triphosphate), 3'-deoxythymidine-5'-0-(1 -thio triphosphate), 3'-azido-2',3'-dideoxythymidine-5'-0-(1 -thio triphosphate), 2',3'- dideoxyuridine-5'-0-(1 -thio triphosphate), 2'-deoxyadenosine-5'-0-(1 -borano triphosphate), 2'- deoxycytidine-5'-0-(1 -borano triphosphate), 2'-deoxyguanosine-5'-0-(1 -borano triphosphate), and 2'-deoxythymidine-5'-0-(1 -borano triphosphate).

[0183] In some embodiments, the mRNA can comprise 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.

[0184] cap structure In some embodiments, the mRNA can comprise a 5' cap structure. The 5' cap can comprise, for example, a triphosphate linkage and a guanine nucleotide with the 7-nitrogen methylated. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')A, G(5')ppp(5')A, and G(5')ppp(5')G. Naturally occurring cap structures include 7-methylguanosine, which is linked via a triphosphate bridge to the 5' end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5')ppp(5')N, where N is any nucleoside. In vivo, the cap is added to the nucleus immediately after transcription initiation by the enzyme guanylyltransferase.

[0185] In some embodiments, the 5' cap can comprise a m7(3' OmeG)(5')ppp(5')(2' OmeA)pG or (CleanCap® 3' OMe) structure. In some embodiments, the 5' cap can comprise m7G(5')ppp(5')G. In some embodiments, an anti -reverse cap analog ("ARCA") or modified ARCA is a 5' cap in which the 2' or 3' OH group is replaced by -OCH3. In some embodiments, the ARCA comprises a 3'-0-Me-m7G(5')ppp(5')G structure. In some embodiments, the 5' cap comprises m7G(5')ppp(5')(2' OmeA)pG. Additional mRNA caps can include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; an unmethylated cap (e.g., GpppG); a methylated cap (e.g., m2'7GpppG), a trimethylated cap analog, or an anti -reverse cap analog (e.g., ARCA; m7,2'0meGpppG, m72'dGpppG, m7'3'0meGpppG, m7,3dGpppG, and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., 'Wove anti-reverse cap analogs with superior translational properties", RNA, 9: 1108-1122 (2003)).

[0186] In some embodiments, a suitable cap is a 7-methylguanylate ("m7G") linked via a triphosphate bridge to the 5' end of the first transcribed nucleotide, resulting in m7G(5')ppp(5')N, where N is any nucleoside. An embodiment of the m7G cap used in embodiments of the present disclosure is m7G(5')ppp(5')G. In some embodiments, the cap is a Cap0 structure. The Cap0 structure lacks a 2'-0-methyl residue of the ribose linked to bases 1 and 2. In some embodiments, the cap is a Capl structure. The Capl structure has a 2'-0-methyl residue at base 2. In some embodiments, the cap is a Cap2 structure. The Cap2 structure has 2'-0-methyl residues linked to bases 2 and 3.

[0187] A variety of m7G cap analogs are known in the art, many of which are commercially available. These include 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. Patent Nos. 8,093,367 and 8,304,529 (including biotinylated cap analogs), incorporated herein by reference.

[0188] In some embodiments, the 5' cap is an inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, m7(3' OmeG)(5')ppp(5')(2' OmeA)pG, CleanCap®, m7(3' OmeG)(5')ppp(5')(2' OmeA)pG, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, Cap2, Cap4, CAP-003, or CAP-225.

[0189] 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.

[0190] tail structure The presence of a "tail" can serve to protect the mRNA from exonuclease degradation. It is believed that poly-A tails stabilize natural messengers and synthetic sense RNAs. Thus, in certain embodiments, a long poly-A tail can be added to the mRNA molecule, thereby making the RNA more stable. The poly-A tail can be added using a variety of art-recognized techniques. For example, a long poly-A tail can be added to synthetic or in vitro transcribed RNA using a poly-A polymerase (Yokoe et al., Nature Biotechnology. 1996; 14: 1252-1256). The transcriptional vector can also encode a long poly-A tail. In addition, the poly-A tail can be added by direct transcription from the PCR product. The poly-A can also be ligated to the 3' end of the sense RNA with an RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., edited by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1991 ed.).

[0191] In some embodiments, the mRNA can comprise a 3' poly(A) tail structure. The poly-A tail can be at least about 10, 50, 100, 200, 300, 400, or at least about 500 nucleotides in length. In some embodiments, the poly-A tail on the 3' end of the mRNA can comprise 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.

[0192] In some embodiments, the mRNA can comprise a 3' poly-C tail structure. The poly-C tail on the 3' end of the mRNA can comprise 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 can be added to or can 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 thus to the translation of the protein. For example, because the length of the poly-A tail can affect the half-life of the sense mRNA molecule, the length of the poly-A tail can be adjusted to alter the level of resistance of the mRNA to nucleases, thereby providing more control over the time course of polynucleotide expression and / or polypeptide production.

[0193] 5' and 3' untranslated regions (UTRs) In some embodiments, the mRNA can include a 5' untranslated region (UTR) and / or a 3' UTR. In some embodiments, the 5' UTR can include one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5' UTR, for example, can include an iron response element. In some embodiments, the length of the 5' UTR can 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 can affect mRNA stability or localization, or a binding site for one or more miRNAs. In some embodiments, the length of the 3' UTR can be between about 0 and about 50 nucleotides, or between about 50 to about 100 nucleotides.

[0194] Exemplary 3' and 5' UTR sequences can be derived from mRNAs having a relatively long half-life (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzymes) to increase the stability of the sense mRNA molecule. For example, the 5' UTR sequence can include a portion of the sequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or to extend the half-life of the polynucleotide. In another example, the 5' UTR can include a sequence of the tobacco etch virus (TEV). In general, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide relative to its unmodified counterpart, and include, for example, modifications made to improve the resistance of such polynucleotides to nucleases in vivo.

[0195] In some embodiments, the UTRs can improve tissue-specific expression in, for example, various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells. In some embodiments, the UTRs can be any of those described in any of PCT Application No. WO2017053297A1 and Patent No. US10519189B2, or functional variants thereof, both of which are incorporated herein in their entirety.

[0196] ionizable lipids In some embodiments of the present disclosure, the LNP can comprise an ionizable lipid, such as SS-OP or an analog thereof, or MC3 or an analog thereof. The charge of the lipid can depend on the pH of the surrounding solution, making it an ionizable lipid. The ionizable lipid can also be cleavable. The ionizable lipid can be cationic at the pH range in the endosome or lysosome of a mammalian cell.

[0197] The ionizable lipid can refer to any of a variety of lipid species that have a net positive charge at a selected pH, such as physiological pH. In some embodiments, the LNP can comprise an ionizable lipid as disclosed in any of WO 2010 / 053572 or WO 2012 / 170930, both of which are incorporated by reference herein in their entirety.

[0198] In some embodiments, the LNP can comprise one or more of MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), DLin-MC3-DMA (4-(dimethylamino)-butanoic acid, (10Z,13Z)-1-(9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester), or an analog thereof, including but not limited to LenMC3, g-LenMC3, MC3MC, MC2C, MC2MC, MC3 thioester, MC3 ether, MC4 ether, MC3 alkyne, MC3 amide, Pan-MC3, Pan-MC4, Pan-MC5, CP-LenMC3, CP-g-LenMC3, CP-MC3, D-Lin-MC2-DMA, Lipid 5, SM-102, ALC-0315, and combinations thereof.

[0199] In some embodiments, the LNP can comprise one or more of cKK-E12 (3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), DLinDAP (1,2-dilinoleyl-3-dimethylammonium-propane), DLin-DMA, DLin-D-DMA, DLin-KC2-DMA (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), and DODMA.

[0200] In some embodiments, the ionizable lipid can comprise SS-OP or an analog thereof. In some embodiments, the ionizable lipid is a compound of Formula (1):

[0201] In Formula (1): R 1a and R 1b each independently represents an alkylene group having 1 to 6 carbon atoms, and can be linear or branched. The alkylene group can have 1 to 4 carbon atoms, or can have 1 to 2 carbon atoms. Specific examples of the alkylene group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a trimethylene group, an isopropylene group, a tetramethylene group, an isobutylene group, a pentamethylene group, and a neopentylene group. R 1a and R 1b may each independently be a methylene group, an ethylene group, a trimethylene group, an isopropylene group, or a tetramethylene group, and can be an ethylene group.

[0202] R 1a may be different or the same as R 1b .

[0203] X a and X b each independently is 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 is a cyclic alkylene having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups and an alkylene tertiary amino group.

[0204] In the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group, the alkyl group having 1 to 6 carbon atoms is branched, although it is linear. The alkyl group can be cyclic. The alkyl group can have 1 to 3 carbon atoms. Specific examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, an isobutyl group, a t-butyl group, a pentyl group, and an isopentyl group. A neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, a cyclohexyl group, and the like can be mentioned.

[0205] Specific structures of the acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group are represented by X 1 .

[0206]

[0207] X 1 of R 5 represents an alkyl group having 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 the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, an isobutyl group, a t-butyl group, a pentyl group, and an isopentyl group. A neopentyl group, a t-pentyl group, a 1,2-dimethylpropyl group, a 2-methylbutyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,2-dimethylbutyl group, a 2,3-dimethylbutyl group, a cyclohexyl group, and the like.

[0208] 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 can 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 aziridinylene group, an azetidinylene group, a pyrrolidinylene group, a piperidinylene group, an imidazolidinylene group, a piperazinylene group, an optional pyrrolidinylene group, a piperidinylene group, or a piperazinylene group.

[0209] Specific structures of the alkylene tertiary amino group having 2 to 5 carbon atoms and containing 1 cyclic tertiary amino group are represented by X 2 .

[0210]

[0211] X 2 is 1 or 2. When p is 1, X 2is a piperidinylene group, and when p is 2, X 2 is a piperidinylene group.

[0212] Specific structures of the cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 2 tertiary amino groups are represented by X 3 .

[0213]

[0214] X 3 is 1 or 2. When w is 1, X 3 is an imidazolidinylene group, and when w is 2, X 3 is a piperazinylene group.

[0215] X a may be different from X b .

[0216] R 2a and R 2b each independently represent an alkylene group having 8 or less carbon atoms or an oxydi-alkylene group, optionally each independently represent an alkylene group having 8 or less carbon atoms.

[0217] The alkylene group having 8 or less carbon atoms can be linear or branched, but is optionally linear. The number of carbon atoms contained in the alkylene group is optionally 6 or less, and is optionally 4 or less. Specific examples of the alkylene group having 8 or less carbon atoms include 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, and the like. In some embodiments, including a methylene group, an ethylene group, a propylene group, and a tetramethylene group.

[0218] The oxydi-alkylene group having 8 or less carbon atoms refers to an alkylene group via an ether bond (alkylene-O-alkylene), and the total number of carbon atoms of the two alkylene groups is 8 or less. Here, the two alkylene groups can be the same or different, but are optionally the same. Specific examples of the oxydi-alkylene group having 8 or less carbon atoms include an oxydi-methylene group, an oxydi-ethylene group, an oxydi-propylene group, and an oxydi-butylene group.

[0219] R 2a may be the same as or different from R 2b .

[0220] Y a and Y beach independently an ester linkage, an amide linkage, a carbamate linkage, an ether linkage, or a urea linkage, optionally each independently an ester linkage, an amide linkage, or a carbamate linkage. While Y a and Y b are not limited in their bonding orientation, if Y a and Y b are an ester linkage, optionally -Z a -CO- -R 2a - and -Z b -CO-O-R 2b - structure.

[0221] Y a may be different or the same as Y b .

[0222] 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. Further, the number of aromatic rings contained in the aromatic compound is optionally one.

[0223] The aromatic rings contained in aromatic compounds having 3 to 16 carbon atoms can be, optionally, benzene, naphthalene, or anthracene rings, including aromatic hydrocarbon rings such as benzene, naphthalene, and anthracene rings, as well as aromatic heterocycles such as imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, triazine, pyrrole, furanothiophene, pyrimidine, pyridazine, pyridine, purine, pteridine, benzimidazole, indole, benzofuran, quinazoline, phthalazine, quinoline, isoquinoline, coumarin, chromone, benzodiazepine, phenoxazine, phenothiazine, and acridine rings. The aromatic rings may have substituents. 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, and acyloxy groups having 2 to 4 carbon atoms, amide groups having 2 to 4 carbon atoms, alkoxycarbonylamide groups having 2 to 4 carbon atoms, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, alkylthioyl groups having 1 to 4 carbon atoms, alkylsulfonyl groups having 4 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, ureoyl groups having 1 to 4 carbon atoms, alkoxy groups having 1 to 4 carbon atoms, aryl groups having 6 to 10 carbon atoms, and aryloxy groups having 6 to 10 carbon atoms. Some examples include acetyl groups, methoxycarbonyl groups, methyl carbonate groups, moyl groups, acetoxy groups, acetamide groups, methoxycarbonylamino groups, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methylthio groups, benzenesulfonyl groups, nitro groups, trifluoromethyl groups, cyano groups, methyl groups, ethyl groups, propyl groups, isopropyl groups, tert-butyl groups, ureo groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups, phenyl groups, and phenoxy groups.

[0224] Z a and Z b The specific structure includes Z 1 .

[0225]

[0226] Where s represents an integer from 0 to 3, t represents an integer from 0 to 3, u represents an integer from 0 to 4, and u-digit R4 represents a substituent independently.

[0227] Z 1 S in the equation can be any integer from 0 to 1.

[0228] Z 1 T in the equation can be any integer from 0 to 2.

[0229] Z 1U in the formula (1) is an integer of 0 to 2.

[0230] Z 1 R 4 in the formula (1) 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 ionizable lipids. Examples of the substituent 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, and 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 of 6 to 10 carbon atoms, nitro groups, trifluoromethyl groups, cyano groups, alkyl groups having 1 to 4 carbon atoms, ureido groups having 1 to 4 carbon atoms, alkoxy groups having 1 to 4 carbon atoms, aryl groups having 6 to 10 carbon atoms, and aryloxy groups having 6 to 10 carbon atoms, and examples include acetyl groups, methoxycarbonyl groups, methylcarbamoyl groups, acetoxy groups, Mido groups, methoxycarbonylamino groups, fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methylthio groups, phenylsulfonyl groups, nitro groups, trifluoromethyl groups, cyano groups, methyl groups, ethyl groups, propyl groups, isopropyl groups, tert-butyl groups, ureido groups, methoxy groups, ethoxy groups, propoxy groups, isopropoxy groups, tert-butoxy groups, phenyl groups, and phenoxy groups. When a plurality of R 4 may be the same or different. 4

[0231] Z a may be different from or even the same as Z b

[0232] R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group with succinic anhydride or glutaric anhydride or a sterol derivative having a hydroxyl group with succinic anhydride or glutaric acid. denotes a residue derived from a reaction product with an anhydride, or a C 12-22 aliphatic hydrocarbon group, and optionally each independently a residue of a reaction product of a fat-soluble vitamin having 12 to 22 carbon atoms and optionally each independently a hydroxyl group with succinic anhydride or glutaric anhydride. or a C 12-22 aliphatic hydrocarbon group, and optionally each independently a C 12-22 aliphatic hydrocarbon group.

[0233] ​​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.

[0234] Examples of sterol derivatives having a hydroxyl group include cholesterol, cholestanol, stigmasterol, β-sitosterol, lanosterol, ergosterol, and the like, optionally cholesterol or cholestanol.

[0235] An aliphatic hydrocarbon group having 12 to 22 carbon atoms can be straight-chained or branched. The aliphatic hydrocarbon group can be saturated or unsaturated. In the case of unsaturated aliphatic hydrocarbon groups, the number of unsaturated bonds contained in the aliphatic hydrocarbon group is typically 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 alkyl groups, alkenyl groups, alkynyl groups, and the like, and optionally includes an alkyl group or an alkenyl group. Specific examples of aliphatic hydrocarbon groups having 12 to 22 carbon atoms include dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, dodecenyI, tridecenyI, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl, octadecatrienyl, eicosa trienyl, eicosa tetraenyl, eicosa pentaenyl, docosa hexaenyl, isostearyl, 1-hexylheptyl, 1-hexylnonyl, 1-octylnonyl, 1-octyIunonyl, 1-decylunonyl, and the like. The aliphatic hydrocarbon group having 12 to 22 carbon atoms is optionally a tridecyl group, a pentadecyl group, a heptadecyl group, a nonadecyl group, a heptadecenyl group, a heptadecadienyl group, or a 1-hexylnonyl group, or a tridecyl group, a heptadecyl group, a heptadecenyl group, and a heptadecadienyl group.

[0236] In one embodiment of the present disclosure, R 3a and R 3bThe aliphatic hydrocarbon group having 12 to 22 carbon atoms represented by R

[0237] R 3a R 3b may be different or the same.

[0238] In one embodiment of the present disclosure, R 1a is the same as R 1b , X a is the same as X b , R 2a is the same as R 2b , Y a is the same as Y b , and Z a is the same as Z b , R 3a is the same as R 3b .

[0239] Preferred examples of the ionizable lipid represented by formula (1) include the following ionizable lipids: the ionizable lipid (1-1); R1aand R1bare each independently an alkylene group having 1 to 6 carbon atoms (for example, a methylene group, an ethylene group); Xaand Xbare each independently an acyclic alkyl tertiary amino group having 1 to 6 carbon atoms and 1 tertiary amino group (for example, -N(CH3)-), or a cyclic alkylene tertiary amino group having 2 to 5 carbon atoms and 1 to 2 tertiary amino groups (for example, a piperidinylene group); R 2a and R 2b are each independently an alkylene group having 8 or less carbon atoms (for example, a methylene group, an ethylene group, a propylene group); Y a and Y b are each independently an ester bond or an amide bond; Z a and Z b are each 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. (for example, -C6H4-CH2-, -CH2-C6H4-CH2-); R 3a and R 3b are each independently a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (for example, a tocopherol) with succinic anhydride or glutaric anhydride, or an aliphatic hydrocarbon group having 12 to 22 carbon atoms (for example, a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group).

[0240] the ionizable lipid (1-2); R1a and R 1b each independently is an alkylene group having 1 to 4 carbon atoms (e.g., a methylene group, an ethylene group); Xaand Xbare 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 piperidylene group); R 2a and R 2b each independently is an alkylene group having 6 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y b each independently is an ester bond or an amide bond; Zaand Zbare 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 independently is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., a tocopherol) with 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).

[0241] ionizable lipid (1-3); R 1a and R 1b each independently is an alkylene group having 1 to 2 carbon atoms (e.g., a methylene group, an ethylene group); X a and X b each independently is X 1 :

[0242] wherein R 5 is an alkyl group having 1 to 3 carbon atoms (e.g., a methyl group), or X 2 :

[0243] wherein p is 1 or 2), R 2a and R 2b each independently is an alkylene group having 4 or less carbon atoms (e.g., a methylene group, an ethylene group, a propylene group); Y a and Y b each independently is an ester bond or an amide bond; Z a and Z b each independently is Z 1 :

[0244] 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. R 3a and R 3b each independently is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group (e.g., a tocopherol) with succinic anhydride, or an aliphatic hydrocarbon group having 13 to 17 carbon atoms (e.g., a heptadecenyl group, a heptadecadienyl group, a 1-hexylnonyl group).

[0245] Specific examples of ionizable lipids of Formula 1 according to 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.

[0246] Ionizable Lipid

[0247]

[0248] Ionizable Lipid

[0249] Lipids having the structure of Formula I are shown in the following table. For example, SS-OP is also referred to as 0-Ph-P4C2. The term “SS-OP analog” as used herein refers to a compound of Formula I.

[0250] Lipid Nomenclature

[0251] cationic lipids In some embodiments, the LNP of the present disclosure comprises a cationic lipid, such as DOTAP or a variant thereof. The cationic lipid can be a “permanent cationic lipid.” The term cationic lipid can be cationic over the pH range present in a mammalian physiological environment, such as blood or interstitial fluid. The cationic lipid can be composed of a cationic amine moiety and a lipid moiety, and the cationic amine moiety and the polyanionic nucleic acid can interact to form a positively charged liposome or lipid membrane structure. Thus, uptake into a cell can be facilitated and the nucleic acid delivered into the cell.

[0252] In some embodiments, the cationic lipid can be selected from one or more of 1,2- dioleoyl-3-trimethylammonium-propane (DOTAP), N,N-distearyl-N,N-dimethylammonium bromide (DABB), or 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC). In some embodiments, the LNP comprises an ionizable lipid, wherein the ionizable lipid is one or more of N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermyl glycine dioctadecylamide (DOGS), 2,3-dioleoyloxy-N-[2(spermine- formylamido)ethyl]-N,N-dimethyl-1-propanaminium (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- dilinoleoyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), dimethyl dioctadecyl ammonium (DDA), 1,2-dilinolenoyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N- dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutane-4-oxy)-l-(cis,cis-9,12-octadecadienyloxy)propane (ClinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-l-(cis,cis-9', 1-2'- octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3- dilinoleoyloxy-N,N-dimethylpropanamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamoyl-3- dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K- DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2-DMA), and mixtures thereof.

[0253] In some embodiments, the cationic lipid refers to a cationic cholesterol lipid. In some embodiments of the disclosure, the LNP comprises imidazol cholesterol ester (ICE). In some embodiments, the ICE structure is substantially similar to:

[0254] In some embodiments of the disclosure, the LNP comprises 25-hydroxycholesterol (25 OH Chol). In some embodiments, the 25 OH Chol structure is substantially similar to:

[0255] In some embodiments of the disclosure, the LNP comprises 20a-hydroxycholesterol 5-cholestene-3a.

[0256] In some embodiments, the 20a-hydroxycholesterol 5-cholestene-3a (also referred to as 20a-diol or 20a chol structure) structure is substantially similar to:

[0257] In some embodiments, the cationic lipid refers to dimethyl dioctadecyl ammonium bromide (DDAB). In some embodiments of the disclosure, the LNP comprises dimethyl dioctadecyl ammonium bromide (DDAB). In some embodiments, the dimethyl dioctadecyl ammonium bromide (DDAB) structure is substantially similar to:

[0258] structural lipids In some embodiments, the LNP comprises a structural lipid. As used herein, a structural lipid is a lipid that contributes physical or chemical properties to the LNP in addition to, or independent of, charge. For example, a structural lipid can tend to have a shape, size, rigidity, hydrophobicity, or other properties that increase the diagnostic and / or therapeutic utility of the LNP, such as by increasing its stability, half-life, deformability, transfection efficiency, tropism, thermal stability, resistance to aggregation, membrane fluidity, or other parameters. In some embodiments, the structural lipid is charge neutral, either due to lack of a charged moiety, or due to being a zwitterion, with balanced charges that total to a net charge of zero.

[0259] In some embodiments, the LNP can comprise a structural lipid selected from one or more of: 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), glycerol- monooleate (GMO), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-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.

[0260] In some embodiments, the LNP can comprise one or more phosphatidyl lipids, such as phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine). In some embodiments, the LNP can comprise a sphingolipid, such as, but not limited to, sphingosine, ceramide, sphingomyelin, cerebrosides, and gangliosides. In some embodiments, the foregoing “structural” lipids contribute to the stability and / or specificity of the LNP composition.

[0261] cholesterol-based lipids In some embodiments, the LNP can comprise one or more cholesterol-based lipids. Cholesterol-based lipids can include, but are not limited to: PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylformamidocholine), l,4-bis(3-N-oleylamino-propyl)piperazine.

[0262] PEGylated lipids In some embodiments of the disclosure, the LNP can comprise one or more pegylated lipids. For example, the use of pegylated phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-octanoyl-sphingosine-l-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide), are contemplated by the present disclosure in combination with one or more ionizable lipids and / or other lipids. In some embodiments, the pegylated lipid comprises a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18). In some embodiments, the pegylated lipid DSPE-PEG-maleimide-lectin can be used. Other contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length covalently linked to a lipid with an alkyl chain of C6-C20 length. Without wishing to be bound by a particular theory, it is contemplated that the addition of pegylated lipids can prevent complex aggregation and increase circulation lifetime to facilitate liposome-encapsulated mRNA delivery to target cells.

[0263] Diagnostic and / or therapeutic methods In certain embodiments, the methods of diagnosis treatment and / or prevention as described herein refer to the transfection of various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells of a subject in need) by administering the LNP of the disclosure comprising one or more mRNA sequences. The compositions and methods of the disclosure can be used to diagnose, prevent, and / or treat a condition in various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells) or a condition involving these tissues and cell types. In some embodiments, the compositions of the disclosure and / or methods of use of the compositions intended for the diagnosis, prevention, and / or treatment of fibrotic conditions (including fibrosis). In some embodiments, the compositions of the disclosure and / or methods of use of the compositions intended for the diagnosis, prevention, and / or treatment of fibrotic conditions (including fibrosis) include encapsulated mRNA expression in various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells). In some embodiments, the compositions of the disclosure and / or methods of use of the compositions intended for the diagnosis, prevention, and / or treatment do not induce cellular, tissue, or systemic toxicity. The compositions can be administered systemically (e.g., intravenously), transdermally, intradermally via a microneedle array, topically (typically in conjunction with a skin barrier permeabilization step such as a microneedle treatment or application of a permeabilizing agent such as a mixture containing proteases, lipases, or other materials known in the art). In some embodiments, the compositions of the disclosure and / or methods of use of the compositions intended for the diagnosis, prevention, and / or treatment of a condition in various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells) or a condition involving these tissues and cell types do not induce cellular or systemic toxicity. Compositions comprising the exemplary LNPs described herein and a nucleic acid encoding an active agent (such as a TERT mRNA) for preventing, treating, or ameliorating a condition or disease in various tissues and cell types of the mammalian body (including stem cells, progenitor cells, germ cells, differentiated or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells) can be administered systemically (e.g., intravenously), transdermally, intradermally via a microneedle array, topically (e.g., in conjunction with a skin barrier permeabilization step such as a microneedle treatment or application of a permeabilizing agent such as a mixture containing proteases, lipases, or other materials known in the art).

[0264] According to embodiments provided herein, there are provided methods of delivering a polynucleotide into one or more of a broad range of different cell types of a subject, comprising administering by intravenous injection a polynucleotide encapsulated in a lipid nanoparticle (LNP), the lipid nanoparticle comprising: (i) a SS-OP or SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 0.6 and 1, wherein the polynucleotide comprises a synthetic RNA that is translated in vivo into a corresponding protein encoded by the synthetic RNA in one or more of the different cell types of the subject at or after administration of the LNP.

[0265] According to embodiments provided herein, there are provided methods of delivering a polynucleotide into one or more of a broad range of different cell types of a subject, comprising administering by intravenous injection a polynucleotide encapsulated in a lipid nanoparticle (LNP), the lipid nanoparticle comprising: (i) a SS-OP or SS-OP analog at a molar percentage of between about 20% and about 60%, (ii) a PEGylated lipid at a molar percentage of between about 0.5% and about 2.5%, and (iii) a cationic lipid at a molar percentage of between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 0.6 and 1, wherein the polynucleotide comprises a synthetic RNA that is translated in vivo into a corresponding protein encoded by the synthetic RNA in one or more of the different cell types of the subject at or after administration of the LNP.

[0266] In the above embodiments, according to embodiments described herein, the PEGylated lipid is DMG-PEG2000. Further in the above embodiments, according to embodiments described herein, the cationic lipid is DOTAP. According to presently contemplated embodiments, the LNP of the above methods and methods described herein are composed of the lipids listed in Table 7.

[0267] According to embodiments described herein, the target cell type is located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 different organs of the subject. According to related embodiments described herein, the target cell type is located in 4 to 6 different organs of the subject. According to related embodiments described herein, the target cell type is located in 5 to 8 different organs of the subject. According to related embodiments described herein, the target cell type is located in 7 to 10 different organs of the subject. According to related embodiments described herein, the target cell type is located in 9 to 12 different organs of the subject. According to related embodiments described herein, the target cell type is located in 10 to 15 different organs of the subject.

[0268] Typically, according to current methods, delivery of polynucleotides is used to diagnose, prevent, and / or treat a condition or disease in various tissues and cell types throughout the body of a mammal. Examples of conditions or diseases include influenza, asthma, type 1 diabetes, type 2 diabetes, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer’s disease, Parkinson’s disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn’s disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, stomach cancer, brain cancer, endometrial cancer, bone cancer, sarcoma, multiple myeloma, skin cancer, basal cell carcinoma, squamous cell carcinoma, tuberculosis, pneumonia, bronchitis, sinusitis, otitis media, urinary tract infection (UTI), hepatitis A, hepatitis B, hepatitis C, HIV / AIDS, syphilis, gonorrhea, chlamydia, herpes simplex virus (HSV), human papillomavirus (HPV), scabies, athlete’s foot, ringworm, lice infestation, measles, mumps, rubella, chickenpox, shingles, pertussis, diphtheria, tetanus, polio, rabies, malaria, dengue fever, yellow fever, Zika virus, Lyme disease, Rocky Mountain Spotted Fever, toxoplasmosis, giardiasis, amebiasis, ascariasis, trichinosis, echinococcosis, leishmaniasis, anthrax, botulism, tetanus, plague, cholera, typhoid fever, salmonellosis, campylobacteriosis, listeriosis, C. difficile infection, norovirus infection, rotavirus infection, cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection, parvovirus infection, West Nile virus infection, hantavirus infection, Ebola virus disease, Marburg virus disease, SARS, MERS, COVID-19, sepsis, cellulitis, osteomyelitis, endocarditis, meningitis, encephalitis, Guillain-Barre syndrome, amyotrophic lateral sclerosis (ALS), Huntington’s disease, cystic fibrosis, Duchenne muscular dystrophy, Becker’s muscular dystrophy, fragile X syndrome, Down syndrome, Turner syndrome, Cri du Chat syndrome, Marfan syndrome, Ehlers-Danlos syndrome, polycystic kidney disease, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson’s disease, Gaucher disease, Fabry disease, Niemann-Pick disease, Tay-Sachs disease, Huntington’s disease, Rett syndrome, Prader-Willi syndrome, Angelman syndrome, Sjogren’s syndrome, Addison’s disease, Cushing’s syndrome, acromegaly, gigantism, pheochromocytoma, hyperparathyroidism, hypoparathyroidism, rickets, osteomalacia, osteoporosis, Paget’s disease of bone, gout, bursitis, tendonitis, tennis elbow, carpal tunnel syndrome, plantar fasciitis, fibromyalgia, chronic fatigue syndrome, migraine, tension headaches,Cluster Headache, Epilepsy, Narcolepsy, Restless Leg Syndrome, Sleep Apnea, Insomnia, Bipolar Disorder, Depression, Anxiety, Schizophrenia, Obsessive-Compulsive Disorder (OCD), Post-Traumatic Stress Disorder (PTSD), Attention Deficit / Hyperactivity Disorder (ADHD), Autism Spectrum Disorder (ASD), Anorexia Nervosa, Bulimia Nervosa, Binge Eating Disorder, Alcohol Use Disorder, Substance Use Disorder, Personality Disorder, Somatic Symptom Disorder, Depersonalization / Derealization Disorder, Hypochondriasis, Munchausen Syndrome, Conversion Disorder, Delirium, Dementia, Fetal Alcohol Syndrome, Neonatal Withdrawal Syndrome, Pulmonary Fibrosis, Idiopathic Pulmonary Fibrosis (IPF), Liver Cirrhosis, Liver Fibrosis, Nonalcoholic Steatohepatitis (NASH), Primary Biliary Cholangitis (PBC), Primary Sclerosing Cholangitis (PSC), Chronic Hepatitis B-Related Fibrosis, Chronic Hepatitis C-Related Fibrosis, Systemic Sclerosis (Scleroderma), Cystic Fibrosis, Myocardial Fibrosis, Hypertrophic Cardiomyopathy, Restrictive Cardiomyopathy, Dilated Cardiomyopathy, Chronic Kidney Disease (CKD), Glomerulonephritis, Diabetic Nephropathy, Interstitial Nephritis, Retroperitoneal Fibrosis, Peritoneal Fibrosis, Dupuytren’s Contracture, Peyronie’s Disease, Myelofibrosis, Bone Marrow Fibrosis, Keloid Formation, Scar Tissue Formation, Adhesive Capsulitis (Frozen Shoulder), Chronic Pancreatitis, Pancreatitis-Associated Fibrosis, Crohn’s Disease-Associated Fibrosis, Ulcerative Colitis-Associated Fibrosis, Cutaneous Fibrosis, Radiation-Induced Fibrosis, Radiation Pneumonitis, Post-Surgical Adhesion, Asbestosis, Silicosis, Sarcoidosis-Associated Fibrosis, and Eosinophilic Fasciitis. Cell types can include, for example, stem cells (e.g., hematopoietic stem cells, progenitor cells), differentiated cells, terminally differentiated cells, endothelial cells, epithelial cells, splenocytes, hepatocytes, renal cells, cancer cells, and / or bone cells of the subject. Moreover generally, the methods of delivering polynucleotides according to the current methods are used to modulate a condition or disease in various tissues and cell types throughout the mammal (including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenocytes, hepatocytes, renal cells, and / or bone cells of the subject). Moreover generally, the methods of delivering polynucleotides according to the current methods are used to increase or initiate expression of a protein (e.g., a therapeutic or supplemental protein) in target cells in various tissues and cell types throughout the mammal (including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, splenocytes, hepatocytes, renal cells, and / or bone cells of the subject).

[0269] When it is desirable to prevent such a condition or disease, the presently described LNP compositions are administered at some time prior to the subject having or being suspected of having the condition or disease.

[0270] In each instance in which a polynucleotide is delivered for the purpose of diagnosis, prevention, and / or treatment of a condition or disease, the presently contemplated LNP is capable of targeting the cell types and tissues in which the condition or disease manifests. In this regard, the presently described LNP has been demonstrated to provide the ability to transfect cells throughout the body of a subject with the encapsulated polynucleotide, including cell types present throughout the body. More specific targeting of cell types and tissues for transfection can be provided using a targeting ligand or moiety, generally as desired according to the purpose of diagnosis, prevention, and / or treatment and the particular type or types of conditions or diseases as the subject of administration to a subject. While the polynucleotide encapsulated in the LNP will vary according to the condition or disease and the purpose of delivery (diagnosis, prevention, or treatment), the presently described LNP is provided as an effective delivery vehicle for such polynucleotides so that it can be delivered to areas previously difficult or impossible to deliver a polynucleotide in vivo in a subject (e.g., a mammal).

[0271] In the compositions and methods described herein, in some embodiments, the LNP pharmaceutical compositions contemplated herein are administered to a subject in need thereof at a dose of about 0.001 mg / kg of the subject's body weight to about 2.0 mg / kg of the subject's body weight. In some embodiments, the targeted LNP is administered to a subject in need thereof at a dose of about 0.01 mg / kg; in some embodiments at a dose of about 0.025 mg / kg; in some embodiments at a dose of about 0.05 mg / kg; in some embodiments at a dose of about 0.075 mg / kg; in some embodiments at a dose of about 0.1 mg / kg; in some embodiments at a dose of about 0.125 mg / kg; in some embodiments at a dose of about 0.150 mg / kg; in some embodiments at a dose of about 0.175 mg / kg; in some embodiments at a dose of about 0.2 mg / kg; in some embodiments at a dose of about 0.5 mg / kg; in some embodiments at a dose of about 0.75 mg / kg; in some embodiments at a dose of about 1.0 mg / kg; in some embodiments at a dose of about 1.25 mg / kg; in some embodiments at a dose of about 1.5 mg / kg; or in some embodiments at a dose of about 2.0 mg / kg. In some embodiments, the LNP is administered to a subject in need thereof at a dose of 0.1 mg / kg. In some embodiments, the LNP is administered to a subject in need thereof at a dose of 0.125 mg / kg.

[0272] In some embodiments, the LNP pharmaceutical compositions contemplated herein are administered to a subject in need thereof in a single dose. In some embodiments, the LNP is administered to a subject in need thereof twice, three times, four times, or five times or more. In some embodiments, the LNP is administered to a subject twice weekly, once weekly, 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 LNP is administered to a subject once a month, once every two months, once every three months, once every six months, once a year, for the duration of administration or as determined by their physician.

[0273] In some embodiments, the LNP pharmaceutical compositions contemplated are delivered in an aerosolized inhalation form, orally, subcutaneously, intravenously, intranasally, intradermally, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly. In exemplary embodiments, the targeted LNP is administered intravenously.

[0274] In some embodiments, exemplary LNP pharmaceutical compositions comprise an excipient or carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline. The composition can contain pharmaceutically acceptable auxiliary substances as required, such as those needed to make the composition near physiological conditions, such as pH and buffering agents, toxicity antagonists, for example, sodium acetate, sodium chloride, sodium citrate, potassium chloride, calcium chloride, and sodium lactate. In some embodiments, the pharmaceutical composition comprises 10 mM sodium citrate buffered to pH 6.4. The composition can contain cryoprotectants, such as glycerol, ethylene glycol, sucrose, propylene glycol, or dimethyl sulfoxide (DMSO). The concentration of active agent in these formulations can vary and will be selected based on fluid volumes, viscosities, and body weights of the patient, and the particular mode of administration selected (e.g., Remington’s Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0275] In other embodiments, the compound or composition is administered without isolating one or more cells, tissues, or organs from the subject (i.e., the 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 a particular cell, cell type, tissue, or organ in the subject.

[0276] Administration of the compounds or compositions of the present disclosure can generally result in transient expression of the mRNA within the LNP in the cell. 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, the Trapeze® Telomerase Detection Kit (Millipore), which provides sensitive detection and quantification of telomerase activity, although other measurement techniques are possible.

[0277] As previously noted, one advantage of the present technology is that expression of the mRNA in the LNP is transient in the transfected cell. In particular, this transient expression is in contrast to previous technologies in which, for example, the telomerase reverse transcriptase gene persists in episomal DNA portions, or is inserted into the genomic sequence of the cell, or otherwise permanently modifies the genetic makeup of the target cell, and results in the inherent activity of the nucleic acid sequence.

[0278] In some embodiments of the methods of the present application, the transient expression is independent of the cell cycle.

[0279] Therapeutic and diagnostic kits Also contemplated herein are diagnostic and / or therapeutic kits comprising a pharmaceutical composition of the LNP contemplated herein, or its lipid components provided in separate containers, along with instructions for manufacture and / or use. In some embodiments, the diagnostic and / or therapeutic kits include devices for administration, including but not limited to syringes, microneedles, inhalers, nebulizers, and vials or containers. In certain embodiments of the kits for the diagnosis, prevention, and / or treatment of a disorder or disease of various tissues and cell types of the mammalian body, including stem cells, progenitor cells, germ cells, differentiated cells or terminally differentiated cells, cancer cells, endothelial cells, epithelial cells, spleen cells, liver cells, kidney cells, and / or bone cells, the kits can include devices for transdermal delivery, devices for intradermal injection via a microneedle array, devices for a skin barrier permeabilization step such as microneedles and / or one or more permeabilizing agents such as mixtures containing proteases, lipases, or other materials known in the art. In some embodiments, the components of the exemplary diagnostic and / or therapeutic kits are provided such that they can be mixed with commercially available microfluidic or vortex mixers. Such kits are typically with or without nucleic acids (e.g., mRNA) that will be encapsulated in the LNP made with the components of the diagnostic and / or therapeutic kits.

[0280] In another aspect, the present disclosure provides ready-to-use kits for diagnosing, preventing, and / or treating a condition or disease, e.g., for extending telomeres in mammalian cells. The kits include any of the above-described compounds or compositions, along with instructions for their use. Such instructions include a package insert approved by the regulatory agency of the country in which the kit is marketed, which directs the dosing regimen and includes contraindications. If a step of combining the agents in the kit is required, such instructions are included in the instructions for use of the kit, including the steps set forth herein for preparing the final product in certain embodiments. Typically, such instructions are conveyed to laboratory technicians and / or professional health care personnel, who possess the requisite expertise to prepare the final composition or formulation for administration to a subject. In some embodiments, the kit further includes packaging material. In some embodiments, the packaging material is airtight. In these embodiments, the packaging material can optionally be filled with an inert gas, such as, for example, nitrogen, argon, and the like. In some embodiments, the packaging material includes a metal foil container, such as, for example, a sealed aluminum bag, and the like. Such packaging materials are well known to those of ordinary skill in the art. The kit can also include a delivery vehicle, such as a lipid as described herein. In some embodiments, one or more components of the formulation are set to be frozen with a cryoprotectant or lyophilized.

[0281] In some embodiments, the kit can also include a desiccant, a culture medium, an RNAse inhibitor, or other such components. In some embodiments, the kit can also include a combination of more than one of these additional components. In some kit embodiments, the components of the kit are sterile.

[0282] Example Figures 1 and 48 depict exemplary pharmacokinetics of the 5-lipid LNPs of Table 1. Time lines of plasma, lung, and liver levels of the 5-lipid LNPs as determined by measuring one of the lipids, DOTAP, are presented. In processing these data, the LNPs were formulated with TERT mRNA (human TERT opti - SEQ ID NO: 1) and lipids in the ratios of Table 1. Male C57B1 / 6 mice were dosed at 1 mg / kg, and plasma and tissues were collected at the time points indicated above. DOTAP in plasma, lung, and liver was measured using LC-MS / MS. The nitrogen:phosphorus N / P charge ratio of the Table 1 compositions was 12.

[0283] Table 1

[0284] N / P ratio (nitrogen:phosphorus charge ratio): 12 Example 2 FIG. 2The ratio of bioluminescent signal from intravenously injected mRNA translated luciferase protein from the indicated organs at the indicated time points following intravenous injection of the 5 lipid LNPs of Table 2 is depicted. During this procedure, the LNPs were formulated with firefly luciferase mRNA and lipids in the ratios of Table 2. Male C57B16 mice were dosed at 1.3 mg / kg and tissues were collected and imaged in vitro at the time points above. The mean radiance values were baseline normalized by subtracting the mean radiance from the negative control (no LNP) treated animals. The relative radiance between the lung, liver, and spleen is plotted in FIG. 2 Table 2. The nitrogen:phosphorus N / P charge ratio of the compositions of Table 2 is 12.

[0285] Table 2

[0286] N / P ratio (nitrogen:phosphorus charge ratio): 12 Example 3 FIGS. 3-17 and FIGS. 20-21 Experiments involving the use of different 5 lipid LNP compositions are described in which pulmonary fibrosis is induced in third generation (G3) TERT knockout (KO) mice, which have a telomere length similar to humans. Bleomycin was administered at 2.0 U / kg by oropharyngeal aspiration. For FIGS. 13-16An initial dose of bleomycin was administered at 0.5 U / kg by oropharyngeal aspiration one week prior to the 2.0 U / kg dose. LNP was 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 alpha-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 in the ratios of Table 3. The DOTAP molar ratio in the 5-lipid LNP of Table 3 was 2.27-fold higher. Mice were dosed twice weekly at a dose of 1.5 mg / kg for two weeks starting on day 7 post-bleomycin. Some mice received bleomycin but not LNP treatment (no LNP), and control mice received saline instead of bleomycin and LNP treatment (no bleomycin). Tissues were harvested on day 24 post-bleomycin. Lungs were fixed with paraffin and formalin-embedded. To measure telomere length, sections were stained with a telomere probe and an anti-surfactant precursor protein C (SPC) antibody to mark alveolar type II (AT2) cells using the Q-FISH method. Telomerase activity in lung tissue lysates was measured using the telomeric repeat amplification protocol (TRAP) assay.

[0287] Table 3

[0288] N / P ratio (nitrogen:phosphorus charge ratio): 26.5 FIG. 3 Representative immunofluorescence micrographs of lung sections for measurement of telomere length by the Q-FISH method known in the art are depicted, in which the intensity of the telomere probe is proportional to the length of the telomere, and the cells are co-stained with an antibody to the AT2 cell marker precursor SPC.

[0289] FIG. 4 Measurement of telomere length in mouse AT2 cells by Q-FISH is depicted, in which mice have been induced to fibrosis with bleomycin ("Bleo") as described above. Treatment with TERT mRNA LNP of Table 3 ("TERT" in the figures) increased the median telomere length and the 10th percentile telomere length of AT2 cells (cells scored as precursor SPC+ by immunofluorescence staining) as measured by Q-FISH relative to mice that received luciferase mRNA LNP ("control" in the figures), nearly reaching the length found in normal mice that did not receive bleomycin ("no bleo" in the figures).

[0290] FIG. 5Depicted is the measurement of telomere length in mouse AT2 cells by Q-FISH, where mice have been induced to fibrose with bleomycin ("Bleo") as described above. Treatment with TERT mRNA LNPs in Table 3 ("TERT" in the figure) increased the median telomere length and the 10th percentile telomere length of alveolar cells as measured by Q-FISH relative to mice that received luciferase mRNA LNPs ("Control" in the figure), nearly reaching the length found in normal mice that did not receive bleomycin ("No Bleo" in the figure). Fields that were predominantly filled with alveoli were used for quantification and included telomere lengths of all cell types present. Typically, alveolar tissue includes epithelial cells, endothelial cells, fibroblasts, immune cells, and other cell types.

[0291] 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 normally ventilated lung volume.

[0292] FIG. 6B Depicted is a timeline of the bleomycin mouse experiment described above. FIGS. 13-16

[0293] FIG. 7 Depicted are false color images of lung sections stained with Sirius Red to quantify fibrotic lesions and fibrosis by the area of tissue covered by collagen deposition. IV infusion of TERT mRNA LNPs of Table 3 reduced fibrosis by 62%.

[0294] FIG. 8 Depicted is the reduction of fibrotic lesions in bleomycin-treated mice caused by treatment with TERT mRNA LNPs of Table 3 as measured by Sirius Red staining.

[0295] FIG. 9 Depicted is the increase in the amount of normally ventilated lung, or useful lung volume, in bleomycin-treated mice caused by treatment with TERT mRNA LNPs of Table 3 as measured by x-ray computed tomography imaging followed by voxel-based quantification of fibrotic tissue based on x-ray intensity.

[0296] FIG. 10 Depicted is the improvement in lung elasticity in bleomycin-treated mice caused by treatment with TERT mRNA LNPs of Table 3 as measured by the Flexivent system.

[0297] FIG. 11 and 12 ​The improvements in forced expiratory volume (FEV1) and forced vital capacity (FVC) induced by TERT mRNA LNP in bleomycin-treated mice, as measured by the Flexivent system, are described respectively.

[0298] FIG. 13 , 14 Tables 1 and 15 respectively depict the improvements in alveolar density, alveolar diameter, and alveolar roundness induced by TERT mRNA LNP treatment in bleomycin-treated mice, as measured by machine vision analysis of lung slice micrographs from Biocellvia.

[0299] FIG. 16 The analysis by Biocellvia depicts FIGS. 13-15 An example of false-color images of lung tissue sections produced during quantitative alveolar structure analysis.

[0300] FIG. 17 The results depict telomerase activity in primary human alveolar epithelial cells isolated from a 50-year-old donor at specified time points after treatment with TERT mRNA LNP at a concentration of 500 ng / ml as shown in Table 3, as measured using the telomere repeat amplification protocol (TRAP) assay.

[0301] FIG. 18 The timeline of experiments measuring the effect of TERT mRNA LNP treatment on the colony-forming ability of primary human alveolar epithelial cells isolated from a 50-year-old donor is depicted. Cells were seeded in colony-forming support medium, treated with TERT mRNA LNP at a concentration of 500 ng / ml TERT mRNA, and incubated for 7 days. Colonies were then counted. FIG. 19 Report.

[0302] FIG. 19 The increased colony-forming capacity of human alveolar epithelial cells from 50-year-old donors was described by TERT mRNA LNP treatment as shown in Table 3.

[0303] Example 4 - Reduction in senescence following telomerase mRNA LNP treatment in mice Method: such as regarding FIGS. 3-12 The experiment was conducted.

[0304] Result: As FIGS. 20-21 As shown, compared with mice treated with luciferase mRNA LNP (control), mice treated with telomerase mRNA LNP showed a 40% reduction in the aging marker P21 in alveolar cells. FIG. 21 In the study, dark-colored cells were positive. Bleomycin treatment increased senescence.

[0305] Example 5 - Effective lung delivery using 2 lipid LNPs Method: According to FIG. 22C The ratio of 2-lipoprotein LNP to firefly luciferase mRNA (Luc) was used to formulate the mixture. Male C57Bl / 6 mice were administered the mixture intravenously (IV) at a dose of 1.25 mg / kg, and organs were imaged ex vivo 21 hours later. The figure shows the mean radiance (photons / s / cm). 2 / sr). FIG. 22A -B、D.

[0306] Results: Bioluminescent signals were observed in the lungs with all formulations, indicating that: 1. Low PEG concentrations are toxic – one mouse died at 0.1% PEG, suggesting that the optimal amount may be between 0.2% and 2% PEG. 2. The signal peaked at 0.5% PEG.

[0307] Example 6 - Titration of lipid:mRNA ratio in 2 lipid LNPs Methods: DOTAP and PEG were formulated with firefly luciferase mRNA (Luc). The lipid ratio was 99% DOTAP and 1% PEG. The lipid:mRNA ratio is expressed as NP; that is, the ratio of nitrogen to phosphate molecules. Each DOTAP molecule has a single nitrogen atom, while each nucleotide on the mRNA has a single phosphate phosphate. C57Bl / 6 male mice were administered 1.5 mg / kg intravenously (IV), and the lungs were removed 23 hours later for ex vivo imaging. FIG. 23 The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0308] Results: Bioluminescent signals were observed in the lungs at all N / P ratios. Increased lung signaling corresponded to increased weight loss—more signal indicated greater toxicity. The N / P ratio was the local minimum for both signaling and weight loss. Example 7 - Time course of protein expression following delivery of lung-targeted mRNA- LNP encoding firefly luciferase Methods: LNP was formulated with firefly luciferase mRNA (Luc) according to the ratios in provisional Table 3. C57Bl / 6 male mice were administered the drug intravenously (IV) at 1.5 mg / kg, and ex vivo organ imaging was performed at 16 hours (“Day 1”), 33 hours (“Day 2”), 73 hours (“Day 3”), and 91 hours (“Day 4”). FIG. 24 The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0309] Results: Given the known half-life of firefly luciferase protein, the bioluminescent signal decreased over time, suggesting that the mRNA half-life is not long and that mRNA-LNP does not circulate in the lungs and retransfect cells over time.

[0310] Example 8 - Titration of SS-OP lipids into 'DOTAP + PEG' LNPs Method: According to FIG. 25D The ratio of LNP to firefly luciferase mRNA (Luc) was used to formulate the mixture. C57Bl / 6 male mice were administered the mixture via intravenous (IV) injection at a dose of 1.5 mg / kg, and organs were imaged ex vivo 22 hours later. FIG. 25A The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0311] result: 1. Encapsulation efficiency is 98% or higher, as measured by the Ribo-Green microplate reader method. FIG. 25B 2. Stable body weight indicates low or no toxicity. FIG. 25C 3. Lung radiation rate peaked at 50% SS-OP lipids. 4.5% SS-OP Local maximum Example 9 - Delivery to lung and liver using 2 LNPs Methods: Liver-targeting LNPs were prepared according to the lipid ratios in Table 4, and lung-targeting LNPs were prepared according to the lipid ratios in Table 3. Both were formulated with firefly luciferase mRNA (Luc). CD1 male mice were administered intravenously (IV) at a dose of 1.0 mg / kg, and organs were imaged ex vivo 19 hours later. Conditions: • Lung-targeted LNPs administered only (“Lung-only”) • Liver-targeted LNPs administered only (“liver-only”) • First administer lung-targeted LNPs, then administer liver-targeted LNPs 1 hour later. "(1) Lung (2) Liver" • First administer liver-targeted LNPs, then administer lung-targeted LNPs 1 hour later. "(1) Liver (2) Lung" • Mix lung-targeting LNPs and liver-targeting LNPs and administer twice, 1 hour apart, as a "liver / lung mixture". • FIG. 26A and 26B Provided average emissivity (photons / s / cm) 2 / sr).

[0312] Table 4

[0313] result: 1. When lung-targeting LNPs are administered, the bioluminescent signal in the lungs is consistent. The highest level is observed in the liver / lung mixture. FIG. 26A 2. Liver BLI signaling is variable, but it is very low when lung LNP is administered first. FIG. 26B .

[0314] 3. The optimal order for these two organs is lungs first, then liver. The second best option is to mix and deliver them.

[0315] Example 10 - Titration of SS-OP lipids into 'DOTAP + PEG' LNPs Method: According to FIG. 27A The ratios in the table represent the ratios of LNP to firefly luciferase mRNA (Luc). C57Bl / 6 male mice were administered the drug intravenously (IV) at a dose of 1.5 mg / kg, and organs were imaged ex vivo 23 hours later. FIG. 27C The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0316] Weight was measured and clinical signs were assessed using a scoring system that included a visual assessment of the mice’s appearance and behavior, with particular attention to movement, posture, and eyes (abbreviated “LPE”) (Nunamaker 2013, PMID: 24209966). FIG. 27D .

[0317] result: 1. When the proportion of SS-OP lipids increased to 75%, tolerance decreased, and both mice needed to be euthanized.

[0318] 2. Based on the data from Example 7, this study demonstrates that, compared to other percentages of SS-OP, 35-65% SS-OP provided relatively lung-specific transfection (as measured by bioluminescent signals) and relatively high tolerability (<10% body weight, no clinical signs (0 LPE), no deaths). 55% SS-OP scored particularly well and can be considered optimal in this respect.

[0319] Example 11 - Stability under frozen storage - long term Methods: LNP was formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. It was prepared and administered 'fresh' on the same day, or frozen at -20°C, -80°C, or in liquid nitrogen and thawed after 70 days. Sucrose was added to 7% as a cryoprotectant before freezing. CD1 male mice were administered 1.0 mg / kg via intravenous (IV) injection, and organs were imaged ex vivo 23 hours later.FIG. 28A Average radiance (photons / s / cm 2 / sr) + / - S.E.M.

[0320] Results: No clinical signs and mild weight loss were observed. FIG. 28B In all cases, lung signals were present in similar amounts, indicating that cryopreservation was effective Example 12 - Stability under frozen storage - short term Methods: LNP were formulated with firefly luciferase mRNA (Luc) in the ratios outlined in Table 3. They were either formulated and dosed 'fresh' on the same day, or cryopreserved at 4C, -20C, -80C, or liquid nitrogen, and thawed after 3 days. Except for the '4C no sucrose' condition, sucrose was added to 15% as a cryoprotectant prior to freezing. CD1 female mice were dosed at 0.4 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 23 hours later. Figure 29A Average radiance (photons / s / cm 2 / sr).

[0321] Results: Under the conditions of storage, lung signals were lower than 'fresh' signals. Based on particle size measured with dynamic light scattering, 4C with sucrose and -20C were out of range (see next slide). Figure 29B .

[0322] Example 13 - Titration of N / P ratio and comparison of 2, 3 and 5 lipid formulations Methods: LNP were formulated with firefly luciferase mRNA (Luc) in the ratios outlined in Table 3. They were either formulated and dosed 'fresh' on the same day, or cryopreserved at 4C, -20C, -80C, or liquid nitrogen, and thawed after 3 days. Except for the '4C no sucrose' condition, sucrose was added to 15% as a cryoprotectant prior to freezing. CD1 female mice were dosed at 0.4 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 23 hours later. Figure 30A Methods: LNP were formulated with firefly luciferase mRNA (Luc) in the ratios outlined in Table 3. They were either formulated and dosed 'fresh' on the same day, or cryopreserved at 4C, -20C, -80C, or liquid nitrogen, and thawed after 3 days. Except for the '4C no sucrose' condition, sucrose was added to 15% as a cryoprotectant prior to freezing. CD1 female mice were dosed at 0.4 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 23 hours later. Figure 30B Average radiance (photons / s / cm 2 / sr).

[0323] Results: 1. Average signals were higher for 3 and 5 lipid (SS and '5 lipid') formulations than for 2 lipid (1% PEG) formulation 2. 10 N / P was optimal in 3 and 5 lipid formulations 3. For 2 lipid, 15 NP was superior to 10 NP 4. All formulations showed promise as lung-targeting LNPs, with the exception of 2 lipid 10 N / P, given high radiance and less than 10% weight loss Figure 30C .

[0324] Example 14 - Titration of N / P ratio (mRNA to lipid ratio) using 3 and 5 lipid formulations Methods: LNP were formulated with firefly luciferase mRNA (Luc) in the ratios outlined in Table 3. They were either formulated and dosed 'fresh' on the same day, or cryopreserved at 4C, -20C, -80C, or liquid nitrogen, and thawed after 3 days. Except for the '4C no sucrose' condition, sucrose was added to 15% as a cryoprotectant prior to freezing. CD1 female mice were dosed at 0.4 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 23 hours later.Figure 31A The ratio of LNP to firefly luciferase mRNA (Luc) was used to formulate the mixture. Bl / 6 male mice were administered the mixture via intravenous (IV) injection at a dose of 1.5 mg / kg, and organs were imaged ex vivo 21 hours later. Figure 31C The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0325] result: 1. Lipid formulations prepared at 3 and 6 N / P conditions both exhibited significant toxicity. 2. For 5-lipid lung-targeting LNPs, the encapsulation efficiency began to drop below 8 N / P. Figure 31B 3. In this study, considering the above factors, lipids 3 and 5 with a N / P ratio of 10 were generally superior. Example 15 - Testing vortexing vs. microfluidic mixing for mRNA-LNP formulations Methods: LNP was formulated with firefly luciferase mRNA (Luc) according to the ratios in Table 3. Balb c / J male mice were administered the solution intravenously (IV) at a dose of 0.1 mg / kg, and organs were imaged ex vivo 21 hours later. Figure 32A The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0326] result: 1. LNPs generated by vortex-mixed mRNA-LNP produced high BLI signals in the lungs and were slightly larger in size (113 nm vs. 86 nm). Figure 32B .

[0327] 2. The encapsulation efficiency of LNPs generated by vortex mixing of mRNA-LNPs was 63%, which is much lower than the 94% of the corresponding product of microfluidic mixing. Figure 32B .

[0328] Example 16 - Titration of NP ratio of 5 lipid mix; comparison of input concentrations Method: According to Figure 33A The ratios in the table represent the ratios of LNP to firefly luciferase mRNA (Luc). C57Bl / 6 male mice were administered the drug intravenously (IV) at a dose of 1.5 mg / kg, and organs were imaged ex vivo 21 hours later. Figure 33B The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0329] For conditions labeled 'dil', mRNA and lipids were diluted to 30% of the original in buffer prior to mixing, but the ratios of the components were kept the same. The mRNA concentration of the input changed from 0.147 mg / ml to 0.44 mg / ml.

[0330] Results: 1. Reducing the DOTAP molar percentage from 49% to 30% increased the signal in 5-lipid formulations by 30% 2. All formulations had encapsulation efficiencies >90% and all mice showed no movement, posture, or eye abnormalities (LPE score) Example 17 - Titration of NP ratio of 5 lipid mix; comparison of 5, 3, 2 lipid LNP formulations Methods: LNP were formulated with Firefly Luciferase mRNA (Luc) at the ratios outlined in Figure 34A . BALB / cJ male mice were dosed via intravenous injection (IV) at 1.5 mg / kg and organs were imaged ex vivo 20 hours later. Figure 34B Average radiance (photons / s / cm 2 / sr) is shown in Figure 1. The formulations used 5 lipids, except for "3-lipid 12 NP" and "1% PEG 15 NP".

[0331] Results: All formulations had encapsulation efficiencies >90%. Figure 34C . For 3 and 5-lipid lung-targeted LNP, the highest signal was observed with 12 NP.

[0332] Example 18 - Testing different total flow rates for 5 lipid formulations Methods: LNP were formulated with Firefly Luciferase mRNA (Luc) at the ratios outlined in Figure 35A . C57B1 / 6 male mice were dosed via intravenous injection (IV) at 2.0 mg / kg and organs were imaged ex vivo 17 hours later. Figure 35C Average radiance (photons / s / cm 2 / sr) is shown in Figure 2. The formulations were the same, the variable changed was the flow rate through the PrecisionNanosystems Nanoassemblr microfluidic mixing platform.

[0333] Results: All 3 flow rates had high bioluminescent signal, encapsulation efficiencies >95%, and no clinical signs (LPE = 0, no deaths). Figure 35B . The optimal flow rate was 12 ml / min.

[0334] Example 19 - Testing different total flow rates for 3 lipid formulation ("PDS") Methods: LNP were formulated with Firefly Luciferase mRNA (Luc) at the ratios outlined in Figure 36AThe LNP was formulated with Firefly Luciferase mRNA (Luc) at the ratios shown in Figure 36B The average radiance (photons / s / cm 2 / sr) is shown in FIG. 2. The total flow rate varied, but the ratio of mRNA (aqueous) to lipid (organic) flow was kept at 3: 1. Thus, the flow rate of the mRNA stream was the total flow rate times 0.75, and the flow rate of the lipid stream was the total flow rate times 0.25.

[0335] Results: 1. The bioluminescent signal was high, the encapsulation efficiency was greater than 98%, and the body weight loss was less than 5% for all three flow rates Figure 36B and 36C ).

[0336] Example 20 - Replacing SS-OP in 3 lipid LNP with different ionizable lipid DLin-MC3-DMA ("MC3"). Titration of MC3 to DOTAP ratio from 9% to 50% MC3.

[0337] Method: The LNP was formulated with Firefly Luciferase mRNA (Luc) at the ratios shown in Figure 37A . Figure 37B The percent encapsulation of the LNP is provided in FIG. 3. The BALB / cJ female mice were dosed via intravenous injection (IV) at 2.0 mg / kg, and the organs were imaged ex vivo 24 hours later. Figure 37C The average radiance (photons / s / cm 2 / sr) is shown in FIG. 3.

[0338] Results: 1. Comparable bioluminescent signals were observed in the lungs for all conditions, and the signal trended downward as the MC3 concentration decreased. Figure 37C .

[0339] 2. The lowest MC3 concentration (3-MC3 0.1) with 9% overall MC3 resulted in a mortality event. Figure 37B .

[0340] 3. The body weight loss trended upward as the MC3 concentration increased, with the highest average body weight loss at 50% MC3. Figure 37D .

[0341] Example 21 - Replacing ionizable lipid SS-OP with DLin-MC3-DMA in 3 lipid LNP.

[0342] Method: The LNP was formulated with Firefly Luciferase mRNA (Luc) at the ratios shown in Figure 38AThe ratio of LNP to firefly luciferase mRNA (Luc) was used. MC3 is an abbreviation for DLin-MC3-DMA. CD1 female mice were administered 2.0 mg / kg via intravenous (IV) injection, and organs were imaged ex vivo 26 hours later. Figure 38C The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0343] result: 1. Death events occurred in LNP containing 70.5% 3-MC, indicating poor tolerability.

[0344] 2. Under all conditions, the encapsulation efficiency is >95%. Figure 38B 3. The average lung radiation rates were comparable, with the LNP formulation containing 33% molar percentage MC3 showing the highest average signal.

[0345] Example 22 - Replacing ionizable lipid SS-OP with DLin-MC3-DMA in 5 lipid LNP.

[0346] Method: According to Figure 39A The ratio of LNP to firefly luciferase mRNA (Luc) was used. MC3 is an abbreviation for DLin-MC3-DMA. CD1 female mice were administered 2.0 mg / kg via intravenous (IV) injection, and organs were imaged ex vivo 26 hours later. Figure 39C The figure shows the average emissivity (photons / s / cm). 2 / sr).

[0347] result: 1. Under all conditions, the encapsulation efficiency is >95%. Figure 39B .

[0348] 2. Under 5 lipid conditions containing SS-OP, the mean lung radiation rate is at least 7.2 times higher than that of any LNP containing DLin-MC3-DMA. Figure 39C .

[0349] Example 23 - Titration of time mRNA is adsorbed to forming LNP in 2 lipid formulation Methods: DOTAP and DMG-PEG2000 were microfluidically mixed with malic acid at a molar ratio of 100:1, then the buffer was exchanged for 0.1M sodium acetate and concentrated. Lipid concentration was measured by HPLC with a UV detector. Firefly luciferase mRNA (Luc) was then added to the lipids to achieve a final N / P ratio of 15. The mRNA was adsorbed before the addition of PBS. Figure 40AThe average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later. Figure 40A The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later. 2 The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later.

[0350] Results: 1. The strongest lung signal was observed under 30 minutes incubation conditions 2. No animal lost more than 5% of their body weight and no deaths were observed, indicating tolerability. Figure 40B For all conditions, the encapsulation efficiency was greater than 98%.

[0351] Example 24 - Titration of mRNA:lipid ratio of 2 lipid LNP Methods: DOTAP and DMG-PEG2000 were subjected to microfluidic mixing with malic acid at a molar ratio of 100: 1, followed by buffer exchange to 0.1 M sodium acetate and concentration. Lipid concentration was measured by HPLC, then firefly luciferase mRNA (Luc) was added to the lipids to an N / P ratio of Figure 41A The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later. Figure 41B The encapsulation percentage for each composition is provided in FIG. 6. mRNA was allowed to adsorb for 30 minutes prior to PBS addition. C57BL / 6 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 27 hours later. Figure 41A The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later. 2 The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later.

[0352] Results: 1. LNP with NP 2 that was not shown did not produce enough mRNA to dose.

[0353] 2. Lung bioluminescent signals were comparable between different conditions Example 25 - Titration of N / P ratio of 3 lipid LNP Methods: LNP “PDS” was formulated with firefly luciferase mRNA (Luc) at the ratios in Table 5. Figure 42B The encapsulation percentage for each composition is provided in FIG. 6. mRNA was allowed to adsorb for 30 minutes prior to PBS addition. C57BL / 6 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 27 hours later. Figure 42A The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later. 2 The average radiance (photons / s / cm2 / sr) is shown in FIG. 6. CD1 female mice were dosed at 2.0 mg / kg via intravenous injection (IV) and organs were imaged ex vivo 24 hours later.

[0354] Table 5

[0355] Results: 1. Death events occurred in MC3 containing LNPs at N / P ratio of 20 and clinical signs (reduced movement and hunched posture) appeared in MC3 LNPs at N / P ratio of 15, indicating that these drugs were not well tolerated.

[0356] 2. Encapsulation efficiency was >98% in all conditions 3. Mean lung radiation decreased with increasing N / P ratio 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 in this case. The yield was lower for NP 2.5 and NP 3, so dosing was performed only for a single animal.

[0357] 5. In this study, LNPs containing DLin-MC3-DMA at N / P ratios of 4 and 5 were the most promising.

[0358] Example 26 - Titration of DMG-PEG 2000 in 2 lipid LNP Methods: DOTAP and DMG-PEG2000 were mixed at a molar percentage of the pegylated lipid to malate as shown in Figure 43A and 43B were subjected to microfluidic mixing, followed by buffer exchange to 0.1 M sodium acetate and concentration. The lipid concentration was measured by HPLC, followed by addition of firefly luciferase mRNA (Luc) to an N / P ratio of 5.0. The mRNA was allowed to adsorb for 30 minutes prior to addition of PBS. C57BL / 6 female mice were dosed at 2.0 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 20 hours later. Figure 43A The mean radiation (photons / s / cm 2 / sr) is shown in Table 6.

[0359] Results: 1. Single cationic lipid - DOTAP - was sufficient to achieve tolerable lung targeting.

[0360] 2. Mean lung radiation decreased with increasing DMG-PEG2000.

[0361] Example 27 - Microfluidic vs. manual mixing of lipids in ethanol and malic acid buffer Methods: DOTAP and DMG-PEG2000 were mixed with malic acid at a 100:1 molar ratio and subjected to microfluidic mixing or manual vortex mixing, followed by buffer exchange into 0.1 M sodium acetate and concentration. Lipid concentration was measured by HPLC, then firefly luciferase mRNA (Luc) was added to an N / P ratio of 5.0. The mRNA was allowed to adsorb for 30 minutes before addition of PBS. C57BL / 6 female mice were dosed at 0.5 mg / kg via intravenous injection (IV), and organs were imaged ex vivo 20 hours later. Figure 44 Average radiance (photons / s / cm2 / sr) is shown in Table 2 / sr).

[0362] Results: 1. Microfluidic mixing increased average lung radiance by 2-fold 2. mRNA encapsulation efficiency was >98% for both conditions Example 28 - Comparison of published MC3 formulation with 5 lipid LNP containing SS-OP Method 1: LNP were formulated with firefly luciferase mRNA (Luc) at the ratios in Figure 45A C57BL / 6J female mice were dosed at the indicated mg / kg via intravenous injection (IV), and organs were imaged ex vivo 20 hours later. Figure 45B Average radiance (photons / s / cm2 / sr) is shown in Table 2 / sr). MC3 formulation is based on Dilliard et al. 2021 PNAS (https: / / doi.org / 10.1073 / pnas.2109256118).

[0363] Results 1: 1. LNP containing 5-lipid SS-OP had 6.3-fold and 5.2-fold higher average lung radiance at 0.5 and 2.0 mg / kg dose groups, respectively, compared to published lung-targeted LNP containing DLin-MC3-DMA. Figure 45B 2. Encapsulation efficiency was >98% for both LNP, and total yield was comparable. Modal particle size by dynamic light scattering was between 35-55 nm.

[0364] Method 2: LNP were formulated with firefly luciferase mRNA (Luc) at the ratios in Figure 45AThe ratio in LNP was also formulated with Cre mRNA. The MC3 formulation is based on Dilliard et al. 2021 PNAS (https: / / doi.org / 10.1073 / pnas.2109256118). Ai14 Tomato fl / fl female mice were dosed intravenously with 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 antibody. Figure 45C The percentage of positive cells in alveolar cells is shown in

[0365] Results 2: 1. 5 lipid LNP containing SS-OP had 4.9-fold higher transfection rate in alveolar cells.

[0366] 2. Representative images of alveolar regions are shown in Example 29 - Cell transfection rates in 3 organs Methods: LNP was formulated using the lipid molar ratios and lipid:mRNA ratios shown in Figure 46A . PD(*) formulations were prepared as described above (Example 26). Ai14 Tomato fl / fl female mice were dosed intravenously with Cre mRNA 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 antibody. Shown are the percentage of positive cells in alveolar spaces Figure 46B ), hepatocytes (non-hepatocytes excluded using nucleus size and roundness) Figure 46C ), and spleen cells Figure 46D . Figure 46E Relevant IHC data is provided in

[0367] Results: 1. 5 lipid LNP (5Lip) and 3 lipid LNP (PDS) had comparable transfection rates in lung and spleen, but 5 lipid LNP had 8-fold higher hepatocyte transfection rate in liver.

[0368] 2. Figure 45D Representative images of alveolar regions are shown in

[0369] Example 30 - Pharmacokinetics using 2 lipid LNP Methods: PD formulations were prepared using mRNA encoding human telomerase as described above (Example 26). C57B1 / 6 male mice were dosed at 1.0 mg / kg and plasma and tissues were collected at the indicated time points. DOTAP was measured using LC-MS / MS as shown in Figure 1.

[0370]

[0371] Results:

[0372] Example 31 - Pharmacokinetics using 5 lipid LNP Methods: LNP were formulated using 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 remaining” is the liver sample excluding the left lobe.

[0373] Results:

[0374] Example 32 - Bio distribution using 5 lipid LNP Methods: LNP were formulated using 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 shown in the figure. Organs were imaged ex vivo 6 hours later. The average radiance (photons / s / cm 2 / sr) for each imaged organ is shown in the figure.

[0375] Results are provided in Figure 49A and 49B .

[0376] Example 33 - Bio distribution using 3 lipid LNP Methods: LNP were formulated using mRNA encoding firefly luciferase using the lipid molar ratios in Table 6. Rhesus macaques were dosed intravenously (IV) at the mg mRNA / kg body weight levels shown in the figure. Organs were imaged ex vivo 6 hours later after addition of luciferin. Figure 50A is the average radiance (photons / s / cm 2 / sr) for each imaged organ. Figure 50B and 50C show the relative radiance between low dose and high dose organs. Figures 50D-50F show representative bioluminescent images of organs from high dose animals. The average radiance (photons / s / cm Table 6

[0377] Figure 50G Biodistribution results of bioluminescence of organs are provided, depicted as fold change in radiance.

[0378] Example 34 - Telomerase activity and telomere elongation in human small airway epithelial cells in the presence of SOC Methods: LNP were formulated using the lipid molar ratios in Table 3 with mRNA encoding human telomerase or mCherry as a control. LNP were added to human small airway epithelial cells (SAEC) obtained from Lonza at 0.5 mg / ml from culture. SAEC were grown in Lonza SAGM until 70% confluent. The day before, drugs representing standard of care (SOC) for pulmonary fibrosis patients, pirfenidone (1 uM) and nintedanib (0.5 uM) were added to the cultures and changed at each media change. To measure telomerase activity, cells were harvested 24 hours after treatment with LNP and lysed in CHAPS buffer for TRAP assay. Lysates were exposed to artificial telomerase single-stranded DNA template followed by PCR amplification to detect telomerase activity. Figure 51B .

[0379] To measure telomere length, cells were harvested and fixed five days after addition of telomerase mRNA LNP. Figure 51A . Q-FISH protocol was performed as follows: cells were spun down onto glass slides, followed by permeabilization and labeling of telomeres with fluorescent probes. Figure 51B . Microscopy and quantitative image analysis were performed to determine the length of individual telomeres. All hTERT LNP samples (+ / - SOC) were pooled for analysis to test for differences in telomere length.

[0380] Results: 1. Telomerase activity was detected in human small airway epithelial cells after addition of hTERT LNP 2. Levels of telomerase activity detected in cells were similar when standard of care drugs were added 3. Median and 20th percentile telomere length were significantly increased by 492 and 435 base pairs, respectively, after addition of a single dose of hTERT LNP.

[0381] Example 35 - Telomerase activity and telomere elongation in human lung fibroblasts in the presence of SOC Methods: LNP were formulated using the lipid molar ratios in Table 3 with mRNA encoding human telomerase or mCherry as a control. LNP were added to fetal human lung fibroblast MRC-5 cells (passage 5) and grown in DMEM + 10% FBS until 70-80% confluent. 24 hours prior to LNP treatment, MRC-5 cells were pre-treated with the standard of care drugs pirfenidone (1 uM) and nintedanib (1 uM) and then treated with 500 ng / ml hTERT LNP or 500 ng / ml mCherry LNP (control). Fresh standard of care drugs were added with each media change.

[0382] To measure telomerase activity, cells were harvested 24 hours after treatment with LNP and lysed in CHAPS buffer for TRAP assay. Lysates were exposed to artificial telomerase single-stranded DNA template and then PCR amplification was used to detect telomerase activity. Figure 52 .

[0383] Results: 1. Telomerase activity was detected in human lung fibroblasts after addition of hTERT LNP.

[0384] 2. When standard of care drugs were added, similar levels of telomerase activity were detected in the cells.

[0385] Example 36 - Lyophilization of 3 and 5 lipid LNP Methods: LNP were formulated using the lipid molar ratios described herein with mRNA encoding firefly luciferase. The LNP ratios used are described in Table 7. LNP were mixed with 15% sucrose and frozen at -80°C. Next, they were placed in the pre-freeze shelf of an L-200 Pro Lyovapor freeze dryer. The vacuum was set to 1 mbar, the condenser to -55°C, and the total run time was 22 hours. LNP were stored at 4°C for 2 days and then resuspended with water. As a control, “PDS” LNP were frozen with 15% sucrose and thawed without lyophilization. Second passage Tert - / - female mice on a C57B1 / 6 background were dosed with 0.3 mg mRNA / kg body weight intravenously (IV) and organs were imaged ex vivo 18 hours later. Figure 55 .

[0386] Results: The in vivo activity of the 5 lipids and PDS were comparable to each other and to the non-lyophilized (freeze-dried) frozen PDS LNP. The lyophilized and reconstituted PD LNP exhibited at least 8-fold higher activity compared to the other LNP.

[0387] Example 37 - Summary of exemplary LNP formulations of the disclosure Table 7 provides the molar percentage composition, N / P ratio, molar ratio of cationic lipid:ionizable lipid, and lipid:mRNA ratio (weight / weight) for exemplary LNP of the disclosure (named“SSOP-DOTAP”,“5Lipid”,“PDS”,“3-MC3”,“PD”, respectively).

[0388] Table 7

[0389] Figure 56 The optimal cationic lipid to ionizable lipid ratio (“C / I ratio”) and N / P ratio determined by the inventors for LNP containing SS-OP family or DLin-MC3-DMA family ionizable lipids were modeled.

[0390] Example 38 - Post-LNP formation addition of mRNA LNP were formulated with Firefly Luciferase mRNA (Luc) according to the ratios in Table 7. For LNP with an asterisk (*), microfluidic mixing was performed with buffer only, no mRNA. Buffer was then exchanged for 0.1 M sodium acetate and concentrated. Firefly Luciferase mRNA (Luc) was added to the lipids to achieve the N / P ratio shown in the figure. mRNA was allowed to adsorb for 2 hours prior to PBS addition, 0.2 µM filtration, and dosing. mRNA LNP were delivered intravenously to C57BL / 6 male mice and organs were imaged ex vivo 17 hours later.

[0391] Results: 1. Addition of mRNA at the end resulted in the following changes in average lung radiance rates Figure 53A ): • PD LNP increased by 85% • PDS LNP decreased by 68% • 5Lipid decreased by 16% 2. Encapsulation efficiency >95% only in the case of PD (*) conditions at the end of the purification process, but not for PDS (*) and 5Lipid (*) conditions. Figure 53B .

[0392] Example 39 - Post-LNP formation addition of mRNA LNP was formulated with firefly luciferase mRNA (Luc) according to the ratios shown in Table 7. For PD(*), microfluidic mixing with buffer only was performed, without mRNA. The buffer was then exchanged for 0.1 M sodium acetate and concentrated. Firefly luciferase mRNA (Luc) was added to the lipids to achieve the N / P ratio shown in the figure. The mRNA was allowed to adsorb for 2 hours before adding PBS, filtration with 0.2 µM, and administration. The mRNA LNP was administered intravenously to male BALB / cJ mice at 1.5 mg / kg, and ex vivo organ imaging was performed 26 hours later.

[0393] result: 1. Adding mRNA at the end of PD preparation resulted in a 31.8-fold increase in bioluminescent signal. Figure 54A .

[0394] 2. The encapsulation efficiency of all formulations is >95%. Figure 54B .

[0395] Example 40 - Transfection of a broad range of cell types Methods: LNPs were formulated with mRNA encoding Cre recombinase using the lipid molar ratios shown in Table 7. Ai14 Tomato fl / fl mice were intravenously administered 1.2 mg / kg of 3-lipoprotein MC3 (3-MC3) or 3-lipoprotein SS-OP (PDS) LNP operably containing Cre mRNA, or PBS as a control.

[0396] Bone marrow cells were collected 6 days after the above administration and used... Figure 57 , 58A Gating schemes for 58B and 58C include gating live cells using DAPI and analyzing them via flow cytometry. For example... Figure 58D As shown, PDS and 3-MC3 LNP preparations were found to transfect bone marrow cells (hematopoietic stem cells) at rates of 5.9% and 12.2%, respectively.

[0397] 120 days later, the same batch of mice were imaged using a Lago whole-body fluorescence imaging system (Spectral Instruments Imaging). Signals were captured in the Cy3 channel. Figure 59A (535nm excitation, 590nm emission, 60-second exposure), and as a control, mice were imaged in the Cy7 channel. Figure 59B(745 excitation, 790 emission, 20-second exposure). Cy3 signaling delineates the location of Cre mRNA LNP transfection in cells, as well as the location of mRNA translation into Cre recombinase, which performs recombination events to remove the stop codon in the tdTomato gene, thereby allowing stable expression of tdTomato. The conclusion is that both 3-MC3 and PDSLNP provide extensive transfection distribution, including the limbs, tail, and entire abdominal region.

[0398] Then, gating was performed on the whole-body fluorescence imaging of the mice to perform more detailed body image analysis, excluding limbs, tails and pelvis. Figure 60B Quantification of whole-body Cy3 images obtained from the ventral side of mice is shown. Average radiance (photons / s / cm² / sr) is shown, after area normalization. 3-MC3 and PDS LNP were found to be 25% and 22% higher, respectively, than in the PBS control group.

[0399] Then, gating was performed on the whole-body fluorescence imager around the limbs, tail, and pelvis of the mice for more detailed image analysis. Figure 61A . Figure 61B Quantitative analysis of Cy3 images of the limbs, tail, and pelvis acquired from the dorsal side of a mouse is shown (500 nm excitation, 590 nm emission, 60 sec exposure time). Average radiance (photons / s / cm² / sr) is shown, normalized for area. Signal distribution along the spine, limbs, tail, and pelvis is observed in both 3-MC3 and PDS images.

[0400] The half-life of Cre mRNA is less than one day, while the half-life of Cre protein is only a few days. Therefore, transfection appears to occur within the first few days, thus permanently activating tdTomato in transfected cells. Previous experiments have demonstrated that tdTomato signaling can be detected within 1–3 days after Cre mRNA injection. The formulation was well-tolerated, as assessed by observing movement, eye appearance, and posture at the cage edge.

[0401] Bone marrow cells were isolated from these mice 126 days after drug administration and subjected to flow cytometry. Let the phylum structure be as follows: Figure 57 and 58A As shown in -58C. Figure 62A The figure shows the percentage of tdTomato+ cells in bone marrow for PBS control, 3-MC3, and PDS.

[0402] Data analysis results from bone marrow cells obtained from mice at day 6 and day 126 post-dosing with PBS control, 3-MC3, and PDS were evaluated. The percentage of tdTomato+ cells in the PBS control was subtracted from all samples at each time point to allow for direct comparison. It was found that the percentage of positive cells in the bone marrow cells decreased by 58% under both 3-MC3 and PDS conditions, indicating that some transfected bone marrow cells were short-lived progenitor cells (day 6), while other cells were long-lived progenitor cells or stem cells (day 126).

[0403] Tissues were fixed in formalin and embedded in paraffin. Sections were stained with anti-tdTomato antibody and digitally scanned. QuPath was used to identify cell nuclei and quantify the percentage of positive cells in each tissue, with quantification results shown in Figure 63A . Results show that Ai14 tdTomato fl / fl mice that received Cre mRNA formulated with 3-MC3 or PDS and injected intravenously at a dose of 1.2 mg / kg resulted in widespread transfection of various tissues of the mice, including those specifically listed. In addition to those tissues listed in Figure 63A , other tissues also appear to have been transfected, and studies are planned to evaluate these additional cells and tissues.

[0404] Immunohistochemistry tissue section images of the liver Figure 63B ), spleen Figure 63C ), kidney tissue Figure 63D ), leg Figure 63E ), and tail Figure 63F ) are shown. In preparing the immunohistochemistry data, tissue sections were collected from the mice and immunostained with an anti-tdTomato antibody conjugated to horseradish peroxidase. Darkening (formerly brown) staining indicates that tdTomato expression was permanently activated in the transfected cells (tdTomato positive) with Cre mRNA.

[0405] Example 41 - Transfection with LNP with antibody targeting ligand Methods: LNP were formulated with 100% N1-methylpseudouridine uridine substituted mRNA encoding Cre recombinase in LNP formulated using standard microfluidic mixing methods (Ignite, PNI), where a 1:3 mix ratio of lipids to mRNA solution was used and included MC3 and maleimide-PEG-lipid conjugated to a monoclonal antibody specific for the stem and progenitor cell marker c-Kit. The antibody was conjugated using methods known in the art, including reduction of the antibody with TCEP, followed by reaction with maleimide-PEG-lipid on the LNP for 1 hour at room temperature with slow rotation, followed by size exclusion column purification (Izon) to remove unconjugated antibody. LNP were dosed at 0.2 mg / kg mRNA to tdTomato mice and bone marrow cells were collected at day 6 post-dose, immunostained with a hematopoietic lineage antibody panel, Sca1 and C-Kit antibodies, and analyzed by flow cytometry according to the gating scheme depicted in Figure 64A Figure 6. LNP formulated with antibody-targeted mRNA successfully transfect a portion of LSK cells. Figure 64B ). Antibody-targeted LNP successfully transfect a portion of LSK cells.

[0406] Example 42 - PDS LNP were formulated with Cre mRNA using the same lipid ratios and procedures as in Example 37, Table 7. Ai14 tdTomato fl / fl male mice were dosed intravenously with 1.5 mg (high dose) or 0.5 mg (low dose) of mRNA per kg body weight. Bone marrow cells were harvested 8 days post-dose. Transfected cells were tdTomato+. To examine hematopoietic stem cells (HSCs), the same gating strategy as in Example 41 was used. Lineage-FITC is a cocktail of antibodies including anti-mouse CD3, CD45R (B220), CD11b, TER-119, and Ly-G6. HSCs were then selected as double positive for Sca1 and cKit. These are referred to as LSK cells. PDS LNP formulations successfully transfect a portion of LSK cells. Figure 65 Transfection of hematopoietic stem cells (lineage-, Sca1+, cKit+ bone marrow cells) is shown using PDS LNP at multiple doses.

[0407] A list of certain exemplary nucleic acid sequences used in the presently described examples 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.

[0408]

[0409]

[0410]

[0411]

[0412] The above examples are for illustrative purposes only and are not intended to limit the scope of the present application. Many variations of the above are possible. Since modifications and changes to the above described embodiments are obvious to one skilled in the art, the present application is not to be limited by the above described embodiments but only by the claims that follow.

[0413] Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

Claims

1. A method for delivering polynucleotides to one or more of a wide range of different cell types in a subject, comprising administering the polynucleotides encapsulated in lipid nanoparticles (LNPs), said lipid nanoparticles comprising: (i) a molar percentage of SS-OP or an SS-OP analogue between about 20% and about 60%, (ii) a molar percentage of polyethylene glycol-modified lipids between about 0.5% and about 2.5%, and (iii) a molar percentage of cationic lipids between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between .6 and 1. The polynucleotides mentioned therein include synthetic RNA, which, upon or after administration of the LNP, is translated in vivo into a corresponding protein encoded by the synthetic RNA in one or more different cell types of the subject.

2. The method of claim 1, wherein the polyethylene glycol-modified lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-maleimide.

3. The method of claim 1, wherein the cationic lipid is DOTAP.

4. The method of claim 1, wherein the polyethylene glycolated lipid is DMG-PEG2000, and wherein the cationic lipid is DOTAP.

5. The method of any one of claims 1 to 4, wherein the one or more different cell types are selected from the group consisting of: lung cells, hematopoietic stem cells, progenitor cells, spleen cells, hepatocytes, kidney cells, endothelial cells, epithelial cells, and cells of limb tissues of the subject.

6. The method of claim 4, wherein the one or more different cell types are located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 different organs of the subject.

7. A method for delivering polynucleotides to one or more of a wide range of different cell types in a subject, comprising administering the polynucleotides encapsulated in lipid nanoparticles (LNPs), said lipid nanoparticles comprising: (i) a molar percentage of DLin-MC3-DMA between about 30% and about 50%, (ii) a molar percentage of polyethylene glycol-modified lipids between about 0.5% and about 2.5%, and (ii) a molar percentage of cationic lipids between about 50% and about 70%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 1 and 2. The polynucleotides mentioned therein include synthetic RNA, which, upon or after administration of the LNP, is translated in vivo into a corresponding protein encoded by the synthetic RNA in one or more different cell types of the subject.

8. The method of claim 7, wherein the polyethylene glycol-modified lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-maleimide.

9. The method of claim 7, wherein the cationic lipid is DOTAP.

10. The method of claim 7, wherein the polyethylene glycol-modified lipid is DMG-PEG2000, and wherein the cationic lipid is DOTAP.

11. The method of any one of claims 7 to 10, wherein the one or more different cell types are selected from the group consisting of: hematopoietic stem cells, progenitor cells, spleen cells, hepatocytes, kidney cells, endothelial cells, epithelial cells, and cells of limb tissues of the subject.

12. The method of claim 11, wherein the one or more different cell types are located in at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 different organs of the subject.

13. A method for delivering polynucleotides to bone marrow cells, comprising administering a polynucleotide encapsulated in lipid nanoparticles (LNPs), said lipid nanoparticles comprising: (i) SS-OP or SS-OP analogues with a molar percentage between about 20% and about 60%, polyethylene glycol-modified lipids with a molar percentage between about 0.5% and about 2.5%, and cationic lipids with a molar percentage between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between .6 and 1; and / or (ii) DLin-MC3-DMA with a molar percentage between about 30% and about 50%, DMG-PEG2000 with a molar percentage between about 0.5% and about 2.5%, and cationic lipids with a molar percentage between about 50% and about 70%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 1 and 2. The polynucleotides mentioned therein include synthetic RNA, which, upon or after administration of the LNP, is translated in vivo into the corresponding protein encoded by the synthetic RNA in the subject's bone marrow cells.

14. The method of claim 13, wherein the polyethylene glycol-modified lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-maleimide.

15. The method of claim 13, wherein the cationic lipid is DOTAP.

16. The method of claim 13, wherein the polyethylene glycol-modified lipid is DMG-PEG2000, and wherein the cationic lipid is DOTAP.

17. A method for delivering polynucleotides to one or more of bone marrow cells, spleen cells, hepatocytes, and / or kidney cells, comprising administering polynucleotides encapsulated in lipid nanoparticles (LNPs), said lipid nanoparticles comprising: (i) SS-OP or SS-OP analogues with a molar percentage between about 20% and about 60%, polyethylene glycol-modified lipids with a molar percentage between about 0.5% and about 2.5%, and cationic lipids with a molar percentage between about 40% and about 50%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between .6 and 1; and / or (ii) DLin-MC3-DMA with a molar percentage between about 30% and about 50%, polyethylene glycol-modified lipids with a molar percentage between about 0.5% and about 2.5%, and cationic lipids with a molar percentage between about 50% and about 70%, wherein the cationic lipid:ionizable lipid (C / I) ratio is between 1 and 2. The polynucleotides include synthetic RNA, which, upon or after administration of the LNP, is translated in vivo into a corresponding protein encoded by the synthetic RNA in one or more of the subject's bone marrow cells, spleen cells, hepatocytes, and / or kidney cells.

18. The method of claim 17, wherein the polyethylene glycol-modified lipid is DMG-PEG2000 or a combination of DMG-PEG2000 and DMG-PEG2000-maleimide.

19. The method of claim 17, wherein the cationic lipid is DOTAP.

20. The method of claim 17, wherein the PEGylated lipid is DMG-PEG2000, and wherein the cationic lipid is DOTAP.

21. The method of claim 17, wherein the corresponding protein encoded by the polynucleotide is expressed in two or more of lung cells, bone marrow cells, spleen cells, hepatocytes and / or kidney cells.

22. The method of claim 17, wherein the corresponding protein encoded by the polynucleotide is expressed in each of the bone marrow cells, spleen cells, hepatocytes and / or kidney cells.

23. The method of any one of claims 17 to 22, wherein the bone marrow cells are bone marrow stem cells and / or progenitor cells.

24. The method of any one of claims 1 to 4, 6 to 10, 12 to 22, wherein the administration is intravenous injection, percutaneous injection, intradermal injection via a microneedle array, or local delivery, wherein the local delivery involves steps of permeating the skin barrier using mechanical means such as microneedling therapy and / or applying a skin permeating agent.

25. The method of claim 23, wherein the administration is intravenous injection, percutaneous injection, intradermal injection via a microneedle array, or local delivery, the local delivery involving steps of using mechanical means such as microneedling therapy and / or applying a skin permeability agent to permeate the skin barrier.

26. The method of any one of claims 1 to 8, wherein the polynucleotide comprises a sequence encoding a diagnostic or therapeutic protein that can be expressed in target cells.

27. The method of any one of claims 1 to 4, 6 to 10, 12 to 22, wherein the polynucleotide comprises 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.

28. The method of claim 27, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1 or a fragment thereof.

29. The method of claim 23, wherein the polynucleotide comprises a sequence encoding a diagnostic or therapeutic protein that can be expressed in the bone marrow stem cells and / or progenitor cells.

30. The method of claim 23, wherein the polynucleotide comprises 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 from another mammalian species.

31. The method of claim 30, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1 or a fragment thereof.

32. The method of any one of claims 1 to 4, 6 to 10, 12 to 22, 25 or 27 to 31, wherein the LNP further comprises a targeting ligand suitable for specifically targeting the cells.

33. The method of claim 32, wherein the suitability for specifically targeting the cell is the binding of the LNP to the cell or other interactions.

34. The method of claim 33, wherein the targeting ligand comprises a targeting group selected from the group consisting of: cell-targeting agents, tissue-targeting agents, lectins, glycoproteins, lipids, proteins, peptides, antibodies suitable for binding to the target cell type, aptamers, small molecules, carbohydrates, lipids, nanobodies, and aptamer-antibody conjugates.

35. The method of any one of claims 1 to 4, 6 to 10, 12 to 22, 25 or 27 to 31, wherein the method is suitable for diagnosing, preventing or treating diseases or ailments involving said cells.

36. The method of claim 35, wherein the disease or ailment is or includes influenza, asthma, type 1 diabetes, type 2 diabetes, hypertension, coronary artery disease, chronic obstructive pulmonary disease (COPD), stroke, Alzheimer's disease, Parkinson's disease, osteoarthritis, rheumatoid arthritis, multiple sclerosis, lupus, Crohn's disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), heart failure, atrial fibrillation, hyperthyroidism, hypothyroidism, anemia, thalassemia, sickle cell disease, hemophilia, leukemia, lymphoma, melanoma, breast cancer, prostate cancer, lung cancer, colorectal cancer, pancreatic cancer, kidney cancer, liver cancer, bladder cancer, cervical cancer, ovarian cancer, testicular cancer, esophageal cancer, gastric cancer, brain cancer, uterine cancer, etc. Endometrial cancer, bone cancer, sarcoma, multiple myeloma, skin cancer, basal cell carcinoma, squamous cell carcinoma, tuberculosis, pneumonia, bronchitis, sinusitis, otitis media, urinary tract infection (UTI), hepatitis A, hepatitis B, hepatitis C, HIV / AIDS, syphilis, gonorrhea, chlamydia, herpes simplex virus (HSV), human papillomavirus (HPV), scabies, tinea pedis, tinea fungus, lice infestation, measles, mumps, rubella, chickenpox, herpes zoster, pertussis, diphtheria, tetanus, poliomyelitis, rabies, malaria, dengue fever, yellow fever, Zika virus, Lyme disease, Rocky Mountain spotted fever, toxoplasmosis, giardiasis, amebiasis, ascariasis, trichinosis, echinococcosis, leishmaniasis, anthrax, botulism, tetanus. Plague, cholera, typhoid fever, salmonellosis, campylobacteriosis, listeriosis, Clostridium difficile infection, norovirus infection, rotavirus infection, cytomegalovirus (CMV) infection, Epstein-Barr virus (EBV) infection, parvovirus infection, West Nile virus infection, Hantavirus infection, Ebola virus disease, Marburg virus disease, SARS, MERS, COVID-19, sepsis, cellulitis, osteomyelitis, endocarditis, meningitis, encephalitis, Guillain-Barré syndrome, amyotrophic lateral sclerosis (ALS), Huntington's disease, cystic fibrosis, Duchenne muscular dystrophy, Becker's muscular dystrophy, fragile X syndrome, Down syndrome, Turner syndrome, Klinefelter syndrome, Marfan syndrome, Ehlers-Donnell syndrome Combination syndrome, polycystic kidney disease, primary biliary cholangitis, primary sclerosing cholangitis, hemochromatosis, Wilson's disease, Gaucher disease, Fabry disease, Niemann-Pick disease, Ty Sachs disease, Huntington's disease, Rett syndrome, Prad-Willi syndrome, Angelman syndrome, Sjögren's syndrome, Addison's disease, Cushing's syndrome, acromegaly, gigantism, pheochromocytoma, hyperparathyroidism, hypoparathyroidism, rickets, osteomalacia, osteoporosis, Paget's disease, gout, bursitis, tendinitis, tennis elbow, carpal tunnel syndrome, plantar fasciitis, fibromyalgia, chronic fatigue syndrome, migraine, tension headache, cluster headache, epilepsy, narcolepsy, restless legs syndrome, sleep apnea, insomnia.Bipolar disorder, depression, anxiety disorder, schizophrenia, obsessive-compulsive disorder (OCD), post-traumatic stress disorder (PTSD), attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), anorexia nervosa, bulimia nervosa, binge eating disorder, alcohol use disorder, substance use disorder, personality disorder, somatic symptom disorder, dissociative identity disorder, hypochondriasis, Munchausen syndrome, conversion disorder, delirium, dementia, fetal alcohol syndrome, neonatal withdrawal syndrome, pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), cirrhosis, liver fibrosis, nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), chronic hepatitis B-related disorders. Fibrosis, chronic hepatitis C-related fibrosis, systemic sclerosis (scleroderma), cystic fibrosis, myocardial fibrosis, hypertrophic cardiomyopathy, restrictive cardiomyopathy, dilated cardiomyopathy, chronic kidney disease (CKD), glomerulonephritis, diabetic nephropathy, interstitial nephritis, retroperitoneal fibrosis, peritoneal fibrosis, Dupuytren's contracture, Peroni's disease, myelofibrosis, keloid formation, scar tissue formation, adhesive capsulitis of the shoulder (frozen shoulder), chronic pancreatitis, pancreatitis-related fibrosis, Crohn's disease-related fibrosis, ulcerative colitis-related fibrosis, cutaneous fibrosis, radiation-induced fibrosis, radiation pneumonitis, postoperative adhesions, asbestosis, silicosis, sarcoidosis-related fibrosis and / or eosinophilic fasciitis.

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