Lipophilic cationic dendrimers and uses thereof

Abstract

The present invention relates to lipophilic cationic dendrimers and uses thereof. Provided herein are modular dendrimers having cationic groups and lipophilic groups. In some aspects, the dendrimers provided herein can be formulated into compositions containing a nucleic acid and one or more auxiliary excipients. In some aspects, these compositions can also be used to treat a disease or disorder with a therapeutic nucleic acid.

Description

[0001] This application is a divisional application of Chinese patent application filed on September 14, 2016, with application number "201680065889.9" and invention title "Lipophilic Cationic Dendritic Polymer and Its Use Thereof". The original application was the Chinese national phase application of international application PCT / US2016 / 051648.

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 218,412, filed September 14, 2015, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention generally relates to the field of dendritic polymers. In particular, it relates to dendritic polymer nanoparticle compositions comprising nucleic acids. More specifically, it relates to dendritic polymer nanoparticle compositions for delivering nucleic acids. More specifically, it relates to dendritic polymer nanoparticle compositions for retrograde delivery of drugs and other excipients. Background Technology

[0005] Since the discovery of RNAi or other nucleic acid agents and the recognition of their therapeutic potential, the search for effective delivery vectors has been ongoing (Whitehead et al., 2009; Kanasty et al., 2013; Akinc et al., 2008; Davis et al., 2010; Love et al., 2010; Siegwart et al., 2011; Jayaraman et al., 2012). Progress has been made in the delivery efficacy of small RNAs to the healthy liver, but existing delivery vectors do not yet meet the clinically desired combination of high efficacy against tumors and low hepatotoxicity to normal cells. Unfortunately, in the past four years, all five Phase III human clinical trials of small molecule drugs for the treatment of hepatocellular carcinoma (HCC) have failed, partly due to the amplified drug toxicity caused by advanced liver dysfunction in patients with debilitated livers (Roberts, LR, 2008; Scudellari, M., 2014). MicroRNAs (miRNAs) represent a promising alternative strategy because they can act as tumor inhibitors by simultaneously targeting multiple pathways involved in cell differentiation, proliferation, and survival. However, these therapeutics require an effective carrier (Ventura and Jacks, 2009; Kasinski and Slack, 2011; Ling et al., 2013; Cheng et al., 2015). The balance between the potency and toxicity of the drug carrier is a useful criterion, particularly in the case of liver cancer, where the carrier's own toxicity can diminish the therapeutic effect of small RNA therapy.

[0006] To achieve this balance between low toxicity and high efficacy, the influence of structural diversity and molecular size on chemical structure of delivery carriers can be used to achieve an effective therapeutic balance. Dendritic polymers are monodisperse macromolecules composed of multiple fully branched monomers radiating radially from a central core. Dendritic polymers therefore possess the same high molecular homogeneity and extensive theoretical space for chemical tuning as polydisperse polymers as small molecules (Bosman et al., 1999; Fréchhet and Tomalia, 2002; Gillies and Frechet, 2002; Grayson and Fréchet, 2001). These inherent characteristics make dendritic polymers unique for a variety of biomedical applications (Stiriba et al., 2002; Lee et al., 2005; Wu et al., 2015) (Murat and Gres, 1996; Percec et al., 2010; Duncan and Izzo, 2005). In gene delivery, most studies have used a limited number of commercial dendritic polymers for further chemical modification. (Kang et al., 2005; Taratula et al., 2009; Khan et al., 2014). Therefore, the expansion of dendritic polymer applications depends on the ability to readily tune the size, chemistry, topology, and ultimately the physical properties of the dendritic polymer through chemical synthesis. Thus, the development of novel dendritic polymers that can serve as carriers for nucleic acids and other drugs is clinically useful. Summary of the Invention

[0007] In some respects, this disclosure provides dendritic polymers of the following formula:

[0008] Core - (repeating unit) n -Terminal group (I)

[0009] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0010] The core has the following formula:

[0011]

[0012] in:

[0013] X1 is an amino or alkylamino group. (C≤12) Dialkylamino (C≤12) heterocyclic alkyl (C≤12) , heteroaryl (C≤12) or its alternative forms;

[0014] R1 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12)or a substituted form of any of these groups; and

[0015] a is 1, 2, 3, 4, 5, or 6; or

[0016] The core has the following formula:

[0017]

[0018] in:

[0019] X2 is N(R5) y ;

[0020] R5 is hydrogen or alkyl (C≤18) Or substituted alkyl (C≤18) ;and

[0021] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0022] R2 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[0023] b is 1, 2, 3, 4, 5, or 6; and

[0024] z is 1, 2, or 3; the condition is that the sum of z and y is 3; or

[0025] The core has the following formula:

[0026]

[0027] in:

[0028] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) , or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) heterocyclic alkane dimethyl (C≤8) , or a substituted form of any of these groups;

[0029] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups; or a group of the following formula: -(CH2CH2N) e (R c )R d ;

[0030] in:

[0031] e is 1, 2, or 3;

[0032] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0033] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[0034] The core is an alkylamine. (C≤8 ), dialkylamine (C≤36) Heterocyclic alkanes (C≤12) , or a substituted form of any of these groups;

[0035] The repeating unit comprises a degradable diacyl group and a linker;

[0036] The degradable diacyl group has the formula:

[0037]

[0038] in:

[0039] A1 and A2 are independently -O- or -NR. a -,in:

[0040] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0041] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (c≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0042]

[0043] in:

[0044] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0045] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0046] R9 is an alkyl group. (C≤8)Or substituted alkyl (C≤8) ;

[0047] The connecting group has the following formula:

[0048]

[0049] in:

[0050] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0051] When the repeating unit contains a linker group, the linker group is connected to degradable diacyl groups on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0052] The terminating group has the following formula:

[0053]

[0054] in:

[0055] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0056] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0057] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0058] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl(C≤6) ;

[0059] The final degradable diacyl group in the chain is attached to a capping group;

[0060] n is 0, 1, 2, 3, 4, 5, or 6;

[0061] Or a pharmaceutically acceptable salt thereof. In some embodiments, the structure of the dendritic polymer is further defined:

[0062] The core has the following formula:

[0063]

[0064] in:

[0065] X1 is an amino or alkylamino group. (C≤12) Dialkylamino (C≤12) heterocyclic alkyl (C≤12) , heteroaryl (C≤12) or its alternative forms;

[0066] R1 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) or a substituted form of any of these groups; and

[0067] a is 1, 2, 3, 4, 5, or 6; and

[0068] The repeating unit comprises a degradable diacyl group and a linker;

[0069] The degradable diacyl group has the formula:

[0070]

[0071] in:

[0072] A1 and A2 are independently -O- or -NR. a -,in:

[0073] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0074] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0075]

[0076] in:

[0077] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0078] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0079] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0080] The connecting group has the following formula:

[0081]

[0082] in:

[0083] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0084] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0085] End-capping groups, wherein the end-capping groups have the formula:

[0086]

[0087] in:

[0088] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0089] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0090] Aryl (C≤12) alkylamino (C≤12)Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0091] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0092] The final degradable diacyl group in the chain is attached to a capping group;

[0093] n is 0, 1, 2, 3, 4, 5, or 6;

[0094] Or a pharmaceutically acceptable salt thereof. In some embodiments, the dendritic polymer has the formula:

[0095] Core - (repeating unit) n -Terminal group (I)

[0096] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0097] The core has the following formula:

[0098]

[0099] in:

[0100] X2 is N(R5) y ;

[0101] R5 is hydrogen or alkyl. (C≤8) , or substituted alkyl (C≤18) ;and

[0102] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0103] R2 is an amino, hydroxyl, or mercapto group, or an alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[0104] b is 1, 2, 3, 4, 5, or 6; and

[0105] z is 1, 2, 3; the condition is that the sum of z and y is 3;

[0106] The repeating unit comprises a degradable diacyl group and a linker;

[0107] The degradable diacyl group has the formula:

[0108]

[0109] in:

[0110] A1 and A2 are independently -O- or -NR. a -,in:

[0111] R a It is ammonia, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0112] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0113]

[0114] in:

[0115] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0116] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0117] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0118] The connecting group has the following formula:

[0119]

[0120] in:

[0121] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0122] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0123] End-capping groups, wherein the end-capping groups have the formula:

[0124]

[0125] in:

[0126] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0127] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0128] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0129] The final degradable diacyl group in the chain is attached to a capping group;

[0130] n is 0, 1, 2, 3, 4, 5, or 6;

[0131] Or a pharmaceutically acceptable salt thereof. In other embodiments, the dendritic polymer has the formula:

[0132] Core - (repeating unit) n -Terminal group (I)

[0133] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0134] The core has the following formula:

[0135]

[0136] in:

[0137] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) heterocyclic alkane dimethyl (C≤8) , or a substituted form of any of these groups;

[0138] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups; or a group of the following formula: -(CH2CH2N) e (R c )R d ;

[0139] in:

[0140] e is 1, 2, or 3;

[0141] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0142] c and d are each independently 1, 2, 3, 4, 5, or 6; and

[0143] The repeating unit comprises a degradable diacyl group and a linker;

[0144] The degradable diacyl group has the formula:

[0145]

[0146] in:

[0147] A1 and A2 are independently -O- or -NR. a -,in:

[0148] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0149] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12), Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0150]

[0151] in:

[0152] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0153] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0154] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0155] The connecting group has the following formula:

[0156]

[0157] Among them:

[0158] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0159] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0160] End-capping groups, wherein the end-capping groups have the formula:

[0161]

[0162] in:

[0163] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18)Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0164] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0165] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0166] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0167] The final degradable diacyl group in the chain is attached to a capping group;

[0168] n is 0, 1, 2, 3, 4, 5, or 6;

[0169] Or a pharmaceutically acceptable salt thereof. In other embodiments, the dendritic polymer has the formula:

[0170] Core - (repeating unit) n -Terminal group (I)

[0171] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0172] The core is an alkylamine. (C≤8） dialkylamine (C≤36) Heterocyclic alkanes (C≤12) or a substituted form of any of these groups; and

[0173] The repeating unit comprises a degradable diacyl group and a linker;

[0174] The degradable diacyl group has the formula:

[0175]

[0176] in:

[0177] A1 and A2 are independently -O- or -NR. a -,in:

[0178] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0179] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0180]

[0181] in:

[0182] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0183] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0184] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0185] The connecting group has the following formula:

[0186]

[0187] in:

[0188] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0189] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0190] End-capping groups, wherein the end-capping groups have the formula:

[0191]

[0192] in:

[0193] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0194] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0195] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12 ), -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0196] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0197] The final degradable diacyl group in the chain is attached to a capping group;

[0198] n is 0, 1, 2, 3, 4, 5, or 6;

[0199] Or a pharmaceutically acceptable salt thereof. In some embodiments, the capping group is further defined by the following formula:

[0200]

[0201] in:

[0202] Y4 is an alkane dimethyl group. (C≤18) Or an alkane dimethyl group in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3 (C≤18) ;and

[0203] R 10 It is hydrogen.

[0204] In other embodiments, the terminating group is further defined by the following formula:

[0205]

[0206] in:

[0207] Y4 is an alkane dimethyl group. (C≤18) ;and

[0208] R 10 It is hydrogen.

[0209] In some implementations, Y4 is an alkane dimethyl group. (C4-18) In other embodiments, the terminating group is further defined by the following formula:

[0210]

[0211] in:

[0212] Y4 is an alkane dimethyl group. (C≤18) Or an alkane dimethyl group in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3 (C≤18) ;

[0213] R 10 It is an alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) .

[0214] In some embodiments, the terminating group is further defined by the following formula:

[0215]

[0216] in:

[0217] Y4 is an alkane dimethyl group. (C≤18) Or an alkane dimethyl group in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3 (C≤18) ;

[0218] R 10 It is a hydroxyl group.

[0219] In some implementations, the core is further defined by the following formula:

[0220]

[0221] in:

[0222] X1 is an alkylamino group. (C≤12) Dialkylamino (C≤12) heterocyclic alkyl (C≤12) , heteroaryl (C≤12) or its alternative form;

[0223] R1 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) or a substituted form of any of these groups; and

[0224] a is 1, 2, 3, 4, 5, or 6;

[0225] In some embodiments, X1 is an alkylamino group. (C≤12) Or substituted alkylamino (C≤12) In some embodiments, X1 is ethylamino. In other embodiments, X1 is dialkylamino. (C≤12) Or substituted dialkylamino (C≤12) In some embodiments, X1 is dimethylamino. In other embodiments, X1 is a heterocyclic alkyl group. (C≤12) Or substituted heterocyclic alkyl (C≤12) In some embodiments, X1 is 4-piperidinyl, N-piperidinyl, N-morpholinyl, N-pyrrolidinyl, 2-pyrrolidinyl, N-piperazinyl, or N-4-methylpiperidinyl. In other embodiments, X1 is a heteroaryl group (…). C≤12) Or replaced heteroaryl groups (C≤12) In some embodiments, X1 is 2-pyridyl or N-imidazolyl. In some embodiments, R1 is hydroxyl. In other embodiments, R1 is amino. In other embodiments, R1 is alkylamino. (C≤12) Or substituted alkylamino (C≤12) In some embodiments, R1 is an alkylamino group. (C≤12) In some embodiments, R1 is methylamino or ethylamino. In some embodiments, a is 1, 2, 3, or 4. In some embodiments, a is 2 or 3. In some embodiments, a is 2. In other embodiments, a is 3. In some embodiments, the nucleus is further defined as a compound of the following formula:

[0226]

[0227] In some implementations, the core is further defined as:

[0228]

[0229] In other embodiments, the core is further defined by the following formula:

[0230]

[0231] in:

[0232] X2 is N(R5) y ;

[0233] R5 is hydrogen or alkyl. (C≤8) , or substituted alkyl (C≤18) ;and

[0234] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0235] R2 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[0236] b is 1, 2, 3, 4, 5, or 6; and

[0237] z is 1, 2, 3; the condition is that the sum of z and y is 3.

[0238] In some embodiments, X2 is N. In other embodiments, X2 is NR5, where R5 is ammonia or alkyl. (C≤8) In some embodiments, R5 is hydrogen. In other embodiments, R5 is methyl. In some embodiments, z is 3. In other embodiments, z is 2. In some embodiments, R2 is hydroxyl. In other embodiments, R2 is amino. In other embodiments, R2 is alkylamino. (C≤12) Or substituted alkylamino (C≤12) In some embodiments, R2 is an alkylamino group. (C≤12) In some embodiments, R2 is a methylamino group. In other embodiments, R2 is a dialkylamino group. (C≤12) Or substituted dialkylamino (C≤12) In some embodiments, R2 is a dialkylamino group. (C≤12) In some embodiments, R2 is dimethylamino. In some embodiments, b is 1, 2, 3, or 4. In some embodiments, b is 2 or 3. In some embodiments, b is 2. In other embodiments, b is 3. In some embodiments, the nucleus is further defined as:

[0239]

[0240]

[0241] In some implementations, the core is further defined as:

[0242]

[0243] In other embodiments, the core is further defined as:

[0244]

[0245] in:

[0246] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) heterocyclic alkane dimethyl (C≤8) , or a substituted form of any of these groups;

[0247] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups; or a group of the following formula: -(CH2CH2N) e (R c )R d ;

[0248] in:

[0249] e is 1, 2, or 3;

[0250] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0251] c and d are each independently 1, 2, 3, 4, 5 or 6.

[0252] In some embodiments, X3 is -O-. In other embodiments, X3 is -NR6-, where R6 is hydrogen or an alkyl group. (C≤8) Or substituted alkyl (C≥8) In some embodiments, X3 is -NH- or -NCH3-. In other embodiments, X3 is an alkylaminodimethyl group. (C≤8) Or substituted alkylamino dimethyl (C≤8) In some embodiments, X3 is -NHCH2CH2NH- or -NHCH2CH2NHCH2CH2NH-. In other embodiments, X3 is an alkoxydiyl group. (C≤8) Or substituted alkoxydiyl (C≤8) In some embodiments, X3 is -OCH2CH2O-. In other embodiments, X3 is an aromatic dimethyl group. (C≤8) Or replaced aromatic dimethyl(C≤8) In some embodiments, X3 is phenylenediol. In other embodiments, X3 is a heterocyclic alkane dimethyl group. (C≤8) Or substituted heterocyclic alkane dimethyl (C≤8) In some embodiments, X3 is N,N′-piperazinediyl.

[0253] In some embodiments, R3 is an amino group. In other embodiments, R3 is a hydroxyl group. In still other embodiments, R3 is an alkylamino group. (C≤12) Or substituted alkylamino (C≤12) In some embodiments, R3 is an alkylamino group. (C≤12) In some embodiments, R3 is a methylamino group. In other embodiments, R3 is a dialkylamino group. (C≤12) Or substituted dialkylamino (C≤12) In some embodiments, R3 is a dialkylamino group. (C≤12) In some implementations, R3 is dimethylamino.

[0254] In some embodiments, R4 is an amino group. In other embodiments, R4 is a hydroxyl group. In still other embodiments, R4 is an alkylamino group. (C≤12) Or substituted alkylamino (C≤12) In some embodiments, R4 is an alkylamino group. (C≤12) In some embodiments, R4 is a methylamino group. In other embodiments, R4 is a dialkylamino group. (C≤12) Or substituted dialkylamino (C≤12) In some embodiments, R4 is a dialkylamino group. (C≤12) In some embodiments, R4 is dimethylamino. In other embodiments, R4 is -(CH2CH2N). e (R c )R d Where: e is 1, 2, or 3; and R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) In some implementations, e is 1 or 2. In some implementations, e is 1. In some implementations, R c It is hydrogen. In some implementations, R d It is hydrogen.

[0255] In some embodiments, c is 1, 2, 3, or 4. In some embodiments, c is 2 or 3. In some embodiments, c is 2. In other embodiments, c is 3. In some embodiments, d is 1, 2, 3, or 4. In some embodiments, d is 2 or 3. In some embodiments, d is 2. In other embodiments, d is 3. In some embodiments, the core is further defined as:

[0256]

[0257]

[0258] In some implementations, the core is further defined as:

[0259]

[0260] In other embodiments, the core is an alkylamine. (C≤18) dialkylamine (C≤36) Heterocyclic alkanes (C≤12) Or a substituted form of any of these groups. In some embodiments, the core is an alkylamine. (C≤18) Or substituted alkylamines (C≤18) In some embodiments, the core is octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, and octadecylamine. In other embodiments, the core is a dialkylamine. (C≤36) Or substituted dialkylamines (C≤36) In some embodiments, the core is N-methyl, N-dodecylamine, dioctylamine, or didecylamine. In other embodiments, the core is a heterocyclic alkane. (C≤12) Or replaced heterocyclic alkanes (C≤12) In some embodiments, the core is 4-N-methylpiperazinyl. In some embodiments, Y1 is an alkanediyl group. (C≤8) Or substituted alkane dimethyl (C≤8) In some implementations, Y1 is an alkane dimethyl group. (C≤8) In some embodiments, Y1 is -CH2CH2-. In some embodiments, Y3 is an alkane dimethyl group. (C≤8) Or substituted alkane dimethyl (C≤8) In some implementations, Y3 is an alkane dimethyl group. (C≤8) In some implementations, Y3 is -CH2CH2-. In other implementations, Y3 is:

[0261]

[0262] in:

[0263] X3 and X4 are alkane dimethyl groups.(C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0264] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups.

[0265] In some implementations, X3 is an alkane dimethyl group. (C≤12) Or substituted alkane dimethyl (C≤12) In some embodiments, X3 is -CH2CH2-. In some embodiments, X4 is an alkane dimethyl group. (C≤12) Or substituted alkane dimethyl (C≤12) In some implementations, X4 is -CH2CH2-. In some implementations, Y5 is a covalent bond. In some implementations, Y3 is:

[0266]

[0267] in:

[0268] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0269] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups.

[0270] In some implementations, X3 is an alkane dimethyl group. (C≤12) Or substituted alkane dimethyl (C≤12) In some embodiments, X3 is -CH2CH2-. In some embodiments, X4 is an alkane dimethyl group. (C≤12) Or substituted alkane dimethyl (C≤12) In some embodiments, X4 is -CH2CH2-. In some embodiments, Y5 is a covalent bond. In some embodiments, Y5 is -CH2- or -C(CH3)2-. In some embodiments, A1 is -O-. In other embodiments, A1 is -NR. a - In some implementations, R a It is hydrogen. In some embodiments, A2 is -O-. In other embodiments, A2 is -NR. a - In some implementations, R aIt is hydrogen. In some embodiments, R9 is an alkyl group. (C≤8) In some embodiments, R9 is methyl. In some embodiments, n is 0, 1, 2, 3, or 4. In some embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0. In other embodiments, n is 1. In other embodiments, n is 2. In other embodiments, n is 3.

[0271] In another aspect, this disclosure provides a composition comprising:

[0272] (a) the dendritic polymers described herein; and

[0273] (b) Nucleic acid.

[0274] In some embodiments, the nucleic acid is a short interfering RNA (e.g., small interfering RNA) (siRNA), microRNA (miRNA), pri-miRNA, messenger RNA (mRNA), CRISPR-associated nucleic acid, single-stranded 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 nucleic acid is siRNA, tRNA, or a nucleic acid that can be used in the CRISPR process. The nucleic acid may be siRNA. In other embodiments, nucleic acids that can be used in the CRISPR process include CRISPR-associated nucleic acids, single-stranded guide RNA (sgRNA), CBISPR-RNA (crRNA), or trans-activating crRNA (tracrRNA). In some embodiments, the nucleic acid is a factor VII-targeting siRNA comprising the following sequences:

[0275] 5′-GGAucAucucAAGucuuAc[dT][dT]-3′ (SEQ ID NO: 1); or

[0276] 3'-GuAAGAcuuGAGAuGAucc[dT][dT]-5' (SEQ ID NO: 2).

[0277] In other embodiments, the nucleic acid is miRNA. In other embodiments, the nucleic acid is mRNA. In other embodiments, the nucleic acid is tRNA. In other embodiments, the nucleic acid is guide RNA. In some embodiments, the guide RNA is used in the CRISPR process. In other embodiments, the nucleic acid is pDNA.

[0278] In some embodiments, the dendritic polymer and the nucleic acid are present in a weight ratio of about 100:1 to about 1:5. In some embodiments, the weight ratio of the dendritic polymer to the nucleic acid is about 50:1 to about 2:1. In some embodiments, the weight ratio of the dendritic polymer to the nucleic acid is 25:1. In other embodiments, the weight ratio of the dendritic polymer to the nucleic acid is 7:1. In some embodiments, the composition further comprises one or more auxiliary lipids. In some embodiments, the auxiliary lipid is selected from steroids, steroid derivatives, PEG lipids, or phospholipids. In some embodiments, the auxiliary lipid is a steroid or a steroid derivative. In some embodiments, the steroid is cholesterol. In some embodiments, the steroid or steroid derivative is present in a molar ratio of about 10:1 to about 1:20 with the dendritic polymer. In some embodiments, the molar ratio of the steroid or steroid derivative to the dendritic polymer is about 1:1 to about 1:10. In some embodiments, the molar ratio of the steroid or steroid derivative to the dendritic polymer is about 38:50. In some embodiments, the molar ratio of steroid or steroid derivative to dendritic polymer is about 1:5.

[0279] In other embodiments, the auxiliary lipid is a PEG lipid. In some embodiments, the PEG lipid is a PEGylated diacylglycerol, such as a compound of the following formula:

[0280]

[0281] in:

[0282] R 12 and R 13 Each is an alkyl group independently. (C≤24) alkenyl (C≤24) , or a substituted form of any of these groups;

[0283] R e It is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) ;and

[0284] x is 1-250.

[0285] In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula:

[0286]

[0287] in:

[0288] n1 is 5-250; and

[0289] n2 and n3 are each independently 2-25.

[0290] In some embodiments, the PEG lipid and the dendritic polymer are present in a molar ratio of about 1:1 to about 1:250. In some embodiments, the molar ratio of PEG lipid to dendritic polymer is about 1:10 to about 1:125. In some embodiments, the molar ratio of PEG lipid to dendritic polymer is about 1:20 to about 1:50.

[0291] In other embodiments, the auxiliary lipid is a phospholipid. In some embodiments, the phospholipid is 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC). In other embodiments, the phospholipid is 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE). In some embodiments, the phospholipid and the dendritic polymer are present in a molar ratio of about 10:1 to about 1:20. In some embodiments, the molar ratio of phospholipid to dendritic polymer is about 1:1 to about 1:10. In some embodiments, the molar ratio of phospholipid to dendritic polymer is about 4:5. In some embodiments, the molar ratio of phospholipid to dendritic polymer is about 1:5. In some embodiments, the composition consists essentially of a dendritic polymer, nucleic acid, and one or more auxiliary lipids.

[0292] In another aspect, this disclosure provides a pharmaceutical composition comprising:

[0293] (a) the compositions or dendritic polymers described herein; and

[0294] (b) Pharmaceutically acceptable carriers.

[0295] In some embodiments, the pharmaceutically acceptable carrier is a solvent or solution. In some embodiments, the pharmaceutical composition is formulated for administration by means of: oral, intrafacial, intra-articular, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intracystic, intravitreal, intravitreal, liposome, local, mucosal, parenteral, rectal, subconjunctival, subcutaneous, sublingual, superficial, buccal, percutaneous, vaginal, as a paste, as a lipid composition, via catheter, via irrigation, 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 intra-arterial injection. In some embodiments, the pharmaceutical composition is formulated as a unit dose.

[0296] On the other hand, this disclosure provides a method for regulating gene expression, comprising delivering nucleic acids to cells, the method comprising contacting the cells with the compositions or pharmaceutical compositions described herein under conditions sufficient to induce nucleic acid uptake into the cells. In some embodiments, the cells are contacted in vitro. In other embodiments, the cells are contacted in vivo. In other embodiments, the cells are contacted ex vivo. In some embodiments, the regulation of gene expression is sufficient to treat a disease or condition. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is liver cancer. In some embodiments, the disease or condition is hepatocellular carcinoma.

[0297] In another aspect, this disclosure provides a method for treating a disease or condition in a patient, comprising administering a pharmaceutically effective amount of the composition or pharmaceutical composition described herein to a patient in need. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is liver cancer. In some embodiments, the disease or condition is hepatocellular carcinoma. In some embodiments, the method further includes administering one or more additional cancer therapies to the patient. In some embodiments, the cancer therapy is a chemotherapy compound, surgery, radiotherapy, 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.

[0298] On the other hand, this disclosure provides dendritic polymers of the following formula:

[0299] Core - (repeating unit) n -Terminal group (I)

[0300] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0301] The core has the following formula:

[0302]

[0303] in:

[0304] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) Heterocyclic olefin dimethyl (C≤8) , or a substituted form of any of these groups;

[0305] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[0306] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[0307] The repeating unit comprises a degradable diacyl group and a linker;

[0308] The degradable diacyl group has the formula:

[0309]

[0310] in:

[0311] A1 and A2 are independently -O- or -NR. a -,in:

[0312] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0313] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0314] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0315] The connecting group has the following formula:

[0316]

[0317] in:

[0318] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0319] When the repeating unit contains a linker group, the linker group is connected to degradable diacyl groups on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0320] The terminating group has the following formula:

[0321]

[0322] in:

[0323] Y4 is an alkane dimethyl group. (C≤18) ;and

[0324] R 10 It is hydrogen;

[0325] The final degradable diacyl group in the chain is attached to a capping group;

[0326] n is 0, 1, 2, 3, 4, 5, or 6;

[0327] Or its pharmaceutically acceptable salt.

[0328] The terms “comprising” (and any form of comprising), “having” (and any form of having), “containing” (and any form of containing), and “including” (and any form of including) are all open-ended linking verbs. Therefore, a method, composition, kit, or system that “comprising,” “having,” “containing,” or “including” one or more of the listed steps or elements has those listed steps or elements, but is not limited to having only those steps or elements; it may have (i.e., cover) elements or steps not listed. Similarly, an element of a method, composition, kit, or system that “comprising,” “having,” “containing,” or “including” one or more of the listed features has those features, but is not limited to having only those features; it may have features not listed.

[0329] Any embodiment of any method, composition, kit, or system of the present invention may consist of or substantially consist of the described steps and / or features, rather than comprising / including / containing / having the described steps and / or features. Therefore, in any claim, the terms "consisting of" or "substantially consisting of" may replace any of the open-ended linking verbs listed above, so that the scope of the given claim is altered compared to the scope in which it would have used the open-ended linking verb.

[0330] The term “or” is used in the claims to mean “and / or” unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, but this disclosure supports the definition of referring only to alternatives and “and / or”.

[0331] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that although specific embodiments of the invention have been pointed out, the detailed description and specific examples are given by way of example only, and various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art based on this detailed description. It should be noted that the fact that a particular compound belongs to one particular general formula does not mean that it cannot also belong to another general formula. Attached Figure Description

[0332] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0333] Figure 1A-1D Small RNA therapy within susceptible liver cancer requires a combination of high potency against tumor cells and low toxicity to normal cells. A modular strategy, employing diverse chemical functional groups and sizes for biocompatible ester-based dendritic polymers, has enabled the discovery of dendritic polymers that balance low toxicity with high in vivo small RNA delivery efficiency. Orthogonal reactions accelerated the synthesis of 1,512 G1 modular degradable dendritic polymers, thereby increasing the number, size, and chemical diversity of molecular structures. The inclusion of degradable ester bonds in each step promotes low toxicity. Figure 1A Small RNAs are too large and anionic to enter cells on their own. To effectively utilize RNAi mechanisms, delivery vectors must accompany small RNAs across numerous extracellular and intracellular barriers. A modular design is envisioned that would allow for fine-tuning of the identity and location of functional groups within dendritic polymer structures. Figure 1BThe library was constructed via a sequential orthogonal reaction. First, amines with a series of initial branch centers (IBCs) reacted quantitatively and selectively with sterically less sterically hindered acrylate groups of AEMA containing two degradable ester groups. The products then underwent DMPP-catalyzed reactions with various thiols. Figure 1C To identify degradable dendritic polymers with optimized topological structures to mediate small RNA overcoming various extracellular and intracellular delivery barriers, the library was divided into four regions: nuclear binding-peripheral stabilization (region I), nuclear binding-peripheral binding (region II), nuclear stabilization-peripheral stabilization (region III), and nuclear stabilization-external binding (region IV). Figure 1D Dendritic polymers were independently modulated for both the core and periphery using chemically diverse amines and thiols. Selected amines were divided into two classes: ionizable amines for RNA binding that produce 1 to 6 branching compounds were labeled 1A-6A, while hydrophobic amines for NP stabilization were labeled 1H-2H. These amines were expected to increase the potency of higher-generation dendritic polymers. Based on hydrocarbon length, hydrophobic alkylamines for NP stabilization were labeled SC1-SC19. The library design included alcohol and carboxylic acid-terminated thiols (SO1-SO9) and amine-functionalized thiols (SN1-SN11) to increase chemical diversity. Higher-generation dendritic polymers with multiple branches, G2-G4, were also synthesized using generational amplification reactions (see [link to generatrix]). Figure 10B and Figure 11 ).

[0334] Figure 2 The high aza-Michael addition selectivity of tris(2-aminoethyl)amine 6A3 with 2-(acryloyloxy)ethyl methacrylate (AEMA) was demonstrated at 50 °C in the presence of 5 mol% butylated hydroxytoluene (BHT). Without the addition of tris(2-aminoethyl)amine, only AEMA remained unreacted after 24 hours, and its conversion was 0%. With the addition of tris(2-aminoethyl)amine, the conversion of AEMA was approximately 82% after 2 hours and approximately 98% after 24 hours, yielding a first-generation dendritic polymer 6A3-G1 with six branches. It should be noted that the addition of excess EAMA (6 × 0.05 equivalents) was intended to facilitate the reaction of EAMA with... 1 HNMR tracing allows for easy monitoring of the reaction. If the EAMA conversion is complete, there should still be 5% H₂ remaining. a and H b Signal.

[0335] Figure 3The high aza-Michael addition selectivity of long-alkyl-chain tetradecylamine 2H4 with 2-(acryloyloxy)ethyl methacrylate (AEMA) was shown at 50 °C in the presence of 5 mol% butylated hydroxytoluene (BHT). At 24 hours, only AEMA remained unreacted with a conversion of 0%. Upon addition of tetradecylamine, the conversion of AEMA was approximately 97% after 24 hours, yielding a first-generation dendritic polymer 2H4-G1 with a long-alkyl-chain core. It should be noted that the addition of excess EAMA (2 × 0.05 equivalents) was intended to facilitate the reaction of EAMA with... 1 H NMR tracing allows for easy monitoring of the reaction. If the EAMA conversion is complete, there should still be 5% H NMR remaining. a and H b Signal.

[0336] Figure 4 The results showed that at 60 °C, in 400 μL DMSO-D6, the thiamike addition of 6A3-G1 (125 mM) with 2-(butylamino)ethanethiol (6 × 1.2 equivalents) or 1-tetradecanethiol (6 × 1.2 equivalents) lasted for 48 hours. Without the addition of the thiol compound, 6A3-G1 remained constant at 60 °C in DMSO-D6 for 48 hours. With the addition of (5 mol%) dimethylphenylphosphine (DMPP) as a catalyst, 6A3-G1 reacted with 1-tetradecanethiol in DMSO-D6 at 60 °C with 100% conversion over 48 hours, while the conversion was only 57% in the absence of DMPP. The conversion of 6A3-G1 with 2-(butylamino)ethanethiol was almost quantitative with or without DMPP, which is likely because the amino group in 2-(butylamino)ethanethiol can act as a catalyst. It should be noted that an excess of thiol (6 × 0.2 equivalents) is added because 6A3-G1 contains (6 × 0.05 equivalents) EAMA, which consumes the thiol reactant (6 × 0.1 equivalents) with its two double bonds.

[0337] Figure 5The results show that at 60 °C, the thiamike addition of 2H4-G1 (125 mM) with 2-(butylamino)ethanethiol (2 × 1.2 equivalents) or 1-tetradecanethiol (2 × 1.2 equivalents) in DMSO-D6 lasts for 48 hours. Without the addition of the thiol compound, 2H4-G1 remains constant at 60 °C in DMSO-D6 for 48 hours. With the addition of (5 mol%) dimethylphenylphosphine (DMPP) as a catalyst, 2H4-G1 reacts with 1-tetradecanethiol in DMSO-D6 at 60 °C with 100% conversion over 48 hours, while the conversion is only 51% in the absence of DMPP. The conversion of 2H4-G1 with 2-(butylamino)ethanethiol is quantitative with or without DMPP, which may be because the amino group in 2-(butylamino)ethanethiol can act as a catalyst. It should be noted that an excess of thiol (2 × 0.2 equivalents) is added because 2H4-G1 contains (2 × 0.05 equivalents) EAMA, which consumes the thiol reactant (2 × 0.1 equivalents) with its two double bonds.

[0338] Figure 6A and Figure 6B This demonstrates the efficient construction of a library of 1,512 first-generation biodegradable dendritic polymers. Figure 6A At 50°C, in the presence of 5 mol% butylated hydroxytoluene (BHT), different amines with various initial branch centers (IBCs) reacted with ethyl 2-(acryloyloxy)methacrylate (AEMA) at an exact 1:1 feed equivalent ratio for 24 hours. The conversion rates of all 42 reactions were determined according to... 1 H NMR is almost quantitative. Figure 6B Each of the 42 CL-G1 compounds was reacted with each of the 36 thiols (P) in 66 μL DMSO at 5% DMPP on a small scale (average approximately 20 mg). The thiol concentration was 750 mM, and the concentrations of 1An&1Hn, 2An&2Hn, 3An, 4An, 5An, and 6An were 750 mM, 275 mM, 250 mM, 187.5 mM, 150 mM, and 125 mM, respectively. All 42 CL-G1 compounds remained stable at 60 °C for 48 hours without the addition of any thiol compounds. Each reaction of all 42 substances with SC4, SN8, and SO9 showed almost quantitative conversion (via...). 1 HNMR measurement).

[0339] Figures 7A-7C Screening for in vitro siRNA delivery of 1,512 G1DDs revealed dendritic polymers capable of overcoming intracellular barriers and establishing structure-activity relationships. Figure 7A ). ( Figure 7BThe heatmap of luciferase silencing in HeLa-Luc cells treated with dendritic polymer nanoparticles (33nMsiLuc, n=3) illustrates the regional activity relationship. Luciferase activity and cell viability were measured to identify the dendritic polymers that balanced high delivery efficiency with low toxicity (see [link to relevant documentation]). Figure 8 Additional data in the document). Figure 7C Analysis of nanoparticle groups achieving greater than 50% silencing identified dendritic polymers with optimized topologies to overcome intracellular delivery barriers. Subregions were further analyzed based on a set of criteria if their hit rate was higher than that of their parent region. The hit rate of the parent region was marked in orange, while higher or lower hit rates in its subgroups were marked in green or blue, respectively. Approximately 6% of the entire library achieved >50% gene silencing. The nuclear-binding-peripheral stabilization region I had a 10% hit rate. Within region I, subregions with SC branches accounted for 15%, while those with SO branches had only a 1% hit rate. Among the subregions with SC branches, dendritic polymers with three to six branches (SC5-8 or SC9-12 branches) had a much higher chance of effectively mediating siRNA delivery.

[0340] Figure 8 Cell viability is shown after incorporating 1,512 first-generation degradable dendritic polymers (G1DD) NPs containing siLuc (33 nM siRNA, values ​​are average for n=3). G1DD was formulated into nanoparticles containing firefly luciferase-targeting siRNA (siLuc) at a weight ratio of 12.5:1 (G1DD:siRNA) and auxiliary lipids cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and lipid PEG2000 at a molar ratio of 50:38:10:2 (G1DD:cholesterol:DSPC:lipid PEG). Cell viability was measured using the ONE-Glo+Tox luciferase reporter gene and cell viability assay (Promega) according to its protocol. Cell viability was obtained by normalization relative to untreated cells. Untreated control (n=6). Experimental samples (n=3).

[0341] Figure 9A and Figure 9B Intracellular siRNA delivery activity of 1,512 first-generation degradable dendritic polymers (G1DD) was demonstrated. G1DD was formulated into nanoparticles containing firefly luciferase-targeting siRNA (siLuc) at a weight ratio of 12.5:1 (G1DD:siRNA) and cofactor cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and lipid PEG2000 at a molar ratio of 50:38:10:2 (G1DD:cholesterol:DSPC:lipid PEG). Figure 9AThe heatmap showing reduced luciferase activity in HeLa cells stably expressing firefly luciferase after treatment with G1DD nanoparticles using 33 nM siRNA was divided into multiple regions and areas to describe the breakdown of the dendritic analysis process (see section). Figure 9B Cell viability and luciferase activity were measured using the ONE-Glo+Tox luciferase reporter gene and cell viability assay (Promega) according to its protocol. Luciferase activity was normalized to untreated cells to obtain a reduction in luciferase activity. Untreated controls (n=6). Experimental samples (n=3). Figure 9B A dendritic polymer-inspired tree analysis process was used to identify degradable dendritic polymers with optimized structures to mediate siRNA overcoming intracellular delivery barriers, analyzed by their hit rates and luciferase activity reductions exceeding 50%. Subregions were further analyzed if their hit rates were higher than those of their parent regions, according to a set of criteria. The hit rate of the parent region is represented by black bars, while higher or lower hit rates in its subregions are indicated by blue or red text. Approximately 6% of the entire library induced >50% gene silencing. The nuclear-binding-peripheral stabilization region (I region) had a 10% hit rate. Within the I region, subregions with SC branches had a 15% hit rate, while those with SO branches had a 1% hit rate. Among subregions with SC branches, dendritic polymers with 3, 4, 5, or 6 branches (SC5-8 or SC9-12 branches) had a significantly higher chance of effectively mediating siRNA overcoming intracellular delivery barriers.

[0342] Figures 10A-10C This study demonstrates systematic in vivo siRNA delivery screening, which further identifies dendritic polymers that can also overcome the extracellular barrier. The analysis provides SAR for designing additional dendritic polymers with predicted activity. Figure 10A Factor VII knockdown in mice was evaluated using 26 first-generation degradable dendritic polymers with diverse structures at a dose of 1 mg / kg siRNA (n=3). PBS control (n=3). Data are shown as mean ± SD. Figure 10B The rational design of biodegradable dendritic polymers with multiple branches is achieved through (I) selecting polyamines with multiple IBCs and (II) increasing branching by generation amplification. Natural polyamines spermidine 5A5 and spermine 6A4 were utilized. Biodegradable dendritic polymers with multiple branches were synthesized by generation amplification using 1A2 (one IBC), 2A2 and 2A11 (two IBCs), 3A3 and 3A5 (three IBCs), and 4A1 and 4A3 (four IBCs) (see also...). Figure 11 ). ( Figure 10CThe knockdown of factor VII by 24 rationally designed degradable dendritic polymers (n=3) using strategies I and II was evaluated in mice at a dose of 1 mg / kg siRNA. PBS control (n=3). Data are shown as mean ± SD. Rationally designed dendritic polymers exhibited activity at high hit rates.

[0343] Figure 11 Synthetic routes for preparing degradable dendritic polymers with multiple branches, namely 2A2 and 2A11 (two IBCs), 3A3 and 3A5 (three IBCs), and 4A1 and 4A3 (four IBCs), are shown by a generational amplification strategy.

[0344] Figures 12A-12E In vivo toxicity assessments of several degradable dendritic polymers (>95% in vivo FVII knockdown) were presented, further identifying dendritic polymers that balance high delivery efficacy with low toxicity. Some degradable dendritic polymers (NPs) exhibited (…) after binding to control siRNA (siCTR) Figure 12A Similar sizes and ( Figure 12B Net surface charge (nanoparticles are described from left to right in the figure, based on...) Figure 12B (Legend of the figure). C12-200 lipid LNPs provide challenging comparisons because they represent the best examples of non-hydrolyzable systems with similar in vivo efficacy. Figure 12C Wild-type mice (p26) were intravenously injected with some NPs (100 mg dendritic polymer / kg or 28 mg control C12-200 / kg) at 4 mg siCTR / kg (n=3). Body weight changes varied between different formulations depending on the dendritic polymer, but all NPs were substantially non-toxic in normal WT mice. Figure 12D Changes in body weight (n=5) in transgenic mice (p32) carrying aggressive MYC-driven tumors after injection of 3 mg siCTR / kg (75 mg / kg 5A2-SC8 and 6A3-SC12 or 21 mg / kg C12-200). Figure 12E Kaplan-Meier survival curves (n=5) of transgenic mice injected with 5A2-SC8 and 6A3-SC12 nanoparticles at days 32, 36, 40, and 44 (75 mg dendritic polymer / kg). In tumor-bearing mice (susceptible hosts), the toxicity of the vector was amplified, and only 5A2-SC8 was adequately tolerated without affecting survival. Data are presented as mean ± sd. Statistical analysis was performed using the (e) Mantel-Cox test; nsP > 0.05; *P < 0.05.

[0345] Figure 13A and Figure 13B This study demonstrates the toxicity and potency of modular, degradable dendritic polymer-delivered miRNAs for tumor growth inhibition in a selected invasive transgenic MYC-driven liver tumor model (Nguyen et al., 2014). Figure 13A This schematically illustrates a LAP-tTa;TRE-MYC transgenic mouse model. When the LAP-tTA transgene is presented, TRE-MYC is switched on or off via a liver-specific LAP promoter in the absence or presence of doxycycline (Dox). Figure 13B Without any treatment, liver tumors are visible at approximately p20-26, then by p32 the liver is covered with small tumors, and eventually the tumors grow and the liver size increases dramatically from p42 to p60.

[0346] Figures 14A-14C Fluorescence imaging confirmed that siRNA was delivered to tumor cells inside the liver. Figure 14A Gross anatomy and fluorescence imaging of transgenic mice carrying aggressive liver tumors at 41 days of age. Fluorescence imaging showed that 5A2-SC8 nanoparticles formulated with Cy5.5-labeled siRNA mediated significant siRNA accumulation throughout the cancerous liver, with smaller accumulations in the spleen and kidneys 24 hours after intravenous injection of 1 mg Cy5.5-siRNA / kg. To further confirm whether 5A2-SC8 NPs could deliver siRNA to tumor cells in vivo, liver tumor tissue was collected, embedded in OTC, and sectioned for H&E staining and confocal imaging 24 hours after intravenous injection. Figure 14B H&E staining confirmed the presence of a tumor in the liver. The same tumor tissue section was scanned using confocal imaging and captured in three channels: DAPI for the nucleus (blue), FITC for actin stained with phalloidin (green), and Cy5.5 for siRNA (red). Figure 14C Confocal imaging of the same region showed that 5A2-SC8 can effectively deliver siRNA to tumor cells inside the liver.

[0347] Figure 15A and Figure 15B The biodistribution of 5A2-SC8 NP formulated with Cy5.5-tagged siRNA is shown in normal wild-type mice and mice carrying liver tumors. Figure 15A ) and H&E staining images of livers from tumor-bearing mice ( Figure 15B5A2-SC8 NP, administered intravenously at 1 mg siRNA / kg for 24 hours, mediated the accumulation of Cy5.5-labeled siRNA in the whole liver of normal mice and mice carrying liver tumors. H&E staining images showed that the livers of tumor-bearing mice were tumor-rich, and the slides used for confocal imaging contained tumor cells. It should be noted that the liver size increased with tumor growth (see [link to image]). Figure 15A (The proportionally proportioned boxes in the text).

[0348] Figures 16A-16H Modular, degradable dendritic polymers demonstrated that therapeutic Lct-7gmiRNA mimics could be delivered to clinically relevant and aggressive MYC-driven genetic tumor models, resulting in significant survival benefits. 5A2-SC8 NP silenced the FVII protein in transgenic mice carrying MYC-driven liver tumors, as seen in blood ( Figure 16A ) and in the collected liver tissue ( Figure 16B Measurements were taken (single injection, 1 mg / kg, p26 mice, 48 hours post-injection) (left side: siCTR, right side: siFVII). Figure 16C The 5A2-SC8 NP technique enabled the delivery of Let-7g to the liver tissue of transgenic mice carrying MYC-driven liver tumors (single injection, 1 mg / kg, p26 mice, 48 hours post-injection). Let-7g expression was significantly increased, while other Let-7 family members were unaffected (siCTR on the left and siFVII on the right). Figure 16D Transgenic mice carrying MYC-driven liver tumors were administered 1 mg / kg Let-7g intravenously once weekly, starting on day 26 (after tumor development began) until day 61. Mice receiving Let-7g had significantly smaller abdomens. Figure 16E Compared to the control group, the treated mice had a smaller abdominal circumference. Figure 16F Representative images of the livers from Let-7g mimicry and control mimicry mice show reduced tumor burden. Figure 16G Weekly delivery of miRNA mimics within the 5A2-SC8 NP range did not affect normal weight gain, while delivery of miRNA mimics within the C12-200LNP range caused weight loss and death. n=5. Figure 16HWeekly delivery of Let-7g from day 26 to day 61 prolonged survival. All untreated control mice (n=9) and mice receiving 5A2-SC8 NP containing the control untargeted mimic (n=5) died around day 60. Mice injected with C12-200LNP died prematurely (n=7). The C12-200+CTR mimic experiment was discontinued because all mice injected with the C12-200+Let-7g mimic had died (n=7). Delivery of Let-7g within 5A2-SC8NP provided a significant survival benefit. Data are shown as mean ± sd. Statistical analysis was performed using a (a, b, c, e) two-tailed Student t-test or (h) Mantel-Cox test; nsP > 0.05; *P < 0.05; **P < 0.0-1; ***P < 0.00-1; ****P < 0.0001.

[0349] Figures 17A-17C This demonstrates the use of cholesterol, phospholipids, and PEG lipids in HeLa-Luc ( Figure 17A ), A549-Luc Figure 17B ) and MDA-MB231-Luc ( Figure 17C Different combinations of dendritic polymer nanoparticles formulated for the delivery of siLuc were used.

[0350] Figure 18A and Figure 18B show( Figure 18A A comparison of different formulations of DSPC lipids versus DOPE lipids versus PEG-DMG in the delivery of siLuc to HeLa-Luc. Figure 18B This study shows a comparison of different formulations of siLuc delivered to HeLa-Luc, with DSPC lipids compared to DOPE lipids and PEG-DHD.

[0351] Figure 19 The delivery of sgRNA was demonstrated using a nanoparticle composition containing dendritic polymers or Z120, and the presence and absence of phospholipid DSPC in the nanoparticle formulation.

[0352] Figure 20A and Figure 20B Showing sgRNA ( Figure 20A The percentage of encapsulation and delivery in HeLa-Luc-Cas9 cells ( ) Figure 20B ).

[0353] Figure 21A and Figure 21B Displays 24-hour incubation ( Figure 21A ) and 48-hour incubation ( Figure 21BThe viability of IGROWV cells that have been delivered with Luc mRNA.

[0354] Figure 22 This image shows a fluorescence microscope photograph of cells treated with mCherry mRNA, demonstrating the delivery of mRNA into the cells. Detailed Implementation

[0355] In some aspects, this disclosure provides lipophilic cationic dendritic polymers that can be used as nucleic acid carriers. In some embodiments, the dendritic polymer contains one or more groups that degrade under physiological conditions. In some embodiments, the dendritic polymer is formulated into a composition comprising the dendritic polymer and one or more nucleic acids. These compositions may further comprise one or more accessory lipids such as cholesterol and / or phospholipids. Finally, in some aspects, this disclosure also provides methods for treating one or more diseases treatable with nucleic acid therapeutic agents using the dendritic polymer compositions.

[0356] A. Chemical definition

[0357] When used in the context of chemical groups: "hydrogen" refers to -H; "hydroxyl" refers to -OH; "oxo" refers to =O; "carbonyl" refers to -C(=O)-; "carboxyl" refers to -C(=O)OH (also written as -COOH or -CO2H); "halogen" 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 case, "phosphate ester" refers to -OP(O)(OH)2 or its deprotonated form; in the divalent case, "phosphate ester" refers to -OP(O)(OH)O- or its deprotonated form; "thiol" refers to -SH; and "thio" refers to =S; "sulfonyl" refers to -S(O)2-; "hydroxysulfonyl" refers to -S(O)2OH; "sulfonamide" refers to -S(O)2NH2; and "sulfinyl" refers to -S(O)-.

[0358] In the case of chemical formulas, the symbol "-" refers to a single bond, and "=" refers to a double bond. This refers to a triple bond. The symbol "----" indicates an optional bond, which can be a single or double bond when present. This indicates a single or double bond. Therefore, for example, the formula... include Furthermore, it should be understood that no single ring atom constitutes part of more than one double bond. Additionally, it should be noted that the covalent bond symbol "-" when connecting one or two stereo atoms does not indicate any preferred stereochemistry. Instead, it encompasses all stereoisomers and mixtures thereof. When perpendicularly passing through the bond (e.g., methyl) The symbol indicates the junction point of the functional groups when drawing the diagram. It should be noted that junction points are typically determined in this way only for larger functional groups to help the reader clearly identify the junction points. This refers to a single bond that connects to the thicker end of the wedge shape and is "outside the page". (Symbol) This refers to a single bond "within the page" that connects to the thicker end of the wedge. (Symbol) This refers to single bonds in which the geometry (e.g., E or Z) surrounding a double bond is indeterminate. Therefore, two options and combinations thereof are contemplated. Any undefined valence on an atom in the structure shown in this application implicitly represents a hydrogen atom bonded to that atom. Black dots on carbon atoms indicate that hydrogen atoms bonded to said carbon are oriented outwards from the paper.

[0359] When the group "R" is described as a "floating group" on a ring system, for example, in the following formula:

[0360]

[0361] R can replace any hydrogen atom attached to any ring atom, including depicted, implied, or explicitly defined hydrogen atoms, as long as a stable structure is formed. When group "R" is depicted as a "floating group" on a fused ring system, such as in the following formula:

[0362]

[0363] Unless otherwise specified, R can substitute for any hydrogen atom bonded to any ring atom of any fused ring. Substituteable hydrogens include the hydrogens depicted (e.g., the hydrogen bonded to nitrogen in the above formula), implicit hydrogens (e.g., hydrogens not shown in the above formula but understood to be present), explicitly defined hydrogens, and optional hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogen bonded to group X when X equals -CH-), as long as a stable structure is formed. In the depicted examples, R can be located on a 5- or 6-membered ring of the fused ring system. In the above formula, the subscript letter "y" immediately following the group "R" enclosed in parentheses represents a numerical variable. Unless otherwise specified, this variable can be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of substituted atoms in the ring or ring system.

[0364] For chemical groups and compound categories, the number of carbon atoms in the group or category is as follows: "Cn" defines the exact number (n) of carbon atoms in the group / category. "C≤n" defines the exact number (n) of possible carbon atoms in the group / category, where the minimum number is as small as the possible number of groups / categories under discussion. For example, it should be understood that the group "alkenyl" (C≤8) "or category "olefins" (C≤8) The minimum number of carbon atoms in "" is 2. This is in contrast to "alkoxy". (C≤10) The symbol “Cn-n′” indicates an alkoxy group having 1 to 10 carbon atoms. “Cn-n′” defines the minimum (n) and maximum (n′) number of carbon atoms in the group. Therefore, “alkyl”... (C2-10) "This indicates alkyl groups having 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class they modify, and may or may not be enclosed in parentheses without indicating any change in meaning. Therefore, the terms 'C5 olefin,' 'C5-olefin,' and 'olefin'..." (C5) "and "olefins" C5 "They are all synonyms."

[0365] When used to modify compounds or chemical groups, the term "saturated" means that the compound or chemical group does not have carbon-carbon double bonds and does not have carbon-carbon triple bonds, unless as shown below. When the term is used to modify atoms, it means that the atom is not part of any double or triple bond. In the case of substituted forms of saturated groups, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. And when such bonds are present, carbon-carbon double bonds that may appear 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 that substance can dissolve in the solution.

[0366] The term "aliphatic," when used without the modifier "substituted," refers to a compound or chemical group that is acyclic or cyclic but non-aromatic hydrocarbon compound or group. In aliphatic compounds / groups, carbon atoms can be straight-chain, branched, or linked together by non-aromatic rings (alicyclic). Aliphatic compounds / groups can be saturated, i.e., linked by carbon-carbon single bonds (alkane / alkyl), or unsaturated, i.e., having one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkynyl / alkynyl).

[0367] The term "aromatic" when used to modify the atoms of a compound or chemical group refers to a planar unsaturated ring containing atoms stabilized by the interactions of bonds that form the ring.

[0368] The term "alkyl" when used without the modifier "substituted" refers to a monovalent saturated aliphatic group having a carbon atom as a linking point, a straight-chain or branched acyclic structure, and no atoms other than carbon and hydrogen. Examples of alkyl groups include -CH3(Me), -CH2CH3(Et), -CH2CH2CH3(n-Pr or propyl), and -CH(CH3)2(i-Pr). i Pr or isopropyl), -CH2CH2CH2CH3(n-Bu), -CH(CH3)CH2CH3(sec-butyl), -CH2CH(CH3)2(isobutyl), -C(CH3)3(tert-butyl, t-Bu or t Bu) and -CH2C(CH3)3 (neopentyl) are non-limiting examples of alkyl groups. The term "alkane diester," when used without the modifier "substituted," refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as connecting points, a straight-chain or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups -CH2- (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkane diesters. "Alkane" refers to the class of compounds having the formula HR, 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 in which hydrogen substitution is not limited to halogen groups (i.e., -F, -Cl, -Br, or -I), and therefore no atoms other than carbon, hydrogen, and halogens are present. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which hydrogen substitution is not limited to fluorine groups, and therefore no atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0369] The term "cycloalkyl" when used without the modifier "substituted" refers to a monovalent saturated aliphatic group having a carbon atom as a linking point, said carbon atom forming part of one or more non-aromatic ring structures, without carbon-carbon double or triple bonds, and without atoms other than carbon and hydrogen. Non-limiting examples include: -CH(CH2)2 (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl (Cy). The term "cycloalkane dimethyl" when used without the modifier "substituted" refers to a divalent saturated aliphatic group having two carbon atoms as linking points, without carbon-carbon double or triple bonds, and without atoms other than carbon and hydrogen. These are non-limiting examples of cycloalkane dimethyl compounds. "Cycloalkane" refers to a class of compounds having the formula HR, 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.

[0370] The term "alkenyl," when used without the modifier "substituted," refers to a monovalent unsaturated aliphatic group having a carbon atom as a linking point, 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 "alkene diel," when used without the modifier "substituted," refers to a divalent unsaturated aliphatic group having two carbon atoms as linking points, 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 alkene dienes. It should be noted that although alkene dienes are aliphatic, once connected at both ends, this does not preclude the group from forming part of an aromatic structure. The terms "chain alkene" and "alkene" are synonyms and refer to a class of compounds having the formula HR, where R is an alkenyl group, as defined above. Similarly, the terms "terminal alkene" and "α-alkene" are synonyms and refer to an alkene having only one carbon-carbon double bond, wherein the bond is part of a vinyl group at the molecule's end. 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 alkenyl groups.

[0371] The term "alkynyl," when used without the modifier "substituted," refers to a monovalent unsaturated aliphatic group having a carbon atom as a linking point, a straight or branched acyclic structure, at least one carbon-carbon triple bond, and no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not exclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH3, and -CH2C≡CCH3 are non-limiting examples of alkynyl groups. "Alkyne" refers to a class of compounds having the formula HR, where R is an alkynyl group. When any of these terms is used with the modifier “replaced”, 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.

[0372] The term "aryl," when used without the modifier "substituted," refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as a connecting point and said carbon atom forming part of one or more six-membered aromatic ring structures, wherein all ring atoms are carbon, and said group is not composed of atoms other than carbon and hydrogen. If more than one ring is present, said ring may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl or aralkyl groups (permitted by carbon number restrictions) connected to a first aromatic ring or any additional aromatic ring present. Non-limiting examples of aryl include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C6H4CH2CH3 (ethylphenyl), naphthyl, and monovalent groups derived from biphenyl. The term "aryl dimethyl" when used without the modifier "substituted" refers to a divalent aromatic group having two aromatic carbon atoms as connecting points and said carbon atoms forming part of one or more six-membered aromatic ring structures, wherein all ring atoms are carbon, and said monovalent group is not composed of atoms other than carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl, aryl, or aralkyl groups (permitted by carbon number restrictions) connected to a first aromatic ring or any additional aromatic ring present. If more than one ring is present, the rings may be fused or unfused. Unfused rings may be connected by one or more of the following: covalent bonds, alkane dienes, or alkene dienes (permitted by carbon number restrictions). Non-limiting examples of alkane dienes include:

[0373]

[0374] "Aromatic hydrocarbons" refers to a class of compounds having the formula HR, where R is an aryl group, as defined herein. Benzene and toluene are non-limiting examples of aromatic hydrocarbons. 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.

[0375] The term "aralkyl" when used without the modifier "substituted" refers to the monovalent group -alkanediyl-aryl, wherein the terms alkanediyl and aryl are 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 alkane diel and / or aryl group 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 aralkyl groups are (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-ethyl-1-yl.

[0376] The term "heteroaryl," when used without the modifier "substituted," refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as a linking point and 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. A heteroaryl ring may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, and sulfur. If more than one ring is present, said ring may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (permitted by carbon number restrictions) attached to an aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazole (Im), isoxazolyl, methylpyridyl, oxazolyl, phenylpyridyl, pyridinyl (pyridinyl / pyridyl), pyrroleyl, pyrimidinyl, pyrazinyl, quinolinyl, quinazolinyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thiopheneyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as a connecting point. The term "heteroaryl diaryl," when used without the modifier "substituted," refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as two connecting points, said atoms forming part of one or more aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and 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 can be fused or unfused. Unfused rings can be linked by one or more of the following: covalent bonds, alkane dienes, or alkene dienes (carbon number restrictions allow). As used herein, the terminology does not exclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number restrictions allow) attached to an aromatic ring or aromatic ring system. Non-limiting examples of heteroaromatic dienes include:

[0377]

[0378] "Heteroaromatic hydrocarbons" refers to a class of compounds having the formula HR, where R is a heteroaryl group. Pyridine and quinoline are non-limiting examples of heteroaromatic hydrocarbons. 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.

[0379] The term "heterocyclic alkyl" when used without the modifier "substituted" refers to a monovalent non-aromatic group having a carbon or nitrogen atom as a linking point, said carbon or nitrogen atom forming part of one or more non-aromatic ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and said heterocyclic alkyl is not composed of atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. The heterocyclic alkyl ring may contain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, or sulfur. If more than one ring is present, said ring may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl groups (permitted by carbon number restrictions) attached to a ring or ring system. Furthermore, the term does not exclude the presence of one or more double bonds in a ring or ring system, provided that the resulting group is still non-aromatic. Non-limiting examples of heterocyclic alkyl groups include aziridinyl, aziridine, pyrrolidine, piperidinyl, piperazine, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, ethylene oxide, and oxacyclobutane. The term "N-heterocyclic alkyl" refers to a heterocyclic alkyl group having a nitrogen atom as a connecting point. N-pyrrolidine is an example of such a group. The term "heterocyclic alkane dimethyl" when used without the modifier "substituted" refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as two connecting points, said atoms forming part of one or more ring structures, wherein at least one ring atom is nitrogen, oxygen, or sulfur, and 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 can be linked by one or more of the following: covalent bonds, alkane dienes, or alkene dienes (carbon number restrictions allow). As used herein, the terminology does not exclude the presence of one or more alkyl groups (carbon number restrictions allow) attached to the ring or ring system. Furthermore, the terminology does not exclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group is still non-aromatic. Non-limiting examples of heterocyclic alkane dienes include:

[0380]

[0381] When these terms are used with the modifier “replaced”, 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.

[0382] The term "acyl" when used without the modifier "substituted" refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl, as defined above. The 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, and -C(O) (imidazolyl) are non-limiting examples of acyl groups. "Thioacyl" is defined similarly, except that the oxygen atom in the group -C(O)R has been replaced by a sulfur atom, i.e., -C(S)R. The term "aldehyde" corresponds to an alkane as defined above, where at least one hydrogen atom has been replaced by a -CHO group. When any of these terms is used with the modifier “replaced”, one or more hydrogen atoms (including hydrogen atoms that may be directly attached to carbon atoms of the carbonyl or thiocarbonyl 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. The groups -C(O)CH2CF3, -CO2H (carboxyl), -CO2CH3 (methylcarboxyl), -CO2CH2CH3, -C(O)NH2 (carbamoyl), and -CON(CH3)2 are non-limiting examples of substituted acyl groups.

[0383] The term "alkoxy" when used without the modifier "substituted" refers to 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," "arylalkoxy," "heteroaryloxy," "heterocyclic alkoxy," and "acyloxy" when used without the modifier "substituted" refer to groups defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heterocyclic alkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent group -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkathioyl" and "acylthioyl" when used without the modifier "substituted" refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced by a hydroxyl group. The term "ether" corresponds to an alkane as defined above, wherein at least one hydrogen atom has been replaced by an alkoxy group. 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.

[0384] The term "alkylamino" when used without the modifier "substituted" refers to the group -NHR, where R is an alkyl group, as defined above. Non-limiting examples include -NHCH3 and -NHCH2CH3. The term "dialkylamino" when used without the modifier "substituted" refers to the group -NRR′, where R and R′ can be the same or different alkyl groups, or R and R′ can be linked together to represent an alkane dimethyl group. Non-limiting examples of dialkylamino include -N(CH3)2 and -N(CH3)(CH2CH3). The terms "cycloalkylamino," "alkenylamino," "alkynylamino," "arylamino," "aralkylamino," "heteroarylamino," "heterocyclic alkylamino," "alkoxyamino," and "alkylsulfonylamino" when used without the modifier "substituted" refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocyclic alkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC6H5. The term "alkylamino dieryl" refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "acylamino" (acylamino) when used without the modifier "substituted" refers to the group -NHR, where R is an acyl group, as defined above. A non-limiting example of an acylamino group is -NHC(O)CH3. The term "alkylimino" when used without the modifier "substituted" refers to the divalent group =NR, 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 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 amide groups.

[0385] When used with the term "comprising" in the claims and / or description, the use of the word "a" or "an" can mean "an," but it also means "one or more," "at least one," and "one or more."

[0386] Throughout this application, the term “about” is used to indicate that a value includes inherent error variations in the apparatus or method used to determine that value, or variations that exist between the subjects under study.

[0387] As used herein, the term "average molecular weight" refers to the relationship between the number of moles of each polymeric substance and the molar mass of that substance. Specifically, each polymer molecule may have different levels of polymerization and therefore different molar masses. Average molecular weight can be used to represent the molecular weight of multiple polymer molecules. Average molecular weight is generally synonymous with 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 this application, unless otherwise stated, average molecular weight represents the number-average molar mass or weight-average molar mass of that formula. In some embodiments, average molecular weight is number-average molar mass. In some embodiments, average molecular weight can be used to describe PEG components present in lipids.

[0388] The terms “comprises,” “has,” and “includes” 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 of “comprises,” “has,” or “includes” one or more steps is not limited to having only those one or more steps, but also covers other steps not listed.

[0389] The term "effective" as used in this specification and / or claims means sufficient to achieve the desired, anticipated, or anticipated results. "Effective amount," "therapeutic effective amount," or "pharmaceutical effective amount," when used in the context of treating a patient or subject with a compound, refers to the amount of compound sufficient to treat a disease when administered to a subject or patient to treat that disease.

[0390] As used in this article, the term "IC" 50 "Inhibitor" refers to the amount of inhibitor that achieves 50% of the maximum response. This quantitative measurement indicates how much of a specific drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical, or chemical process (or a component of the process, i.e., enzyme, cell, cell receptor, or microorganism) by half.

[0391] An "isomer" of the first compound is a separate compound in which each molecule contains the same component atoms as the first compound, but the three-dimensional configurations of these atoms are different.

[0392] 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 some embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, adolescents, infants, and fetuses.

[0393] As is commonly used in this article, "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for use in contact with human and animal tissues, organs, and / or bodily fluids without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0394] "Pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable as defined above and has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or acid addition salts formed with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthylsulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-en-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-en-1-carboxylic acid, acetic acid, aliphatic monocarboxylic acids and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzene Formic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheponic 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, mucoconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanic acids, propionic 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, which can be formed when existing acidic protons can react with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, thioglycolic acid, N-methylglucosamine, etc. It should be recognized that the specific anion or cation constituting any salt of the present invention is not important, as long as the salt is generally pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and their preparation and use are presented in the Handbook of Pharmaceutical Salts: Properties, and Use (edited by PHStahl and CGWermuth, Verlag Helvetica Chimica Acta, 2002).

[0395] As used herein, the term “pharmaceuticalally acceptable carrier” refers to a pharmaceutically acceptable material, composition, or medium involved in the delivery or transport of a chemical agent, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.

[0396] "Prevention" includes: (1) suppressing the onset of disease in subjects or patients who may be at risk of disease and / or susceptible to disease but have not yet experienced or shown any or all of the pathology or symptoms of disease, and / or (2) slowing the onset of the pathology or symptoms of disease in subjects or patients who may be at risk of disease and / or susceptible to disease but have not yet experienced or shown any or all of the pathology or symptoms of disease.

[0397] A "repeating unit" is the simplest structural entity of a particular material, such as a skeleton and / or polymer, whether organic, inorganic, or organometallic. In the case of polymer chains, the repeating units are linked together sequentially along the chain, like beads on a necklace. For example, in polyethylene -[-CH2CH2-] n In this formula, the repeating unit is -CH2CH2-. The subscript "n" indicates the degree of polymerization, i.e., the number of repeating units linked together. When the value of "n" is undefined or "n" does not exist, it simply indicates the repetition of the expression within the parentheses and the polymeric nature of the material. The concept of repeating units also applies to places where the connectivity between repeating units extends three-dimensionally, such as in metal-organic frameworks, modified polymers, thermosetting polymers, etc. In the case of dendritic polymers, repeating units can also be described as branching units, inner layers, or generations. Similarly, end-capping groups can also be described as surface groups.

[0398] "Stereoisomers" or "optical isomers" are isomers of a given compound in which the same atoms are bonded to the same other atoms, but in different three-dimensional configurations. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other (e.g., left-hand and right-hand). "Diabeta-isomers" are stereoisomers of a given compound that are not diastereomers. Chiral molecules contain a chiral center, also called a stereocenter or stereo-center, which is any point in a molecule with groups, but not necessarily an atom, such that the exchange of any two groups results in a stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus, or sulfur atom, but other atoms can also be stereocenters in both organic and inorganic compounds. A molecule can have multiple stereocenters, resulting in many stereoisomers. In compounds whose stereoisomerism is attributed to a tetrahedral stereocenter (e.g., tetrahedral carbon), it is assumed that the total number of possible stereoisomers will not exceed 2. n, where n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer than the maximum possible number of stereoisomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Optionally, a mixture of enantiomers can be enantiomer-enriched, such that one enantiomer is present in an amount greater than 50%. Typically, techniques known in the art can be used to resolve or separate enantiomers and / or diastereomers. It is anticipated that for any stereocenter or chiral axis for which stereochemistry is not yet defined, the stereocenter or chiral axis may exist in its R form, S form, or a mixture of R and S forms (including racemic and non-racemic mixtures). As used herein, the phrase “substantially free of other stereoisomers” means that the composition contains ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of another stereoisomer.

[0399] "Treatment" includes (1) suppressing the disease (e.g., preventing further development of the pathology and / or symptoms) in a subject or patient experiencing or displaying the pathology or symptoms of the disease, (2) improving the disease (e.g., reversing the pathology and / or symptoms) in a subject or patient experiencing or displaying the pathology or symptoms of the disease, and / or (3) achieving any measurable reduction of the disease in a subject or patient experiencing or displaying the pathology or symptoms of the disease.

[0400] The foregoing definitions take precedence over any conflicting definitions in any references incorporated herein by reference. However, the fact that some terms are defined should not be construed as indicating that any undefined term is indeterminate. Rather, all terms used are intended to describe the invention in a manner that would allow those skilled in the art to understand the scope and practice of the invention.

[0401] B. Dendritic polymers and dendritic structures

[0402] In some aspects of this disclosure, dendritic polymers containing lipophilic and cationic components are provided. A dendritic polymer is a polymer exhibiting regular dendritic branching, formed by adding branching layers sequentially or generationally to or from a core, and characterized by a core, at least one internal branching layer, and a surface branching layer. (See Petar R. Dvornic and Donald A. Tomalia in Chem. UK, 641-645, August 1994.) In other embodiments, the term "dendritic polymer," as used herein, is intended to include, but is not limited to, a molecular structure having a core, an inner layer (or "generation") of repeating units regularly connected to the starting core, and an outer surface with terminal groups connected to the outermost generation. A "dendritic" is a dendritic polymeric material having branches emanating from a focal point, which is directly or via a connecting portion connected to or may be connected to the core to form a larger dendritic polymer. In some embodiments, the dendritic polymer structure has repeating groups radiating from a central core, which are doubled for each branch by each repeating unit. In some embodiments, the dendritic polymers described herein may be described as small molecules, medium molecules, lipids, or lipid-like substances. These terms can be used to describe compounds described herein that have a dendritic appearance (e.g., molecules radiated from a single focal point).

[0403] Although dendritic polymers are polymers, they are superior to conventional polymers due to their controllable structure, single molecular weight, numerous and controllable surface functional groups, and their traditionally spherical conformation after reaching a certain generation. Dendritic polymers can be prepared by sequentially reacting each repeating unit to produce monodisperse, dendritic, and / or generational polymer structures. A single dendritic polymer consists of a central core molecule, with dendritic wedges attached to one or more functional sites on this core. Depending on the assembly monomers used in the preparation process, the surface layer of the dendritic polymer can have a variety of functional groups disposed thereon, including anionic, cationic, hydrophilic, or lipophilic groups.

[0404] The physical properties of dendritic polymers can be tuned by altering the functional groups and / or chemical properties of the core, repeating units, and surface or capping groups. Some properties that can be modified include, but are not limited to, solubility, toxicity, immunogenicity, and bioadhesion ability. Dendritic polymers are often described by their generation or the number of repeating units in their branches. A dendritic polymer consisting only of a core molecule is called generation 0, while each consecutive repeating unit along all branches is generation 1, generation 2, etc., up to the capping or surface groups. In some embodiments, a half-generation may be produced solely by a first condensation reaction with an amine rather than a second condensation reaction with a thiol.

[0405] The preparation of dendritic polymers requires a level of synthetic control achieved through a series of stepwise reactions, including the construction of dendritic polymers through each successive group. Dendritic polymer synthesis can be convergent or divergent. During divergent dendritic polymer synthesis, molecules assemble from the core to the periphery in a stepwise process, including linking one generation to the previous generation and then altering the functional groups of the next stage of the reaction. Functional group transformation is necessary to prevent uncontrolled polymerization. Such polymerization will result in highly branched molecules that are not monodisperse and are also known as hyperbranched polymers. Due to steric effects, the repeating units of the dendritic polymer continue to react to produce spherical or globular molecules until steric overcrowding prevents the complete reaction of a particular generation and destroys the monodispersity of the molecule. Therefore, in some embodiments, dendritic polymers of generations G1–G10 are specifically considered. In some embodiments, the dendritic polymer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating units, or any range derived therefrom. In some embodiments, the dendritic polymers used herein are G0, G1, G2, or G3. However, the number of possible generations (e.g., 11, 12, 13, 14, 15, 20, or 25) can be increased by reducing the number of spacer units in the branched polymer.

[0406] Furthermore, dendritic polymers possess two main chemical environments: one generated by specific surface groups on the capped ends, and the interior of the dendritic structure itself, which may be shielded from both the bulk medium and the surface groups due to its higher-order structure. Because of these distinct chemical environments, dendritic polymers have been found to have many different potential applications, including in therapeutic applications.

[0407] In some aspects, the dendritic polymers of this disclosure are assembled using the differential reactivity of acrylate and methacrylate groups with amines and thiols. Dendritic polymers that can be used herein include secondary or tertiary amines and thioethers formed by the reaction of acrylate groups with primary or secondary amines and methacrylates with mercapto groups. Furthermore, the repeating units of the dendritic polymers described herein may contain groups that are biodegradable under physiological conditions. In some embodiments, these repeating units may contain one or more germinating diether, ester, amide, or disulfide groups. In some embodiments, the core molecule is a monoamine that allows dendritic polymerization to occur only in one direction. In other embodiments, the core molecule is a polyamine having multiple different dendritic branches, each branch may contain one or more repeating units. The dendritic polymer can be formed by removing one or more hydrogen atoms from the core. In some embodiments, these hydrogen atoms are located on heteroatoms such as nitrogen atoms. In some embodiments, the end-capping groups are lipophilic groups such as long-chain alkyl or alkenyl groups. In other embodiments, the end-capping groups are long-chain haloalkyl or haloalkenyl groups. In other embodiments, the capping group is an aliphatic or aromatic group containing an ionizable group such as an amine (-NH2) or a carboxylic acid (-CO2H). In other embodiments, the capping group is an aliphatic or aromatic group containing one or more hydrogen bond donors such as a hydroxyl group, an amide group, or an ester.

[0408] The dendritic polymers provided in this disclosure are illustrated, for example, in the above summary section and in the claims below. They can be prepared using the methods outlined in the Examples section. These methods can be further modified and optimized using organic chemistry principles and techniques applicable to those skilled in the art. Such principles and techniques are taught, for example, in March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (2007), which is incorporated herein by reference.

[0409] The dendritic polymers of this disclosure may contain one or more asymmetrically substituted carbon or nitrogen atoms and may be separable in optically active or racemic forms. Therefore, all chiral, diastereomeric, racemic, epimeric, and geometrically isomeric forms of the chemical formula are contemplated unless a specific stereochemical or isomeric form is explicitly specified. The dendritic polymers may exist as racemic mixtures and racemic mixtures, as single enantiomers, diastereomeric mixtures, and single diastereomeric forms. In some embodiments, a single diastereomeric isomer is obtained. The chiral center of the dendritic polymers of this disclosure may have an S or R configuration. Furthermore, it is contemplated that one or more dendritic polymers may exist as constitutive isomers. In some embodiments, the compounds have the same chemical formula but differ in their connection to the nitrogen atom of the nucleus. Without wishing to be bound by any theory, it is believed that such dendritic polymers exist because the starting monomer reacts first with a primary amine and then statistically with any secondary amine present. Thus, the constitutive isomers may present a fully reacted primary amine and then a mixture of reacted secondary amines.

[0410] The chemical formula used to represent the dendritic polymers of this disclosure will generally show only one of several possible different tautomers. For example, many types of ketone groups are known to exist in equilibrium with corresponding enol groups. Similarly, many types of imine groups exist in equilibrium with enamine groups. Regardless of which tautomer is depicted for a given chemical formula, and regardless of which tautomer is most prevalent, all tautomers of a given chemical formula are expected.

[0411] The dendritic polymers disclosed herein may also have the advantages of being more effective, less toxic, having a longer duration of action, higher potency, producing fewer side effects, being more easily absorbed, and / or having better pharmacokinetic characteristics (e.g., higher oral bioavailability and / or lower clearance) compared to compounds known in the prior art, and / or having other useful pharmacological, physical, or chemical properties, whether or not for the indications described herein or otherwise.

[0412] Furthermore, the atoms constituting the dendritic polymers of this disclosure are intended to include all isotopic forms of these atoms. As used herein, isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and not limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include 13 C and 14 C.

[0413] It should be recognized that the specific anion or cation forming part of any salt form of the dendritic polymers presented herein is not important, as long as the salt as a whole is pharmacologically acceptable. Other examples of pharmaceutically acceptable salts and their preparation and use are presented in the Handbook of Pharmaceutical Salts: Properties, and Use (2002), which is incorporated herein by reference.

[0414] C. Assisting lipids

[0415] In some aspects of this disclosure, one or more auxiliary lipids are mixed with the polymers of this disclosure to produce compositions. In some embodiments, the polymer is mixed with 1, 2, 3, 4, or 5 different types of auxiliary lipids. It is contemplated that the polymer can be mixed with multiple different lipids of a single type. In some embodiments, the lipids may be steroids or steroid derivatives. In other embodiments, the lipids are PEG lipids. In other embodiments, the lipids are phospholipids. In other embodiments, the dendritic polymer composition comprises a steroid or steroid derivative, PEG lipids, and phospholipids.

[0416] 1. Steroids and steroid derivatives

[0417] In some aspects of this disclosure, polymers are mixed with one or more steroids or steroid derivatives to produce dendritic polymer compositions. In some embodiments, the steroids or steroid derivatives include any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a tetracyclic 17-carbon ring structure, which may further comprise one or more substitutions, said substitutions including alkyl, alkoxy, hydroxyl, oxo, acyl, or double bonds between two or more carbon atoms. In one aspect, the cyclic structure of the steroid comprises three fused cyclohexyl rings and a fused cyclopentyl ring, as shown in the following formula:

[0418]

[0419] In some embodiments, the steroid derivative comprises the aforementioned ring structure having one or more non-alkyl substituted components. In some embodiments, the steroid or steroid derivative is a sterol, wherein the formula is further defined as:

[0420]

[0421] In some embodiments of this disclosure, the steroid or steroid derivative is a cholesterane or a cholesterane derivative. In cholesteranes, the ring structure is further defined by the following formula:

[0422]

[0423] As described above, cholesterane derivatives include one or more non-alkyl substitutions in the aforementioned ring system. In some embodiments, cholesterane or cholesterane derivatives are cholesterene or cholesterene derivatives, or sterols or sterol derivatives. In other embodiments, cholesterane or cholesterane derivatives are cholesterene and sterols or derivatives thereof.

[0424] In some embodiments, the composition may further comprise a steroid to dendritic polymer molar ratio of about 1:10 to about 1:20. In some embodiments, the molar ratio is about 1:20, 1:18, 1:16, 1:14, 1:12, 1:10, 1:8, 1:6, 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, 8:1 to about 10:1 or any range derived therefrom. In some embodiments, the molar ratio is about 38:50 or about 1:5.

[0425] 2. PEG or PEGylated lipids

[0426] In some aspects of this disclosure, a polymer is mixed with one or more PEGylated lipids (or PEG lipids) to produce a dendritic polymer composition. In some embodiments, this disclosure includes the use of any lipid already linked with a PEG group. In some embodiments, the PEG lipid is a diglyceride that also contains a PEG chain linked to a 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 linked to a linker group and a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-bound ceramides, PEG-modified dialkylamines and PEG-modified 1,2-diacoxypropyl-3-amine, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearylphosphatidylethanolamine or PEG-modified myristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. PEG-modified molecules are 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 invention, which are incorporated herein by reference.

[0427] In another aspect, PEG lipids have the formula:

[0428]

[0429] Where: R 12 and R 13 Each is an alkyl group independently. (C≤24) alkenyl (C≤24) or a substituted form of any of these groups; R e It is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) And x is 1-250. In some implementations, R e It is an alkyl group (C≤8) For example, methyl. R 12 and R13 Each is an alkyl group independently. (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, methoxy polyethylene glycol.

[0430] In another aspect, PEG lipids have the formula:

[0431]

[0432] Wherein: n1 is an integer from 1 to 100, and n2 and n3 are each independently selected from integers from 1 to 29. In some embodiments, n1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derived therefrom. In some embodiments, n1 is about 30 to about 50. In some embodiments, n2 is 5 to 23. In some embodiments, n2 is 11 to about 17. In some embodiments, n3 is 5 to 23. In some embodiments, n3 is 11 to about 17.

[0433] In some embodiments, the composition may further comprise a molar ratio of PEG lipid to dendritic polymer of about 1:1 to about 1:250. In some embodiments, the molar ratio is about 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:125, 1:150, 1:175, 1:200, 1:225 to about 1:250 or any range derived therefrom. In some embodiments, the molar ratio is about 1:25 or about 3:100.

[0434] 3. Phospholipids

[0435] In some aspects of this disclosure, polymers are mixed with one or more phospholipids to produce dendritic polymer compositions. In some embodiments, any lipid 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 distearylphosphatidylcholine or dioleoylphosphatidylethanolamine.

[0436] In some embodiments, the composition may further comprise a molar ratio of phospholipid to dendritic polymer of about 1:10 to about 1:20. In some embodiments, the molar ratio is about 1:20, 1:18, 1:16, 1:14, 1:12, 1:10, 1:8, 1:6, 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, 8:1 to about 10:1 or any range derived therefrom. In some embodiments, the molar ratio is about 38:50 or about 1:5.

[0437] D. Nucleic acids and nucleic acid-based therapeutic agents

[0438] 1. Nucleic acid

[0439] In some aspects of this disclosure, the dendritic polymer composition comprises one or more nucleic acids. In some embodiments, the dendritic polymer composition comprises one or more nucleic acids present with the dendritic polymer in a weight ratio of about 5:1 to about 1:100. In some embodiments, the weight ratio of nucleic acid to dendritic polymer 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 derived therefrom. In some embodiments, the weight ratio is about 1:25 or about 1:7. Furthermore, it should be understood that this disclosure is not limited to the specific nucleic acids disclosed herein. The scope of this invention is not limited to any particular source, sequence, or type of nucleic acid; however, as can be readily identified by those skilled in the art, related homologues in nucleic acids from various other sources, including nucleic acids from non-human species (e.g., mice, rats, rabbits, dogs, monkeys, gibbons, chimpanzees, apes, baboons, cattle, pigs, horses, sheep, cats, and other species). Nucleic acids intended for use in this disclosure may comprise sequences based on naturally occurring sequences. Considering the degeneracy of the genetic code, a sequence 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 is 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 complementary at 75%, 80%, 85%, 90%, 95%, and 100%.

[0440] In some respects, nucleic acids are sequences that silence, complement, or replace another sequence present in the body. A sequence of 17 base pairs in length should appear only once in the human genome, thus sufficient to specify a unique target sequence. While shorter oligomers are easier to prepare and increase in vivo accessibility, determining the specificity of hybridization involves many other factors. The binding affinity and sequence specificity of oligonucleotides to their complementary targets increase with increasing length. Exemplary oligonucleotides with 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs are expected to be used, but others are also considered. Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or longer were also considered.

[0441] The nucleic acids used herein can be derived from genomic DNA, i.e., directly cloned from the genome of a specific organism. However, in a preferred embodiment, the nucleic acids will contain complementary DNA (cDNA). Also considered 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 invention can be used as molecular weight standards, for example, in gel electrophoresis.

[0442] The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as a template. Compared to genomic DNA or DNA polymerized from genomic, unprocessed, or partially processed RNA templates, the advantage of using cDNA is that cDNA primarily contains the coding sequence of the corresponding protein. Sometimes, a complete or partial genomic sequence may be preferred, for example when non-coding regions are required for optimal expression, or when non-coding regions such as introns will be targeted in an antisense strategy.

[0443] In some implementations, the nucleic acid contains one or more antisense segments that suppress gene or gene product expression. The antisense approach leverages the fact that nucleic acids tend to pair with "complementary" sequences. Complementarity means that the polynucleotide is a polynucleotide capable of base pairing according to the standard Watson-Crick complementarity rule. That is, a larger purine will pair with a smaller pyrimidine base, forming a combination of guanine (G:C) with cytosine and adenine (A:T) with thymine in the case of DNA, or adenine (A:U) with uracil in the case of RNA. The presence of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine in the hybridization sequence does not interfere with pairing.

[0444] Targeting double-stranded (ds) DNA with polynucleotides leads to triple helix formation; targeting RNA leads to double helix formation. Antisense polynucleotides, upon introduction into target cells, specifically bind to their target polynucleotides 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, either in vitro or in vivo (e.g., in host animals, including human subjects).

[0445] Antisense constructs can be designed to bind to gene promoters and other control regions, exons, introns, or even exon-intron boundaries. The most efficient antisense constructs are expected to include regions complementary to intron / exon splicing sites. Therefore, preferred embodiments are proposed that include antisense constructs with complementarity to regions within 50-200 bases of the intron-exon splicing site. It has been observed that some exon sequences can be included in the construct without significantly affecting its target selectivity. The amount of exon material included will vary depending on the specific exon and intron sequences used. Whether excessive exon DNA is included can be easily tested by testing the construct in vitro to determine whether normal cellular function is affected or the expression of related genes with complementary sequences is affected.

[0446] As mentioned above, "complementary" or "antense" refers to a polynucleotide sequence that is substantially complementary throughout its length and has very few base mismatches. For example, a 15-base sequence can be called complementary when it has complementary nucleotides at 13 or 14 positions. Naturally, a perfectly complementary sequence would be one that is completely complementary throughout its length and has no base mismatches. Other sequences with lower homology are also considered. For example, antisense constructs can be designed with a limited region of high homology but also containing non-homologous regions (e.g., ribozymes; see below). These molecules, while having less than 50% homology, will bind to the target sequence under appropriate conditions.

[0447] Combining portions of genomic DNA with cDNA or synthetic sequences to form siRNA or generate specific constructs can be advantageous. For example, when the final construct requires introns, genomic cloning will be necessary. eDNA, siRNA, or synthetic polynucleotides can provide more convenient restriction sites for the remainder of the construct, and therefore can be used for the rest of the sequence. Other implementations include dsRNA or ssRNA, which can be used to target genomic sequences or coding / non-coding transcripts.

[0448] In other embodiments, the dendritic polymer composition may comprise a nucleic acid containing one or more expression vectors for use in gene therapy. Expression requires the provision of appropriate signals within the vector, and it includes various regulatory elements, such as enhancers / promoters from viral and mammalian sources that drive the expression of genes of interest in host cells. Elements designed to optimize the stability and translatability of messenger RNA in host cells are also defined. Conditions for establishing permanently stable cell clones of expression products using a variety of advantageous drug-selective markers are also provided, as well as elements for linking the expression of drug-selective markers to the expression of peptides.

[0449] Throughout this application, the term "expression construct" is intended to include any type of genetic construct containing nucleic acids encoding gene products, wherein some or all of the nucleic acid coding sequences can be transcribed. The transcript may be translated into a protein, but is not required to do so. In some embodiments, expression includes both gene transcription and mRNA translation into a gene product. In other embodiments, expression includes only the transcription of nucleic acids encoding the gene of interest.

[0450] The term "vector" is used to refer to a vector nucleic acid molecule in which a nucleic acid sequence can be inserted to introduce it into a cell that can be replicated. The nucleic acid sequence can be "exogenous," meaning it is foreign to the cell into which the vector is introduced, or the sequence is homologous to a sequence in the cell but located in a position within the host cell's nucleic acid that is not normally present. Vectors include plasmids, granules, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YAC). Those skilled in the art will be able to construct vectors using standard recombination techniques described in Sambrook et al. (1989) and Ausubel et al. (1994), both of which are incorporated herein by reference.

[0451] The term "expression vector" refers to a vector containing at least a portion of a nucleic acid sequence encoding a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, such as in the production of antisense molecules or ribozymes. Expression vectors may contain various "control sequences," which refer to nucleic acid sequences necessary for transcription and possibly translation of operationally linked coding sequences in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that also serve other functions, as described below.

[0452] 2. siRNA

[0453] As described above, the present invention contemplates the use of one or more repressive nucleic acids to reduce the expression and / or activation of genes or gene products. Examples of repressive nucleic acids include, but are not limited to, molecules that target nucleic acid sequences, such as siRNA (small interfering RNA), short hairpin RNA (shRNA), double-stranded RNA, antisense oligonucleotides, ribozymes, and molecules that target genes or gene products, such as aptamers.

[0454] Repressive nucleic acids can inhibit gene transcription or prevent the translation of gene transcripts in cells. Repressive nucleic acids can be 16 to 1000 nucleotides in length, and in some embodiments, they can be 18 to 100 nucleotides in length.

[0455] Inhibitory nucleic acids are well known in the art. For example, siRNA, shRNA, and double-stranded RNA are described in U.S. Patents 6,506,559 and 6,573,099 and U.S. Patent Publications 2003 / 0051263, 2003 / 0055020, 2004 / 0265839, 2002 / 0168707, 2003 / 0159161, and 2004 / 0064842, all of which are incorporated herein by reference in their entirety.

[0456] Since Fire and colleagues discovered RNAi in 1998, the biochemical mechanisms have been rapidly characterized. Double-stranded RNA (dsRNA) is cleaved by Dicer, a family of RNase III ribonucleases. This process produces siRNAs approximately 21 nucleotides in length. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC), which is directed to the target mRNA. The RISC cleaves the target mRNA in the middle of its complementary region. In mammalian cells, the associated microRNAs (miRNAs) are found to be short RNA fragments (approximately 22 nucleotides). miRNAs are produced following Dicer-mediated cleavage of a longer (approximately 70 nucleotides) precursor with an incomplete hairpin RNA structure. These miRNAs are incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of the target mRNA.

[0457] Several factors need to be considered when designing nucleic acids capable of producing RNAi effects, such as the nature of the siRNA, the persistence of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA introduced into the organism will typically contain exon sequences. Furthermore, the RNAi process is homology-dependent, therefore, sequences must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Specifically, siRNAs exhibit greater than 80%, 85%, 90%, 95%, 98%, or even 100% identity between the siRNA sequence and a portion of the EphA nucleotide sequence. Sequences with less than approximately 80% identity to the target gene are generally less potent. Therefore, the greater the identity between the siRNA and the gene to be suppressed, the less likely the expression of unrelated genes will be affected.

[0458] Furthermore, the size of the siRNA is an important consideration. In some embodiments, this disclosure relates to siRNA molecules comprising at least about 19-25 nucleotides and capable of regulating gene expression. In the context of this disclosure, the length of the siRNA is particularly less than 500, 200, 100, 50, 25, or 20 nucleotides. In some embodiments, the length of the siRNA is about 25 to about 35 nucleotides or about 19 to about 25 nucleotides.

[0459] To improve the effectiveness of siRNA-mediated gene silencing, guidelines for target site selection on mRNA have been developed for optimal siRNA design (Soutschek et al., 2004; Wadhwa et al., 2004). These strategies allow for rational methods for selecting siRNA sequences to achieve maximum gene knockdown. Various vectors, including plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus, have been used to facilitate siRNA entry into cells and tissues (Wadhwa et al., 2004).

[0460] Within a repressive nucleic acid, the components of the nucleic acid do not need to be of the same type or always homogeneous (e.g., a repressive nucleic acid may contain nucleotides and nucleic acids or nucleotide analogs). Typically, repressive nucleic acids form a double-stranded structure; said double-stranded structure can be generated by two separate nucleic acids that are partially or completely complementary. In some embodiments of the invention, the repressive nucleic acid may contain only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by being complementary to itself (e.g., forming a hairpin loop). The double-stranded structure of the repressive nucleic acid may contain 16-500 or more consecutive nucleobases, including all ranges that can be derived therefrom. The repressive nucleic acid may contain 17 to 35 consecutive nucleobases, more particularly 18 to 30 consecutive nucleobases, more particularly 19 to 25 consecutive nucleobases, more particularly 20 to 23 consecutive nucleobases, or 20 to 22 consecutive nucleobases, or 21 consecutive nucleobases, which hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.

[0461] siRNA can be obtained from commercial sources, natural sources, or synthesized using any of a number of techniques well known to those skilled in the art. For example, commercial sources of pre-designed siRNA include Invitrogen's Stealth. TM Select technology (Carlsbad, CA) (Austin, TX) and (Valencia, CA). The repressive nucleic acid that can be used in the compositions and methods of the present invention can be any nucleic acid sequence that has been found to be an effective downregulator of a gene or gene product from any source.

[0462] In some embodiments, the present invention provides isolated siRNA molecules having at least 19 nucleotides having at least one strand substantially complementary to at least ten but no more than thirty consecutive nucleotides of the nucleic acid encoding a gene, and having a depressant effect on the expression of a gene or gene product. In one embodiment of this disclosure, the siRNA molecule has at least one strand substantially complementary to at least ten but no more than thirty consecutive nucleotides of the mRNA encoding a gene or gene product.

[0463] In one embodiment, the siRNA molecule has at least 75%, 80%, 85%, or 90% homology, particularly at least 95%, 99%, or 100% similarity or identity, or any percentage therebetween, to at least 10 consecutive nucleotides of any nucleic acid sequence encoding a target therapeutic protein (e.g., the invention covers more than 75%, more than 80%, more than 85%, etc., and the range is intended to include all integers therebetween).

[0464] siRNA may also contain alterations to one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as to the ends or interior of RNA of 19 to 25 nucleotides (at one or more nucleotides of the RNA). In some respects, the RNA molecule contains a 3′-hydroxyl group. The nucleotides in the RNA molecule of the present invention may also contain non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, such as a phosphate thioester, a dithiophosphate ester, or other modified backbones known in the art, or may contain non-natural nucleoside internucleotides. Other modifications to siRNA (e.g., 2′-O-methylribonucleotide, 2′-deoxy-2′-fluororibonucleotide, "universal base" nucleotide, 5-C-methyl nucleotide, one or more phosphate thioester nucleotide internucleotides, and inverted deoxy base residues are incorporated herein by reference) in U.S. Publication 2004 / 0019001 and U.S. Patent 6,673,611 (each of which is incorporated herein by reference in its entirety). In summary, all of the above-described modified nucleic acids or RNAs are referred to as modified siRNAs.

[0465] In one embodiment, the siRNA is capable of reducing the expression of a specific genetic product by at least 10%, at least 20%, at least 30%, or at least 40%, at least 50%, at least 60%, or at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or more, or any range thereof.

[0466] 3. CRISPRCAS

[0467] CRISPR (Regularly Clustered Interspaced Short Palindromic Repeats) are short, repetitive DNA loci containing a sequence of bases. Each repeat is followed by a short segment of "spacer DNA" from a previously exposed virus. CRISPR is present in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaea. CRISPR is typically associated with cas genes encoding CRISPR-related proteins. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and bacteriophages and provides a form of acquired immunity. CRISPR spacer sequences in eukaryotes recognize and silence these foreign genetic elements, such as RNAi.

[0468] The repetitive sequences of the bacterium *Escherichia coli* were first described in 1987. In 2000, similar clustered repetitive sequences were identified in other bacteria and archaea and termed short regularly spaced repeats (SRSRs). SRSRs were renamed CRISPR in 2002. A group of genes (some encoding putative nucleases or helicases) have been found to be associated with CRISPR repeat sequences (cas, or CRISPR-associated genes).

[0469] In 2005, three independent researchers demonstrated that the CRISPR spacer sequence is homologous to several phage DNAs and extrachromosomal DNAs such as plasmids. This suggests that the CRISPR / Cas system may play a role in bacterial adaptive immunity. Koonin and colleagues proposed that the spacer sequence serves as a template for RNA molecules, similar to eukaryotic cells using a system called RNA interference.

[0470] In 2007, Barrangou, Horvath (a food industry scientist at Danisco), and others demonstrated that they could alter the resistance of *Streptococcus thermophilus* to phage attack via spacer DNA. Doudna and Charpentier had independently studied CRISPR-related proteins to understand how bacteria deploy spacer sequences in their immune defenses. Together, they investigated a simpler CRISPR system that relies on a protein called Cas9. They found that bacteria respond to invading phages by transcribing the spacer sequence and palindromic DNA into long RNA molecules, which the cell then cleaves into fragments called crRNA using tracrRNA and Cas9.

[0471] CRISPR was first used as a genome engineering / editing tool in human cell culture in 2012. It has been widely used in various organisms, including baker's yeast (Saccharomyces cerevisiae), zebrafish, nematodes (C. elegans), plants, mice, and several other organisms. Furthermore, CRISPR has been modified into a programmable transcription factor, enabling scientists to target and activate or silence specific genes. Libraries of thousands of guide RNAs are now available.

[0472] In March 2014, MIT researchers cured a rare liver disease in mice, providing the first evidence that CRISPR can reverse disease symptoms in living animals. Since 2012, the CRISPR / Cas system has been used for gene editing (silencing, enhancing, or altering specific genes), even in eukaryotes such as mice and primates. By inserting a plasmid containing the cas gene and a specially designed CRISPR, the genome of an organism can be cut at any desired location.

[0473] CRISPR repeat sequences range in size from 24 to 48 base pairs. They typically exhibit some binary symmetry, meaning they form secondary structures such as hairpins, but are not true palindromes. Repeat sequences are separated by spacer sequences of similar length. Some CRISPR spacer sequences perfectly match sequences from plasmids and phages, but some spacer sequences match the genomes of prokaryotes (self-targeting spacer sequences). New spacer sequences can be rapidly added in response to phage infection.

[0474] CRISPR-associated (Cas) genes are typically associated with CRISPR repeat spacer sequence arrays. As of 2013, more than forty different Cas protein families have been described. Among these families, Cas1 appears to be ubiquitous across different CRISPR / Cas systems. Specific combinations of Cas genes and repeat sequence structures have been used to define eight CRISPR isotypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with another gene module encoding a mysterious repeat-associated protein (RAMP). More than one CRISPR isotype can occur within a single genome. The sporadic distribution of CRISPR / Cas isotypes suggests that this system has been influenced by horizontal gene transfer during microbial evolution.

[0475] The exogenous DNA is apparently processed into small elements (approximately 30 base pairs in length) by proteins encoded by the Cas gene, and then inserted into a CRISPR locus near the leader sequence. RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs consisting of single exogenous-derived sequence elements with flanking repeats. The RNA directs other Cas proteins to silence the exogenous genetic elements at the RNA or DNA level. There is evidence of functional diversity among CRISPR isoforms. The Cse (Cas isoform E. coli) protein (called CasA-E in E. coli) forms the functional complex Cascade, which processes CRISPR RNA transcripts into Cascade-preserved spacer-repetitive sequence units. In other prokaryotes, Cas6 processes CRISPR transcripts. Interestingly, in E. coli, CRISPR-based phage inactivation requires Cascadc and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) protein, found in *Pyrococcus furiosus* and other prokaryotes, forms a functional complex with small CRISPR RNA that recognizes and cleaves complementary target RNA. RNA-guided CRISPR enzymes are classified as type V restriction enzymes.

[0476] See also U.S. Patent Publication 2014 / 0068797, the entire contents of which are incorporated herein by reference.

[0477] i.Cas9

[0478] Cas9 is a nuclease specifically designed to cut DNA, possessing two active cleavage sites, one on each double helix. It has been shown that one or both sites can be disabled while retaining Cas9's homing ability to locate its target DNA. Jinek combined tracrRNA and spacer RNA into a "guide RNA" molecule, which, when mixed with Cas9, can find and cleave the correct DNA target. Jinek et al. proposed that this synthetic guide RNA may be useful for gene editing.

[0479] Cas9 proteins are highly enriched in both pathogenic and symbiotic bacteria. CRISPR / Cas-mediated gene regulation may help modulate endogenous bacterial genes, particularly during bacterial-eukaryotic host interactions. For example, the Cas9 protein of *Francisella novicida* uses a unique small CRISPR / Cas-associated RNA (scaRNA) to repress endogenous transcripts that encode bacterial lipoproteins crucial to *F. novicida*, suppressing host responses and promoting virulence.

[0480] ii gRNA or sgRNA

[0481] As an RNA guide protein, Cas9 requires a short RNA to guide the recognition of DNA targets (Mali et al., 2013a). Although Cas9 preferentially queries DNA sequences containing the PAM sequence NGG, it can bind even without a protospacer sequence target. However, the Cas9-gRNA complex requires a tight match with the gRNA to generate a double-strand break (Cho et al., 2013; Hsu et al., 2013). In bacteria, CRISPR sequences are expressed in various RNAs and then processed to produce guide strands of RNA (Bikard et al., 2013). Because eukaryotic systems lack some of the proteins required to process CRISPR RNA, synthetic construct gRNAs are produced to assemble basic fragments of RNA for Cas9 targeting into a single RNA expressed using the RNA polymerase type III promoter U6 (Mali et al., 2013a, b). The synthetic gRNA is slightly over 100 bp in minimum length and contains a portion targeting the 20 protospacer sequence nucleotides immediately preceding the PAM sequence NGG; the gRNA does not contain the PAM sequence.

[0482] 4. Modification of nucleobases

[0483] In some embodiments, the nucleic acids of this disclosure comprise one or more modified nucleosides containing a modified sugar moiety. Such compounds containing one or more sugar-modified nucleosides may possess desired properties, such as enhanced nuclease stability or increased binding affinity to target nucleic acids, relative to oligonucleotides containing only nucleosides with 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 substitute. Such a sugar substitute may contain one or more substitutions corresponding to the substituted sugar moiety.

[0484] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridged sugar substituents, including but not limited to substituents at the 2′ and / or 5′ positions. Examples of suitable sugar substituents at the 2′ position include, but are not limited to, 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE”). In some embodiments, the sugar substituent at the 2′ position is selected from allyl, amino, azide, thio, O-allyl, O-C1-C. 10 Alkyl, O--C1-C 10 Substituted alkyl groups; OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn) and O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H, or a substituted or unsubstituted C1-C. 10 Alkyl group. 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-bridged sugar substituent, such as TF-5′-methyl sugar moiety (for additional 5′,2′-disubstituted sugar moieties and nucleosides, see, for example, PCT International Application WO 2008 / 101157).

[0485] Nucleosides containing a 2′-substituted sugar moiety are referred to as 2′-substituted nucleosides. In some embodiments, the 2′-substituted nucleosides contain a 2′-substituent selected from: halogroup, allyl group, amino group, azidogroup, SH group, CN group, OCN group, CF3 group, OCF3 group, O group, S group, or N(R group). m )-alkyl; O, S or N(R) m )-Alkenyl; O, S or N(R) m -Alkyne; O-alkylalkenyl-O-alkyl, alkyne, alkylaryl, aralkyl, O-alkylaryl, 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 Independently, it is H, an amino protecting group, or a substituted or unsubstituted C1-C. 10 Alkyl groups. These 2′-substituents may be further substituted by one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxyl, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0486] In some embodiments, the 2′-substituted nucleoside comprises a 2′-substituent selected from the following: 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 acetamides (O--CH2--C(=O)--N(R) m (R) n ), where each R m and R n It is independently of H, amino protecting group, or substituted or unsubstituted C1-C 10 alkyl.

[0487] 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.

[0488] In some embodiments, the 2′-substituted nucleoside comprises a sugar moiety containing a 2′-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0489] Some modified sugar moieties contain bridging sugar substituents that form a second ring leading to the bicyclic sugar moieties. In some such embodiments, the bicyclic sugar moieties contain bridges 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 analogues (see, for example, U.S. Patent 7,399,845); 4′-C(CH3)(CH3)--O-2′ and their analogues (see, for example, WO 2009 / 006478); 4′-CH2--N(OCH3)-2′ and its analogues (see, for example, WO2008 / 150729); 4′-CH2--O--N(CH3)-2′ (see, for example, US2004 / 0171570, disclosed on 2 September 2004); 4′-CH2--O--N(R)-2′ and 4′-CH2--N(R)--O-2′, wherein each R is independently H, a protecting group or C1-C 12 Alkyl; 4′-CH2--N(R)--O-2′, where R is H, C1-C 12 Alkyl or protecting group (see U.S. Patent 7,427,672); 4′-CH2--C(H)(CH3)-2′ (see, for example, Chattopadhyaya et al., Journal of Organic Chemistry, 2009, 74, 118-134); and 4′-CH2--C(=CH2)-2′ and analogues thereof (see PCT International Application WO 2008 / 154401).

[0490] In some embodiments, such a 4′ to 2′ bridge independently comprises 1 to 4 linking groups independently selected from: --[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 )--;in:

[0491] x is 0, 1, or 2;

[0492] n is 1, 2, 3 or 4;

[0493] Each R a and R b Independently, it is H, protecting group, hydroxyl group, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 alkenyl, C2-C 12 Alkyne group, substituted C2-C 12 alkynyl group, C5-C 20 Aryl, substituted C5-C 20 Aryl, heterocyclic, substituted heterocyclic, heteroaryl, substituted heteroaryl, C5-C7 alicyclic, substituted C5-C7 alicyclic, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)--H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxide (S(=O)-J1); and

[0494] 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 Alkyne group, substituted C2-C 12 alkynyl group, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)--H), substituted acyl, heterocyclic, substituted heterocyclic, C1-C 12 Aminoalkyl, substituted C1-C 12 Aminoalkyl groups, or protecting groups.

[0495] Nucleosides containing a bicyclic sugar moiety are called bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to: (A) α-L-methyleneoxy(4′-CH2--O-2′)BNA, (B) β-D-methyleneoxy(4′-CH2--O-2′)BNA (also known as locked nucleic acid or LNA), (C) ethyleneoxy(4′-(CH2)2--O-2′)BNA, (D) aminooxy(4′-CH2--O--N(R)-2′)BNA, (E) oxyamino(4′-CH2--N(R)--O-2′)BNA, and (F) methyl(methyleneoxy)(4′-CH(C (H3)--O-2′)BNA (also known as restricted ethyl or cEt), (G) methylene-thio (4′-CH2--S-2′)BNA, (H) methylene-amino (4′-CH2-N(R)-2′)BNA, (I) methylcarbocyclic (4′-CH2--CH(CH3)-2′)BNA, (J) propylenecarbocyclic (4′-(CH2)3-2′)BNA, and (K) methoxy (ethyleneoxy) (4′-CH(CH2OMe)-O-2′)BNA (also known as restricted MOE or cMOE).

[0496] Other bicyclic sugar moieties are known in the art, for example: Singh et al., *Chem. Commun.*, 1998, 4, 455-456; Koshkin et al., *Tetrahedron*, 1998, 54, 3607-3630; Wahlestedt et al., *Proceedings of the National Academy of Sciences of the United States of America*, 2000, 97, 5633-5638; Kumar et al., *Bioorganic Chemistry and Medicine*. Medicinal Chemistry Communications (Bioorg. Med. Chem. Lett.), 1998, 8, 2219-2222; Singh et al., Journal of Organic Chemistry (J. Org. Chem.), 1998, 63, 10035-10039; Srivastava et al., Journal of the American Chemical Society (J. Am. Chcm. Soc.), 129(26) 8362-8379 (July 4, 2007); Elayadi et al., Research Drug Opinions (Curr. Opinion) Invens. Drugs, 2001, 2, 5561; Braasch et al., Chemical Biology, 2001, 8, 1-7; Orum et al., New Insights in Molecular Therapy, 2001, 3, 239-243; US 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; US ​​Patent Publication No. US 2004 / 0171570, US US 2007 / 0287831 and US 2008 / 0039618; US 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.

[0497] In some embodiments, the bicyclic sugar moiety and the nucleotide containing such a bicyclic sugar moiety are further defined by isomer configuration. For example, a nucleotide containing a 4′-2′ methylene-oxygen bridge can be in the α-L configuration or the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2--O-2′) bicyclic nucleotides have been incorporated into antisense oligonucleotides exhibiting antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0498] In some embodiments, the substituted sugar moiety comprises one or more non-bridged sugar substituents and one or more bridged sugar substituents (e.g., 5′-substituted and 4′-2′-bridged sugars; PCT International Application WO 2007 / 134181, wherein LNA is substituted, for example, 5′-methyl or 5′-vinyl).

[0499] In some embodiments, the modified sugar moiety is a sugar substitute. In some such embodiments, the oxygen atom of a naturally occurring sugar is replaced by, for example, a sulfur, carbon, or nitrogen atom. In some such embodiments, the modified sugar moiety also contains bridging and / or non-bridging substituents as described above. For example, some sugar substitutes contain a 4′-sulfur atom and a substitution at the 2′ position (see, for example, published U.S. Patent Application US 2005 / 0130923) and / or the 5′ position. As further examples, carbocyclic bicyclic nucleosides with 4′-2′ bridges have been described (see, for example, Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443; and Albaek et al., Journal of Organic Chemistry, 2006, 71, 7731-7740).

[0500] In some embodiments, the sugar substitute comprises a ring having a number of atoms other than five. For example, in some embodiments, the sugar substitute comprises a six-membered tetrahydropyran. Such a tetrahydropyran can be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, but are not limited to, hexotol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C., *J. Bioorg. & Med. Chem.*, (2002) 10: 841-854), and fluorinated HNA (F-HNA).

[0501] In some embodiments, a modified THP nucleoside of formula VII is provided, wherein q1, q2, q3, q4, q5, q6, and q7 are each H. In some 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, a THP nucleoside of formula VII is provided, wherein one of R1 and R2 is F. In some embodiments, R1 is fluorine and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.

[0502] Many other bicyclic and tricyclic sugar substitute ring systems are also known in the art, which can be used to modify nucleosides for incorporation into antisense compounds (see, for example, review article: Leumann, JC, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

[0503] 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 epoxy atom with S and further substitution at the 2′ position (see US Patent Publication US 2005 / 0130923), or optionally 5′-substituted bicyclic nucleic acids (see PCT International Application WO 2007 / 134181, wherein the 4′-CH2--O-2′ bicyclic nucleoside is further substituted at the 5′ position with 5′-methyl or 5′-vinyl). The synthesis and preparation of carbocyclic bicyclic nucleosides, as well as their oligomerization and biochemical studies, have also been described (see, for example, Srivastava et al., 2007).

[0504] In some embodiments, the present invention provides oligonucleotides comprising modified nucleosides. Those modified nucleotides may include modified sugars, modified nucleobases, and / or modified bonds. Specific modifications are selected to give the resulting oligonucleotides desired characteristics. In some embodiments, the oligonucleotides comprise one or more RNA-like nucleosides. In some embodiments, the oligonucleotides comprise one or more DNA-like nucleotides.

[0505] In some embodiments, the nucleosides of the present invention comprise one or more unmodified nucleobases. In some embodiments, the nucleosides of the present invention comprise one or more modified nucleobases.

[0506] In some embodiments, the modified nucleobase is selected from: universal bases, hydrophobic bases, hybrid bases, size-enhancing 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-halogenated uracil and cytosine, 5-propynyl(CH3)uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azouracil. Cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine, 3-deadenine and 3-deadenine, universal bases, hydrophobic bases, mixed bases, size-enhanced bases and fluorinated bases. Other modified nucleobases include tricyclic pyrimidines such as phenoxazincytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenthiazincytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clasts such as substituted phenoxazincytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), and carbazolecytidine ( 2 H-pyrido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrido-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced by another heterocycle, such as 7-deadenine, 7-deadenanine, 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*, ed. Kroschwitz, JI, John Wiley & Sons, 1990, pp. 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, YS, 1993.

[0507] Representative U.S. patents teaching the preparation of certain of the above-mentioned modified nucleosides and other modified nucleosides include, but are not limited to, U.S. patents 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; 5,5 02,177; 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.

[0508] In some embodiments, the present invention provides oligonucleotides comprising linked nucleosides. In these embodiments, the nucleosides can be linked together using any internucleotide bond. Two main classes of internucleotide linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleotide bonds include, but are not limited to, phosphate diesters (P=O), phosphate triesters, methylphosphonates, aminophosphates, and thiophosphates (P=S). Representative phosphorus-free internucleotide 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 natural phosphodiester bonds, modified bonds can be used to alter (and typically increase) the nuclease resistance of oligonucleotides. In some embodiments, the internucleotide bonds with chiral atoms can be prepared as racemic mixtures or as individual enantiomers. Representative chiral bonds include, but are not limited to, alkylphosphonates and thiophosphates. Methods for preparing phosphorus-containing and phosphorus-free internucleotide bonds are well known to those skilled in the art.

[0509] The oligonucleotides described herein contain one or more asymmetric centers, thus producing enantiomers, diastereomers, and other stereoisomers, which can be defined in absolute stereochemistry as (R) or (S), α or β (e.g., for glycoterminal isomers), or (D) or (L) (e.g., for amino acids, etc.). All these possible isomers, as well as their racemic and optically pure forms, are included in the antisense compounds provided herein.

[0510] Neutral nucleoside bonds include, but are not limited to, phosphate triesters, 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′), methyl acetal (3′-O--CH2--O-5′), and thiomethyl acetal (3′-S--CH2--O-5′). Other neutral nucleoside bonds include nonionic bonds, which include siloxanes (dialkylsiloxanes), carboxylic esters, carboxamides, sulfides, sulfonates, and amides (see, for example: Carbohydrate Modifications in Antisense Research; eds. YSSanghvi and PDCook, ACS Symposium Series, 580; Chapters 3 and 4, 40–65). Other neutral nucleoside bonds include nonionic bonds containing mixed N, O, S, and CH2 components.

[0511] Further modifications can be made at other positions on the oligonucleotide, particularly at the 3′ position of the 3′-terminal nucleotide of the sugar and the 5′ position of the 5′-terminal nucleotide. For example, one further modification of the ligand-bound oligonucleotide of the present invention involves chemically linking the oligonucleotide to one or more additional non-ligand moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. These 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-triphenylmethylthiol (Manoharan et al., 1992; Manoharan et al., 1993), thiocholesterol (Oberhauser et al., 1992), and aliphatic chains such as dodecyl glycol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanov et al., 1990; Svinarchu). K et al., 1993), phospholipids such as di-hexadecyl-rac-glycerol or 1,2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate triethylammonium (Manoharan et al., 1995; Shea et al., 1990), polyamines or polyethylene glycol chains (Manoharan et al., 1995), or adamantaneacetic acid (Manoharan et al., 1995), palmitoyl moiety (Mishra et al., 1995), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996).

[0512] Representative U.S. patents teaching the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. patents 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; and 5,118,8 02;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 The patents mentioned herein are each incorporated herein by reference: 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.

[0513] E. Reagent kit

[0514] This disclosure also provides a kit. Any of the components disclosed herein can be combined in kit form. In some embodiments, the kit comprises a dendritic polymer or composition as described above or in the claims.

[0515] The kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container in which components can be placed, and preferably suitably aliquoted. In cases where more than one component is present in the kit, the kit typically also contains a second, third, or other additional container in which additional components can be placed individually. However, various combinations of components can be contained in containers. In some embodiments, all nucleic acid delivery components are combined in a single container. In other embodiments, some or all of the dendritic polymer delivery components are provided with the polymer in separate containers.

[0516] The kits of this invention will also typically include packaging for containing various containers, which is tightly sealed for commercial sale. Such packaging may include cardboard or injection-molded or blow-molded plastic packaging in which the desired containers are held. The kit may also include instructions for using the kit components. The instructions may include variations that can be implemented.

[0517] F. Example

[0518] The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following embodiments represent technologies discovered by the inventors that function well in the practice of the present invention and can therefore be considered as preferred modes of practice. However, based on this disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed and similar or analogous results can still be obtained without departing from the spirit and scope of the present invention.

[0519] Example 1: Materials and Instruments

[0520] 1. Materials used in chemical synthesis

[0521] All amines, thiols, and other unspecified chemicals were purchased from Sigma-Aldrich. 1,2-Distearayoyl-sn-glycerol-3-phosphocholine (DSPC) was purchased from Avanti Lipids. The lipid PEG2000 was chemically synthesized as described below. C12-200 was synthesized according to a reported procedure (Love et al., 2010). All organic solvents were purchased from Fisher Scientific and purified using a solvent purification system (Innovative Technology).

[0522] 2. Nucleic acids and other materials used in in vitro and in vivo experiments

[0523] All siRNAs were purchased from Sigma-Aldrich. Let-7g miRNA mimics and their control mimics were purchased from Ambion, Life Technologies. Dulbecco's Modified Eagle Media (DMEM) and fetal bovine serum (FBS) were purchased from Sigma-Aldrich. OptiMEM, DAPI, and Alexa Fluor 488 phalloidin were purchased from Life Technologies. ONE-Glo+Tox was purchased from Promega. Biophen FVII was purchased from Aniara Corporation.

[0524] The sequences of the sense and antisense strands of siRNA are as follows:

[0525] siLuc (siRNA targeting luciferase). dT is a DNA base. All others are RNA bases.

[0526] Justice: 5′-GAUUAUGUCCGGUUAUGUA[dT][dT]-3′(SEQ ID NO: 3)

[0527] Antonym: 3′-UACAUAACCGGACAUAAUC[dT][dT]-5′(SEQ ID NO: 4)

[0528] siFVII (siRNA targeting FVII). 2′-fluorinated nucleotides are lowercase letters.

[0529] Justice: 5′-GGAucAucuCAAGucuuAc[dT][dT]-3′(SEQ ID NO: 1)

[0530] Antonym: 3′-GuAAGAcuuGAGAuGAucc[dT][dT]-5′(SEQ ID NO: 2)

[0531] siCTR (siRNA used as a control)

[0532] Justice: 5′-GCGCGAUAGCGCGAAUAUA[dT][dT]-3′(SEQ ID NO: 5)

[0533] Antonym: 3′-UAUAUUCGCGCUAUCGCGC[dT][dT]-5′(SEQ ID NO: 6)

[0534] In control experiments, Sigma-Aldrich MISSION siRNA universal negative control #1 (catalog number: SIC001) was used as the non-targeted siRNA. A 2′OMe-modified control siRNA (Sigma-Aldrich, proprietary modification) was used in in vivo studies to reduce immune stimulation.

[0535] Cy5.5-tagged siRNA (siRNA for imaging)

[0536] Justice: 5′-Cy5.5-GAUUAUGUCCGGUUAUGUA[dT][dT]-3′(SEQ ID NO: 3)

[0537] Antonym: 3′-UACAUAACCGGACAUAAUC[dT][dT]-5′(SEQ ID NO: 4)

[0538] Let-7g miRNA mimic

[0539] Ambion (Life Technologies) mirVaha miRNA mimic (Catalogue No.: 4464070, Product ID: MC11758, Name: has-let-7g). The exact sequence and modifications are not disclosed by Ambion. The mimic is mature human Let-7g.

[0540] Negative control (CTR) miRNA mimic

[0541] Ambion (Life Technologies) mirVana miRNA mimic, negative control #1 (catalog number: 4464061). The exact sequence and modifications have not been disclosed by Ambion.

[0542] 3. Robot Automation

[0543] Nanoparticle (NP) formulation and in vitro screening were performed on a Tecan Freedom EVO 200 fluid handling robot equipped with an 8-channel liquid handling arm (LiHa), a multi-channel arm (MCA) with a 96-channel head, a robotic manipulator arm (RoMa), and an integrated InfiniTe F / M200 Pro microplate reader (Tecan). Two integrated custom-designed heated and stirred chemical reaction stations (V&P Scientific 710E-3HM series drum stirrers) provided reaction and mixing support. All operations were programmed in EVOware Standard software (Tecan).

[0544] 4. Synthesis and Characterization

[0545] 1 H and 13 C10 NMR was performed on a Varian 500 MHz spectrometer. MS was performed on a Voyager DE-Pro MALDI TOF. Rapid chromatography was performed on a Teledyne Isco CombiFlashh Rf-200i chromatographic system equipped with a UV-vis and evaporative light scattering detector (ELSD). Particle size and zeta potential were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS (He-Ne laser, λ = 632 nm).

[0546] 5. Nanoparticle formulations for in vivo studies

[0547] The dendritic polymer nanoparticles formulated for in vivo studies were prepared using a microfluidic mixing instrument (Precision Nanosystems NanoAssemblr) with a herringbone rapid mixing feature. As described below, an ethanol solution of dendritic polymer, DSPC, cholesterol, and lipid PEG2000 was rapidly combined with an acidic solution of siRNA. The typical ratio of aqueous solution to EtOH was 3:1 (volume), and the typical flow rate was 12 mL / min.

[0548] 6. Automated in vitro delivery screening of modular biodegradable dendritic polymers

[0549] Nanoparticle (NP) formulation and in vitro screening were performed on a Tecan Freedom EVO 200 fluid handling robot equipped with an 8-channel liquid handling arm (LiHa), a multi-channel arm (MCA) with a 96-channel head, a robotic manipulator (RoMa), and an integrated InfiniTe F / M200 Pro microplate reader (Tecan).

[0550] Stable transfection of the luciferase gene was achieved using lentiviral infection, followed by clonal selection to obtain HeLa cells (HeLa-Luc) stably expressing firefly luciferase from HeLa cells (ATCC). HeLa-Luc cells were seeded (10,000 cells / well) into each well of opaque white 96-well plates (Corning) and allowed to attach overnight in DMEM supplemented with 5% FBS and free of phenol red. The medium was replaced with fresh FBS-containing medium the day before transfection.

[0551] G1DD-siLuc nanoparticles were formulated using an automated fluid handling robot to accelerate the discovery process. All operations were programmed in EVOware Standard software. First, the dendritic polymer reaction solution was diluted from its initial reaction concentration to 12.5 mM in ethanol. Next, the dendritic polymer solution was diluted a second time in ethanol from 12.5 mM to 1 mM using a LiHa arm. Then, 89.2 μL of a lipid mixture in ethanol was added to a 96-well clear plate. The lipid mixture consisted of DSPC (0.0690 mM), cholesterol (0.2622 mM), and lipid PEG2000 (0.0138 mM) in ethanol. Subsequently, 30.8 μL of each dendritic polymer (1 mM) was added to the lipid mixture in the 96-well plate via LiHa, followed by rapid mixing (15 times; 75 μL mixing volume; speed 250 μL / s). The LiHa was added and mixed to 8 tips at a time. 50 μL of siLuc (20 ng / μL) in citrate buffer (pH 4.3) was added to a second clear 96-well plate via LiHa. Then, 30 μL of an ethanol mixture (dendritic polymer, DSPC, cholesterol, lipid PEG2000) was added to the 50 μL siLuc solution, followed by rapid mixing (15 times; 75 μL mixing volume; speed 250 μL / s) to form dendritic polymer nanoparticles. Next, 120 μL of sterile PBS (1×) was added and mixed with LiHa to dilute the NP and increase the pH. The plate was then reformatted to allow for easy transfer to growing cells. Finally, 20 μL of the NP solution was added to the cultured cells using a sterile disposable needle tip through an MCA96 nozzle to avoid contamination. The cells were ultimately treated with 100 ng of siLuc (33 nM). During this screening phase, the molar ratio of dendritic polymer to siLuc was 100:1. The final composition of the formulation was G1DD:cholesterol:DSPC:lipid PEG2000 = 50:38:10:2 (in moles). Cells were incubated at 37°C and 5% CO2 for 24 hours, and then the activity and viability of firefly luciferase were analyzed using the One Glo+Tox assay kit (Promega).

[0552] 7. Dendritic polymer-small RNA formulations for in vivo studies

[0553] Dendritic polymer nanoparticles formulated for in vivo studies were prepared using a microfluidic mixing instrument (Precision Nanosystems NanoAssemblr) with a herringbone rapid mixing feature. An ethanol solution of dendritic polymer, DSPC, cholesterol, and lipid PEG2000 (molar ratio 50:38:10:2) was rapidly combined with an acidic solution of small RNA to obtain a final weight ratio of 25:1 (dendritic polymer:small RNA). The typical ratio of aqueous solution to EtOH is 3:1 (volume), and the typical flow rate is 12 mL / min. C12-200 LNPs were prepared according to a reported procedure (Love et al., 2010). An ethanol solution of C12-200, DSPC, cholesterol, and lipid PEG2000 (molar ratio 50:38.5:10:1.5) was rapidly combined with an acidic solution of small RNA to obtain a final weight ratio of 7:1 (C12-200:small RNA). All prepared NPs were purified by dialysis in sterile PBS with a 3.5 kDa cutoff and their size was measured by dynamic light scattering (DLS) prior to in vivo studies. Encapsulation of small RNAs was measured using the Ribogreen binding assay (Invitrogen) when applicable, by taking small amounts of solution and following the protocol.

[0554] 8. Animal Research

[0555] All experiments were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center and complied with applicable local, state, and federal regulations. Female C57BL / 6 mice were purchased from Harlan Laboratories (Indianapolis, IN). Transgenic mice carrying MYC-driven liver tumors were generated by crossing the TRE-MYC strain with the LAP-tTA strain. Mice carrying the LAP-tTA and TRE-MYC genotypes were fed 1 mg / mL of dox, and MYC was induced by dox withdrawal. Power analysis was performed to predict the number of animals required to achieve statistical significance.

[0556] 9. Silencing of factor VII in mice

[0557] For in vivo delivery screening, female C57BL / 6 mice received intravenous tail injections of either PBS (negative control, n=3) or dendritic polymer NPs containing non-targeting siRNA (siCTR, negative control, n=3) or dendritic polymer NPs containing anti-factor VII siRNA (siFVII, n=3) diluted in PBS (total volume less than 200 μL). Forty-eight hours later, weight gain / loss was measured, and mice were anesthetized by isoflurane inhalation to collect blood samples via retro-orbital bleeding. Serum was separated using serum separation tubes (Becton Dickinson), and factor VII protein levels were analyzed by a colorimetric assay (Biophen FVII, Aniara Corporation). Standard curves were constructed using samples from PBS-injected mice, and relative factor VII expression was determined by comparing the treated groups with the untreated PBS control.

[0558] For therapeutic studies, FVII knockdown in transgenic mice was validated using the aforementioned blood assays and by qPCR of collected liver tissue. To assess statistical significance, a two-tailed Student's test was performed with a confidence level of 95%.

[0559] 10. Biological Distribution

[0560] Female C57BL / 6 mice or transgenic mice carrying liver tumors were administered 200 μL of a dendritic polymer NP containing Cy5.5-siRNA at a dose of 1 mg / kg via the tail vein. Mice were euthanized and organs removed 24 hours post-injection. Biodistribution of the entire organ was assessed by imaging with an IVIS Lumina system (Caliper Life Sciences) equipped with a Cy5.5 filter.

[0561] For confocal imaging, frozen tissue sections (7 μm) were fixed with 4% paraformaldehyde at room temperature for 10 minutes. Slides were washed three times with PBS and blocked for 30 minutes in PBS containing 1% albumin. The sections were then incubated for 30 minutes with Alexa Fluor 488 phalloidin (1:200 dilution, Life Technologies) in PBS containing 1% albumin. Slides were washed three times with 0.1% Tween 20 and fixed with ProLong Gold Antifade (Life Technologies). The sections were imaged using an LSM 700 point scanning confocal microscope (Zeiss) equipped with 25x objectives.

[0562] 11. In vivo toxicity assessment and Let-7g treatment study

[0563] Wild-type or transgenic mice carrying liver tumors were randomly assigned to different groups. Mice received an intravenous injection via tail vein into a dendritic polymer NP containing siCTR. Their weight was monitored daily. For transgenic mice carrying liver tumors, multiple tail vein injections were administered to simulate repeated dosing.

[0564] For the Let-7g treatment study, transgenic mice carrying liver tumors received weekly intravenous injections of a dendritic polymer NP containing either a Let-7g mimic or a CTR mimic, at a dose of 1 mg / kg in 200 μL PBS, from 26 to 61 days of age. Randomization was performed using the treatment order. No blinding was conducted. Body weight, abdominal size, and survival were carefully monitored. Two-tailed t-tests or Mantel-Cox tests with 95% confidence levels were performed to assess statistical significance.

[0565] Example 2: Synthesis and Characterization of PEG Lipids and Dendritic Polymers

[0566] 1. Synthesis of a library containing 1,512 first-generation biodegradable dendritic polymers (G1DD)

[0567] G1DD was synthesized via two sequential orthogonal reactions. First, amines with different initial branch centers (IBCs) were reacted with the acrylate groups of 2-(acryloyloxy)ethyl methacrylate (AEMA), wherein the molar ratio of amine to AEMA was equal to the IBC number (e.g., 2A amine: two equivalents of AEMA; six equivalents of AEMA). The reaction was carried out at 50°C with 5 mol% butylated hydroxytoluene (BHT) for 24 hours. Next, each first-step adduct was reacted with a thiol, wherein the molar ratio of thiol to adduct was equal to the amine IBC number (e.g., 2A amine first-step adduct: two equivalents of each thiol; 6A amine first-step adduct: six equivalents of each thiol). The reaction was carried out at 60°C with 5 mol% dimethylphenylphosphine (DMPP) catalyst for 48 hours. The synthesis of a library of 1,512 members was accelerated by conducting the reaction in glass vials and an aluminum reaction block. A custom-made heated and stirred chemical reaction station (V&P Scientific 710E-3HM series drum stirrer) was used.

[0568] Initial in vitro delivery screening experiments were conducted using crude G1DD. Further studies were performed using purified dendritic polymers to validate activity.

[0569] All in vivo animal experiments were performed using purified G1DD. Purified G1DD was obtained by rapid column chromatography on a neutral alumina column using a Teledyne Isco chromatographic system with gradient elution of hexane and ethyl acetate.

[0570]

[0571] Higher-generation biodegradable dendritic polymers were prepared according to the previous method (Ma et al., 2009). 1A2-G1 was directly prepared by reacting 1A2 amine with 1 equivalent of AEMA in the presence of 5 mol% BHT at 50 °C for 24 hours. 1A2-G1 (4.00 g, 11.7 mmol) was dissolved in 10 mL of DMSO. 2-Aminoethanethiol (1.37 g, 17.5 mmol) was added to the above solution, and the reaction was stirred at room temperature for 30 min. Immediately afterwards, 300 mL of dichloromethane was added to the reaction solution, and the mixture was washed with cold brine (50 mL × 3) to remove excess 2-aminoethanethiol. The organic phase was dried with magnesium sulfate and concentrated by rotary evaporation for direct use in the next step. AEMA (4.75 g, 25.8 mmol) and BHT (227 mg, 1.08 mmol) were added to the above solution. The reaction was stirred at 50 °C and monitored by 1H NMR. After the reaction was complete, the solution was repeatedly washed with 20 mL of hexane until TLC analysis showed no EAMA. The washed solution was dried under vacuum to obtain a viscous liquid 1A3-G2, which was directly used in the next step. Following the above two-step synthetic procedure, 1A3-G2 was reacted to obtain a viscous liquid 1A3-G3, which was directly used in the next step. After dissolving 0.5 g (0.3 mmol) of 1A2-G3 in 0.5 mL of DMSO, 1-octylthiol (216 μL, 1.22 mmol) and dimethylphenylphosphine (DMPP) (8.6 μL, 0.061 mmol) were added. The reaction was stirred at 60 °C for 48 hours, and then purified by operating a neutral alumina column with a gradient elution of hexane and ethyl acetate. A pale yellow viscous liquid 1A2-G3-SC8 was obtained.

[0572] 3. Synthesis of lipid PEG2000

[0573]

[0574] PEG 44 -OH (80 g, 40 mmol) and pyridine (6.5 mL, 80 mmol) were dissolved in 250 mL of anhydrous DCM and cooled at 0 °C. 50 mL of DCM containing methanesulfonyl chloride (15.5 mL, 200 mmol) was added over 30 minutes, and the mixture was stirred overnight at room temperature. Another 100 mL of DCM was added, and the organic phase was washed with saturated NaHCO3 solution (50 mL × 3), followed by washing with brine (50 mL × 3). The resulting solution was concentrated, and the residue was recrystallized in isopropanol and dried to give a white powder PEG2000-Ms (74 g, 93%).

[0575] PEG2000-Ms (35.41 g, 17.7 mmol) was dissolved in 250 mL of DMF. Then, NaN3 (12.4 g, 19.0 mmol) was added to the solution. The reaction was stirred at 50 °C for 2 days under nitrogen atmosphere. After removing the DMF, the residue was dissolved in 300 mL of DCM and washed with brine (50 mL × 3). After removing the solvent, the residual oily substance was dissolved in 50 mL of methanol, and the product was precipitated three times with 300 mL of diethyl ether to obtain the desired compound (25.55 g, 72%) as a white powder of PEG2000-N3.

[0576] Proprynneamine (0.50 g, 9.1 mmol), BHT (191 mg, 0.91 mmol), and EAMA (2.73 g, 18.2 mmol) were added to a 25 mL reaction vial. The mixture was stirred at 50 °C for 48 hours. The reaction mixture was cooled to give a colorless oily product T3-G1, which was used for the next step of the reaction without purification.

[0577] 4. Characterization of the selected dendritic polymer

[0578]

[0579] 1 H NMR (400MHz, CDCl3, δ): 4.38-4.19 (br, 28H, -OCH2CH2O-), 2.90-2.80 (br, 7H, -C(O)CH(CH3)CH2S-), 2.75-2 .71(br, 14H, -NCH2CH2C(O)-), 2.70-2.49(br, 28H, -C(O)CH(CH3)CH2S-, -SCH2-), 2.49-2.39(br, 36H, -N(CH 3)2, -NCH2CH2N(CH2CH2)2NCH2-, -CH2N(CH2-)2), 1.57-1.48(m, 8H, -SCH2CH2CH2-), 1.37-1.28(br, 8H, SCH2 CH2CH2-), 1.28-1.16(br, 53H, -SCH2CH2(CH2)4CH3, -CHC(CH3)CH2S-), 0.85(t, J=7.1Hz, 12H, -(CH2)4CH3). 13C NMR (400MHz, CDCl3, δ): 174.92, 172.03, 62.22, 62.17, 62.13, 62.07, 49.06, 40.23, 4 0.14, 35.36, 32.68, 32.56, 31.76, 29.60, 29.14, 28.82, 22.58, 16.85, 16.81, 14.04. MS(MALDI-TOF, m / z)C 109 H 196 N6O 28 Calculated value of S7: 2261.21, experimental value: 2262.43.

[0580]

[0581] 1 H NMR (400MHz, CDCl3, δ): 4.34-4.21 (br, 16H, -OCH2CH2O-), 2.82-2.76 (m, 4H, -SCH2CH(CH3)-), 2.73 (t, J=7.1Hz, 8H, -C(O)CH2CH2N-), 2 .70-2.64(m, 4H, -SCH2CH(CH3)-), 2.58-2.51(m, 4H, -SCH2CH(CH3)-), 2.51-2.46(m, 8H, -CH2CH2S-), 2.45-2.40(m, 18H, (-C(O)CH2CH2 )2NCH2CH2CH2N(CH2-)2), 2.35-2.26(br, 4H, -CH2CH2N(CH2-)2), 1.65-1.58(br, 4H, -NCH2CH2CH2N-), 1.57-1.49(m, 8H, -SCH2CH2CH2- ), 1.37-1.28(br, 8H, -SCH2CH2CH2-), 1.28-1.16 (br, 44H, -SCH2CH2(CH2)4CH3, -CHC(CH3)CH2S-), 0.85(t, J=7.0Hz, 12H, -(CH2)4CH3). 13 CNMR (400MHz, CDCl3, δ): 174.90, 172.18, 62.18, 62.05, 49.05, 40.14, 35.37, 32.68, 32.40, 31.76, 29.60, 29.15, 28.83, 22.60, 16.81, 14.08. MS(MALDI-TOF, m / z)C 78 H 144 N4O 16 Calculated value of S4: 1520.95, experimental value: 1521.32.

[0582]

[0583] 1 1H NMR (400 MHz, CDCl3, δ): 4.32 - 4.21 (br, 16H, -OCH2CH2O-), 2.82 - 2.76 (m, 4H, -SCH2CH(CH3)-), 2.73 (t, J = 7.0 Hz, 8H, -C(O)CH2CH2N-), 2.69 - 2.62 (m, 4H, -SCH2CH(CH3)-), 2.58 - 2.50 (m, 4H, -SCH2CH(CH3)-), 2.50 - 2.45 (m, 8H, -CH2CH2S-), 2.45 - 2.38 (m, 12H, (-C(O)CH2CH2)2NCH2-), 2.34 - 2.24 (br, 4H, -CH2N(CH3)CH2-), 2.24 - 2.00 (br, 3H, -CH2N(CH3)CH2-), 1.66 - 1.57 (br, 4H, -NCH2CH2CH2N-), 1.57 - 1.48 (m, 8H, -SCH2CH2CH2-), 1.37 - 1.28 (br, 8H, -SCH2CH2CH2-), 1.28 - 1.16 (br, 45H, -SCH2CH2(CH2)4CH3, -CHC(CH3)CH2S-), 0.85 (t, J = 7.0 Hz, 12H, -(CH2)4CH3). 13 13C NMR (400 MHz, CDCl3, δ): 174.90, 172.18, 62.18, 62.05, 49.00, 40.13, 35.36, 32.68, 32.35, 31.76, 29.60, 29.15, 28.83, 22.60, 16.81, 14.04. MS (MALDI-TOF, m / z) C 75 H 139 N3O 16 Calculated for C

[0584]

[0585] 1H NMR (400MHz, CDCl3, δ): 4.34-4.20 (br, 20H, -OCH2CH2O-), 2.82-2.76 (m, 5H, -SCH2CH(CH3)-), 2.75-2.70 (br, 10H, -C (O)CH2CH2N-), 2.69-2.62(m, 5H, -SCH2CH(CH3)-), 2.60-2.52(m, 5H, -SCH2CH(CH3)-), 2.52-2.49(m, 10H, -CH2CH2S-) , 2.49-2.45(br, 16H, -NCH2CH2N-), 2.45-2.40(br, 10H, -CH2N-), 1.57-1.48(br, 10H, -SCH2CH2CH2-), 1.37-1.28(br , 10H, -SCH2CH2CH2-), 1.28-1.16(br, 55H, -SCH2CH2(CH2)4CH3, -CHC(CH3)CH2S-), 0.87-0.79(br, 15H, -(CH2)4(H3). 13 C NMR (400MHz, CDCl3, δ): 174.93, 172.13, 62.28, 62.01, 49.04, 40.13, 35.36, 32.68, 32.35, 31.76, 29.60, 29.15, 28.83, 22.59, 16.82, 14.05. MS(MALDI-TOF, m / z)C 93 H 173 N5O 20 The calculated value for S5 is 1840.13, and the experimental value is 1841.37. 5A2-SC8 has also been prepared with 6 arms (structure shown below).

[0586]

[0587]

[0588] 1H NMR (400MHz, CDCl3, δ): 4.32-4.21(br, 20H, -OCH2CH2O-), 2.82-2.76(m, 5H, -SCH2CH(CH3)-), 2.76-2.70(br, 10H, -C(O)CH 2CH2N-), 2.69-2.62(m, 5H, -SCH2CH(CH3)-), 2.58-2.50(m, 5H, -SCH2CH(CH3)-), 2.50-2.45(m, 10H, -CH2CH2S-), 2.45-2.20 (br, 20H, (-(CH2)2NCH2-, -CH2NHCH2-), 1.66-1.57 (br, 6H, -NCH2CH2CH2N-), 1.57-1.48 (br, 10H, -SCH2CH2CH2-), 1.37-1. 28(br, 10H, -SCH2CH2CH2-), 1.28-1.16 (br, 55H, -SCH2CH2(CH2)4CH3, -CHC(CH3)CH2S-), 0.82-0.75(br, 15H, -(CH2)4CH3). 13 C NMR (400MHz, CDCl3, δ): 174.98, 172.13, 62.28, 62.01, 49.04, 40.13, 35.36, 32.68, 32.35, 31.76, 29.60, 29.15, 28.83, 22.61, 16.85, 14.14. MS(MALDI-TOF, m / z)C 94 H 174 N4O 20 Calculated value of S5: 1839.13, experimental value: 1838.97.

[0589]

[0590] 1H NMR (400MHz, CDCl3, δ): 4.33-4.20(br, 24H, -OCH2CH2O-), 2.82-2.77(m, 6H, -SCH2CH(CH3)-), 2.77-2.71(br, 12H, -C( O)CH2CH2N-), 2.68-2.62(m, 6H, -SCH2CH(CH3)-), 2.60-2.52(m, 6H, -SCH2CH(CH3)-), 2.52-2.48(br, 12H, -CH2CH2S-), 2.48-2.46(br, 12H, -NCH2CH2N-), 2.45-2.40(br, 12H, (-CH2)2N-), 1.57-1.47(br, 12H, -SCH2CH2CH2-), 1.37-1.28(br , 12H, -SCH2CH2CH2-), 1.28-1.16 (br, 108H, -SCH2CH2(CH2)8CH3, -CHC(CH3)CH2S-), 0.87-0.80(br, 18H, -(CH2)8CH3). 13 C NMR (400MHz, CDCl3, δ): 174.87, 172.07, 62.16, 62.04, 49.48, 40.47, 40.11, 35.34, 32.6 9, 32.42, 31.86, 29.61, 29.58, 29.57, 29.50, 29.29, 29.21, 28.85, 22.62, 16.81, 14.06. MS(MALDI-TOF, m / z)C 132 H 246 N4O 24 Calculated value of S6: 2463.65, experimental value: 2464.52.

[0591] Example 3: Library design and synthesis of first-generation biodegradable dendritic polymer (G1DD)

[0592] Hepatocellular carcinoma is a challenging host for therapeutic interventions because drug-induced hepatotoxicity can exacerbate underlying liver disease (Boyerinas et al., 2010). To achieve effective RNAi-mediated therapy, a balance between high efficacy and low toxicity of the delivery vector must be maintained. This requires a universal strategy to easily modulate the delivery vector in terms of size, chemical structure, and final physical properties. Figure 1AIn some embodiments, the dendritic polymer is designed to exhibit one or more of the following characteristics: optimal monodisperse material for chemical and size manipulation (Wu et al., 2004; Carlmark et al., 2009; Killops et al., 2008; Ma et al., 2009; Franc and Kakkar, 2010). First-generation degradable dendritic polymers (G1DDs) are diversified by sequentially reacting with 2-(acryloyloxy)ethyl methacrylate (AEMA) using orthogonal reactions, with various parameters including: core (C), bonded or repeating unit (L), and peripheral or capped groups (P). Figure 1B In some implementations, esters are chosen as the starting degradable bond because polyesters are used in FDA-approved products and have minimal toxicity. The ester value increases with each growth step, providing an opportunity to identify degradable dendritic polymers with a balanced potency and toxicity.

[0593] Previous results have shown that these orthogonal reactions can construct polyester dendritic polymers with a series of generations (Ma et al., 2009). However, prior to utilizing this strategy, it had been demonstrated that this method could generate diverse dendritic polymers using a variety of chemically different amines and thiols without purification. To investigate the robustness of this chemistry, the structural limitations of the orthogonal Michael addition reaction were tested using the most challenging starting materials: tri(2-aminoethyl)amine with six NH bonds as the initial branching center (IBC) and tetradecylamine with a 14-carbon alkyl chain. After 24 hours at 50 °C in the presence of 5 mol% butylated hydroxytoluene (BHT) (to suppress free radical formation), both tri(2-aminoethyl)amine and tetradecylamine reacted quantitatively and selectively with the acrylate functional groups in AEMA, while AEMA itself remained unreacted under these conditions. Figure 2 and Figure 3 In the second orthogonal reaction (thiamiker addition), dimethylphenylphosphine is required as a catalyst to achieve the final product under low concentration (minimum 125 mM) or small-scale (average about 20 mg) conditions and to achieve high conversion (according to...). 1 The H NMR is 100%, so that the material can be used for subsequent testing or generation amplification without purification. Figure 4 and Figure 5 Some dendritic polymers were resynthesized on a larger scale and purified by rapid chromatography before being studied in vivo.

[0594] Due to multiple delivery barriers, the effectiveness of small RNA vectors via nanoencapsulation is affected by various factors, including pK. aTopology / structure and hydrophobicity (Siegwart et al., 2011; Jayaraman et al., 2012; Schaffert et al., 2011; Whitehead et al., 2014). To facilitate the identification of degradable dendritic polymers with high delivery efficiency, a G1DD library was designed with four regions: core-binding-peripheral stabilization (region I), core-binding-peripheral binding (region II), core-stabilization-peripheral stabilization (region III), and core-stabilization-peripheral binding (region IV). Figure 1C and Figure 1D In regions I and II, RNA binding is regulated by amines having one (1An) to six (6An) initial branching centers (IBCs). Therefore, the corresponding dendritic polymers contain one to six branches. In regions III and IV, the stabilization of the RNA-dendritic polymer NP varies primarily with different alkyl chain lengths (1Hn and 2Hn). In regions II and IV, the binding capacity of aminothiols (SNn) is primarily regulated by different amines, while in regions I and III, stabilization varies with alkylthiol (SCn) length and carboxyl- and hydroxy-alkylthiols (SOn). The efficacy of the dendritic polymers throughout the compound library was tested (Figure 6).

[0595] Example 4: In vitro G1DD screening via intracellular siRNA delivery

[0596] Delivery vectors must overcome a series of extracellular and intracellular barriers to enable small RNAs to be active within tumor cells. G1DD was identified as a medium capable of mediating siRNA overcoming intracellular barriers by screening a 1,512-member G1DD library for in vitro delivery of siRNA to HeLa cells stably expressing luciferase. G1DD was formulated into nanoparticles (NPs) containing luciferase-targeting siRNA (siLuc) and cofactor lipids cholesterol, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), and lipid PEG2000 (Akinc et al., 2008; Semple et al., 2010). Intracellular delivery potential was assessed by quantifying luciferase reduction and cell viability (Figures 7–9).

[0597] To extract SAR from in vitro data, we utilize a tree analysis process inspired by dendritic polymers. Figure 7B and Figure 9BOf the 1,512 dendritic polymers, 88 mediated >50% luciferase silencing, and the overall library hit rate was 6%. When analyzing the hit rates for all four regions (I-IV), the hit rate for region I was 10%, while the hit rates for regions II, III, and IV were 0%, 2%, and 3%, respectively. This result indicates that these dendritic polymers with siRNA-binding cores and stabilizing peripheries (region I) have a much higher potential for intracellular siRNA delivery. Among the branching types in region I, dendritic polymers with SO peripheries had hit rates as low as 1%, while those with SC peripheries had hit rates as high as 15%. Without being bound by any theory, it is believed that hydrophobic stabilization from the dendritic polymer periphery is crucial for the efficient delivery of siRNA into cells via nanoencapsulation. This may lead to increased hydrophobic packaging, thus providing additional NP stability (Leung et al., 2012). After further investigation into the number and length of branches of these dendritic polymers with binding nuclei and SC periphery, dendritic polymers with binding nuclei and three, four, five, or six SC5-8 or SC9-12 branches had a >25% chance of delivering siLuc to HeLa cells, with luciferase knockdown >50%. Through in vitro screening of complete G1DD libraries and dendritic analysis, groups of dendritic polymers exhibiting increased intracellular siRNA delivery were identified: groups with binding nuclei / SC periphery and groups with binding nuclei and 3-6 SC5-8 or SC9-12 branches.

[0598] Example 5: Identification of degradable dendritic polymers for effective in vivo siRNA delivery and design of G2-G4 dendritic polymers

[0599] Dendritic polymers capable of overcoming intracellular barriers have been identified, followed by those capable of overcoming extracellular barriers to efficiently deliver siRNA in vivo. By separating these two processes, the chemical functions of barrier overcoming can be identified, including blood stability, liver (tumor) localization, cellular uptake, and release of active siRNA. The ability of dendritic polymers to silence factor VII in hepatocytes was evaluated, as this clotting factor can be readily quantified from small serum samples (Akinc et al., 2008; Semple et al., 2010). Twenty-six of the target degradable dendritic polymers were selected to maximize chemical diversity: 22 were selected based on their optimized chemical structures from the dendritic analysis process, while the other four (2A2-SC14, 2A6-SC14, 2A9-SC14, and 6A1-SO9) were selected based on their high intracellular siRNA retrograde ability. Dendritic polymers were formulated with anti-factor VII siRNA (siFVII) and administered intravenously to mice at a dose of 1 mg siFVII / kg. FVII activity was quantified 3 days post-injection. Despite high in vitro potency, 2A2-SC14, 2A6-SC14, 2A9-SC14, 6A1-SO9, and most tribranched dendritic polymers showed only minimal in vivo FVII knockdown. Figure 10A Dendritic polymers containing a binding core and four, five, or six SC8 or SC12 branches exhibit higher knockdown rates. Based on these studies, SC8-branched dendritic polymers are generally more effective than SC12-branched compounds.

[0600] Based on in vitro and in vivo high-throughput screening results, we inquired whether SAR information could now be used to rationally design dendritic polymers with predicted activity to validate our method. A series of degradable dendritic polymers were prepared using two strategies: (I) by selecting polyamines with five or six IBCs; and (II) by increasing branching through dendritic polymer generation (Figure 10). Two natural amines, spermidine (5 IBCs) and spermine (6 IBCs), were selected for strategy I. For strategy II, 1A2 (one IBC), 2A2 and 2A11 (two IBCs), 3A3 and 3A5 (three IBCs), and 4A1 and 4A3 (four IBCs) were selected to generate degradable dendritic polymers with multiple branches through generation. Figure 10C and Figure 11 An evaluation was conducted on another 24 biodegradable dendritic polymers. Figure 10CTo further investigate in vivo SAR, after amplification, higher-generation dendritic polymers with four or six SC branches (1A2 (one IBC), 2A2 (two IBCs), and 3A3 and 3A5 (three IBCs)) exhibited good in vivo siRNA delivery to hepatocytes, while dendritic polymers with eight branches showed lower activity. This process transformed inactive amine nuclei in in vitro screening, and then rationally designed higher-generation dendritic polymers exhibiting in vivo activity.

[0601] Example 6: In vivo toxicity assessment of degradable dendritic polymers in mice carrying MYC-driven liver tumors

[0602] To identify degradable dendritic polymers that offer the desired balance of low toxicity and high potency for liver cancer treatment, degradable dendritic polymers were selected to assess their in vivo toxicity. In parallel, we analyzed C12-200 lipid LNPs, selecting them as the best example of a non-hydrolyzed system previously demonstrated to be potent in mice and non-human primates (Love et al., 2010). Lipids, as a class, are benchmark materials at the forefront of clinical research (Kanasty et al., 2013; Love et al., 2010; Sahay et al., 2013). A non-immunogenic control siRNA was used to best assess the toxicity of the individual dendritic polymers themselves. The dendritic polymer NPs were formulated at a weight ratio of 25:1 (dendritic polymer:siCTR), higher than the amount required for better probing of toxicity. C12-200 LNPs were prepared using the same formulation parameters as previously reported (Love et al., 2010). The size and zeta potential of each NP in PBS buffer were characterized. They all had similar sizes, with diameters of 64–80 nm, and their surface charges were nearly neutral (…). Figure 12A and Figure 12B Each formulated NP was administered intravenously to wild-type mice at a dose of 4 mg siCTR / kg (100 mg dendritic polymer / kg or 28 mg C12-200 / kg). Among the many different ways to assess in vivo toxicity, weight loss can be used as a simple informative parameter. In normal mice, the selected NPs (including the C12-200 control LNP) showed the least weight change. However, among the candidates, mice injected with 5A2-SC8 and 6A3-SC12 experienced faster recovery and normal weight gain after day one.

[0603] Based on these results, 5A2-SC8 and 6A3-SC12 were selected to further evaluate their in vivo toxicity in chronically ill transgenic mice with aggressive liver tumors, including single and multiple injections. A well-established Tet-On MYC-induced transgenic liver cancer model was selected. Figure 13A(Nguyen et al., 2014). Since tumors are more aggressive when MYC is overexpressed at early developmental time points, MYC is induced immediately after birth (p0), leading to rapidly growing liver tumors. At 32 days of age (p32), these morbid transgenic mice carrying aggressive liver tumors were injected with 3 mg siCTR / kg (75 mg dendritic polymer / kg or 21 mg C12-200 / kg) of 5A2-SC8 or 6A3-SC12 NP. Mice receiving 5A2-SC8 injections lost approximately 5% of their body weight on day 1 and rapidly recovered to their starting weight on day 2, while those receiving 6A3-SC12 injections still had lost 10% of their body weight by day 3 and could not recover. Figure 12D Following multiple injections, these mice died 7 days earlier than untreated mice due to the toxicity of the 6A3-SC12 vector. Figure 12E Compared to WT mice, although receiving approximately 3 times less lipids than 5A2-SC8 injected mice, mice with aggressive tumors injected with C12-200LNP experienced a >20% weight loss after one day. Figure 12D These data demonstrate that small changes in chemical structure can produce significant changes in toxicity. It also shows that tumor-bearing mice are more sensitive to intervention than healthy mice. Based on these results, 5A2-SC8 emerged as a degradable dendritic polymer that balances low toxicity (tolerable up to 75 mg / kg in tumor-bearing mice) with effective in vivo FVII knockdown (>95% at 1 mg siFVII / kg). In addition to being less toxic than the benchmark compound, 5A2-SC8 NP is more effective because these dendritic polymers reduce clinical concerns about dose-limiting toxicities and allow for a wider therapeutic window.

[0604] Example 7: Systemic administration of Let-7g miRNA mimic effectively inhibits liver tumor growth.

[0605] To evaluate the ability of the degradable dendritic polymer NP to deliver therapeutic miRNA mimics without inducing additional toxicity, the p0-induced aggressive MYC transgenic hepatocellular carcinoma model was again used (Nguyen et al., 2014). These mice developed rapidly growing cancer similar to pediatric hepatoblastoma (HB), a tumor type sharing many molecular characteristics with HCC. After 20 days, abdominal distension was clearly visible from the mass effect, and the tumor grew rapidly. Without intervention, the mice died within 60 days of birth. Given the speed and lethality of this model, the chances of successful treatment are limited.

[0606] Because 5A2-SC8 balances low in vivo toxicity with its effectiveness in silencing hepatocyte target FVII, the study first investigated whether 5A2-SC8 NP could deliver siRNA to tumor cells. At 41 days of age (p41), the livers of these transgenic mice were filled with tumors. Figure 14A At p40, mice were intravenously injected with 5A2-SC8 NPs containing Cy5.5-labeled siRNA at a dose of 1 mg / kg. Twenty-four hours post-injection, fluorescence imaging showed that 5A2-SC8-mediated siRNA accumulation was observed in the cancerous liver, while only small amounts accumulated in the spleen and kidneys. Figures 14A-14B 5A2-SC8 NP delivers siRNA to both normal and transgenic livers, even in cancerous livers larger than normal livers. Figure 15A ).

[0607] To further confirm whether 5A2-SC8 NP can deliver siRNA into tumor cells in vivo, liver tumor tissue was collected and imaged 24 hours after intravenous injection. H&E staining showed that the tumor tissue contained numerous cell nuclei and exhibited a cancerous phenotype. Figure 15B Confocal imaging confirmed that 5A2-SC8 NP can effectively deliver labeled siRNA into tumor cells. Figure 14C ).

[0608] The therapeutic benefits of 5A2-SC8-mediated small RNA delivery in these chronically diseased transgenic mice were then evaluated. One of the most important miRNAs is Let-7, a family of tumor suppressor proteins downregulated in many tumor types (Boyerinas et al., 2010; Roush and Slack, 2008). Because endogenous Let-7g is known to be downregulated in liver hepatocellular carcinoma (HB) (Nguyen et al., 2014), tests were conducted to determine whether delivery of Let-7g mimics in this aggressive genetically engineered mouse model could inhibit the development of liver cancer.

[0609] The 5A2-SC8 NP was confirmed to deliver siRNA in this model. Blood assays were used in collected liver tissue samples. Figure 16A ) and via qPCR, intravenous delivery of a single dose of siFVII showed effective silencing of the FVII protein. Figure 16B This silencing was achieved at p26, after the onset of tumor development. Next, 1 mg / kg Let-7g was intravenously delivered to tumor-bearing mice (p26) via 5A2-SC8 NP. Forty-eight hours post-injection, Let-7g expression in liver tissue increased sevenfold. Figure 16C ).

[0610] Then, the treatment regimen began at p26, which involved weekly administration of 5A2-SC8 NP containing either Let-7g mimicry or control mimicry at a dose of 1 mg / kg. At p50, mice receiving Let-7g mimicry had significantly smaller abdomens and reduced tumor burden. Figure 16D-16F Let-7g causes abdominal circumference reduction and quantifies tumor growth (). Figure 16E The effect on tumor growth was confirmed by ex vivo liver imaging. Figure 16F Most importantly, weekly delivery of Let-7g from day 26 to day 61 did not affect weight gain. Figure 16G and significantly prolong survival ( Figure 16H All untreated control mice and mice receiving 5A2-SC8 NP containing the CTR mimic died at approximately 60 days of age. C12-200LNP (Let-7g or the control mimic) caused premature death and required discontinuation of the experiment. Delivery of Let-7g within 5A2-SC8 NP provided a significant survival benefit, with one mouse surviving to 100 days. These results suggest that 5A2-SC8 can balance high delivery efficacy with low toxicity, providing significant therapeutic benefits in chronically ill transgenic mice by effectively inhibiting liver tumor growth.

[0611] Example 8: Evaluation of different lipid compositions for siRNA delivery

[0612] To evaluate which lipid compositions in dendritic polymer nanoparticles led to improved siRNA delivery, the identities and concentrations of different phospholipids and PEG-lipids were varied. Three different cell lines (HeLa-Luc, A549-Luc, and MDA-MB231-Luc) were used. Cells were present at 10K cells per well and incubated for 24 hours. Readings were determined 24 hours post-transfection. In the nanoparticles, DSPC and DOPE were used as phospholipids, and PEG-DSPE, PEG-DMG, and PEG-DHD were used as PEG-lipids. The compositions contained lipids or dendritic polymers:cholesterols:phospholipids:PEG-lipids in a molar ratio of 50:38:10:2. The molar ratio of lipids / dendritic polymers to siRNA was 100:1, with a dose of 100 ng used. The efficacy of these compositions was determined using RiboGreen, Cell-titer Fluor, and OneGlo assays. Results showed that HeLa-Luc cells ( Figure 17A ), A549-Luc Figure 17B ) and MDA-MB231-Luc ( Figure 17CThe relative luciferase activity in the samples was determined. The six formulations used in the study included: dendritic polymer (lipid) + cholesterol + DSPC + PEG-DSPE (formulation 1), dendritic polymer (lipid) + cholesterol + DOPE + PEG-DSPE (formulation 2), dendritic polymer (lipid) + cholesterol + DSPC + PEG-DMG (formulation 3), dendritic polymer (lipid) + cholesterol + DOPE + PEG-DMG (formulation 4), resinous polymer (lipid) + cholesterol + DSPC + PEG-DSPE (formulation 5), and dendritic polymer (lipid) + cholesterol + DOPE + PEG-DHD (formulation 6).

[0613] Further experiments were conducted to determine which phospholipids showed increased siRNA molecule delivery. Using the HeLa-Luc cell line, 10K cells per well were incubated for 24 hours and readouts were performed 24 hours post-transfection. The compositions contained DOPE or DOPC as phospholipids and PEG-DHD as a PEG-lipid. The molar ratio of lipid (or dendritic polymer):cholesterol:phospholipid:PEG-lipid was 50:38:10:2, and the molar ratio of resinous polymer (or lipid) to siRNA was 200:1. These compositions were tested at 50 ng doses using the Cell-titerFluor and OneGlo assays. These results are presented in… Figure 18A and Figure 18B middle.

[0614] Example 9: Evaluation of dendritic polymer nanoparticles for delivering sgRNA and other CRISPR nucleic acids

[0615] To evaluate the nucleic acid delivery composition for CRISPR / Cas gene editing, the delivery of sgRNA and mRNA was tested. Cell lines that allow for rapid screening of dendritic polymers NP and Z120 for sgRNA delivery were established. For example, HeLa (cervical cancer) and A549 (lung cancer) cells were established to co-express luciferase and Cas9. Selection and quality control were validated. Guide RNAs were designed according to previously reported methods targeting the first exon of the desired target gene. Targets with the highest scores in indicative cleavage activity and sequence specificity were used for sgRNA preparation using a defined protocol. Commercially synthesized DNA oligonucleotides were annealed, cloned by BsbI digestion, and ligated into a plasmid backbone containing Cas9. In vitro transcription was able to isolate the sgRNA, which could then be packaged in dendritic polymer NPs for delivery. A series of five different guide sequences were designed for luciferase. These guide sequences were validated by sgLuc-Cas9 pDNA transfection using commercial reagents to select the optimal sgRNA sequence. Next, we packaged sgLuc into dendritic polymer NPs and evaluated sgRNA delivery in HeLa-Luc-Cas9 cells. After a defined number of hours of exposure, luciferase activity was measured compared to untreated cells using One Glo+Tox (Promega). In a typical experiment, 10K cells were seeded per well, followed by 24 hours of incubation, addition of dendritic polymer nanoparticles containing sgLuc, and readouts were performed 24–48 hours post-transfection. These compositions contained a combination of dendritic polymer, DSPC or DOPE, cholesterol, and PEG-lipids. Additionally, the compositions contained varying concentrations of MgCl2. The molar ratio of lipids (or dendritic polymer):cholesterol:PEG-lipids was 50:38.5:0.5, the molar ratio of lipids to nucleic acid (sgRNA) was 200:1, and the dose was 50 ng. Results were obtained using Cell-titer Fluor and OneGlo assays. Results under phospholipid-free conditions are shown in [Figure / Table / Insert Table ... Figure 19 Similar studies were conducted using existing phospholipids. In these compositions, phospholipid DSPC was used in the formulation. Phospholipid-containing compositions were used in the same ratio as the phospholipid-free compositions described above, with a molar ratio of 50:38.5:10:0.5 (lipid / dendritic polymer: cholesterol: phospholipid: PEG-lipid), and the same dosage was used. These compositions were tested using RiboGreen, Cell-titerFluor, and OneGlo at two time points (24 hours and 72 hours). Data obtained at 24 hours showed that... Figure 20A In the middle, and the data obtained within 72 hours showed that Figure 20B middle.

[0616] Example 10: Evaluation of dendritic polymer nanoparticles for mRNA delivery

[0617] Similar to studies on siRNA, mRNA molecule delivery was tested using the dendritic polymer and Z120 described herein. IGR.OV1 cell lines were used at a concentration of 4K cells per well, incubated for 24 hours, and readouts were obtained at 24 and 48 hours post-transfection. These compositions contained DSPC, DOPE, or phospholipid-free and PEG-DHD as PEG-lipids. The molar ratio of lipid (or dendritic polymer):cholesterol:phospholipid:PEG-lipid was 50:38.5:0(10):2, the weight ratio of dendritic polymer to nucleic acid (mRNA) was 5:1, 10:1, 20:1, 30:1, or 40:1, and two different dosages: 50 ng and 100 ng. Results were obtained using the Cell-titer Fluor and OneGlo assays. These results are shown in Figure 21A (24 hours) and Figure 21B (48 hours). Furthermore, a nanoparticle composition using N / P and DSPC as phospholipids and PEG-DHD as a PEG-lipid in a 20:1 ratio was employed. Figure 22 The image shows the delivery of mCherry mRNA.

[0618] ************************************

[0619] Based on this disclosure, all compositions and methods disclosed and claimed herein can be performed and carried out without excessive experimentation. Although the compositions and methods of the present invention have been described according to certain embodiments, it will be apparent to those skilled in the art that variations can be made to the compositions and methods, and the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted for the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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[0856] This application also relates to the following technical solutions:

[0857] 1. A dendritic polymer of the following formula:

[0858] Core - (repeating unit) n -Terminal group (I)

[0859] The nucleus is connected to the repeating unit by removing one or more ammonia atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0860] The core has the following formula:

[0861]

[0862] in:

[0863] X1 is an amino or alkylamino group. (C≤12) Dialkylamino (C≤12) heterocyclic alkyl (C≤12) , heteroaryl (C≤12) or its alternative forms;

[0864] R1 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) or a substituted form of any of these groups; and

[0865] a is 1, 2, 3, 4, 5, or 6; or

[0866] The core has the following formula:

[0867]

[0868] in:

[0869] X2 is N(R5) y ;

[0870] R5 is hydrogen or alkyl (C≤18) Or substituted alkyl (C≤18) ;and

[0871] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0872] R2 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[0873] b is 1, 2, 3, 4, 5, or 6; and

[0874] z is 1, 2, or 3; the condition is that the sum of z and y is 3; or

[0875] The core has the following formula:

[0876]

[0877] in:

[0878] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) heterocyclic alkane dimethyl (C≤8) , or a substituted form of any of these groups;

[0879] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups; or a group of the following formula: -(CH2CH2N) e (R c )R d ;

[0880] in:

[0881] e is 1, 2, or 3;

[0882] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0883] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[0884] The core is an alkylamine. (C≤18） dialkylamine (C≤36) Heterocyclic alkanes (C≤12) , or a substituted form of any of these groups;

[0885] The repeating unit comprises a degradable diacyl group and a linker;

[0886] The degradable diacyl group has the formula:

[0887]

[0888] in:

[0889] A1 and A2 are independently -O- or -NR. a -,in:

[0890] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0891] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0892]

[0893] in:

[0894] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0895] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0896] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0897] The connecting group has the following formula:

[0898]

[0899] in:

[0900] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0901] When the repeating unit contains a linker group, the linker group is connected to degradable diacyl groups on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0902] The terminating group has the following formula:

[0903]

[0904] in:

[0905] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0906] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0907] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0908] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0909] The final degradable diacyl group in the chain is attached to a capping group;

[0910] n is 0, 1, 2, 3, 4, 5, or 6;

[0911] Or its pharmaceutically acceptable salt.

[0912] 2. The dendritic polymer according to technical solution 1, wherein:

[0913] The core has the following formula:

[0914]

[0915] in:

[0916] X1 is an amino or alkylamino group. (C≤12) Dialkylamino (C≤12) heterocyclic alkyl (C≤12) , heteroaryl (C≤12) or its alternative forms;

[0917] R1 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) or a substituted form of any of these groups; and

[0918] a is 1, 2, 3, 4, 5, or 6; and

[0919] The repeating unit comprises a degradable diacyl group and a linker;

[0920] The degradable diacyl group has the formula:

[0921]

[0922] in:

[0923] A1 and A2 are independently -O- or -NR. a -,in:

[0924] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0925] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0926]

[0927] in:

[0928] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0929] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0930] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0931] The connecting group has the following formula:

[0932]

[0933] in:

[0934] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0935] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0936] End-capping groups, wherein the end-capping groups have the formula:

[0937]

[0938] in:

[0939] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0940] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0941] Aryl (C≤12)alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0942] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0943] The final degradable diacyl group in the chain is attached to a capping group;

[0944] n is 0, 1, 2, 3, 4, 5, or 6;

[0945] Or its pharmaceutically acceptable salt.

[0946] 3. The dendritic polymer according to technical solution 1, wherein the dendritic polymer has the formula:

[0947] Core - (repeating unit) n -Terminal group (I)

[0948] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0949] The core has the following formula:

[0950]

[0951] in:

[0952] X2 is N(R5) y ;

[0953] R5 is hydrogen or alkyl. (C≤8) , or substituted alkyl (C≤18) ;and

[0954] y is 0, 1, or 2, provided that the sum of y and z is 3;

[0955] R2 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[0956] b is 1, 2, 3, 4, 5, or 6; and

[0957] z is 1, 2, 3; the condition is that the sum of z and y is 3;

[0958] The repeating unit comprises a degradable diacyl group and a linker;

[0959] The degradable diacyl group has the formula:

[0960]

[0961] in:

[0962] A1 and A2 are each independently -O- or -NRa-, where:

[0963] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0964] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[0965]

[0966] in:

[0967] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[0968] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0969] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[0970] The connecting group has the following formula:

[0971]

[0972] in:

[0973] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[0974] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[0975] End-capping groups, wherein the end-capping groups have the formula:

[0976]

[0977] in:

[0978] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[0979] R 10 It is hydrogen, carboxyl, hydroxyl, or

[0980] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[0981] The final degradable diacyl group in the chain is attached to a capping group;

[0982] n is 0, 1, 2, 3, 4, 5, or 6;

[0983] Or its pharmaceutically acceptable salt.

[0984] 4. The dendritic polymer according to technical solution 1, wherein the dendritic polymer has the formula:

[0985] Core - (repeating unit) n -Terminal group (I)

[0986] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[0987] The core has the following formula:

[0988]

[0989] in:

[0990] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) heterocyclic alkane dimethyl (C≤8) , or a substituted form of any of these groups;

[0991] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups; or a group of the following formula: -(CH2CH2N) e (R c )R d ;

[0992] in:

[0993] e is 1, 2, or 3;

[0994] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[0995] c and d are each independently 1, 2, 3, 4, 5, or 6; and

[0996] The repeating unit comprises a degradable diacyl group and a linker;

[0997] The degradable diacyl group has the formula:

[0998]

[0999] in:

[1000] A1 and A2 are independently -O- or -NR. a -,in:

[1001] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl(C≤6) ;

[1002] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[1003]

[1004] in:

[1005] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[1006] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[1007] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[1008] The connecting group has the following formula:

[1009]

[1010] in:

[1011] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[1012] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[1013] End-capping groups, wherein the end-capping groups have the formula:

[1014]

[1015] in:

[1016] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl(C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[1017] R 10 It is hydrogen, carboxyl, hydroxyl, or

[1018] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[1019] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[1020] The final degradable diacyl group in the chain is attached to a capping group;

[1021] n is 0, 1, 2, 3, 4, 5, or 6;

[1022] Or its pharmaceutically acceptable salt.

[1023] 5. The dendritic polymer according to technical solution 1, wherein the dendritic polymer has the formula:

[1024] Core - (repeating unit) n -Terminal group (I)

[1025] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[1026] The core is an alkylamine. (C≤18) dialkylamine (C≤36) Heterocyclic alkanes (C≤12) or a substituted form of any of these groups; and

[1027] The repeating unit comprises a degradable diacyl group and a linker;

[1028] The degradable diacyl group has the formula:

[1029]

[1030] in:

[1031] A1 and A2 are independently -O- or -NR. a -,in:

[1032] Ra is hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[1033] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups; or a group of the following formula:

[1034]

[1035] in:

[1036] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[1037] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[1038] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[1039] The connecting group has the following formula:

[1040]

[1041] in:

[1042] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[1043] Wherein, when the repeating unit contains a linker group, the linker group is connected to a degradable diacyl group on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[1044] End-capping groups, wherein the end-capping groups have the formula:

[1045]

[1046] in:

[1047] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤18) Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;

[1048] R 10 It is hydrogen, carboxyl, hydroxyl, or

[1049] Aryl (C≤12) alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) -C(O)N(R) 11 )-Alkyl (C≤6) - Heterocyclic alkyl (C≤12) -C(O)-alkylamino (C≤12) -C(O)-dialkylamino (C≤12) -C(O)-N-heterocyclic alkyl (C≤12) ,in:

[1050] R 11 It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[1051] The final degradable diacyl group in the chain is attached to a capping group;

[1052] n is 0, 1, 2, 3, 4, 5, or 6;

[1053] Or its pharmaceutically acceptable salt.

[1054] 6. The dendritic polymer according to any one of technical solutions 1 to 5, wherein the end-capping group is further defined by the following formula:

[1055]

[1056] in:

[1057] Y4 is an alkane dimethyl group. (C≤18) Or alkane dimethyl (C≤C18)Alkyl groups in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3, or -OC(O)CH3 (C≤18) ;and

[1058] R 10 It is hydrogen.

[1059] 7. The dendritic polymer according to any one of technical solutions 1 to 6, wherein the end-capping group is further defined by the following formula:

[1060]

[1061] in:

[1062] Y4 is an alkane dimethyl group. (C≤18) ;and

[1063] R 10 It is hydrogen.

[1064] 8. The dendritic polymer according to any one of technical solutions 1 to 7, wherein Y4 is an alkane dimethyl group. (C4-18) .

[1065] 9. The dendritic polymer according to any one of technical solutions 1 to 5, wherein the end-capping group is further defined by the following formula:

[1066]

[1067] in:

[1068] Y4 is an alkane dimethyl group. (C≤18) Or an alkane dimethyl group in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3 (C≤8) ;

[1069] R 10 It is an alkylamino (C≤12) Dialkylamino (C≤12) N-heterocyclic alkyl (C≤12) .

[1070] 10. The dendritic polymer according to any one of technical solutions 1 to 5, wherein the end-capping group is further defined by the following formula:

[1071]

[1072] in:

[1073] Y4 is an alkane dimethyl group. (C≤18)Or an alkane dimethyl group in which one or more hydrogen atoms have been replaced by -OH, -F, -Cl, -Br, -I, -SH, -OCH3, -OCH2CH3, -SCH3 or -OC(O)CH3 (C≤18) ;

[1074] R 10 It is a hydroxyl group.

[1075] 11. The dendritic polymer according to any one of technical solutions 1 to 2 and 6 to 10, wherein the core is further defined by the following formula:

[1076]

[1077] in:

[1078] X1 is an alkylamino group. (C≤12) Dialkylamino (C≤12) heterocyclic alkyl (C≤12) , heteroaryl (C≤12) or its alternative forms;

[1079] R1 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) or a substituted form of any of these groups; and

[1080] a is 1, 2, 3, 4, 5 or 6.

[1081] 12. The dendritic polymer according to technical solution 11, wherein X1 is a dialkylamino group. (C≤12) Or substituted dialkylamino (C≤12) .

[1082] 13. The dendritic polymer according to technical solution 11, wherein X1 is a heterocyclic alkyl group. (C≤12) Or substituted heterocyclic alkyl (C≤12) .

[1083] 14. The dendritic polymer according to technical solution 13, wherein X1 is 4-piperidinyl, N-piperidinyl, N-morpholinyl, N-pyrrolidinyl, 2-pyrrolidinyl, N-piperazinyl or N-4-methylpiperidinyl.

[1084] 15. The dendritic polymer according to any one of technical solutions 11 to 14, wherein R1 is an amino group.

[1085] 16. The dendritic polymer according to any one of technical solutions 11 to 15, wherein a is 1, 2, 3 or 4.

[1086] 17. The dendritic polymer according to any one of claims 11 to 16, wherein the core is further defined as a compound of the following formula:

[1087]

[1088] 18. The dendritic polymer according to technical solution 17, wherein the core is further defined as:

[1089]

[1090] 19. The dendritic polymer according to any one of claims 1, 3, and 6 to 10, wherein the core is further defined by the following formula:

[1091]

[1092] in:

[1093] X2 is N(R5) y ;

[1094] R5 is hydrogen or alkyl. (C≤8) , or substituted alkyl (C≤18) ;and

[1095] y is 0, 1, or 2, provided that the sum of y and z is 3;

[1096] R2 is an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[1097] b is 1, 2, 3, 4, 5, or 6; and

[1098] z is 1, 2, 3; the condition is that the sum of z and y is 3.

[1099] 20. The dendritic polymer according to technical solution 19, wherein X2 is N.

[1100] 21. The dendritic polymer according to technical solution 19, wherein X2 is NR5, and R5 is hydrogen or alkyl. (C≤8) .

[1101] 22. The dendritic polymer according to technical solution 21, wherein R5 is hydrogen.

[1102] 23. The dendritic polymer according to technical solution 21, wherein R5 is methyl.

[1103] 24. The dendritic polymer according to technical solution 19 or technical solution 20, wherein z is 3.

[1104] 25. The dendritic polymer according to any one of technical solutions 19 and 21 to 23, wherein z is 2.

[1105] 26. The dendritic polymer according to any one of claims 19 to 25, wherein R2 is an amino group.

[1106] 27. The dendritic polymer according to any one of claims 19 to 25, wherein R2 is an alkylamino group. (C≤12) Or substituted alkylamino (C≤12) .

[1107] 28. The dendritic polymer according to technical solution 27, wherein R2 is methylamino.

[1108] 29. The dendritic polymer according to any one of claims 19 to 25, wherein R2 is a dialkylamino group. (C≤12) Or substituted dialkylamino (C≤12) .

[1109] 30. The dendritic polymer according to technical solution 29, wherein R2 is dimethylamino.

[1110] 31. The dendritic polymer according to any one of technical solutions 19 to 30, wherein b is 1, 2, 3 or 4.

[1111] 32. The dendritic polymer according to any one of technical solutions 19 to 31, wherein the core is further defined as:

[1112]

[1113] 33. The dendritic polymer according to technical solution 32, wherein the core is further defined as:

[1114]

[1115] 34. The dendritic polymer according to any one of technical solutions 1, 4, and 6 to 10, wherein the core is further defined as:

[1116]

[1117] in:

[1118] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) heterocyclic alkane dimethyl (C≤8) , or a substituted form of any of these groups;

[1119] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups; or a group of the following formula: -(CH2CH2N) e (R c )R d ;

[1120] in:

[1121] e is 1, 2, or 3;

[1122] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) ;

[1123] c and d are each independently 1, 2, 3, 4, 5 or 6.

[1124] 35. The dendritic polymer according to technical solution 34, wherein X3 is an alkylamino diester. (C≤8) Or substituted alkylamino dimethyl (C≤8) .

[1125] 36. The dendritic polymer according to technical solution 35, wherein X3 is -NHCH2CH2NH- or -NHCH2CH2NHCH2CH2NH-.

[1126] 37. The dendritic polymer according to technical solution 34, wherein X3 is a heterocyclic alkane dimethyl group. (C≤8) Or substituted heterocyclic alkane dimethyl (C≤8) .

[1127] 38. The dendritic polymer according to technical solution 37, wherein X3 is N,N′-piperazinediyl.

[1128] 39. The dendritic polymer according to any one of technical solutions 34 to 38, wherein R3 is an amino group.

[1129] 40. The dendritic polymer according to any one of technical solutions 34 to 38, wherein R3 is an alkylamino group. (C≤12) Or substituted alkylamino (C≤12) .

[1130] 41. The dendritic polymer according to technical solution 40, wherein R3 is methylamino.

[1131] 42. The dendritic polymer according to any one of technical solutions 34 to 41, wherein R4 is an amino group.

[1132] 43. The dendritic polymer according to any one of technical solutions 34 to 41, wherein R4 is an alkylamino group. (C≤12) Or substituted alkylamino (C≤12) .

[1133] 44. The dendritic polymer according to technical solution 43, wherein R4 is methylamino.

[1134] 45. The dendritic polymer according to any one of technical solutions 34 to 41, wherein R4 is -(CH2CH2N). e (R c Rd:

[1135] in:

[1136] e is 1, 2, or 3;

[1137] R c and R d Each is independently hydrogen or alkyl (C≤6) Or substituted alkyl (C≤6) .

[1138] 46. ​​The dendritic polymer according to any one of technical solutions 34 to 45, wherein the core is further defined as:

[1139]

[1140] 47. The dendritic polymer according to technical solution 46, wherein the core is further defined as:

[1141]

[1142] 48. The dendritic polymer according to any one of claims 1 to 10, wherein the core is an alkylamine. (C≤18) dialkylamine (C≤36) Heterocyclic alkanes (C≤12) , or a substituted form of any of these groups.

[1143] 49. The dendritic polymer according to technical solution 48, wherein the core is octylamine, decylamine, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, N-methyl, N-dodecylamine, dioctylamine, didecylamine or 4-N-methylpiperazinyl.

[1144] 50. The dendritic polymer according to any one of technical solutions 1 to 49, wherein Y1 is an alkane dimethyl group. (C≤8) Or substituted alkane dimethyl (C≤8) .

[1145] 51. The dendritic polymer according to technical solution 50, wherein Y1 is -CH2CH2-.

[1146] 52. The dendritic polymer according to any one of technical solutions 1 to 53, wherein Y3 is an alkane dimethyl group. (C≤8) Or substituted alkane dimethyl (C≤8) .

[1147] 53. The dendritic polymer according to technical solution 52, wherein Y3 is -CH2CH2-.

[1148] 54. The dendritic polymer according to any one of technical solutions 1 to 51, wherein Y3 is:

[1149]

[1150] in:

[1151] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[1152] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) Or, or a substituted form of any of these groups; or

[1153] Y3 is:

[1154]

[1155] in:

[1156] X3 and X4 are alkane dimethyl groups. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups;

[1157] Y5 is a covalent bond, an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) , or a substituted form of any of these groups.

[1158] 55. The dendritic polymer according to any one of technical solutions 1 to 54, wherein A1 is -O- or -NH-.

[1159] 56. The dendritic polymer according to any one of technical solutions 1 to 55, wherein A2 is -O- or -NH-.

[1160] 57. The dendritic polymer according to any one of claims 1 to 56, wherein R9 is an alkyl group. (C≤8) .

[1161] 58. The dendritic polymer according to technical solution 57, wherein R9 is methyl.

[1162] 59. The dendritic polymer according to any one of technical solutions 1 to 58, wherein n is 1, 2 or 3.

[1163] 60. A composition comprising:

[1164] (A) The dendritic polymer according to any one of technical solutions 1 to 59; and

[1165] (B) Nucleic acid.

[1166] 61. The composition according to technical solution 60, wherein the nucleic acid is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), regularly clustered short palindromic repeats (CRISPR) related nucleic acid, single-stranded 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).

[1167] 62. The composition according to technical solution 61, wherein the nucleic acid is siRNA, tRNA, or a nucleic acid that can be used in the CRISPR process.

[1168] 63. The composition according to any one of claims 60 to 62, wherein the dendritic polymer and the nucleic acid are present in a weight ratio of about 100:1 to about 1:5.

[1169] 64. The composition according to any one of claims 60 to 63, wherein the composition further comprises one or more auxiliary lipids.

[1170] 65. The composition according to technical solution 64, wherein the auxiliary lipid is selected from steroids, steroid derivatives, PEG lipids or phospholipids.

[1171] 66. The composition according to claim 65, wherein the auxiliary lipid is a steroid or a steroid derivative.

[1172] 67. The composition according to claim 65, wherein the steroid is cholesterol.

[1173] 68. The composition according to claim 66, wherein the steroid or steroid derivative is present with the dendritic polymer in a molar ratio of about 10:1 to about 1:20.

[1174] 69. The composition according to claim 65, wherein the auxiliary lipid is a PEG lipid.

[1175] 70. The composition according to claim 69, wherein the PEG lipid is PEGylated diacylglycerol.

[1176] 71. The composition according to claim 70, wherein the PEG lipid is further defined by the following formula:

[1177]

[1178] in:

[1179] R 12 and R 13 Each is an alkyl group independently. (C≤24) alkenyl (C≤24) , or a substituted form of any of these groups;

[1180] R e It is hydrogen, alkyl (C≤8) Or substituted alkyl (C≤8) ;and

[1181] x is 1-250.

[1182] 72. The composition according to claim 69, wherein the PEG lipid is dimyristoyl-sn-glycerol or a compound of the following formula:

[1183]

[1184] in:

[1185] n1 is 5-250; and

[1186] n2 and n3 are each independently 2-25.

[1187] 73. The composition according to claim 69, wherein the PEG lipid and the dendritic polymer are present in a molar ratio of about 1:1 to about 1:250.

[1188] 74. The composition according to claim 65, wherein the auxiliary lipid is a phospholipid.

[1189] 75. The composition according to claim 65, wherein the phospholipid is 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE).

[1190] 76. The composition according to claim 74, wherein the phospholipid and the dendritic polymer are present in a molar ratio of about 10:1 to about 1:20.

[1191] 77. The composition according to any one of claims 60 to 76, wherein the composition is substantially composed of the dendritic polymer, the nucleic acid, and one or more auxiliary lipids.

[1192] 78. A pharmaceutical composition comprising:

[1193] (A) The composition according to any one of technical solutions 60 to 77; and

[1194] (B) Pharmaceutically acceptable carriers.

[1195] 79. The pharmaceutical composition according to claim 78, wherein the pharmaceutically acceptable carrier is a solvent or solution.

[1196] 80. The pharmaceutical composition according to technical solution 78 or technical solution 79, wherein the pharmaceutical composition is formulated for administration by means of: oral, intrafacial, intra-articular, intra-articular, intracranial, intradermal, intralesional, intramuscular, intranasal, intraocular, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrarectal, intrathecal, intratracheal, intratumoral, intraumbilical, intravaginal, intravenous, intracystic, intravitreal, via liposomes, via local, via mucosa, via parenteral, via rectum, subconjunctival, subcutaneous, sublingual, via surface, via buccal, via skin, via vagina, as an ointment, as a lipid composition, via catheter, via irrigation, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, or via local perfusion.

[1197] 81. The pharmaceutical composition according to claim 80, wherein the pharmaceutical composition is formulated for intravenous or intra-arterial injection.

[1198] 82. The pharmaceutical composition according to any one of claims 78 to 81, wherein the pharmaceutical composition is formulated as a unit dose.

[1199] 83. A method for regulating gene expression, comprising delivering nucleic acids into a cell, the method comprising contacting the cell with a composition or pharmaceutical composition according to any one of claims 60 to 82 under conditions sufficient to induce the uptake of the nucleic acids into the cell.

[1200] 84. The method according to technical solution 83, wherein the cells are in contact in vitro or outside the body.

[1201] 85. The method according to technical solution 83, wherein the cells are in contact in vivo.

[1202] 86. The method according to any one of technical solutions 83 to 85, wherein the regulation of gene expression is sufficient to treat a disease or symptom.

[1203] 87. The method according to technical solution 86, wherein the disease or symptom is cancer.

[1204] 88. A method of treating a disease or ailment in a patient, comprising administering to a patient in need a pharmaceutically effective amount of the composition or pharmaceutical composition according to any one of claims 60 to 82.

[1205] 89. The method according to technical solution 88, wherein the disease or symptom is cancer.

[1206] 90. The method according to technical solution 88 or technical solution 89, wherein the method further comprises administering one or more other cancer therapies to the patient.

[1207] 91. The method according to technical solution 90, wherein the cancer therapy is a chemotherapy compound, surgery, radiotherapy, or immunotherapy.

[1208] 92. The method according to any one of technical solutions 88 to 91, wherein the composition or pharmaceutical composition is administered to the patient once.

[1209] 93. The method according to any one of technical solutions 88 to 91, wherein the composition or pharmaceutical composition is administered to the patient two or more times.

[1210] 94. The method according to any one of technical solutions 88 to 93, wherein the patient is a mammal.

[1211] 95. The method according to technical solution 94, wherein the patient is a human being.

[1212] 96. A dendritic polymer of the following formula:

[1213] Core - (repeating unit) n -Terminal group (I)

[1214] The nucleus is connected to the repeating unit by removing one or more hydrogen atoms from the nucleus and replacing the atoms with the repeating unit, and wherein:

[1215] The core has the following formula:

[1216]

[1217] in:

[1218] X3 is -NR6-, where R6 is hydrogen or alkyl. (C≤8) Or substituted alkyl (C≤8) -O- or alkylaminodimethyl (C≤8) alkoxydiyl (C≤8) , Aromatic dimethyl (C≤8) heteroaryl dimethyl (C≤8) Heterocyclic olefin dimethyl (C≤8) , or a substituted form of any of these groups;

[1219] R3 and R4 are each independently an amino, hydroxyl, mercapto, or alkylamino group. (C≤12) Dialkylamino (C≤12) , or a substituted form of any of these groups;

[1220] c and d are each independently 1, 2, 3, 4, 5, or 6; or

[1221] The repeating unit comprises a degradable diacyl group and a linker;

[1222] The degradable diacyl group has the formula:

[1223]

[1224] in:

[1225] A1 and A2 are independently -O- or -NR. a -,in:

[1226] R a It is hydrogen, alkyl (C≤6) Or substituted alkyl (C≤6) ;

[1227] Y3 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[1228] R9 is an alkyl group. (C≤8) Or substituted alkyl (C≤8) ;

[1229] The connecting group has the following formula:

[1230]

[1231] in:

[1232] Y1 is an alkane dimethyl group. (C≤12) olefin dimethyl (C≤12) , Aromatic dimethyl (C≤12) or a substituted form of any of these groups; and

[1233] When the repeating unit contains a linker group, the linker group is connected to degradable diacyl groups on 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 group contains two degradable diacyl groups connected to the nitrogen atom of the linker group; and where n is the number of linker groups present in the repeating unit; and

[1234] The terminating group has the following formula:

[1235]

[1236] in:

[1237] Y4 is an alkane dimethyl group. (C≤18) ;and

[1238] R 10 It is hydrogen;

[1239] The final degradable diacyl group in the chain is attached to a capping group;

[1240] n is 0, 1, 2, 3, 4, 5, or 6;

[1241] Or its pharmaceutically acceptable salt.

Claims

1. A compound, which is a compound of formula (I-1): Core-repeating unit-terminated group (I-1) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is 2, 3, 4, 5, or 6. in: The core has the following formula: (IV) in: X3 is -NR6- or a heterocyclic alkane dimethyl group. C≤8 R6 is hydrogen or alkyl C≤8 ; R3 and R4 are independently amino and alkylamino groups, respectively. C≤12 or dialkylamino C≤12 The alkylamino or dialkylamino group is optionally substituted with -NH2. c is 1, 2, 3, or 4; d is 1, 2, 3, or 4; Each repeating unit contains a degradable diacyl group, having the formula: (VII) in: A1 and A2 are each independently -O-. Y3 is an alkane dimethyl group. C≤12 ;and R9 is an alkyl group. C≤8 ;and The terminating group has the following formula: (VIII) in: Y4 is an alkane dimethyl group. C≤18 ;and R 10 It is hydrogen; Or its pharmaceutically acceptable salt.

2. The compound according to claim 1, wherein R3 is an alkylamino group. (C≤12) .

3. The compound according to claim 1, wherein R3 is an alkylamino group substituted with -NH2. (C≤12) .

4. The compound according to claim 1, wherein R3 is an amino group.

5. The compound according to any one of claims 1 to 4, wherein R4 is an alkylamino group. (C≤12) .

6. The compound according to any one of claims 1 to 4, wherein R4 is an alkylamino group substituted with -NH2. (C≤12) .

7. The compound according to any one of claims 1 to 4, wherein R4 is an amino group.

8. The compound according to any one of claims 1 to 4, wherein R6 is an alkyl group. (C≤8) .

9. The compound according to any one of claims 1 to 4, wherein X3 is N,N′-piperazinediyl.

10. The compound according to any one of claims 1 to 4, wherein X3 is -NH-.

11. The compound according to any one of claims 1 to 4, wherein X3 is -NCH3-.

12. The compound according to any one of claims 1 to 4, wherein c is 2.

13. The compound according to any one of claims 1 to 4, wherein c is 3.

14. The compound according to any one of claims 1 to 4, wherein d is 2.

15. The compound according to any one of claims 1 to 4, wherein d is 3.

16. A compound that is of formula (I-2): Core-repeating unit-terminated group (I-2) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing the hydrogen atoms with the repeating unit, wherein the removed hydrogen atoms are on the nitrogen atoms of the nucleus, and the number of repeating units is 2, wherein the nucleus has the structure of formula (II): (II) in, In equation (II): X1 is a dialkylamino group. (C≤12) Or heteroaryl (C≤12) ; R1 is an amino group; and a is 1, 2, 3, 4, 5, or 6; Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is an alkane dimethyl group. (C≤12) ,and R9 is an alkyl group. (C≤8) ;and The terminating group is: (VIII) Where Y4 is an alkane dimethyl group. (C≤18) And R 10 It is hydrogen; Or its pharmaceutically acceptable salt.

17. The compound according to claim 16, wherein a is 1, 2, 3 or 4.

18. The compound according to claim 16, wherein a is 3.

19. The compound according to claim 16, wherein a is 2.

20. The compound according to any one of claims 16 to 19, wherein X1 is a dialkylamino group. (C≤12) .

21. The compound according to any one of claims 16 to 19, wherein X1 is dimethylamino.

22. The compound according to any one of claims 16 to 19, wherein X1 is a heteroaryl group. (C≤12) .

23. The compound according to any one of claims 16 to 19, wherein X1 is 2-pyridyl or N-imidazolyl.

24. The compound of claim 16, wherein the nucleus is selected from: , and .

25. A compound that is of formula (I-3): Core-repeating unit-terminated group (I-3) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is four, wherein the nucleus has the following structure: ； Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is an alkane dimethyl group. (C≤12) ,and R9 is an alkyl group. (C≤8) ;and The terminating group is: (VIII) Where Y4 is an alkane dimethyl group. (C≤18) And R 10 It is hydrogen; Or its pharmaceutically acceptable salt.

26. Compounds, that are compounds of formula (I-4): Core-repeating unit-terminated group (I-4) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is 4, 5, or 6, wherein the nucleus has the following structure: ； Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is an alkane dimethyl group. (C≤12) ,and R9 is an alkyl group. (C≤8) ;and The terminating group is: (VIII) Where Y4 is an alkane dimethyl group. (C≤18) And R 10 It is hydrogen; Or its pharmaceutically acceptable salt.

27. The compound of claim 26, wherein the core is connected to six repeating units.

28. Compounds that are of formula (I-5): Core-repeating unit-terminated group (I-5) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is four or five, wherein the nucleus has the following structure: ； Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is an alkane dimethyl group. (C≤12) ,and R9 is an alkyl group. (C≤8) ;and The terminating group is: (VIII) Where Y4 is an alkane dimethyl group. (C≤18) And R 10 It is hydrogen; Or its pharmaceutically acceptable salt.

29. The compound of claim 28, wherein the core is connected to five repeating units.

30. A compound that is of formula (I-6): Core-repeating unit-terminated group (I-6) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is two, wherein the nucleus has the following structure: ； Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is an alkane dimethyl group. (C≤12) ,and R9 is an alkyl group. (C≤8) ;and The terminating group is: (VIII) Where Y4 is an alkane dimethyl group. (C≤18) And R 10 It is hydrogen; Or its pharmaceutically acceptable salt.

31. The compound according to any one of claims 1 to 4, 16 to 19 and 25 to 30, wherein Y3 is -CH2CH2- and R9 is -CH3.

32. The compound according to any one of claims 1 to 4, 16 to 19, and 25 to 30, wherein the end-capping group is selected from: , , , , , , , , , , , and .

33. The compound according to any one of claims 1 to 4, 16 to 19 and 25 to 30, wherein the compound is a compound of formula (I-1), (I-2), (I-3), (I-4), (I-5) or (I-6).

34. The compound according to any one of claims 1 to 4, 16 to 19 and 25 to 30, wherein the compound is a salt of a compound of formula (I-1), (I-2), (I-3), (I-4), (I-5) or (I-6).

35. A compound of formula (I-2a): Core-repeating unit-terminated group (I-2a) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is two, wherein the nucleus is selected from: , and ; Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is selected from: , , , , , , , , , , , and ; Or its pharmaceutically acceptable salt.

36. Compounds of formula (I-2a): Core-repeating unit-terminated group (I-2a) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is two, wherein the nucleus is selected from: , and ; Each repeating unit is a degradable diacyl group, having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is selected from: , , , , , , , , , , , and .

37. The compound according to claim 35 or 36, wherein the core is: .

38. The compound according to claim 35 or 36, wherein the core is: .

39. The compound according to claim 35 or 36, wherein the core is: .

40. The compound according to any one of claims 1 to 4, 16 to 19, 25 to 30, and 35 to 36, wherein the end-capping group is selected from: , , , , , and .

41. The compound according to any one of claims 1 to 4, 16 to 19, 25 to 30, and 35 to 36, wherein the end-capping group is: or .

42. A compound of formula (I-1a): Core-repeating unit-terminated group (I-1a) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is four, wherein the nucleus has the following structure: ； The repeating unit is a degradable diacyl group having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is: or ; Or its pharmaceutically acceptable salt.

43. Compounds of formula (I-1a): Core-repeating unit-terminated group (I-1a) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is four, wherein the nucleus has the following structure: ； The repeating unit is a degradable diacyl group having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is: or .

44. Compounds that are of formula (I-1b): Core-repeating unit-terminated group (I-1b) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is 6, wherein the nucleus has the following structure: ； The repeating unit is a degradable diacyl group having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is: or ; Or its pharmaceutically acceptable salt.

45. Compounds of formula (I-1b): Core-repeating unit-terminated group (I-1b) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is 6, wherein the nucleus has the following structure: ； The repeating unit is a degradable diacyl group having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is: or .

46. ​​Compounds that are of formula (I-1c): Core-repeating unit-terminated group (I-1c) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is 5, wherein the nucleus has the following structure: ； The repeating unit is a degradable diacyl group having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is: or ; Or its pharmaceutically acceptable salt.

47. Compounds of formula (I-1c): Core-repeating unit-terminated group (I-1c) The nucleus is connected to the repeating unit by removing hydrogen atoms from the nucleus and replacing them with repeating units, wherein the removed hydrogen atoms are located on nitrogen atoms of the nucleus, and the number of repeating units is 5, wherein the nucleus has the following structure: ； The repeating unit is a degradable diacyl group having the formula: (VII) In equation (VII): A1 and A2 are both -O-. Y3 is -CH2CH2-, and R9 is -CH3; and The terminating group is: or .

48. The compound according to any one of claims 1 to 4, 16 to 19, 25 to 30, 35 to 36 and 42 to 47, wherein the end-capping group is: 。 49. The compound according to any one of claims 1 to 4, 16 to 19, 25 to 30, 35 to 36 and 42 to 47, wherein the end-capping group is: 。

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