Compositions and methods for organ-specific delivery of nucleic acids

Abstract

The present disclosure provides compositions that exhibit preferential targeting or delivery of nucleic acid compositions to specific organs. In some embodiments, the compositions comprise steroids or sterols, ionizable cationic lipids, phospholipids, PEG lipids, and permanent cationic lipids, which can be used to deliver nucleic acids. The disclosure also provides pharmaceutical compositions containing the compositions, methods of modulating gene expression and methods of treating diseases using the compositions, and methods of preparing related lipid nanoparticles.

Description

[0001] This application is a divisional application of a Chinese patent application with application number 201980071318.X, filing date September 4, 2019, and invention title "Compositions and Methods for Organ-Specific Delivery of Nucleic Acids".

[0002] This application claims the priority benefit of U.S. Provisional Application Serial No. 62 / 726,770, filed September 4, 2018, the entire content of which is hereby incorporated by reference. Technical Field

[0003] The present disclosure generally relates to the field of molecular biology. More specifically, it relates to the tissue-specific delivery of therapeutic agents such as nucleic acids, proteins, or small molecule therapeutic agents in lipid nanoparticles. Background Art

[0004] CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (Cas)) technology can edit the genome in a precise, sequence-dependent manner, resulting in permanent changes. Due to the ability to target disease-causing mutations, it has the incredible promise of curing genetic diseases once and many other applications in different fields. To date, successful editing has been mainly mediated by viral vectors, which require laborious customization for each target and pose challenges to clinical translation due to immunogenicity, the production of antibodies that prevent repeated administration, and concerns about rare but dangerous integration events. There is clearly still a need to accomplish CRISPR / Cas editing via synthetic nanoparticles (NPs) to expand the safe and effective applications of gene editing.

[0005] CRISPR / Cas achieves sequence-specific DNA editing through RNA-guided CRISPR-associated protein 9 (Cas9) nuclease or its homologs that form double-stranded breaks (DSBs) in genomic DNA and related CRISPR-associated (CAS) proteins. Cas9 is guided by a programmable RNA called single-guide RNA (sgRNA). The Cas9 / sgRNA complex recognizes complementary genomic sequences with a 3′ protospacer adjacent motif (PAM) sequence. After DNA cleavage, DSB repair pathways effect targeted mutagenesis, or insertions / deletions (indels) that disrupt the target gene. When delivered with donor DNA, cells can utilize homology-directed repair (HDR) to correct gene mutations. For therapeutic utility, transient Cas9 expression is preferred to limit off-target genomic alterations. Since both the Cas9 protein and sgRNA must be present in the same cell, co-delivery of Cas9 mRNA and the sgRNA for the target sequence in a NP is an attractive approach, especially for in vivo use where tissue penetration and cellular uptake are more challenging. Similarly, direct delivery of sgRNA Cas9 protein ribonucleoprotein (RNP) is also attractive for gene editing. CRISPR / Cas editing using viral, membrane-disrupting, and hydrodynamic injection is functional but has limitations that impede in vivo therapeutic use clinically, including persistent expression of Cas9 and off-target editing. Additionally, these delivery systems are generally not selective for the specific organ in which editing is desired. For example, most lipid nanoparticles accumulate through biological processes in the liver, reducing the efficacy of the composition for delivery into the target organ.

[0006] Similarly, other therapeutic agents such as proteins and small molecule therapeutics can benefit from organ-specific delivery. Many different types of compounds such as chemotherapeutic agents exhibit significant cytotoxicity. If these compounds could be better targeted to the desired organ, fewer off-target effects would be seen.

[0007] Accordingly, there remains a need to develop new lipid nanoparticles that exhibit preferential delivery to a specific organ. SUMMARY OF THE INVENTION

[0008] In some aspects, the present disclosure provides lipid compositions that exhibit organ-specific delivery of the lipid composition. These compositions can be used to deliver nucleic acid components to a specific organ.

[0009] In some aspects, the present disclosure provides a composition comprising:

[0010] (A) a therapeutic agent; and

[0011] (B) a lipid nanoparticle composition comprising:

[0012] (1) Permanent cationic lipid;

[0013] (2) Ionizable cationic lipid; and

[0014] (3) Phospholipid;

[0015] Wherein the composition preferentially delivers nucleic acids to target organs selected from: lung, heart, brain, spleen, lymph node, bone, bone marrow, 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. In some embodiments, the target organs are selected from lung, heart, brain, spleen, lymph node, bone, bone marrow, skeletal muscle, stomach, small intestine, large intestine, kidney, bladder, breast, testis, ovary, uterus, spleen, thymus, brain stem, cerebellum, spinal cord, eye, ear, tongue or skin.

[0016] 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.

[0017] In some embodiments, the selective organ-targeting compound is a permanent cationic lipid. In some embodiments, the permanent cationic lipid is present in a molar percentage of the lipid nanoparticle composition of from about 5% to about 20%. In some embodiments, the molar percentage of the permanent cationic lipid is from about 12% to about 18%. In some embodiments, the molar percentage of the permanent cationic lipid is about 15%. In some embodiments, the permanent cationic lipid is present in a molar percentage of the lipid nanoparticle composition of from about 20% to about 65%. In some embodiments, the molar percentage of the permanent cationic lipid is from about 40% to about 61%. In some embodiments, the molar percentage of the permanent cationic lipid is about 50%.

[0018] In some embodiments, the permanent cationic lipid contains quaternary ammonium ions. In some embodiments, the permanent cationic lipid is further defined as:

[0019]

[0020] Wherein:

[0021] R1 and R2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;

[0022] R3, R3′ and R3″ are each independently an alkyl (C≤6) or a substituted alkyl (C≤6) ;

[0023] X - is a monovalent anion.

[0024] In some embodiments, R1 is an alkenyl (C8-C24) or a substituted alkenyl (C8-C24) . In some embodiments, R2 is an alkenyl (C8-C24) or a substituted alkenyl (C8-C24) . In other embodiments, R1 is an alkyl (C8-C24) or a substituted alkyl (C8-C24) . In other embodiments, R2 is an alkyl (C8-C24) or a substituted alkyl (C8-C24) . In some embodiments, both R1 and R2 are the same. In some embodiments, each of R3, R3′, and R3″ is the same. In some embodiments, each of R3, R3′, and R3″ is methyl. In some embodiments, X - is a halide anion such as bromide or chloride. In some embodiments, the permanent cationic lipid is further defined as:

[0025]

[0026] In other embodiments, the permanent cationic lipid is further defined as:

[0027]

[0028] Wherein:

[0029] R4 and R4′ are each independently an alkyl (C6-C24) , an alkenyl (C6-C24) or a substituted form of either group;

[0030] R4″ is an alkyl (C≤24) , an alkenyl (C≤24) or a substituted form of either group;

[0031] R4″′ is an alkyl (C1-C8) , an alkenyl (C2-C8) or a substituted form of either group; and

[0032] X2 is a monovalent anion.

[0033] In some embodiments, R4 is an alkyl (C6-C24) or a substituted alkyl (C6-C24) such as octadecyl. In some embodiments, R4′ is an alkyl (C6-C24) or a substituted alkyl (C6-C24) such as octadecyl. In some embodiments, R4″ is an alkyl (C≤24) or a substituted alkyl(C≤24) In some embodiments, R4″ is an alkyl group (C≤8) or a substituted alkyl group (C≤8) such as methyl. In some embodiments, R4″′ is an alkyl group (C1-C8) or a substituted alkyl group (C1-C8) such as methyl. In some embodiments, X2 is a halide such as chloride or bromide. In some embodiments, the permanent cationic lipid is further defined as:

[0034]

[0035] In some embodiments, the permanent cationic lipid is further defined as:

[0036]

[0037] wherein:

[0038] R1 and R2 are each independently an alkyl group (C8-C24) , an alkenyl group (C8-C24) or a substituted form of either group;

[0039] R3, R3′ and R3″ are each independently an alkyl group (C≤6) or a substituted alkyl group (C≤6) ;

[0040] R4 is an alkyl group (C≤6) or a substituted alkyl group (C≤6) ; and

[0041] X - is a monovalent anion.

[0042] In some embodiments, R1 is an alkenyl group (C8-C24) or a substituted alkenyl group (C8-C24) . In some embodiments, R2 is an alkenyl group (C8-C24) or a substituted alkenyl group (C8-C24) . In other embodiments, R1 is an alkyl group (C8-C24) or a substituted alkyl group (C8-C24) . In other embodiments, R2 is an alkyl group (C8-C24) or a substituted alkyl group (C8-C24) . In some embodiments, both R1 and R2 are the same.

[0043] In some embodiments, R3, R3′ and R3″ are each the same, such as R3, R3′ and R3″ are each methyl. In some embodiments, R4 is an alkyl group (C≤6) such as ethyl. In some embodiments, X - is a halide anion such as bromide or chloride.

[0044] In some embodiments, the permanent cationic lipid is further defined as:

[0045]

[0046] In other embodiments, the selective organ targeting compound is a permanent anionic lipid. In some embodiments, the permanent anionic lipid is present in a molar percentage of the lipid nanoparticle composition of from about 5% to about 50%. In some embodiments, the molar percentage of the permanent anionic lipid is from about 10% to about 45%. In some embodiments, the molar percentage of the permanent anionic lipid is about 30%. In some embodiments, the permanent anionic lipid comprises a phosphate group.

[0047] In some embodiments, the permanent anionic lipid is further defined as:

[0048]

[0049] Wherein:

[0050] R1 and R2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;

[0051] R3 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) or -Y1-R4, wherein:

[0052] Y1 is an alkanediyl (C≤6) or a substituted alkanediyl (C≤6) ; and

[0053] R4 is an acyloxy (C≤8-24) or a substituted acyloxy (C≤8-24) .

[0054] In some embodiments, R1 is alkenyl (C8-C24) or substituted alkenyl (C8-C24) . In other 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.

[0055] In some embodiments, R3 is hydrogen. In other embodiments, R3 is -Y1-R4, wherein:

[0056] Y1 is an alkanediyl (C≤6) or a substituted alkanediyl (C≤6) ; and

[0057] R4 is an acyloxy (C≤8-24) or a substituted acyloxy (C≤8-24) .

[0058] In some embodiments, Y1 is a substituted alkanediyl (C≤6) such as 2-hydroxypropanediyl. In some embodiments, R4 is an acyloxy (C≤8-24) such as octadecenoate. In some embodiments, the permanent anionic lipid is further defined as:

[0059]

[0060] In other embodiments, the selective organ targeting compound is a C6-C 24 diacylphosphatidylcholine. In some embodiments, the diacylphosphatidylcholine is present in a molar percentage of from about 5% to about 50% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the diacylphosphatidylcholine is from about 10% to about 45% such as about 30%.

[0061] In some embodiments, the selective organ targeting compound comprises at least two fatty acid chains, a quaternary amine, and an anionic phosphate group. In some embodiments, the diacylphosphatidylcholine is further defined as:

[0062]

[0063] Wherein:

[0064] R1 and R2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;

[0065] R3, R3′, and R3″ are each independently an alkyl (C≤6) or a substituted alkyl (C≤6) ; and

[0066] X - is a monovalent anion.

[0067] In some embodiments, R1 is an alkenyl (C8-C24) or a substituted alkenyl (C8-C24) . In some embodiments, R2 is an alkenyl (C8-C24) or a substituted alkenyl (C8-C24) . In other embodiments, R1 is an alkyl (C8-C24) or a substituted alkyl (C8-C24)。In other embodiments, R2 is an alkyl group (C8-C24) or a substituted alkyl group (C8-C24) 。In some embodiments, both R1 and R2 are the same.

[0068] 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 diacyl phosphatidylcholine is further defined as:

[0069]

[0070] In some embodiments, the ionizable cationic lipid is present in the lipid nanoparticle composition at a molar percentage of about 5% to about 30%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 7.5% to about 20%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 11.9%. In some embodiments, the ionizable cationic lipid is present in the lipid nanoparticle composition at a molar percentage of about 15% to about 30%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 15% to about 25%. In some embodiments, the molar percentage of the ionizable cationic lipid is about 20.3%.

[0071] In some embodiments, the ionizable cationic 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 ionizable cationic lipid is a dendrimer or a dendron. In some embodiments, the ionizable cationic lipid contains at least two C6-C24 alkyl or alkenyl groups. In some embodiments, the ionizable cationic lipid contains at least two C8-C24 alkyl groups. In some embodiments, the ionizable cationic lipid is a dendrimer further defined by the following formula:

[0072] Core - repeating unit - capping group (I)

[0073] wherein the core is connected to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeating unit, and wherein:

[0074] the core has the following formula:

[0075]

[0076] wherein:

[0077] X1 is amino or alkylamino (C≤12) , dialkylamino (C≤12) , heterocycloalkyl (C≤12) , heteroaryl (C≤12) or a substituted form thereof;

[0078] R1 is amino, hydroxy or mercapto or alkylamino (C≤12) , dialkylamino (C≤12) or a substituted form of any of these groups; and

[0079] a is 1, 2, 3, 4, 5 or 6; or

[0080] the core has the following formula:

[0081]

[0082] wherein:

[0083] X2 is N(R5) y ;

[0084] R5 is hydrogen, alkyl (C≤18) or substituted alkyl (C≤18) ; and

[0085] y is 0, 1 or 2, provided that the sum of y and z is 3;

[0086] R2 is amino, hydroxy or mercapto or alkylamino (C≤12) , dialkylamino (C≤12) or a substituted form of any of these groups;

[0087] b is 1, 2, 3, 4, 5 or 6; and

[0088] z is 1, 2, 3; provided that the sum of z and y is 3; or

[0089] the core has the following formula:

[0090]

[0091] wherein:

[0092] X3 is -NR6-, where 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;

[0093] R3 and R4 are each independently an amino group, a hydroxyl group, a mercapto group, or an alkylamino group (C≤12) , a dialkylamino group (C≤12) or a substituted form of any of these groups; or a group of the formula: -N(R f ) f (CH2CH2N) e (R c )R d ;

[0094] wherein:

[0095] e and f are each independently 1, 2, or 3; provided that the sum of e and f is 3;

[0096] R c 、R d and R f are each independently hydrogen, an alkyl group (C≤6) or a substituted alkyl group (C≤6) ;

[0097] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[0098] the core is an alkylamine (C≤18) 、a dialkylamine (C≤36) 、a heterocycloalkane (C≤12) or a substituted form of any of these groups;

[0099] wherein the repeating unit contains a degradable diacyl group and a linker;

[0100] the degradable diacyl group has the formula:

[0101]

[0102] wherein:

[0103] A1 and A2 are each independently -O- or -NR a -, wherein:

[0104] R a is hydrogen, an alkyl group (C≤6) or a substituted alkyl group (C≤6) ;

[0105] Y3 is an alkanediyl group (C≤12) 、an alkenediyl group (C≤12) 、an arenediyl group (C≤12) or a substituted form of any of these groups; or a group of the formula:

[0106]

[0107] wherein:

[0108] X3 and X4 are alkanediyl (C≤12) , alkenediyl (C≤12) , arenediyl (C≤12) or a substituted form of any of these groups;

[0109] Y5 is a covalent bond, alkanediyl (C≤12) , alkenediyl (C≤12) , arenediyl (C≤12) or a substituted form of any of these groups; and

[0110] R9 is alkyl (C≤8) or substituted alkyl (C≤8) ;

[0111] The linker group has the following formula:

[0112]

[0113] Wherein:

[0114] Y1 is alkanediyl (C≤12) , alkenediyl (C≤12) , arenediyl (C≤12) or a substituted form of any of these groups; and

[0115] When the repeating unit contains a linker group, if n is greater than 1, the linker group contains an independent degradable diacyl group that is connected to both the nitrogen and sulfur atoms of the linker group, wherein the first group in the repeating unit is a degradable diacyl group, and for each linker group, the next repeating unit contains two degradable diacyl groups that are connected to the nitrogen atoms of the linker group; and wherein n is the number of linker groups present in the repeating unit; and

[0116] The capping group has the following formula:

[0117]

[0118] Wherein:

[0119] Y4 is alkanediyl (C≤18) or alkanediyl (C≤18) where one or more hydrogen atoms on the alkanediyl (C≤18) have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3;

[0120] R 10 is hydrogen, carboxyl, hydroxyl, or

[0121] 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:

[0122] R 11 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) ;

[0123] wherein the final degradable diacyl in the chain is linked to a capping group;

[0124] n is 0, 1, 2, 3, 4, 5 or 6;

[0125] or a pharmaceutically acceptable salt thereof. In some embodiments, the capping group is further defined by the formula:

[0126]

[0127] wherein:

[0128] Y4 is alkanediyl (C≤18) ; and

[0129] R 10 is hydrogen.

[0130] In some embodiments, the core is further defined by the formula:

[0131]

[0132] wherein:

[0133] X2 is N(R5) y ;

[0134] R5 is hydrogen or alkyl (C≤8) or substituted alkyl (C≤18) ; and

[0135] y is 0, 1 or 2, provided that the sum of y and z is 3;

[0136] R2 is amino, hydroxy, mercapto or alkylamino (C≤12) , dialkylamino (C≤12) or a substituted form of any of these groups;

[0137] b is 1, 2, 3, 4, 5 or 6; and

[0138] z is 1, 2, 3; provided that the sum of z and y is 3.

[0139] In some embodiments, the core is further defined by the following formula:

[0140]

[0141] Where:

[0142] X3 is -NR6-, where R6 is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) , -O- or alkylamino diyl (C≤8) , alkoxy diyl (C≤8) , arene diyl (C≤8) , heteroarene diyl (C≤8) , heterocycloalkane diyl (C≤8) or a substituted form of any of these groups;

[0143] 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 a group of the following formula: -N(R f ) f (CH2CH2N) e (R c )R d ;

[0144] Where:

[0145] e and f are each independently 1, 2 or 3; provided that the sum of e and f is 3;

[0146] R c , R d and R f are each independently hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) ;

[0147] c and d are each independently 1, 2, 3, 4, 5 or 6.

[0148] In some embodiments, the core is further defined as:

[0149]

[0150]

[0151] In some embodiments, the degradable diacyl is further defined as:

[0152] In some embodiments, the linker is further defined as:

[0153]

[0154] Wherein:

[0155] Y1 is an alkanediyl (C≤8) or a substituted alkanediyl (C≤8) .

[0156] In some embodiments, the dendrimer is further defined as:

[0157]

[0158]

[0159]

[0160] or a pharmaceutically acceptable salt thereof.

[0161] In some embodiments, the phospholipid is present in the lipid nanoparticle composition at a molar percentage of about 8% to about 20%. 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 in the lipid nanoparticle composition at a molar percentage of about 20% to about 23%. In some embodiments, the molar percentage of the phospholipid is about 20% to about 21%. 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.

[0162] In some embodiments, the composition further comprises a steroid. In some embodiments, the steroid is present in the lipid nanoparticle composition at a molar percentage of about 39% to about 46%. 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 in the lipid nanoparticle composition at a molar percentage of about 15% to about 39%. In some embodiments, the molar percentage of the steroid is about 20% to about 27.5%. In some embodiments, the molar percentage of the steroid is about 23.8%. In some embodiments, the steroid is cholesterol.

[0163] In some embodiments, the composition further comprises a PEGylated lipid. In some embodiments, the PEGylated lipid is present at a molar percentage of from about 0.5% to about 10.0% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the PEGylated lipid is from about 2.0% to about 2.8%. In some embodiments, the molar percentage of the PEGylated lipid is about 2.4%. In other embodiments, the PEGylated lipid is present at a molar percentage of from about 3.9% to about 4.6% of the lipid nanoparticle composition. In some embodiments, the molar percentage of the PEGylated lipid is from about 4.0% to about 4.3%. 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 from about 1000 to about 10,000 Daltons. In some embodiments, the PEG lipid is a PEGylated diacylglycerol. In some embodiments, the PEG lipid is further defined by the following formula:

[0164]

[0165] Wherein:

[0166] R 12 and R 13 are each independently an alkyl (C≤24) an alkenyl (C≤24) or a substituted form of any of these groups;

[0167] R e is hydrogen, an alkyl (C≤8) or a substituted alkyl (C≤8) ; and

[0168] x is 1 - 250.

[0169] In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula:

[0170]

[0171] Wherein:

[0172] n1 is 5 - 250; and

[0173] n2 and n3 are each independently 2 - 25.

[0174] 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 with five tails or 5A2-SC8 with 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.

[0175] In some embodiments, the therapeutic agent is a small molecule such as a small molecule selected from: anticancer agents, antifungal agents, psychiatric agents such as analgesics, level-of-consciousness-altering agents such as anesthetics or hypnotics, non-steroidal anti-inflammatory drugs (NSAIDs), anthelmintics, anti-acne agents, anti-anginal agents, anti-arrhythmic agents, anti-asthma agents, antibacterial agents, anti-benign prostatic hyperplasia agents, anticoagulants, antidepressants, anti-diabetic agents, anti-emetics, anti-epileptic agents, anti-gout agents, anti-hypertensive agents, anti-inflammatory agents, antimalarial agents, anti-migraine agents, antimuscarinic agents, anti-tumor agents, anti-obesity agents, anti-osteoporosis agents, anti-Parkinson's syndrome agents, anti-proliferative agents, anti-protozoal agents, anti-thyroid agents, antitussives, anti-incontinence agents, antiviral agents, anti-anxiety agents, appetite suppressants, beta-blockers, cardiac inotropes, chemotherapeutic drugs, cognitive enhancers, contraceptives, corticosteroids, Cox-2 inhibitors, diuretics, erectile dysfunction improvers, expectorants, gastrointestinal agents, histamine receptor antagonists, immunosuppressants, keratolytics, lipid regulators, leukotriene inhibitors, macrolides, muscle relaxants, neuroleptics, nutritional agents, narcotic analgesics, protease inhibitors or sedatives. 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, primary-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (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 in a lipid nanoparticle composition:nucleic acid ratio of about 1:1 to about 1:100. In some embodiments, the ratio is about 1:10 to about 1:60. In some embodiments, the ratio is about 1:40.

[0176] 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, homologs thereof, or modified forms thereof. In some embodiments, the protein is Cas9. In some embodiments, the protein and the nucleic acid are present in a molar ratio of about 1:1 to about 1:20. In some embodiments, the molar ratio is about 1:1 to about 1:10. In some embodiments, the molar ratio is about 1:3 to about 1:8.

[0177] In some embodiments, the composition has a negative ζ potential. In some embodiments, the ζ potential is from -0.25 mV to about -10 mV. In some embodiments, the ζ potential is about -0.5 mV to about -2 mV. In some embodiments, the composition comprises both a protein and a nucleic acid. In some embodiments, the composition comprises a Cas9 protein and a single-guide nucleic acid. In some embodiments, the composition comprises a Cas9 protein, a single-guide nucleic acid, and donor DNA.

[0178] In another aspect, the present disclosure provides a pharmaceutical composition comprising:

[0179] (A) the composition described herein; and

[0180] (B) an excipient.

[0181] In some embodiments, the pharmaceutical composition is formulated for administration: orally, intralipidly, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularly, pericardially, intraperitoneally, intrapleurally, intraprostatically, rectally, intrathecally, intratracheally, intratumorally, intraumbilically, vaginally, intravenously, intravesically, intravitreally, in liposomal form, topically, mucosally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, superficially topically, buccally, transdermally, vaginally, in the form of crèmes, in lipid composition form, via a catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via local perfusion. In some embodiments, the pharmaceutical composition is formulated for intravenous or intraarterial injection. In some embodiments, the excipient is a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a solvent or a solution. In some embodiments, the pharmaceutical composition is formulated as a unit dose.

[0182] In another aspect, the present disclosure provides a method of modulating gene expression that includes delivering a nucleic acid to a cell, the method comprising: contacting the cell with a composition or pharmaceutical composition described herein under conditions sufficient to effect uptake of the nucleic acid into the cell.

[0183] 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 modulation of gene expression is sufficient to treat a disease or disorder such as cancer.

[0184] In yet another aspect, the present disclosure provides a method of treating a disease or disorder in a patient, the method comprising administering to a patient in need thereof 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.

[0185] 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.

[0186] In other aspects, the present disclosure provides a method of preparing lipid nanoparticles, which comprises:

[0187] (A) Dissolve a permanent cationic lipid, an ionizable cationic lipid, and a phospholipid in a first solution to form a lipid solution, wherein the lipid solution is formed in an organic solvent;

[0188] (B) Dissolve a therapeutic agent in a buffer, wherein the buffer is a buffer having a pH of about 6.8 to about 7.6 to form a buffered therapeutic agent solution; and

[0189] (C) Mix the lipid solution with the buffered therapeutic agent solution to form lipid nanoparticles.

[0190] 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 method has an encapsulation efficiency greater than 80%.

[0191] In another aspect, the present disclosure provides a composition comprising:

[0192] (A) A therapeutic agent;

[0193] (B) A lipid nanoparticle composition comprising:

[0194] (1) An ionizable cationic lipid;

[0195] (2) A phospholipid; and

[0196] (3) A selective organ targeting compound;

[0197] wherein the organ targeting ligand causes preferential delivery of the composition to organs other than the liver.

[0198] In yet another aspect, the present disclosure provides a composition comprising:

[0199] (A) A therapeutic agent;

[0200] (B) A lipid nanoparticle composition comprising:

[0201] (1) An ionizable cationic lipid;

[0202] (2) A phospholipid;

[0203] (3) A selective organ targeting compound;

[0204] (4) A steroid; and

[0205] (5) A PEG lipid;

[0206] wherein the organ targeting ligand causes preferential delivery of the composition to organs other than the liver.

[0207] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0208] (A) an ionizable cationic lipid;

[0209] (B) a phospholipid; and

[0210] (C) a selective organ targeting compound;

[0211] wherein the composition has an apparent pK of about 8 to about 13 a and the composition predominantly delivers nucleic acids to the lung.

[0212] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0213] (A) an ionizable cationic lipid;

[0214] (B) a phospholipid;

[0215] (C) a selective organ targeting compound;

[0216] (D) a steroid; and

[0217] (E) a PEG lipid;

[0218] wherein the composition has an apparent pK of about 8 to about 13 a and the composition predominantly delivers nucleic acids to the lung.

[0219] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0220] (A) an ionizable cationic lipid;

[0221] (B) a phospholipid; and

[0222] (C) a selective organ targeting compound;

[0223] wherein the composition has an apparent pK of about 3 to about 6 a and the composition predominantly delivers nucleic acids to the spleen.

[0224] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0225] (A) a steroid;

[0226] (B) an ionizable cationic lipid;

[0227] (C) Phospholipids;

[0228] (D) PEG lipids; and

[0229] (E) Selective organ targeting compounds;

[0230] wherein the composition has an apparent pK of about 3 to about 6 a and the composition primarily delivers nucleic acids to the spleen.

[0231] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0232] (A) Ionizable cationic lipid;

[0233] (B) Phospholipids; and

[0234] (C) C6-C 24 diacyl phosphatidylcholine;

[0235] wherein the composition primarily delivers nucleic acids to the lymph nodes.

[0236] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0237] (A) Steroid;

[0238] (B) Ionizable cationic lipid;

[0239] (C) Phospholipids;

[0240] (D) PEG lipids; and

[0241] (E) C6-C 24 diacyl phosphatidylcholine;

[0242] wherein the composition primarily delivers nucleic acids to the lymph nodes.

[0243] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0244] (A) Ionizable cationic lipid;

[0245] (B) Phospholipids; and

[0246] (C) Selective organ targeting compounds;

[0247] wherein the surface of the composition interacts with vitronectin and the composition primarily delivers nucleic acids to the lungs.

[0248] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0249] (A) an ionizable cationic lipid;

[0250] (B) a phospholipid;

[0251] (C) a selective organ targeting compound;

[0252] (D) a steroid; and

[0253] (E) a PEG lipid;

[0254] wherein the surface of the composition interacts with vitronectin and the composition predominantly delivers nucleic acids to the lung.

[0255] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0256] (A) an ionizable cationic lipid;

[0257] (B) a phospholipid; and

[0258] (C) a selective organ targeting compound;

[0259] wherein the surface of the composition interacts with Apo H and the composition predominantly delivers nucleic acids to the spleen.

[0260] In another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, the lipid nanoparticle composition comprising:

[0261] (A) a steroid;

[0262] (B) an ionizable cationic lipid;

[0263] (C) a phospholipid;

[0264] (D) a PEG lipid; and

[0265] (E) a selective organ targeting compound;

[0266] wherein the surface of the composition interacts with Apo H and the composition predominantly delivers nucleic acids to the spleen.

[0267] In yet another aspect, the present disclosure provides a composition comprising a therapeutic agent and a lipid nanoparticle composition, wherein the targeting protein binding in the protein corona present at the surface of the composition binds predominantly to a target protein present in the target organ, wherein the target organ is not the liver.

[0268] In some embodiments, the targeting protein is selected from vitronectin or β2-glycoprotein I (Apo H). In some embodiments, the targeting protein is vitronectin and the target organ is the lung. In other embodiments, the targeting protein is Apo H and the target organ is the spleen.

[0269] 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, the selective organ targeting compound is further selected from sugars, lipids, small molecule therapeutics, vitamins, or proteins. In some embodiments, the selective organ targeting compound is a lipid such as a permanent cationic lipid, a permanent anionic lipid, or phosphatidylcholine. In some embodiments, the lipid nanoparticle composition further comprises an ionizable cationic 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.

[0270] 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 results in a lipid nanoparticle composition having an apparent pK of about 3 to about 6 a of the lipid nanoparticle composition.

[0271] 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 results in a lipid nanoparticle composition having an apparent pK of about 8 to about 13 a of the lipid nanoparticle composition.

[0272] With respect to a particular component, as used herein, "substantially free" is used herein to mean that the particular component is not deliberately formulated in the composition and / or is present only as a contaminant or in trace amounts. The total amount of the particular component caused by any unintentional contamination of the composition is preferably less than 0.01%. Most preferably, the composition is one in which the amount of the particular component is undetectable by standard analytical methods.

[0273] As used herein in the specification and claims, "a" or "an" can mean one or more. As used herein in the specification and claims, when used in conjunction with the word "comprising", the word "a" or "an" can mean one or more than one. As used herein, in the specification and claims, "another" or "a further" can mean at least a second or more.

[0274] As used herein in the specification and claims, the term "about" is used to indicate that a value includes the inherent variability of error of the device and method used to measure that value, or the variations that exist among study subjects.

[0275] Other objects, features, and advantages of the present disclosure will be apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate certain embodiments of the present disclosure, they are given by way of illustration only, since 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0276] The following drawings form a part of this specification and are included to further confirm certain aspects of the present disclosure. By referring to one or more of these drawings, in conjunction with the detailed description of the specific embodiments presented herein, the present disclosure can be better understood.

[0277] Figures 1A-C: DOTAP mDLNP formulations mediate excellent mRNA delivery efficacy and exhibit tissue-specific delivery characteristics with a given percentage of DOTAP after low-dose intravenous injection. (Figure 1A) Schematic of DOTAP mDLNP formation and the structure of DOTAP. The molar ratio of 5A2-SC8 / DOPE / Chol / DMG-PEG was fixed at 15 / 15 / 30 / 3 (named mDLNP). Then only the DOTAP ratio was adjusted from 0 to 1200 to generate a series of DOTAP mDLNP formulations and named DOTAPY, where Y represents the percentage of DOTAP in the total lipids. (Figure 1B) Ex vivo images of luciferase in major organs 6 h after intravenous injection with a dose of 0.1 mg / kg Luc mRNA (n = 2). As the molar percentage of DOTAP increased, luciferase protein expression shifted from the liver to the spleen and then to the lung. (Figure 1C) Quantitative data confirmed that the DOTAP percentage was the most important factor for tissue-specific delivery, mDLNP (0%) was the best for the liver, DOTAP10-15 (similar between them) was the best for the spleen, and DOTAP50 was the best for the lung. Since luciferase expression was detected only in the liver, spleen, and lung after intravenous injection, the relative expression in each organ was calculated. Apparently, the higher the percentage of DOTAP (permanent cationic lipid) in the formulation, the less luminescence in the liver, and it approached 0 when >70%. However, the higher the DOTAP percentage, the more luminescence in the lung, and it approached 100% when >70%. DOTAP5-20 showed a higher percentage in the spleen, and DOTAP10 seemed to be the highest.

[0278] Figure 2A1-2F2: The structure of lipids determines the mRNA expression profile after intravenous injection. Generally, as the percentage increases, quaternary lipids alter mRNA delivery from the liver to the spleen and then to the lungs, and zwitterionic lipids at higher percentages help deliver mRNA into the spleen. However, tertiary amine lipids do not change the mRNA expression organ but improve the delivery efficacy in the liver. To further confirm the delivery trend of quaternary lipid mDLNPs, two additional quaternary lipids, DDAB and EPC, were selected for in vivo mRNA delivery, and their modification strategies were the same as DOTAP. (Figure 2A1, Figure 2B1) DDAB and EPC exhibited large structural differences between themselves and with DOTAP, including at three levels of comparison: the length of the hydrophobic tail, saturated and unsaturated bonds, and the chemical structure of the headgroup. Formulations containing 5%, 15%, 40%, and 50% of the quaternary lipid were formed to examine the size distribution and in vivo evaluation (0.1 mg / kg, 6 h, n = 2). (Figure 2A2, Figure 2B2) Similar to DOTAP mDLNPs, DDAB and EPC also presented a similar mRNA delivery profile. A lower cationic percentage (5%) delivered mRNA to the liver and spleen, and then more was delivered to the spleen when increased to 15%. Once increased to 40%, almost no mRNA expression was observed in the liver and spleen, but the lungs showed high luciferase signals, and then decreased at 50%. These results were very similar to DOTAP mDLNPs, which suggested that mDLNPs functionalized with quaternary lipids were a general and scalable strategy for tissue-targeted mRNA delivery. Then, similar to the DOTAP strategy, the representative zwitterionic lipids DSPC and DOCPe were used to evaluate in vivo mRNA delivery (Figure 2C1, Figure 2D1). The structures of DSPC and DOCPe lipids belong to the general class of zwitterionic lipids. Before intravenous injection, the size distribution of DSPC and DOCPe mDLNP formulations was tested by DLS. In this article, DSPC and DOCPe showed two levels of comparison in structure: saturated versus unsaturated hydrophobic tails, and the position of the charge relative to that in the headgroup. (Figure 2C2, Figure 2D2) Interestingly, a similar mRNA expression profile to that of the quaternary lipid formulations was not observed. Instead, DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% in DSPC and less than 50% in DOCPe), and no signal was seen in the lungs regardless of the percentage (0.1 mg / kg, 6 h, n = 2). Inspired by these results, the ionizable tertiary amine lipids, DODAP and C12-200, were further tested with the same strategy. Except for the headgroup (quaternary amine versus tertiary amine), DODAP has the same structure as DOTAP, and C12-200 is an effective lipid for siRNA or mRNA delivery and has a completely different structure from DODAP.(Figure 2E1, Figure 2F1) Similarly, the size distributions of the two modified mDLNPs remain good at certain percentages (less than 80%). (Figure 2E2, Figure 2F2) Surprisingly, DODAP and C12-200 cannot alter the mRNA expression profile (different effects compared to quaternary lipids or zwitterionic lipids). Instead, DODAP and C12-200 increase the delivery of mRNA to the liver. Supporting this is that DODAP20 and C12-200 show much better delivery efficacy (0.1 mg / kg, 6 h, n = 2) than the original mDLNP formulation. As the percentage of DODAP or C12-200 increases (50% or 80%), the luciferase signal drops significantly, but the liver remains the main organ rather than the spleen or lung.

[0279] Figure 3A-C: To determine why various lipids can induce large differences in mRNA expression in organs, biodistribution assays and pK a measurements were then performed. Both biodistribution and pK a play important roles in the mRNA expression profile in organs. (Figure 3A) Organ biodistribution of Cy5.5-Luc mRNA formulations delivered by three modified mDLNPs (DOTAP (quaternary lipid), DSPC (zwitterionic lipid), and DODAP (tertiary amine lipid)). C57BL / 6 mice were intravenously injected at a dose of 0.5 mg / kg and imaged 6 hours after injection (n = 2). Compared with the original mDLNP formulation (without DOTAP), DOTAP altered the organ biodistribution of mRNA. Both DOTAP10 and DOTAP50 could deliver mRNA to the lung, and DOTAP50 increased more, which could partly explain why the DOTAP formulation mediated mRNA expression in the lung at higher percentages. However, even at 80% (DSPC) or 50% (DODAP) percentages, DSPC and DODAP could not alter the mRNA biodistribution more. It was also noted that, as shown in Figures 1 and 2, for DOTAP50 and DSPC80, the mRNA remained in the liver, the former being an NP targeting the lung and the latter being an NP targeting the spleen. Therefore, biodistribution is not the only factor explaining this mechanism. (Figure 3B) Then the pK a of all tested and effective formulations was measured, including the original mDLNP, DOTAP, DDAB, EPC, DSCP, DOCPe, DODAP, and C12-200-modified formulations. (Figure 3C) Finally, based on defined rules, the relationship between pK a and tissue-specific mRNA delivery was plotted. Here, as shown in the table, 8 scoring rules were designed. Obviously, all liver-targeting formulations have a narrow pK a(~6 - 7), the spleen - targeted preparation has no obvious range, but lung - targeted delivery requires a high pK a (>9.25).

[0280] Figure 4A-4C : Gene editing in the liver and lungs was achieved in both Td - Tomato and C57BL / 6 mice. (Figure 4A) The schematic shows that co - delivery of Cas9 mRNA and sgTom1 activated Td - Tomato expression in Td - Tomato mice. (Figure 4B) When treated with mDLNP and DOTAP50 preparations respectively, Td - Tomato expression was induced in the liver and lungs. mDLNP and DOTAP50 preparations were intravenously injected into mice to co - deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, weight / weight) at a total dose of 2.5 mg / kg (50 μg each), and then the fluorescence of major organs was detected on the 10th day after treatment. (Figure 4C) The T7E1 assay showed that tissue - specific characteristics were further confirmed by in - vivo PTEN editing. C57BL6 mice were intravenously injected with mDLNP, DODAP20 or DOTAP50 for tissue - specific gene editing at a total dose of 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 the 10th day after treatment.

[0281] Figure 5A - C: Characterization of DOTAP mDLNP preparations (n = 3). Size, PDI (Figure 5A) and ζ - potential (Figure 5B) were detected by dynamic light scattering (DLS). (Figure 5C) The encapsulation efficiency (EE%) was tested by Ribogreen RNA assay.

[0282] Figures 6A-C: DOTAP formulations show excellent mRNA delivery efficiency and exhibit delivery potential for cargoes (such as proteins) that are not well tolerated by ethanol or acidic buffers. (Figure 6A) DOTAP mDLNPs mediated high Luc mRNA expression in Huh-7 cells and A549 cells and revealed that 5%-50% DOTAP percentages were better for mRNA delivery and 10% was the best. At 24 h post transfection, Luc mRNA expression and cell viability were tested at a dose of 50 ng / well of mRNA (n = 4). Here, DOTAP mDLNPs were formed in PBS rather than citrate buffer (10 mM, pH 4.0). (Figures 6B and 6C) Decreasing ethanol volume percentages did not affect the characterization and mRNA delivery efficacy. To test the effect of ethanol on mRNA delivery, DOTAP25 was selected as a model and four formulations with different volume ratios of ethanol to PBS (1:3, 1:5, 1:7.5, and 1:10) were formed. All four formulations showed similar EE, size, and PDI in FaDu cells (Figure 6B) and exhibited equal mRNA delivery efficacy (Figure 6C) (50 ng / well of mRNA, 24 h, n = 4). Thus, the formulation was optimized using 1X PBS (pH 7.4) instead of acidic buffer (10 mM pH 4.0), and the ethanol percentage was significantly reduced, which provides the possibility that DOTAP formulations deliver those cargoes that are not well tolerated at high ethanol concentrations or in acidic buffers, such as proteins.

[0283] Figure 7 : Quantitative data on biodistribution in major organs. C57BL6 mice were intravenously injected with various Cy5.5-Luc mRNA formulations at a dose of 0.5 mg / kg (n = 2). After 6 h, the heart, lungs, liver, spleen, and kidneys were isolated, imaged, and quantified.

[0284] Figure 8A-8C: For DOTAP10 formulations formed by PBS or citrate buffer, there were no significant differences in size distribution and Luc mRNA delivery efficacy, but it was not applicable to DSPC50 and DODAP50. To test the effect of buffer on in vivo mRNA delivery efficacy, DOTAP10 (quaternary lipid), DSPC50 (zwitterionic lipid), and DODAP50 (tertiary amine lipid) were selected. Each Luc mRNA formulation was intravenously injected into C57 BL6 mice at a dose of 0.1 mg / kg. After 6 hours, the major organs were isolated and imaged (n = 2). (Figure 8A) There was no significant change in size and delivery efficacy between DOTAP10 formed by PBS and citrate buffer (10 mM, pH 4.0). (Figures 8B and 8C) However, DSPC50 and DODAP50 formed by citrate buffer significantly improved the mRNA delivery effect, although there was no significant difference in size distribution. DOTAP (or another permanent cationic lipid) is an essential requirement for the formation of LNPs at neutral pH (such as 7.4 PBS buffer).

[0285] Figure 9 : Western blot results of the quality test of IVT Cas9 mRNA delivered by mDLNP. To achieve tissue-specific gene editing, Cas9 mRNA and sgRNA were designed for co-delivery. First, Cas9 mRNA was prepared by IVT and its quality was tested by Western blot analysis. In this assay, Cas9 pDNA was delivered by both Lipofectamine 2000 and commercial Cas9 mRNA (TriLink). mDLNP was the positive control and mCherry mDLNP was the negative control. One day before transfection, 293T cells were seeded in 12-well plates and treated with cells for 24 hours under each condition before performing Western blot. The effect of IVT Cas9 mRNA was much better than that of commercial mRNA. Therefore, IVT Cas9 mRNA was used for in vivo gene editing.

[0286] Figures 10A and 10B: sgRNA screening and weight ratio (Cas9 mRNA / sgRNA) optimization in Td-Tomato mice. To achieve maximum gene editing in Td-Tomato mice, sgRNA sequences were screened and the weight ratio of Cas9 mRNA to sgRNA was optimized. (Figure 10A) Size distribution of Cas9 / sgTom1, Cas9 / sgTom2, and Cas9 / sgLoxP mDLNP formulations and td-tomato expression in the liver. Three mDLNPs were intravenously injected into mice at a total dose of 3 mg / kg (Cas9 / sgRNA, 4 / 1, weight / weight), and the organs were imaged on day 7. It seems that sgTom1 is the leader among the three candidates. (Figure 10B) SgTom1 was selected and further tested for liver gene editing with different weight ratios (Cas9 / sgRNA) of 2 / 1, 4 / 1, and 6 / 1. The mDLNP was used for intravenous injection at a dose of 3 mg / kg, and td-tomato expression was detected on day 7. In this example, the effect of 4 / 1 was better than that of 2 / 1 and 6 / 1.

[0287] Figure 11A-11G: Characterization of Cas9 / sgRNA complexes and DOTNP lipid nanoparticles after encapsulation. Size (Figure 11A) and ζ potential (Figure 11B) of Cas9 / sgLUC complexes (mol / mol = 1 / 1) in PBS (pH 7.4) and in citrate buffer (pH 4.2). The size of Cas9 / sgLUC complexes prepared in citrate buffer was very large (greater than 100 nm), and the ζ potential was positively charged, so it was impossible to be encapsulated by lipid nanoparticles. However, the size of Cas9 / sgLUC complexes prepared in PBS was compact (less than 20 nm) and negatively charged, so it could be encapsulated by lipid nanoparticles. Size (Figure 11C) and ζ potential (Figure 11D) of Cas9 / sgLUC complexes prepared with different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with Cas9 / sgLUC complexes (1 / 1, mol / mol), higher molar ratios (1 / 3 and 1 / 5, mol / mol) showed smaller sizes and more negative charges, which were beneficial for lipid nanoparticle encapsulation. Size (Figure 11E) and ζ potential (Figure 11F) of DOTNP10 lipid nanoparticles (named DOTNP10-L) encapsulating Cas9 / sgLUC complexes when prepared at different molar ratios (1 / 1, 1 / 3, 1 / 5). (Figure 11G) TEM image of DOTNP10-L (1 / 3, mol / mol). DOTAP lipid nanoparticles are composed of five components (including 5A2-SC8, cholesterol, DOPE, DMG-PEG, and DOTAP). The molar ratios of 5A2-SC8, cholesterol, DOPE, and DMG-PEG are fixed (15:15:30:5, mol / mol), and DOTNP X refers to DOTNP with different molar percentages of DOTAP. Here, different sgRNAs were used, including sgLUC, sgGFP, sgTOM, sgPTEN, etc. To distinguish them, the first letter of each gene was added at the end of DOTNP. For example, DOTNP10-L refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgLUC complexes; DOTNP10-G refers to DOTNP10 lipid nanoparticles encapsulating Cas9 / sgGFP complexes.

[0288] Figure 12A-12F: DOTNP lipid nanoparticles can deliver the Cas9 / sgRNA complex into the nucleus and exhibit effective gene editing in vitro. (Figure 12A) Confocal images of Hela-Luc cells after incubation with DOTNP10 encapsulating Cas9-EGFP / sgLUC complex (1 / 3, mol / mol) for 1 h, 3 h, 6 h, and 24 h (using 9 nM sgRNA). Green: EGFP-fused Cas9 protein; Blue: Nucleus stained with Hoechst 33342. Red arrows indicate the process of DOTNP10 entering the nucleus. (Figure 12B) Indel percentages at the LUC locus after incubation with DOTNP10-L at different molar ratios for 3 days analyzed by TIDE sequencing (using 24 nM sgRNA). DOTNP10 lipid nanoparticles encapsulating Cas9 / sgGFP (DOTNP10-G) were used as a negative control. Here, two commercial Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used. (Figure 12C) T7EI cleavage assay of Hela-Luc cells incubated with different formulations (using 24 nM sgRNA). 1. 100 bp DNA ladder; 2. PBS; 3. DOTNP10-G (1 / 3); 4. DOTNP10-L (1 / 1); 5. DOTNP10-L (1 / 3); 6. DOTNP10-L (1 / 5); 7. DOTNP10-L (1 / 3) prepared in citrate buffer. Two commercial Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used. Among them, when using Truecut Cas9 protein, a 1 / 3 molar ratio showed the best gene editing. (Figure 12D) Fluorescence microscopy images of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G (using 24 nM sgRNA). Here, DOTNP10-L was used as a negative control. (Figure 12E) Flow cytometry analysis of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G. (Figure 12F) Mean fluorescence intensity of SKOV3-GFP cells incubated with DOTNP10-L and DOTNP10-G measured by flow cytometry.

[0289] Figures 13A and 13B: DOTNP shows tissue-specific gene editing in vivo. (Figure 13A) Ex vivo images of tdTomato fluorescence in major organs 7 days after intravenous injection of different formulations (1.5 mg / kg sgRNA / mouse). DOTNP5-T refers to DOTNP5 lipid nanoparticles encapsulating the Cas9 / sgTom complex; DOTNP10-T refers to DOTNP10 lipid nanoparticles encapsulating the Cas9 / sgTom complex; DOTNP50-T refers to DOTNP50 lipid nanoparticles encapsulating the Cas9 / sgTom complex. tdTomato fluorescence was observed only in the liver in the DOTNP5-T treatment group; in the DOTNP10-T group, slight fluorescence was visible in the lung, and if the DOTAP dose was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lung. (Figure 13B) T7EI cleavage assays in liver and lung organs after incubation with DOTNP5-P (DOTNP5 lipid nanoparticles encapsulating the Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 lipid nanoparticles encapsulating the Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 lipid nanoparticles encapsulating the Cas9 / sgPTEN complex) (2 mg / kg sgRNA / mouse). The results were consistent with those obtained by ex vivo imaging. Gene editing was detected only in the liver after treatment with DOTNP5-P; gene editing was obtained in both the liver and lung after incubation with DOTNP10-P; while in the DOTNP50-P treatment group, most of the gene editing was observed in the lung.

[0290] Figures 14A and 14B show details of the MC3 LNP and DOTAP-modified MC3 formulations, including (Figure 14A) the structure of each component, (Figure 14B) the molar ratio, the weight ratio of total lipid to mRNA, size, and PDI.

[0291] Figures 15A and 15B show the selection of DLin-MC3-DMA (Figure 15A) and C12-200 (Figure 15B) and evaluation with the DOTAP strategy at a dose of 0.1 mg / kg (6 h, n = 2). As the DOTAP percentage increased from 0 to 50%, MC3- and C12-200-based LNPs showed the same mRNA expression profile, like mDLNP, where the luciferase signal transferred from the liver to the spleen and finally to the lung.

[0292] Figures 16A and 16B show details of the C12-200 LNP and DOTAP-modified C12-200 formulations, including (Figure 16A) the structure of each component, (Figure 16B) the molar ratio, the weight ratio of total lipid to mRNA, size, and PDI.

[0293] Figures 17A and 17B show a further optimization of (Figure 17A) mDLNP. As a key lipid in mDLNP, 5A2-SC8 was used as the "fifth" lipid to modify mDLNP to form four formulations with an additional percentage of 10% to 30%. (Figure 17B) Ex vivo luciferase images and quantitative data show that an additional 15% to 25% of 5A2-SC8 significantly improved mRNA delivery efficacy, and 20% had the highest signal (0.05 mg / kg, 6 h, n = 2).

[0294] Figure 18 Show the structures of 5A2-SC8, DOPE, cholesterol, and DMG-PEG. mDLNP is an effective and safe mRNA delivery vector for liver-targeted therapeutics developed in previous work and is composed of 5A2-SC8, DOPE, cholesterol, and DME-PEG at a molar ratio of 15 / 15 / 30 / 3.

[0295] Figure 19A-19GDisplaying selective organ targeting (SORT) allows for the systematic and predictable engineering of lipid nanoparticle (LNP) systems to precisely edit cells in specific organs. (19A) Adding supplementary components (termed SORT lipids) to conventional LNPs systematically alters the in vivo delivery profile and mediates tissue-specific delivery according to the percentage and biophysical properties of the SORT lipids. This general approach has successfully redirected multiple classes of nanoparticles. Bioluminescence images of mice intravenously injected with 0.1 mg / kg luciferase mRNA are shown here, which was within lung- and spleen-specific DLin-MC3-DMA LNPs (Onpattro SNALP) and liver-enhanced 5A2-SC8 degradable dendrimer-based LNPs (DLNPs). Inclusion of SORT lipids in 4-component 5A2-SC8, DLin-MC3-DMA, and C12-200 LNPs created 5-component SORT LNPs. (19B) 5A2-SC8 SORT LNPs were formulated at a molar ratio of 5A2-SC8 / DOPE / Chol / DMG-PEG / SORT lipid = 15 / 15 / 30 / 3 / X (mol / mol), where X was adjusted from 0 to 1200 to prepare a series of LNPs with 0% to 100% SORT lipid (fraction of total lipid). Here, inclusion of the permanent cationic lipid (DOTAP) systematically shifted luciferase protein expression from the liver to the spleen and then to the lung according to the DOTAP percentage (0.1 mg / kg Luc mRNA, 6 h). (19C) Quantitative data confirm that the SORT lipid percentage is the most important factor for tissue-specific delivery; 0% (mDLNP) is optimal for the liver; 5 - 15% is optimal for the spleen; and 50% is optimal for the lung. (19D) Relative luciferase expression in each organ confirms that fractional expression is predictably tunable. (19E) Inclusion of anionic SORT lipids enables selective delivery of mRNA to the spleen. When the 18PA lipid was introduced into mDLNP up to 40%, luciferase expression was only observed in the spleen (0.1 mg / kg Luc mRNA, 6 h). (19F) Ex vivo luminescence images of major organs 6 hours after intravenous injection of DLin-MC3-DMA SORT LNPs at a dose of 0.1 mg / kg Luc mRNA. As the molar percentage of DOTAP increased, luciferase expression shifted from the liver to the lung. Exclusive delivery of Luc mRNA to the spleen mediated by 18PA. The same trend was observed for modified C12-200 LNPs (0.1 mg / kg, 6 h). (19G) Details of selected SORT lipid formulations.

[0296] Figure 20A-20CShows details of (20A) DOTAP and 18PA SORT LNP, including molar ratio, mole percentage, total lipid to mRNA weight ratio, size, PDI, and ζ potential. (20B) LNP was formulated using a modified ethanol dilution method. The SORT lipid was included in the ethanol phase, and sgRNA / mRNA was encapsulated during LNP formation. (20C) Shows the chemical structures of the lipids used in standard mDLNP and DOTAP / 18PA SORT formulations. For the development of SORT, an ionizable cationic lipid based on a degradable dendrimer (termed 5A2-SC8) was the focus of LNP that could deliver siRNA / miRNA to extend survival in a genetically engineered mouse model of MYC-driven liver cancer and trigger polyploidy in the liver (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b). The LNP molar composition optimized for mRNA delivery was focused on liver-named mDLNP (Cheng et al., 2018). This liver-targeted basal mRNA formulation of 5A2-SC8 / DOPE / cholesterol / DMG-PEG2000 = 15 / 15 / 30 / 3 (mol) was prepared and supplemented with SORT lipid to prepare SORT LNP (details in 20A). For further clarity, traditional 4-component LNP consists of an ionizable cationic lipid (defined herein as containing an amino group with pK a <8), an amphiphilic phospholipid (defined as a lipid with an equal number of positive and negative charges), cholesterol, and a poly(ethylene glycol) (PEG) lipid (most commonly PEG2000-DMG). SORT LNP contains a 5th lipid, such as a permanent cationic lipid (defined as having a positive charge, no pK a or pK a >8) or a permanent anionic lipid (defined as having a negative charge).

[0297] Figures 21A and 21B show the in vitro luciferase (Luc) mRNA delivery results of DOTAP-modified SORT mDLNP in (Figure 21A) Huh-7 hepatocytes and (Figure 21B) A549 lung cells as a function of the percentage of incorporated DOTAP. The Luc mRNA delivery results show that LNPs with 5%-50% DOTAP percentage delivered the most mRNA in Huh-7 hepatocytes and A549 lung cells. SORT LNPs with 10% DOTAP were much more effective in vitro than the previously reported basic mDLNP. No detectable cytotoxicity was observed for any of the formulations, and all were uniform (low PDI), with diameters in the range of 90 nm to 150 nm (Figure 20). Measurement of the surface charge revealed that DOTAP was encapsulated inside together with the mRNA, rather than on the surface of the LNP, because when DOTAP was less than 60%, the ζ potential was close to 0. The surface charge became positive only at percentages higher than 65% (Figure 20), which revealed that PEG lipid-coated SORT LNPs with selective tissue tropism could be found to have a nearly neutral surface charge, which is an important property for clinical translation. Cells were seeded into 96-well plates at a density of 1×10 4 cells / well one day before transfection. Luc mRNA expression and cell viability (n = 4) were measured at a dose of 50 ng / well of Luc mRNA 24 hours after treatment.

[0298] Figure 22A-22C The chemical structures of the lipids used in (22A) DLin-MC3-DMA SNALP (Jayaraman et al., 2012) and (22B) C12-200 LLNP (Love et al., 2010) are shown. The basic mRNA formulations targeting the liver of DLin-MC3-DMA / DSPC / cholesterol / DMG-PEG2000 = 50 / 10 / 38.5 / 1.5 (mol) and C12-200 / DOPE / cholesterol / DMG-PEG2000 = 35 / 16 / 46.5 / 2.5 (mol) were prepared and supplemented with SORT lipids later to prepare SORT LNPs. (22C) Tables and results of other SORT formulations using DLin-MC3-DMA and C12-200. For all DLin-MC3-DMA and C12-200 LNPs, the weight ratio of total lipid / mRNA was 20 / 1 (weight / weight).

[0299] Figure 23A-23CThe display SORT depends on general biophysical properties rather than exact chemical structures. (23A) SORT lipids can be divided into specific groups with defined biophysical properties. The permanent cationic SORT lipids (DDAB, EPC, and DOTAP) all produce the same mRNA delivery profile (liver to spleen to lung based on SORT lipid percentage, 0.1 mg / kg LucmRNA, 6 h). (23B) The anionic SORT lipids (14PA, 18BMP, 18PA) all produce the same mRNA delivery profile (spleen only based on SORT lipid percentage). (23C) The ionizable cationic SORT lipids with tertiary amines (DODAP, C12-200) enhanced liver delivery with no luciferase expression in the lung (0.1 mg / kg Luc mRNA, 6 h).

[0300] Figures 24A and 24B show further application of SORT to further enhance mDLNP liver delivery using ionizable cationic lipids as SORT lipids. (24A) Schematic of SORT. (24B) 5A2-SC8 was used as the SORT lipid, and the basal mRNA mDLNP formulation (5A2-SC8 / DOPE / cholesterol / DMG-PEG2000 = 15 / 15 / 30 / 3 (mol)) was supplemented with additional 5A2-SC8 essentially using the SORT method. Ex vivo luciferase images and quantitative data showed that when an additional 15%-25% SORT lipid was added, the mRNA delivery efficacy was sharply improved. Incorporation with 20% produced the maximum expression (0.05 mg / kg, 6 h, n = 2). Thus, SORT allows the development of a second-generation mDLNP with increased potency.

[0301] Figures 25A and 25B show the evaluation of the role of zwitterionic SORT lipids. With the increase in SORT lipid incorporation, the inclusion of zwitterionic SORT lipids in mDLNPs targeting the liver altered the expression from the liver to the spleen. After intravenous injection, 80% DSPC and 50% DOCPe SORT LNP delivered mRNA only to the spleen. (25A) Schematic of the SORT method. (25B) Ex vivo luminescence images in major organs 6 h after intravenous injection. DSPC and DOCPe (zwitterionic lipids with different structures) improved the delivery of Luc mRNA into the spleen in increasing percentages (0.1 mg / kg, 6 h, n = 2).

[0302] Figures 26A and 26B SORT was evaluated as a potential strategy to “activate” inactive LNP formulations. (26A) Schematic of supplementing an inactive C1 formulation with SORT lipids to test whether SORT can confer activity. (26B) Details of C1 LNP (inactive LNP) and DOTAP (or DODAP) C1 SORT LNP, including lipid molar ratio, mole percentage, total lipid to mRNA weight ratio, size, and PDI. C1 LNP was prepared in a way that allows mRNA encapsulation and favorable biophysical properties (uniform <200 nm size). However, there was no protein expression at all after intravenous injection of C1 LNP. Thus, it was asked whether SORT can “activate” the dead LNP. DODAP and DOTAP SORT lipids were evaluated. DODAP@C1 LNP delivered mRNA to the spleen and liver, and DOTAP@C1 LNP delivered mRNA to the lung and spleen (0.1 mg / kg, 6 h, n = 2). Thus, SORT can activate dead LNP and provide tissue selectivity.

[0303] Figures 27A and 27B show that SORT alters LNP biodistribution and reveals the association between relative apparent pK a and organ specificity. (27A) Fluorescent Cy5-labeled mRNA was used to track the biodistribution of SORT LNP. Inclusion of DOTAP as the SORT lipid increased mRNA accumulation in the lung, which partly explains the ability to deliver RNA to the mouse lung. 18PA increased uptake into the spleen. DODAP slightly increased liver accumulation and decreased spleen accumulation (0.5 mg / kg, 6 h). It should be noted that this data describes the location of SORT LNP, rather than the ability to effectively deliver mRNA intracellularly. (27B) The relative apparent pK a of all 67 active mRNA formulations was measured by the TNS assay and plotted against the in vivo delivery efficacy in different organs (functional delivery by mRNA translated into protein). As expected, all liver-targeted formulations had a narrow pK a (6 - 7). Surprisingly, lung-targeted delivery required a high pK a (>9), and a lower pK a (<6) facilitated spleen delivery. It should be noted that all SORT LNP contain ionizable cationic lipids (for endosomal escape) as well as a mixture of other charged and uncharged lipids that together mediate tissue tropism.

[0304] Figures 28A and 28B show that Cy5-labeled mRNA was used to track the biodistribution of SORT LNPs. Organ distribution of DSPC mDLNPs after intravenous injection. (28A) Schematic of SORT. (28B) Cy5 fluorescence and quantification data of major organs treated with DSPC mDLNPs (0.5 mg / kg, 6 h, n = 2).

[0305] Figure 29 Show that the modified TNS assay was used to measure the overall / apparent pK of mRNA formulations a . A total of 67 NP successful formulations (high in vivo potency) were evaluated. When generating 50% of the normalized signal, the relative pK was estimated compared to the basal LNP formulation (without addition of SORT lipid) a . The TNS assay has historically been used to measure LNPs with a single ionizable cationic lipid and neutral (non-ionizable) co-lipids, yielding values for the ionization behavior of the cationic lipid trapped within the self-assembled LNP. Here, a modified method was used because SORT LNPs (e.g., DOTAP) with a high percentage (>40%) of permanently cationic lipids do not buffer charge well even though they contain ionizable cationic lipids. Due to the complexity of SORT LNPs containing multiple charged lipids (as opposed to a single ionizable cationic lipid in traditional LNPs), the focus was on the 50% relative signal, which is related to the in vivo tissue specific activity.

[0306] Figures 30A and 30B show that the inclusion of ionizable lipids (e.g., 5A2-SC8) is required for potency. LNPs containing SORT lipids but no ionizable cationic lipids are inactive. (30A) Schematic of SORT C2 LNP. (30B) Details of C2 and SORT lipid C2 LNPs. Ex vivo luciferase images show that neither DODAP nor DOTAP could achieve significant mRNA delivery of C2 LNPs. These results indicate that successful mRNA delivery requires ionizable amino lipids (0.1 mg / kg, 6 h, n = 2).

[0307] Figure 31A-31EShows tissue-specific gene editing achieved by Cre mRNA delivery with SORT LNP in Td-Tomato mice. (31A) Schematic shows that delivery of Cre mRNA activates Td-Tom expression in Td-Tom transgenic mice. (31B) mDLNP and 20% DODAP LNP specifically induced Td-Tom fluorescence in the liver, and 50% DOTAP LNP selectively edited the lung. Td-Tom fluorescence in major organs was detected 2 days after intravenous injection of Cre mRNA-loaded LNP (0.3 mg / kg). (31C) 30% 18PA SORT LNP induced gene editing in the spleen (note the high liver background fluorescence in PBS-injected mice). (31D) Effective tissue editing was further verified using confocal microscopy. Scale bars = 20 μm and 100 μm. (31E) The percentage of TdTom + cells was quantified in defined cell type populations in the liver, lung, and spleen (day 2, 0.3 mg / kg).

[0308] Figure 32 B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J (Ai9) mice showed some autofluorescence in the area of TdTom uptake. Additionally, there were large differences in TdTom autofluorescence between different organs (n = 2). The liver and kidney showed the highest signal, while the spleen showed the lowest. Although this did not interfere with the detection of editing in most organs (when excitation settings were appropriately adjusted to eliminate the background), it did complicate the detection of splenic TdTom expression because the background spleen was so much lower than the other organs.

[0309] Figure 33A-33CIt was shown that by co-delivering Cas9 mRNA and sgRNA, CRISPR / Cas gene editing in the spleen was achieved in both Td-Tom transgenic mice and wild-type C57 / BL6 mice. (33A) The schematic diagram shows that co-delivery of Cas9 mRNA and sgTom1 activates Td-Tom expression in Td-Tom mice. (33B) Td-Tom expression was induced in the spleen and liver by the spleen-targeting formulation 30% 18PA SORTLNP. Quantitative data showed that the editing in the spleen was higher than that in the liver. Td-Tom fluorescence in major organs was detected on the 2nd day after intravenous treatment with co-delivery (2 / 1, weight / weight) of Cas9 mRNA and modified sgTom1 at a total dose of 4 mg / kg. (33C) T7E1 assay indicated that specific PTEN editing in the spleen was obtained by co-delivery of Cas9mRNA (IVT) and sgPTEN. C57 / BL6 mice were intravenously injected with 30% 18PA SORT LNP (Cas9 mRNA / sgPTEN, 2 / 1, weight / weight) at a total dose of 4 mg / kg, and PTEN editing was detected on the 2nd day. In this case, no liver editing was observed, suggesting that spleen-specific editing could be achieved.

[0310] Figure 34 It was shown that by administering a single 0.3 mg / kg dose of Cre mRNA, DODAP-20SORT LNP achieved almost 100% TdTom editing in hepatocytes. As shown in the flow cytometry histogram, there was a complete separation between TdTom-control mice and mice treated with 20% DODAP. After liver perfusion, the excised livers of mice treated with 20% DODAP SORT LNP were surprisingly bright red compared to the control livers. Even without fluorescence excitation, the livers glowed red due to the complete activation of TdTom expression. TdTom mice were injected with 0.3 mg / kg Cre mRNA and then sacrificed after 2 days (n = 3). Hepatocytes were isolated by two-step collagenase perfusion and TdTom fluorescence was analyzed by flow cytometry.

[0311] Figure 35 The FACS gating strategy for analyzing TdTom+ expression in lung cells was shown. Ghost Red 780 was used to distinguish live cells from dead cells. EpCam+ was used to define epithelial cells, CD45+ and CD31- were used to define immune cells, and CD45- and CD31+ were used to define endothelial cells. Gating of Td-Tom+ in cell types was plotted based on control mice injected with PBS. Td-Tom mice were injected with Cre mRNA formulation and Td-Tom+ in the given cell types was detected by flow cytometry after 2 days (n = 3).

[0312] Figure 36 Shows the FACS gating strategy for analyzing TdTom+ expression in splenocytes. Ghost Red 780 was used to distinguish live cells from dead cells. CD44+ was used to distinguish immune cells, and then CD3+ and CD11b- for T cells, CD3- and CD11b+ for macrophages, and CD19+ and CD11b- for B cells. Based on control mice injected with PBS, the gating of Td-Tom+ in cell types was plotted. Td-Tom mice were injected with Cre mRNA preparation, and Td-Tomato+ in the given cell type was detected by flow cytometry 2 days later (n = 3).

[0313] Figure 37A-37GShows tissue-specific CRISPR / Cas gene editing in Td-Tom transgenic mice and C57 / BL6 wild-type mice mediated by SORTLNP through co-delivery of Cas9 mRNA and sgRNA and through delivery of Cas9 RNP. (37A) Schematic shows that co-delivery of Cas9 mRNA (or Cas9 protein) and sgTom1 activates Td-Tom expression in Td-Tom transgenic mice. (37B) mDLNP and 20% DODAP LNP specifically induce Td-Tom fluorescence in the liver, and 50% DOTAP LNP selectively edits the lung. Td-Tom fluorescence was detected 10 days after intravenous injection of Cas9 mRNA and modified sgTom1 (4 / 1, weight / weight) at a total dose of 2.5 mg / kg. (37C) tdTom expression was confirmed by confocal imaging of tissue sections. Scale bars = 20 μm and 100 μm. (37D) Cas9 mRNA and sgPTEN were co-delivered in SORT LNP to selectively edit the liver, lung, and spleen of C57 / BL6 mice (total dose of 2.5 mg / kg (Cas9 mRNA / sgPTEN, 4 / 1, weight / weight; measured 10 days after a single injection). T7E1 assay indicates that tissue-specific PTEN editing was achieved. (37E) H&E sections and IHC further confirmed successful PTEN editing. Clear cytoplasm indicates lipid accumulation in H&E sections and PTEN loss in IHC images. Scale bars = 60 μm. (37F) Delivery of Cas9 / sgTom1 ribonucleoprotein (RNP) complexes in 7% DOTAP or 55% DOTAP SORT LNP specifically induces Td-Tom fluorescence in the liver and lung, respectively. Td-Tom fluorescence was detected 7 days after intravenous injection of Cas9 / sgTom1 RNP at a dose of 1.5 mg / kg sgTom1. tdTom expression was confirmed by confocal imaging of tissue sections. Scale bars = 20 μm and 100 μm. (37G) Liver- and lung-tropic SORT LNP also delivered Cas9 / sgPTEN RNP to selectively edit the liver and lung of C57 / BL6 mice (1.5 mg / kg sgPTEN; measured 7 days after a single injection). T7E1 assay indicates that tissue-specific PTEN editing was achieved.

[0314] Figure 38 Evaluates IVT Cas9 mRNA by western blot. 293T cells were seeded in 12-well plates one day before transfection and treated with each condition for 24 hours before performing western blot. Cas9 pDNA was delivered by Lipofectamine2000 and mRNA was delivered by mDLNP.

[0315] Figures 39A and 39B show the optimization of the weight ratio of IVT Cas9 mRNA to sgTom1 by a co-delivery strategy of Cas9 mRNA and sgRNA. (39A) Schematic illustration shows that co-delivery of Cas9 mRNA and sgTom1 activates Td-Tom expression in transgenic mice. (39B) Td-Tom fluorescence imaging of major organs on day 7 after intravenous injection indicates that a Cas9 / sgTom1 of 2 / 1 (weight / weight) is optimal. The total RNA dose is 1 mg / kg, and IVT Cas9 mRNA and modified sgTom1 are co-encapsulated by mDLNP.

[0316] Figure 40A-40I A modular scheme is shown that was developed to achieve systemic nanoparticle delivery of CRISPR / Cas9 ribonucleoprotein (RNP) for tissue-specific genome editing. (40A) Addition of a permanent cationic supplement component (such as DOTAP) to traditional LNP formulations enables encapsulation and protection of the Cas9 / sgRNA complex using a neutral buffer during nanoparticle formation. Precise tuning of tissue-specific gene editing mediated by the DOTAP percentage. (40B) Size distribution of Cas9 / sgLuc RNP prepared in PBS buffer (pH 7.4) and citrate buffer (pH 4.0). The increase in size may be due to denaturation. (40C) Size distribution of 5A2-DOT-10 encapsulating Cas9 / sgLuc RNP prepared in PBS and citrate buffer. 5A2-DOT-10 prepared without RNP is used as a control. (40D) Size distribution of Cas9 / sgRNA RNP with Cas9 / sgLuc molar ratios of 1 / 1, 1 / 3, and 1 / 5. (40E) Size distribution of 5A2-DOT-10 encapsulating Cas9 / sgLuc with molar ratios of 1 / 1, 1 / 3, and 1 / 5. (40F) ζ-potential of Cas9 / sgRNA RNP shows a decrease in charge. (40G) No significant difference in ζ-potential is observed for 5A2-DOT-10 encapsulating Cas9 / sgLuc with different molar ratios. (40H) Time-dependent cellular uptake of 5A2-DOT-10LNP encapsulating EGFP-fused Cas9 / sgRNA shows cytoplasmic release and gradual entry into the nucleus. (40I) Inhibition of 5A2-DOT-10LNP uptake was studied using specific endocytosis inhibitors. AMI: inhibitor of macropinocytosis; CMZ: inhibitor of clathrin-mediated endocytosis; GEN: inhibitor of caveolae-mediated endocytosis; MβCD: lipid raft-mediated endocytosis; 4 degrees: energy-mediated endocytosis.

[0317] Figures 41A-C show a table of 5A2-DOT-X LNPs (41A) that shows the molar ratios and percentages used to formulate 5A2-DOT-5 (5 mol% DOTAP), 5A2-DOT-10, 5A2-DOT-20, 5A2-DOT-30, 5A2-DOT-40, 5A2-DOT-50, and 5A2-DOT-60 (60 mol% DOTAP) LNPs. The total lipid / sgRNA ratio for all LNPs was 40:1 (weight). (41B) Gene editing in HeLa-Luc cells was detected using the T7EI assay after treatment with different 5A2-DOT-X Cas9 / sgLuc RNP formulations. (41C) Gene editing was analyzed using Sanger sequencing and ICE analysis.

[0318] Figure 42 Representative TEM images of 5A2-DOT-10 encapsulating the Cas9 / sgLuc RNP complex at a molar ratio of 1 / 3 are shown. 5A2-DOT-10 Cas9 / sgLuc was prepared at a total lipid concentration of 2 mg / mL in PBS buffer. 3 μL of the nanoparticle solution was dropped onto a carbon TEM grid and allowed to deposit for 1 minute, then blotted with filter paper. The TEM grid was then imaged using transmission electron microscopy (FEI Tecnai G2 Spirit Biotwin).

[0319] Figure 43 Confocal images are shown that show cellular uptake of PBS (control), free Cas9 / sgLuc complex (control), and 5A2-DOT-10 Cas9 / sgLuc in Hela-Luc cells 20 hours after treatment. The Cas9-EGFP fusion protein was used to track the subcellular distribution of the Cas9 / sgRNA complex. The Cas9 / sgLuc complex did not exhibit detectable green fluorescence above background (PBS) inside the cells, while bright green signals were detected after treatment with 5A2-DOT-10.

[0320] Figure 44A-44HShows that gene editing occurs rapidly and efficiently in vitro. (44A) T7EI cleavage assay of DNA isolated from HeLa-Luc cells treated with various nanoparticles and controls. Efficient gene editing was mediated by 5A2-DOT-10 delivering Cas9 / sgLucRNP (1 / 3 and 1 / 5). Insertion-deletion (%) at the Luc locus was quantified by ICE analysis. It should be noted that gene editing with LNP prepared using low pH citrate buffer (current and established methods) was 0%. (44B) Fluorescence microscopy images of HeLa-GFP cells after treatment with various formulations. Scale bar = 100 μm. Treatment with 5A2-DOT-10 Cas9 / sgGFP significantly reduced GFP fluorescence. (44C) Flow cytometry analysis of HeLa-GFP cells after treatment with various formulations. Only for the 5A2-DOT-10 Cas9 / sgGFP group, the peak of GFP-positive cells completely shifted to the left, indicating that almost all GFP-positive cells turned black. (44D) Time-dependent GFP fluorescence intensity of HeLa-GFP cells after various treatments. Permanent loss of GFP fluorescence was observed after day 2 with treatment with 5A2-DOT-10 Cas9 / sgGFP, while ICE analysis of Sanger sequencing data showed that insertion-deletion remained above 90% after day 2. (44E and 44F) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP alone, 5A2-SC8 loaded with Cas9 / sgGFP, C12-200, DLin-MC3-DMA LNP formulation containing 10% complementary DOTAP, traditional C12-200 and DLin-MC3-DMA LNP nanoparticle formulations loaded with Cas9 / sgGFP, and RNAiMAX loaded with Cas9 / sgGFP. GFP fluorescence was significantly reduced after treatment with all three DOTAP-modified formulations. ICE analysis of Sanger sequencing data further confirmed that the highest gene editing efficiency was obtained with 5A2-DOT-10 LNP. Mean ± standard deviation of the mean (n = 3). Statistical significance was determined using two-tailed Student's t-test. t value = 42.69, degrees of freedom (df) = 4 (P < 0.0001); t value = 16.75, degrees of freedom (df) = 4 (P < 0.0001); t value = 37.53, degrees of freedom (df) = 4 (P < 0.0001). A P value < 0.05 was considered statistically significant. (44G) 5A2-DOT-10Cas9 / sgGFP LNP was stored at 4 °C for 2 months. The nanoparticle diameter and PDI were monitored over time. (44H) Periodic treatment of HeLa-GFP cells with the stored LNP showed no loss of activity, indicating long-term LNP and RNP stability as well as transfection potential. All cells in the above experiments were treated with 24 nM sgRNA.

[0321] Figures 45A and 45B show gene editing by different nanopreparations in Hela-GFP cells. (45A) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with Cas9 / sgGFP alone, 5A2-DOT-10Cas9 / sgLuc, and 5A2-DOT-10Cas9 / sgGFP (at a total lipid / sgGFP weight ratio of 40:1). (45B) Mean fluorescence intensity (%) of Hela-GFP cells after treatment with 5A2-DOT-10Cas9 / sgGFP prepared at total lipid / sgGFP weight ratios of 10:1, 20:1, 30:1, and 40:1.

[0322] Figure 46A-46KShows that the generalizable RNP delivery strategy (Figure 40A) is universal for ionizable cationic lipid nanoparticles (DLNP, LLNP, SNALP) and for other cationic lipids that are positively charged at pH 7.4 and for other neutral buffers. (46A) Protocols for LNP formulations with different ionizable lipids. (46B) Details of LNP formulations with different ionizable lipids, including determination of the molar ratio and percentage of each component and the weight ratio of total lipid to sgRNA. (46C) Chemical structures of ionizable cationic lipids used in the formulations, including 5A2-SC8, C12-200, and Dlin-MC3-DMA. (46D) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP RNP encapsulated in 5A2-DOT-10, C12-200-DOT-10, and MC3-DOT-10. GFP fluorescence was significantly reduced after treatment with all three formulations. (46E) Preparation protocols for LNP formulations with different permanent cationic lipids. (46F) Details of LNP formulations with different permanent cationic lipids, including determination of the molar ratio and percentage of each component and the weight ratio of total lipid to sgRNA. (46G) Chemical structures of permanent cationic lipids used in the formulations, including DOTAP, DDAB, and EPC. (46H) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with Cas9 / sgGFP RNP encapsulated in 5A2-DOT-10, 5A2-DDAB-10, and 5A2-EPC-10. Instead of DOTAP, other cationic lipids (DDAB and EPC) were also able to achieve efficient gene editing. (46I) Protocols for LNP formulations in different buffers. (46J) Mean fluorescence intensity (%) of HeLa-GFP cells after treatment with 5A2-DOT-10 formulated with different buffers, including PBS, Opti-MEM, HEPES, and citrate buffer. RNP encapsulation and delivery require a neutral buffer. (46K) Indels (%) at the GFP locus in genomic DNA isolated from HeLa-GFP cells after treatment with 5A2-DOT-10 Cas9 / sgGFP LNP prepared with different buffers were measured using ICE analysis. All neutral buffers showed high gene editing in cells, indicating the importance of neutral buffers in nanoparticle preparation. Note that Figures 44E and 44F have been reproduced above in Figure 45 to combine relevant data for increased clarity.

[0323] Figure 47A-47JDemonstrated efficient multiplex genome editing in vivo. (47A) Schematic shows how delivery of Cas9 / sgTOM RNP activates Td-Tom expression in Td-Tomato transgenic mice. 5A2-DOT-X LNPs were injected locally (by intramuscular or intracerebral injection) and systemically (by intravenous injection via the tail vein) into Td-Tom mice. In vivo imaging of Td-Tom mice after intramuscular (1 mg / kg sgTom) (47B) or intracerebral (0.15 mg / kg sgTOM) (47D) injection of 5A2-DOT-10Cas9 / sgTOM showed bright red fluorescence in leg muscle or brain tissue, respectively. Successful CRISPR / Cas gene editing was further confirmed by confocal imaging of (47C) muscle and (47E) brain tissue sections. 5A2-DOT-10 had higher gene editing efficiency compared to the positive control RNAiMAX previously used for local RNP injection. (47F) In vivo imaging of Td-Tom mice after intravenous (IV) injection of 5A2-DOT-X Cas9 / sgTOM LNPs with different molar percentages of DOTAP. Td-Tom fluorescence (as a downstream readout of DNA editing) showed that low DOTAP percentages facilitated liver editing, while high DOTAP percentages facilitated lung editing (1.5 mg / kg sgTOM, IV). (47G) Successful CRISPR / Cas gene editing was further confirmed by confocal imaging. (47H) T7EI cleavage assays were performed on DNA isolated from liver and lung tissues after systemic intravenous treatment with 5A2-DOT-5, 5A2-DOT-10, 5A2-DOT-50, and 5A2-DOT-60 encapsulating Cas9 / sgPTEN. Indels (%) were calculated and reported. (47I) 5A2-DOT-50LNP (5A2-DOT-50-combination) containing a pooled sgRNA (sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1) with 6 targets was administered intravenously to td-Tom mice at a total RNA dose of 2 mg / kg (0.33 mg / kg per sgRNA). Gene editing at the TOM locus was confirmed by in vivo imaging, and (47J) editing at the other 5 loci was confirmed using T7EI cleavage assays on lung tissue.

[0324] Figure 48A-48HThe 5A2-DOT-X LNP simplifies the generation of complex mouse models. (48A) To establish an orthotopic liver-specific cancer model, 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP was injected into adult C57BL / 6 mice weekly (3 injections, total sgRNA of 2.5 mg / kg, intravenously, n = 4). At 12, 15, and 20 weeks, the mice were sacrificed and the livers were collected for tumor analysis. (48B) T7EI cleavage results from genomic DNA extracted from the liver confirmed gene editing at all three loci. (48C) Representative photographs of mouse livers containing tumors excised 20 weeks after injection. (48D) H&E and Ki67 staining further confirmed progressive tumor formation. Higher expression of the tumor proliferation biomarker Ki67 was detected in tumor lesions. Scale bar = 100 μm. (48E) To create an orthotopic lung-specific cancer model, 5A2-DOT-50LNP encapsulating Cas9 / sgEml4 / sgAlk RNP was injected into adult C57BL / 6 mice once (2 mg / kg) or twice (1.5 mg / kg per week for 2 weeks) (intravenously, n = 5). At 10, 16, and 24 weeks, the mice were sacrificed and the lungs were collected for tumor analysis. (48F) T7EI cleavage results from genomic DNA extracted from the lungs confirmed gene editing at the Eml4 and Alk loci. PCR amplicons of the Eml4-Alk rearrangement were also detected in all lungs treated with 5A2-DOT-50LNP. (48G) The Eml4-Alk rearrangement was further confirmed by subcloning and DNA sequencing (predicted = SEQ ID NO:50; clone 1 = SEQ ID NO:51; clone 2 = SEQ ID NO:52; clone 3 = SEQ ID NO:53; clone 4 = SEQ ID NO:54; clone 5 = SEQ ID NO:55; clone 6 = SEQ ID NO:56; clone 7 = SEQ ID NO:57; clone 8 = SEQ ID NO:58). (48H) H&E and Ki67 staining further confirmed progressive tumor formation. Higher expression of the tumor proliferation biomarker Ki67 was detected in lung tumor lesions. Scale bar = 100 μm.

[0325] Figures 49A and 49B show the gene editing efficiency of unmodified sgRNAs synthesized by in vitro transcription (IVT) compared to chemically modified and synthetic sgRNAs (2'-O-methyl 3'-thio-phosphate modification in the first 3 and last 3 nucleotides). (49A) Relative luciferase activity in Hela-Luc-Cas9 cells after treatment with IVT sgRNAs and chemically modified sgRNAs encapsulated in nanoparticles. (49B) T7EI assay to detect the gene editing efficiency of nanoparticles encapsulating Cas9 / IVT sgRNA and Cas9 / chemically modified sgRNA. Cleavage bands at 536 bp and 184 bp were clearly observed in the group treated with modified sgRNAs.

[0326] Figure 50 Shows gene editing of P53, PTEN, and RB1 genes in mouse liver after treatment with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP. After two weeks of weekly treatment, gene editing of liver genomic DNA at the PTEN, P53, and RB1 genomic loci was detected by T7EI assay. The PBS-treated group was used as a control. Cleavage bands were detected at 261 bp and 215 bp of the PCR amplicon targeting P53; cleavage bands were detected at 345 bp and 293 bp of the PCR amplicon targeting PTEN; cleavage bands were detected at 395 bp and 207 bp of the PCR amplicon targeting RB1.

[0327] Figure 51 Shows gene editing of P53, PTEN, and RB1 genes in mouse liver detected by T7EI assay after treatment with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP. The PBS-treated and 5A2-DOT-5 (without Cas9 / sgRNA) treatment groups alone were used as controls. T7EI results of genomic DNA extracted from mouse tumors 20 weeks after treatment with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP showed that knockout of these three genes induced tumorigenesis as cleavage bands were detected at all three genomic loci.

[0328] Figure 52 Shows representative photographs of mouse liver and excised tumors dissected from mice in the group treated with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP for 15 weeks.

[0329] Figure 53A-53CShows H&E and Ki67 staining images of mouse liver after 15 and 20 weeks of treatment with 5A2-DOT-5LNP alone (without Cas9 / sgRNA) (control) (53A), and tumors excised from mice in the group treated with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP for 20 weeks (53B). No morphological changes were detected in the treatment with 5A2-DOT-5LNP alone, indicating that the nanocarrier alone cannot cause tumors. Scale bar: 100 μm. (53C) Large view image of mouse liver tumor generation after 20 weeks of treatment with 5A2-DOT-5LNP encapsulating Cas9 / sgP53 / sgPTEN / sgRB1 RNP. Scale bar: 500 μm.

[0330] Figure 54A-54D Shows the generation of Eml4-Alk rearrangement in mouse lungs after 7 days of treatment with 5A2-DOT-50LNP (total sgRNA at 2 mg / kg) encapsulating Cas9 / sgEml4 / sgAlk RNP. Eml4 editing (54A) and Alk editing (54B) were detected in genomic DNA extracted from mouse lungs by T7EI assay. (54C) PCR analysis of genomic DNA extracted from mouse lungs to determine Eml4-Alk inversion. (54D) The PCR amplicons were subcloned, and the sequences of 6 independent clones were listed, and a representative chromatogram is presented above. The chromatogram is identical to that predicted for the Eml4-Alk rearrangement. (Predicted = SEQ ID NO:59; Clone 1 = SEQ ID NO:59; Clone 2 = SEQ ID NO:60; Clone 4 = SEQ ID NO:61; Clone 5 = SEQ ID NO:61; Clone 6 = SEQ ID NO:62)

[0331] Figure 55 Shows H&E and Ki67 staining images of mouse liver after 10 and 16 weeks of treatment with 5A2-DOT-50LNP alone (without Cas9 / sgRNA) (LNP dose equal to total sgRNA at 1 mg / kg). No morphological changes were detected in animals injected with 5A2-DOT-50LNP alone. Scale bar: 100 μm.

[0332] Figure 56 Shows a large view image of mouse lung tumor generation after 24 weeks of treatment with 5A2-DOT-50LNP encapsulating Cas9 / sgEml4 / sgAlk RNP. Scale bar: 500 μm. Several tumor lesions (highlighted) were observed from the H&E and Ki67 staining images.

[0333] Figure 57It is shown that 5A2-DOT-10LNP can effectively deliver ovalbumin (OVA) protein into the cytoplasm of HeLa-Luc cells. Before imaging by confocal microscopy, the cells were treated with free rhodamine-labeled OVA protein and 5A2-DOT-10LNP encapsulating rhodamine-labeled OVA for 22 h. Specific embodiments

[0334] Lipid nanoparticles (LNPs) are described herein, which are composed of 1) a permanent cationic lipid, 2) an ionizable cationic lipid, and 3) a phospholipid, and may optionally contain cholesterol and lipid PEG or both. The inclusion of the permanent cationic lipid is used to direct the LNP to specific organs such as the lung, lymph nodes, or spleen. The data presented herein indicate that this effect is general and the components are modular, indicating that 5A2-SC8 can be replaced by any ionizable cationic lipid, DOTAP can be replaced by any cationic lipid, and DOPE can be replaced by any phospholipid. In some embodiments, cholesterol and lipid PEG are also included, but formulations without cholesterol or lipid PEG are feasible. These vectors can deliver mRNA, sgRNA, and protein to specific organs in the body, thus addressing a major challenge. It is expected that these vectors can also deliver other nucleic acids (such as siRNA, miRNA, crRNA, trRNA, tRNA, etc.) and drugs (such as small molecules) to specific organs in the body.

[0335] A. Chemical definitions

[0336] When used in the context of chemical groups: "hydrogen" refers to -H; "hydroxy" refers to -OH; "oxo" refers to =O; "carbonyl" refers to -C(=O)-; "carboxy" refers to -C(=O)OH (also written as -COOH or -CO2H); "halo" independently refers to -F, -Cl, -Br, or -I; "amino" refers to -NH2; "hydroxyamino" refers to -NHOH; "nitro" refers to -NO2; imino refers to =NH; "cyano" refers to -CN; "isocyanate" refers to -N=C=O; "azido" refers to -N3; in the monovalent context, "phosphate" refers to -OP(O)(OH)2 or its deprotonated form; in the divalent context, "phosphate" refers to -OP(O)(OH)O- or its deprotonated form; "mercapto" refers to -SH; and "thio" refers to =S; "sulfonyl" refers to -S(O)2-; "hydroxy sulfonyl" refers to -S(O)2OH; "sulfonamide" refers to -S(O)2NH2; and "sulfinyl" refers to -S(O)-.

[0337] In the context of chemical formulas, the symbol "-" refers to a single bond, "=" refers to a double bond, and "≡" refers to a triple bond. The symbol "----" represents an optional bond which, if present, is either a single or a double bond. The symbol represents a single or a double bond. Thus, for example, the formula includes and it should be understood that none of these ring atoms forms part of more than one double bond. Further, it should be noted that when connecting one or two stereogenic atoms, the covalent bond symbol "-" does not indicate any preferred stereochemistry. Instead, it encompasses all stereoisomers and mixtures thereof. When drawn perpendicular through the bond (e.g., for a methyl group, ), the symbol indicates the point of attachment of the group. It should be noted that typically only larger groups are identified in this way to help the reader clearly identify the point of attachment. The symbol refers to a single bond where the group attached to the thick end of the wedge "comes out of the plane of the paper". The symbol refers to a single bond where the group attached to the thick end of the wedge "goes into the plane of the paper". The symbol refers to a single bond where the geometry around the double bond (e.g., E or Z) is not determined. Thus, both options and their combinations are contemplated. Any undefined valency on an atom of the structures shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen attached to that carbon is out of the plane of the paper.

[0338] When the group "R" is described as a "floating group" on a ring system, e.g., in the following formula:

[0339]

[0340] then R can replace any hydrogen atom attached to any of the ring atoms, including those depicted, implied or explicitly defined, so long as a stable structure is formed. When the group "R" is described as a "floating group" on a fused ring system, e.g., in the following formula:

[0341]

[0342] Then R can replace any hydrogen attached to any ring atom of any one of the fused rings, unless otherwise indicated. Replaceable hydrogens include the depicted hydrogens (e.g., the hydrogen attached to nitrogen in the above formula), implicit hydrogens (e.g., the hydrogens in the above formula that are not shown but are understood to be present), explicitly defined hydrogens, and optional hydrogens whose presence depends on the identity of the ring atom (e.g., when X is equal to -CH-, the hydrogen attached to group X), provided that a stable structure is formed. In the example shown, R can be located on the 5-membered or 6-membered ring of the fused ring system. In the above formula, the subscript letter "y" following the group "R" enclosed in parentheses represents a numerical variable. Unless otherwise indicated, this variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of replaceable hydrogen atoms of the ring or ring system.

[0343] 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, and the minimum number is as small as possible for the group / class being discussed. For example, it should be understood that the minimum number of carbon atoms in the group "alkenyl" (C≤8) " or the class "alkene" (C≤8) " is 2. In contrast, "alkoxy" (C≤10) " indicates an alkoxy group having 1 - 10 carbon atoms. "Cn-n′" defines the minimum number (n) and the maximum number (n′) of carbon atoms in the group. Thus, "alkyl" (C2-10) " represents those alkyl groups having 2 - 10 carbon atoms. These carbon number indicators can be before or after the chemical group or class they modify, and it can be included or not included in parentheses, without indicating any change in meaning. Thus, the terms "C5 alkene", "C5-alkene", "alkene" (C5) " and "alkene" C5 " are all synonymous.

[0344] When used to modify a compound or chemical group, the term "saturated" means that the compound or chemical group does not have carbon-carbon double bonds and carbon-carbon triple bonds, unless otherwise stated. When the 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 a substituted form of a saturated group, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds can be present. And when such bonds are present, carbon-carbon double bonds that may occur as part of keto-enol tautomerism or imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that no more of the substance can be dissolved in the solution.

[0345] The term "aliphatic", when used without the modifier "substituted", means 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 (which is linked by single carbon-carbon bonds (alkanes / alkyls)), or unsaturated (which has one or more carbon-carbon double bonds (alkenes / alkenyls) or has one or more carbon-carbon triple bonds (alkynes / alkynyls)).

[0346] The term "aromatic", when used to modify a compound or chemical group atom, means that the compound or chemical group contains a planar unsaturated ring of atoms that is stabilized by the interaction of bonds forming the ring.

[0347] The term "alkyl", when used without the modifier "substituted", means a monovalent saturated aliphatic group that has a carbon atom as a point of attachment, has a straight-chain or branched-chain acyclic structure, and has 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, tert-butyl, t-Bu or tBu) and -CH2C(CH3)3 (neopentyl) are non-limiting examples of alkyl groups. The term "alkanediyl" when used without the modifier "substituted" denotes a divalent saturated aliphatic group having 1 or 2 saturated carbon atoms as attachment points, having a straight-chain or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. Groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkanediyl groups. "Alkane" denotes a class of compounds having the formula H-R, where R is an alkyl group as defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms have been 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 a subset of substituted alkyl groups where the replacement of hydrogen atoms is limited to halogenation (i.e., -F, -Cl, -Br, or -I) such that there are no other atoms other than carbon, hydrogen, and halogen. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups where the replacement of hydrogen atoms is limited to fluorination such that there are no other atoms other than carbon, hydrogen, and fluorine. Groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0348] The term "cycloalkyl" when used without the modifier "substituted" denotes a monovalent saturated aliphatic group having a carbon atom as an attachment point, said carbon atom forming part of one or more non-aromatic ring structures, no carbon-carbon double or triple bonds, and 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" denotes a divalent saturated aliphatic group having 2 carbon atoms as attachment points, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group 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 group as defined above. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms have been 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.

[0349] The term "alkenyl", when used without the modifier "substituted", denotes a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, having a straight-chain 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", denotes a divalent unsaturated aliphatic group having 2 carbon atoms as the points of attachment, having a straight-chain or branched, straight-chain 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 should be noted that although alkenediyl groups are aliphatic, once attached at both ends, it does not exclude the group from forming part of an aromatic structure. The terms "olefin" and "alkene" are synonymous and denote a class of compounds having the formula H-R, where R is an alkenyl as defined above. Similarly, the terms "terminal olefin" and "α-olefin" are synonymous and denote an olefin having exactly one carbon-carbon double bond, where 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 have been 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 alkenyls.

[0350] The term "alkynyl", when used without the modifier "substituted", denotes a monovalent unsaturated aliphatic group having a carbon atom as the point of attachment, having a straight-chain or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. The term alkynyl as used herein 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. "Alkyne" denotes a class of compounds having the formula H-R, where R is an alkynyl. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms have been 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.

[0351] The term "aryl", when used without the modifier "substituted", denotes a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, the carbon atom forming part of one or more 6-membered aromatic ring structures, where the ring atoms are all carbon and where the group is not composed of atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. The term as used herein does not exclude the presence of one or more alkyl or aralkyl groups (permitted carbon number limitations) attached to the first aromatic ring or any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and monovalent groups derived from biphenyl. The term "arylene", when used without the modifier "substituted", denotes a divalent aromatic group having 2 aromatic carbon atoms as the points of attachment, the carbon atoms forming part of one or more 6-membered aromatic ring structures, where the ring atoms are all carbon and where the divalent group is not composed of atoms other than carbon and hydrogen. The term as used herein does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (permitted carbon number limitations) attached to the first aromatic ring or any additional aromatic rings present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be joined by one or more of the following: covalent bonds, alkanediyl, or alkenediyl groups (permitted carbon number limitations). Non-limiting examples of arylene groups include:

[0352]

[0353] "Arene" refers to a class of compounds having the formula H-R, where R is an aryl group, as 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 have been 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.

[0354] The term "aralkyl", when used without the modifier "substituted", refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided 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 groups have been 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 aralkyls are: (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-eth-1-yl.

[0355] The term "heteroaryl", when used without the modifier "substituted", denotes a monovalent aromatic group having an aromatic carbon or nitrogen atom as a point of attachment, said carbon or nitrogen atom forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein said heteroaryl is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. The heteroaryl ring may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. The use of this term herein does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (carbon number limitations permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl include furyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridyl (pyridinyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl and triazolyl. The term "N-heteroaryl" denotes a heteroaryl having a nitrogen atom as a point of attachment. The term "heteroarylene", when used without the modifier "substituted", denotes a divalent aromatic group having 2 aromatic carbon atoms, 2 aromatic nitrogen atoms, or 1 aromatic carbon atom and 1 aromatic nitrogen atom as 2 points of attachment, said atoms forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein said divalent group is not composed of atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be linked by one or more of the following: covalent bond, alkanediyl or alkenediyl groups (carbon number limitations permitting). The use of this term herein does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (carbon number limitations permitting) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroarylene include:

[0356]

[0357] "Heteroarene" denotes 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 have been 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.

[0358] The term "heterocycloalkyl", when used without the modifier "substituted", denotes a monovalent non-aromatic group having a carbon atom or a nitrogen atom as a point of attachment, which carbon atom or nitrogen atom forms part of one or more non-aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein said heterocycloalkyl is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. The heterocycloalkyl ring may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen or sulfur. If more than one ring is present, the rings may be fused or unfused. The term as used herein does not exclude the presence of one or more alkyl groups (within carbon number limitations) attached to the ring or ring system. Also, the 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 include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyranyl, oxiranyl and oxetanyl. The term "N-heterocycloalkyl" denotes a heterocycloalkyl 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", denotes a divalent cyclic group having 2 carbon atoms, 2 nitrogen atoms, or 1 carbon atom and 1 nitrogen atom as 2 points of attachment, said atoms forming part of one or more ring structures, wherein at least one ring atom is nitrogen, oxygen or sulfur, and wherein said divalent group is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be linked by one or more of the following: covalent bond, alkanediyl or alkenediyl groups (within carbon number limitations). The term as used herein does not exclude the presence of one or more alkyl groups (within carbon number limitations) attached to the ring or ring system. Also, the 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 heterocycloalkanediyl include:

[0359]

[0360] When these terms are used with the modifier "substituted", one or more hydrogen atoms have been 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.

[0361] The term "acyl", when used without the modifier "substituted", denotes the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl or heteroaryl, as 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 has been replaced by a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkane as defined above, in which at least one hydrogen atom has been replaced by a -CHO group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms (including the hydrogen atom directly attached to the carbon atom of the carbonyl or thiocarbonyl group, if any) have been 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.

[0362] The term "alkoxy", when used without the modifier "substituted", denotes the group -OR, where R is an alkyl group 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", "aralkyloxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the modifier "substituted", denote groups defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkanediyl" denotes the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio", when used without the modifier "substituted", denote the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane as defined above, where at least one hydrogen atom has been replaced with a hydroxy group. The term "ether" corresponds to an alkane as defined above, where at least one hydrogen atom has been replaced with an alkoxy group. When any of these terms is used with the modifier "substituted", one or more hydrogen atoms have been independently replaced with -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.

[0363] The term "alkylamino", when used without the modifier "substituted", denotes 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", denotes the group -NRR′, where R and R′ may be the same or different alkyl groups, or R and R′ may together represent an alkanediyl group. 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", denote groups defined as -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" denotes the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "acylamino" (acylamino group), when used without the modifier "substituted", denotes the group -NHR, where R is acyl, as defined above. A non-limiting example of an acylamino group is -NHC(O)CH3. The term "alkylimino", when used without the modifier "substituted", denotes 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 have been 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 acylamino groups.

[0364] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one", but it is also consistent with the meanings of "one or more", "at least one", and "more than one".

[0365] Throughout the present application, the term "about" is used to indicate that a value includes the inherent variability of the error of the device and method used to measure that value, or the variability that exists among study subjects.

[0366] As used in the present application, the term "average molecular weight" represents the relationship between the number of moles of each polymeric substance and the molar mass of that substance. Specifically, each polymer molecule can have a different level of polymerization and thus a different molar mass. The average molecular weight can be used to represent the molecular weights of a plurality of polymer molecules. The average molecular weight is generally synonymous with the average molar mass. Specifically, there are three main types of average molecular weight: number average molar mass, weight (mass) average molar mass, and z-average molar mass. In the context of the present application, unless otherwise indicated, the average molecular weight represents 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.

[0367] The terms "comprising", "having", and "including" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises", "comprising", "has", "having", "includes", and "including", is also open-ended. For example, any method that "comprises", "has", or "includes" one or more steps is not limited to having only those one or more steps, and also encompasses other unlisted steps.

[0368] When used in this specification and / or the claims, the term "effective" means sufficient to achieve a desired, expected, or intended result. When used in the context of treating a patient or subject with a compound, an "effective amount", "therapeutically effective amount", or "pharmaceutically effective amount" means the amount of the compound that is sufficient to effect such treatment of the disease when administered to the subject or patient to treat the disease.

[0369] The term "IC 50 " represents the inhibitory dose that achieves 50% of the maximum response. This quantitative measure indicates the amount of a particular drug or other substance (inhibitor) 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 half.

[0370] An "isomer" of a first compound is a separate compound in which each molecule contains the same component atoms as the first compound, but in which the three-dimensional configuration of those atoms is different.

[0371] 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.

[0372] As used herein, "pharmaceutically acceptable" generally refers to those compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for contact with the tissues, organs, and / or bodily fluids of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0373] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present disclosure that are pharmaceutically acceptable as defined above and have the desired pharmacological activity. Such salts include acid addition salts formed with the following acids: inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropanoic 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 sulfates, aromatic sulfates, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropanoic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfate, 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, propanoic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tert-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 an inorganic base or an organic base. 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 recognized that the particular anion or cation forming part of any salt of the present disclosure is not critical, so long as the salt as a whole is pharmaceutically acceptable. Other examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (edited by P. H. Stahl & C. G. Wermuth, Verlag Helvetica Chimica Acta, 2002).

[0374] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a chemical agent.

[0375] "Prevention" or "preventing" includes: (1) inhibiting the onset of a disease in a subject or patient who may be at risk of and / or predisposed to the disease but has not yet experienced or exhibited any or all symptoms or signs of the disease; and / or (2) slowing the onset of symptoms or signs of a disease in a subject or patient who may be at risk of and / or predisposed to the disease but has not yet experienced or exhibited any or all symptoms or signs of the disease.

[0376] "Repeating unit" is the simplest structural entity of certain materials, e.g., frameworks and / or polymers, whether organic, inorganic, or organometallic. In the case of a polymer chain, the repeating units are connected together in sequence along the chain, like the beads of a necklace. For example, in polyethylene -[-CH2CH2-] n -, the repeating unit is -CH2CH2-. The subscript "n" represents the degree of polymerization, i.e., the number of repeating units connected together. When the value of "n" is undefined or in the absence of "n", it merely indicates the repetition of the formula within the brackets and the polymeric nature of the material. The concept of repeating unit is equally applicable to cases where the connectivity between repeating units extends three-dimensionally, such as in metal-organic frameworks, modified polymers, thermosetting polymers, etc. In the context of dendrimers, the repeating unit can also be described as a branching unit, inner layer, or generation. Similarly, the end groups can also be described as surface groups.

[0377] "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 configuration of those atoms is different. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other like left and right hands. "Diastereoisomers" 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 (although not necessarily an atom) in a molecule carrying multiple groups such that the interchange of any two groups produces a stereoisomer. In organic compounds, chiral centers are usually carbon, phosphorus, or sulfur atoms, although other atoms can also become stereogenic centers in organic and inorganic compounds. A molecule can have multiple stereogenic centers, giving rise to many stereoisomers of it. In compounds whose stereoisomerism is attributed to tetrahedral stereogenic centers (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers does not exceed 2n , wherein n is the number of tetrahedral stereocenters. Molecules with symmetry often have a smaller number than the maximum possible number of stereoisomers. 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%. Generally, using techniques known in the art, enantiomers and / or diastereomers can be resolved or separated. It is contemplated that for any stereocenter or chiral axis for which the stereochemistry has not been defined, the stereocenter or chiral axis can exist in its R form, S form, or as a mixture of the R and S forms (including racemic and non-racemic mixtures). The phrase "substantially free of other stereoisomers" as used herein means that the composition contains ≤ 15%, more preferably ≤ 10%, even more preferably ≤ 5%, or most preferably ≤ 1% of one or more additional stereoisomers.

[0378] "Treatment" or "treating" includes (1) inhibiting the disease in a subject or patient who is experiencing or exhibiting the symptoms or manifestations of the disease (e.g., preventing further development of the symptoms and / or manifestations), (2) ameliorating the disease in a subject or patient who is experiencing or exhibiting the symptoms or manifestations of the disease (e.g., reversing the symptoms and / or manifestations), and / or (3) achieving any measurable alleviation of the disease in a subject or patient who is experiencing or exhibiting the symptoms or manifestations of the disease.

[0379] The above definitions supersede any conflicting definitions in any of the references incorporated herein by reference. However, the fact that certain terms are defined should not be taken to mean that any term that is not defined is uncertain. On the contrary, all terms used are considered to describe the present disclosure in a manner that would be understood by one of ordinary skill in the art and enable the practice of the present disclosure.

[0380] B. Cationic Ionizable Lipids

[0381] In some aspects of the present disclosure, there are provided compositions comprising compounds containing lipophilic and cationic components, wherein the cationic component is ionizable. In some embodiments, the cationic ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated and uncharged at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group can contain one or more protonatable amines capable of forming a cationic group at physiological pH. The cationic ionizable lipid compound can further comprise one or more lipid components, such as two or more having C6-C 24Fatty acids of alkyl or alkenyl carbon groups. These lipid groups can be linked by ester bonds or can be further added to sulfur atoms via Michael addition. In some embodiments, these compounds can be dendrimers, dendrons, polymers, or combinations thereof.

[0382] In some aspects of the present disclosure, there are provided compositions comprising compounds containing lipophilic and cationic components, wherein the cationic component is ionizable. In some embodiments, the ionizable cationic lipid represents a lipid and lipid-like molecule having a nitrogen atom that can acquire a charge (pK a ). These lipids are known in the literature as cationic lipids. These molecules having an amino group typically have 2-6 hydrophobic chains, typically alkyl or alkenyl such as C6-C24 alkyl or alkenyl, but can have at least 1 or more than 6 tails. In some embodiments, these cationic ionizable lipids are dendrimers, which are polymers that exhibit regular dendritic branching and are formed by sequentially or generationally adding branching layers to or from a core and are characterized by a single core, at least one internal branching layer, and a surface branching 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, molecular architectures having an inner core, an inner layer (or "generation") of repeating units regularly attached to the starting core, and an outer surface capped with end groups attached to the outermost generation. A "dendron" is a dendritic polymeric substance having branches emanating from a focal point, which is the core, or can be directly or through a linking moiety attached to the core to form a larger dendrimer. In some embodiments, the dendrimer structure has repeating groups radiating from a central core, which are doubled for each branch with each repeating unit. In some embodiments, the dendrimers described herein can be described as small molecules, medium-sized molecules, lipids, or lipid-like substances. These terms can be used to describe compounds having a dendron-like appearance as described herein (e.g., molecules radiating from a single focal point).

[0383] Although dendrimers are polymers, they may be superior to traditional polymers because they have a controllable structure, a single molecular weight, numerous and controllable surface functional groups, and a traditionally spherical conformation after reaching a certain generation. Dendrimers can be prepared by the sequential reaction of each repeating unit to produce a monodisperse, dendritic and / or generation-structured polymer structure. A single dendrimer consists of a central core molecule to which dendritic wedges are attached at one or more functional sites. Depending on the assembly monomers used in the preparation process, the surface layer of the dendrimer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic or lipophilic groups.

[0384] Changing the functional groups and / or chemical properties of the core, repeating units, and surface or capping groups can modulate their physical properties. Some of the properties that can be changed include, but are not limited to, solubility, toxicity, immunogenicity, and bioadhesive ability. Dendrimers are often described by their generation or the number of repeating units in the branches. A dendrimer consisting only of the core molecule is called generation 0, and each successive repeating unit along all branches is generation 1, generation 2, and so on, until the capping or surface groups. In some embodiments, a half-generation may be produced only by the first condensation reaction with an amine rather than the second condensation reaction with a thiol.

[0385] The preparation of dendrimers requires a level of synthetic control achieved through a series of stepwise reactions that involve building the dendrimer through each successive group. Dendrimer synthesis can be convergent or divergent. During divergent dendrimer synthesis, the molecule is assembled from the core to the periphery in a stepwise process that involves attaching one generation to the previous generation and then changing the functional groups for the next reaction stage. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization would result in highly branched molecules that are not monodisperse and are otherwise called hyperbranched polymers. Due to steric effects, the dendrimer repeating units continue to react to produce spherical or globular molecules until steric overcrowding prevents complete reaction at a particular generation and disrupts the monodispersity of the molecule. Thus, in some embodiments, dendrimers of generations G1 - G10 are specifically contemplated. In some embodiments, the dendrimer contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therefrom. In some embodiments, the dendrimers used herein are G0, G1, G2, or G3. However, the possible number of generations (such as 11, 12, 13, 14, 15, 20, or 25) can be increased by reducing the spacer units in the branched polymer.

[0386] In addition, dendrimers have two main chemical environments: the environment created by specific surface groups on the capping generation, and the interior of the dendrimer structure that may be shielded from the bulk medium and surface groups due to higher-order structures. Due to these different chemical environments, dendrimers have found many different potential uses, including in therapeutic applications.

[0387] In some aspects, the differential reactivity of acrylate and methacrylate groups with amines and thiols is used to assemble dendrimers that can be used in the present compositions. The dendrimers can include secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines, and methacrylate with thiol groups. In addition, the repeating units of the dendrimers can contain groups that are degradable under physiological conditions. In some embodiments, these repeating units can contain one or more geminal diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendrimerization in only one direction. In other embodiments, the core molecule is a polyamine having multiple different dendritic branches, each branch can contain one or more repeating units. The dendrimers can be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are on heteroatoms such as nitrogen atoms. In some embodiments, the capping group is a lipophilic group such as a long-chain alkyl or alkenyl group. In other embodiments, the capping group is a long-chain haloalkyl or haloalkenyl group. In other embodiments, the capping group is an aliphatic or aromatic group containing an ionizable group such as amine (-NH2) or carboxylic acid (-CO2H). In other embodiments, the capping group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as hydroxyl groups, amide groups, or esters.

[0388] The cationic ionizable lipids of the present disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be isolated in optically active or racemic forms. 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 meant. The cationic ionizable lipids may exist as racemates 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 may have the S or R configuration. In addition, one or more of the cationic ionizable lipids are expected to exist as structural isomers. In some embodiments, the compounds have the same chemical formula but 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 may present a fully reacted primary amine and then a mixture of reacted secondary amines.

[0389] The chemical formulas used to represent the cationic ionizable lipids of the present disclosure will generally show only one of the several different possible tautomers. For example, many types of keto 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 regardless of which tautomer is the most prevalent, all tautomers of the given chemical formula are meant.

[0390] The cationic ionizable lipids of the present disclosure may also have the following advantages: compared to compounds known in the prior art (whether for the indications described herein or otherwise), they may be more effective, less toxic, have a longer duration of action, be more potent, produce fewer side effects, be more readily absorbed, and / or have a better pharmacokinetic profile (e.g., higher oral bioavailability and / or lower clearance rate), and / or have other useful pharmacological, physical, or chemical properties.

[0391] In addition, 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 those atoms having the same atomic number but different mass numbers. As a general example and not by way of limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C.

[0392] It should be recognized that the particular anion or cation forming part of any salt form of the cationic ionizable lipid provided herein is not critical, provided that the salt as a whole is pharmaceutically acceptable. Other 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.

[0393] In some embodiments, the ionizable cationic 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 therefrom. 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 therefrom.

[0394] C. Selective Organ Targeting (SORT) Compounds

[0395] In some aspects, the present disclosure encompasses one or more selective organ targeting (SORT) compounds that result in the selective delivery of the composition to a particular organ. The compound can be a lipid, small molecule therapeutic agent, sugar, vitamin, peptide, or protein.

[0396] In some embodiments, the selective organ targeting (SORT) compound is present in the composition in a molar ratio 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 therefrom. In some embodiments, the SORT compound can 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%.

[0397] In some embodiments, the SORT compound can be a lipid. A lipid is a small molecule having two or more C6-C 24 alkyl or alkenyl chains. A small molecule therapeutic agent is a compound containing fewer than 100 non-hydrogen atoms and having a weight less than 2,000 daltons. A sugar is a molecule containing the molecular formula C n H 2n O na molecule of (where n is from 3 to 7), or a combination of multiple molecules of this formula. A protein is an amino acid sequence containing at least 3 amino acid residues. A protein without a formal tertiary structure can also be called a peptide. A protein can also contain a complete protein with a tertiary structure. A vitamin is a macronutrient and is composed 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 compounds of vitamin C, vitamin D, vitamin E, and vitamin K.

[0398] 1. Permanent cationic lipid

[0399] In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic components and a permanent cationic group. The permanent cationic lipid can contain a group having a positive charge (regardless of pH). One type of permanent cationic group that can be used in the permanent cationic lipid is a quaternary ammonium group. These permanent cationic lipids include structures such as those described in the following formulas:

[0400]

[0401] where:

[0402] Y1, Y2, or Y3 are each independently X1C(O)R1 or X2N + R3R4R5; provided that at least one of Y1, Y2, and Y3 is X2N + R3R4R5;

[0403] R1 is C1-C 24 alkyl, C1-C 24 substituted alkyl, C1-C 24 alkenyl, C1-C 24 substituted alkenyl;

[0404] X1 is O or NR a , where R a is hydrogen, C1-C4 alkyl, or C1-C4 substituted alkyl;

[0405] X2 is C1-C6 alkanediyl or C1-C6 substituted alkanediyl;

[0406] 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;

[0407] A1 is an anion having a charge equal to that of X2N in the compound +The number of R3R4R5 groups.

[0408] In another embodiment, the permanent cationic lipid is further defined by the following formula:

[0409]

[0410] Wherein:

[0411] R6 - R9 are each independently C1 - C 24 alkyl, C1 - C 24 substituted alkyl, C1 - C 24 alkenyl, C1 - C 24 substituted alkenyl; provided that at least one of R6 - R9 is a C8 - C 24 group; and

[0412] A2 is a monovalent anion.

[0413] In another embodiment, the permanent cationic lipid is further defined by the following formula:

[0414]

[0415] Wherein:

[0416] R1 and R2 are each independently alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;

[0417] R3, R3′ and R3″ are each independently alkyl (C≤6) or substituted alkyl (C≤6) ;

[0418] R4 is alkyl (C≤6) or substituted alkyl (C≤6) ; and

[0419] X - is a monovalent anion.

[0420] In some embodiments, the permanent cationic lipid is present in an amount of about 4 to about 16 mole percent of the total lipid composition. The composition can contain about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mole percent or any range derivable therefrom. In other embodiments, the composition can comprise about 18 to about 66 mole percent of the total lipid composition. In some embodiments, the composition can 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 therefrom.

[0421] 2. Permanent anionic lipids

[0422] In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic components and permanent anionic groups. One anionic group that can be used in the permanent anionic lipid is a phosphate group. The phosphate group can be a deprotonated compound having a negative charge at a pH below 8, 9, 10, 11, 12, 13 or 14. The hydrophobic component can be one or more C6-C 24 alkyl or alkenyl groups. The compound can have one hydrophobic group, two hydrophobic groups or three hydrophobic groups.

[0423] In some embodiments, the permanent anionic lipid has a structure of the following formula:

[0424]

[0425] Wherein:

[0426] R1 and R2 are each independently an alkyl (C8-C24) , alkenyl (C8-C24) or a substituted form of either group;

[0427] R3 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) or -Y1-R4, wherein:

[0428] Y1 is an alkanediyl (C≤6) or a substituted alkanediyl (C≤6) ; and

[0429] R4 is an acyloxy (C≤8-24) or a substituted acyloxy (C≤8-24) .

[0430] 3. Phosphatidylcholine

[0431] In some aspects, the present disclosure provides one or more lipids having one or more hydrophobic components, a cationic amino group and a negatively charged phosphate group. The cationic amino group can be a quaternary amine having three methyl groups attached to the nitrogen atom. The hydrophobic component can be one or more C6-C 24 alkyl or alkenyl groups. The compound can have one hydrophobic group, two hydrophobic groups or three hydrophobic groups. In some embodiments, the phosphatidylcholine compound is further defined as:

[0432]

[0433] Wherein:

[0434] R1 and R2 are each independently an alkyl(C8-C24) 、alkenyl (C8-C24) or a substituted form of any one of the groups;

[0435] R3, R3′ and R3″ are each independently an alkyl group (C≤6) or a substituted alkyl group (C≤6) ; and

[0436] X - is a monovalent anion.

[0437] D. Additional Lipids in Lipid Nanoparticles

[0438] In some aspects of the present disclosure, a composition containing one or more lipids is mixed with a cationic ionizable lipid to produce a composition. In some embodiments, the cationic ionizable lipid is mixed with 1, 2, 3, 4, or 5 different types of lipids. It is contemplated that the cationic ionizable lipid can be mixed with multiple different lipids of a single type. In some embodiments, the cationic ionizable lipid composition comprises at least a steroid or steroid derivative, a PEG lipid, and a phospholipid.

[0439] In some embodiments, the lipid nanoparticles are preferentially delivered to a 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, brain stem, 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 a part of one of these organ systems. As used herein, the term "preferential delivery" is used to mean a composition that is delivered to a target organ or organ system at at least 25% of the administered dose. This term is used to mean a composition that is delivered to a target organ or organ system at at least 25%, 50%, or at least 75% of the administered dose.

[0440] 1. Steroids and Steroid Derivatives

[0441] In some aspects of the present disclosure, a cationic ionizable lipid is mixed with one or more steroids or steroid derivatives to produce a composition. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a four-ring 17-carbon cyclic structure, which may further contain one or more substitutions, including alkyl, alkoxy, hydroxy, oxo, acyl, or double bonds between two or more carbon atoms. In one aspect, the ring structure of the steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:

[0442]

[0443] In some embodiments, the steroid derivative comprises the above ring structure having one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:

[0444]

[0445] In some embodiments of the present disclosure, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure is further defined by the following formula:

[0446]

[0447] As described above, cholestane derivatives include one or more non-alkyl substitutions of the above ring systems. In some embodiments, the cholestane or cholestane derivative is cholestene or a cholestene derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is cholestere and a sterol or its derivative.

[0448] In some embodiments, the composition may further comprise a molar percentage of steroid of from about 40 to about 46 of the total lipid composition. In some embodiments, the molar percentage is about 40, 41, 42, 43, 44, 45, to about 46 or any range derivable therein. In other embodiments, the molar percentage of steroid relative to the total lipid composition is from about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 or any range derivable therein.

[0449] 2. PEG or PEGylated lipid

[0450] In some aspects of the present disclosure, the polymer is mixed with one or more polyethylene glycolylated lipids (or PEG lipids) to produce a lipid composition. In some embodiments, the present disclosure includes the use of any lipid that has a PEG group attached thereto. In some embodiments, the PEG lipid is a diglyceride that also contains a PEG chain attached to the glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl 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-conjugated ceramide, PEG-modified dialkylamine, and PEG-modified 1,2-diacyl-oxypropane-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 moiety 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. U.S. Patent 5,820,873, WO 2010 / 141069, or U.S. Patent 8,450,298 teach some non-limiting examples of lipids that can be used in the present disclosure, which are incorporated herein by reference.

[0451] In another aspect, the PEG lipid has the following formula:

[0452]

[0453] Wherein: R 12 and R 13 are each independently an alkyl (C≤24) , alkenyl (C≤24) or a substituted form of any of these groups; R e is hydrogen, an alkyl (C≤8) or a substituted alkyl (C≤8) ; and x is 1 - 250. In some embodiments, R e is an alkyl (C≤8)Such as methyl. R 12 and R 13 are each independently an alkyl group (C≤4-20) . In some embodiments, x is 5 - 250. In one embodiment, x is 5 - 125 or x is 100 - 250. In some embodiments, the PEG lipid is 1,2 - dimyristoyl - sn - glycerol, methoxypolyethylene glycol.

[0454] In another aspect, the PEG lipid has the following formula:

[0455]

[0456] where: n1 is an integer between 1 and 100, and n2 and n3 are each independently selected from integers between 1 and 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 5 - 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 - 23. In some embodiments, n3 is 11 to about 17.

[0457] In some embodiments, the composition may further comprise a molar percentage of PEG lipid to the total lipid composition of 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 therefrom. In other embodiments, the molar percentage is 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 therefrom.

[0458] 3. Phospholipid

[0459] In some aspects of the present disclosure, the polymer is mixed with one or more phospholipids to produce a composition. In some embodiments, any lipid that also contains a phosphate ester group. In some embodiments, the phospholipid is a structure containing one or two long-chain C6-C24 alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate ester 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 such as 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, where zwitterionic lipids are defined as lipids and lipid-like molecules having a positive charge and a negative charge.

[0460] In some embodiments, the composition can further comprise a molar percentage of phospholipid in 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 about 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, to about 20 or any range derivable therein.

[0461] E. Nucleic Acids and Nucleic Acid-Based Therapeutic Agents

[0462] 1. Nucleic Acids

[0463] 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 in a weight ratio of from about 5:1 to about 1:100 with the lipid composition. In some embodiments, the weight ratio of the nucleic acid to the 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 therefrom. In some embodiments, the weight ratio is about 1:40. Additionally, it should be clear that the present disclosure is not limited to the specific nucleic acids disclosed herein. The present disclosure is not limited in scope to any particular source, sequence, or type of nucleic acid, however, one of ordinary skill in the art can readily identify related homologs in a variety of other nucleic acid sources, including nucleic acids from non-human species (e.g., mouse, rat, rabbit, dog, monkey, gibbon, chimpanzee, ape, baboon, cow, pig, horse, sheep, cat, and other species). Nucleic acids contemplated for use in the present disclosure may comprise sequences based on naturally occurring sequences. Given the degeneracy of the genetic code, sequences having at least about 50%, typically at least about 60%, more typically about 70%, most typically about 80%, preferably at least about 90%, and most preferably about 95% of the nucleotides are identical to the nucleotide sequence of the naturally occurring sequence. In another embodiment, the nucleic acid is a sequence complementary to the naturally occurring sequence, or is 75%, 80%, 85%, 90%, 95%, and 100% complementary. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or longer are encompassed herein.

[0464] The nucleic acids used herein may be derived from genomic DNA, i.e., directly cloned from the genome of a particular organism. However, in a preferred embodiment, the nucleic acid will comprise complementary DNA (cDNA). Also encompassed are cDNA plus natural introns or introns from another gene; such engineered molecules are sometimes referred to as "mini-genes". At a minimum, these and other nucleic acids of the present disclosure can be used as molecular weight standards in, for example, gel electrophoresis.

[0465] The term "cDNA" is intended to mean DNA prepared using messenger RNA (mRNA) as a template. The advantage of using cDNA, as compared to genomic DNA or DNA polymerized from genomic, non-processed or partially processed RNA templates, is that cDNA contains primarily the coding sequences of the corresponding protein. There may be instances where all or part of the genomic sequence is preferred, such as when non-coding regions are required for optimal expression, or when non-coding regions such as introns are to be targeted in an antisense strategy.

[0466] In some embodiments, the nucleic acid comprises one or more antisense segments that inhibit the expression of a gene or gene product. Antisense methods utilize the fact that nucleic acids tend to pair with "complementary" sequences. Complementary means that a polynucleotide is a polynucleotide capable of base pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine pairing with cytosine (G:C) and adenine pairing with thymine (A:T) in the case of DNA, or adenine pairing with uracil (A:U) in the case of RNA. The inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. in the hybridizing sequences does not interfere with base pairing.

[0467] Targeting double-stranded (ds) DNA with a polynucleotide results in triple helix formation; targeting RNA results in double helix formation. Antisense polynucleotides specifically bind to their target polynucleotides when introduced into target cells and interfere with transcription, RNA processing, transport, translation, and / or stability. Antisense RNA constructs or DNA encoding such antisense RNA can be used to inhibit gene transcription or translation or both in host cells in vitro or in vivo (such as within a host animal including a human subject).

[0468] Antisense constructs can be designed to bind to the promoter and other control regions, exons, introns, or even exon-intron boundaries of a gene. It is expected that the most effective antisense constructs will include regions complementary to intron / exon splice junctions. Thus, a preferred embodiment is proposed to include an antisense construct having complementarity to a region within 50 - 200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without severely affecting its target selectivity. The amount of exon material included will vary depending on the specific exon and intron sequences used. Whether too much exon DNA is included can be easily tested simply by testing the construct in vitro to determine whether normal cell function is affected or the expression of the relevant gene with the complementary sequence is affected.

[0469] As described above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, when sequences of 15 bases in length have complementary nucleotides at 13 or 14 positions, they can be referred to as complementary. Naturally, completely complementary sequences will be sequences that are completely complementary over their entire length and have no base mismatches. Other sequences with a lower degree of homology are also encompassed. For example, antisense constructs can be designed with limited regions of high homology but also containing non-homologous regions (e.g., ribozymes; see below). These molecules, although having less than 50% homology, will bind to the target sequence under appropriate conditions.

[0470] 2. Modified nucleobases

[0471] In some embodiments, the nucleic acids of the present disclosure comprise one or more modified nucleosides, which nucleosides comprise modified sugar moieties. Such compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity to a target nucleic acid relative to oligonucleotides comprising only nucleosides having naturally occurring 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 may comprise one or more substitutions corresponding to the substitutions of the substituted sugar moiety.

[0472] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents, which non-bridging sugar substituents include, but are not limited to, substituents at the 2' and / or 5' positions. 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 selected from allyl, amino, azido, thio, O-allyl, O--C1-C 10 alkyl, O--C1-C 10 substituted alkyl; 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 a 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 more than one non-bridging sugar substituent, for example, a T-F-5'-methyl sugar moiety (for additional 5',2'-disubstituted sugar moieties and nucleosides, see, for example, PCT International Application WO 2008 / 101157).

[0473] Nucleosides comprising 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 the group consisting of: halogen, allyl, amino, azido, 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-alkenyl-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 a substituted or unsubstituted C1-C 10 alkyl. These 2'-substituents may be further substituted by one or more substituents independently selected from hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), mercapto, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0474] 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 ), where each R m and R n is independently H, an amino protecting group, or a substituted or unsubstituted C1-C 10 alkyl.

[0475] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing 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.

[0476] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety containing a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0477] Certain modified sugar moieties contain a bridging sugar substituent that forms a second ring, thereby producing a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety contains a bridge between the 4' and 2' furanose ring atoms. Examples of such 4' to 2' sugar substituents include, but are not limited to: --[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 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 September 2, 2004); 4'-CH2--O--N(R)-2', and 4'-CH2--N(R)--O-2'-, where each R is independently H, a protecting group or a 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 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).

[0478] In some embodiments, such 4' to 2' bridges independently contain 1 to 4 linking groups independently selected from the following: --[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 )--; wherein:

[0479] x is 0, 1 or 2;

[0480] n is 1, 2, 3 or 4;

[0481] Each R a and R b is independently H, a protecting group, a hydroxyl group, 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, a heterocyclic residue, a substituted heterocyclic residue, a heteroaryl, a substituted heteroaryl, a C5-C7 cycloaliphatic residue, a substituted C5-C7 cycloaliphatic residue, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, an acyl group (C(=O)--H), a substituted acyl group, CN, a sulfonyl group (S(=O)2-J1) or a sulfinyl group (S(=O)-J1); and

[0482] 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, an acyl group (C(=O)--H), a substituted acyl group, a heterocyclic residue, a substituted heterocyclic residue, C1-C 12 aminoalkyl, substituted C1-C 12 aminoalkyl or a protecting group.

[0483] Nucleosides containing bicyclic sugar moieties are referred to as bicyclic nucleosides or BNA. Bicyclic nucleosides include, but are not limited to: (A) α-L-methylenoxy (4'-CH2--O-2') BNA, (B) β-D-methylenoxy (4'-CH2--O-2') BNA (also known as locked nucleic acid or LNA), (C) ethylenoxy (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 (methylenoxy) (4'-CH(CH3)--O-2') BNA (also known as constrained ethyl or cEt), (G) methylene-sulfur (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 (ethylenoxy) (4'-CH(CH2OMe)-O-2') BNA (also known as constrained MOE or cMOE).

[0484] Other bicyclic sugar moieties are known in the art, e.g., 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 (July 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. Patents 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 Publication Nos. US2004 / 0171570, US 2007 / 0287831, and US2008 / 0039618; U.S. Serial 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.

[0485] In some embodiments, the bicyclic sugar moiety and the nucleoside incorporating such a bicyclic sugar moiety are further defined by the isomeric configuration. For example, a nucleoside containing a 4'-2' methylene-oxy bridge can be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4'-CH2--O-2') bicyclic nucleosides have been incorporated into antisense oligonucleotides that exhibit antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0486] In some embodiments, the substituted sugar moiety comprises 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, e.g., with 5'-methyl or 5'-vinyl).

[0487] In some embodiments, the modified sugar moiety is a sugar surrogate. In some such embodiments, the oxygen atom of a naturally occurring sugar is replaced with, e.g., a sulfur, carbon, or nitrogen atom. In some such embodiments, such a modified sugar moiety also comprises bridging and / or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4'-sulfur atom and substitutions at the 2'-position (see, e.g., published U.S. patent application US2005 / 0130923) and / or the 5'-position. As another 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).

[0488] In some embodiments, the sugar surrogate comprises a ring having other than 5 atoms. For example, in some embodiments, the sugar surrogate comprises a 6-membered tetrahydropyran. Such a tetrahydropyran can be further modified or substituted. Nucleosides comprising such a modified tetrahydropyran include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. and Med. Chem. (2002) 10:841-854), and fluoro-HNA (F-HNA).

[0489] In some embodiments, there is provided a modified THP nucleoside of formula VII, where 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 not 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, where 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.

[0490] Many other bicyclic and tricyclic sugar surrogate ring systems are also known in the art and can be used to modify nucleosides for incorporation into antisense compounds (see, e.g., the review article: Leumann, J.C, Bioorganic and Medicinal Chemistry, 2002, 10, 841 - 854).

[0491] Combinations of modifications are also provided, not limited to, for example, 2'-F-5'-methyl substituted nucleosides (for other disclosed 5',2'-disubstituted nucleosides, see PCT International Application WO 2008 / 101157) and substitution of the ribosyl epoxide atom with S and further substitution at the 2'-position (see U.S. Patent Publication US 2005 / 0130923), or alternatively 5'-substitution of bicyclic nucleic acids (see PCT International Application WO 2007 / 134181, in which 4'-CH2--O-2' bicyclic nucleosides are further substituted at the 5'-position with 5'-methyl or 5'-vinyl). The synthesis and preparation of carbocyclic bicyclic nucleosides and their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., 2007).

[0492] 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 has the desired characteristics. In some embodiments, the oligonucleotide comprises one or more RNA-like nucleosides. In some embodiments, the oligonucleotide comprises one or more DNA-like nucleotides.

[0493] In some embodiments, the nucleosides of the present disclosure comprise one or more unmodified nucleobases. In certain embodiments, the nucleosides of the present disclosure comprise one or more modified nucleobases.

[0494] In some embodiments, the modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, expanded-size bases, and fluorinated bases as defined herein. 5-Substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines as defined herein, including 2-aminopropyladenine, 5-propynyluracil; 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-halouracils and cytosines, 5-propynyl(CH3)uracils and cytosines, and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-thioalkyl, 8-hydroxy, 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, universal bases, hydrophobic bases, promiscuous bases, expanded-size bases, and fluorinated bases. Other 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), G-clamps such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine ( 2 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. Other nucleobases include those disclosed in U.S. Patent 3,687,808; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., ed., John Wiley and Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, Y.S., 1993.

[0495] Representative U.S. patents that teach the preparation of certain of the above-described modified nucleobases and other modified nucleobases include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653; and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0496] In some embodiments, the present disclosure provides oligonucleotides comprising linked nucleosides. In such embodiments, the nucleosides can be linked together using any internucleoside bond. Two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative phosphorus-free 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 native phosphodiester bond, modified bonds can be used to alter (usually increase) the nuclease resistance of the oligonucleotide. In some embodiments, internucleoside bonds having chiral atoms can be prepared as racemic mixtures or as individual enantiomers. Representative chiral bonds include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free internucleoside bonds are well known to those of skill in the art.

[0497] The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which can be defined as (R) or (S), α or β (e.g., for anomeric isomers of sugars), or (D) or (L) (e.g., for amino acids, etc.) in terms of absolute stereochemistry. All such possible isomers, as well as their racemic and optically pure forms, are included in the antisense compounds provided herein.

[0498] Neutral internucleoside linkages include, but are not limited to, 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'). Other neutral internucleoside linkages include nonionic linkages, which include siloxanes (dialkylsiloxanes), carboxylates, carboxamides, sulfides, sulfonates 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). Other neutral internucleoside linkages include nonionic linkages containing mixed N, O, S and CH2 components.

[0499] 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 the 5'-position of the 5'-terminal nucleotide. For example, an additional modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically linking one or more additional non-ligand moieties or conjugates to the oligonucleotide, which 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), bile acids (Manoharan et al., 1994), thioethers such as hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), cholesteryl thioether (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-glycerol-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).

[0500] Representative U.S. patents teaching 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.

[0501] 3. Protein

[0502] In some embodiments, the composition can further comprise one or more proteins. Some proteins can include enzymes such as nucleases. The compositions described herein can comprise one or more CRISPR-related proteins (e.g., CRISPR enzymes), including Cas proteins. 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 their modified forms. These enzymes are known; for example, the amino acid sequence of the Cas9 protein from Streptococcus pyogenes can be found in the SwissProt database under accession number Q99ZW2.

[0503] The protein in the compositions described herein can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae). The CRISPR enzyme can direct cleavage of one or both strands at the target sequence position, such as within the target sequence and / or within the complementary sequence of the target sequence. The CRISPR enzyme can be mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, an aspartic acid-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves single strand). In some embodiments, the Cas9 nickase can be used in combination with guide sequences (e.g., two guide sequences) that respectively target the sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0504] In some embodiments, the present disclosure provides compounds containing one or more therapeutic proteins. Therapeutic proteins that can be included in the compositions 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 alpha, 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 alpha, darbepoetin alpha, follicle-stimulating factor (FSH), and factor VIII.

[0505] 4. Small molecule therapeutic agents

[0506] In some aspects, the present disclosure provides a composition comprising a therapeutic agent. The therapeutic agent can be a small molecule such as 7-methoxypteridine, 7-methylpteridine, abacavir, abafungin, abarelix, acebutolol, acenaphthene, acetaminophen, acetanilide, acetazolamide, acetohexamide, acitretin, acrivastine, adenine, adenosine, alafloxacin, albendazole, salbutamol, alclofenac, aldesleukin, alemtuzumab, alfuzosin, alitretinoin, alobarbital, allopurinol, all-trans retinoic acid (ATRA), aloprin, alprazolam, alprenolol, hexamethylmelamine, amifostine, amiloride, aminoglutethimide, aminopyrine, amiodarone hydrochloride, amitriptyline, amlodipine, amobarbital, amodiaquine, amoxapine, amphetamine, amphetamine, amphotericin, amphotericin B, ampicillin, amprenavir, amsacrine, amyl nitrate, amobarbital, anastrozole, anrinone, anthracene, anthracycline antibiotics, aprobital, arsenic trioxide, asparaginase, aspirin, astemizole, atenolol, atorvastatin, atovaquone, atrazine, atropine, atropine azathioprine, auranofin, azacitidine, azapropazone, azathioprine, azintamide, azithromycin, aztreonam, baclofen, barbiturates, live bacillus Calmette-Guérin, beclomethasone, bendrofluazide, benezepril, benidipine, benolate, benperidol, benzazepam, benzodiazepine amide, benzanthracene, benzathine penicillin, benzhexol hydrochloride, benznidazole, benzodiazepines, benzoic acid, hydroxynaphthofenine, betamethasone, bevacizumab (atorvastatin), bexarotene, bezafibrate, bicalutamide, bifonazole, biperiden, bisacodyl, bisantrene, bleomycin, bleomycin, bortezomib, brinzolamide, bromazepam, bromocriptine mesylate, bromperidol, brotizolam, budesonide, bumetanide, bupropion, busulfan, butalbital, butamben, butenafine hydrochloride, butalbital, butalbital (n-butalbital), butoconazole, butoconazole nitrate, butylparaben, caffeine, calcifediol, calcipotriene (calcip rotriene), calcitriol, captestosterone, canbendazole, camphor, camptothecin, camptothecin analogs, candesartan, capecitabine, capsaicin, captopril, carbamazepine, carbimazole, cypermethrin, carboplatin, carbromide, carimazole, carmustine, cefmandole, cefazolin, cefixime, ceftazidime, cefuroxime axetil, celecoxib, cephradine, cerivastatin, cetrizine, cetuximab, chlorambucil, chloramphenicol, chlordiazepoxide, clomethiazole, chloroquine, chlorothiazide, chlorpheniramine, chlorproguanil hydrochloride, chlorpromazine, chlorsulfuronamide, chlorprothixene, chlorpyrifos, chlortetracycline, chlorthalidone, chlorzoxazone, cholecalciferol, Cilostazol, cimetidine, cinnarizine, cinoxacin, ciprofibrate, ciprofloxacin hydrochloride, cisapride, cisplatin, citalopram, cladribine, clarithromycin, clemastine fumarate, clioquinol, clobazam, clofarabine, clofazimine, clofibrate, clomiphene citrate, clomipramine, clonazepam, clopidogrel, clotiazepam, clotrimazole, clotrimazole, cloxacillin, clozapine, cocaine, codeine, colchicine, colistin, conjugated estrogens, corticosterone, cortisone, cortisone acetate, cyclizine, cyclohexylbarbital, cyclobenzaprine, cyclobutane-spirobarbiturate, cyclohexane-spirobarbiturate, cycloheptane-spirobarbiturate, cyclohexane-spirobarbiturate, cyclopentane-spirobarbiturate, cyclophosphamide, cyclopropane - Spirobarbiturate, cycloserine, cyclosporine, cyproheptadine, cyproheptadine hydrochloride, cytarabine, cytosine, dacarbazine, dactinomycin, danazol, dananthrone, dantrolene sodium, dapsone, erythropoietin alfa, darodipine, daunorubicin, decoquinate, dehydroepiandrosterone, delavirdine, demethylchlorocycline, denileukin, deoxycorticosterone, deoxymethasone, dexamethasone, dextroamphetamine, dexchlorpheniramine, dexfenfluramine, dexrazoxane, dextropropoxyphene, diatrizoate, diazepam, diazoxide, dichlorophen, 2,4-dichloropropionic acid, diclofenac, dicoumarol, didanosine, diflunisal, digitoxin, digoxin, dihydrocodeine, dihydroequilin, dihydroergotamine mesylate, diiodohydroxyquinoline, diltiazem hydrochloride, diloxanide furoate, Dimenhydrinate, Demopramine, Dinitolamide, Diosgenin, Diphenoxylate Hydrochloride, Biphenyl, Dipyridamole, Dirithromycin, Disopyramide, Disulfiram, Diuron, Docetaxel, Domperidone, Donepezil, Doxazosin, Doxazosin Hydrochloride, Doxorubicin (Neutral), Doxorubicin Hydrochloride, Doxycycline, Drostanolone Propionate, Droperidol, Diprophylline, Echinocandin, Econazole, Econazole Nitrate, Efavirenz, Eli Lilly, Enalapril, Enmoumab, Enoximone, Epinephrine, Epipodophyllotoxin Derivatives, Epirubicin, Epoetin Alfa, Iprosartan (eposartan), Dehydroequilin, Equilin, Ergocalciferol, Ergotamine Tartrate, Erlotinib, Erythromycin, Estradiol, Estramustine, Estriol, Estrone, ethacrynic acid, ethambutol, ethinyl acetamide, ethionamide, profenamide hydrochloride, ethyl 4-aminobenzoate (benzocaine), ethyl parahydroxybenzoate, ethinyl estradiol, etodolac, etomidate, etoposide, acitretin, exemestane, felbamate, felodipine, fenbendazole, fenbuconazole, fenbufen, fenfos, fenclorac, fenfluramine, fenofibrate, fenoldepam, fenoprofen calcium, fenoxycarb, fenpicrol, fentanyl, fenticonazole, fexofenadine, filgrastim, finasteride, flecainide acetate, floxuridine, fludarabine, fluconazole, fluconazole, flucytosine, fludioxonil, fludrocortisone, fludrocortisone acetate, flufenamic acid,Flunanisone, Flunarizine Hydrochloride, Flunisolide, Flunitrazepam, Fluocortolone, Fluometuron, Fluorene, Fluorouracil, Fluoxetine Hydrochloride, Fluoxymesterone, Flupenthixol Decanoate, Fluphenthixol Decanoate, Flurazepam, Flurbiprofen, Fluticasone Propionate, Fluvastatin, Folic Acid, Fosinopril, Fosphenytoin Sodium, Frovatriptan, Furosemide, Fulvestrant, Furazolidone, Gabapentin, G-BHC (Lindane), Gefitinib, Gemcitabine, Gemfibrozil, Gemtuzumab, Glafenine, Glibenclamide, Gliclazide, Glimepiride, Glipizide, Glutethimide, Glibenclamide, Nitroglycerin, Goserelin Acetate, Grepafloxacin, Griseofulvin, Guaifenesin, Guanabenz Acetate, Guanine, Halofantrine Hydrochloride, Haloperidol, Hydrochlorothiazide, Heptabarbital, Hesperetin, Hexachlorobenzene, Hexobarbital, Histrelin Acetate, Hydrocortisone, Hydroflumethiazide, Hydroxyurea, Hyoscyamine, Hypoxanthine, Ibritumomab Tiuxetan, Ibuprofen, Idarubicin, Allylbutabarbital, 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 Barbiturate, 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, Leuprorelin Acetate, Levamisole, Levofloxacin, Lidocaine, Linuron, Lisinopril, Lomefloxacin, Lomustine, Loperamide, Loratadine, Lorazepam, Lomefloxacin (Lorefloxacin), Chloromethiazole, Losartan Potassium, Lovastatin, Lysergide Maleate, Maprotiline Hydrochloride, Mazindol, Mebendazole, Meclizine Hydrochloride, Meclofenamic Acid, Medazepam, Metildigoxin, Medroxyprogesterone Acetate, Mefenamic Acid, Mefloquine Hydrochloride, Megestrol Acetate, Melphalan, Methscopolamine Bromide, Meprobamate, Meptazinol, Mercaptopurine, Mesalazine, Mesna, Mesoridazine, Mestranol, Methadone, Methaqualone, Methocarbamol, Mephenytoin, Methotrexate, Methoxsalen, Methsuximide, Methyclothiazide, Methylphenidate, Methylphenobarbital, Methylparaben, Methylprednisolone, Methyltestosterone, Methyprylon, Methysergide Maleate, Metoclopramide, Metolazone, Metoprolol, Metronidazole, Mianserin Hydrochloride, Miconazole, Midazolam, Mifepristone, Miglitol, Minocycline, Minoxidil, Mitomycin C, Mitotane, Mitoxantrone, Mycophenolate Mofetil, Morindone, Montelukast, Morphine, Moxifloxacin Hydrochloride,Nabumetone, Nadolol, Nalbuphine, Nalidixic Acid, Nandrolone, Tetracene, Naphthalene, Naproxen, Naratriptan Hydrochloride, Natamycin, Nelarabine, Nelfinavir, Nevirapine, Nicardipine Hydrochloride, Niacinamide, Niacin, 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Ravuconazole, Repaglinide, Cyclobarbital, Reserpine, Retinoids, Rifabutin, Rifampicin, Rifapentine, Lymecycline, Risperidone, Ritonavir, Rituximab, Rizatriptan Benzoate, Rofecoxib, Ropinirole Hydrochloride, Rosiglitazone, Saccharin, Salbutamol, Salicylamide, Salicylic Acid, Saquinavir, Sargramostim, Secbutabarbital, Secobarbital, Sertaconazole, Sertindole, Sertraline Hydrochloride, Simvastatin, Sirolimus, Sorafenib, Sparfloxacin, Spiramycin, Spironolactone, Dihydrotestosterone, Stanozolol, Stavudine, Diethylstilbestrol, Streptozocin, Strychnine, Sulconazole, Sulconazole Nitrate,Sulfacetamide, sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfanilamide, sulfathiazole, sulindac, sulphabenzamide, sulphacetamide, sulphadiazine, sulphadoxine, sulfafurazole, sulphamerazine, sulpha-methoxazole, sulphapyridine, sulfasalazine, sulfinpyrazone, sulpiride, thiazide, sumatriptan succinate, sunitinib malate, tacrine, tacrolimus, tabubarbital, tamoxifen citrate, tamulosin, targretin, 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hydrochloride, vigabatrin, vinylbital, vinblastine, vincristine, vinorelbine, voriconazole, xanthine, zileuton, zoledronate, zoledronic acid, zolmitriptan, zolpidem and zopiclone.

[0507] F. Kit

[0508] 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 contains the composition as described above or in the claims.

[0509] The kit will typically include at least one vial, tube, flask, bottle, syringe, or other container in which the components can be placed and preferably appropriately aliquoted. In the case where there is more than one component present in the kit, the kit will typically also contain a second, third, or other additional container in which additional components can be separately placed. However, various combinations of components can be included in the container. In some embodiments, all 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.

[0510] The kits of the present disclosure will typically also include a packaging for containing the various containers, which is tightly sealed for commercial sale. Such packaging can include cardboard or injection or blow molded plastic packaging that holds the desired containers. The kit can also include instructions regarding the use of the components of the kit. The instructions can include the variants that can be achieved.

[0511] F. Examples

[0512] The following examples are included to demonstrate the preferred embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present disclosure and thus can be considered to constitute a preferred mode of its practice. However, those skilled in the art should understand, upon considering the present disclosure, that many changes can be made in the specific embodiments disclosed and still obtain similar or analogous results without departing from the spirit and scope of the present disclosure.

[0513] Example 1 - Preparation of DOTAP-Modified Lipid Nanoparticles

[0514] Lipid nanoparticles (LNPs) are the most effective class of carriers for in vivo nucleic acid delivery. Historically, effective LNPs have consisted of four components: ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and lipid poly(ethylene glycol) (PEG). However, these LNPs only result in general delivery of nucleic acids, rather than delivery to a targeted organ or tissue. LNPs typically only deliver RNA to the liver. Therefore, new LNP formulations are sought to attempt to provide targeted nucleic acid delivery.

[0515] Four standard types of lipids were mixed at a molar ratio of 15:15:30:3, with or without a permanent cationic lipid. Briefly, LNPs were prepared by mixing 5A2-SC8 (ionizable cationic), DOPE (zwitterionic), cholesterol, DMG-PEG, and DOTAP (permanent cation) at the ratios shown in Table 1.

[0516]

[0517] To prepare the mDLNP formulation, 5A2-SC8, DOPE, cholesterol, and DMG-PEG were dissolved in ethanol at a given molar ratio (15:15:30:3). The mRNA was dissolved in citrate buffer (10 mM, pH 4.0). Then the mRNA was diluted into the lipid solution to achieve a weight ratio of 40:1 (total lipid:mRNA) by rapidly mixing the mRNA into the lipid solution at a volume ratio of 3:1 (mRNA:lipid, v / v). Then the solution was incubated at room temperature for 10 min. 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 into ethanol containing 5A2-SC8, DOPE, cholesterol, DMG-PEG, and DOTAP, fixing the weight ratio of 40:1 (total lipid:mRNA) and the volume ratio of 3:1 (mRNA:lipid). As shown in Table 1, each formulation was designated as DOTAPX, where X represents the molar percentage of DOTAP in the total lipid.

[0518] Example 2 - Characterization of DOTAP-modified mDLNP formulations.

[0519] To characterize the different mDLNP formulations, the size, polydispersity index, and ζ potential were examined by dynamic light scattering, and each formulation was measured three times separately. The size and polydispersity index are shown in Figure 5A, indicating that all formulations fell within the size range of approximately 90 nm to approximately 160 nm regardless of the DOTAP concentration, while the polydispersity index varied from approximately 0.1 to approximately 0.3, indicating relative uniformity in size. The ζ potential of each formulation is shown in Figure 5B, and it shows that the ζ potential generally increased with the concentration of DOTAP.

[0520] Next, the 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 by mDLNP without DOTAP with an efficiency of approximately 85% (Figure 5C). A low pH is required to protonate the ionizable amines in ionizable cationic lipids (such as 5A2-SC8, C12-200, DLin-MC3-DMA) to allow electrostatic complexation of the negatively charged mRNA. For all other formulations in this figure, mRNA dissolved in PBS was mixed at pH 7.4. Apparently, for low concentrations of DOTAP, the encapsulation efficiency is low, but when the molar percentage of DOTAP is higher than 25%, the encapsulation efficiency increases to >80% (Figure 5C). For all formulations with a molar percentage of DOTAP greater than 25%, the encapsulation efficiency is between approximately 80% and 95%. Thus, the potential to use neutral pH PBS mixing is a feature of the permanent cationic lipid strategy. This strategy allows for tissue-specific delivery and high Cas9 protein encapsulation. The addition of the permanent cationic lipid allows for the formation of LNPs at neutral pH. These encapsulation results are those when using PBS as the buffer. When using an acidic buffer (such as citrate buffer (10 mM, pH 4.0)), then for all formulations of 0-100% DOTAP, the encapsulation efficiency is high (>90%).

[0521] Finally, the pK was determined using the 2-(p-toluidino)-6-naphthalene sulfonic acid (TNS) assay a (Figure 3B) (Zhao et al., 2016). The relationship between pK a and tissue-specific mRNA delivery was plotted based on defined rules. As shown in the table, 8 rules were designed here for scoring. Obviously, the liver-targeting formulations have a narrow pK a (~6 - 7), the spleen-targeting formulations do not have an obvious range, but lung-targeted delivery requires a high pK a (>9.25). Considering both distribution and pK a detection, the conclusion is that the internal charge of the NPs is a factor affecting mRNA distribution, and the overall / apparent LNP pK a is another factor determining the mRNA-mediated protein expression profile in organs.

[0522] Example 3 - Potency of Permanently Cationic Lipid-Modified mDLNP for mRNA Delivery

[0523] To examine the delivery efficacy of LNPs including permanent cationic lipids to deliver active cargo in vitro, DOTAP-modified mDLNPs were loaded with mRNA encoding luciferase and transfected into Huh-7 hepatocytes and A549 adenocarcinoma human alveolar basal epithelial cells with 50 ng / well of mRNA. These cells were cultured for 24 h and then luciferase expression and cell viability were examined. As shown in Figure 6A, for mRNA delivery and expression in Huh-7 hepatocytes in vitro, 5%-50% DOTAP percentages were better, and 10% DOTAP appeared to have the greatest luciferase delivery and expression (Figure 6A). It should be noted that in vivo delivery characteristics may be different. Generally, due to other in vivo barriers, organ distribution, and cell properties of SORT LNPs, these studies may not be useful for predicting in vivo activity or tissue tropism. Additionally, cell viability was examined and it was found that 10% DOTAP produced high viability within mDLNPs that exhibited robust luciferase expression (Figure 6A). Examination of the same transfection with the A549 lung cancer cell line showed similar results, with cells transfected with the DOTAP10 formulation showing almost twice the fluorescence of any other formulation and maintaining high cell viability (Figure 6A). DOTAP SORT LNPs form in PBS (pH 7.4) rather than in citrate buffer (10 mM, pH 4.0), but can form in either buffer system. This is an attribute as it allows encapsulation and delivery of cargo unstable in ethanol or acidic buffers, such as proteins.

[0524] To determine the effect of ethanol concentration in the formulation, DOTAP25 was selected and prepared with various ethanol-to-PBS ratios (1:3, 1:5, 1:7.5, and 1:10) (Figure 6B). All four formulations showed similar encapsulation efficiencies, sizes, and PDIs (Figure 2B1 and Figure 2B2). mRNA delivery efficiency was also measured by transfecting FaDu hypopharyngeal cancer cells with 50 ng / well of mRNA in each formulation. The delivery efficiency of each formulation was similar and the effect on cell viability was also very small (Figure 6C). Thus, these formulations appear to be applicable to many cell types, including liver, lung, and larynx. Further, the formulation has also been successfully modified to use 1×PBS (pH 7.4) instead of acidic buffer (pH 4.0), and these data show that the ethanol percentage can be significantly reduced, which provides the possibility that the DOTAP formulation can deliver cargo (such as proteins) much more sensitive to high ethanol concentrations and acidic buffers.

[0525] Next, to test the ability of these mDLNPs to deliver mRNA in vivo, mice were injected with a 0.1 mg / kg dose of Luc mRNA in each formulation. Figure 1B shows ex vivo images of luciferase in the major organs 6 hours after intravenous injection of each formulation. Intriguingly, as the molar percentage of DOTAP increased, luciferase expression shifted from the liver to the spleen and then to the lung, demonstrating organ-specific delivery. Quantification of these data revealed that the DOTAP percentage was a factor in tissue-targeted delivery, and mDLNP (0% DOTAP) was optimal for liver delivery, while 5 - 15% DOTAP was optimal for spleen delivery, and DOTAP50 (50%) was optimal for lung delivery (Figure 1B). Assuming that luciferase expression was detected only in the liver, spleen, and lung after intravenous injection, the percentage of luciferase expressed in each organ could be calculated (Figure 1C). These data clearly indicated that as the molar percentage of DOTAP in the formulation increased, delivery to and expression in the liver decreased, and when the DOTAP percentage was greater than 70%, near-zero expression was seen in the liver (Figure 1B, 1C). However, the greater the DOTAP percentage, the more luminescence was seen in the lung tissue, and when the DOTAP percentage was greater than 80%, nearly 100% luminescence was seen in the lung (Figure 1C). Concentrations of 5 and 30 molar percentages of DOTAP showed higher percentages of luminescence in the spleen tissue, while DOTAP10 showed the highest relative luminescence in the spleen compared to other tissues (Figure 1C). These results indicated that after injection, the lipid concentration could be adjusted for delivery to specific tissues.

[0526] To more deeply test the organ biodistribution of specific DOTAP formulations, PBS or liver-targeted NPs (mDLNP), 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 of Cy5-Luc mRNA (dye-labeled mRNA to track RNA LNP). Six hours after injection, the major organs were collected and imaged (Figure 3A). The organ distribution of the formulations changed with the amount of DOTAP, and as the DOTAP percentage increased, accumulation in the liver gradually shifted to the lung, but NPs remained in the liver regardless of the DOTAP percentage ( Figure 7 , Figure 8). Considering this data together with the data in Figure 1, it was clear that organ distribution was insufficient to analyze the delivery efficacy (mRNA translation into protein) to the targeted tissue. Further considering the similar size distributions and EE, ζ potential, and pK a among these formulations might play a role in mRNA expression in the targeted tissue.

[0527] To understand whether the addition of DOTAP to mDLNP is of limited effect or whether the distributions shown above are general for mDLNP formulated with permanent cationic lipids, mDLNP containing another popular cationic lipid, didodecyldimethylammonium bromide (DDAB) (Figure 2A1), was generated. DDAB has two hydrophobic tails with 18 carbon atoms and no unsaturated bonds and has a headgroup 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). Similar to the DOTAP formulations above, even though the percentage of DDAB in the NPs varied 10-fold (5% to 50%), the size distributions were almost identical (Figure 2A1). Similar to DOTAP NPs, in vivo luciferase expression showed the following trend: as the percentage of DDAB increased, luminescence shifted from the liver to the spleen and then to the lungs (Figure 2A2).

[0528] mDLNP was formed with a third permanent cationic lipid having a headgroup of 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride, which has a structure similar to DOTAP but has shorter 14-carbon hydrophobic tails ((14:0)EPC) (Figure 2B1). Similar to the DDAB strategy, (14:0)EPC5, (14:0)EPC15, (14:0)EPC40, and (14:0)EPC50 formulations were prepared and the size distributions (Figure 2B1) and in vivo Luc mRNA delivery (0.1 mg / kg, 6 h, n = 2) (Figure 2B2) were examined. Similar to the mDLNP analyzed above, the particle sizes were generally uniform (Figure 2B1), and as expected, as the EPC mole percentage increased (14:0), luminescence shifted from the liver to the spleen and then to the lungs (Figure 2B2). Taking all this data together, including different hydrophobic tails, saturated and unsaturated bonds, and different headgroups, it seems that cationic lipid-formulated mDLNP is general for mRNA delivery to targeted tissues.

[0529] To understand whether the effects of DOTAP addition to LNP are specific to permanent cationic lipids, the effects of adding zwitterionic lipids instead of permanent cationic lipids in the mDLNP formulation were examined. Two representative zwitterionic lipids, phospholipids with different chemical structures: DSPC and DOCPe, were tested. Additionally, tests were also conducted to determine whether the addition of zwitterionic lipids (instead of permanent cationic lipids) would affect 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 both the positively and negatively charged functional headgroups as well as the positions of the hydrophobic domains (saturated versus unsaturated), suggesting that any observed effects regarding zwitterionic lipids are general / common. The mDLNPs formulated with DSPC or DOCPe were similar (Figures 2C1, 2D1). Interestingly, the inclusion of zwitterionic lipids in the 5-component modified DLNP did not alter the protein expression profile from liver to lung as did DOTAP and other permanent cationic lipids. Instead, both DSPC and DOCPe improved mRNA delivery to the spleen within a given range (less than 80% in DSPC and less than 50% in DOCPe). For any percentage, there was no protein expression in the lung (0.1 mg / kg, 6 h, n = 2) (Figures 2C2, 2D2). Thus, the inclusion of additional zwitterionic lipids can assist spleen delivery, but cannot modulate the delivery efficacy from liver to spleen to lung as does the inclusion of permanent cationic lipids.

[0530] To understand whether the effect of DOTAP addition to LNP is specific to permanent cationic lipids, the effect of adding ionizable cationic lipids instead of permanent cationic lipids to the mDLNP formulation was examined. Two representative ionizable cationic lipids with different chemical structures were tested: C12-200 and DODAP. Except for the head group (quaternary amine vs. tertiary amine), DODAP has the same structure as DOTAP. C12-200 is an effective lipid for siRNA and mRNA delivery containing an ionizable tertiary amine (also without a quaternary amine), which has a completely different structure from DODAP. (Figure 2E1, 2F1) Similarly, the size distribution of the two modified mDLNPs remained uniform at certain percentages (less than 80%). (Figure 2E1, 2F1) Surprisingly, the inclusion of ionizable cationic lipids in the 5-component modified DLNP did not change the protein expression profile from liver to spleen to lung as did DOTAP and other permanent cationic lipids. Instead, the inclusion of ionizable cationic lipids into DLNP increased the mRNA delivery efficacy of mRNA to the liver. Compared to the original mDLNP (only 5A2-SC8) without additional ionizable cationic lipids (0.1 mg / kg, 6 h, n = 2), these agents showed much better delivery efficacy. As the percentage of DODAP or C12-200 increased (50% or 80%), the luciferase signal decreased significantly, but the liver remained the main organ rather than the spleen or lung. Therefore, the conclusion is that organ-specific effects can be attributed to the inclusion of a specific ratio of permanent cationic lipids. In addition, the data show that permanent cationic lipids produce different effects from ionizable cationic lipids. These data further show that these trends are general across lipid classes.

[0531] Example 4 - CRISPR / Cas9 Gene Editing Using Modified mDLNPs Co-Delivering Cas9 mRNA and sgRNA

[0532] First, three sgRNAs targeting Td-Tomato mice were compared to determine which sgRNA was the most effective in subsequent experiments. These sgRNAs were sgTom1, sgTom2, and sgLoxP. As shown in Figure 10A, sgTom1 and sgLoxP were delivered and expressed with similar results and were more successful in inducing TdTomato than sgTom2 (Figure 10A). Considering the weak PAM of sgLoxP (NAG), sgTom1 was ultimately selected for further experiments.

[0533] In view of the tissue-specific mRNA (luc mRNA) delivery shown by DOTAP NPs, and the similar delivery trends shown by both DDAB- and EPC-modified NPs, DOTAP-modified mDLNPs were then used for co-delivery of Cas9 mRNA / sgRNA, aiming to achieve tissue-specific gene editing. To examine co-delivery in vivo, genetically engineered mice containing a homozygous ROSA26 promoter Lox-Stop-Lox tdTomato (tdTO) cassette present in all cells were used (Figure 4A). Co-delivery of DOTAP-modified mDLNPs containing Cas9-mRNA and sgRNA targeting LoxP or targeting Tom enabled deletion of the stop cassette and induction of tdTO expression (Figure 4B). Mice were intravenously injected with mDLNP and DOTAP50 formulations at a total dose of 2.5 mg / kg (50 μg each) to co-deliver IVT Cas9 mRNA and modified sgTom1 (4 / 1, weight / weight), and then fluorescence in major organs was detected on day 10 after treatment (Figure 4B). Liver- and lung-specific CRISPR / Cas gene editing was achieved. Splenic-specific editing was also achieved. However, due to very high background red autofluorescence, quantification of splenic editing could not be performed using this TdTomato reporter mouse.

[0534] To further examine tissue-specific editing, PTEN was selected as an endogenous target. C57BL / 6 mice were intravenously injected with mDLNP, DODAP20 or DOTAP50 to achieve tissue-specific gene editing. 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 day 10 after treatment. An sgRNA targeting PTEN was used. T7E1 assays showed that tissue-specific characteristics were further confirmed with in vivo PTEN editing. (Figure 4C).

[0535] Example 5 - CRISPR / Cas9 Gene Editing Using Modified mDLNPs Delivering Cas9 Protein / sgRNA Ribonucleoprotein (RNP)

[0536] Based on the following discovery: the inclusion of a permanent cationic lipid (such as DOTAP) into a conventional LNP formulation containing an ionizable cationic lipid, zwitterionic lipid, cholesterol, and a polyethylene glycolylated lipid, it was investigated whether this formulation method could also deliver other cargoes sensitive to ethanol and / or low pH acidic aqueous buffers. A key element of the DOTAP strategy is that the formulation can be prepared using PBS at neutral pH. Therefore, it was examined whether this method could also encapsulate and deliver large proteins (such as Cas9) for gene editing applications. Thus, DOTNP lipid nanoparticles consist of five components: the modular inclusion of an ionizable cationic lipid (such as 5A2-SC8), a zwitterionic lipid (such as DOPE), cholesterol, DMG-PEG, and a permanent cationic lipid (such as DOTAP). The molar ratio of 5A2-SC8, cholesterol, DOPE, and DMG-PEG is fixed (15:15:30:5, mol / mol), and DOTNPX refers to DOTNP with different molar percentages of DOTAP.

[0537] Check whether the initially characterized Cas9 / sgRNA complex is sensitive to acidic pH. The size (diameter) (Figure 11A) and ζ potential (Figure 11B) of the Cas9 / sgLUC complex (mol / mol = 1 / 1) were measured in PBS (pH 7.4) and in citrate buffer (pH 4.2). The size of the Cas9 / sgLUC complex prepared in citrate buffer was large (greater than 100 nm), and the ζ potential was positively charged. These two properties (size greater than typical effective LNPs) and positive charge (charge incompatible with complexes having positively charged lipids) made it impossible to be effectively encapsulated by lipid nanoparticles. However, the size of the Cas9 / sgLUC complex prepared in PBS was compact (less than 20 nm) and had a negative charge. Therefore, it could be encapsulated by lipid nanoparticles when formulated at neutral pH. Next, Cas9 / sgRNA complexes with different Cas9 protein to sgRNA molar ratios were prepared and characterized. The size (Figure 11C) and ζ potential (Figure 11D) of the Cas9 / sgLUC complexes prepared with different Cas9 / sgRNA molar ratios (1 / 1, 1 / 3, and 1 / 5). Compared with the Cas9 / sgLUC complex (1 / 1, mol / mol), higher molar ratios (1 / 3 and 1 / 5, mol / mol) showed smaller sizes and more negative charges, which might be favorable for lipid nanoparticle encapsulation. Compositions were prepared and the DOTNP lipid nanoparticles encapsulating the Cas9 / sgRNA complex were characterized. The size (Figure 11E) and ζ potential (Figure 11F) of the DOTNP10 lipid nanoparticles encapsulating the Cas9 / sgLUC complex (named DOTNP10-L) when prepared at different molar ratios (1 / 1, 1 / 3, 1 / 5). This data indicated encapsulation of the Cas9 / sgRNA RNP into monodisperse LNPs. Figure 11G shows a TEM image of the DOTNP10-L (1 / 3, mol / mol) LNP with encapsulated RNP. After this preliminary study, different sgRNAs were used, including sgLUC, sgGFP, sgTOM, and sgPTEN. To distinguish them, the first letter of each gene was added at the end of DOTNP. DOTNP10-L refers to the DOTNP10 lipid nanoparticles encapsulating the Cas9 / sgLUC complex; DOTNP10-G refers to the DOTNP10 lipid nanoparticles encapsulating the Cas9 / sgGFP complex.

[0538]

[0539] Table 7. Characterization of DOTAP10, DSPC50, and DODAP50 formulations formed with PBS and citrate buffer, including size, PDI, and encapsulation efficiency.

[0540]

[0541] To examine whether DOTNP lipid nanoparticles could deliver the Cas9 / sgRNA RNP complex into the nucleus and mediate efficient gene editing in vitro, a series of experiments were conducted. First, DOTNP containing Cas9 / sgRNA RNP labeled with green fluorescent EGFP was tracked by confocal microscopy (Figure 12A). After incubation with DOTNP10 encapsulating Cas9-EGFP / sgLUC complex (1 / 3, mol / mol) (using 9 nM sgRNA) for 1 h, 3 h, 6 h, and 24 h, images of Hela-Luc cells showed that DOTNP was internalized into the cells and the Cas9 RNP was transported into the nucleus. Green: EGFP-fused Cas9 protein; blue: nucleus stained with Hoechst 33342. Red arrows indicate the process of DOTNP10 entering the nucleus. (Figure 12B)

[0542] Next, it was examined whether DOTNP lipid nanoparticles could deliver the Cas9 / sgRNA RNP complex and whether it could cleave the targeted luciferase DNA. After incubation with DOTNP10-L at different molar ratios (using 24 nM sgRNA) for 3 days, the percentage of DNA indels (insertions and deletions) at the LUC locus was quantified using the TIDE assay. DOTNP10 lipid nanoparticles encapsulating Cas9 / sgGFP (DOTNP10-G) were used as a negative control. Here, two commercial Cas9 proteins (GeneArt Cas9 and Truecut Cas9) were used (Figure 12B). Next, a T7EI cleavage assay of Hela-Luc cells incubated with different formulations (24 nM sgRNA) was performed to demonstrate DNA editing (Figure 12C). Among the tested conditions, when using the Truecut Cas9 protein, a 1 / 3 molar ratio showed the best gene editing. Next, the editing of GFP was tested using fluorescence microscopy (Figure 12D). Images of SKOV3-GFP cells incubated with DOTNP10-L (control, not targeting GFP) and DOTNP10-G (targeting GFP) (24 nM sgRNA) showed target editing confirmed 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 obtained by flow cytometry showed CRISPR / Cas editing of GFP.

[0543] Next, it was examined whether DOTNP lipid nanoparticles could deliver the Cas9 / sgRNA RNP complex in vivo to achieve CRISPR / Cas-mediated gene editing. As before, a genetically engineered TdTomato mouse model was used. Each mouse was delivered 1.5 mg / kg of sgRNA with the following formulations: DOTNP5-T refers to DOTNP5 LNP encapsulating the Cas9 / sgTom complex; DOTNP10-T refers to DOTNP10 LNP encapsulating the Cas9 / sgTom complex; DOTNP50-T refers to DOTNP50 LNP encapsulating the Cas9 / sgTom complex. After intravenous injection of these formulations, tdTomato fluorescence was quantified ex vivo in the major organs 7 days after injection (Figure 13A). tdTomato fluorescence was observed only in the liver of the DOTNP5-T treatment group; in the DOTNP10-T group, slight fluorescence was seen in the lung, and if the dose of DOTAP was further increased to 50% (DOTNP50-T), most of the tdTomato fluorescence was observed in the lung. Thus, similar to the mRNA delivery experiment outlined above, the DOTAP method also allows tissue-specific gene editing of the Cas9 / sgRNA ribonucleoprotein (RNP) complex. To further examine delivery, LNPs containing sgRNA against PTEN were delivered. Using the T7EI cleavage assay of liver and lung organs, intravenous injection (2 mg / kg sgRNA / mouse) of DOTNP5-P (DOTNP5 LNP encapsulating the Cas9 / sgPTEN complex), DOTNP10-P (DOTNP10 LNP encapsulating the Cas9 / sgPTEN complex), and DOTNP50-P (DOTNP50 LNP encapsulating the Cas9 / sgPTEN complex) was determined to mediate gene editing (Figure 13B). The results were consistent with those obtained by ex vivo imaging. Gene editing was detected only in the liver after treatment with DOTNP5-P; gene editing was obtained in both the liver and the lung when incubated with DOTNP10-P; while in the DOTNP50-P treatment group, most gene editing was observed in the lung.

[0544] The data provided herein show that lipid nanoparticles can be formulated with different compositions to specifically target payloads to specific tissues. Specifically, lipid nanoparticles with a low concentration of permanent cationic lipid (≤10%) effectively deliver nucleic acids to the liver, while LNPs with less than 30% permanent cationic lipid effectively deliver nucleic acids to the spleen, and LNPs with more than 30% permanent cationic lipid effectively deliver nucleic acids to the lung. These findings appear to be general, with little effect of the headgroup, saturation, and tail length.

[0545] Example 6 - Addition of Another Lipid to Four Known Lipid Combinations Results in Altered Delivery Targets

[0546] The generality of the approach of including a "fifth" lipid in an established 4-component LNP was then explored.

[0547] To examine whether the inclusion of a permanently cationic lipid (such as DOTAP) would alter the tissue specificity of other ionizable cationic lipids, two well-known and well-established ionizable cationic lipid LNP systems were selected. DLin-MC3-DMA was chosen and formulated with DSPC, cholesterol, and PEG-DMG. The same molar composition as Patisiran / Onpattro (Alnylam Pharmaceuticals) was generated and supplemented with 15% or 50% DOTAP (the additional 5th lipid in the Onpattro 4-lipid formulation) (Figure 14). DLin-MC3-DMA LNPs are considered the "gold standard" for siRNA and mRNA delivery. To date, they have only been shown to deliver to the liver after intravenous administration. As shown in Figure 15A, DOTAP altered the mRNA expression profile of DLin-MC3-DMA-based LNPs in organs (0.1 mg / kg luciferase mRNA, 6 h). As the percentage of DOTAP increased, the luciferase signal shifted from the liver to the spleen and finally to the lung, which was exactly the same phenomenon as that for 5A2-SC8 mDLNPs. To further investigate the generality of this approach, we included DOTAP in C12-200 LNPs (Figure 16). While DLin-MC3-DMA is a two-tailed lipid with a single dimethylamine headgroup and is considered a stable nucleic acid lipid nanoparticle (SNALP), C12-200 is a representative "lipidoid" that can be formulated into lipid-like LNPs. All three are ionizable cationic lipids. Similar to the results with 5A2-SC8 and DLin-MC3-DMA, the inclusion of 15% or 50% DOTAP in C12-200 LNPs altered the luciferase protein expression after mRNA delivery from the liver to the spleen to the lung (Figure 15B). Thus, the fifth lipid approach, such as the addition of a permanently cationic lipid, can be generalized to other ionizable cationic lipid LNPs.

[0548] Example 7 - Addition of Another Lipid to Four Known Lipid Combinations Results in Improved Delivery

[0549] In addition, the generality of this approach was explored by investigating whether an additional ionizable cationic lipid would improve liver delivery.

[0550] To examine whether ionizable cationic lipids generally promote hepatic delivery, an additional 5A2-SC8 ionizable cationic lipid was included as a “fifth” lipid into LNPs containing appropriate ratios of 5A2-SC8, DOPE, cholesterol, and PEG DMG. The additional 5A2-SC8 was included at percentages of 10-30 (Figure 17A and Figure 18 ). To avoid saturation luminescence, a low dose of 0.05 mg / kg mRNA dose (intravenous, 6 h) was tested. As shown in Figure 17B, ex vivo images and quantitative data confirmed that an increased additional 15% to 25% of 5A2-SC8 did contribute to improving mRNA delivery efficacy in the liver. 5A2-SC8^20 (5A2-SC8 LNP + 20% additional 5A2-SC8) increased luciferase to 2-3-fold that of the original mDLNP formulation.

[0551] Example 8 - Studies related to selective organ targeting compositions

[0552] The present disclosure describes a strategy called selective organ targeting (SORT) that allows for the systematic engineering of nanoparticles to accurately deliver a variety of cargoes, including mRNA, Cas9 mRNA / sgRNA, and Cas9 ribonucleoprotein (RNP) complexes, to the lungs, spleen, and liver of mice after intravenous (IV) administration (Figure 19A). Conventional LNPs are composed of ionizable cationic lipids, zwitterionic phospholipids, cholesterol, and poly(ethylene glycol) (PEG) lipids. The present disclosure shows that the addition of a supplementary component, called a SORT compound or selective organ targeting compound, precisely alters the in vivo RNA delivery profile and mediates tissue-specific gene delivery and editing, which varies with the percentage and biophysical properties of the added SORT lipid. The present disclosure shows evidence of the theory for tissue-specific delivery, establishes that the method can be used for various nanoparticle systems, and provides a method for LNP design to edit therapeutically relevant cells.

[0553] Effective intracellular delivery materials generally rely on an optimal balance of ionizable amine binding and release of RNA (pK abetween 6.0 - 6.5) and nanoparticles with stable hydrophobicity (Kanasty et al., 2013; Jayaraman et al., 2012; Nelson et al., 2013; Hao et al., 2015). Without wishing to be bound by any theory, it is believed that internal and / or external charges may be factors that regulate tissue tropism. Intravenous administration of the developed SORT LNPs enables a high level of tissue-specific gene editing. SORT is compatible with various methods that employ gene editing mechanisms, including mRNA, Cas9 mRNA / sgRNA, and Cas9 RNP (systemic RNP delivery). SORT LNPs targeting the lung edit 40% of epithelial cells and 65% of endothelial cells; SORT LNPs targeting the spleen edit 13% of B cells and 10% of T cells; and enhanced SORT LNPs targeting the liver edit 93% of hepatocytes after a single low-dose injection.

[0554] A. Discovery and Development of SORT

[0555] To test the hypothesis that internal charge modulation can mediate tissue-specific delivery, a strategy was conceived to add a 5th lipid to an established LNP composition that had been validated for efficacy in hepatocytes. The rationale was to adjust an effective LNP formulation without disrupting the core 4-component ratio that is commonly used to mediate RNA encapsulation and endosomal escape (Wittrup et al., 2015; Cheng et al., 2018).

[0556] Examined the addition of a permanent cationic lipid (defined as having a positive charge without a pK a or pK a > 8) to a degradable dendritic polymerizable ionizable (pK a<8) Role in cationic lipids (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b), the 5A2-SC8 effectively delivered fumarylacetoacetate hydrolase (FAH) mRNA to hepatocytes and prolonged survival in FAH knockout mice (Cheng et al., 2018). This initial 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, weight / weight) (Figure 20). Then a series of LNPs were formed by systematically increasing the percentage of additional permanent cationic lipid from 5% to 100% of the total lipid (Figure 19B and 20). Initially, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a well-known quaternary ammonium lipid, was selected as the SORT lipid to add to the LNP formulation. Thus, the DOTAP-modified SORT formulation contained 5 lipid components, where 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 prepare a titration series of the formulation (Figure 20).

[0557] Then, the effect of SORT modification was evaluated by intravenously (IV) delivering luciferase (Luc) mRNA at a dose of 0.1 mg / kg. As the molar percentage of DOTAP increased, the resulting luciferase protein expression gradually shifted from the liver to the spleen and then to the lung, demonstrating a distinct and precise organ-specific delivery trend with a threshold that allows exclusive lung delivery (Figure 19B). The DOTAP percentage is a key factor in tuning tissue specificity. The basal LNPs (0% DOTAP) were optimal for liver delivery, as expected since they had been previously optimized for hepatocyte delivery (Cheng et al., 2018). By adding 10 - 15% DOTAP, the resulting SORT LNPs could now deliver mRNA to cells in the spleen. Further increasing the permanently cationic SORT lipid, it was found that 50% DOTAP was optimal for lung delivery (Figure 19C). It is worth noting that although the 50% DOTAP SORT LNPs effectively delivered mRNA to the lung in vivo, they were less effective for in vitro delivery (Figure 21). Additionally, the 50% DOTAP SORT LNPs had a neutral ζ-potential surface charge (-0.52 mV) (Figure 20), indicating that tissue tropism was not due to MPS uptake related to positive charge. Calculating the relative expression in each organ, the application of DOTAP as a SORT lipid completely altered the delivery from the liver to the lung (Figure 19D). It is estimated that over 99% of current intravenous nanomedicines are sequestered by the MPS (Wilhelm et al., 2016; Gustafson et al., 2015), so these new SORT nanoparticles overcome a long-standing challenge in nanomedicines.

[0558] The functional role of the permanently cationic SORT lipid was elucidated, and without wishing to be bound by any theory, it is believed that the inclusion of other lipids may also alter tissue tropism. To explore this potential, the negatively charged 1,2-dioleoyl-sn-glycero-3-phosphate (18PA) was incorporated as a SORT lipid in a manner similar to DOTAP (Figure 20). At 10 - 40% 18PA incorporation, the SORT LNPs now mediated completely selective delivery to the spleen with no luciferase expression in any other organ (Figure 19E). Thus, the negatively charged SORT lipid allows its precise delivery to the spleen. These results suggest that the SORT lipid percentage can be adjusted for specific tissue mRNA delivery by intravenous injection.

[0559] B. SORT can be generalized to other LNP types and lipid classes

[0560] Then, it was explored whether the SORT method could be applied to other classes of established 4-component LNPs to test the generality of SORT. First, DLin-MC3-DMA was formulated with DSPC, cholesterol, and PEG-DMG, which had the same molar composition as the FDA-approved Onpattro (Patisiran) (30) (Figure 22), and was considered the “gold standard” for siRNA and mRNA delivery. To date, they have only shown delivery to the liver after intravenous administration, which was also confirmed here (Figure 19F). As expected, supplementing DLin-MC3-DMA LNPs with DOTAP altered 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 and then to the lung, which was exactly the same as the phenomenon of the initially tested 5A2-SC8 DLNP. To further investigate the generality of this protocol, DOTAP was included in C12-200 LNPs (Figure 19G and 22), which also well validated the delivery of RNA to the liver (Kove et al., 2010; Kauffman et al., 2015). The same as the results of 5A2-SC8 and DLin-MC3-DMA LNPs, the inclusion of 15% or 50% DOTAP in C12-200 LNPs altered the luciferase protein expression after mRNA delivery from the liver to the spleen and then to the lung (Figure 19G and 22). Additionally, the inclusion of 18PA as the SORT lipid reflected the results of 5A2-SC8 and mediated the exclusive delivery of DLin-MC3-DMA SNALP and C12-200 LLNP for Luc mRNA to the spleen (Figure 19F-G). DLin-MC3-DMA is a two-tailed lipid with a single dimethylamine headgroup that forms stable nucleic acid lipid nanoparticles (SNALPs), and C12-200 is a representative lipidoid that forms lipid-like LNPs (LLNPs). Thus, it was shown that the SORT method could be generalized to other types of ionizable cationic lipid LNPs, which would enable existing liver-targeted LNPs to be easily altered to deliver mRNA to the spleen or lung. Specifically, the SORT technology could enable the rapid redevelopment of the FDA-approved Onpattro for the treatment of diseases of the lung and spleen.

[0561] To understand whether the observed tissue tropism profile is specific to the exact chemical structure or can be generalized to defined chemical classes, multiple permanent cationic, anionic, zwitterionic, and ionizable cationic SORT lipids were evaluated (Figure 23). First, 5A2-SC8 LNPs were generated with two additional permanent cationic lipids: didodecyldimethylammonium bromide (DDAB) and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine chloride (EPC). These lipids all contain a quaternary ammonium group, but have major chemical differences in the polar headgroup, linker region, and hydrophobic domain (e.g., saturation). LNPs containing 5%, 15%, 40%, 50%, and 100% DDAB or EPC were formulated and characterized. The in vivo luciferase expression profile matched that of DOTAP LNPs, where as the percentage of DDAB or EPC increased, their luminescence activity systematically shifted from the liver to the spleen and then to the lungs (0.1 mg / kg, 6 h). Once the percentage increased to 40%, high luciferase signals were only observed in the lungs (Figure 23A). 1,2-Dimyristoyl-sn-glycero-3-phosphate (14PA) and sn-(3-oleoyl-2-hydroxy)-glycerol-1-phosphate-sn-3'-(1',2'-dioleoyl)-glycerol (18BMP) were generated as representative anionic lipids, which have very different structures compared to 18PA. All anionic SORT lipids promoted exclusive delivery to the spleen (Figure 23B). This flexibility provides a means to balance multiple factors, including efficacy, selectivity, and tolerability, by optimizing SORT compounds.

[0562] Inspired by these findings, other ionizable cationic lipids were added to the established formulation. As expected, the addition of DODAP or C12-200 to 5A2-SC8 LNPs did not significantly alter the tissue tropism, but surprisingly did enhance liver delivery >10-fold at 20% incorporation (Figure 23C). Supplementing the established 5A2-SC8 LNPs with additional 5A2-SC8 as a SORT lipid significantly improved liver mRNA delivery, generating 10 7 photons / sec / cm 2 . Thus, SORT provides a new strategy to further improve LNP systems targeted to the liver (Figure 24). The effects of using zwitterionic lipids (DOCPe and DSPC) as SORT lipids were also evaluated. Although the tissue tropism was found to shift from the liver to the spleen, it was less selective compared to the application of cationic or anionic SORT lipids (Figure 25).

[0563] To test the limitations of the SORT method, it was examined whether SORT could “activate” an otherwise inactive formulation. Indeed, supplementation of a completely inactive formulation with DODAP or DOTAP resulted in tissue-specific delivery to the spleen and lungs (Figure 26). Collectively, these results suggest that SORT is a modular and versatile strategy for achieving tissue-targeted delivery.

[0564] C. SORT alters the protein corona, LNP biodistribution, and apparent pK a to mediate organ-specific delivery

[0565] Mechanistic experiments were performed to explore how and why including additional lipids in defined classes controls the delivery of mRNA to different organs. Logically, LNPs delivered to cells within the lung should biodistribute (accumulate) in the lung. The in vivo distribution of Cy5-labeled mRNA was tracked to follow the biodistribution of SORT lipid-containing 5A2-SC8 LNPs with lung (DOTAP quaternary amino lipid), spleen (18PA anionic lipid and DSPC zwitterionic lipid), and liver (DODAP ionizable tertiary amino lipid) tropism (Figures 27A and 28). All LNPs were injected intravenously at a dose of 0.5 mg / kg of Cy5-labeled mRNA and imaged 6 hours later. As shown in Figure 27A, DOTAP altered the biodistribution, and as a function of the percentage of DOTAP, lung accumulation gradually increased. Incorporation of 18PA increased uptake into the spleen. DODAP slightly increased liver accumulation and decreased spleen accumulation. Intriguingly, even though lung- and spleen-specific SORT LNPs still accumulated in the liver, there was no protein expression in the liver at all. This suggests that organ biodistribution is necessary for organ-specific potency but is not the only factor explaining the mechanism of tissue-targeted delivery.

[0566] Without wishing to be bound by any theory, it is believed that changes in biodistribution and activity in a defined cell population may be due to alterations in the protein corona, where the binding of specific proteins gives rise to a biological identity of functional activity. Using quantitative mass spectrometry analysis, it was found that the addition of the SORT molecule greatly altered the specific proteins that bind most tightly and the overall protein corona composition. Without wishing to be bound by any theory, it is believed that lung-specific SORT LNPs selectively and most abundantly bind vitronectin, which can interact with positively charged lipids and bind to ανβ3 integrin highly expressed on endothelial and epithelial cells of the lung. This mechanism of endogenous targeting stands in direct contrast to the use of ανβ5 integrin to target adenovirus to bronchial epithelium. Spleen-specific SORT LNPs bind most tightly to β2-glycoprotein I, which has been shown to interact with negatively charged lipids and may play a role in the spleen localization of immune cell populations in the spleen. It is worth noting that the complex mixture of bound proteins may also play a role. This collective effect is also seen as a potential mechanism for targeting multiple cell types and is a method for further enriching specificity for a particular cell type in an organ using alternative SORT molecules. It has previously been shown that apolipoprotein E binds to DLin-MC3-DMA Onpattro LNPs and that efficacy is lost in Apo E knockout animals. Thus, there is strong evidence that Apo E is required for receptor-mediated targeting and hepatocyte uptake (presumably via the LDL receptor), and that the protein corona mechanism described can control cell specificity and efficacy. Thus, it is encouraging to see that mDLNPs are also closely associated with Apo E, providing further evidence of their hepatocyte efficacy and enhancing the validity of the protein corona assay. Liver-enhanced SORT LNPs retain Apo E binding but are also enriched in albumin, suggesting possible sequestration of cell types within the liver. These data cumulatively show that the chemical structure of the SORT molecule can target specific protein coronas for altering organ tropism and cell specificity. Without wishing to be bound by any theory, it is believed that the identity of the SORT molecule can control the protein corona identity, suggesting that SORT molecules can include sugars, lipids, small molecule therapeutics, vitamins, small molecules, hydrophilic molecules, hydrophobic molecules, amphiphilic molecules, peptides, proteins, etc.

[0567] The apparent / total pK a was examined because it has been identified as a parameter correlating LNPs with functional activity. For example, it has been shown that a pK a around 6.4 is optimal for hepatocyte delivery (Jayaraman et al., 2012). For all effective in vivo formulations (67 LNPs), the apparent pK was analyzed using the TNS assay a(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 the more complex mixed LNP. Thus, when generating 50% of the normalized signal, the relative pK is alternatively estimated. a . When the relative pK is plotted against tissue tropism a , the SORT LNPs are grouped into defined apparent pK a ranges (Figure 27B). As expected, all effective liver-targeting formulations have a very narrow pK within a very defined range of 6 - 7 a (Jayaraman et al., 2012). All formulations targeting the lung are located within a high pK a range (>9). In contrast, the spleen-tropic SORT LNPs are grouped into a low pK a range (2 - 6). These results confirm that 6 - 7 is optimal for delivery to the liver, but reveal the discovery that high pK a mediates lung delivery and low pK a mediates spleen delivery. It should be noted that all SORT LNPs still contain ionizable cationic lipids, which are considered useful for endosomal escape due to their ability to acquire charge (Wittrup et al., 2015). Control experiments were performed and it was confirmed that the inclusion of ionizable cationic lipids is required for potency (Figure 30). Thus, SORT allows the retention of molecules with the specific micro-substance pK a required for potency in the desired molar ratio, while the inclusion of SORT lipids alters the apparent pK a . Without wishing to be bound by any theory, it is believed that a bipartite mechanism may be at work. SORT LNPs selectively bind to specific proteins in serum, which enables receptor-mediated potency in lung or spleen cells, very similar to the way lipoprotein particles (such as LDL) naturally transport cholesterol. This controlled and predictable endogenous targeting mechanism enables SORT LNPs to reach non-liver targets. The second part involves how the SORT molecule alters the properties of non-liver-targeting SORT LNPs such that they no longer have the physiochemical properties (e.g., overall / apparent pK a 6.4) of liver potency, which provides precision. It should also be noted that other and more complex factors, such as cell-specific endocytic transport differences, may also play a role. Considering the results, it is proposed that the internal charge of the LNP nanostructure mediates biodistribution and that the apparent pK a is related to the protein expression profile in a specific organ. This specific value can be used to continue the development of other organ-specific nanoparticles.

[0568] Table 1: Details of mDLNP formulations (SORT LNP) modified with DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe, including the molar ratio and percentage of each component, the weight ratio of total lipid to mRNA, size, and PDI.

[0569]

[0570] a X represents DDAB, EPC, 14PA, 18BMP, DODAP, C12-200, 5A2-SC8, DSPC, and DOCPe.

[0571] D. SORT enables lung-, liver-, and spleen-specific gene editing after intravenous administration

[0572] Given the ability of SORT LNPs to target specific organs, these findings were then applied to tissue-specific gene editing by intravenous injection. CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein (Cas)) technology can edit the genome in a precise and sequence-dependent manner and has been rapidly developed for various applications, including the potential correction of pathogenic 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 achieved by local administration of injections (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, in which case specific cells need to be corrected to cure the disease. Such correction can be best achieved by systemic administration. It has recently been reported that the intravenous co-delivery of Cas9 mRNA and sgRNA is a safe and effective strategy for achieving gene editing (Miller et al., 2017; Yin et al., 2017; Finn et al., 2018). However, to date, there have been no reports on the rational engineering of LNPs to edit cells in organs other than the liver.

[0573] To examine and quantify the ability of SORT LNPs to mediate organ-specific gene editing, genetically engineered tdTomato (tdTom) reporter mice containing a LoxP-flanked stop cassette (Tabebordbar et al., 2016), which prevents the expression of tdTom protein (Staahl et al., 2017), were utilized. Once the stop cassette is deleted, tdTom fluorescence is turned on, allowing the detection of gene-edited cells (Figure 31A). Cre recombinase mRNA (Cre mRNA) was initially delivered to activate tdTom in the edited cells. In the selected organs treated with liver-, lung-, and spleen-selective SORT LNPs, fluorescent tissues were readily apparent (Figure 31B). It should be noted that a separate control must be used for each experiment because these mice have some background organ fluorescence, with the weakest fluorescence in the spleen compared to other organs (Figures 31C and 32). This makes spleen-specific detection more challenging to distinguish in the tdTom mouse model. When subsequently editing endogenous PTEN, spleen-specific SORT LNPs showed clean DNA cleavage only in the spleen by T7E1 assay (Figure 33C), with no DNA cleavage in the liver or lung. Nevertheless, tdTom-positive cells were readily visible by confocal imaging of tissue sections (Figure 31D).

[0574] E. SORT achieves high levels of editing in specific and therapeutically relevant cell populations

[0575] Flow cytometry using single cells isolated from the edited organs was used to quantify gene editing of specific cell types in the liver, lung, and spleen (Figure 31E). After a single injection of 0.3 mg / kg Cre mRNA, liver-specific SORT (20% DODAP) 5A2-SC8 LNPs edited approximately 93% of all hepatocytes in the liver (Figure 31E and 34). This is the highest level of hepatocyte gene editing reported to date. At the same dose, lung-specific SORT (50% DOTAP) 5A2-SC8 LNPs edited approximately 40% of all epithelial cells, approximately 65% of all endothelial cells, and approximately 20% of immune cells in the lung (Figure 31E and 35). Given that epithelial cells are the main target for correcting mutations in CFTR that cause cystic fibrosis, this result establishes lung-specific SORT LNPs as a compelling delivery system and immediate application for correcting CFTR mutations. Finally, spleen-specific SORT (30% 18PA) 5A2-SC8 LNPs edited approximately 13% of all B cells, approximately 10% of all T cells, and approximately 20% of all macrophages (Figure 31E and 36). Due to the increased selectivity compared to previous studies, spleen-specific SORT LNPs can be used to treat non-Hodgkin B cell lymphoma and other immune disorders. Although the initial focus was on quantification with a single low-dose injection, higher levels of editing can be achieved by administering higher doses or multiple injections.

[0576] F. SORT allows tissue-specific gene editing by intravenous co-delivery of Cas9 mRNA / sgRNA and delivery by Cas9 RNP

[0577] Next, the ability of SORT LNPs to achieve tissue-specific CRISPR / Cas gene editing through intravenous co-delivery of Cas9 mRNA and sgRNA in single nanoparticles was examined (Figures 37A, 38, and 39, Table 2). SORT LNPs targeting the liver and lung were injected at a dose of 2.5 mg / kg total RNA (4:1 mRNA:sgRNA, wt:wt), and gene editing was quantified 10 days after a single intravenous injection. As shown in Figure 37B, strong tdTom fluorescence was observed in the liver for mice treated with the basal LNP and 20% DODAP SORT LNPs, and strong fluorescence was observed in the lungs of mice treated with 50% DOTAP SORT LNPs. All results were consistent with the Luc mRNA delivery results. Due to the rapid turnover of splenic immune cells in mice (Kamath et al., 2000), the weight ratio of Cas9 / sgRNA was optimized to 2 / 1 (Figure 39), and spleen editing was tested two days after injection. Considering background autofluorescence, bright tdTom fluorescence was observed in the spleens of mice treated with 30% 18PA, and clear T7E1 cleavage bands were detected only in the DNA isolated from the spleen (no editing in the liver or lung) (Figure 33). Fluorescence was then confirmed by imaging tissue sections with confocal microscopy (Figure 37C).

[0578] Next, direct delivery of Cas9 RNPs was explored, which is the most challenging strategy for synthetic vectors. The application of permanently cationic SORT lipids enabled the encapsulation of the Cas9 protein / sgtdTom complex and control of tissue tropism. Intravenous injection of 7% DOTAP SORT LNPs achieved liver editing, while 55% DOTAP SORT LNPs achieved exclusive lung editing (Figure 37F). These data indicate that the described method is capable of achieving liver, lung, and spleen-specific CRISPR / Cas gene editing.

[0579] To surpass reported mice, the ability of tissue-specific LNPs to edit endogenous targets was tested. PTEN was chosen because it is a well-defined 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. The generation of insertions and deletions (indels) was quantified 10 days after a single intravenous injection. As shown in Figure 37D, clear DNA cleavage bands were observed in specific tissues by T7E1 assay, indicating that the basal LNP and 20% DODAP SORT LNP mediated efficient PTEN editing in the liver, while no mediation was observed in the lung or spleen. Notably, 50% DOTAP SORT LNP showed PTEN editing only 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, which is a known phenotype of PTEN loss due to lipid accumulation (Xue et al., 2014). In addition, negative staining of PTEN was observed in both IHC sections of liver and lung tissues, providing clear evidence of PTEN editing. Although it was more challenging to distinguish spleen-specific 18PA SORT LNP editing in the tdTom mouse model, clear spleen PTEN editing could be observed in wild-type mice with an optimized Cas9 / sgPTEN weight ratio (2 / 1) and detection time (2 days). No editing of DNA in the liver or lung was observed by T7E1 assay on mice injected with 18PA SORT LNP (Figure 33). Finally, SORT was applied to Cas9 RNP and endogenous editing of PTEN was examined. As before, 7% and 55% of DOTAP SORT LNPs containing Cas9 protein / sgPTEN were able to perform liver- and lung-specific editing, respectively (Figure 37G). These results of targeting endogenous genes demonstrated rationally directed tissue-selective gene editing achieved by synthetic vectors.

[0580] G. Materials and Methods

[0581] I. Materials

[0582] 5A2-SC8 (Zhou et al., 2016), DLin-MC3-DMA (Jayaraman et al., 2012), and C12-200 (Love et al., 2010) were synthesized and purified following the 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-phosphate-sn-3'-(1',2'-dioleoyl)-glycerol (ammonium salt) (18:1Hemi 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 ethyl phosphate (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-glycero-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 kit (MWCO, 3.5 kDa) was purchased from Sigma-Aldrich. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from ThermoFisher Scientific. Cas9 mRNA was generated 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.

[0583]

[0584] II. Nanoparticle Formation

[0585] RNA-loaded LNP formulations were 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), and the base formulation was prepared as previously described (Jayaraman et al., 2012; Love et al., 2010). Unless otherwise specified, all lipids with the designated molar ratios were dissolved in ethanol, and the RNA was dissolved in 10 mM citrate buffer (pH 4.0). The two solutions were rapidly mixed at a water-to-ethanol volume ratio of 3:1 (3:1, water:ethanol, vol:vol) to achieve a final weight ratio of 40:1 (total lipid:mRNA), and then incubated at room temperature for 10 min. To prepare SORT LNP formulations containing anionic SORT lipids such as 18PA, 14PA, and 18BMP, the anionic lipids were first dissolved in tetrahydrofuran (THF), then mixed with other lipid components in ethanol, and finally a formulation containing the mRNA buffer (10 mM, pH 3.0) as described above was obtained. All formulations were named based on the additional lipids. Taking DOTAP mDLNP as an example, the internal molar ratio of mDLNP was fixed as reported in the published paper, with 5A2-SC8 / DOPE / cholesterol / DMG-PEG of 15 / 15 / 30 / 3 (Cheng et al., 2018). DOTAP was dissolved in the above ethanol-lipid mixture in a specified amount to make the molar ratio of 5A2-SC8 / DOPE / cholesterol / DMG-PEG / DOTAP equal to 15 / 15 / 30 / 3 / X, and then rapidly mixed with the aqueous mRNA solution following the above standard protocol, finally producing SORT LNP named Y% DOTAP, where Y refers to the molar percentage of DOTAP in the total lipids. Formulations with other additional lipids were formed similarly by the above method (Figure 20 and Table 3). For Cas9 / sgRNA ribonucleoprotein (RNP) encapsulation, it was formulated using 1×PBS, and the molar ratio of Cas9 and sgRNA was fixed at 1:3. After SORT LNP formation, the fresh LNP formulation was diluted with 1×PBS to 0.5 ng / μL mRNA (final ethanol concentration <5%) for in vitro assays and size detection. For in vivo experiments, the formulation was dialyzed against 1×PBS (Pur-A-Lyzer Midi dialysis kit, WMCO 3.5 kDa, Sigma-Aldrich) for 2 h and diluted with PBS to 15 μL / g for intravenous (IV) injection.

[0586] Table 3: sgRNA sequences

[0587]

[0588] III. Characterization of mRNA formulations

[0589] The size distribution and polydispersity index (PDI) were measured using dynamic light scattering (DLS, Malvern MicroV; He-Ne laser, λ = 632 nm), and the ζ potential was measured after dilution with 1×PBS. To measure the apparent pK a of the mRNA formulations, a 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) assay (Cheng et al., 2018; McLaughlin and Harary, 1976; Bailey and Cullis, 1994; Heyes et al., 2005) was employed with some modifications. The mRNA formulation (60 μM total lipid) and the TNS probe (2 μM) were incubated for 5 min with a series of buffers (pH range 2.5 - 11) containing 10 mM HEPES, 10 mM MES (4-morpholineethanesulfonic acid), 10 mM ammonium acetate, and 130 mM NaCl. At λ Ex = 321 nm and λ Em = 445 nm, the average fluorescence intensity of each well (black-bottom 96-well plate) was measured using a Tecan plate reader, and the data were normalized against the value at pH 2.5. Typically, the apparent pK a is defined by the pH at half-maximal fluorescence. Although this method can be used to estimate the overall / apparent pK a of most all LNPs, it cannot be used for SORT LNPs containing >40% permanently cationic lipid, as these LNPs are always charged. Thus, when 50% of the normalized signal is produced, instead the relative pK a is estimated relative to the basal LNP formulation (without addition of SORT lipid). This alternative calculation does not change the pK a of most LNPs, but does allow estimation of permanently cationic SORT LNPs consistent with experimental results of tissue-selective RNA delivery. Thus, it can be recommended to use the standard TNS assay when the LNP contains a single ionizable cationic lipid, and the alternative 50% normalized signal method for systems such as SORT, which contain a complex mixture of multiple lipids carrying multiple charge states.

[0590] IV. In vitro luciferase expression and cell viability testing

[0591] One day prior to transfection, Huh-7 or A549 cells were seeded at 1×10 4Cells were seeded at a density of into white 96-well plates. The medium was replaced with 150 μL of fresh DMEM medium (5% FBS), and then 50 μL of the Luc mRNA preparation was added, with 25 ng of mRNA fixed per well. After incubating for an additional 24 hours, mRNA expression and cytotoxicity were detected using the ONE-Glo + Tox kit based on the standard protocol of Promega.

[0592] V. Animal experiments

[0593] All animal experiments were approved by the Institution Animal Care and Use Committees of The University of Texas Southwestern Medical Center and were in accordance with applicable local, state, and federal regulations. C57BL / 6 mice were obtained from the UTSW Mouse Breeding Core Facility. B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze / J mice (also known as Ai9 or Ai9(RCL-tdT) mice) were obtained from The Jackson Laboratory (007909) and bred to maintain homozygous expression of the Cre reporter allele, which has a stop cassette flanked by loxP that blocks transcription of the CAG promoter-driven red fluorescent tdTomato protein. After Cre-mediated recombination, Ai9 mice will express tdTomato fluorescence. Ai9 mice are congenic on the C57BL / 6J genetic background.

[0594] VI. In vivo Luc mRNA delivery and biodistribution

[0595] C57BL / 6 mice weighing 18 - 20 g were intravenously injected with various Luc mRNA preparations at a dose of 0.1 or 0.05 mg / kg. n = 2 - 4 per group. After 6 hours, the mice were intraperitoneally (IP) injected with D-luciferin (150 mg / kg) and imaged using the IVIS Lumina system (Perkin Elmer). For biodistribution, C57BL / 6 mice were intravenously injected with the Cy5-Luc mRNA preparation at a dose of 0.5 mg / kg. Ex vivo imaging (Cy5 channel) was performed 6 hours after injection.

[0596] VII. mRNA synthesis

[0597] Optimized Cre recombinase mRNA and Cas9 mRNA were generated by in vitro transcription (IVT). Briefly, the NLS-Cre fragment and Cas9 fragment were prepared by a PCR procedure 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 region (UTR) and polyadenylation sequence. The IVT reaction was carried out following a standard protocol, but replacing the typical UTP 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 study.

[0598] Table 4: Primers, including the length of the PCR products and their purposes

[0599]

[0600]

[0601] The coding sequences of NLS-Cre and Cas9 are as follows:

[0602] NLS-Cre:

[0603]

[0604] SV40 NLS-Cas9-Nucleoplasmin NLS:

[0605]

[0606] VIII. Western Blot

[0607] The quality of IVT Cas9 mRNA was analyzed by Western blot. One day before transfection, 293T cells were seeded into 12-well plates at a density of 1×10 5 cells / well. The cells were treated with various formulations in a total volume of 600 μL for an additional 24 h. The formulations included 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 protease inhibitor cocktail (100×, ThermoFisher) were added to each well and shaken at room temperature for 20 min. The cell lysates were collected into 1.6 mL tubes and centrifuged at 4 °C for 10 min (13,000 g). If not used immediately, the supernatants were collected into new tubes and stored at -80 °C. Before performing Western blot, the protein concentration was measured using a BCA assay kit (ThermoFisher). 15 μg of total protein was loaded and separated by a 4-20% polyacrylamide gel (ThermoFisher). The separated proteins were then transferred onto a polyvinylidene fluoride membrane (BioRad) and blocked with 5% BSA (dissolved in PBST) at room temperature for 1 h. The primary antibody was applied overnight at 4 °C. After washing four times with PBST, the membrane was incubated with the secondary antibody at room temperature for 1 h, and then imaged with an ECL substrate after washing four times with PBST (ThermoFisher).

[0608] IX. Gene Editing in Td-Tomato Mouse Model (Cre mRNA)

[0609] The Cre mRNA formulation was prepared as described above and administered intravenously (0.3 mg / kg Cre mRNA). Two days later, the mice (n = 4 per group) were sacrificed and the major organs were imaged by the IVIS Lumina system (Perkin Elmer).

[0610] X. Cell Isolation and Staining for Flow Cytometry

[0611] To test for Td-Tomato + cells in each organ's cell type, cell isolation and staining were performed 2 days after treatment with Cre mRNA preparation (0.3 mg / kg), followed by analysis by flow cytometry.

[0612] For hepatocyte isolation, two-step collagenase perfusion was performed as previously described (Cheng et al., 2018). Briefly, mice were anesthetized and fixed with isoflurane. Perfusion was started with liver perfusion medium (Thermo Fisher Scientific, 17701038) for 7 - 10 min, then switched to liver digestion medium (Thermo Fisher Scientific, 17703034) for an additional 7 - 10 min. The liver was collected into a plate containing 10 mL of liver digestion medium and cut to release hepatocytes. The released hepatocytes were then collected, washed twice with hepatocyte wash medium (Thermo Fisher Scientific, 17704024), and once with 1×PBS. After further separation by coarse filtration and low-speed (50×g) centrifugation, hepatocytes were analyzed by a FACS Aria II SORP machine (BD Biosciences).

[0613] For the isolation and staining of splenocyte types, the excised spleen was minced with a sterile scalpel and homogenized in 250 μL of 1× digestion medium (45 units / μL collagenase I, 25 units / μL DNase I, and 30 units / μL hyaluronidase). The spleen solution was transferred into a 15 mL tube containing 5 - 10 mL of 1× digestion medium. Subsequently, the spleen solution was filtered through a 70 μm filter and washed once with 1× PBS. The cell pellet was obtained by centrifuging at 300×g for 5 min. 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 min. After incubation, 4 mL of cell staining buffer (BioLegend) was added to stop RBC lysis. The solution was centrifuged again at 300×g for 5 min to obtain the cell pellet. The single cells were resuspended in cell staining buffer and added to a flow tube containing antibodies (100 μL total volume). The cells were incubated with the antibodies in the dark at 4 °C for 20 min. 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). Ghost Dye Red780 (Tonbo Biosciences, 13-0865-T500) was used to distinguish live cells.

[0614] For the isolation and staining of lung cell types, the isolated lungs were minced with a sterile scalpel and then transferred into 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 h with shaking. After incubation, any 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), AlexaFluor 488 anti-mouse CD31 (BioLegend, 102414), and Alexa Fluor 647 anti-mouse CD326 (Ep-CAM) (BioLegend, 118212). Ghost Dye Red 780 (Tonbo Biosciences, 13-0865-T500) was used to distinguish live cells.

[0615] XI. Gene Editing in the Td-Tomato Mouse Model (Cas9 mRNA / sgRNA and Cas9 / sgRNA RNP)

[0616] To evaluate gene editing in vivo, Td-Tom mice of comparable body weight and the same sex were selected. Cas9 mRNA and sgRNA were co-delivered to tdTomato (td-Tom) mice. Cas9mRNA / sgTom1 (4 / 1, weight / weight) was co-delivered via various formulations with a total RNA dose equal to 2.5 mg / kg. At 10 days post-intravenous injection, major organs were removed and imaged on an IVIS Lumina system. For the formulation targeting the spleen, the total RNA dose was 4 mg / kg, and the weight ratio of Cas9 mRNA to sgTom1 was 2 / 1, with the detection time at 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 at day 7 post-injection (n = 2 - 4 per 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) on a Cryostat instrument (Leica Biosystems). The mounted tissue sections were stained with 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) and then imaged by confocal microscopy on a Zeiss LSM 700.

[0617] XII. Gene Editing in C57BL / 6 Mice (Cas9 mRNA / sgPTEN and Cas9 / sgRNA RNP)

[0618] To examine endogenous gene editing in vivo, PTEN was selected. Wild-type C57BL / 6 mice were intravenously injected with various vectors (n = 2 - 4 per group) by co-delivering Cas9 mRNA and modified sgPTEN (4 / 1, mRNA / sgRNA, weight / weight) at a total dose of 2.5 mg / kg. Ten days later, tissues were collected and genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (ThermoFisher). For the formulation targeting the spleen, the total RNA dose was 4 mg / kg, Cas9 mRNA / sgTom1 was 2 / 1 (weight / weight), 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 per group). After obtaining the PTEN PCR product, the T7E1 assay (NEB) was performed to confirm gene editing efficacy by a standard protocol. In addition, the evaluation of PTEN editing was performed on tissue sections by H&E staining and immunohistochemistry (IHC). Briefly, paraformaldehyde (PFA)-fixed tissues were embedded in paraffin, sectioned, and H&E stained by the Molecular Pathology Core at UTSW. 4-μm sections were made in a standard manner, and IHC was detected using the Elite ABC Kit and DAB Substrate (Vector Laboratories).

[0619] Example 9: Formulations Using Neutral Buffer

[0620] It was observed that Cas9 RNP denatured in acidic buffer, resulting in an increase in hydrodynamic size from 10 nm to 150 nm (Figure 40B). This made it difficult, if not impossible, to encapsulate RNP into monodisperse nanoparticles. These studies focused on lipid nanoparticles (LNPs) because they are one of the most efficient classes of RNA delivery vectors in preclinical models and in humans (Wood, 2018; Wang et al., 2017; Doudna and Charpentier, 2014; Hajj and Whitehead, 2017; Sander and Joung, 2014). Among the four components of LNPs [ionizable cationic lipid, zwitterionic phospholipid, cholesterol, and poly(ethylene glycol) (PEG) lipid], pK aIonizable cationic lipids with a pKa of about 6.4 are useful for activity because they bind negatively charged RNA at a mixed pH (e.g., pH 4 when the amine is protonated), lose charge at neutral pH prior to cellular uptake, and then regain charge as the pH in the endosome decreases to fuse with the endosomal membrane and enable cargo release into the cytoplasm. However, this feature prevents efficient encapsulation of cargo at neutral pH because ionizable cationic lipids are uncharged at neutral pH. To overcome this challenge, a fifth component is added, specifically a cationic lipid that is positively charged at neutral pH, which allows encapsulation of RNA and protein using a neutral buffer (instead of an acidic buffer), thereby preserving the tertiary structure and stability of the RNP (Figure 40A).

[0621] To evaluate this strategy, 5A2-SC8 was selected as the ionizable cationic lipid because 5A2-SC8 LNPs safely deliver short siRNA / miRNA and long mRNA to mice with impaired liver function, including MYC-driven hepatocellular carcinoma (Zhou et al., 2016; Zhang et al., 2018a; Zhang et al., 2018b) or gene knockout of fumarylacetoacetate hydrolase (FAH) (Cheng et al., 2018). The introduction of a permanent cationic lipid (e.g., DOTAP) into a conventional 4-component 5A2-SC8 LNP formulation actually allows for controlled self-assembly to occur by mixing an ethanolic solution of the lipid with a PBS solution of the RNP (1 / 3, v / v). The incorporation of DOTAP was evaluated from 5-60 mol% of the total lipid (Figure 41), which revealed higher gene editing levels of 10-20% in vitro and the formation of stable RNP-loaded nanoparticles with a size of <200 nm( Figure 42)。Initially, using sgRNA targeting reporter luciferase (sgLuc), the size of LNP with 10 mol% DOTAP incorporation (5A2-DOT-10: 5A2-SC8 / DOPE / Chol / DMG-PEG / DOTAP = 15 / 15 / 30 / 3 / 7 (mol / mol)) was observed. The particles prepared using PBS buffer were slightly larger than the nanoparticles without RNP loading. The same LNP prepared using low pH buffer did not change in size, suggesting that RNP was not encapsulated (Figure 40C). To determine the optimal molar ratio between Cas9 protein and sgRNA, Cas9 / sgRNA complexes at 1 / 1, 1 / 3, and 1 / 5 (mol / mol) were prepared, which decreased the RNP size (Figure 40D) and increased the negative charge (Figure 40F). After encapsulation, the RNP ratio did not change the size or ζ-potential of the resulting LNP (Figure 40E, 40G). The surface charge of all LNPs was neutral, which not only indicated successful encapsulation but also could be used to minimize in vivo uptake by the mononuclear phagocyte system (MPS). To further examine whether 5A2-DOT-10 could successfully mediate the delivery of RNP into the nucleus, LNPs with encapsulated fluorescent EGFP-fused Cas9 protein were tracked. Free RNP alone could not enter the cells as no green fluorescence above background was detected ( Figure 43 ). After 3 hours of treatment with 5A2-DOT-10, bright green fluorescence was observed in the cytoplasm of the cells. Then, the EGFP-fused Cas9 protein was observed to gradually enter the nucleus within 6 hours due to the presence of a nuclear localization signal on Cas9 (Figure 40H). Endocytosis is energy-dependent and mainly relies on lipid rafts, as treatment with MβCD (an inhibitor of lipid raft-based endocytosis) significantly inhibited the cellular uptake of the nanoparticles (Figure 40I).

[0622] To quantify gene editing efficacy, HeLa-Luc and HeLa-GFP reporter cells were used. Different Cas9 / sgLuc ratios were examined and gene editing was higher at 1 / 3 and 1 / 5 (Figure 44A). Results from the T7 endonuclease I (T7EI) assay confirmed that most all target DNA bands (720 bp) were cleaved into two cleavage bands (536 bp and 184 bp). No cleavage bands were observed with the control treatment group. To test the hypothesis that a neutral pH buffer is required to encapsulate the RNP and maintain Cas9 function, the gene editing efficiency of 5A2-DOT-10 prepared with pH 4 citrate buffer was also evaluated. No cleavage bands were observed at all (Figure 44A). The negative results were further confirmed by Sanger sequencing, providing additional evidence that conventional acid-based formulation methods cannot produce effective NPs. Switching to cells expressing GFP, 5A2-DOT-10 encapsulating Cas9 / sgGFP induced indels in the GFP DNA and knocked out nearly all GFP expression. The control group showed similar fluorescence intensity to PBS-treated cells (Figure 44B), which was confirmed by flow cytometry (Figures 44C and 45). Permanent gene editing was manifested by the loss of GFP in growing cells and confirmed by Sanger sequencing, where CRISPR editing inference (ICE) analysis showed indels reaching 95% (Figure 44D). Looking towards clinical translation, the stability of RNP-loaded 5A2-DOT-10 was monitored at 4 °C for 2 months. The LNP did not change in size and remained homogeneous (PDI < 0.2) (Figure 44G). Continuous testing of the 5A2-DOT-10 nanoparticles revealed constant gene editing activity even after 60 days of storage (Figure 44H).

[0623] The strategy of adding a permanent cationic lipid to the classic 4-component LNP to achieve efficient RNP delivery is not limited to the dendrimer-based ionizable lipid, 5A2-SC8. To demonstrate this, complementary DOTAP was included in the nanoparticle formulations prepared using other types of ionizable materials: the well-known DLin-MC3-DMA lipid used in FDA-approved Onpattro (Wood, 2018) and the C12-200 lipidoid (Figure 46A-B). Even though they have very different chemical structures compared to 5A2-SC8 (Figure 46C), all DOTAP-modified nanoparticles could efficiently edit cells, while the previously established C12-200 or MC3 formulations without DOTAP showed low editing efficiency (Figure 44E). 5A2-DOT-10 also achieved a higher editing efficiency than the positive control RNAiMAX. Since the 5A2-DOT-10 LNP was more effective than the MC3-DOT-10 and C12-200-DOT-10, all subsequent experiments were performed using 5A2-SC8. In addition to DOTAP, other cationic lipids (including DDAB and EPC) were introduced into the LNP formulations (Figure 46E-G). The results were similar for all three cationic lipids with different chemical structures (Figure 46H). These results indicate that this strategy is universal for ionizable cationic lipid nanoparticles (DLNP, LLNP, SNALP) and other cationic lipids that are positively charged at pH 7.4. Because this method allows the adjustment of the FDA-approved Onpattro formulation to achieve RNP delivery, this protocol provides different directions for the treatment of clinically translatable human diseases.

[0624] The key to successful RNP delivery is to replace the standard acidic buffer with PBS buffer to maintain protein stability. To test whether this method is compatible with other neutral buffers, LNPs were formulated in PBS, Opti-MEM medium, and HEPES. The formulation prepared in citrate buffer (pH 4) was used as a control (Figure 46I). Significant and equivalent gene editing (>90%) was achieved using LNPs prepared under all three neutral buffer conditions but not in the acidic buffer (Figure 44F). ICE analysis of the sequencing results was consistent with the results shown by flow cytometry (Figure 46K).

[0625] To examine in vivo gene editing, 5A2-DOT-10 encapsulating the Cas9 / sgTOM complex was delivered to the Td-Tomato mouse model (Figure 47A). In these mice, deletion of the CRISPR-mediated Lox-Stop-Lox cassette turned on downstream tdTom expression in successfully edited cells. 5A2-DOT-10 LNPs loaded with Cas9 / sgTOM RNP were injected into the left leg of mice by intramuscular injection at a dose of 1 mg / kg sgTOM. Since RNAiMAX, which was previously used for direct injection gene editing (Zuris et al., 2015), was complexed with Cas9 / sgTOM RNP for comparison. Higher Td-Tom fluorescence was observed in muscles treated with 5A2-DOT-10 than in mice treated with RNAiMAX (Figure 47B). Imaging of tissue sections further confirmed that gene editing produced brighter red fluorescence in the 5A2-DOT-10 treatment group (Figure 47C). 5A2-DOT-10 was injected into the brain of Td-Tom mice (0.15 mg / kg sgTOM). Again, bright red signals were observed near the injection site, thus confirming the editing of the mouse brain (Figure 47D-E).

[0626] It was evaluated that the improved stability and potency of 5A2-DOT-10 could mediate successful systemic gene editing in tissues. To examine this RNP delivery strategy, LNPs with different molar percentages of DOTAP (5-60%) were prepared and the RNP was delivered to Td-Tom mice IV (1.5 mg / kg sgTOM). Seven days after injection of 5A2-DOT-5, Td-Tom fluorescence was observed only in the liver. Increasing the percentage of incorporated DOTAP from 5% to 60% resulted in a gradual fluorescence (CRISPR-guided gene editing) from the liver to the lungs. 5A2-DOT-60 mainly achieved lung editing (Figure 47F). These results indicate that deep tissue editing can be achieved in a tissue-specific manner by modulating the internal lipid component chemistry and molar ratio. Tissue-specific editing was further confirmed by confocal imaging of tissue sections (Figure 47G). Then, editing of the endogenous target Pten was evaluated by systemically injecting LNPs encapsulating Cas9 / sgPTEN RNP into wild-type C57BL / 6 mice. Clear T7EI cleavage bands were detected only in the livers of mice treated with 5A2-DOT-5 and in the lungs of mice treated with 5A2-DOT-50 and 5A2-DOT-60 (Figure 47H).

[0627] To evaluate the possibility of simultaneous in vivo editing of multiple genes, Cas9 protein and six different sgRNAs were loaded into 5A2-DOT-50. sgTOM, sgP53, sgPTEN, sgEml4, sgALK, and sgRB1 were loaded into the Cas9 protein for encapsulation. Then, Td-Tom mice were treated with 5A2-DOT-50 (the combination) via tail vein injection (0.33 mg / kg of each sgRNA). One week later, bright Td-Tom fluorescence was detected in the lungs, indicating gene editing of TOM (Figure 47I). Clear T7EI cleavage bands were observed at all other 5 genomic loci, indicating that 5A2-DOT-50 was able to simultaneously and efficiently edit multiple genes at low doses (Figure 47J). Quantitative analysis revealed a target editing efficiency of up to 22% in the lungs (Figures 47 and 48). In this study, sgRNAs with terminal modifications of the first and last 3 nucleotides were used to enhance sgRNA stability and reproducibility (Figure 49) (Finn et al., 2018; Hendel et al., 2015). Reports have shown that compared with terminally modified sgRNAs, precise modification of other nucleotides can increase in vivo gene editing by 2-4-fold (Finn et al., 2018; Yin et al., 2017), indicating that the editing efficiency reported in this study may be higher with further sgRNA optimization. Nevertheless, the high efficacy and tissue specificity of 5A2-DOT-50 allow simultaneous editing of 6 targets in the lungs with a single injection.

[0628] Traditionally, animal models have been generated by transgenesis or genetic engineering in embryonic stem cells, which is both time-consuming and expensive. The direct mutation of tumor and other disease-related genes in adult mice using CRISPR / Cas provides a feasible solution for the rapid generation of models. This can only be achieved using expensive lentiviruses that must be engineered for each target and by hydrodynamic injection into the liver (Xue et al., 2014; Maddalo et al., 2014). Since mutations in multiple genes are usually required to generate functional cancer models, there is a great need to develop an inexpensive and effective non-viral nanoparticle-based multiplexing scheme. Since 5A2-DOT-X LNPs are effective, can simultaneously edit multiple targets, can be repeatedly administered, and provide tissue specificity, they offer a way to generate a variety of animal models.

[0629] 5A2-DOT-5 was used to selectively knock out three tumor suppressor genes (P53, PTEN, and RB1) in the liver simultaneously. These genes have been identified in many human cancers, including liver cancer. C57BL / 6 mice were treated by intravenous injection of 2.5 mg / kg total sgRNA weekly for 3 weeks, and the gene editing efficiency in the mouse liver was detected (Figure 48A). Clear cleavage bands were observed at all three gene loci at 2, 12, 15, and 20 weeks after treatment by T7EI assay (Figures 48B, 50, and 51). Over time, the cleavage bands were much brighter, indicating tumor growth. When the mice were sacrificed at 15 and 20 weeks, visible tumors were found on the liver, and several metastatic tumors were found in the abdominal cavity (Figures 48C and 52). Tumor formation was also detected by H&E staining and IHC staining targeting the tumor proliferation biomarker Ki67 at different time points (Figures 48D and 53).

[0630] To generate a challenging lung cancer mouse model, focus was placed on the Eml4-Alk chromosomal rearrangement, a complex mutation found in many solid human tumors, especially non-small cell lung cancer (Maddalo et al., 2014; Blasco et al., 2014). The Eml4-Alk fusion protein produced after rearrangement between Eml4 and Alk promotes cancer development. Utilizing the high efficacy and lung-targeting specificity of 5A2-DOT-50, one (total sgRNA at a dose of 2 mg / kg) or two (total sgRNA at a dose of 1.5 mg / kg once a week) intravenous doses were injected and the tumorigenesis process was evaluated (Figure 48E). Indel generation was detected in the extracted lung DNA from both groups of mice at all time points examined (Figures 48F and 54). Clear gene rearrangement bands were detected in the lungs of 5A2-DOT-50-treated mice, thus confirming the successful generation of chromosomal rearrangement (Figures 48F and 54). Over time, the Eml4-Alk rearrangement bands were much brighter, indicating the proliferation of edited cells. Sequencing results after subcloning these PCR amplicons further confirmed the Eml4-Alk rearrangement (Figures 48G and 54). Several tumor lesions were observed in the lungs at 16 and 24 weeks after H&E staining and Ki67 staining (Figures 48H, 55, and 56). These results showed that a single injection of 5A2-DOT-50 LNP could successfully generate chromosomal rearrangement and lead to lung tumorigenesis in adult mice. Therefore, these LNPs were positioned to accelerate the in situ creation of multiple disease models.

[0631] B. Materials and Methods

[0632] I. Materials.

[0633] 5A2-SC8 (Zhou et al., 2016), DLin-MC3-DMA (Jayaraman et al., 2012), and C12-200 (Love et al., 2010) were synthesized and purified following the published protocols. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Sigma-Aldrich. 1,2-Dimyristoyl-sn-glycero-methoxy(poly((ethylene glycol) MW 2000) (DMG-PEG2000) was purchased from NOF America Corporation. ONE-Glo + Tox luciferase reporter assay kit was purchased from Promega Corporation. Pur-A-Lyzer Midi dialysis kit (MWCO, 3.5 kDa) was purchased from Sigma-Aldrich. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), Hoechst 33342, DLS Ultramicro cuvettes, Lipofectamine RNAiMAX transfection reagent, and Lab-Tek chambered coverglass units were purchased from Thermo Fisher Scientific. Cas9 protein and Ki-67 monoclonal antibody were purchased from Thermofisher. GenCrispr NLS Cas9-EGFP nuclease was purchased from GenScript. Modified sgRNA was purchased from Synthego.

[0634] II. Cas9 / sgRNA Complex Preparation.

[0635] Separate solutions of Cas9 protein and sgRNA were mixed together in equal volumes in a labeled buffer. After mixing, the mixture was incubated at room temperature for 5 minutes to allow for the formation of RNP for the self-assembly of the intact Cas9 / sgRNA complex. The molar ratios of Cas9 protein to sgRNA used were 1 / 1, 1 / 3, and 1 / 5.

[0636] III. Optimized Nanoparticle Formulation and Characterization.

[0637] Dissolve the ionizable cationic lipids (5A2-SC8, C12-200, or DLin-MC3-DMA) (Zhou et al., 2016; Jayaraman et al., 2012; Love et al., 2010), zwitterionic lipids (DOPE or DSPC), cholesterol, DMG-PEG, and permanent cationic lipids (DOTAP, DDAB, or EPC) in ethanol at a given molar ratio. Dissolve the Cas9 / sgRNA complex in 1×PBS buffer. Rapidly pipette the Cas9 / sgRNA RNP complex solution in PBS buffer into the ethanol solution of lipids at a volume ratio of 3:1 (Cas9 / sgRNA RNP:lipids, v / v) such that the weight ratio of total lipids to sgRNA is 40:1 (weight), and then incubate at room temperature for 15 min. After that, directly characterize the fresh formulation and use it for in vitro assays. For animal experiments, dialyze the formulation against 1×PBS (Pur-A-Lyzer Midi dialysis kit, MWCO 3.5 kDa) for 1 h to remove ethanol before local injection (intramuscular or intracerebral injection) or systemic injection (intravenous injection). Measure the size distribution and ζ-potential of the nanoscale formulation using dynamic light scattering (DLS, Malvern; He-Ne laser, λ = 632 nm; detection angle = 173°).

[0638] IV. RNAiMAX Formulation.

[0639] To prepare the RNAiMAX-complexed RNP, prepare the Cas9 / sgRNA complex in Opti-MEM and gently mix it with Lipofectamine RNAiMAX transfection reagent diluted in Opti-MEM (at a dose of 1 μL RNAiMAX / μg sgRNA). Incubate the mixture solution at room temperature for 30 minutes to complete the complexation.

[0640] V. Standard LNP Formulation.

[0641] To prepare C12-200 and MC3 LNPs encapsulating RNP, the Cas9 / sgRNA RNP complex solution in citrate buffer (pH 4.0) was rapidly mixed by pipette into the ethanol solution of lipids at a volume ratio of 3:1 (Cas9 / sgRNA RNP: total lipid, v / v), such that the weight ratio of total lipid to sgRNA was 40:1 (weight / weight), and then incubated at room temperature for 15 min. For C12-200 LNP, the molar ratio of C12-200 / DOPE / Chol / DMG-PEG was 35 / 16 / 46.5 / 2.5; for MC3 LNP, the molar ratio of DLin-MC3-DMA / DSPC / Chol / DMG-PEG was 50 / 10 / 38.5 / 1.5.

[0642] VI.5A2-DOT-10Cas9 / sgLuc-treated cell uptake and uptake mechanism

[0643] To examine cell uptake, HeLa-Luc cells were seeded at a density of 2×10 4 cells / well into Lab-Tek Chambered Coverglass (8-well) and incubated overnight at 37 °C. Then, the old medium was replaced with 150 μL of fresh DMEM containing 10% FBS, and the cells were treated with 50 μL of 5A2-DOT-10 encapsulating Cas9-EGFP / sgLuc RNP (9 nM sgRNA / well). At 1 h, 3 h, 6 h, and 24 h after treatment, the cells were washed three times with PBS and stained with Hoechst (0.1 mg / mL) for 15 min at 37 °C, and then imaged by confocal microscopy (Zeiss LSM 700).

[0644] To examine the uptake mechanism, assays for specific inhibition of endocytic pathways were evaluated using Hela-Luc cells. Only 5A2-DOT10 treatment was used as a control. HeLa-Luc cells were seeded at 5×10 5Cells were seeded at a density of 1.5×10

[0645] VII. T7EI assay for detecting genome editing

[0646] For in vitro genomic DNA editing analysis, HeLa-Luc cells were seeded at a density of 1.5×10 5Cells were seeded at a density of cells / well into a 12-well plate and incubated overnight. Then, different nanoplatforms containing 24 nM sgRNA were added to the cells. After 3 days, the cells were harvested, washed, and resuspended in 50 μL of 1× Passive Lysis Buffer (Promega) together with 2 μL of Proteinase K (Thermofisher). Subsequently, a lysis PCR program (hold at 65 °C for 15 min, hold at 95 °C for 10 min) was run to obtain cell lysates. Then, the targeted genomic locus was amplified using the following PCR amplification program (hold at 95 °C for 5 min; (hold at 95 °C for 30 s; hold at 60 - 64 °C for 30 s; hold at 72 °C for 1 min) for a total of 40 cycles; hold at 72 °C for 7 min, and then hold at 4 °C). The cell lysate was used as the DNA template. Then, the amplicons were purified using a PCR purification kit (Qiagen), and 200 ng of the purified DNA was added to 19 μL of an annealing reaction mixture containing 1× NEBuffer 2. Then, the PCR products were annealed in a thermal cycler using the following conditions (hold at 95 °C for 5 min, then cool the mixture from 95 °C to 85 °C at a ramp rate of -2 °C / s, subsequently cool from 85 °C to 25 °C at a ramp rate of -0.1 °C / s, and then hold at 4 °C) to form heteroduplex DNA. Then, 1 μL of T7EI (NEB) was added and incubated at 37 °C for 15 min. Then, the cleavage reaction was stopped by adding 1.5 μL of 0.25 M EDTA. Subsequently, the digested DNA was analyzed using 2.5% agarose gel electrophoresis. All primers used for the T7EI assay are listed in Table 5.

[0647] Table 5: sgRNA sequences

[0648]

[0649] For in vivo genomic DNA editing analysis, genomic DNA was extracted from tissues using the PureLink Genomic DNA Mini Kit (Invitrogen) according to the manufacturer's instructions. Subsequently, the aforementioned procedure was followed as described above for the T7EI assay.

[0650] The fragmented PCR products were analyzed, and the indel percentage was calculated based on the following formula:

[0651] % Gene modification = 100 × (1 - (1 - cleavage fraction) 1 / 2 )

[0652] where the cleavage fraction is the sum of the cleavage product peaks divided by the sum of the cleavage product peaks and the parental peak. 5

[0653] VIII. Sanger Sequencing for Detecting Genomic Editing.

[0654] The purified PCR amplicons of the T7EI assay along with their forward primers were sequenced by the McDermott Center Sequencing core facility at the University of Texas Southwestern Medical Center. Finally, the sequencing data were analyzed using the online analysis software ICE Analysis, a web tool provided by Synthego.

[0655] IX. In vitro gene editing in Hela-GFP cells.

[0656] HeLa-GFP reporter cells were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin at 37 °C / 5% CO2. For experiments, HeLa-GFP cells were seeded into 12-well plates at a cell density of 1.5×10 5 cells / well and incubated overnight. Then, the medium was replaced with 0.5 mL of fresh complete DMEM, and then 100 μL of the nano...

Claims

1. A composition comprising: (A) A therapeutic agent; and (B) A lipid nanoparticle composition comprising: (1) A selective organ targeting compound; (2) An ionizable cationic lipid; and (3) A phospholipid; wherein the composition preferentially delivers nucleic acid to a target organ selected from the group consisting of: 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, brain stem, cerebellum, spinal cord, eye, ear, tongue, or skin.

2. The composition of claim 1, wherein the selective organ targeting compound is a permanent cationic lipid.

3. The composition of claim 1, wherein the selective organ targeting compound is a permanent anionic lipid.

4. The composition of claim 1, wherein the selective organ targeting compound is C6-C 24 diacyl phosphatidylcholine.

5. The composition according to any one of claims 1 - 4, wherein the ionizable cationic lipid is a dendrimer further defined by the formula: Core - repeating unit - capping group (I) wherein the core is connected to the repeating unit by removing one or more hydrogen atoms from the core and replacing the atoms with the repeating unit, and wherein: The core has the formula: wherein: X1 is an amino group or an alkylamino group (C≤12) , a dialkylamino group (C≤12) , a heterocycloalkyl group (C≤12) , a heteroaryl group (C≤12) or a substituted form thereof; R1 is an amino group, a hydroxyl group, a mercapto group or an alkylamino group (C≤12) , a dialkylamino group (C≤12) or a substituted form of any one of these groups; and a is 1, 2, 3, 4, 5, or 6; or The core has the formula: wherein: X2 is N(R5) y ; R5 is hydrogen, an alkyl group (C≤18) or a substituted alkyl group (C≤18) ; and y is 0, 1, or 2, provided that the sum of y and z is 3; R2 is amino, hydroxy, 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, 3; provided that the sum of z and y is 3; or The core has the formula: wherein: X3 is -NR6-, where R6 is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8) , -O- or alkylamino diyl (C≤8) , alkoxy diyl (C≤8) , arene diyl (C≤8) , heteroarene diyl (C≤8) , heterocycloalkane diyl (C≤8) or a substituted form of any of these groups; R3 and R4 are each independently an amino group, a hydroxyl group, a mercapto group or an alkylamino group (C≤12) , a dialkylamino group (C≤12) or a substituted form of any of these groups; or a 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 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; wherein the repeating unit contains a degradable diacyl and a linker; The degradable diacyl has the formula: wherein: A1 and A2 are each independently -O- or -NR a -, where: R a is hydrogen, an alkyl group (C≤6) or a substituted alkyl group (C≤6) ; Y3 is an alkanediyl (C≤12) , an alkenediyl (C≤12) , an arenediyl (C≤12) or a substituted form of any of these groups; or a group of the formula: 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, an alkanediyl (C≤12) , an alkenediyl (C≤12) , an arenediyl (C≤12) or a substituted form of any of these groups; and R9 is an alkyl group (C≤8) or a substituted alkyl group (C≤8) ; The linker group has the formula: 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 wherein when the repeating unit contains a linker group, if n is greater than 1, the linker group contains an independent degradable diacyl that is attached to both the nitrogen and sulfur atoms of the linker group, wherein the first group in the repeating unit is a degradable diacyl, and wherein for each linker group, the next repeating unit contains two degradable diacyls that are attached to the nitrogen atoms of the linker group; and wherein n is the number of linker groups present in the repeating unit; and The capping group has the formula: wherein: Y4 is an alkanediyl (C≤18) or an alkanediyl in which (C≤18) one or more hydrogen atoms on the alkanediyl have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3 (C≤18) ; R 10 is hydrogen, a carboxyl group, a hydroxyl group, or Aryl (C≤12) 、Alkylamino (C≤12) 、Dialkylamino (C≤12) 、N-Heteroalkyl (C≤12) 、-C(O)N(R 11 )-Alkylene (C≤6) -Heteroalkyl (C≤12) 、-C(O)-Alkylamino (C≤12) 、-C(O)-Dialkylamino (C≤12) 、-C(O)-N-Heteroalkyl (C≤12) wherein: R 11 is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6) ; wherein the final degradable diacyl in the chain is attached to the capping group; n is 0, 1, 2, 3, 4, 5, or 6; or a pharmaceutically acceptable salt thereof.

6. The composition according to claim 5, wherein the dendrimer is further defined as: or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition comprising: (A) The composition according to any one of claims 1 - 6; and (B) An excipient.

8. A method of regulating gene expression comprising delivering a nucleic acid to a cell, the method comprising: Contacting the cell with the composition or pharmaceutical composition according to any one of claims 1 - 7 under conditions sufficient to cause the nucleic acid to be taken up by the cell.

9. A method of treating a disease or disorder in a patient, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of a composition or pharmaceutical composition according to any one of claims 1 - 7, wherein the composition or pharmaceutical composition comprises a therapeutic nucleic acid for the disease or disorder.

10. A method of preparing lipid nanoparticles, comprising: (A) dissolving a permanent cationic lipid, an ionizable cationic 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, wherein the buffer is a buffer having a pH of from about 6.8 to about 7.6 to form a buffered therapeutic agent solution; and (C) mixing the lipid solution with the buffered therapeutic agent solution to form lipid nanoparticles.

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