Compositions and methods for organ-specific delivery of nucleic acids

Abstract

To provide: compositions which exhibit preferential targeting or delivery of a nucleic acid composition to a particular organ; methods of modulating the expression of a gene comprising a step of delivering a nucleic acid to a cell; and methods of preparing lipid nanoparticles.SOLUTION: A composition comprises a steroid or sterol, an ionizable cationic lipid, a phospholipid, a PEG lipid and a permanently cationic lipid which may be used to deliver a nucleic acid.SELECTED DRAWING: None

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 726,770, filed on Sep. 4, 2018, the entire content of which is incorporated herein by reference.

[0002] 1. Field The present disclosure generally relates to the field of molecular biology. More specifically, the present disclosure relates to the tissue-specific delivery of therapeutic agents, such as nucleic acids, proteins, or small molecule therapeutic agents, within lipid nanoparticles.

Background Art

[0003] 2. Description of Related Art Using CRISPR / Cas (clustered regularly interspaced short palindromic repeat / CRISPR-associated protein) technology, the genome can be accurately and sequence-dependently edited and permanently altered. Since CRISPR / Cas technology can target mutations that cause diseases, it is highly promising for single-shot genetic disease treatment and many other applications in various fields. To date, successful editing has mainly been mediated by viral vectors. This requires time-consuming customization for each target, presenting challenges for clinical translation due to immunogenicity, the generation of antibodies that prevent repeated administration, and concerns about rare but dangerous integration events. To expand the safe and effective use of gene editing, it has clearly been necessary to achieve CRISPR / Cas editing via synthetic nanoparticles (NPs).

[0004] Using CRISPR / Cas enables sequence-specific DNA editing by an RNA-guided CRISPR-associated protein 9 (Cas9) nuclease or its homologs, and a double-strand break (DSB) is formed in genomic DNA. Cas9 is guided by a programmable RNA called single-guide RNA (sgRNA). The Cas9 / sgRNA complex recognizes a complementary genomic sequence with a 3' protospacer adjacent motif (PAM) sequence. After DNA cleavage, directed mutagenesis, i.e., insertions / deletions (indels) that delete the target gene, occurs by the DSB repair pathway. When donor DNA is delivered, cells can utilize homologous recombination repair (HDR) to correct gene mutations. For therapeutic utility, transient Cas9 expression that limits off-target genomic changes is preferred. Since both the Cas9 protein and sgRNA must be present in the same cell, co-delivery of Cas9 mRNA and sequence-targeted sgRNA in one NP is an attractive approach, especially for in vivo applications where tissue penetration and cell uptake are more difficult. Similarly, direct delivery of the sgRNA Cas9 protein ribonucleoprotein (RNP) is also attractive for gene editing. CRISPR / Cas editing using viruses, membrane deformation, and hydrodynamic injection functions, but there are limitations that can prevent in vivo therapeutic use in the clinic, including persistent Cas9 expression and off-target editing. Furthermore, these delivery systems generally lack selectivity for the specific organs where editing is required. For example, most lipid nanoparticles accumulate in the liver via biological processes, and thus the efficacy of the composition is reduced when delivered to the target organ.

[0005] Similarly, from organ-specific delivery, other therapeutic agents such as proteins and small molecule therapeutics may benefit. Many different types of compounds, such as chemotherapeutic agents, exhibit significant cytotoxicity. If these compounds can be better directed by delivery to the desired organ, off-target effects will be reduced.

[0006] Therefore, there is still a need to develop new lipid nanoparticles that exhibit preferential delivery to specific organs. SUMMARY OF THE INVENTION

[0007] Summary In some aspects, the present disclosure provides lipid compositions that exhibit organ-specific delivery of lipid compositions. These compositions can be used to deliver nucleic acid components to specific organs.

[0008] In some aspects, the present disclosure provides (A) a therapeutic agent, and (B) (1) a permanently cationic lipid; (2) a cationic ionizable lipid; and (3) a phospholipid comprising a lipid nanoparticle composition and a composition comprising a nucleic acid that preferentially delivers to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. In some embodiments, the target organ is selected from the lungs, heart, brain, spleen, lymph nodes, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin.

[0009] In some embodiments, the target organ is the lung, lymph node, or spleen. In some embodiments, the target organ is the lung. In other embodiments, the target organ is the spleen. In other embodiments, the target organ is the liver. In other embodiments, the target organ is the lymph node.

[0010] In some embodiments, the selectively organ-targeted compound is a permanent cationic lipid. In some embodiments, the permanent cationic lipid is present at a molar percentage of about 5% to about 20% relative to the lipid nanoparticle composition. In some embodiments, a molar percentage of about 12% to about 18% of the permanent cationic lipid is present. In some embodiments, the molar percentage of the permanent cationic lipid is about 15%. In some embodiments, the permanent cationic lipid is present at a molar percentage of about 20% to about 65% relative to the lipid nanoparticle composition. In some embodiments, a molar percentage of about 40% to about 61% of the permanent cationic lipid is present. In some embodiments, the molar percentage of the permanent cationic lipid is about 50%.

[0011] In some embodiments, the permanent cationic lipid contains a quaternary ammonium ion. In some embodiments, the permanent cationic lipid is further defined as TIFF2025108518000001.tif24128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; X - is a monovalent anion.

[0012] In some embodiments, R1 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In some embodiments, R2 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In other embodiments, R1 is alkyl (C8~C24) or substituted alkyl (C8~C24) . In other embodiments, R2 is alkyl (C8~C24) or substituted alkyl (C8~C24)is. In some embodiments, both R1 and R2 are the same. In some embodiments, R3, R3', and R3'' are each identical. In some embodiments, R3, R3', and R3'' are each methyl. In some embodiments, X - is a halide anion, for example, bromide or chloride. In some embodiments, the permanent cationic lipid is further defined as TIFF2025108518000002.tif22128.

[0013] In other embodiments, the permanent cationic lipid is further defined as TIFF2025108518000003.tif15128, wherein R4 and R4' are each independently alkyl (C6~C24) alkenyl (C6~C24) or a substituted form of any group; R4'' is alkyl (C≦24) alkenyl (C≦24) or a substituted form of any group; R4''' is alkyl (C1~C8) alkenyl (C2~C8) or a substituted form of any group; and X2 is a monovalent anion.

[0014] In some embodiments, R4 is alkyl (C6~C24) or substituted alkyl (C6~C24) for example, octadecyl. In some embodiments, R4' is alkyl (C6~C24) or substituted alkyl (C6~C24) for example, octadecyl. In some embodiments, R4'' is alkyl (C≦24) or substituted alkyl (C≦24) is. In some embodiments, R4'' is alkyl (C≦8) or substituted alkyl (C≦8) for example, methyl. In some embodiments, R4''' is alkyl (C1~C8) or substituted alkyl (C1~C8), for example, is methyl. In some embodiments, X2 is a halide, for example, chloride or bromide. In some embodiments, the permanent cationic lipid is further defined as TIFF2025108518000004.tif10128.

[0015] In some embodiments, the permanent cationic lipid is further defined as TIFF2025108518000005.tif25128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; R4 is alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion.

[0016] In some embodiments, R1 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In some embodiments, R2 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In other embodiments, R1 is alkyl (C8~C24) or substituted alkyl (C8~C24) . In other embodiments, R2 is alkyl (C8~C24) or substituted alkyl (C8~C24) . In some embodiments, R1 and R2 are both the same.

[0017] In some embodiments, R3, R3', and R3'' are each the same, for example, R3, R3', and R3'' are each methyl. In some embodiments, R4 is alkyl (C≦6) , for example, ethyl. In some embodiments, X -is a halide anion, for example, bromide or chloride.

[0018] In some embodiments, the permanent cationic lipid is further defined as TIFF2025108518000006.tif28128.

[0019] In other embodiments, the selective organ targeting compound is a permanent anionic lipid. In some embodiments, the permanent anionic lipid is present at a molar percentage of about 5% to about 50% relative to the lipid nanoparticle composition. In some embodiments, a molar percentage of about 10% to about 45% of the permanent anionic lipid is present. In some embodiments, the molar percentage of the permanent anionic lipid is about 30%. In some embodiments, the permanent anionic lipid contains a phosphate group.

[0020] In some embodiments, the permanent anionic lipid is further defined as TIFF2025108518000007.tif25128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) or a substituted form of any group; R3 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) or -Y1-R4, wherein Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) is.

[0021] In some embodiments, R1 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) is. In other embodiments, R2 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) is. In other embodiments, R1 is alkyl (C8~C24)or substituted alkyl (C8~C24) is. In other embodiments, R2 is alkyl (C8~C24) or substituted alkyl (C8~C24) is. In some embodiments, both R1 and R2 are the same.

[0022] In some embodiments, R3 is hydrogen. In other embodiments, R3 is -Y1-R4, wherein Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) is; and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) is.

[0023] In some embodiments, Y1 is substituted alkanediyl (C≦6) , for example, 2-hydroxypropanediyl. In some embodiments, R4 is acyloxy (C≦8~24) , for example, octadecenoate. In some embodiments, the permanent anionic lipid is further defined as TIFF2025108518000008.tif107128.

[0024] In other embodiments, the selectively organ-targeted compound is C6-C 24 diacyl phosphotidylcholine. In some embodiments, the diacyl phosphotidylcholine is present in a molar percentage of about 5% to about 50% relative to the lipid nanoparticle composition. In some embodiments, a molar percentage of about 10% to about 45%, for example, about 30% of diacyl phosphotidylcholine is present.

[0025] In some embodiments, the selectively organ-targeted compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. In some embodiments, the diacyl phosphotidylcholine is further defined as TIFF2025108518000009.tif25128, In the formula, R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion.

[0026] In some embodiments, R1 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In some embodiments, R2 is alkenyl (C8~C24) or substituted alkenyl (C8~C24) . In other embodiments, R1 is alkyl (C8~C24) or substituted alkyl (C8~C24) . In other embodiments, R2 is alkyl (C8~C24) or substituted alkyl (C8~C24) . In some embodiments, both R1 and R2 are the same.

[0027] In some embodiments, R3, R3', and R3'' are each the same. In some embodiments, R3, R3', and R3'' are each methyl. In some embodiments, X - is a halide anion such as bromide or chloride. In some embodiments, the diacylphosphatidylcholine is further defined as TIFF2025108518000010.tif22128.

[0028] In some embodiments, the cationic ionizable lipid is present at a molar percentage of about 5% to about 30% based on the lipid nanoparticle composition. In some embodiments, a molar percentage of about 7.5% to about 20% of the cationic ionizable lipid is present. In some embodiments, the molar percentage of the cationic ionizable lipid is about 11.9%. In some embodiments, the cationic ionizable lipid is present at a molar percentage of about 15% to about 30% based on the lipid nanoparticle composition. In some embodiments, a molar percentage of about 15% to about 25% of the cationic ionizable lipid is present. In some embodiments, the molar percentage of the cationic ionizable lipid is about 20.3%.

[0029] In some embodiments, the cationic ionizable lipid contains an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. In some embodiments, the ammonium group is positively charged at a pH of about 6 to about 8. In some embodiments, the cationic ionizable lipid is a dendrimer or dendron. In some embodiments, the cationic ionizable lipid contains at least two C6-C24 alkyl or alkenyl groups. In some embodiments, the cationic ionizable lipid contains at least two C8-C24 alkyl groups. In some embodiments, the cationic ionizable lipid has the following formula: Core - repeating unit - end group (I) is a dendrimer further defined by or a pharmaceutically acceptable salt thereof, by removing one or more hydrogen atoms from the core and replacing said atoms with repeating units, the core is linked to the repeating unit, and the core has the following formula: TIFF2025108518000011.tif10128, wherein, X1 is amino or alkylamino (C≦12) , dialkylamino (C≦12) , heterocycloalkyl (C≦12) , heteroaryl (C≦12) , or a substituted form thereof; R1 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has the following formula: TIFF2025108518000012.tif12128, and wherein X2 is N(R5) y ; R5 is hydrogen, alkyl (C≦18) , or substituted alkyl (C≦18) ; y is 0, 1, or 2, provided that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3, provided that the sum of z and y is 3; or the core has the following formula: TIFF2025108518000013.tif11128, and wherein X3 is -NR6-, wherein R6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiy (C≦8) , arenediyl (C≦8) , heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) , or a substituted form of any of these groups; R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12), dialkylamino (C≦12) , or a substituted form of any of these groups; or the formula: -N(R f )) f (CH2CH2N) e (R c )R d ; wherein e and f sum to 3, e and f are each independently 1, 2, or 3; R c , R d , and R f are each independently hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is alkylamine (C≦18) , dialkylamine (C≦36) , heterocycloalkane (C≦12) , or a substituted form of any of these groups; the repeating unit contains a cleavable diacyl and a linker; the cleavable diacyl group has the following formula: TIFF2025108518000014.tif18128, wherein A1 and A2 are each independently -O- or -NR a -; wherein R a is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; Y3 is alkanediyl (C≦12) , alkenediyl (C≦12) , arenediyl (C≦12) , or a substituted form of any of these groups; or the formula: TIFF2025108518000015.tif13128; wherein X3 and X4 are alkanediyl (C≦12) , alkenediyl (C≦12), an arenediyl (C≦12) or a substituted form of any of these groups; Y5 is a covalent bond, an alkanediyl (C≦12) an alkenediyl (C≦12) an arenediyl (C≦12) or a substituted form of any of these groups; and R9 is alkyl (C≦8) or substituted alkyl (C≦8) ; The linker group has the following formula: TIFF2025108518000016.tif16128 and wherein Y1 is an alkanediyl (C≦12) an alkenediyl (C≦12) an arenediyl (C≦12) or a substituted form of any of these groups; and When the repeating unit contains a linker group, if n is greater than 1, the linker group contains an independent degradable diacyl group bonded to both the nitrogen atom and the sulfur atom of the linker group, the first group in the repeating unit is a degradable diacyl group, and for each linker group, the adjacent repeating unit contains two degradable diacyl groups bonded to the nitrogen atom of the linker group; n is the number of linker groups present in the repeating unit; and The terminal group has the following formula: TIFF2025108518000017.tif13128 and Y4 is an alkanediyl (C≦18) or an alkanediyl (C≦18) where one or more of the hydrogen atoms on it are replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH 3、 or alkanediyl substituted with -OC(O)CH3 (C≦18) ; R 10 is hydrogen, carboxy, hydroxy, or aryl (C≦12) alkylamino (C≦12) dialkylamino (C≦12) N - heterocycloalkyl (C≦12) , -C(O)N(R 11)-alkanediyl (C≦6) -heterocycloalkyl (C≦12) 、-C(O)-alkyl-amino (C≦12) 、-C(O)-dialkylamino (C≦12) 、-C(O)-N-heterocyclo-alkyl (C≦12) and wherein R 11 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; the last degradable diacyl in the chain is attached to the terminal group; n is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, the terminal group is of the following formula: TIFF2025108518000018.tif13128 and is further defined by wherein Y4 is alkanediyl (C≦18) and R 10 is hydrogen. In some embodiments, the core is of the following formula: TIFF2025108518000019.tif12128 and is further defined by wherein X2 is N(R5) y ; R5 is hydrogen, or alkyl (C≦8) or substituted alkyl (C≦18) ; and y and z sum to 3, where y is 0, 1, or 2; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) dialkylamino (C≦12) or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z and y sum to 3, where z is 1, 2, 3. In some embodiments, the core is of the following formula: TIFF2025108518000020.tif11128 and is further defined by wherein X3 is -NR6- wherein R6 is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8) -O-, or alkylaminodiyl (C≦8) alkoxydiy (C≦8) arenediyl (C≦8) heteroarenediyl (C≦8) heterocycloalkanediyl (C≦8) or a substituted form of any of these groups; R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) dialkylamino (C≦12) or a substituted form of any of these groups; or the group of the formula: -N(R f ) f (CH2CH2N) e (R c )R d ; wherein e and f are each independently 1, 2, or 3, provided that the sum of e and f is 3; R c R d and R f are each independently hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; c and d are each independently 1, 2, 3, 4, 5, or 6. In some embodiments, the core is further defined as TIFF2025108518000021.tif123148. In some embodiments, the degradable diacyl is further defined as TIFF2025108518000022.tif15128. In some embodiments, the linker is further defined as TIFF2025108518000023.tif16128, wherein Y1 is alkanediyl (C≦8) or substituted alkanediyl (C≦8)It is. In some embodiments, the dendrimer is further defined as TIFF2025108518000024.tif219147TIFF2025108518000025.tif187141 or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the phospholipid is present at a molar percentage of about 8% to about 20% with respect to the lipid nanoparticle composition. In some embodiments, the molar percentage of the phospholipid is about 10% to about 14%. In some embodiments, the molar percentage of the phospholipid is about 11.9%. In other embodiments, the phospholipid is present at a molar percentage of about 20% to about 23% with respect to the lipid nanoparticle composition. In some embodiments, there is a molar percentage of about 20% to about 21% of the phospholipid. In some embodiments, the molar percentage of the phospholipid is about 20.3%. In some embodiments, the phospholipid is further defined as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or 1,2-distearoyl-sn-glycero-3-phosphocholine. In some embodiments, the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.

[0031] In some embodiments, the composition further comprises a steroid. In some embodiments, the steroid is present at a molar percentage of about 39% to about 46% with respect to the lipid nanoparticle composition. In some embodiments, the molar percentage of the steroid is about 40% to about 43%. In some embodiments, the molar percentage of the steroid is about 40.5%. In other embodiments, the steroid is present at a molar percentage of about 15% to about 39% with respect to the lipid nanoparticle composition. In some embodiments, there is a molar percentage of about 20% to about 27.5% of the steroid. In some embodiments, the molar percentage of the steroid is about 23.8%. In some embodiments, the steroid is cholesterol.

[0032] In some embodiments, the composition further comprises a PEGylated lipid. In some embodiments, the PEGylated lipid is present in a molar percentage of about 0.5% to about 10.0% relative to the lipid nanoparticle composition. In some embodiments, a molar percentage of about 2.0% to about 2.8% of the PEGylated lipid is present. In some embodiments, the molar percentage of the PEGylated lipid is about 2.4%. In other embodiments, the PEGylated lipid is present in a molar percentage of about 3.9% to about 4.6% relative to the lipid nanoparticle composition. In some embodiments, a molar percentage of about 4.0% to about 4.3% of the PEGylated lipid is present. In some embodiments, the molar percentage of the PEGylated lipid is about 4.1%. In some embodiments, the PEGylated lipid comprises a PEG moiety of about 1000 to about 10,000 Daltons. In some embodiments, the PEG lipid is PEGylated diacylglycerol. In some embodiments, the PEG lipid is further defined by the following formula: TIFF2025108518000026.tif18128, wherein R 12 and R 13 are each independently alkyl (C≦24) , alkenyl (C≦24) , or a substituted form of any of these groups; R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) ; and x is from 1 to 250. In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula: TIFF2025108518000027.tif39128, wherein n1 is from 5 to 250; and n2 and n3 are each independently from 2 to 25.

[0033] In some embodiments, the composition comprises cholesterol and DMG-PEG. In some embodiments, the composition comprises DOPE. In other embodiments, the composition comprises DSPC. In some embodiments, the composition further comprises DLin-MC3-DMA. In other embodiments, the composition further comprises C12-200. In some embodiments, the composition further comprises 3A5-SC8, 3A3-SC8, 4A1-SC8, 4A3-SC8, 5A2-SC8 having five tails, or 5A2-SC8 having six tails. In some embodiments, the composition further comprises 5A2-SC8. In some embodiments, the composition further comprises DOTAP. In some embodiments, the composition comprises cholesterol, DMG-PEG, DSPC, DLin-MC3-DMA, and DOTAP.

[0034] In some embodiments, the therapeutic agent is a small molecule, such as an anti-cancer agent, an anti-fungal agent, a psychiatric agent, such as an analgesic, an agent that changes the level of consciousness, such as an anesthetic or a sleep agent, a non-steroidal anti-inflammatory drug (NSAIDs), an anthelmintic, an anti-acne agent, an anti-angina agent, an anti-arrhythmic agent, an anti-asthma agent, an antibacterial agent, an anti-benign prostatic hyperplasia agent, an anticoagulant, an antidepressant, an anti-diabetic agent, an anti-emetic, an anti-epileptic agent, an anti-gout agent, an antihypertensive agent, an anti-inflammatory agent, an anti-malarial agent, an anti-migraine agent, an anti-muscarinic agent, an anti-neoplastic agent, an anti-obesity agent, an anti-osteoporosis agent, an anti-Parkinson's disease agent, an anti-proliferative agent, an anti-protozoal agent, an anti-thyroid agent, an antitussive, an anti-incontinence agent, an anti-viral agent, an anti-anxiety agent, an appetite suppressant, a β-blocker, a cardiac inotropic agent, a chemotherapeutic agent, a cognition enhancer, a contraceptive, a corticosteroid, a Cox-2 inhibitor, a diuretic, an erectile dysfunction improver, an expectorant, a gastrointestinal agent, a histamine receptor antagonist, an immunosuppressant, a keratolytic, a lipid regulator, a leukotriene inhibitor, a macrolide, a muscle relaxant, a nerve blocker, a nutrient, an opioid analgesic, a protease inhibitor, or a sedative. In other embodiments, the therapeutic agent is a protein. In other embodiments, the therapeutic agent is a nucleic acid, such as a therapeutic nucleic acid. In some embodiments, the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA). In some embodiments, the composition comprises a first nucleic acid and a second nucleic acid. In some embodiments, the first nucleic acid is messenger RNA. In some embodiments, the second nucleic acid is single guide RNA. In some embodiments, the first nucleic acid is messenger RNA (mRNA) and single guide RNA (sgRNA).In some embodiments, the nucleic acid is present at a ratio of lipid nanoparticle composition to nucleic acid of about 1:1 to about 1:100. In some embodiments, the ratio is from about 1:10 to about 1:60. In some embodiments, the ratio is about 1:40.

[0035] In some embodiments, the composition further comprises a protein. In some embodiments, the protein is a protein related to translation or transcription. In some embodiments, the protein is related to the CRISPR process. In some embodiments, the protein is a CRISPR-related protein. In some embodiments, the protein is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, its homolog, or a modified version thereof. In some embodiments, the protein is Cas9. In some embodiments, the protein and the nucleic acid are present at a molar ratio of about 1:1 to about 1:20. In some embodiments, the molar ratio is from about 1:1 to about 1:10. In some embodiments, the molar ratio is from about 1:3 to about 1:8.

[0036] In some embodiments, the composition has a negative zeta potential. In some embodiments, the zeta potential is from -0.25 mV to about -10 mV. In some embodiments, the zeta potential is from about -0.5 mV to about -2 mV. In some embodiments, the composition contains both a protein and a nucleic acid. In some embodiments, the composition contains a Cas9 protein and a single guide nucleic acid. In some embodiments, the composition contains a Cas9 protein, a single guide nucleic acid, and donor DNA.

[0037] In another aspect, the present disclosure (A) a composition described herein, and (B) an excipient to provide a pharmaceutical composition.

[0038] In some embodiments, the pharmaceutical composition is formulated for oral, intraadiposal, intraarterial, intraarticular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intrapericardial, intraperitoneal, intrapleural, intraprostatic, rectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intracellular, intravitreal, liposomally, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, locally, transbuccally, transdermally, vaginally, in a cream, in a lipid composition, via a catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via topical delivery, or via topical perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous injection or intraarterial injection. In some embodiments, the excipient is a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical composition is formulated as a unit dose.

[0039] In yet another aspect, the present disclosure provides a method of regulating gene expression, comprising the step of delivering a nucleic acid to a cell, the method comprising contacting the cell with a composition or a pharmaceutical composition described herein under conditions sufficient to cause uptake of the nucleic acid by the cell.

[0040] In some embodiments, the cell is contacted in vitro or ex vivo. In some embodiments, the cell is contacted in vivo. In some embodiments, the regulation of gene expression is sufficient to treat a disease or disorder, such as cancer.

[0041] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a pharmaceutically effective amount of a composition or pharmaceutical composition described herein, wherein the composition or pharmaceutical composition comprises a therapeutic nucleic acid or protein for the disease or disorder.

[0042] In some embodiments, the disease or disorder is cancer. In some embodiments, the method further comprises administering to the patient one or more additional cancer therapies. In some embodiments, the cancer therapy is a chemotherapeutic compound, surgery, radiation therapy, or immunotherapy. In some embodiments, the composition or pharmaceutical composition is administered to the patient once. In other embodiments, the composition or pharmaceutical composition is administered to the patient two or more times. In some embodiments, the patient is a mammal such as a human.

[0043] In yet another aspect, the present disclosure provides a method of preparing lipid nanoparticles, comprising: (A) dissolving a permanent cationic lipid, a cationic ionizable lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent; (B) dissolving a therapeutic agent in a buffer to form a buffered therapeutic agent solution, wherein the buffer is a buffer having a pH of about 6.8 to about 7.6; and (C) mixing the lipid solution with the buffered therapeutic agent solution to form lipid nanoparticles and providing a method.

[0044] In some embodiments, the organic solvent is a C1-C4 alcohol solvent such as ethanol. In some embodiments, the buffer is an aqueous PBS buffer. In some embodiments, the encapsulation efficiency of the method exceeds 80%.

[0045] In yet another aspect, the present disclosure provides (A) a therapeutic agent, and (B) (1) Cationic ionizable lipid; (2) Phospholipid; and (3) Selective organ targeting compound A lipid nanoparticle composition comprising A composition comprising, wherein the organ targeting ligand causes preferential delivery of the composition to organs other than the liver.

[0046] In yet another aspect, the present disclosure relates to (A) A therapeutic agent, and (B) (1) Cationic ionizable lipid; (2) Phospholipid; (3) Selective organ targeting compound; (4) Steroid; and (5) PEG lipid A lipid nanoparticle composition comprising A composition comprising, wherein the organ targeting ligand causes preferential delivery of the composition to organs other than the liver.

[0047] In yet another aspect, the present disclosure relates to a therapeutic agent and (A) Cationic ionizable lipid; (B) Phospholipid; and (C) Selective organ targeting compound A lipid nanoparticle composition comprising A composition comprising, having an apparent pK of about 8 to about 13 a and mainly delivering nucleic acids to the lungs.

[0048] In another aspect, the present disclosure relates to a therapeutic agent and (A) Cationic ionizable lipid; (B) Phospholipid; (C) Selective organ targeting compound; (D) Steroid; and (E) PEG lipid; A lipid nanoparticle composition comprising A composition comprising, having an apparent pK of about 8 to about 13 aProvided is a composition that has and mainly delivers nucleic acids to the lung.

[0049] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; and (C) a selective organ targeting compound in a lipid nanoparticle composition, the composition having an apparent pK of about 3 to about 6 and mainly delivering nucleic acids to the spleen. a a Provided is a composition that has and mainly delivers nucleic acids to the spleen.

[0050] In still yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a steroid; (B) a cationic ionizable lipid; (C) a phospholipid; (D) a PEG lipid; and (E) a selective organ targeting compound in a lipid nanoparticle composition, the composition having an apparent pK of about 3 to about 6 and mainly delivering nucleic acids to the spleen. a a Provided is a composition that has and mainly delivers nucleic acids to the spleen.

[0051] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; and (C) C6-C 24 diacyl phosphatidylcholine in a lipid nanoparticle composition, the composition mainly delivering nucleic acids to lymph nodes.

[0052] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and (A) a steroid; (B) a cationic ionizable lipid; (C) a phospholipid; (D) a PEG lipid; and (E) C6-C 24Diacylphosphatidylcholine a lipid nanoparticle composition comprising and a composition that mainly delivers nucleic acids to lymph nodes is provided.

[0053] In yet another aspect, the present disclosure provides a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; and (C) a selective organ targeting compound a lipid nanoparticle composition comprising and a composition, wherein the surface of the composition interacts with vitronectin and the composition mainly delivers nucleic acids to the lungs.

[0054] In yet another aspect, the present disclosure provides a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; (C) a selective organ targeting compound; (D) a steroid; and (E) a PEG lipid a lipid nanoparticle composition comprising and a composition, wherein the surface of the composition interacts with vitronectin and the composition mainly delivers nucleic acids to the lungs.

[0055] In yet another aspect, the present disclosure provides a therapeutic agent and (A) a cationic ionizable lipid; (B) a phospholipid; and (C) a selective organ targeting compound a lipid nanoparticle composition comprising and a composition, wherein the surface of the composition interacts with Apo H and the composition mainly delivers nucleic acids to the spleen.

[0056] In another aspect, the present disclosure provides a therapeutic agent and (A) a steroid; (B) a cationic ionizable lipid; (C) a phospholipid; (D) PEG lipid; and (E) a selective organ targeting compound a lipid nanoparticle composition comprising the same, and a composition comprising the same, wherein the surface of the composition interacts with Apo H and the composition mainly delivers nucleic acid to the spleen.

[0057] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein a targeting protein present in the protein corona on the surface of the composition binds to a target protein substantially present in the target organ, and the target organ is not the liver.

[0058] In some embodiments, the targeting protein is selected from vitronectin or β2-glycoprotein I (ApoH). In some embodiments, the targeting protein is vitronectin and the target organ is the lung. In other embodiments, the targeting protein is ApoH and the target organ is the spleen.

[0059] In some embodiments, the lipid nanoparticle composition further comprises a selective organ targeting compound that modifies the binding of proteins on the protein corona. In some embodiments, further, the selective organ targeting compound is selected from sugars, lipids, small molecule therapeutic agents, vitamins, or proteins. In some embodiments, the selective organ targeting compound is a lipid, for example, a permanent cationic lipid, a permanent anionic lipid, or phosphatidylcholine. In some embodiments, the lipid nanoparticle composition further comprises a cationic ionizable lipid. In some embodiments, the lipid nanoparticle composition further comprises a phospholipid. In some embodiments, the lipid nanoparticle composition further comprises a steroid. In some embodiments, the lipid nanoparticle composition further comprises a PEG lipid.

[0060] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ targeting compound, and the selective organ targeting compound has an apparent pK of about 3 to about 6a To provide a composition that results in a lipid nanoparticle composition having

[0061] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the lipid nanoparticle composition comprises a selective organ targeting compound, and the selective organ targeting compound has an apparent pK of about 8 to about 13 a To provide a composition that results in a lipid nanoparticle composition having

[0062] As used herein, “essentially free of” with respect to a specified component means that none of the specified components are intentionally formulated into the composition and / or are present only as contaminants or in trace amounts. The total amount of the specified component due to unintentional contamination of the composition is preferably less than 0.01%. Most preferably, the composition is one in which the amount of the specified component cannot be detected using standard analytical methods.

[0063] As used herein and in the claims herein, “a” or “an” may mean one or more. As used herein and in the claims herein, when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more. As used herein and in the claims herein, “another” or “further” may mean at least second or more.

[0064] As used herein and in the claims, the term “about” is used to indicate that a value includes the inherent variability of error of the device, method used to determine that value, or variability that exists between test subjects.

[0065] [Invention 1001] (A) A therapeutic agent, and (B) (1) A selective organ targeting compound; (2) Cationic ionizable lipid; and (3) Phospholipid A lipid nanoparticle composition comprising A composition comprising, which preferentially delivers a nucleic acid to a target organ selected from the lung, heart, brain, spleen, lymph node, bone marrow, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. [Invention 1002] The composition of Invention 1001, wherein the target organ is the lung, lymph node, or spleen. [Invention 1003] The composition of Invention 1001 or 1002, wherein the selective organ targeting compound is a permanently cationic lipid. [Invention 1004] The composition of Invention 1003, wherein the permanently cationic lipid is present at a molar percentage of about 5% to about 20% with respect to the lipid nanoparticle composition. [Invention 1005] The composition of any one of Inventions 1001 to 1004, wherein the permanently cationic lipid is present at a molar percentage of about 20% to about 65% with respect to the lipid nanoparticle composition. [Invention 1006] The composition of any one of Inventions 1001 to 1005, wherein the permanently cationic lipid contains a quaternary ammonium ion. [Invention 1007] The permanently cationic lipid is Further defined as TIFF2025108518000028.tif24128, Wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) Or a substituted form of any group; R3, R3', and R3'' are each independently alkyl (C≦6) Or substituted alkyl (C≦6) ; X - Is a monovalent anion, The composition of any one of Inventions 1004 to 1006. [Invention 1008] The permanent cationic lipid is the composition of Invention 1007, further defined as TIFF2025108518000029.tif21128. [Invention 1009] The permanent cationic lipid is further defined as TIFF2025108518000030.tif14128, and wherein R4 and R4' are each independently alkyl (C6~C24) , alkenyl (C6~C24) , or a substituted form of any of the groups; R4'' is alkyl (C≦24) , alkenyl (C≦24) , or a substituted form of any of the groups; R4''' is alkyl (C1~C8) , alkenyl (C2~C8) , or a substituted form of any of the groups; and X2 is a monovalent anion, the composition of any of Inventions 1004 - 1006. [Invention 1010] The permanent cationic lipid is the composition of Invention 1009, further defined as TIFF2025108518000031.tif9128. [Invention 1011] The permanent cationic lipid is further defined as TIFF2025108518000032.tif24128, and wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any of the groups; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; R4 is alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion, The composition of any one of the present inventions 1004 to 1006. [The present invention 1012] The permanent cationic lipid is The composition of the present invention 1011, further defined as TIFF2025108518000033.tif27128. [The present invention 1013] The composition of the present invention 1001 or 1002, wherein the selective organ targeting compound is a permanent anionic lipid. [The present invention 1014] The composition of the present invention 1013, wherein the permanent anionic lipid is present in a molar percentage of about 5% to about 50% with respect to the lipid nanoparticle composition. [The present invention 1015] The composition of the present invention 1013 or 1014, wherein the permanent anionic lipid contains a phosphate group. [The present invention 1016] The permanent anionic lipid is Further defined as TIFF2025108518000034.tif24128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any group; R3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y1-R4, wherein Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) ; and R4 is acyloxy (C≦8~24) or substituted acyloxy (C≦8~24) is The composition of any one of the present inventions 1013 to 1015. [The present invention 1017] The permanent anionic lipid is The composition of the present invention 1016, further defined as TIFF2025108518000035.tif103128. [The present invention 1018] The selected organ-targeting compound is C6-C 24 The composition of the present invention 1001 or 1002, which is diacyl phosphotidylcholine. [The present invention 1019] The composition of the present invention 1018, wherein diacyl phosphotidylcholine is present in a molar percentage of about 5% to about 50% based on the lipid nanoparticle composition. [The present invention 1020] The composition of the present invention 1018 or 1019, wherein the selected organ-targeting compound contains at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. [The present invention 1021] Diacyl phosphotidylcholine is Further defined as TIFF2025108518000036.tif24128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion, The composition of any one of the present inventions 1018 to 1020. [The present invention 1022] Diacyl phosphotidylcholine is Further defined as TIFF2025108518000037.tif21128, the composition of the present invention 1022. [The present invention 1023] The composition of any one of the present inventions 1001 to 1022, wherein the cationic ionizable lipid is present in a molar percentage of about 5% to about 30% based on the lipid nanoparticle composition. [The present invention 1024] The composition of any one of the present inventions 1001 to 1022, wherein the cationic ionizable lipid is present in a molar percentage of about 15% to about 30% based on the lipid nanoparticle composition. [The present invention 1025] A composition according to any one of 1001 to 1024 of the present invention, wherein the cationic ionizable lipid contains an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups. [The present invention 1026] The cationic ionizable lipid is represented by the following formula: Core - repeating unit - terminal group (I) which is a dendrimer further defined by or a pharmaceutically acceptable salt thereof, wherein the core is linked to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeating unit, and the core has TIFF2025108518000038.tif10128, wherein (C≦12) X1 is amino or alkylamino (C≦12) , dialkylamino (C≦12) , heterocycloalkyl (C≦12) , heteroaryl or a substituted form thereof; (C≦12) R1 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino or a substituted form of any of these groups; and a is 1, 2, 3, 4, 5, or 6; or the core has TIFF2025108518000039.tif11128, wherein y X2 is N(R5) where (C≦18) R5 is hydrogen, alkyl (C≦18) or substituted alkyl; y is 0, 1, or 2 provided that the sum of y and z is 3; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12)or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3, provided that the sum of z and y is 3; or the core has the following formula: TIFF2025108518000040.tif11128, wherein, X3 is -NR6-, wherein, R6 is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8) -O-, or alkylaminodiyl (C≦8) alkoxydiylyl (C≦8) arenediyl (C≦8) heteroarenediyl (C≦8) heterocycloalkanediyl (C≦8) or a substituted form of any of these groups; R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) dialkylamino (C≦12) or a substituted form of any of these groups; or the formula: -N(R f ) f (CH2CH2N) e (R c )R d is a group; wherein, e and f are each independently 1, 2, or 3, provided that the sum of e and f is 3; R c R d and R f are each independently hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; c and d are each independently 1, 2, 3, 4, 5, or 6; or the core is alkylamine (C≦18) dialkylamine (C≦36) heterocycloalkane (C≦12)or a substituted form of any of these groups; The repeating unit contains a cleavable diacyl and a linker; The cleavable diacyl group has the following formula: TIFF2025108518000041.tif17128 having wherein, A1 and A2 are each independently -O- or -NR a -, wherein, R a is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; Y3 is alkanediyl (C≦12) alkenediyl (C≦12) arenediyl (C≦12) or a substituted form of any of these groups; or a group of the formula: TIFF2025108518000042.tif12128; wherein, X3 and X4 are alkanediyl (C≦12) alkenediyl (C≦12) arenediyl (C≦12) or a substituted form of any of these groups; Y5 is a covalent bond, alkanediyl (C≦12) alkenediyl (C≦12) arenediyl (C≦12) or a substituted form of any of these groups; and R9 is alkyl (C≦8) or substituted alkyl (C≦8) ; The linker group has the following formula: TIFF2025108518000043.tif15128 having, wherein, Y1 is alkanediyl (C≦12) alkenediyl (C≦12) arenediyl (C≦12) or a substituted form of any of these groups; and When the repeating unit contains a linker group, if n is greater than 1, the linker group contains an independent degradable diacyl group bonded to both the nitrogen atom and the sulfur atom of the linker group, the first group in the repeating unit is the degradable diacyl group, and for each linker group, the adjacent repeating unit contains two degradable diacyl groups bonded to the nitrogen atom of the linker group; n is the number of linker groups present in the repeating unit; and the terminal group has the following formula: TIFF2025108518000044.tif12128, and wherein, Y4 is alkanediyl (C≦18) or alkanediyl (C≦18) where one or more of the hydrogen atoms thereon are -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH 3、 or -OC(O)CH3-substituted alkanediyl (C≦18) ; R 10 is hydrogen, carboxy, hydroxy, or aryl (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , N-heterocycloalkyl (C≦12) , -C(O)N(R 11 )-alkanediyl (C≦6) -heterocycloalkyl (C≦12) , -C(O)-alkylamino (C≦12) , -C(O)-dialkylamino (C≦12) , -C(O)-N-heterocycloalkyl (C≦12) ; wherein, R 11 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; the last degradable diacyl in the chain is bonded to the terminal group; n is 0, 1, 2, 3, 4, 5, or 6, any composition of the present invention from 1001 to 1025. [The present invention 1027] the terminal group has the following formula: It is further defined by TIFF2025108518000045.tif12128, Y4 is alkanediyl (C≦18) and; R 10 is hydrogen, the composition of the present invention 1026. [The present invention 1028] The core has the following formula: It is further defined by TIFF2025108518000046.tif11128, wherein, X2 is N(R5) y and; R5 is hydrogen, or alkyl (C≦8) or substituted alkyl (C≦18) and; under the condition that the sum of y and z is 3, y is 0, 1, or 2; R2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) dialkylamino (C≦12) or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and under the condition that the sum of z and y is 3, z is 1, 2, 3, the composition of the present invention 1026 or 1027. [The present invention 1029] The core has the following formula: It is further defined by TIFF2025108518000047.tif11128, wherein, X3 is -NR6-, wherein, R6 is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8) -O-, or alkylaminodiyl (C≦8) alkoxydiyl (C≦8) arenediyl (C≦8) heteroarenediyl (C≦8) heterocycloalkanediyl (C≦8) or a substituted form of any of these groups; R3 and R4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; or of the formula: -N(R f ) f (CH2CH2N) e (R c )R d ; where in the formula e and f are each independently 1, 2, or 3, provided that the sum of e and f is 3; or R c , R d , and R f are each independently hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) ; c and d are each independently 1, 2, 3, 4, 5, or 6, a composition of the present invention 1026 or 1027. [Present invention 1030] wherein the core is further defined as TIFF2025108518000048.tif122144, a composition of any one of the present inventions 1026 to 1029. [Present invention 1031] wherein the dendrimer is further defined as TIFF2025108518000049.tif157143TIFF2025108518000050.tif149137 or TIFF2025108518000051.tif82131, or a pharmaceutically acceptable salt thereof, a composition of any one of the present inventions 1026 to 1030. [Present invention 1032] wherein the phospholipid is present at a molar percentage of about 8% to about 20% relative to the lipid nanoparticle composition, a composition of any one of the present inventions 1001 to 1031. [Present invention 1033] wherein the phospholipid is present at a molar percentage of about 20% to about 23% relative to the lipid nanoparticle composition, a composition of any one of the present inventions 1001 to 1031. [Invention 1034] A composition according to any one of Inventions 1001 to 1033, further comprising a steroid. [Invention 1035] The composition of Invention 1034, wherein the steroid is present at a molar percentage of about 39% to about 46% based on the lipid nanoparticle composition. [Invention 1036] The composition of Invention 1035, wherein the steroid is present at a molar percentage of about 15% to about 39% based on the lipid nanoparticle composition. [Invention 1037] A composition according to any one of Inventions 1001 to 1036, further comprising a PEGylated lipid. [Invention 1038] The composition of Invention 1037, wherein the PEGylated lipid is present at a molar percentage of about 0.5% to about 10.0% based on the lipid nanoparticle composition. [Invention 1039] The PEG lipid is further defined by the following formula: TIFF2025108518000052.tif18128, wherein R 12 and R 13 are each independently alkyl (C≦24) , alkenyl (C≦24) , or a substituted form of any of these groups; R e is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) ; and x is from 1 to 250, The composition of Invention 1037 or 1038. [Invention 1040] The PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula: TIFF2025108518000053.tif38128, wherein n1 is from 5 to 250; and n2 and n3 are each independently from 2 to 25, The composition of Invention 1037 or 1038. [Invention 1041] A composition according to any one of inventions 1001 to 1040, wherein the therapeutic agent is a nucleic acid. [Invention 1042] A composition according to invention 1041, wherein the nucleic acid is a therapeutic nucleic acid. [Invention 1043] A composition according to invention 1041, wherein the nucleic acid is siRNA, miRNA, pri - miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR) - associated nucleic acid, single - guide RNA (sgRNA), CRISPR RNA (crRNA), trans - activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double - stranded DNA (dsDNA), single - stranded DNA (ssDNA), single - stranded RNA (ssRNA), and double - stranded RNA (dsRNA). [Invention 1044] A composition according to any one of inventions 1041 to 1043, wherein the nucleic acid is present at a ratio of lipid nanoparticle composition to nucleic acid of about 1:1 to about 1:100. [Invention 1045] A composition according to any one of inventions 1001 to 1044, further comprising a protein. [Invention 1046] A composition according to any one of inventions 1001 to 1045, wherein the protein and the nucleic acid are present at a molar ratio of about 1:1 to about 1:20. [Invention 1047] A composition according to any one of inventions 1001 to 1046, comprising both a protein and a nucleic acid. [Invention 1048] (A) A composition according to any one of inventions 1001 to 1047, and (B) an excipient A pharmaceutical composition comprising the same. [Invention 1049] A method for regulating gene expression, comprising the step of delivering a nucleic acid to a cell, The method comprising the step of contacting the cell with any one of the compositions or pharmaceutical compositions of the present invention 1001 to 1048 under conditions sufficient to cause the uptake of nucleic acid into the cell. [The present invention 1050] A method for treating a disease or disorder in a patient, comprising the step of administering to a patient in need of treatment of the disease or disorder a pharmaceutically effective amount of any one of the compositions or pharmaceutical compositions of the present invention 1001 to 1048, The method, wherein the composition or pharmaceutical composition comprises a therapeutic nucleic acid for the disease or disorder. [The present invention 1051] The method of the present invention 1050, wherein the disease or disorder is cancer. [The present invention 1052] A method for preparing lipid nanoparticles, the method comprising the following steps: (A) Dissolving a permanent cationic lipid, a cationic ionizable lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent; (B) Dissolving a therapeutic agent in a buffer to form a buffered therapeutic agent solution, wherein the buffer is a buffer having a pH of about 6.8 to about 7.6; and (C) Mixing the lipid solution with the buffered therapeutic agent solution to form lipid nanoparticles. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, while specific embodiments of the present disclosure are shown, it is to be understood that various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description, and thus the detailed description and specific examples are given by way of illustration only.

Brief Description of the Drawings

[0066] The drawings form a part of this specification and are included to further demonstrate specific aspects of the present disclosure. The present disclosure can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

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Modes for Carrying Out the Invention

[0067] Description of Exemplary Embodiments 1) Lipid nanoparticles (LNPs) composed of a permanent cationic lipid, 2) a cationic ionizable lipid, and 3) a phospholipid are described herein and may optionally contain cholesterol and / or lipid PEG. The inclusion of the permanent cationic lipid serves to direct the LNP to specific organs such as the lung, lymph nodes, or spleen. The data presented herein show that this effect is universal and that the components are modular, indicating that 5A2-SC8 can be exchanged for any cationic ionizable lipid, DOTAP can be exchanged for any cationic lipid, and DOPE can be exchanged for any phospholipid. In some embodiments, cholesterol and lipid PEG are also included, but formulations without either cholesterol or lipid PEG are achievable. These carriers can deliver mRNA, sgRNA, and proteins to specific organs in vivo, thus solving a major problem. These carriers are also intended to be able to deliver other nucleic acids (e.g., siRNA, miRNA, crRNA, trRNA, tRNA, etc.) and drugs (e.g., small molecules) to specific organs in vivo.

[0068] A. Chemical Definition When used in the context of chemical groups: "hydrogen" means -H; "hydroxy" means -OH; "oxo" means =O; "carbonyl" means -C(=O)-; "carboxy" means -C(=O)OH (also denoted as -COOH or -CO2H); "halo" independently means -F, -Cl, -Br or -I; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" means -NO2; imino means -NH; "cyano" means -CN; "isocyanate" means -N=C=O; "azide" means -N3; in a monovalent context, "phosphate" means -OP(O)(OH)2 or its deprotonated form; in a divalent context, "phosphate" means -OP(O)(OH)O- or its deprotonated form; "mercapto" means -SH; "thio" means =S; "sulfonyl" means -S(O)2-; "hydroxysulfonyl" means -S(O)2OH; "sulfonamide" means -S(O)2NH2; and "sulfinyl" means -S(O)-.

[0069] In the context of chemical formulas, the symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. The symbol "----" represents an optional bond, which, if present, is either a single or a double bond. The symbol: TIFF2025108518000054.tif5170 indicates a single or double bond. Thus, for example, the formula: TIFF2025108518000055.tif10128 is TIFF2025108518000056.tif12128 is included. It is understood that such ring atoms do not form part of more than one double bond. Further, note that the covalent bond symbol "-", when connecting one or two stereogenic atoms, does not indicate any preferred stereochemistry. Rather, it encompasses all stereoisomers and mixtures thereof. The symbol: TIFF2025108518000057.tif4140 when drawn perpendicular across the bond TIFF2025108518000058.tif shows the bond points based on 8154. Note that in order to assist the reader in clearly identifying the bond points, the bond points are typically only specified in this way for larger groups. Symbol: TIFF2025108518000059.tif 5128 means a single bond where the group attached to the thick end of the wedge is "outside the page". Symbol: TIFF2025108518000060.tif 5128 means a single bond where the group attached to the thick end of the wedge is "inside the page". Symbol: TIFF2025108518000061.tif 4128 means a single bond where the geometry around the double bond (e.g., either E or Z) is not defined. Therefore, both options, and combinations thereof, are intended. Any undefined valence on an atom of the structures shown in this application implies a hydrogen atom bonded to that atom. The thick dot on a carbon atom indicates that the hydrogen bonded to that carbon is oriented out of the plane of the paper.

[0070] The group "R", for example, if in the formula: TIFF2025108518000062.tif 14128 is depicted as a "floating group" on the ring system, then R can replace any hydrogen atom bonded to any ring atom, including the depicted, implied, or explicitly defined hydrogens, as long as a stable structure is formed. The group "R", for example, in the formula: As in TIFF2025108518000063.tif17128, if depicted as a "floating group" on a condensed ring system, R can replace any hydrogen bonded to any ring atom of any of the condensed rings, unless otherwise specified. Substitutable hydrogens include depicted hydrogens (e.g., hydrogens bonded to nitrogen in the above formula), implied hydrogens (e.g., hydrogens in the above formula that are not shown but are understood to be present), clearly defined hydrogens, and any hydrogen whose existence depends on the identity of the ring atom (e.g., when X is equal to -CH-, the hydrogen bonded to group X). In the depicted examples, R can be present in either the 5-membered or 6-membered ring of the condensed ring system. In the above formula, the subscript "y" immediately following the group "R" enclosed in parentheses represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of substitutable hydrogen atoms in the ring or ring system.

[0071] For chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number being as small as possible for the group / class in question. For example, it is understood that the minimum number of carbon atoms in the group "alkenyl" (C≦8) or class "alkene" (C≦8) is 2. Compare with "alkoxy" (C≦10) which indicates an alkoxy group having 1 to 10 carbon atoms. "Cn-n'" defines both the minimum number (n) and the maximum number (n') of carbon atoms in the group. Thus, "alkyl" (C2~10) indicates an alkyl group having 2 to 10 carbon atoms. These carbon number indicators can be before or after the chemical group or class they modify and may or may not be enclosed in parentheses without indicating any change in meaning. Thus, "C5 olefin", "C5-olefin", "olefin" (C5) and "olefin" C5The terms "」" are all synonyms.

[0072] The term "saturated", when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and no carbon-carbon triple bonds, except as described below. When this term is used to modify an atom, it means that the atom is not part of any double or triple bond. In the case of substitution patterns of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. If such bonds are present, carbon-carbon double bonds that can arise as part of keto-enol tautomerism or imine / enamine tautomerism are not excluded. The term "saturated", when used to modify a solution of a substance, means that the substance can no longer dissolve in that solution.

[0073] The term "aliphatic", when used without the modifier "substituted", indicates that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic hydrocarbon compound or group. In an aliphatic compound / group, carbon atoms can be linked together in a straight chain, branched chain, or non-aromatic ring (alicyclic). An aliphatic compound / group can be saturated (alkane / alkyl) linked by single carbon-carbon bonds, or unsaturated by one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0074] The term "aromatic", when used to modify a compound or chemical group atom, means a compound or chemical group containing a planar unsaturated ring of atoms stabilized by the interaction of the bonds forming the ring.

[0075] The term "alkyl", when used without the modifier "substituted", refers to a monovalent saturated aliphatic group having a carbon atom as the point of attachment, a straight-chain or branched acyclic structure, and no atoms other than carbon and hydrogen. Groups -CH3 (Me), -CH2CH3 (Et), -CH2CH2CH3 (n-Pr or propyl), -CH(CH3)2 (i-Pr, i Pr or isopropyl), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl, t-butyl, t-Bu or tBu), and -CH2C(CH3)3 (neo-pentyl) are non-limiting examples of alkyl groups. The term "alkanediyl", when used without the modifier "substituted", refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as bonding points, a straight-chain or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. "Alkane" refers to a class of compounds having the formula H-R, where R is alkyl as defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, and -CH2CH2Cl. The term "haloalkyl" is part of substituted alkyl, and the replacement of hydrogen atoms is limited to halo (i.e., -F, -Cl, -Br, or -I) so that no other atoms other than carbon, hydrogen, and halogen are present. The group -CH2Cl is a non-limiting example of haloalkyl. The term "fluoroalkyl" is part of substituted alkyl, and the replacement of hydrogen atoms is limited to fluoro so that no other atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0076] The term "cycloalkyl", when used without the modifier "substituted", refers to a monovalent saturated aliphatic group having a carbon atom as a point of attachment, which carbon atom forms part of one or more non-aromatic ring structures, has no carbon-carbon double or triple bonds, and has no atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkanediyl", when used without the modifier "substituted", refers to a divalent saturated aliphatic group having two carbon atoms as points of attachment, having no carbon-carbon double or triple bonds, and having no atoms other than carbon and hydrogen. The group: TIFF2025108518000064.tif8128 is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to a class of compounds having the formula H-R where R is a cycloalkyl as defined above for this term. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0077] The term "alkenyl", when used without the modifier "substituted", refers to a monovalent unsaturated aliphatic group having a carbon atom as a point of attachment, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH2 (vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. The term "alkenediyl", when used without the modifier "substituted", refers to a divalent unsaturated aliphatic group having two carbon atoms as points of attachment, straight or branched, straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH3)CH2-, -CH=CHCH2-, and -CH2CH=CHCH2- are non-limiting examples of alkenediyl groups. It is known that although an alkenediyl group is aliphatic, when linked at both ends, this group is not excluded from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonyms and refer to the class of compounds having the formula H-R where R is an alkenyl as defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonyms and refer to an alkene having only one carbon-carbon double bond and the bond is part of a vinyl group at the end of the molecule. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0078] The term "alkynyl", when used without the modifier "substituted", refers to a monovalent unsaturated aliphatic group having a carbon atom as a point of attachment, a straight-chain or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term "alkynyl" does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to a class of compounds having the formula H-R where R is alkynyl. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0079] The term "aryl", when used without the modifier "substituted", refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a point of attachment, which carbon atom forms part of one or more 6-membered aromatic ring structures, all ring atoms are carbon, and the group does not consist of atoms other than carbon and hydrogen. When two or more rings are present, the rings may or may not be fused. As used herein, this term does not exclude the presence of one or more alkyl or aralkyl groups (where carbon number limitations permit) attached to the first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl groups include monovalent groups derived from phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and biphenyl. The term "arenediyl", when used without the modifier "substituted", refers to a divalent aromatic group having two aromatic carbon atoms as points of attachment, which carbon atoms form part of one or more 6-membered aromatic ring structures, all ring atoms are carbon, and the monovalent groups do not consist of atoms other than carbon and hydrogen. As used herein, this term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (where carbon number limitations permit) attached to the first aromatic ring or any additional aromatic ring present. When two or more rings are present, the rings may or may not be fused. Non-fused rings may be linked via one or more of the following: a covalent bond, an alkanediyl, or an alkenediyl group (where carbon number limitations permit). Non-limiting examples of arenediyl groups include TIFF2025108518000065.tif37147.

[0080] "Arene" refers to a class of compounds having the formula H-R, where R is aryl as the term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0081] The term "aralkyl", when used without the modifier "substituted", refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are used in the manner consistent with their respective definitions above. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the modifier "substituted", one or more hydrogen atoms from the alkanediyl and / or aryl group are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Non-limiting examples of substituted aralkyl are (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-ethan-1-yl.

[0082] The term "heteroaryl", when used without the modifier "substituted", refers to a monovalent aromatic group having an aromatic carbon atom or a nitrogen atom as a point of attachment, said carbon atom or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heteroaryl group consisting of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. The heteroaryl ring may contain one, two, three or four ring atoms selected from nitrogen, oxygen and sulfur. When two or more rings are present, the rings may or may not be fused. As used herein, this term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where carbon number limitations permit) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a point of attachment. The term "heteroarylene", when used without the modifier "substituted", refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two points of attachment, said atoms forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the divalent group consisting of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. When two or more rings are present, the rings may or may not be fused. Non-fused rings may be linked via one or more of the following: covalent bond, alkanediyl, or alkenediyl group (where carbon number limitations permit). As used herein, this term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (where carbon number limitations permit) attached to the aromatic ring or aromatic ring system.Non-limiting examples of heteroarylene groups include. TIFF2025108518000066.tif15128. "Heteroarene" refers to a class of compounds having the formula H-R where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0083] The term "heterocycloalkyl", when used without the modifier "substituted", refers to a monovalent non-aromatic group having a carbon or nitrogen atom as a point of attachment, said carbon or nitrogen atom forming part of one or more non-aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heterocycloalkyl group consisting of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. The heterocycloalkyl ring can contain one, two, three or four ring atoms selected from nitrogen, oxygen or sulfur. When two or more rings are present, the rings may or may not be fused. As used herein, this term does not exclude the presence of one or more alkyl groups (where carbon number limitations permit) attached to the ring or ring system. Similarly, this term does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as a point of attachment. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl", when used without the modifier "substituted", refers to a divalent ring group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two points of attachment, said atoms forming part of one or more ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the divalent group consisting of no atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. When two or more rings are present, the rings may or may not be fused. Non-fused rings can be linked via one or more of the following: covalent bonds, alkanediyl, or alkenediyl groups (where carbon number limitations permit). As used herein, this term does not exclude the presence of one or more alkyl groups (where carbon number limitations permit) attached to the ring or ring system.Similarly, this term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting radical remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include. TIFF2025108518000067.tif14128. When these terms are used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0084] The term "acyl", when used without the modifier "substituted", refers to the group -C(O)R where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl as those terms are defined above. Groups -CHO, -C(O)CH3 (acetyl, Ac), -C(O)CH2CH3, -C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, -C(O)CH2C6H5, -C(O)(imidazolyl) are non-limiting examples of acyl groups. "Thioacyl" is defined in a similar manner except that the oxygen atom of the group -C(O)R is replaced by a sulfur atom and is -C(S)R. The term "aldehyde" corresponds to an alkane as defined above where at least one hydrogen atom is replaced by a -CHO group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms (if any, including hydrogen atoms directly bonded to the carbon atom of the carbonyl or thiocarbonyl group) are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. Groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methyl carboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0085] The term "alkoxy", when used without the modifier "substituted", refers to a group -OR where R is alkyl as defined above. Non-limiting examples include -OCH3 (methoxy), -OCH2CH3 (ethoxy), -OCH2CH2CH3, -OCH(CH3)2 (isopropoxy), -OC(CH3)3 (tert-butoxy), -OCH(CH2)2, -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the modifier "substituted", refer to groups -OR where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to a divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio", when used without the modifier "substituted", refer to groups -SR where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane as defined above where at least one hydrogen atom is replaced by a hydroxy group. The term "ether" corresponds to an alkane as defined above where at least one hydrogen atom is replaced by an alkoxy group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2.

[0086] The term "alkylamino", when used without the modifier "substituted", refers to the group -NHR where R is alkyl as defined above. Non-limiting examples include -NHCH3 and -NHCH2CH3. The term "dialkylamino", when used without the modifier "substituted", refers to the group -NRR' where R and R' can be the same or different alkyl groups or R and R' together can represent alkanediyl. Non-limiting examples of dialkylamino groups include -N(CH3)2 and -N(CH3)(CH2CH3). The terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino", and "alkylsulfonylamino", when used without the modifier "substituted", refer to the group -NHR where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "alkylaminodiyl" refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amide" (acylamino), when used without the modifier "substituted", refers to the group -NHR where R is acyl as defined above. A non-limiting example of an amide group is -NHC(O)CH3. The term "alkylimino", when used without the modifier "substituted", refers to the divalent group =NR where R is alkyl as defined above.When any of these terms is used with the modifier "substituted", one or more hydrogen atoms attached to a carbon atom are independently replaced by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -C(O)NHCH3, -C(O)N(CH3)2, -OC(O)CH3, -NHC(O)CH3, -S(O)2OH, or -S(O)2NH2. The groups NHC(O)OCH3 and -NHC(O)NHCH3 are non-limiting examples of substituted amide groups.

[0087] The use of the words "a" or "an" can mean "one" when used in the claims and / or the specification with the term "comprising", but is also consistent with the meanings of "one or more", "at least one", and "one or two or more".

[0088] Throughout this application, the term "about" is used to indicate that a value includes the inherent variations of the error of the device, method, or variations that exist among the test subjects used to obtain that value.

[0089] As used in this application, the term "average molecular weight" refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule can have different levels of polymerization and thus can have different molar masses. The average molecular weight can be used to represent the molecular weights of a plurality of polymer molecules. The average molecular weight is typically synonymous with the average molar mass. In particular, there are three main types of average molecular weights: number average molar mass, weight (mass) average molar mass, and Z average molar mass. In the context of this application, unless otherwise specified, the average molecular weight refers to either the number average molar mass or the weight average molar mass of the formula. In some embodiments, the average molecular weight is the number average molar mass. In some embodiments, the average molecular weight can be used to describe the PEG component present in the lipid.

[0090] The terms "comprise", "have" and "include" are conjunctive verbs without limitation. Any form or tense of one or more of these verbs, such as "comprises", "comprising", "has", "having", "includes" and "including", is without limitation. For example, any method that "comprises", "has" or "includes" one or more steps is not limited to having only those one or more steps, but also encompasses other unenumerated steps as well.

[0091] As used in this specification and / or the claims, the term "effective" means sufficient to achieve the desired, expected, or intended result. An "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount", when used in the context of treating a patient or subject with a compound, means an amount of the compound that is sufficient to effect such treatment of the disease when administered to the subject or patient for treating the disease.

[0092] As used herein, "IC" 50 " refers to the inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is required to inhibit a given biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism) by only half.

[0093] An "isomer" of a first compound is another compound in which each molecule contains the same constituent atoms as the first compound, but the three-dimensional arrangement of those atoms is different.

[0094] As used herein, the terms "patient" or "subject" refer to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.

[0095] As generally used herein, "pharmaceutically acceptable" means a compound, material, composition, and / or dosage form that, within the scope of sound medical judgment, is suitable for use in contact with human and animal tissues, organs, and / or body fluids without excessive toxicity, irritation, allergic response, or other problems or complications, corresponding to a reasonable benefit-risk ratio.

[0096] "Pharmaceutically acceptable salts", as defined above, mean salts of the compounds of the present disclosure that are pharmaceutically acceptable and possess the desired pharmacological activity. Such salts include salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butylacetic acid, trimethylacetic acid, etc. Pharmaceutically acceptable salts also include base addition salts that can be formed when the acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. It should be understood that the specific anions or cations forming part of any salt of the present disclosure are not critical as long as the salt as a whole is pharmaceutically acceptable. Further examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0097] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or medium such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting a chemical substance.

[0098] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who has a high risk of and / or is susceptible to the disease but has not yet experienced or presented any or all of the pathological conditions or general symptoms of the disease, and / or (2) delaying the onset of the pathological conditions or general symptoms of a disease in a subject or patient who has a high risk of and / or is susceptible to the disease but has not yet experienced or presented any or all of the pathological conditions or general symptoms of the disease.

[0099] A "repeating unit" is the simplest structural entity of a backbone and / or polymer of a particular material, for example, any of an organic material, an inorganic material or an organometal. In the case of a polymer chain, the repeating units are continuously linked along the chain like the beads of a necklace. For example, in polyethylene -[-CH2CH2-] n -, the repeating unit is -CH2CH2-. The subscript "n" indicates the degree of polymerization, i.e., the number of repeating units linked together. If the value of "n" remains undefined or there is no "n", it simply designates the polymer nature of the material as well as the repetition of the formula within the parentheses. The concept of repeating units is equally applicable where the linkages between repeating units extend three-dimensionally, such as in metal-organic frameworks, modified polymers, thermosetting polymers, etc. In the context of dendrimers, repeating units can also be described as branching units, inner layers, or generations. Similarly, end groups can also be described as surface groups.

[0100] "Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms, but the three-dimensional arrangement of those atoms is different. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. A chiral molecule contains a chiral center, also known as a stereogenic center or stereocenter, which is any point in a molecule that bears a group such that an exchange of any two groups leads to a stereoisomer, but is not necessarily an atom. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, although in organic and inorganic compounds other atoms can also be stereocenters. A molecule can have multiple stereocenters and give rise to many stereoisomers. In compounds where the stereoisomerism is due to tetrahedral stereogenic centers (e.g., tetrahedral carbon), the total number of hypothetically possible stereoisomers is 2 n raised to the power of n, where n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer stereoisomers than the maximum possible number. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or axis of chirality where the stereochemistry is not defined, it is contemplated that the stereocenter or axis of chirality can exist as a mixture of the R and S forms, including its R-form, S-form, or racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains ≦15%, more preferably ≦10%, even more preferably ≦5%, or most preferably ≦1% of another stereoisomer.

[0101] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or presenting a disease state or overall symptoms of the disease (e.g., stopping further occurrence of the disease state and / or overall symptoms), (2) ameliorating a disease in a subject or patient experiencing or presenting a disease state or overall symptoms of the disease (e.g., reversing the disease state and / or overall symptoms), and / or (3) effecting any measurable reduction of a disease in a subject or patient experiencing or presenting a disease state or overall symptoms of the disease.

[0102] The foregoing definitions supersede any contrary definitions in any incorporated by reference document herein. However, the fact that a particular term is defined should not be construed to indicate that any undefined term is unclear. Rather, all terms used are believed to be such that one of ordinary skill in the art would understand the scope of the disclosure and be able to practice the disclosure.

[0103] B. Cationic Ionizable Lipids In some aspects of the disclosure, there are provided compositions containing compounds containing a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, the cationic ionizable lipid is protonated at physiological pH but can deprotonate at a pH greater than 8, 9, 10, 11, or 12 and contains one or more groups that are uncharged. The ionizable cationic group may contain one or more protonatable amines capable of forming a cationic group at physiological pH. The cationic ionizable lipid compound may also contain one or more lipid components, e.g., C6-C 24 Two or more fatty acids having an alkyl or alkenyl carbon group may further be included. These lipid groups may be attached via an ester bond or may be further attached by Michael addition to a sulfur atom. In some embodiments, these compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0104] In some aspects of the present disclosure, there is provided a composition comprising a compound containing a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, the cationic ionizable lipid refers to a lipid and lipid-like molecule having a nitrogen atom capable of obtaining a charge (pKa). These lipids may be known as cationic lipids in the literature. These molecules having an amino group typically have 2 to 6 hydrophobic chains, often alkyl or alkenyl, for example, C6-C24 alkyl or alkenyl groups, but may have at least one, or more than 6 tails. In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers showing regular dendritic branching and are formed by sequentially or generationally adding branched layers to or from the core, and are characterized by a core, at least one inner branched layer, and a surface branched layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. in Britain, 641-645, August 1994). In other embodiments, the term "dendrimer" as used herein is intended to include, but is not limited to, a molecular structure having an inner core, an inner layer (or "generation") of repeating units regularly bonded to the inner core, and an outer surface of end groups bonded to the outermost generation. A "dendron" is a type of dendrimer having branches emanating from a focal point that may or may form a larger dendrimer by being directly or through a linking moiety linked to a core. In some embodiments, the dendrimer structure has a radial repeating group from a central core that serves as each repeating unit for each branch. In some embodiments, the dendrimers described herein may be described as small molecules, medium-sized molecules, lipids, or lipid-like materials. These terms may be used to describe compounds described herein having a dendron-like appearance (e.g., molecules radiating from a single focal point).

[0105] Dendrimers are polymers, but dendrimers are preferred over conventional polymers because they have a controllable structure, a single molecular weight, a large number of controllable surface functional groups, and conventionally assume a spherical structure after reaching a specific generation. Dendrimers can be prepared by sequential reactions of each repeating unit to produce a monodisperse, dendritic and / or generation-structured polymer structure. Each individual dendrimer consists of a central core molecule having dendritic wedges attached to one or more functional sites on its central core. The dendrimer surface layer can have various functional groups including anionic, cationic, hydrophilic or lipophilic groups placed thereon by the assembly monomers used during preparation.

[0106] By modifying the core, the functional groups and / or chemical properties of the repeating units, and the surface or end groups, their physical properties can be adjusted. Some of the properties that can be varied include, but are not limited to, solubility, toxicity, immunogenicity, and biological adhesion ability. Dendrimers are often described by the number of repeating units in their generations or branches. A dendrimer consisting only of the core molecule is said to be generation 0, while each successive repeating unit along all branches is generation 1, generation 2, etc. up to the end groups or surface groups. In some embodiments, half generations are possible that result only from the first condensation reaction with an amine rather than the second condensation reaction with a thiol.

[0107] The preparation of dendrimers requires a certain level of synthetic control, which is achieved by a series of stepwise reactions involving the construction of the dendrimer by each successive group. Dendrimer synthesis can be either convergent or divergent. During various dendrimer syntheses, molecules are assembled from the core to the periphery by an iterative process of attaching one generation to the previous generation and subsequently changing the functional groups for the next step of the reaction. The conversion of functional groups is necessary to prevent uncontrolled polymerization. Such polymerization results in highly branched molecules that are not monodisperse but are otherwise known as hyperbranched polymers. Continuing to react the dendrimer repeating units results in the formation of globular or spherical molecules due to steric effects, until a steric overcrowding at a particular generation hinders complete reaction and destroys the monodispersity of the molecule. Thus, in some embodiments, dendrimers of generations G1 to G10 are particularly contemplated. In some embodiments, the dendrimer includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein. In some embodiments, the dendrimer used herein is G0, G1, G2, or G3. However, by reducing the spacer units in the branched polymer, the possible number of generations (such as 11, 12, 13, 14, 15, 20, or 25, etc.) can be increased.

[0108] Furthermore, dendrimers have two major chemical environments: an environment created by specific surface groups at the time of termination and the interior of the dendritic structure that can be shielded from the bulk medium and surface groups by the higher-order structure. Due to these different chemical environments, dendrimers have found numerous different potential applications, including therapeutic uses.

[0109] In some aspects, the dendrimers that can be used in the compositions of the present invention are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. Dendrimers include secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines and methacrylate with mercapto groups. Further, the repeating units of the dendrimer can include groups that are degradable under physiological conditions. In some embodiments, these repeating units can include one or more initial (germinal) diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows for dendritic polymerization in only one direction. In other embodiments, the core molecule is a polyamine having a plurality of different dendritic branches, each of which can include one or more repeating units. The dendrimer can be formed by removing one or more hydrogen atoms from this core. In some embodiments, these hydrogen atoms are on a heteroatom such as a nitrogen atom. In some embodiments, the end groups are lipophilic groups such as long-chain alkyl or alkenyl groups. In other embodiments, the end groups are long-chain haloalkyl or haloalkenyl groups. In other embodiments, the end groups are aliphatic or aromatic groups containing ionizable groups such as amine (-NH2) or carboxylic acid (-CO2H). In yet other embodiments, the end groups are aliphatic or aromatic groups containing one or more hydrogen bond donors such as hydroxyl groups, amide groups, or esters.

[0110] The cationic ionizable lipids of the present disclosure contain one or more asymmetrically substituted carbon or nitrogen atoms and can be isolated in optically active form or racemic form. Thus, unless a specific stereochemistry or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, epimeric forms, and all geometric isomeric forms of the chemical formula are contemplated. The cationic ionizable lipids can occur as racemic compounds and racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers. In some embodiments, a single diastereomer is obtained. The chiral centers of the cationic ionizable lipids of the present disclosure can have the S configuration or the R configuration. Further, it is contemplated that one or more of the cationic ionizable lipids can exist as structural isomers. In some embodiments, the compounds have the same formula but have different connectivity to the core nitrogen atom. Without wishing to be bound by any theory, it is believed that such cationic ionizable lipids exist because the starting monomer first reacts with the primary amine and then statistically with any secondary amine present. Thus, the structural isomers can present a mixture of fully reacted primary amines and then reacted secondary amines.

[0111] The chemical formulas used to represent the cationic ionizable lipids of the present disclosure will typically show only, perhaps, one of several different tautomers. For example, many types of ketone groups are known to exist in equilibrium with the corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Whichever tautomer is depicted for a given formula, and whichever is the most common, all tautomers of a given chemical formula are contemplated.

[0112] The cationic ionizable lipids of the present disclosure also, whether used in the indications described herein or otherwise, have the advantages that they are more effective than compounds known in the prior art, have low toxicity, act for a long time, are potent, cause few side effects, are readily absorbed, and / or have a good pharmacokinetic profile (e.g., high oral bioavailability and / or low clearance), and / or may have other useful pharmacological, physical, or chemical properties compared to compounds known in the prior art.

[0113] Furthermore, the atoms constituting the cationic ionizable lipids of the present disclosure are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include atoms having the same atomic number but different mass numbers. As a general example, but not by way of limitation, the isotopes of hydrogen include tritium and deuterium, and the isotopes of carbon include 13 C and 14 C.

[0114] It should be recognized that the particular anions or cations forming part of any salt form of the cationic ionizable lipids provided herein are not critical as long as the salt is pharmaceutically acceptable as a whole. Further examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0115] In some embodiments, the cationic ionizable lipid is present in an amount of about 20 to about 23. In some embodiments, the mole percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the mole percentage is about 7.5 to about 20. In some embodiments, the mole percentage is about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0116] C. Selective Organ Targeting (SORT) Compounds In some aspects, the disclosure includes one or more selective organ targeting (SORT) compounds that lead to the selective delivery of the composition to a particular organ. The compound may be a lipid, a small molecule therapeutic agent, a sugar, a vitamin, a peptide, or a protein.

[0117] In some embodiments, the selective organ targeting (SORT) compound is present in the composition in a molar ratio in the range of about 2%, 4%, 5%, 10%, 15%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 45%, 50%, 55%, 60%, 65% to about 70%, or any range derivable therein. In some embodiments, the SORT compound may be present in an amount of about 5% to about 40%, about 10% to about 40%, about 20% to about 35%, about 25% to about 35%, or about 28% to about 34%.

[0118] In some embodiments, the SORT compound may be a lipid. A lipid is a small molecule having two or more alkyl or alkenyl chains of C6 - C 24 A small molecule therapeutic agent is a compound containing less than 100 non-hydrogen atoms and having a weight of less than 2,000 daltons. A sugar has the molecular formula C n H 2n O nA molecule containing the same or a combination of multiple molecules of the formula, where n is from 3 to 7. A protein is an amino acid sequence containing at least three amino acid residues. A protein without a regular tertiary structure may sometimes be called a peptide. Proteins may also include intact proteins with a tertiary structure. Vitamins are macronutrients and consist of one or more compounds selected from vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12 and one or more compounds selected from vitamin C, vitamin D, vitamin E, and vitamin K.

[0119] 1. Permanent cationic lipid In some aspects, the present disclosure provides one or more hydrophobic components and one or more types of lipids having a permanent cationic group. The permanent cationic lipid can contain a group having a positive charge regardless of pH. One of the permanent cationic groups that can be used in the permanent cationic lipid is a quaternary ammonium group. These permanent cationic lipids have the following formula: including a structure as described in TIFF2025108518000068.tif14128, wherein, at least one of Y1, Y2, and Y3 is X2N + under the condition of Y1, Y2, or Y3 being each independently X1C(O)R1 or X2N + R3R4R5; R1 is C1-C 24 alkyl, C1-C 24 substituted alkyl, C1-C 24 alkenyl, C1-C 24 substituted alkenyl; X1 is O or NR a ; wherein, R a is hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl; X2 is C1-C6 alkanediyl or C1-C6 substituted alkanediyl; R3, R4, and R5 are each independently C1-C 24 alkyl, C1-C 24 substituted alkyl, C1-C 24 alkenyl, C1-C 24 substituted alkenyl; A1 is an anion having a charge equal to the number of X2N + R3R4R5 groups in the compound.

[0120] In another aspect, the permanent cationic lipid is defined further by the following formula: TIFF2025108518000069.tif14128, wherein, at least one of R6-R9 is a C8-C 24 group, provided that R6-R9 are each independently C1-C 24 alkyl, C1-C 24 substituted alkyl, C1-C 24 alkenyl, C1-C 24 substituted alkenyl; and A2 is a monovalent anion.

[0121] In another aspect, the permanent cationic lipid is defined further by the following formula: TIFF2025108518000070.tif25128, wherein, R1 and R2 are each independently alkyl (C8~C24) or alkenyl (C8~C24) or a substituted form of any of the groups; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; R4 is alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion.

[0122] In some embodiments, the permanent cationic lipid is present in an amount of about 4 to about 16 mole percent relative to the total lipid composition. The composition may contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mole percent, or any range derivable therein. In other embodiments, the composition may comprise about 18 to about 66 mole percent relative to the total lipid composition. In some embodiments, the composition may contain about 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, or 66 mole percent, or any range derivable therein.

[0123] 2. Permanent anionic lipid In some aspects, the disclosure provides one or more hydrophobic components and one or more lipids having a permanent anion group. One of the anion groups that can be used in the permanent anionic lipid is a phosphate group. The phosphate group may be a deprotonated and negatively charged compound at a pH less than 8, 9, 10, 11, 12, 13 or 14. The hydrophobic component may be one or more C6-C 24 alkyl group or alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups.

[0124] In some embodiments, the permanent anionic lipid has the following formula: TIFF2025108518000071.tif25128 and has a structure of wherein R1 and R2 are each independently alkyl (C8~C24) alkenyl (C8~C24) or a substituted form of any group; R3 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) or -Y1-R4, wherein Y1 is alkanediyl (C≦6) or substituted alkanediyl (C≦6) and; R4 is acyloxy(C≦8~24) or substituted acyloxy (C≦8~24) is.

[0125] 3. Phosphatidylcholine In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic components, a cationic amine group, and a negatively charged phosphate group. The cationic amine group may be a quaternary amine having three methyl groups bonded to a nitrogen atom. The hydrophobic component may be one or more C6-C 24 alkyl group or alkenyl group. The compound may have one hydrophobic group, two hydrophobic groups, or three hydrophobic groups. In some embodiments, the phosphatidylcholine compound is further defined as TIFF2025108518000072.tif25128, wherein R1 and R2 are each independently alkyl (C8~C24) , alkenyl (C8~C24) , or a substituted form of any group; R3, R3', and R3'' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion.

[0126] D. Further Lipids in Lipid Nanoparticles In some aspects of the present disclosure, to create a composition, a composition containing one or more lipids is mixed with a cationic ionizable lipid. In some embodiments, the cationic ionizable lipid is mixed with 1, 2, 3, 4, or 5 different types of lipids. The cationic ionizable lipid is intended to be able to be mixed with multiple different lipids of one type. In some embodiments, the cationic ionizable lipid composition includes at least a steroid or steroid derivative, a PEG lipid, and a phospholipid.

[0127] In some embodiments, the lipid nanoparticles are preferentially delivered to the target organ. In some embodiments, the target organ is selected from the lung, heart, brain, spleen, bone marrow, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, lymph node, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brainstem, cerebellum, spinal cord, eye, ear, tongue, or skin. Alternatively, the composition can be preferentially delivered to a target organ system such as the nervous system, cardiovascular system, or respiratory system, or to a part of one of these organ systems. As used herein, the term "preferentially delivered" is used to refer to a composition in which at least 25% of the administered amount is delivered to the target organ or organ system. This term is used to refer to a composition in which at least 25%, 50%, or at least 75% of the administered amount is delivered to the target organ or organ system.

[0128] 1. Steroids and Steroid Derivatives In some aspects of the present disclosure, a cationic ionizable lipid is mixed with one or more steroids or steroid derivatives to create a composition. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure that may further include one or more substitutions including an alkyl group, an alkoxy group, a hydroxy group, an oxo group, an acyl group, or a double bond between two or more carbon atoms. In one aspect, the ring structure of the steroid includes three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula: TIFF2025108518000073.tif17128. In some embodiments, the steroid derivative includes the above ring structure having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, the formula of which is further defined as TIFF2025108518000074.tif19128.

[0129] In some aspects of the present disclosure, the steroid or steroid derivative is a cholestane or cholestane derivative. In cholestane, the ring structure is defined by the formula: TIFF2025108518000075.tif36128. As described above, the cholestane derivative includes one or more non-alkyl substitutions of the above ring system. In some aspects, the cholestane or cholestane derivative is a cholestene or cholestene derivative or a sterol or sterol derivative. In other aspects, the cholestane or cholestane derivative is both cholestene and a sterol or its derivative.

[0130] In some aspects, the composition may further include a molar percentage of steroid relative to the total lipid composition of from about 40 to about 46. In some aspects, the molar percentage is from about 40, 41, 42, 43, 44, 45, to about 46 or any range derivable therein. In other aspects, the molar percentage of steroid relative to the total lipid composition is from about 15 to about 40. In some aspects, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein.

[0131] 2. PEG or PEGylated lipid In some aspects of the present disclosure, the polymer is mixed with one or more PEGylated lipids (or PEG lipids) to create a lipid composition. In some embodiments, the present disclosure includes using any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride that also includes a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound that includes one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group with a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugated PEG-modified dialkylamine and PEG-modified 1,2-diacetyloxypropane-3-amine, PEG-modified diacylglycerol and dialkylglycerol. In some embodiments, PEG-modified distearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present disclosure are taught by U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0132] In another aspect, the PEG lipid has the following formula: TIFF2025108518000076.tif18128 wherein R12 and R 13 are each independently alkyl (C≦24) alkenyl (C≦24) or a substituted form of any of these groups; R e is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8) and x is from 1 to 250. In some embodiments, R e is alkyl such as methyl (C≦8) R 12 and R 13 are each independently alkyl (C≦4~20) In some embodiments, x is from 5 to 250. In one embodiment, x is from 5 to 125, or x is from 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0133] In another aspect, the PEG lipid has the following formula: TIFF2025108518000077.tif39128wherein n1 is an integer from 1 to 100, and n2 and n3 are each independently selected from integers from 1 to 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therefrom. In some embodiments, n1 is from about 30 to about 50. In some embodiments, n2 is from 5 to 23. In some embodiments, n2 is from 11 to about 17. In some embodiments, n3 is from 5 to 23. In some embodiments, n3 is from 11 to about 17.

[0134] In some embodiments, the composition may further comprise a molar percentage of PEG lipid relative to the total lipid composition of from about 4.0 to about 4.6. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 to about 4.6, or any range derivable therein. In other embodiments, the molar percentage is from about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 to about 4.0 or any range derivable therein.

[0135] 3. Phospholipids In some aspects of the present disclosure, to create the composition, the polymer is mixed with one or more phospholipids. In some embodiments, any lipid that also contains a phosphate group. In some embodiments, a phospholipid has a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups and optionally a small organic molecule. In some embodiments, the small organic molecule is an amino acid, a sugar, or an amino-substituted alkoxy group, for example, choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, and zwitterionic lipids define lipids and lipid-like molecules that have both a positive charge and a negative charge.

[0136] In some embodiments, the composition may further comprise a molar percentage of phospholipid relative to the total lipid composition of from about 20 to about 23. In some embodiments, the molar percentage is about 20, 20.5, 21, 21.5, 22, 22.5 to about 23, or any range derivable therein. In other embodiments, the molar percentage is from about 7.5 to about 60. In some embodiments, the molar percentage is from about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20, or any range derivable therein.

[0137] E. Nucleic Acids, and Nucleic Acid-Based Therapeutic Agents 1. Nucleic Acids In some aspects of the present disclosure, the lipid composition comprises one or more nucleic acids. In some embodiments, the lipid composition comprises one or more nucleic acids present at a weight ratio of about 5:1 to about 1:100 relative to the lipid composition. In some embodiments, the weight ratio of nucleic acid to lipid composition is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any range derivable therein. In some embodiments, the weight ratio is about 1:40. Further, it should be apparent that the present disclosure is not limited to the specific nucleic acids disclosed herein. However, one of ordinary skill in the art can readily identify related homologs in various other sources of nucleic acids, including nucleic acids from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, rhesus monkeys, chimpanzees, apes, baboons, cows, pigs, horses, sheep, cats, and other species), so the present disclosure is not limited in scope to any particular source, sequence, or type of nucleic acid. The nucleic acids used in the present disclosure are contemplated to include sequences based on naturally occurring sequences. Considering the degeneracy of the genetic code, sequences having at least about 50%, generally at least about 60%, more generally about 70%, most generally about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotide sequence of the natural sequence. In another aspect, the nucleic acid is a complementary sequence to the natural sequence, or is complementary to 75%, 80%, 85%, 90%, 95%, and 100%. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or more are contemplated in the present invention.

[0138] The nucleic acids used herein may be derived from genomic DNA, i.e., may be directly cloned from the genome of a particular organism. However, in a preferred embodiment, the nucleic acids will include complementary DNA (cDNA). cDNA with natural introns or introns derived from another gene are also contemplated; molecules so engineered are sometimes referred to as "mini-genes." At a minimum, these and other nucleic acids of the present disclosure can be used, for example, as molecular weight standards in gel electrophoresis.

[0139] The term "cDNA" is intended to mean DNA prepared using messenger RNA (mRNA) as a template. In contrast to genomic DNA, or DNA polymerized from non-processing or partially processed RNA templates, the advantage of using cDNA is that cDNA mainly contains the coding sequence of the corresponding protein. In cases where non-coding regions are required for optimal expression, or where full or partial genomic sequences may be preferred, such as when non-coding regions such as introns are targeted in antisense strategies.

[0140] In some embodiments, the nucleic acid includes one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. Complementary means that a polynucleotide is capable of base pairing according to the standard Watson-Crick complementarity rules. That is, a larger purine will form a base pair with a smaller pyrimidine, forming combinations of guanine paired with cytosine (G:C), adenine paired with thymine in the case of DNA (A:T), or adenine paired with uracil in the case of RNA (A:U). Incorporation of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others into the hybridizing sequence does not interfere with base pairing.

[0141] Targeting double-stranded (ds) DNA with a polynucleotide results in triple helix formation; targeting RNA will result in double helix formation. When introduced into a target cell, an antisense polynucleotide specifically binds to its target polynucleotide and interferes with transcription, RNA processing, transport, translation, and / or stability. An antisense RNA construct, or DNA encoding such antisense RNA, can be utilized to inhibit gene transcription or translation or both, either in vitro or in vivo, for example, in a host animal including a human subject.

[0142] Antisense constructs can be designed to bind to promoters and other regulatory regions, exons, introns, or even exon-intron boundaries of a gene. The most effective antisense constructs are contemplated to include regions complementary to intron / exon splice junctions. Accordingly, a preferred embodiment is proposed to include an antisense construct having complementarity to a region within 50 to 200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without significantly affecting its target selectivity. The amount of exon material included will vary depending on the specific exon and intron sequences used. Simply by testing the construct in vitro to determine whether normal cell function is affected or whether the expression of related genes having complementary sequences is affected, it is possible to easily test whether there is too much exon DNA included.

[0143] As described above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have few base mismatches. For example, a sequence 15 bases in length can be termed complementary if it has complementary nucleotides at position numbers 13 or 14. Of course, a completely complementary sequence would be one that is completely complementary over its entire length and has no base mismatches. Other sequences with a lower degree of homology are also contemplated. For example, antisense constructs (e.g., ribozymes; see below) can be designed that have limited regions of high homology and also include non-homologous regions. These molecules have less than 50% homology but will bind to the target sequence under appropriate conditions.

[0144] 2. Modified nucleobases In some embodiments, the nucleic acids of the present disclosure include one or more modified nucleosides that include a modified sugar moiety. Such compounds that include one or more sugar-modified nucleosides can have desirable properties such as increased nuclease stability or increased binding affinity to the target nucleic acid as compared to oligonucleotides that include only nucleosides with natural sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates can include one or more substitutions corresponding to the substitution of the substituted sugar moiety.

[0145] In some embodiments, the modified sugar moiety is a substituted sugar moiety that includes one or more non-bridging sugar substituents including, but not limited to, substituents at the 2'-position and / or 5'-position. Examples of sugar substituents suitable for the 2'-position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2'-position is allyl, amino, azido, thio, O-allyl, O--C1~C 10 alkyl, O--C1~C 10Substituted alkyl; selected from OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C 10 alkyl. Examples of sugar substituents at the 5'-position include, but are not limited to, 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar comprises two or more non-bridging sugar substituents, e.g., a T-F-5'-methyl sugar moiety (see, e.g., PCT International Application WO 2008 / 101157 for further 5',2'-bis-substituted sugar moieties and nucleosides).

[0146] Nucleosides containing a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O, S, or N(R m )-alkyl; O, S, or N(R m )-alkenyl; O, S or N(R m )-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(R m )(R n ) or O--CH2--C(=O)--N(R m )(R n ), where each R m and R n is, independently, H, an amino protecting group or substituted or unsubstituted C1-C 10 alkyl. These 2'-substituents can be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0147] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2-CH=CH2, O--CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(R m )(R n ), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O--CH2--C(=O)--N(R m )(R n ), and each R m and R n is independently H, an amino protecting group or a substituted or unsubstituted C1-C 10 alkyl.

[0148] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0149] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0150] Certain modified sugar moieties include a bridged sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety includes a bridge between the 4' and 2' furanose ring atoms. Examples of such 4'-2' sugar substituents include --[C(R a )(R b )] n --, --[C(R a )(R b )] n --O--, --C(R a R b )--N(R)--O-- or --C(R a R b)--O--N(R)--; 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)--O-2' (LNA); 4'-(CH2)--S-2'; 4'-(CH2)2--O-2' (ENA); 4'-CH(CH3)--O-2' (cEt) and 4'-CH(CH2OCH3)--O-2', and their analogs (see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)--O-2' and its analogs (see, e.g., WO 2009 / 006478); 4'-CH2--N(OCH3)-2' and its analogs (see, e.g., WO2008 / 150729); 4'-CH2--O--N(CH3)-2' (see, e.g., US2004 / 0171570 published on September 2, 2004); 4'-CH2--O--N(R)-2', and 4'-CH2--N(R)--O-2'- are included, but not limited to, where each R is independently H, a protecting group, or C1-C 12 alkyl; 4'-CH2--N(R)--O-2', where R is H, C1-C 12 alkyl, or a protecting group (see U.S. Patent No. 7,427,672); 4'-CH2--C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2--C(=CH2)-2' and its analogs (see PCT International Application WO 2008 / 154401).

[0151] In some embodiments, such 4'-2' linkages are independently --[C(R a )(R b )] n --, --C(R a )=C(R b )--, --C(R a )=N--, --C(=NR a )--, --C(=O)--, --C(=S)--, --O--, --Si(R a )2--, --S(=O) x --, and --N(R a)-- includes 1 to 4 linking groups independently selected from; wherein, x is 0, 1, or 2; n is 1, 2, 3, or 4; Each R a and R b is independently H, a protecting group, hydroxyl, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C 12 alkynyl, substituted C2-C 12 alkynyl, C5-C 20 aryl, substituted C5-C 20 aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and Each J1 and J2 is independently H, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C 12 alkynyl, substituted C2-C 12 alkynyl, C5-C 20 aryl, substituted C5-C 20 aryl, acyl (C(=O)--H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C 12 aminoalkyl, substituted C1-C 12 aminoalkyl, or a protecting group.

[0152] Nucleosides containing a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNA. Bicyclic nucleosides include (A) α-L-methyleneoxy (4'-CH2--O-2') BNA, (B) β-D-methyleneoxy (4'-CH2--O-2') BNA (also referred to as locked nucleic acid or LNA), (C) ethyleneoxy (4'-(CH2)2--O-2') BNA, (D) aminooxy (4'-CH2--O--N(R)-2') BNA, (E) oxyamino (4'-CH2--N(R)--O-2') BNA, (F) methyl(methyleneoxy) (4'-CH(CH3)--O-2') BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2--S-2') BNA, (H) methylene-amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2--CH(CH3)-2') BNA, (J) propylene carbocyclic (4'-(CH2)3-2') BNA, and (K) methoxy(ethyleneoxy) (4'-CH(CH2OMe)-O-2') BNA (also referred to as constrained MOE or cMOE), but are not limited thereto.

[0153] Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 5561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004 / 106356, WO 1994 / 14226, WO 2005 / 021570, and WO 2007 / 134181; U.S. Patent Application Publication Nos. 2004 / 0171570, 2007 / 0287831, and 2008 / 0039618; U.S. Patent Applications Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844; and PCT International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922.

[0154] In some embodiments, bicyclic sugar moieties, and nucleosides incorporating such bicyclic sugar moieties, are further defined by their stereoconfiguration. For example, nucleosides containing a 4'-2'methylene-oxy bridge can be in the α-L or β-D configuration. To date, α-L-methyleneoxy (4'-CH2--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides exhibiting antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0155] In some embodiments, the substituted sugar moiety includes one or more non-bridging sugar substituents and one or more bridging sugar substituents (e.g., 5'-substituted and 4'-2' bridged sugars; PCT International Application WO 2007 / 134181, where LNA is substituted, for example, with a 5'-methyl or 5'-vinyl group).

[0156] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atoms of the natural sugar are replaced, for example, with sulfur, carbon or nitrogen atoms. In some such embodiments, such modified sugar moieties also include bridging and / or non-bridging substituents as described above. For example, certain sugar surrogates include a 4'-sulfur atom and substitutions at the 2'-position (see, e.g., published U.S. Patent Application No. 2005 / 0130923) and / or the 5'-position. As a further example, carbocyclic bicyclic nucleosides having a 4'-2' bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).

[0157] In some embodiments, the sugar substitute contains a ring other than a 5-atom ring. For example, in some embodiments, the sugar substitute contains a 6-membered tetrahydropyran. Such tetrahydropyrans can be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoro HNA (F-HNA).

[0158] In some embodiments, there is provided a modified THP nucleoside of Formula VII wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In some embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In some embodiments, there is provided a THP nucleoside of Formula VII wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.

[0159] Many other bicyclic and tricyclic sugar substitute ring systems that can be used to modify nucleosides for incorporation into antisense compounds are also known in the art (see, for example, review: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0160] Optionally, 2'-F-5'-methyl-substituted nucleosides (see PCT International Application WO 2008 / 101157 for other disclosed 5',2'-bis-substituted nucleosides), substitution of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see US Patent Application No. 2005 / 0130923), or 5'-substitution of bicyclic nucleic acids (reference is made to PCT International Application WO 2007 / 134181, and 4'-CH2--O-2' bicyclic nucleosides are further substituted at the 5'-position with a 5'-methyl or 5'-vinyl group), etc., combinations of modifications are also provided. Also, the synthesis and preparation of carbocyclic bicyclic nucleosides are described along with their oligomerization and biochemical studies (see, for example, Srivastava et al., 2007).

[0161] In some embodiments, the present disclosure provides oligonucleotides comprising modified nucleosides. Those modified nucleotides can include modified sugars, modified nucleobases, and / or modified linkages. Specific modifications are selected such that the resulting oligonucleotide possesses the desired characteristics. In some embodiments, the oligonucleotide includes one or more RNA-like nucleosides. In some embodiments, the oligonucleotide includes one or more DNA-like nucleotides.

[0162] In some embodiments, the nucleosides of the present disclosure include one or more unmodified nucleobases. In certain embodiments, the nucleosides of the present disclosure include one or more modified nucleobases.

[0163] In some embodiments, the modified nucleobases include universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein, 2-aminopropyladenine, 5-propynyluracil, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines including 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2Included are G-clamps such as H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases can also include those in which the purine or pyrimidine base is replaced by another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, Y. S., 1993.

[0164] Representative U.S. patents that teach some of the preparations of the above-described modified nucleobases and other modified nucleobases include, but are not limited to, U.S. Patent No. 3,687,808; No. 4,845,205; No. 5,130,302; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5,594,121; No. 5,596,091; No. 5,614,617; No. 5,645,985; No. 5,681,941; No. 5,750,692; No. 5,763,588; No. 5,830,653 and No. 6,005,096, each of which is hereby incorporated by reference in its entirety.

[0165] In some embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides can be linked to each other using any internucleoside linkage. Two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing internucleoside linking groups include, but are not limited to, methylene methylimino (--CH2--N(CH3)--O--CH2--), thiodiester (--O--C(O)--S--), thiocarbamate (--O--C(O)(NH)--S--); siloxane (--O--Si(H)2--O--); and N,N'-dimethylhydrazine (--CH2--N(CH3)--N(CH3)--). Compared to the natural phosphodiester bond, modified bonds can be used to alter, typically increase, the nuclease resistance of the oligonucleotide. In some embodiments, internucleoside linkages having chiral atoms can be prepared as a racemic mixture or as separate enantiomers. Representative chiral bonds include, but are not limited to, alkylphosphonate and phosphorothioate. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those of skill in the art.

[0166] The oligonucleotides described herein contain one or more asymmetric centers and thus can give rise to enantiomers, diastereomers, and other stereoisomeric arrangements that can be defined as (R) or (S) with respect to absolute stereochemistry, α or β in the case of sugar anomers, or (D) or (L) in the case of amino acids, etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.

[0167] Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3'-CH2--N(CH3)--O-5'), amide-3 (3'-CH2--C(=O)--N(H)-5'), amide-4 (3'-CH2--N(H)--C(=O)-5'), formacetal (3'-O--CH2--O-5'), and thioformacetal (3'-S--CH2--O-5'). Additional neutral internucleoside linkages include nonionic linkages including siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters and amides (see, for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside linkages include nonionic linkages containing mixed N, O, S and CH2 components.

[0168] Additional modifications can also be made at other positions on the oligonucleotide, particularly at the 3'-position of the sugar on the 3'-terminal nucleotide and at the 5'-position of the 5'-terminal nucleotide. For example, one additional modification of the ligand-binding oligonucleotide of the present disclosure involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., 1989), cholic acid (Manoharan et al., 1994), thioethers such as hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), thiocolesterol (Oberhauser et al., 1992), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchuk et al., 1993), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995; Shea et al., 1990), polyamines or polyethylene glycol chains (Manoharan et al., 1995), or adamantane acetic acid (Manoharan et al., 1995), palmitoyl moieties (Mishra et al., 1995), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., 1996).

[0169] Representative U.S. patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of which is incorporated herein by reference.

[0170] 3. Protein In some embodiments, the composition may further comprise one or more proteins. Some proteins may include enzymes such as nuclease enzymes. The compositions described herein may include one or more CRISPR - associated proteins (e.g., CRISPR enzymes) including a Cas protein. Non - limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are known. For example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0171] The protein in the composition described herein may be Cas9 (e.g., derived from S. pyogenes or S. pneumoniae). The CRISPR enzyme can induce cleavage of one or both strands at the location of the target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The CRISPR enzyme can be mutated relative to the corresponding wild-type enzyme such that the ability to cleave one or both strands of the target polynucleotide containing the target sequence is lost. For example, a substitution of aspartic acid to alanine (D10A) in the RuvC catalytic domain of Cas9 derived from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (that cleaves one strand). In some embodiments, Cas9 nickase may be used in combination with a guide sequence, e.g., two guide sequences that target the sense and antisense strands of a DNA target. This combination can be used to introduce breaks in both strands and induce NHEJ or HDR.

[0172] In some embodiments, the present disclosure provides a compound containing one or more therapeutic proteins. Therapeutic proteins that may be included in the composition include a wide range of molecules, such as cytokines, chemokines, interleukins, interferons, growth factors, coagulation factors, anticoagulants, blood factors, bone morphogenetic proteins, immunoglobulins, and enzymes. Some non-limiting examples of specific therapeutic proteins include erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), α-galactosidase A, α-L-iduronidase, thyrotropin α, N-acetylgalactosamine-4-sulfatase (rhASB), dornase alfa, tissue plasminogen activator (TPA), activase, glucocerebrosidase, interferon (IF) β-1a, interferon β-1b, interferon γ, interferon α, TNF-α, IL-1 to IL-36, human growth hormone (rHGH), human insulin (BHI), human chorionic gonadotropin α, darbepoetin alfa, follicle-stimulating hormone (FSH), and factor VIII.

[0173] 4. Small molecule therapeutic agents In some aspects, the present disclosure provides a composition comprising a therapeutic agent. The therapeutic agent is a small molecule, such as 7-methoxypteridine, 7-methylpteridine, abacavir, avafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acethexamide, acitretin, acrivastine, adenine, adenosine, alatrophloxacin, albendazole, albuterol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, allobarbital, allopurinol, all-trans retinoic acid (ATRA), allopurinol, alprazolam, alprenolol, altretamine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone HCl, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphotericin, amphotericin B, ampicillin, amprenavir, amsacrine, amyl nitrate, amylobarbital, anastrozole, anrinone, anthracene, anthracycline, aprobarbital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, atropine azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbital, BCG live, beclamide, beclomethasone, bendroflumethiazide, benazepril, benidipine, benorylate, benperidol, benzodiazepam, benzamide, benzanthracene, benzathine penicillin, benzhexol HCl, benznidazole, benzodiazepine, benzoic acid, bephenium hydroxynaphthoate, betamethasone, bevacizumab (Avastin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide, bupropion,Busulfan, Butalbital, Butamben, Butenafine HCl, Butobarbital, Butobarbital (Butethal), Butoconazole, Butoconazole Nitrate, Butylparaben, Caffeine, Calcifediol, Calciprotriene, Calcitriol, Calusterone, Candesartan, Capecitabine, Capsaicin, Captopril, Carbamazepine, Carbimazole, Carbofuran, Carboplatin, Carbroromal, Carimazole, Carmustine, Cefamandole, Cefazolin, Cefixime, Ceftazidime, Cefuroxime Axetil, Celecoxib, Cefradine, Cerivastatin, Cetrizine, Cetuximab, Chlorambucil, Chloramphenicol, Chlordiazepoxide, Chloromethiazole, Chloroquine, Chlorothiazide, Chlorpheniramine, Chlorproguanil HCl, Chlorpromazine, Chlorpropamide, Chlorprothixene, Chlorpyrifos, Chlorotetracycline, Chloroxuron, Chlorzoxazone, Colecalciferol, Chrysene, Cilostazol, Cimetidine, Cinnalidine, Cinoxacin, Ciprofibrate, Ciprofloxacin HCl, Cisapride, Cisplatin, Citalopram, Cladribine, Clarithromycin, Clemastine Fumarate, Clioquinol, Clobazam, Clofarabine, Clofazimine, Clofibrate, Clomiphene Citrate, Clomipramine, Clonazepam, Clopidogrel, Chlorthiazepamide, Clotrimazole, Clotrimazole, Cloroxacillin, Clozapine, Cocaine, Codeine, Colchicine, Colistin, Conjugated Estrogens, Cortosterone, Cortisone, Cortisone Acetate, Cyclizine, Cyclobarbital, Cyclobenzaprine, Cyclobutane-spirobarbiturate, Cycloethane-spirobarbiturate, Cycloheptane-spirobarbiturate, Cyclohexane-spirobarbiturate, Cyclopentane-spirobarbiturate, Cyclophosphamide, Cyclopropane-spirobarbiturate, Cycloserine, Cyclosporine, Cyproheptadine, Cyproheptadine HCl, Cytarabine, Cytosine, Dacarbazine, Dactinomycin, Danazol, Dansylon,Dantrolene sodium, dapsone, darbepoetin alfa, darodipine, daunorubicin, dechoxolate, dehydroepiandrosterone, delavirdine, demeclocycline, denileukin, deoxycorticosterone, deoxymethasone, dexamethasone, dextroamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diamorphine, diatrizoic acid, diazepam, diazoxide, dichlorophene, dichlorprop, diclofenac, dicumarol, didanosine, diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroechin, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem HCl, diloxanide furoate, dimenhydrinate, dimorpholamine, dinitolmide, diosgenin, diphenoxylate HCl, diphenyl, dipyridamole, dirithromycin, disopyramide, disulfiram, diuron, docetaxel, domperidone, donepezil, doxazosin, doxazosin HCl, doxorubicin (neutral), doxorubicin HCl, doxycycline, drostanolone propionate, droperidol, dyphylline, echinocandin, econazole, econazole nitrate, efavirenz, ellipticine, enalapril, enlimomab, enoximone, epinephrine, epipodophyllotoxin derivative, epirubicin, epoetin alfa, eposartan, ekirenin, echin, ergocalciferol, ergotamine tartrate, erlotinib, erythromycin, estradiol, estramustine, estriol, estrone, ethacrynic acid, ethambutol, ethamidate, etoposide, etretinate, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenchlorphos, fenclofenac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, phenoxycarb, fenpiclonil, fentanylFeniconazole, Fexofenadine, Filgrastim, Finasteride, Flecamide acetate, Floxuridine, Fludarabine, Fluconazole, Fluconazole, Flucytosine, Fluoxastrobin, Fludrocortisone, Fludrocortisone acetate, Flufenamic acid, Flunalisone, Flunarizine HCl, Flunisolide, Flunitrazepam, Fluocortolone, Fluometuron, Fluorene, Fluorouracil, Fluoxetine HCl, Fluoxymesterone, Flupentixol decanoate, Flupentixol decanoate, Flurazepam, Flurbiprofen, Fluticasone propionate, Fluvastatin, Folic acid, Hoselenopril, Phenytoin sodium, Frovatriptan, Furosemide, Fluvestrant, Furazolidone, Gabapentin, G-BHC (Lindane), Gefitinib, Gemcitabine, Gemfibrozil, Gemtuzumab, Graphenin, Glybenclamide, Glyclazide, Glimepiride, Glypidide, Glutetimide, Gliquidone, Glyceryl trinitrate (Nitroglycerin), Goserelin acetate, Grepafloxacin, Glyceofulvin, Guaiiphenesin, Guanabenz acetate, Guanine, Halofantrine HCl, Haloperidol, Hydrochlorothiazide, Heptabarbital, Heroin, Hesperetin, Hexachlorobenzene, Hexetal, Histrelin acetate, Hydrocortisone, Hydroflumethiazide, Hydroxyurea, Hyosthiamine, Hypoxanthine, Ibritumomab, Ibuprofen, Idarubicin, Idobutal, Ifosfamide, Ihydroequilenin, Imatinib mesylate, Imipenem, Indapamide, Indinavir, Indomethacin, Indoprofen, Interferon α-2a, Interferon α-2b, Iodamide, Iopanoic acid, Iprodione, Irbesartan, Irinotecan, Isavuconazole, Isocarboxazid, Isoconazole, Isoguanine, Isoniazid, Isopropyl barbital, Isoproturon, Isosorbide dinitrate, Isosorbide mononitrate, Isradipine, Itraconazole, Itraconazole, Itraconazole (Itra), Ivermectin, Ketoconazole, Ketoprofen, Ketorolac, Khellin, Labetalol, Lamivudine, Lamotrigine, Lanatoside C, Lansoprazole,L-Dopa, Leflunomide, Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Levofloxacin, Lidocaine, Linuron, Lisinopril, Lomefloxacin, Lomustine, Loperamide, Loratadine, Lorazepam, Lomefloxacin, Lormetazepam, Losartan Mesylate, Lovastatin, Lisuride Maleate, Maprotiline HCl, Mazindol, Mebendazole, Mecamylamine HCl, Mefenamic Acid, Medazepam, Medigoxin, Medroxyprogesterone Acetate, Mefenamic Acid, Mefloquine HCl, Megestrol Acetate, Melphalan, Mepenzolate Bromide, Meprobamate, Meptazinol, Mercaptopurine, Mesalazine, Mesna, Mesoridazine, Mestranol, Methadone, Metacalone, Methocarbamol, Methoin, Methotrexate, Methoxsalen, Methsuximide, Methyclothiazide, Methylphenidate, Methylphenobarbital, Methyl-p-hydroxybenzoate, Methylprednisolone, Methyltestosterone, Meprylon, Methylcellulose Maleate, Metoclopramide, Metolazone, Metoprolol, Metronidazole, Mianserin HCl, Miconazole, Midazolam, Mifepristone, Miglitol, Minocycline, Minoxidil, Mitomycin C, Mitotane, Mitoxantrone, Mycophenolate Mofetil, Molindone, Montelukast, Morphine, Moxifloxacin HCl, Nabumetone, Nadolol, Nalbuphine, Nalidixic Acid, Nandrolone, Naphthacene, Naphthalene, Naproxen, Naratriptan HCl, Natamycin, Nelarabine, Nelfinavir, Nevirapine, Nicardipine HCl, Nicotinamide, Nicotinic Acid, Nicoumalone, Nifedipine, Nilutamide, Nimodipine, Nimorazole, Nisoldipine, Nitrazepam, Nitrofurantoin, Nitrofurazone, Nizatidine, Nofetumomab, Norethisterone, Norfloxacin, Norgestrel, Nortriptyline HCl, Nystatin, Estradiol, Ofloxacin, Olanzapine, Omeprazole, Omoconazole, Ondansetron HCl, Oprelvekin, Ornidazole, Oxaliplatin, Oxamniquine, Oxantelembonate, Oxaprozin, Oxatomide, Oxazepam, Oxcarbazepine, Oxfendazole, Oxiconazole, Oxprenolol, Oxyphenbutazone, Oxyphencyclimine HCl, Paclitaxel, Parifermin, Pamidronate, p-Aminosalicylic Acid, Pantoprazole, Paramethadione, Paroxetine HCl, Pegademase, Pegaspargase, Pegfilgrastim, Pemetrexed Disodium, Penicillamine, Pentaerythritol Trinitrate, Pentazocine, Pentazocine, Pentobarbital, Pentobarbital, Pentostatin, Pentoxifylline, Perphenazine, Perphenazine Pimozide, Perylene, Phenacemide, Phenacetin, Phenanthrene, Phenindione, Phenobarbital, Phenolbarbitone, Phenolphthalein, Phenoxybenzamine, Phenoxybenzamine HCl, Phenoxymethylpenicillin, Phensuximide, Phenylbutazone, Phenytoin, Pindolol, Pioglitazone, Pipoproman, Piroxicam, Pizotifen Maleate, Platinum Compound, Puromycin, Polyene, Polymyxin B, Porfimer Sodium, Posaconazole (Posa), Pramipexole, Plastron, Pravastatin, Pradicantel, Prazosin, Prazosin HCl, Prednisolone, Prednisone, Primidone, Probarbital, Probenecid, Probucol, Procarbazine, Prochlorperazine, Progesterone, Proguanil HCl, Promethazine, Propofol, Propoxur, Propranolol, Propylparaben, Propylthiouracil, Prostaglandin, Pseudophedrine, Pteridine-2-Methyl-Thiol, Pteridine-2-Thiol, Pteridine-4-Methyl-Thiol, Pteridine-4-Thiol, Pteridine-7-Methyl-Thiol, Pteridine-7-Thiol, Pyrantel Embonate, Piratinamide, Pyrene, Pyridostigmine, Pyrimethamine, Quetiapine, Quinacrine, Quinapril, Quinidine, Quinidine Sulfate, Quinine, Quinine Sulfate, Rabeprazole Sodium, Ranitidine HCl, Rasburicase, Rabuconazole, Repaglinide, Reposar, Reserpine, Retinoid, Rifabutin, Rifampicin, Rifapentine, Rimexolone, Risperidone,Ritonavir, Rituximab, Rizatriptan Benzoate, Rofecoxib, Ropinirole HCl, Rosiglitazone, Saccharin, Salbutamol, Salicylamide, Salicylic Acid, Saquinavir, Sargramostim, Secbutabarbital, Secobarbital, Ciclopirox, Cilindol, Citalopram HCl, Simvastatin, Sirolimus, Sorafenib, Sparfloxacin, Spiramycin, Spironolactone, Stanozolol, Stanozolol, Stibozine, Stilbestrol, Streptozocin, Strychnine, Sulconazole, Sulconazole Nitrate, Sulfacetamide, Sulfadiazine, Sulfamerazine, Sulfamethazine, Sulfamethoxazole, Sulfanilamide, Sulfathiazole, Sulindac, Sulfabenzamide, Sulfacetamide, Sulfadiazine, Sulfadoxine, Sulfafurazole, Sulfamerazine, Sulfamethoxazole, Sulfapyridine, Sulfasalazine, Flufenpyrazone, Sulpiride, Sulthiame, Sumatriptan Succinate, Sunitinib Malate, Tacrine, Tacrolimus, Talbutal, Tamoxifen Citrate, Tamulosin, Targretin, Taxane, Tazarotene, Telmisartan, Temazepam, Temozolomide, Teniposide, Tenoxicam, Terazosin, Terazosin HCl, Terbinafine HCl, Terbutaline Sulfate, Terconazole, Trifluoperazine, Testosterone, Testosterone, Tetracycline, Tetrahydrocannabinol, Tetroxoprim, Thalidomide, Thebaine, Theobromine, Theophylline, Thiabendazole, Thiamphenicol, Thioguanine, Thioridazine, Thiotepa, Thotoin, Thiamine, Tiagabine HCl, Tibolone, Ticlopidine, Tinidazole, Thioconazole, Tirofiban, Tizanidine HCl, Tramadol, Trastuzumab, Trazodone HCl, Tretinoin, Triamcinolone, Triamterene, Triazolam, Triazole, Trifluoperazine, Trimethoprim, Trimipramine Maleate, Triphenylene, Troglitazone, Tromethamine, Tropicamide, Trovafloxacin, Cibamate, Ubiquinone (Coenzyme Q10),It may also be undecenoic acid, uracil, uracil mustard, uric acid, valproic acid, valrubicin, valsartan, vancomycin, venlafaxine HCl, vigabatrin, vinbarbital, vincristine, vinblastine, vinorelbine, voriconazole, xanthine, zafirlukast, zidovudine, zileuton, zoledronate, zoledronic acid, sumatriptan, zolpidem, and zopiclone.,

[0174] F. Kit The present disclosure also provides a kit. Any of the components disclosed herein can be combined in the form of a kit. In some embodiments, the kit includes the compositions described above or in the claims.

[0175] Kits generally include at least one vial, test tube, flask, bottle, syringe or other container in which the components are disposed and preferably can be appropriately dispensed. If there are two or more components in the kit, the kit generally also includes a second, third or other additional container in which additional components can be separately disposed. However, various combinations of components can be included in the container. In some embodiments, all of the lipid nanoparticle components are combined in a single container. In other embodiments, some or all of the lipid nanoparticle components are provided in separate containers.

[0176] The kits of the present disclosure also typically include a packaging for enclosing the various containers for commercial sale. Such packaging can include cardboard or injection molded or blow molded plastic packaging in which the desired containers are held. The kit can also include instructions for using the kit components. The instructions can include executable variations.

Examples

[0177] F. Examples The following examples are included to demonstrate preferred embodiments of the present disclosure. The techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure, and thus, it should be understood by those skilled in the art that they can be considered to constitute preferred modes for its practice. However, those skilled in the art should recognize that, in light of the present disclosure, many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the invention, and still obtain similar or analogous results.

[0178] Example 1: Preparation of Lipid Nanoparticles Modified with DOTAP Lipid nanoparticles (LNPs) are the most effective carrier class for in vivo nucleic acid delivery. Historically, effective LNPs are composed of four components: a cationic ionizable lipid, a zwitterionic phospholipid, cholesterol, and a lipid poly(ethylene glycol) (PEG). However, these LNPs deliver nucleic acids only systemically without organ-targeted delivery or tissue-targeted delivery. LNPs typically deliver RNA only to the liver. Therefore, new LNP formulations are being explored in attempts to provide targeted nucleic acid delivery.

[0179] Four classical types of lipids were mixed at a molar ratio of 15:15:30:3 with or without the addition of a permanent cationic lipid. Briefly, LNPs were prepared by mixing 5A2-SC8 (cationic ionizable), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanent cationic) in the ratios shown in Table 1.

[0180] (Table 1) Molar Ratios and Molar Percentages of Lipids in Modified LNPs TIFF2025108518000078.tif229130

[0181] To prepare the mDLNP formulation, 5A2-SC8, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at a predetermined molar ratio (15:15:30:3). The mRNA was dissolved in citrate buffer (10 mM, pH 4.0). Then, the mRNA was diluted with the lipid solution by mixing the mRNA with the lipid solution at a volume ratio of 3:1 (mRNA:lipid) such that the weight ratio was 40:1 (total lipid:mRNA). This solution was then incubated at room temperature for 10 minutes. To form the DOTAP-modified mDLNP formulation, the mRNA was dissolved in 1×PBS or citrate buffer (10 mM, pH 4.0) and rapidly mixed with ethanol containing 5A2-SC8, DOPE, cholesterol, DMG-PEG, and DOTAP, fixing the weight ratio at 40:1 (total lipid:mRNA) and the volume ratio at 3:1 (mRNA:lipid). As shown in Table 1, each formulation was named DOTAPX. Wherein, X represents the molar percentage of DOTAP in the total lipid.

[0182] Example 2: Characterization of mDLNP Formulations Modified with DOTAP To characterize the various mDLNP formulations, the size, polydispersity index, and zeta potential were examined three times for each formulation by dynamic light scattering. The size and polydispersity index are shown in Figure 5A. From this, it can be seen that regardless of the DOTAP concentration, all the formulations fall within a size range of approximately 90 nm to approximately 160 nm, while the polydispersity index indicating relative size uniformity varies from approximately 0.1 to approximately 0.3. The zeta potential of each formulation is shown in Figure 5B. From this, it can be seen that the zeta potential generally increases with the DOTAP concentration.

[0183] Next, encapsulation efficiency was tested using the Ribogreen RNA assay (Zhao et al., 2016). Briefly, when mRNA was dissolved in acidic buffer (10 mM citrate, pH 4), the mRNA was encapsulated into mDLNP without DOTAP with approximately 85% efficiency (Figure 5C). A low pH is required to protonate the ionizable amines in cationic ionizable lipids (e.g., 5A2-SC8, C12-200, DLin-MC3-DMA) to enable electrostatic complex formation with negatively charged mRNA. For all other formulations in this figure, mixing was performed using mRNA dissolved in PBS at pH 7.4. Clearly, when using low concentrations of DOTAP, the encapsulation efficiency was low, but when the molar percentage of DOTAP exceeded 25%, it increased to >80% (Figure 5C). The encapsulation efficiency was approximately 80% to approximately 95% for all formulations using a molar percentage of DOTAP exceeding 25%. Thus, the potential to use neutral pH PBS mixing is a feature of the permanent cationic lipid strategy. This strategy enables tissue-specific delivery and encapsulation of a large amount of Cas9 protein. The addition of permanent cationic lipids enables LNP formation at neutral pH. These encapsulation results are those when PBS was used as the buffer. When using an acidic buffer (e.g., citrate buffer (10 mM, pH 4.0)), the encapsulation efficiency was high (>90%) for all formulations with 0 to 100% DOTAP.

[0184] Finally, pKa was determined using the 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS) assay (Figure 3B) (Zhao et al., 2016). Based on the defined rules, the relationship between pKa and tissue-specific mRNA delivery was plotted. Here, as shown in the table, eight rules were designed for scoring. Apparently, the pKa of the liver-targeted formulation was narrow (about 6 - 7), there was no significant range for the spleen-targeted formulation, while lung-targeted delivery required a high pKa (>9.25). Considering distribution and pKa detection together, it was concluded that the internal charge of the NPs is one factor affecting mRNA distribution, and the overall / apparent LNP pKa is another factor determining the protein expression profile via mRNA in organs.

[0185] Example 3: Potency of mDLNPs Modified with Permanently Cationic Lipids for mRNA Delivery To examine the delivery efficacy of LNPs containing permanent cationic lipids for delivering active cargo in vitro, DOTAP-modified mDLNPs were loaded with mRNA encoding luciferase, and Huh-7 liver cells and A549 adenocarcinoma human alveolar basal epithelial cells were transfected with 50 ng / well of mRNA. After culturing these cells for 24 hours, luciferase expression and cell viability were examined. As shown in Figure 6A, 5% - 50% DOTAP percentage was better for mRNA delivery and expression in Huh-7 liver cells in vitro, and 10% DOTAP seemed to show the maximum luciferase delivery and expression (Figure 6A). Note that the delivery characteristics in vivo may be different. In general, because SORT LNPs have additional in vivo barriers, organ distribution, and cellular specificity, these studies may not be useful for predicting in vivo activity or tissue tropism. Furthermore, it was found that 10% DOTAP among mDLNPs that examined cell viability and showed robust luciferase expression resulted in high viability (Figure 6A). Similar results were shown when the same transfection was examined using the A549 lung cancer cell line. Cells transfected with the DOTAP10 formulation showed almost twice the fluorescence of other formulations and maintained high cell viability (Figure 6A). DOTAP SORT LNPs were formed in PBS (pH 7.4) rather than citrate buffer (10 mM, pH 4.0), but can be formed in either buffer system. This is a property that enables the encapsulation and delivery of cargo that is not stable in ethanol or acidic buffers, such as proteins.

[0186] To confirm the effect of ethanol concentration in the formulation, DOTAP25 was selected and prepared using various ethanol:PBS ratios (1:3, 1:5, 1:7.5, and 1:10) (Figure 6B). All four formulations showed similar encapsulation efficiency, size, and PDI (Figure 2B). mRNA delivery efficiency was also measured by transfecting FaDu hypopharyngeal carcinoma cells with 50 ng / well of mRNA in each formulation. The delivery efficiency of each formulation was similar and had little effect on cell viability (Figure 6C). Therefore, these formulations appear to be applicable to a number of cell types, including the liver, lung, and pharynx. Furthermore, this formulation was also successfully modified to use 1×PBS (pH 7.4) instead of acidic buffer (pH 4.0). From these data, it can be seen that the percentage of ethanol can be drastically reduced. From this, it was obtained that the DOTAP formulation has the potential to deliver cargos that are highly sensitive to high ethanol concentrations and acidic buffers, such as proteins.

[0187] Next, to test the ability of these mDLNPs to deliver mRNA in vivo, mice were injected with a dose of 0.1 mg / kg of Luc mRNA in each formulation. Figure 1B shows ex vivo images of luciferase in major organs 6 hours after IV injection of each formulation. Interestingly, as the molar percentage of DOTAP increased, luciferase expression shifted from the liver to the spleen and then to the lungs, demonstrating organ-specific delivery. Quantifying these data, it became clear that the DOTAP percentage is a factor in tissue-targeted delivery, with mDLNP (0% DOTAP) being optimal for liver delivery, 5 - 15% DOTAP being optimal for spleen delivery, and DOTAP50 (50%) being optimal for lung delivery (Figure 1B). Assuming that luciferase expression was only detected in the liver, spleen, and lungs after IV injection, the percentage of luciferase expressed in each organ could be calculated (Figure 1C). From these data, it is clearly seen that as the molar percentage of DOTAP in the formulation increases, delivery to and expression in the liver decrease, and near-zero expression in the liver is observed when the DOTAP percentage exceeds 70% (Figures 1B, 1C). However, the higher the DOTAP percentage, the more luminescence is observed in the lung tissue, and near-100% luminescence in the lungs is observed when the DOTAP percentage exceeds 80% (Figure 1C). Concentrations of 5 and 30 molar percentages of DOTAP showed a high percentage of luminescence in the spleen tissue, while DOTAP10 showed the highest relative luminescence in the spleen compared to other tissues (Figure 1C). These results indicate that the lipid concentration can be adjusted according to specific tissue delivery after injection.

[0188] To further test the in vivo organ distribution of a specific DOTAP formulation, PBS, or liver-targeted NPs (mDLNPs), spleen-targeted NPs (DOTAP10), and lung-targeted NPs (DOTAP50) were injected into C57BL / 6 mice (n = 2) at a dose of 0.5 mg / kg Cy5-Luc mRNA (a dye-labeled mRNA to track the RNA LNPs). The major organs were collected and imaged 6 hours after injection (Figure 3A). The organ distribution of the formulations changed with the amount of DOTAP, and as the percentage of DOTAP increased, the accumulation in the liver gradually shifted to the lungs, but the NPs were still present in the liver regardless of the DOTAP percentage (Figures 7 and 8). Considering this data together with the data in Figure 1, it is clear that organ distribution is not sufficient to analyze tissue-targeted delivery efficacy (mRNA translation into protein). Furthermore, considering the similar size distributions and EE among these formulations, zeta potential and pKa may play a role in tissue-targeted mRNA expression.

[0189] In an attempt to understand whether the effect of adding DOTAP to mDLNP is limited or whether the distribution shown above is universal for mDLNP formulated with a permanently cationic lipid, mDLNPs were prepared including another popular cationic lipid, didodecyldimethylammonium bromide (DDAB) (Figure 2A1). DDAB has 18 carbons, two hydrophobic tails without unsaturated bonds, and a head that is completely different from DOTAP (Figure 1C). DDAB5, DDAB15, DDAB40, and DDAB50 formulations were selected for in vivo delivery (0.1 mg / kg, 6 h, n = 2). There was little difference in size distribution as in the above DOTAP formulations (Figure 2A1), but the percentage of DDAB in the NPs changed 10-fold (5% - 50%). From in vivo luciferase expression, a tendency was shown that as the percentage of DDAB increased, luminescence moved from the liver to the spleen and then to the lungs, similar to DOTAP NPs (Figure 2A2).

[0190] A third permanent cationic lipid was used to form mDLNP, which has a structure similar to DOTAP but has a shorter 14-carbon hydrophobic tail ((14:0)EPC), the head group being 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride (Figure 2B1). Similar to the DDAB strategy, (14:0)EPC5, (14:0)EPC15, (14:0)EPC40, and (14:0)EPC50 formulations were prepared and their size distribution (Figure 2B1) and in vivo Luc mRNA delivery (0.1 mg / kg, 6 h, n = 2) were examined (Figure 2B2). Similar to the mDLNP analyzed above, the particle sizes were generally uniform (Figure 2B1), and as expected, luminescence migrated from the liver to the spleen and then to the lungs as the (14:0)EPC molar percentage increased (Figure 2B2). Collectively, all of these data, including different hydrophobic tails, saturated and unsaturated linkages, and different head groups, suggest that mDLNP formulated with cationic lipids for tissue-targeted mRNA delivery is universal.

[0191] In an attempt to understand whether the effect of DOTAP addition to LNP is specific to permanent cationic lipids, the effect of adding zwitterionic lipids to the mDLNP formulation instead of permanent cationic lipids was investigated. Two representative zwitterionic lipids, phosplolipids with different chemical structures: DSPC and DOCPe, were tested. Furthermore, this was also tested to confirm whether the addition of zwitterionic lipids (instead of permanent cationic lipids) affects the tissue-specific delivery efficacy. Figures 2C1 and 2D1 show the chemical structures of DSPC and DOCPe lipids (zwitterionic lipids). There are differences in the positions of the positively charged functional head and the negatively charged functional head and the hydrophobic domain (saturated vs. unsaturated). This suggests that the observed effect is general / universal to zwitterionic lipids. The mDLNPs formulated with DSPC or DOCPe were similar (Figures 2C1, 2D1). Interestingly, including zwitterionic lipids in the 5-component improved DLNP did not change the protein expression profile from the liver to the lung as in the case of DOTAP and other permanent cationic lipids. Instead, both DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% for DSPC and less than 50% for DOCPe). There was no protein expression in the lung at any percentage (0.1 mg / kg, 6 h, n = 2) (Figures 2C2, 2D2). Therefore, further inclusion of zwitterionic lipids can assist spleen delivery, but cannot regulate the delivery efficacy from the liver to the spleen to the lung as in the case of permanent cationic lipid inclusion.

[0192] In an attempt to understand whether the effect of adding DOTAP to LNP is specific to permanent cationic lipids, the effect of adding cationic ionizable lipids to the mDLNP formulation was investigated instead of permanent cationic lipids. Two representative cationic ionizable lipids with different chemical structures, C12-200 and DODAP, were tested. DODAP has the same structure as DOTAP except for the head (quaternary amine vs. tertiary amine). C12-200 contains an ionizable tertiary amine (which is not a quaternary amine either) and is an effective lipidoid used for siRNA or mRNA delivery and has a completely different structure from DODAP. (Figs. 2E1, 2F1) Similarly, the size distribution of both improved mDLNPs remained uniform at a certain percentage (less than 80%). (Figs. 2E1, 2F1) Surprisingly, even when cationic ionizable lipids were included in the 5-component improved DLNP, the protein expression profile from the liver to the spleen to the lung did not change as in the case of DOTAP and other permanent cationic lipids. Instead, including cationic ionizable lipids in the DLNP increased the mRNA delivery efficacy of mRNA to the liver. These showed significantly better delivery efficacy than the original mDLNP (0.1 mg / kg, 6 h, n = 2) without further cationic ionizable lipids (only 5A2-SC8). As the percentage of DODAP or C12-200 increased (50% or 80%), the luciferase signal decreased significantly, but the liver remained the major organ instead of the spleen or lung. Therefore, it was concluded that the organ-specific effect could be due to the inclusion of a specific ratio of permanent cationic lipids. Furthermore, from this data, it can be seen that permanent cationic lipids produce different effects from cationic ionizable lipids. Additionally, from these data, it can be seen that these trends are universal with respect to lipid classes.

[0193] Example 4: CRISPR / Cas9 Gene Editing Using Improved mDLNP for Simultaneous Delivery of Cas9 mRNA and sgRNA First, to determine which sgRNA was most effective in subsequent experiments, three sgRNAs targeting Td-Tomato mice were compared. These sgRNAs were sgTom1, sgTom2, and sgLoxP. As shown in Figure 10A, sgTom1 and sgLoxP were delivered and expressed, and similar results were obtained. These performed better than sgTom2 in the induction of TdTomato (Figure 10A). Considering the weak PAM of sgLoxP(NAG), sgTom1 was finally selected for further experiments.

[0194] Given that DOTAP NPs have been shown to deliver tissue-specific mRNA (luc mRNA) and NPs modified with DDAB and EPC showed similar delivery trends, next, aiming to achieve tissue-specific gene editing, DOTAP-modified mDLNPs were used to co-deliver Cas9 mRNA / sgRNA. To examine in vivo co-delivery, transgenic mice containing a homozygous Rosa26 promoter Lox-Stop-Lox tdTomato (tdTO) cassette present in all cells were used (Figure 4A). Co-delivery of Cas9-mRNA and DOTAP-modified mDLNPs containing sgRNA against LoxP or Tom deleted the Stop cassette and induced tdTO expression (Figure 4B). Mice were IV injected with mDLNP and DOTAP50 formulations to co-deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, wt / wt) at a total dose of 2.5 mg / kg (50 μg each). Fluorescence in major organs was then detected on day 10 after treatment. (Figure 4B) Liver-specific and lung-specific CRISPR / Cas gene editing was achieved. Spleen-specific editing was also achieved. However, spleen editing could not be quantified using this TdTomato reporter mouse due to very strong background red autofluorescence.

[0195] PTEN was selected as an endogenous target to further investigate tissue-specific editing. To achieve tissue-specific gene editing, C57BL / 6 mice were injected intravenously (IV) with mDLNP, DODAP20, or DOTAP50. The total dose was 2.5 mg / kg (50 μg each), the weight ratio of IVT Cas9 mRNA to modified sgPTEN was 4 / 1, and the detection time was 10 days after treatment. PTEN-targeting sgRNA was used. From the T7E1 assay, it was found that tissue-specific characteristics were further confirmed by in vivo PTEN editing (Figure 4C).

[0196] Example 5: CRISPR / Cas9 Gene Editing Using Modified mDLNP to Deliver Cas9 Protein / sgRNA Ribonucleoprotein (RNP) Based on the discovery that conventional LNP formulations containing cationic ionizable lipids, zwitterionic lipids, cholesterol, and PEGylated lipids were incorporated with a permanent cationic lipid (e.g., DOTAP), it was investigated whether this formulation methodology could also deliver other cargos that are sensitive to ethanol and / or low pH acidic buffered aqueous solutions. An important element of the DOTAP strategy is that the formulation can be prepared using PBS at neutral pH. Therefore, this methodology was also investigated to determine whether it could encapsulate and deliver large proteins such as Cas9 for gene editing applications. Thus, DOTNP lipid nanoparticles consist of a modular composition of five components: a cationic ionizable lipid (e.g., 5A2-SC8), a zwitterionic lipid (e.g., DOPE), cholesterol, DMG-PEG, and a permanent cationic lipid (e.g., DOTAP). The molar ratios of 5A2-SC8, cholesterol, DOPE, and DMG-PEG were fixed (15:15:30:5, mol / mol). DOTNPX refers to DOTNP with different molar percentages of DOTAP.

[0197] It was investigated whether the initially characterized Cas9 / sgRNA complex is sensitive to acidic pH. The size (diameter) (Figure 11A) and zeta potential (Figure 11B) of the Cas9 / sgLUC complex (mol / mol = 1 / 1) were measured in PBS (pH 7.4) and citrate buffer (pH 4.2). The size of the Cas9 / sgLUC complex prepared in citrate buffer was large (greater than 100 nm), and the zeta potential was positively charged. Due to these two characteristics (a size larger than typical effective LNPs) and a positive charge (a charge not compatible with complexing with positively charged lipids), it is impossible to be effectively encapsulated by lipid nanoparticles. However, the size of the Cas9 / sgLUC complex prepared in PBS was small (less than 20 nm) and had a negative charge. Therefore, it can be encapsulated by lipid nanoparticles when formulated at neutral pH. Next, Cas9 / sgRNA complexes with different Cas9 protein:sgRNA molar ratios were prepared and characterized. The size (Figure 11C) and zeta potential (Figure 11D) of the Cas9 / sgLUC complexes prepared at different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with the Cas9 / sgLUC complex (1 / 1, mol / mol), the larger the molar ratio (1 / 3 and 1 / 5, mol / mol), the smaller the size and the larger the negative charge. This may be beneficial for lipid nanoparticle encapsulation. These compositions were prepared, and the DOTNP lipid nanoparticles after encapsulating the Cas9 / sgRNA complex were characterized. The size (Figure 11E) and zeta potential (Figure 11F) of the DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complex (named DOTNP10-L) when prepared at different molar ratios (1 / 1, / 3, 1 / 5). This data indicates the encapsulation of the Cas9 / sgRNA RNP into monodisperse LNPs. Figure 11G shows a TEM image of the DOTNP10-L (1 / 3, mol / mol) LNP containing the encapsulated RNP. After this initial study, different sgRNAs including sgLUC, sgGFP, sgTOM, and sgPTEN were used. To distinguish them, the first letter of each gene was added at the end of DOTNP.For example, DOTNP10-L means a DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgLUC complex. DOTNP10-G means a DOTNP10 lipid nanoparticle-encapsulated Cas9 / sgGFP complex.

[0198] (Table 7) Characterization of DOTAP10, DSPC50, and DODAP50 formulations formed by PBS and citrate buffer, including size, PDI, and encapsulation efficacy TIFF2025108518000079.tif45153

[0199] (Table 8) Enumeration of all primers used in this study, including the length and purpose of the PCR products (Cas9 = SEQ ID NO: 3-4; Ca9 Seq-1 = SEQ ID NO: 5; Ca9 Seq-2 = SEQ ID NO: 6; Ca9 Seq-3 = SEQ ID NO: 7; Ca9 Seq-4 = SEQ ID NO: 8; Ca9 Seq-5 = SEQ ID NO: 9; Ca9 Seq-6 = SEQ ID NO: 10; Ca9 Seq-7 = SEQ ID NO: 11; PTEN = SEQ ID NO: 14-15; IVT sgTom1 = SEQ ID NO: 44-45; IVT sgTom2 = SEQ ID NO: 46-47; IVT sgLoxP = SEQ ID NO: 48-49) TIFF2025108518000080.tif213125

[0200] A series of experiments were conducted to investigate whether DOTNP lipid nanoparticles can deliver the Cas9 / sgRNA RNP complex to the nucleus in vitro and mediate efficient gene editing. First, DOTNP containing Cas9 / sgRNA RNP tagged with green fluorescent EGFP was tracked by confocal microscopy (Figure 12A). From images of Hela-Luc cells after incubation with DOTNP10 encapsulating the Cas9-EGFP / sgLUC complex (1 / 3, mol / mol) for 1 hour, 3 hours, 6 hours, and 24 hours (using 9 nM sgRNA), it was found that DOTNP translocated into the cells and the Cas9 RNP was transported to the nucleus. Green: EGFP-fused Cas9 protein; Blue: Nucleus stained with Hoechst33342. The red arrow indicates the process by which DOTNP10 enters the nucleus. (Figure 12B)

[0201] Next, it was investigated whether DOTNP lipid nanoparticles could deliver the Cas9 / sgRNA RNP complex and whether the Cas9 / sgRNA RNP complex could cleave the target luciferase DNA. The percentage of DNA indels (insertions and deletions) at the LUC locus after incubating with different molar ratios of DOTNP10-L for 3 days was quantified using the TIDE assay (using 24 nM sgRNA). DOTNP10 lipid nanoparticle encapsulated Cas9 / sgGFP (DOTNP10-G) was used as a negative control. Here, two commercially available Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used. (Figure 12B) Next, to demonstrate DNA editing, a T7EI cleavage assay (24 nM sgRNA) of Hela-Luc cells incubated with different formulations was performed (Figure 12C). Among the conditions tested, a 1 / 3 molar ratio showed the best gene editing when using the Truecut Cas9 protein. Next, GFP editing was tested using a fluorescence microscope (Figure 12D). Images of SKOV3-GFP cells incubated with DOTNP10-L (control, not targeting GFP) and DOTNP10-G (targeting GFP) (24 nM sgRNA). Editing on the target was demonstrated by the disappearance of GFP protein expression. Finally, flow cytometry was used to analyze SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G (Figure 12E). (Figure 12F) The mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G by flow cytometry indicated GFP editing by CRSPR / Cas.

[0202] Next, it was investigated whether DOTNP lipid nanoparticles could deliver the Cas9 / sgRNA RNP complex in vivo to achieve gene editing via CRISPR / Cas. As before, a genetically engineered TdTomato mouse model was used. 1.5 mg / kg of sgRNA was delivered per mouse using the following formulations. DOTNP5-T means the DOTNP5 LNP-encapsulated Cas9 / sgTom complex. DOTNP10-T means the DOTNP10 LNP-encapsulated Cas9 / sgTom complex. DOTNP50-T means the DOTNP50 LNP-encapsulated Cas9 / sgTom complex. After IV injection of these formulations, 7 days after injection, tdTomato fluorescence was quantified ex vivo in major organs (Figure 13A). In the DOTNP5-T treatment group, tdTomato fluorescence was detected only in the liver. In the DOTNP10-T group, slight fluorescence was observed in the lung, and when the DOTAP dose was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lung. Thus, similar to the mRNA delivery experiments summarized above, the DOTAP methodology also enables tissue-specific gene editing of the Cas9 / sgRNA ribonucleoprotein (RNP) complex. To further investigate delivery, LNPs containing sgRNA against PTEN were delivered. Using the T7EI cleavage assay of liver and lung organs, IV injection of DOTNP5-P (DOTNP5 LNP-encapsulated Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 LNP-encapsulated Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 LNP-encapsulated Cas9 / sgPTEN complex) (2 mg / kg of sgRNA / mouse) was confirmed to mediate gene editing (Figure 13B). The results were consistent with those obtained by ex vivo imaging. Gene editing was observed only in the liver after treatment with DOTNP5-P. When incubated with DOTNP10-P, gene editing was obtained in both the liver and the lung. In contrast, in the DOTNP50-P treatment group, most of the gene editing was observed in the lung.

[0203] It can be seen from the data presented herein that lipid nanoparticles can be prepared using a variety of compositions to specifically target the payload to specific tissues. In particular, lipid nanoparticles containing low concentrations of permanent cationic lipids (≤10%) are effective in delivering nucleic acids to the liver, LNPs containing less than 30% of permanent cationic lipids are effective in delivering nucleic acids to the spleen, and LNPs containing more than 30% of permanent cationic lipids effectively deliver nucleic acids to the lung. These findings appear to be universal, and the head, saturation, and tail length have little effect.

[0204] Example 6: Changing the delivery target by adding another lipid to four known lipid compositions Next, the generalizability of an approach (methodology) of including a "fifth" lipid in an established four-component LNP was explored.

[0205] To investigate whether the inclusion of a permanent cationic lipid (e.g., DOTAP) can alter the tissue specificity of other cationic ionizable lipids, two well-known and well-established cationic ionizable lipid LNP systems were selected. DLin-MC3-DMA was selected and formulated with DSPC, cholesterol, and PEG-DMG. The same molar composition as Patisiran / Onpattro (Alnylam Pharmaceuticals) was generated and 15% or 50% DOTAP (the extra fifth lipid in the Onpattro 4-lipid formulation) was added (Figure 14). DLin-MC3-DMA LNPs are considered the "gold standard" for siRNA and mRNA delivery. To date, these have only been shown to deliver to the liver after IV administration. As shown in Figure 15A, DOTAP changed the mRNA expression profile in organs of DLin-MC3-DMA-based LNPs (0.1 mg / kg luciferase mRNA, 6 hours). As the percentage of DOTAP increased, the luciferase signal shifted from the liver to the spleen and finally to the lung. This was exactly the same phenomenon as that of 5A2-SC8 mDLNP. To further investigate the generality of this approach, the inventors included DOTAP in C12-200 LNPs (Figure 16). DLin-MC3-DMA is a lipid with two tails and one dimethylamine head that is considered a stable nucleic acid lipid nanoparticle (SNALP), whereas C12-200 is a representative "lipidoid" that can be formulated into lipid-like LNPs. All three are cationic ionizable lipids. Although identical to the results using 5A2-SC8 and DLin-MC3-DMA, inclusion of 15 or 50% DOTAP in C12-200 LNPs changed luciferase protein expression from the liver to the spleen and then to the lung after mRNA delivery (Figure 15B). Thus, the fifth lipid methodology (e.g., adding a permanent cationic lipid) is generalizable to other cationic ionizable lipid LNPs.

[0206] Example 7: Delivery is improved by adding another lipid to four known lipid compositions Furthermore, the generalizability of the approach was explored by asking whether additional cationic ionizable lipids improve liver delivery.

[0207] Generally, to investigate whether cationic ionizable lipids promote liver delivery, an additional 5A2-SC8 cationic ionizable lipid was included as the "fifth" lipid in LNPs containing 5A2-SC8, DOPE, cholesterol, and PEG DMG in appropriate ratios. Excess 5A2-SC8 was included at 10 - 30 percent (Figures 17A and 18). To avoid saturation of luminescence, a low 0.05 mg / kg dose of the mRNA dose was tested (IV, 6 hours). As shown in Figure 17B, from both ex vivo images and quantitative data, an increased 15% - 25% of excess 5A2-SC8 was proven to contribute to the improvement of mRNA delivery efficacy in the liver. 5A2-SC8^20 (5A2-SC8 LNP + 20% excess 5A2-SC8) increased luciferase 2 - 3 fold more than the original mDLNP formulation.

[0208] Example 8: Studies related to selectively organ-targeted compositions The present disclosure describes a strategy called Selective Organ Targeting (SORT) that enables the systematic engineering of nanoparticles to accurately deliver diverse cargos, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes, to the lungs, spleens, and livers of mice after intravenous (IV) administration (Figure 19A). Conventional LNPs are composed of cationic ionizable lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. The present disclosure shows that the addition of an auxiliary component (referred to as a SORT compound or selective organ targeting compound) precisely alters the in vivo RNA delivery profile and mediates tissue-specific gene delivery and editing as a function of the percent of added SORT lipid and biophysical properties. The present disclosure provides evidence of the theory for tissue-specific delivery, establishes that this methodology is useful for various nanoparticle systems, and provides an LNP design method for editing therapeutically relevant cells.

[0209] Effective intracellular delivery materials have relied on the conventional optimal balance of ionizable amines (pKa 6.0 - 6.5) for binding and releasing RNA and nanoparticles for stabilizing hydrophobicity (Kanasty et al., 2013; Jayaraman et al., 2012; Nelson et al., 2013; Hao et al., 2015). Without being bound by any theory, it is thought that internal and / or external charges may be a factor in regulating tissue tropism. Intravenous administration of the developed SORT LNPs enabled high levels of tissue-specific gene editing. SORT is compatible with various methods of arranging gene editing machinery, including mRNA, Cas9 mRNA / sgRNA, and Cas9 RNP (systemic RNP delivery). Lung-targeted SORT LNPs edited 40% of epithelial cells and 65% of endothelial cells. Spleen-targeted SORT LNPs edited 13% of B cells and 10% of T cells. Enhanced liver-targeted SORT LNPs edited 93% of hepatocytes after a single low-dose injection.

[0210] A. Discovery and Development of SORT To investigate the hypothesis that tissue-specific delivery can be mediated by modulating internal charge, a strategy of adding a fifth lipid to an established LNP composition was devised and its efficacy in hepatocytes was verified. This principle was to adjust an effective LNP formulation without disrupting the core four-component ratio commonly used to mediate RNA encapsulation and endosome avoidance (Wittrup et al., 2015; Cheng et al., 2018).

[0211] The effect of adding a permanent cationic lipid (defined as being positively charged without a pKa or with a pKa > 8) to a degradable dendrimer ionizable (pKa < 8) cationic lipid named 5A2-SC8 used in mDLNP (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) was investigated. This effectively delivered fumarylacetoacetate hydrolase (FAH) mRNA to hepatocytes and extended the survival period in FAH knockout mice (Cheng et al., 2018). This first basic mDLNP formulation consisted of 5A2-SC8, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, DMG-PEG (15 / 15 / 30 / 3, mol / mol), and mRNA (5A2-SC8 / mRNA, 20 / 1, wt / wt) (Figure 20). A series of LNPs were then formed by systematically increasing the percentage of the additional permanent cationic lipid from 5 to 100% relative to the total lipids (Figures 19B and 20). First, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a well-known quaternary amino lipid, was selected as the SORT lipid to be added to the LNP formulation. Thus, the DOTAP-modified SORT formulation contained five lipid components. 5A2-SC8 / DOPE / Chol / DMG-PEG was fixed at 15 / 15 / 30 / 3 (mol / mol), and DOTAP was added at a molar ratio of 0 to 1200 to create a titrated series of formulations (Figure 20).

[0212] Next, the effect of the SORT modification was evaluated by delivering luciferase (Luc) mRNA intravenously (IV) at a dose of 0.1 mg / kg. As the molar percentage of DOTAP increased, the resulting luciferase protein expression continuously shifted from the liver to the spleen and then to the lungs. This demonstrated a clear and accurate organ-specific delivery trend with a threshold enabling exclusive lung delivery (Figure 19B). The DOTAP percentage was an important factor in regulating tissue specificity. The basic LNP (0% DOTAP) was optimal for liver delivery, as expected since it had been previously optimized for hepatocyte delivery (Cheng et al., 2018). Adding 10 - 15% DOTAP enabled the resulting SORT LNPs to deliver mRNA to cells within the spleen. Further increasing the permanently cationic SORT lipid revealed that 50% DOTAP was optimal for lung delivery (Figure 19C). It is notable that 50% DOTAP SORT LNPs were effective at delivering mRNA to the lungs in vivo but were not as effective as in vitro delivery (Figure 21). Additionally, 50% DOTAP SORT LNPs had a neutral zeta potential surface charge (-0.52 mV) (Figure 20). This indicates that tissue tropism is not due to macrophage uptake related to positive charge. Calculating the relative expression in each organ, using DOTAP as the SORT lipid resulted in a complete shift in delivery from the liver to the lungs (Figure 19D). Thus, considering that >99% of the latest IV nanomedicines were estimated to be sequestered by the MPS (Wilhelm et al., 2016; Gustafson et al., 2015), these new SORT nanoparticles overcome a longstanding challenge in nanomedicine.

[0213] Although not bound by any theory, since the functional role of the permanent cationic SORT lipid has been elucidated, it is thought that the inclusion of other lipids may also change tissue tropism. To explore this potential, negatively charged 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) was incorporated as the SORT lipid, similar to DOTAP (Figure 20). At 10–40% 18PA incorporation, SORT LNPs mediated completely selective delivery to the spleen with no luciferase expression in other organs (Figure 19E). Thus, negatively charged SORT lipids enable clear delivery to the spleen. These results indicate that the SORT lipid percentage can be adjusted for specific tissue mRNA delivery via IV injection.

[0214] B. SORT is generalizable to other LNP types and lipid classes Next, to test whether SORT is universal, we explored whether the SORT methodology could be applied to other established four-component LNPs. First, we formulated DLin-MC3-DMA with DSPC, cholesterol, and PEG-DMG using the same molar composition as the FDA-approved Onpattro (Patisiran) (30), which is considered the “gold standard” for siRNA and mRNA delivery (Figure 22). To date, these have only been shown to deliver to the liver after IV administration, which was also confirmed here (Figure 19F). As expected, adding DOTAP to the DLin-MC3-DMA LNP changed the protein expression profile of the Onpattro formulation. As the percentage of SORT lipid increased, the luciferase signal shifted from the liver to the spleen to the lung. This was the exact same phenomenon as the 5A2-SC8 DLNP tested first. To further investigate the universality of this approach, DOTAP was included in the C12-200 LNP (Figures 19G and 22). C12-200 LNP is also well-established for RNA delivery to the liver (Kove et al., 2010; Kauffman et al., 2015). Identical to the results with 5A2-SC8 and DLin-MC3-DMA LNPs, including 15% or 50% DOTAP in the C12-200 LNP changed the luciferase protein expression from the liver to the spleen to the lung after mRNA delivery (Figures 19G and 22). Furthermore, the inclusion of 18PA as the SORT lipid reproduced the results with 5A2-SC8 and mediated exclusive Luc mRNA delivery to the spleen for both DLin-MC3-DMA SNALP and C12-200 LLNP (Figures 19F–G). DLin-MC3-DMA is a lipid with two tails and one dimethylamine head that forms stable nucleic acid lipid nanoparticles (SNALP), whereas C12-200 is a representative lipidoid that forms lipid-like LNPs (LLNP). Thus, the SORT methodology was shown to be generalizable to other classes of cationic ionizable lipid LNPs. This allows for easy modification of existing liver-targeted LNPs to deliver mRNA to the spleen or lung.Specifically, using the SORT technology may enable the rapid redevelopment of Onpattro, which is approved by the FDA, to treat diseases in the lungs and spleen.

[0215] To understand whether the observed tissue tropism profiles are specific to the exact chemical structure or can be generalized to defined chemical classes, multiple permanent cationic, anionic, zwitterionic, and cationic ionizable SORT lipids were evaluated (Figure 23). First, 5A2-SC8 LNPs were prepared with two additional permanent cationic lipids: didecyldimethylammonium bromide (DDAB) and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride (EPC). All of these lipids contain quaternary amino groups but have significant chemical differences (e.g., saturation) in the polar head, linker region, and hydrophobic domain. LNPs containing 5%, 15%, 40%, 50%, and 100% DDAB or EPC were formulated and characterized. The in vivo luciferase expression profiles were consistent with those of DOTAP LNPs, and the luminescence activity systematically shifted from the liver to the spleen and then to the lungs as the DDAB or EPC percentage increased (0.1 mg / kg, 6 h). When the percentage increased up to 40%, a high luciferase signal was observed exclusively in the lungs (Figure 23A). As representative anionic lipids with different structures compared to 18PA, 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (18BMP) were prepared. All anionic SORT lipids promoted exclusive delivery to the spleen (Figure 23B). This flexibility opens the way to balance multiple factors, including efficacy, selectivity, and tolerance, by optimizing the SORT compounds.

[0216] Triggered by these findings, other cationic ionizable lipids were added to the established formulations. As expected, addition of DODAP or C12-200 to 5A2-SC8 LNP did not significantly change the tissue tropism, but surprisingly, liver delivery increased >10-fold at 20% incorporation (Figure 23C). Addition of additional 5A2-SC8 as a SORT lipid to the established 5A2-SC8 LNP dramatically improved liver mRNA delivery, achieving 10 7 photons / sec / cm 2 at an extremely low dose of 0.05 mg / kg. Thus, SORT provides a new strategy to further improve liver-targeted LNP systems (Figure 24). The effect of using zwitterionic lipids (DOCPe and DSPC) as SORT lipids was also evaluated. The tissue tropism was found to shift from the liver to the spleen but was not selective compared to the use of cationic or anionic SORT lipids (Figure 25).

[0217] To test the limits of the SORT methodology, it was investigated whether SORT could “activate” an inert formulation. Indeed, addition of DODAP or DOTAP to a completely inert formulation resulted in tissue-specific delivery to the spleen and lungs (Figure 26). Collectively, these results indicate that SORT is a modular and universal strategy for achieving tissue-targeted delivery.

[0218] C. SORT modifies the protein corona, LNP biodistribution, and apparent pK to mediate organ-specific delivery a to change Mechanistic experiments were performed to explore how and why mRNA delivery to different organs is controlled by including extra lipids in defined categories. It is natural that LNPs delivered to cells in the lung must biodistribute (accumulate) in the lung. Cy5-labeled mRNA was delivered to track the in vivo distribution of 5A2-SC8 LNPs containing SORT lipids with lung (DOTAP quaternary amino lipid), spleen (18PA anionic lipid and DSPC zwitterionic lipid), and liver (DODAP ionizable tertiary amino lipid) tropisms (Figures 27A and 28). All LNPs were injected IV at a dose of 0.5 mg / kg Cy5-labeled mRNA and imaged 6 hours later. As shown in Figure 27A, DOTAP altered biodistribution and accumulation in the lung increased progressively as a function of the DOTAP percent. Incorporation of 18PA increased uptake into the spleen. DODAP slightly increased liver accumulation and decreased spleen accumulation. Interestingly, there was no protein expression in the liver for lung-specific and spleen-specific SORT LNPs, but these LNPs still accumulated in the liver. This suggests that organ biodistribution is necessary for organ-specific efficacy but is not the only factor explaining the mechanism of tissue-targeted delivery.

[0219] Although not bound by any theory, changes in in vivo distribution and activity in defined cell populations may be due to changes in the protein corona. When specific proteins bind in the protein corona, a biologically functional identity is created. Using quantitative mass spectrometry, it was found that the addition of the SORT molecule caused a drastic change in both the most closely bound specific proteins and the overall protein corona composition. Although not bound by any theory, lung-specific SORT LNPs are thought to bind selectively and most abundantly to vitronectin. Vitronectin can interact with positively charged lipids and binds to the αvβ3 integrin, which is highly expressed on the surface of lung endothelial and epithelial cells. This endogenous targeting mechanism is directly compared to adenoviruses that utilize the αvβ5 integrin to target bronchial epithelium. Spleen-specific SORT LNPs have been shown to interact with negatively charged lipids and may be most closely bound to β2-glycoprotein I, which perhaps plays a role in spleen localization to immune cell populations in the spleen. It is noteworthy that the complex mixture of bound proteins may also play a role. This ensemble effect is also a potential mechanism for targeting multiple cell types and was considered one way to further enhance specificity to a particular cell type within an organ using alternative SORT molecules. Apolipoprotein E binds to DLin-MC3-DMA Onpattro LNPs and it has previously been shown that efficacy is lost in ApoE knockout animals. Thus, there is strong evidence that ApoE is required for receptor-mediated targeting and uptake by the LDL receptor in hepatocytes, and that the described protein corona mechanism can control cell specificity and efficacy. Therefore, it is encouraging to find that mDLNPs also bind strongly to ApoE, providing further evidence of these hepatocyte efficacies and strengthening the validity of the protein corona assay. Liver-enhanced SORT LNPs retain ApoE binding but are also abundant in albumin.This suggests that there may be cell type proliferation in the liver. These data cumulatively indicate that the chemical structure of the SORT molecule can direct specific protein coronas that alter organotropism and cell specificity. Without being bound by any theory, it is believed that the identity of the SORT molecule can control protein corona identity. This suggests that the SORT molecule can include sugars, lipids, small molecule therapeutics, vitamins, small molecules, hydrophilic molecules, hydrophobic molecules, amphiphilic molecules, peptides, proteins, etc.

[0220] Apparent / overall pK as a parameter correlating LNP with functional activity a has been demonstrated, so apparent / overall pK a was examined. For example, a pK of approximately 6.4 has been shown to be optimal for delivery to hepatocytes (Jayaraman et al., 2012). Using the TNS assay, apparent pK a was analyzed for all effective in vivo formulations (67 types of LNP) (Figures 27B and 29, Table 1). Since SORT involves the inclusion of additional charged lipids, the resulting TNS titration curves capture the ionization behavior of more complex mixed-species LNPs. Therefore, instead, the relative pK a at which 50% of the normalized signal occurred was estimated. When plotting relative pK a against tissue tropism, SORT LNPs were divided into defined apparent pK a ranges (Figure 27B). As expected, the pK a of all effective liver-targeted formulations was very narrow and within the well-established range of 6 - 7 (Jayaraman et al., 2012). All lung-targeted formulations were located in the high pK a range (>9). In contrast, spleen-tropic SORT LNPs were divided into the low pK a range (2 - 6). These results support that 6 - 7 is optimal for liver delivery, while high pK a mediates lung delivery and low pK a a ​has revealed the discovery of mediating spleen delivery. It should be noted that all SORT LNPs still contain a cationic ionizable lipid. Cationic ionizable lipids are considered useful for endosome avoidance because of their ability to acquire a charge (Wittrup et al., 2015). Control experiments were conducted. It was confirmed that the inclusion of a cationic ionizable lipid is necessary to obtain efficacy (Figure 30). Therefore, SORT enables the retention of molecules with a specific microspecies pK a while including SORT lipids modifies the apparent pK a . Without being bound by any theory, it is thought that a two-part mechanism may play a role. SORT LNPs selectively bind to specific proteins in the serum that enable receptor-mediated efficacy in cells in the lung or spleen. This is very similar to the way lipoprotein particles (e.g., LDL) naturally transport cholesterol. Using this controlled and predictable endogenous targeting mechanism enables SORT LNPs to reach non-liver targets. The second part involves a method of modifying the properties of non-liver-targeted SORT LNPs so that the SORT molecule no longer has the physiochemical properties (e.g., overall / apparent pK a 6.4) required to obtain liver efficacy. This provides precision. It should also be noted that other more complex factors, such as differences in cell-specific endocytic transport, may also play a role. Considering the results, it is suggested that the internal charge of the LNP nanostructure mediates biodistribution and that the apparent pK a is correlated with the protein expression profile in specific organs. This special value can be used to continue developing other organ-specific nanoparticles.

[0221] Details of mDLNP formulations (SORT LNP) modified by DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe, including the molar ratio and molar percentage of each component, the weight ratio of total lipid to mRNA, size, and PDI TIFF2025108518000081.tif162156 a X represents DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe.

[0222] D.SORT enables lung-specific, liver-specific, and spleen-specific gene editing after intravenous administration Assuming that SORT LNPs have the ability to target specific organs, these findings were then applied to tissue-specific gene editing via IV injection. CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (Cas)) technology can precisely edit the genome in a sequence-dependent manner and has rapidly evolved for use in a wide variety of applications, including the potential correction of disease-causing mutations (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Hendel et al., 2015; Yin et al., 2016; Yin et al., 2017; Wang et al., 2018; Amoasii et al., 2018). Gene editing can be accomplished by local administration injection (Zuris et al., 2015; Sun et al., 2015; Chew et al., 2016; Staahl et al., 2017). However, many severe genetic disorders are caused by mutations in cells deep within organs, and correcting specific cells is necessary to cure the disease. Such correction may be best accomplished by systemic administration. Recently, it was reported that IV co-delivery of Cas9 mRNA and sgRNA is a safe and effective strategy for gene editing (Miller et al., 2017; Yin et al., 2017; Finn et al., 2018). However, to date, there have been no reports of LNPs rationally engineered to edit cells in organs other than the liver.

[0223] To examine and quantify the ability of SORT LNPs to mediate organ-specific gene editing, we utilized a genetically engineered tdTomato (tdTom) reporter mouse containing a LoxP-flanked stop cassette (Tabebordbar et al., 2016) that blocks the expression of the tdTom protein (Staahl et al., 2017). When the stop cassette is deleted, tdTom fluorescence is turned on, enabling the detection of gene-edited cells (Figure 31A). To activate tdTom in the edited cells, Cre recombinase mRNA (Cre mRNA) was first delivered. Fluorescent tissues were readily visible in the selected organs treated with liver-selective SORT LNP, lung-selective SORT LNP, and spleen-selective SORT LNP (Figure 31B). It should be noted that separate controls had to be used for each experiment because these mice had some background organ fluorescence. The background organ fluorescence in the spleen was the weakest compared to other organs (Figures 31C and 32). This made it difficult to distinguish spleen specificity detection in the tdTom mouse model. When endogenous PTEN was later edited, the spleen-selective SORT LNP showed clear DNA cleavage only in the spleen by the T7E1 assay (Figure 33C), and no DNA cleavage was shown in the liver or lung. Nevertheless, tdTom-positive cells were readily observed by confocal imaging of tissue sections (Figure 31D).

[0224] E. SORT enables high levels of editing in specific and therapeutically relevant cell populations Flow cytometry of single cells isolated from edited organs was used to quantify gene editing of specific cell types in the liver, lung, and spleen (Figure 31E). The liver-specific SORT (20% DODAP) 5A2-SC8 LNP edited approximately 93% of all hepatocytes in the liver after a single injection of 0.3 mg / kg Cre mRNA (Figures 31E and 34). This is the highest level of hepatocyte gene editing reported to date. The lung-specific SORT (50% DOTAP) 5A2-SC8 LNP edited approximately 40% of all epithelial cells, approximately 65% of all endothelial cells, and approximately 20% of immune cells in the lung at the same dose (Figures 31E and 35). Considering that epithelial cells are the primary target for correcting CFTR mutations that cause cystic fibrosis, this result establishes lung-specific SORT LNP as a compelling delivery system for immediate application to correct CFTR mutations. Finally, the spleen-specific SORT (30% 18PA) 5A2-SC8 LNP edited approximately 13% of all B cells, approximately 10% of all T cells, and approximately 20% of all macrophages (Figures 31E and 36). Due to improved selectivity compared to previous studies, spleen-specific SORT LNP may be applicable for treating non-Hodgkin B-cell lymphoma and other immune disorders. Although the initial focus was on quantification of single low-dose injections, higher levels of editing may be achievable by administering higher doses or multiple injections.

[0225] F. SORT enables tissue-specific gene editing by IV co-delivery of Cas9 mRNA / sgRNA and delivery of Cas9 RNP Next, by co - delivering Cas9 mRNA and sgRNA in a single nanoparticle via IV (Figures 37A, 38, and 39, Table 2), the ability of SORT LNPs to achieve tissue - specific CRISPR / Cas gene editing was examined. Liver - targeted SORT LNPs and lung - targeted SORT LNPs were injected at a dose of 2.5 mg / kg total RNA (4:1 mRNA:sgRNA, wt:wt) via a single IV injection, and gene editing was quantified 10 days later. As shown in Figure 37B, strong tdTom fluorescence was observed in the liver for both the basal LNP - treated mice and the 20% DODAP SORT LNP - treated mice, and strong fluorescence was observed in the lungs of the 50% DOTAP SORT LNP - treated mice. All results were consistent with the Luc mRNA delivery results. Since splenic immune cells are rapidly replaced in mice (Kamath et al., 2000), the weight ratio of Cas9 / sgRNA was optimized to 2 / 1 (Figure 39), and splenic editing was tested 2 days after injection. Taking into account background autofluorescence, bright tdTom fluorescence was observed in the spleens of 30% 18PA - treated mice. Exclusively, distinct T7E1 cleavage bands were detected in the DNA isolated from the spleens (no liver or lung editing) (Figure 33). Fluorescence was then confirmed by imaging tissue sections via confocal microscopy (Figure 37C).

[0226] Next, direct delivery of Cas9 RNPs, the most challenging strategy for synthetic carriers, was explored. Using a permanently cationic SORT lipid, tissue tropism could be controlled to encapsulate the Cas9 protein / sgtdTom complex. Liver editing was achieved by IV injection of 7% DOTAP SORT LNPs, whereas exclusive lung editing was achieved by 55% DOTAP SORT LNPs (Figure 37F). From these data, it can be seen that tissue - specific CRISPR / Cas gene editing of the liver, lung, and spleen is enabled by the described methodology.

[0227] We tested the ability of tissue-specific LNPs to edit endogenous targets in go beyond reporter mice. We selected PTEN because it is a well-established tumor suppressor expressed in most cells. Wild-type C57BL / 6 mice were injected with SORT LNPs (2.5 mg / kg total RNA) co-loaded with Cas9 mRNA and sgPTEN. Ten days after a single IV injection, the occurrence of insertions and deletions (indels) was quantified. As shown in Figure 37D, distinct DNA cleavage bands were observed in specific tissues by the T7E1 assay. This demonstrated that both the base LNP and 20% DODAP SORT LNP mediated effective PTEN editing in the liver, but not in the lung or spleen at all. Notably, 50% DOTAP SORT LNP showed PTEN editing exclusively in the lung. To further confirm PTEN editing, H&E staining and immunohistochemistry (IHC) of tissue sections were performed. As shown in Figure 37E, cells in the tissue sections clearly showed clear cytoplasm, a known phenotype of PTEN loss due to lipid accumulation (Xue et al., 2014). Furthermore, negative staining of PTEN was observed in both IHC sections of liver and lung tissues. Thus, clear evidence of PTEN editing was obtained. In the tdTom mouse model, spleen-specific 18PA SORT LNP editing was difficult to distinguish, but clear spleen PTEN editing could be observed in wild-type mice using the optimized weight ratio of Cas9 / sgPTEN (2 / 1) and detection time (2 days). No DNA editing was observed in the liver or lung by the T7E1 assay performed on 18PA SORT LNP-injected mice (Figure 33). Finally, SORT was applied to Cas9 RNP to examine endogenous editing of PTEN. Similar to before, 7% and 55% DOTAP SORT LNPs containing Cas9 protein / sgPTEN enabled tissue-specific editing in the liver and lung, respectively (Figure 37G). From these results targeting endogenous genes, it was demonstrated that rationally guided tissue-selective gene editing was achieved by synthetic carriers.

[0228] G. Materials and Methods I. Materials 5A2-SC8 (Zhou et al., 2016), DLin-MC3-DMA (Jayaraman et al., 2012), and C12-200 (Love et al., 2010) were synthesized and purified according to the following published protocols. 1,2-Dioleoyl-3-trimethylammonium-propane ( DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (18PA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14PA), sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (ammonium salt) (18:1 Hemi BMP, 18BMP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethylphosphate (DOCPe), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Sigma-Aldrich. 1,2-Dimyristoyl-sn-glycerol-methoxy(poly((ethylene glycol) MW 2000) (DMG-PEG2000) was purchased from NOF America Corporation. Cas9 protein was purchased from Thermo Fisher. ONE-Glo + Tox Luciferase Reporter assay kit was purchased from Promega Corporation. Pur-A-Lyzer Midi Dialysis Kits (WMCO, 3.5 kDa) were purchased from Sigma-Aldrich. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Thermo Fisher Scientific. Cas9 mRNA was produced by in vitro translation (IVT).Cy5-labeled firefly luciferase mRNA (Cy5-Luc mRNA), unlabeled firefly luciferase mRNA (Luc mRNA), and mCherry mRNA were purchased from TriLink BioTechnologies. D-Luciferin (sodium salt) was purchased from Gold Biotechnology. Modified sgTom1 and sgPTEN (Table 2) were purchased from Synthego.

[0229] (Table 2) Apparent relative pK of SORT LNPs measured by the TNS assay a value TIFF2025108518000082.tif243163

[0230] II. Nanoparticle formation The LNP formulation loaded with RNA was formed using the ethanol dilution method (Zhou et al., 2016). A liver-targeted mRNA formulation (mDLNP) was developed and reported in a previous paper (Cheng et al., 2018). As previously described (Jayaraman et al., 2012; Love et al., 2010), the basic formulation was prepared. Unless otherwise specified, total lipids with the designated molar ratio were dissolved in ethanol, and RNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were quickly mixed in a 3:1 aqueous solution:ethanol ratio (3:1, aqueous solution:ethanol, vol:vol) by volume to achieve a final weight ratio of 40:1 (total lipids:mRNA), and then incubated at room temperature for 10 minutes. To prepare SORT LNP formulations containing anionic SORT lipids (e.g., 18PA, 14PA, and 18BMP), the anionic lipid was first dissolved in tetrahydrofuran (THF), then mixed with other lipid components in ethanol, and finally, the formulation was obtained using the mRNA buffer (10 mM, pH 3.0) as described above. All formulations were named based on the additional lipid. Taking DOTAP mDLNP as an example, as reported in the published paper, the internal molar ratio of mDLNP was fixed with 5A2-SC8 / DOPE / cholesterol / DMG-PEG at 15 / 15 / 30 / 3 (Cheng et al., 2018). As an additional lipid, DOTAP was dissolved in the above ethanol lipid mixture in a specified amount such that the molar ratio of 5A2-SC8 / DOPE / cholesterol / DMG-PEG / DOTAP was equal to 15 / 15 / 30 / 3 / X, quickly mixed with the mRNA aqueous solution according to the above standard protocol, and finally SORT LNP was obtained. This was named Y%DOTAP. Y means the molar percentage of DOTAP in the total lipids. Similarly, formulations containing other additional lipids were formed using the above method (Figure 20 and Table 3). For Cas9 / sgRNA ribonucleoprotein (RNP) encapsulation, 1×PBS was used in the formulation, and the molar ratio of Cas9 to sgRNA was fixed at 1:3.After the formation of SORT LNPs, for in vitro assays and size detection, fresh LNP formulations were diluted to 0.5 ng / μL mRNA (final ethanol concentration < 5%) using 1× PBS. For in vivo experiments, the formulations were dialyzed against 1× PBS for 2 hours (Pur-A-Lyzer Midi Dialysis Kits, WMCO 3.5 kDa, Sigma-Aldrich) and diluted to 15 μL / g with PBS for intravenous (IV) injection.

[0231] (Table 3) sgRNA sequences TIFF2025108518000083.tif30151

[0232] III. Characterization of mRNA formulations Size distribution and polydispersity index (PDI) were measured using dynamic light scattering (DLS, Malvern MicroV model; He-Ne laser, λ = 632 nm), and zeta potential was measured after dilution with 1× PBS. The apparent pK of the mRNA formulations a To measure, some modifications were made to use the 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS) assay (Cheng et al., 2018; McLaughlin and Harary, 1976; Bailey and Cullis, 1994; Heyes et al., 2005). The mRNA formulation (60 μM total lipid) and the TNS probe (2 μM) were incubated for 5 minutes with a series of buffers containing 10 mM HEPES, 10 mM MES (4-morpholineethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCl (pH range of 2.5 - 11). The average fluorescence intensity of each well (black bottom 96-well plate) was measured by a Tecan plate reader at λ Ex = 321 nm and λ Em = 445 nm, and the data were normalized to the value at pH 2.5. Typically, the apparent pK a was defined as the pH at which the fluorescence maximum was halved. This method was used for most LNPs to determine the overall / apparent pK of the LNP aAlthough it was useful for estimating >40% permanently cationic lipids, it could not be used for SORT LNPs containing >40% permanently cationic lipids because these LNPs are always charged. Therefore, instead, the relative pK when 50% of the normalized signal occurred was estimated compared to the basic LNP formulation (without added SORT lipid). a This alternative calculation could estimate pK a without changing it for most LNPs and was consistent with the experimental results of tissue-selective RNA delivery for permanently cationic SORT LNPs. Therefore, it can be suggested that the standard TNS assay is used when the LNP contains one type of cationic ionizable lipid, and an alternative 50% normalized signal method is used for systems such as SORT that contain a complex mixture of multiple lipids with various charge states.

[0233] IV. In Vitro Luciferase Expression and Cell Viability Assays Huh-7 or A549 cells were seeded in white 96-well plates at a density of 1×10 4 cells / well the day before transfection. The medium was replaced with 150 μL of fresh DMEM medium (5% FBS), and then 50 μL of the Luc mRNA formulation was added, fixing at 25 ng mRNA per well. After further incubation for 24 hours, mRNA expression and cytotoxicity were detected using the ONE-Glo+Tox kit according to the standard protocol of Promega.

[0234] V. Animal Experiments All animal experiments were approved by the Institution Animal Care and Use Committees of The University of Texas Southwestern Medical Center and are in compliance with local, state, and federal government regulations where applicable. C57BL / 6 mice were obtained from the UTSW Mouse Breeding Core Facility. B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)HzeThe Ai9 mouse (also known as the Ai9 or Ai9(RCL-tdT) mouse) was obtained from the Jackson Laboratory (007909) and bred to maintain homozygous expression of a Cre reporter allele with a loxP-flanked STOP cassette that blocks transcription of the red fluorescent tdTomato protein driven by the CAG promoter. After Cre-mediated recombination, Ai9 mice express tdTomato fluorescence. Ai9 mice are congenic on a C57BL / 6J genetic background.

[0235] VI. In Vivo Luc mRNA Delivery and Biodistribution C57BL / 6 mice weighing 18 - 20 g were injected intravenously (IV) with various Luc mRNA formulations at a dose of 0.1 mg / kg or 0.05 mg / kg. n = 2 - 4 mice / group. Six hours later, the mice were injected intraperitoneally (IP) with D-luciferin (150 mg / kg) and imaged using an IVIS Lumina system (Perkin Elmer). For biodistribution, C57BL / 6 mice were injected IV with a Cy5-Luc mRNA formulation at a dose of 0.5 mg / kg. Ex vivo imaging (Cy5 channel) was performed 6 hours after injection.

[0236] VII. mRNA Synthesis Optimized Cre recombinase mRNA and Cas9 mRNA were generated by in vitro transcription (IVT). Briefly, the NLS-Cre fragment and the Cas9 fragment were prepared by PCR programs using pCAG-CreERT2 and pSpCas9(BB)-2A-GFP (PX458) as PCR templates, respectively. These fragments were then cloned into the pCS2+MT vector with optimized 5'(3') untranslated regions (UTRs) and polyA sequences. The IVT reaction was performed according to a standard protocol, but typical UTP was replaced with N1-methylpseudouridine-5'-triphosphate. Finally, the mRNA was capped (Cap-1) with vaccinia capping enzyme and 2'-O-methyltransferase (NEB). Table 4 shows the primers used in this specification.

[0237] (Table 4) Length of the PCR product and primers containing its purpose TIFF2025108518000084.tif123156

[0238] The coding sequences of NLS-Cre and Cas9 are as follows. TIFF2025108518000085.tif150150TIFF2025108518000086.tif228150TIFF2025108518000087.tif82150

[0239] VIII. Western blot The quality of IVT Cas9 mRNA was analyzed by Western blot. One day before transfection, 293T cells were seeded at 1×10 5Seeded at cell / well density. With a total volume of 600 μL, the cells were further treated for 24 hours with various formulations containing mCherry mDLNP (0.5 μg mRNA / well), mCherry mDLNP (1.0 μg mRNA / well), IVT Cas9 mDLNP (0.5 μg mRNA / well), IVT Cas9 mDLNP (1.0 μg mRNA / well), and Lipofectamine 2000 / Cas9 pDNA (0.5 μg pDNA / well). After washing three times with 1×PBS, 100 μL of lysis buffer (50 mM Tris HCl, pH 7.4 containing 150 mM NaCl, 1 mM EDTA, and 1% TRITON X-100) and 1 μL of protein inhibitor cocktail (100×, Thermo Fisher) were added to each well and shaken at RT for 20 minutes. The cell lysates were collected and placed in 1.6 mL tubes and centrifuged at 4 °C for 10 minutes (13,000 g). The supernatant was collected and placed in a new tube and stored at -80 °C if not used immediately. Before performing Western blot, the protein concentration was measured using a BCA assay kit (ThermoFisher). 15 micrograms of total protein was loaded and separated on a 4–20% polyacrylamide gel (ThermoFisher). The separated proteins were then transferred to a polyvinylidene membrane (BioRad) and blocked with 5% BSA (dissolved in PBST) at RT for 1 hour. The primary antibody was applied overnight at 4 °C. After washing four times with PBST, the membrane was incubated with the secondary antibody at RT for 1 hour and then imaged using an ECL substrate and washed four times with PBST (ThermoFisher).

[0240] IX. Gene Editing (Cre mRNA) in the IX.Td-Tomato Mouse Model As described above, Cre mRNA formulations were prepared and IV injection (0.3 mg / kg Cre mRNA) was performed. Two days later, the mice (n = 4 per group) were sacrificed and the major organs were imaged by an IVIS Lumina system (Perkin Elmer).

[0241] X. Cell Isolation and Staining for Flow Cytometry Td-Tomato in cell types of each organ + To test the cells, they were treated with Cre mRNA preparation (0.3 mg / kg), and 2 days later, cell isolation and staining were performed, followed by analysis by flow cytometry.

[0242] For hepatocyte isolation, as previously described (Cheng et al., 2018), two-step collagenase perfusion was performed. Briefly, mice were anesthetized and fixed with isofluorane. Perfusion was initiated with liver perfusion medium (Thermo Fisher Scientific, 17701038) for 7 - 10 minutes and then switched to liver digestion medium (Thermo Fisher Scientific, 17703034) for an additional 7 - 10 minutes. The liver was collected in a plate containing 10 mL of liver digestion medium, cut, and dissociated to obtain hepatocytes. Then, the dissociated hepatocytes were collected and washed twice with hepatocyte wash medium (Thermo Fisher Scientific, 17704024) and once with 1×PBS. After further isolation by filtration and low-speed (50×g) centrifugation, the hepatocytes were analyzed using a FACS Aria II SORP machine (BD Biosciences).

[0243] For isolation and staining of spleen cell types, the excised spleen was minced finely with a sterile blade and placed into 250 μL of 1× digestion medium (45 units / μL collagenase I, 25 units / μL DNase I, and 30 units / μL hyaluronidase) and homogenized. The spleen solution was transferred to a 15 mL tube containing 5 - 10 mL of 1× digestion medium. Next, the spleen solution was filtered through a 70 μm filter and washed once with 1× PBS. A cell pellet was obtained by centrifuging at 300×g for 5 minutes. The supernatant was removed, and the cell pellet was resuspended in 2 mL of 1× RBC lysis buffer (BioLegend, 420301) and incubated on ice for 5 minutes. After incubation, 4 mL of cell staining buffer (BioLegend) was added to stop RBC lysis. The solution was centrifuged at 300×g for 5 minutes to obtain a cell pellet. Single cells were resuspended in cell staining buffer and added to a flow tube containing antibody (100 μL total volume). The cells were incubated with the antibody at 4 °C in the dark for 20 minutes. The stained cells were washed twice with 1 mL of 1× PBS and then resuspended in 500 μL of 1× PBS for flow cytometry analysis. The antibodies used were Pacific Blue anti-mouse CD45 (BioLegend, 103126), Alexa Fluor 488 anti-mouse / human CD11b (BioLegend, 101217), Alexa Fluor 647 anti-mouse CD19 (BioLegend, 115522), and PerCP-Cyanine5.5 anti-mouse CD3e (145-2C11) (Tonbo Biosciences, 65-0031). Live cells were identified using Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500).

[0244] For the isolation and staining of lung cell types, the isolated lungs were minced finely with a sterile blade and then transferred to a 15 mL tube containing 10 mL of 2× digestion medium (90 units / μL collagenase I, 50 units / μL DNase I, and 60 units / μL hyaluronidase), and incubated at 37 °C for 1 hour with shaking. After incubation, the remaining lung tissue was homogenized. The following steps were similar to the above spleen protocol. The antibodies used here were Pacific Blue anti-mouse CD45 (BioLegend, 103126), Alexa Fluor 488 anti-mouse CD31 (BioLegend, 102414), and Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (BioLegend, 118212). Live cells were identified using Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500).

[0245] XI. Gene Editing in XI.Td-Tomato Mouse Model (Cas9 mRNA / sgRNA and Cas9 / sgRNA RNP) To evaluate in vivo gene editing, Td-Tom mice of the same weight and sex were selected. Cas9 mRNA and sgRNA were co-delivered to tdTomato (td-Tom) mice. Cas9 mRNA / sgTom1 (4 / 1, wt / wt) was co-delivered by various formulations at a total RNA dose equal to 2.5 mg / kg. Major organs were harvested 10 days after IV injection and imaged by the IVIS Lumina system. For the spleen-targeted formulation, the total RNA dose was 4 mg / kg, the weight ratio of Cas9 mRNA to sgTom1 was 2 / 1, and the detection time was 2 days. For RNP delivery, the molar ratio of Cas9 protein to sgRNA was fixed at 1:3, the injection dose was 1.5 mg / kg RNA, and the detection time was 7 days after injection (n = 2 - 4 mice / group). To confirm Td-Tom expression, tissue sections were further prepared and imaged by confocal microscopy. Briefly, tissue blocks were embedded in optimal cutting temperature compound (OCT) (Sakura Finetek) and cryosectioned (8 μm) using a cryostat instrument (Leica Biosystems). Mounted tissue slices were stained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then imaged by confocal microscopy using a Zeiss LSM 700.

[0246] XII. Gene Editing in C57BL / 6 Mice (Cas9 mRNA / sgPTEN and Cas9 / sgRNA RNP) PTEN was selected to examine endogenous gene editing in vivo. Various carriers were IV injected (n = 2 - 4 mice / group) by co-delivering Cas9 mRNA and modified sgPTEN (4 / 1, mRNA / sgRNA, wt / wt) at a total dose of 2.5 mg / kg to wild-type C57BL / 6 mice. Tissues were collected after 10 days, and genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher). For spleen-targeted formulations, the total RNA dose was 4 mg / kg, Cas9 mRNA / sgTom1 was 2 / 1 (wt / wt), and the detection time was 2 days after injection. For RNP delivery, the molar ratio of Cas9 protein to sgRNA was fixed at 1:3, the injection dose was 1.5 mg / kg RNA, and the detection time was 7 days after injection (n = 2 - 4 mice / group). After obtaining the PTEN PCR product, the T7E1 assay (NEB) was performed to confirm gene editing efficacy by standard protocols. Furthermore, PTEN editing was evaluated on tissue sections by H&E staining and immunohistochemistry (IHC). Briefly, the Molecular Pathology Core at UTSW embedded paraformaldehyde (PFA)-fixed tissues in paraffin, sectioned them, and stained them with H&E. 4-μm sections were prepared by standard methods and detected using the Elite ABC Kit and DAB Substrate (Vector Laboratories) for IHC.

[0247] Example 9: Formulations Using Neutral Buffer Cas9 ribonucleoprotein (RNP) denatures in acidic buffer, and it was observed that its hydrodynamic size increases from 10 nm to 150 nm accordingly (Figure 40B). Therefore, encapsulation of RNP into monodisperse nanoparticles is difficult, if not impossible. These studies have focused on lipid nanoparticles (LNPs) because they are the most effective class of RNA delivery carriers in preclinical models and humans (Wood, 2018) (Wang et al., 2017; Doudna & Charpentier, 2014; Hajj & Whitehead, 2017; Sander & Joung, 2014). Among the four LNP components [cationic ionizable lipid, zwitterionic phospholipid, cholesterol, and poly(ethylene glycol) (PEG) lipid], the cationic ionizable lipid with a pK a of approximately 6.4 binds to negatively charged RNA at the mixing pH (e.g., pH 4, at which time the amine is protonated), loses its charge at neutral pH, is taken up by cells, and then regains its charge when the endosomal pH decreases to release the cargo into the cytoplasm, which is useful for activity because it fuses with the endosomal membrane. However, this feature hinders effective encapsulation of the cargo at neutral pH because the cationic ionizable lipid is uncharged at neutral pH. To overcome this challenge, adding a fifth component, specifically a cationic lipid that is positively charged at neutral pH, enables encapsulation of RNA and protein using neutral buffer (instead of acidic buffer), thus preserving the tertiary structure and stability of the RNP (Figure 40A).

[0248] To evaluate this strategy, 5A2-SC8 was selected as the cationic ionizable lipid because 5A2-SC8 LNPs can safely deliver short siRNA / miRNA and long mRNA to mice with liver cancer driven by MYC (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) or impaired liver function with gene knockout of fumarylacetoacetate hydrolase (FAH) (Cheng et al., 2018). Indeed, introduction of a permanent cationic lipid (e.g., DOTAP) into the conventional four-component 5A2-SC8 LNP formulation resulted in controlled self-assembly by mixing an ethanol solution of the lipid with a PBS solution of the RNP (1 / 3, v / v). Incorporation of 5 - 60 mol% DOTAP relative to total lipid was evaluated (Figure 41). From this, high levels of gene editing in vitro and formation of stable RNP-loaded nanoparticles with sizes <200 nm were revealed at 10 - 20% (Figure 42). First, the size of LNPs (5A2-DOT-10: 5A2-SC8 / DOPE / Chol / DMG-PEG / DOTAP = 15 / 15 / 30 / 3 / 7 (mol / mol)) with 10 mol% DOTAP incorporation, prepared using PBS buffer and using a reporter luciferase-targeted sgRNA (sgLuc), was observed. This was slightly larger than nanoparticles without RNP loading. The same LNPs prepared using a low pH buffer did not change in size, which means that the RNP was not encapsulated (Figure 40C). To determine the optimal molar ratio of Cas9 protein to sgRNA, Cas9 / sgRNA complexes at 1 / 1, 1 / 3, and 1 / 5 (mol / mol) were prepared. This decreased the size of the RNP (Figure 40D) and increased the negative charge (Figure 40F). This RNP ratio did not change the size or zeta potential of the resulting LNPs after encapsulation (Figure 1E, 1G). The surface charge of all LNPs was neutral, indicating that not only was encapsulation successful but it was also useful for minimizing in vivo uptake by the mononuclear phagocyte system (MPS).To further investigate whether 5A2-DOT-10 can successfully mediate the delivery of RNP to the nucleus, an LNP with encapsulated fluorescent EGFP-fused Cas9 protein was tracked. Since only free RNP could not be detected with green fluorescence above the background, it could not enter the cells (Figure 43). After treatment with 5A2-DOT-10 for 3 hours, bright green fluorescence was observed in the cytoplasm of the cells. Subsequently, since Cas9 has a nuclear localization signal, it was observed that the EGFP-fused Cas9 protein gradually entered the nucleus within 6 hours (Figure 40H). Endocytosis is energy-dependent and mainly depends on lipid rafts because treatment with MβCD, an inhibitor of lipid raft-based endocytosis, significantly inhibited the cellular uptake of nanoparticles (Figure 40I).

[0249] To quantify gene editing efficacy, HeLa-Luc and HeLa-GFP reporter cells were used. When various Cas9 / sgLuc ratios were examined, gene editing was high at 1 / 3 and 1 / 5 (Figure 44A). From the results of the T7 endonuclease I (T7EI) assay, it was demonstrated that most of the target DNA bands (720 bp) were cleaved into two cleavage bands (536 bp and 184 bp). No cleavage bands were observed in the control treatment group. To test the hypothesis that a neutral pH buffer is required to encapsulate RNP while protecting Cas9, the gene editing efficiency of 5A2-DOT-10 prepared using a pH 4 citrate buffer was also evaluated. No cleavage bands were observed at all (Figure 44A). Furthermore, negative results were confirmed by Sanger sequencing. This provided additional evidence that effective NPs do not occur with conventional acid-based formulation methods. When switched to GFP-expressing cells, 5A2-DOT-10 encapsulated Cas9 / sgGFP induced indels in GFP DNA and knocked out almost all GFP expression. The control group showed a fluorescence intensity similar to that of PBS-treated cells (Figure 44B). This was confirmed by flow cytometry (Figures 44C and 45). Permanent gene editing was revealed by the unclear disappearance of GFP in growing cells and confirmed by Sanger sequencing. From the Inference of CRISPR Edits (ICE) analysis, it was found that the indels reached 95% (Figure 44D). Looking towards clinical translation, the stability of 5A2-DOT-10 loaded with RNP w...

Claims

**Claim 1** (A) A therapeutic agent and (B) (1) A selective organ-targeting compound; (2) A cationic ionizable lipid; and (3) A phospholipid comprising a lipid nanoparticle composition and comprising a composition for preferentially delivering a nucleic acid to a target organ selected from the lungs, heart, brain, spleen, lymph nodes, bone marrow, bone, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, liver, testis, ovary, uterus, spleen, thymus, brain stem, cerebellum, spinal cord, eye, ear, tongue, or skin. **Claim 2** The composition according to claim 1, wherein the target organ is the lung, lymph node, or spleen. **Claim 3** The composition according to claim 1 or 2, wherein the selective organ-targeting compound is a permanently cationic lipid. **Claim 4** The composition according to claim 3, wherein the permanently cationic lipid is present at a molar percentage of about 5% to about 20% based on the lipid nanoparticle composition. **Claim 5** The composition according to any one of claims 1 to 4, wherein the permanently cationic lipid is present at a molar percentage of about 20% to about 65% based on the lipid nanoparticle composition. **Claim 6** The composition according to any one of claims 1 to 5, wherein the permanently cationic lipid contains a quaternary ammonium ion. **Claim 7** The permanently cationic lipid is further defined as wherein, in the formula The composition according to any one of claims 4 to 6. R 1 and R 2 are each independently alkyl (C8～C24) alkenyl (C8～C24) or a substituted form of any of the groups; R 3 、R 3 ', and R 3 '' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; X - is a monovalent anion, **Claim 8** The permanently cationic lipid is further defined as The composition according to claim 7. **Claim 9** The permanently cationic lipid is further defined as wherein, in the formula The composition according to any one of claims 4 to 6. **Claim 10** R 4 and R 4 ' are each independently alkyl (C6～C24) alkenyl (C6～C24) or a substituted form of any of the groups; R 4 '' is alkyl (C≦24) , alkenyl (C≦24) , or a substituted form of any group; R 4 is alkyl (C1～C8) alkenyl (C2～C8) or a substituted form of any group; and X 2 is a monovalent anion, The permanently cationic lipid is further defined as The composition according to claim 9. **Claim 11** The permanently cationic lipid is further defined as wherein, in the formula The composition according to any one of claims 4 to 6. **Claim 12** The permanently cationic lipid is further defined as R 1 and R 2 each independently is alkyl (C8～C24) alkenyl (C8～C24) or a substituted form of any of the groups; R 3 、 R 3 ', and R 3 '' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; R 4 is alkyl (C≦6) or substituted alkyl (C≦6) and X - is a monovalent anion, The composition according to claim 11. **Claim 13** The composition according to claim 1 or 2, wherein the selective organ-targeting compound is a permanently anionic lipid. **Claim 14** The composition according to claim 13, wherein the permanently anionic lipid is present at a molar percentage of about 5% to about 50% based on the lipid nanoparticle composition. **Claim 15** The composition according to claim 13 or 14, wherein the permanently anionic lipid contains a phosphate group. **Claim 16** The permanently anionic lipid is further defined as wherein, in the formula wherein, in the formula The composition according to any one of claims 13 to 15. **Claim 17** The permanently anionic lipid is further defined as R 1 and R 2 are each independently alkyl (C8～C24) alkenyl (C8～C24) or a substituted form of any group; R 3 is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) , or -Y 1 -R 4 and The composition according to claim 16. Y 1 is an alkanediyl (C≦6) or a substituted alkanediyl (C≦6) ; and R 4 is acyloxy (C≦8～24) or substituted acyloxy (C≦8～24) and **Claim 18** ​ ​ ​ ​ The selected organ-targeting compound is C 6 ~C 24 The composition according to claim 1 or 2, which is diacyl phosphotidylcholine.

19. The composition according to claim 18, wherein the diacylphosphatidylcholine is present in a molar percentage of about 5% to about 50% with respect to the lipid nanoparticle composition.

20. The composition according to claim 18 or 19, wherein the selectively organ-targeted compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group.

21. The diacylphosphatidylcholine is further defined as wherein R 1 and R 2 each independently is alkyl (C8～C24) alkenyl (C8～C24) or a substituted form of any group; R 3 and R 3 ', and R 3 '' are each independently alkyl (C≦6) or substituted alkyl (C≦6) ; and X - is a monovalent anion, The composition according to any one of claims 18 to 20.

22. The diacylphosphatidylcholine is further defined as, the composition according to claim 22.

23. The composition according to any one of claims 1 to 22, wherein the cationic ionizable lipid is present in a molar percentage of about 5% to about 30% with respect to the lipid nanoparticle composition.

24. The composition according to any one of claims 1 to 22, wherein the cationic ionizable lipid is present in a molar percentage of about 15% to about 30% with respect to the lipid nanoparticle composition.

25. The composition according to any one of claims 1 to 24, wherein the cationic ionizable lipid contains an ammonium group that is positively charged at physiological pH and contains at least two hydrophobic groups.

26. The cationic ionizable lipid is a dendrimer further defined by the following formula: Core - repeating unit - end group (I) or a pharmaceutically acceptable salt thereof, wherein the core is linked to the repeating unit by removing one or more hydrogen atoms from the core and substituting the atom with the repeating unit, and the core has the following formula: wherein a is 1, 2, 3, 4, 5, or 6; X 1 is amino or alkylamino (C≦12) , dialkylamino (C≦12) heterocycloalkyl (C≦12) heteroaryl (C≦12) or a substituted form thereof; R 1 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; and or alternatively the core has the following formula: wherein wherein X 2 is N(R 5 ) y and; R 5 is hydrogen, alkyl (C≦18) or substituted alkyl (C≦18) ; y is 0, 1, or 2 provided that the sum of y and z is 3; R 2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, or 3 provided that the sum of z and y is 3; or alternatively the core has the following formula: wherein X 3 is -NR 6 - and is wherein R 6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiylyl (C≦8) , arenediyl (C≦8) , heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) , or a substituted form of any of these groups; R 3 and R 4 each independently represents amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; or a group of the formula: -N(R f ) f (CH 2 CH 2 N) e (R c )R d ; wherein e and f are each independently 1, 2, or 3 provided that the sum of e and f is 3; R c 、R d 、and R f are each independently hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; c and d are each independently 1, 2, 3, 4, 5, or 6; or The core is an alkylamine (C≦18) , a dialkylamine (C≦36) , a heterocycloalkane (C≦12) , or a substituted form of any of these groups; the repeating unit contains a degradable diacyl and a linker; the degradable diacyl group has the following formula: wherein wherein A 1 and A 2 each independently represents -O- or -NR a - and wherein R a is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; Y 3 is an alkanediyl (C≦12) an alkenediyl (C≦12) an arenediyl (C≦12) or a substituted form of any of these groups; or the formula: is a group of wherein X 3 and X 4 is an alkanediyl (C≦12) an alkenediyl (C≦12) an arenediyl (C≦12) or a substituted form of any of these groups; Y 5 is a covalent bond, an alkanediyl (C≦12) , an alkenediyl (C≦12) , an arenediyl (C≦12) , or a substituted form of any of these groups; and R 9 is alkyl (C≦8) or substituted alkyl (C≦8) ; and the linker group has the following formula: wherein wherein Y 1 is an alkanediyl (C≦12) an alkenediyl (C≦12) an arenediyl (C≦12) or a substituted form of any of these groups; and When the repeating unit contains a linker group, if n is greater than 1, the linker group contains an independent degradable diacyl group bonded to both the nitrogen atom and the sulfur atom of the linker group, the first group in the repeating unit is a degradable diacyl group, and for each linker group, the adjacent repeating unit contains two degradable diacyl groups bonded to the nitrogen atom of the linker group; n is the number of linker groups present in the repeating unit; and The terminal group has the following formula: having wherein Y 4 is an alkanediyl (C≦18) or an alkanediyl (C≦18) wherein one or more of the hydrogen atoms on the alkanediyl is / are -OH, -F, -Cl, -Br, -I, -SH, -OCH 3 , -OCH 2 CH 3 , -SCH 3、 or -OC(O)CH 3 substituted alkanediyl (C≦18) ; R 10 is hydrogen, carboxy, hydroxy, or aryl (C≦12) , alkylamino (C≦12) , dialkylamino (C≦12) , N - heterocycloalkyl (C≦12) , -C(O)N(R 11 )-alkanediyl (C≦6) -heterocycloalkyl (C≦12) , -C(O)-alkylamino (C≦12) , -C(O)-dialkylamino (C≦12) , -C(O)-N - heterocycloalkyl (C≦12) and wherein R 11 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; the last degradable diacyl in the chain is bonded to the terminal group; n is 0, 1, 2, 3, 4, 5, or 6, The composition according to any one of claims 1 to 25.

27. The terminal group is further defined by the following formula: and Y 4 is an alkanediyl (C≦18) and; and R 10 wherein R is hydrogen The composition according to claim 26.

28. The core is further defined by the following formula: and wherein X 2 is N(R 5 ) y and; R 5 is hydrogen, or alkyl (C≦8) or substituted alkyl (C≦18) and y is 0, 1, or 2, provided that the sum of y and z is 3; R 2 is amino, hydroxy, or mercapto, or alkylamino (C≦12) or dialkylamino (C≦12) or a substituted form of any of these groups; b is 1, 2, 3, 4, 5, or 6; and z is 1, 2, 3, provided that the sum of z and y is 3, The composition according to claim 26 or 27.

29. The core is further defined by the following formula: and wherein X 3 is -NR 6 - and is wherein R 6 is hydrogen, alkyl (C≦8) , or substituted alkyl (C≦8) , -O-, or alkylaminodiyl (C≦8) , alkoxydiylyl (C≦8) , arenediyl (C≦8) , heteroarenediyl (C≦8) , heterocycloalkanediyl (C≦8) , or a substituted form of any of these groups; R 3 and R 4 are each independently amino, hydroxy, or mercapto, or alkylamino (C≦12) , dialkylamino (C≦12) , or a substituted form of any of these groups; or the formula: -N(R f ) f (CH 2 CH 2 N) e (R c )R d wherein; wherein e and f are each independently 1, 2, or 3, provided that the sum of e and f is 3; or R c 、R d 、and R f are each independently hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) ; c and d are each independently 1, 2, 3, 4, 5, or 6, The composition according to claim 26 or 27.

30. The core is The composition according to any one of claims 26 to 29, further defined as

31. The dendrimer is or or a pharmaceutically acceptable salt thereof, and the composition according to any one of claims 26 to 30 is further defined as

32. The composition according to any one of claims 1 to 31, wherein the phospholipid is present in a molar percentage of about 8% to about 20% with respect to the lipid nanoparticle composition.

33. The composition according to any one of claims 1 to 31, wherein the phospholipid is present in a molar percentage of about 20% to about 23% with respect to the lipid nanoparticle composition.

34. The composition according to any one of claims 1 to 33, further comprising a steroid.

35. The composition according to claim 34, wherein the steroid is present in a molar percentage of about 39% to about 46% with respect to the lipid nanoparticle composition.

36. The composition according to claim 35, wherein the steroid is present in a molar percentage of about 15% to about 39% with respect to the lipid nanoparticle composition.

37. The composition according to any one of claims 1 to 36, further comprising a PEGylated lipid.

38. The composition according to claim 37, wherein the PEGylated lipid is present at a molar percentage of about 0.5% to about 10.0% based on the lipid nanoparticle composition.

39. The PEG lipid is further defined by the following formula: wherein in the formula, R 12 and R 13 are each independently alkyl (C≦24) alkenyl (C≦24) or a substituted form of any of these groups; R e is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8) ; and x is from 1 to 250, The composition according to claim 37 or 38.

40. The PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula: and in the formula, n 1 is from 5 to 250; and n 2 and n 3 each independently is from 2 to 25, The composition according to claim 37 or 38.

41. The composition according to any one of claims 1 to 40, wherein the therapeutic agent is a nucleic acid.

42. The composition according to claim 41, wherein the nucleic acid is a therapeutic nucleic acid.

43. The nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-associated nucleic acid, single guide RNA (sgRNA), CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA), The composition according to claim 41.

44. The composition according to any one of claims 41 to 43, wherein the nucleic acid is present at a ratio of nucleic acid to lipid nanoparticle composition of about 1:1 to about 1:

100.

45. The composition according to any one of claims 1 to 44, further comprising a protein.

46. The composition according to any one of claims 1 to 45, wherein the protein and the nucleic acid are present at a molar ratio of about 1:1 to about 1:

20.

47. The composition according to any one of claims 1 to 46, comprising both a protein and a nucleic acid.

48. (A) The composition according to any one of claims 1 to 47, and (B) an excipient A pharmaceutical composition comprising.

49. A method for regulating gene expression, comprising the step of delivering a nucleic acid to a cell, The method, comprising the step of contacting the cell with the composition according to any one of claims 1 to 48 or the pharmaceutical composition under conditions sufficient to cause uptake of the nucleic acid into the cell.

50. A method of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a pharmaceutically effective amount of a composition or pharmaceutical composition according to any one of claims 1 to 48, wherein the composition or pharmaceutical composition comprises a therapeutic nucleic acid for the disease or disorder, said method. **Claim 51** The method according to claim 50, wherein the disease or disorder is cancer. **Claim 52** A method of preparing lipid nanoparticles, the method comprising the following steps: (A) Dissolving a permanent cationic lipid, a cationic ionizable lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent; (B) Dissolving a therapeutic agent in a buffer to form a buffered therapeutic agent solution, wherein the buffer is a buffer having a pH of about 6.8 to about 7.6; and (C) Mixing the lipid solution with the buffered therapeutic agent solution to form lipid nanoparticles.

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