Biodegradable polymers containing side chains having polyamine and polyalkylene oxide groups

Abstract

Provided are polymers comprising a hydrolyzable polymer backbone, the polymer backbone comprising (i) monomeric units comprising hydrophobic side chains; (ii) monomeric units comprising side chains comprising polyamine groups and polyalkylene oxide groups; and, optionally, (iii) monomeric units comprising side chains that comprise polyamine groups and that do not comprise polyalkylene oxide groups, as well as methods for preparing such polymers and methods for using such polymers to deliver nucleic acids and / or polypeptides to cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to U.S. Provisional Patent Application No. 63 / 202,919, filed June 11, 2021, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Background of the Invention Peptide-, protein-, and nucleic acid-based technologies have myriad applications for preventing, curing, and treating disease. However, the safe and efficient delivery of large molecules (e.g., polypeptides and nucleic acids) to their target tissues remains problematic. Thus, there is a continuing need for new compositions and methods useful for the delivery of therapeutic molecules. Summary of the Invention [Means for solving the problem]

[0003] Brief summary of the invention Provided herein are polymers comprising a hydrolyzable polymer backbone, the polymer backbone comprising (i) monomeric units comprising hydrophobic side chains, (ii) monomeric units comprising side chains comprising polyamine groups and polyalkylene oxide groups, and, optionally, (iii) monomeric units comprising side chains comprising polyamine groups but not polyalkylene oxide groups. Also provided are methods for preparing the polymers and methods for using the polymers to deliver nucleic acids and / or polypeptides to cells.

[0004] As used herein, formula 1:

[0005] [ka]

[0006] During the ceremony: m 1 , m 2 , m 3 and m4 Each of m is an integer from 0 to 1000, 1 + m 2 + m 3 + m 4 The sum of is greater than 2, and m 3 + m 4 The sum of is at least 1; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is at least 1, The symbol " / " indicates that the units separated by it are combined randomly or in any order; R in each case 3a are independently a methylene or ethylene group; R in each case 3b are independently a methylene or ethylene group; R in each case 11 are independently hydrogen or a C1-C4 alkyl group or a C2-C4 alkenyl group, any of which may be substituted with one or more substituents; each X 1 are independently -C(O)O- and -C(O)NR 11 -, -C(O)-, -S(O)(O)- or a bond; X in each case 2 is a hydrophobic side chain; A in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 and B in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 4 -R5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and In the formula, p1 to p3 each independently represent an integer of 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; s2 is an integer from 0 to 5; R in each case 2 are independently hydrogen or C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 a cycloalkenyl group, or R 2 If applicable, the second R 2in combination with to form a heterocyclic group; Z may be present, and when present, is -C(O)-, -C(O)O-, -S(O)(O)-, or -C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or optionally substituted aryl or heteroaryl; R in each case 4 are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, —S(O)(O)— or a bond; and R 5 is a group containing polyalkylene oxide Also provided is a polymer comprising the structure:

[0007] The polymer of Formula 1 above can be prepared by reacting a polymer of Formula 2:

[0008] [ka]

[0009] In the formula, m 1 and m 2 Each of m is an integer from 0 to 1000, 1 + m 2 The sum of is greater than 2; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is at least 1, The symbol " / " indicates that the units separated by it are combined randomly or in any order; R in each case 3a are independently a methylene or ethylene group; R in each case 3b are independently a methylene or ethylene group; R in each case 11are independently hydrogen or a C1-C4 alkyl group or a C2-C4 alkenyl group, any of which may be optionally substituted with one or more substituents, optionally hydrogen or a C1-C3 alkyl group; each X 1 are independently -C(O)O- and -C(O)NR 11 -, -C(O)-, -S(O)(O)- or a bond; X in each case 2 is a hydrophobic side chain; A in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 During the ceremony: Each of p1 to p3 independently represents an integer of 1 to 5; r1 is an integer from 0 to 5; and R in each case 2 are independently hydrogen or C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 cycloalkenyl group or, where applicable, R 2 is the second R 2 in combination with to form a heterocyclic group; Group A of the polymer 1 can be easily prepared by modifying the moiety

[0010] The present disclosure also provides compositions containing polymers and nucleic acids and / or polypeptides comprising the structure of Formula 1. Additional polymers, compositions containing the polymers, and methods for preparing and using the polymers are also provided herein. [Brief explanation of the drawings]

[0011] [Figure 1]FIG. 1 provides the amino acid sequence of Cas9 from Streptococcus pyogene (SEQ ID NO: 1). [Figure 2] FIG. 2 provides the amino acid sequence of Cpf1 from Francisella tularensis subsp. Novicida U112 (SEQ ID NO: 2). [Figure 3] FIG. 3 provides the sequence of AsCpf1 (SEQ ID NO: 19). [Figure 4] FIG. 4 provides the sequence of LbCpf1 (SEQ ID NO: 20). [Figure 5] FIG. 5 is a schematic diagram of the mouse ai9 reporter construct. [Figure 6] FIG. 6 shows ex vivo epifluorescence imaging of the brain and spinal cord of a luciferase-expressing mouse treated with polymer nanoparticles containing Cre mRNA. [Figure 7] Figure 7 shows ex vivo epifluorescence imaging of a brain section from a luciferase-expressing mouse treated with polymer nanoparticles containing Cre mRNA. The bottom right image shows a sagittal section of the mouse brain highlighting the cerebellum. The bottom left image shows the orientation of the section with respect to anterior (A), posterior (P), dorsal (D), and ventral (V) regions. [Figure 8] FIG. 8 shows the delivery of nucleic acid cargo to the brain using the polymers provided herein. [Figure 9] FIG. 9 illustrates the delivery of nucleic acid cargo to the spinal cord using the polymers provided herein. [Figure 10] FIG. 10 shows the ratio of brain / spinal delivery of nucleic acid cargo using the polymers provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Invention Provided herein is a polymer comprising a hydrolyzable polymer backbone, the polymer backbone comprising (i) a monomer unit comprising a hydrophobic side chain; and (ii) a monomer unit comprising a side chain comprising a polyamine group and a polyalkylene oxide group. The polymer may optionally further comprise (iii) a monomer unit comprising a side chain comprising a polyamine group but not a polyalkylene oxide group.

[0013] A hydrolyzable polymer backbone can be a polymer backbone having bonds that are susceptible to cleavage under physiological conditions (e.g., physiological pH, physiological temperature, or by naturally occurring factors (e.g., enzymes) in certain in vivo tissues such as blood, serum, etc.). Generally, hydrolyzable polymer backbones comprise polyamides, poly-N-alkylamides, polyesters, polycarbonates, polycarbamates, or combinations thereof. In some embodiments, the hydrolyzable polymer backbone comprises polyamides.

[0014] The polymer comprises a monomer unit having a side chain comprising a hydrophobic group (e.g., a hydrophobic side chain). The monomer can comprise any hydrophobic group. In some embodiments, the hydrophobic group is an aryl or aliphatic group, including cyclic, straight-chain, or branched aliphatic. In some embodiments, the hydrophobic group is C1-C 12 (e.g., C1-C 10 , C1-C8, C1-C6, C1-C3;C2-C 12 , C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , or C8-C 10 ) alkyl groups, C2-C 12 (For example, C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12, C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 ,C6-C8, C8-C 12 , C8-C 10 ) alkenyl group, or C3-C 12 (C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , C8-C 10 ) cycloalkyl or cycloalkenyl groups. In some embodiments, the hydrophobic group is a C-C 12 The hydrophobic group may comprise an alkyl, alkenyl, cycloalkyl, or cycloalkenyl group. In some embodiments, the hydrophobic group comprises fewer than 10 carbons or fewer than 8 carbons. For example, the hydrophobic group may comprise a C2-C 10 , C2-C8, or C2-C6 (e.g., C3-C8 or C3-C6) alkyl groups. The alkyl or alkenyl groups can be branched or straight-chained. In any of the foregoing embodiments, the hydrophobic group can be linked to the polymer backbone directly or via a linkage comprising, for example, an ester, amide, or ether group, optionally further comprising an alkylene linker (e.g., a methylene or ethylene linker).

[0015] The polymer comprises monomer units having side chains containing polyamine groups and polyalkylene oxide groups, and optionally monomer units having side chains containing polyamine groups but not polyalkylene oxide groups. As used herein, the term "polyamine" encompasses any chemical moiety containing two, three, or more amine groups (e.g., oligoamines) or larger polyamines containing four or more amine groups (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more). Examples of polyamines include, for example, polyalkylamines. The amine groups can be primary amine groups, secondary amine groups, tertiary amine groups, or any combination thereof. The polyamine can be linear or branched. In some embodiments, the polyamine is linear.

[0016] As used herein, the term "polyalkylene oxide" includes any chemical moiety containing two, three, or more alkylene oxide groups (e.g., oligoalkylene oxides) or polyalkylene oxides containing four or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 50, 75, 100, or more) alkylene oxide groups. Examples of polyalkylene oxide groups include polyethylene oxide (PEG), polypropylene oxide (PPO), or mixtures thereof (EO / PO). It will be understood by those skilled in the art that the number of alkylene oxide groups can exist as a range of species having different amounts of alkylene oxide groups, such that the reported number of alkylene oxide groups is the average number of such groups.

[0017] In side chains comprising polyamines and polyalkylene oxides, the polyalkylene oxide group can be linked to the polyamine and / or polymer backbone in any suitable manner, such as directly or via a bond comprising, for example, an ester, amide, or ether group, optionally further comprising an alkylene linker (e.g., a C1-C6 or C2-C4 alkylene group, such as a methylene or ethylene linker). In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group comprise at least one polyethylene glycol group (also known as a polyethylene oxide group) having a total of 2 to 200 ethylene oxide units (e.g., 2 to 150 units, 2 to 100 units, 2 to 50 units, 10 to 200 units, 10 to 150 units, 10 to 100 units, 10 to 50 units, 25 to 200 units, 25 to 150 units, 25 to 100 units, 25 to 50 units, 50 to 200 units, 50 to 150 units, or 50 to 100 units). In some embodiments, the side chains comprising a polyamine group and a polyalkylene oxide group comprise at least one polypropylene oxide group having a total of 2 to 200 propylene oxide units (e.g., 2 to 150 units, 2 to 100 units, 2 to 50 units, 10 to 200 units, 10 to 150 units, 10 to 100 units, 10 to 50 units, 25 to 200 units, 25 to 150 units, 25 to 100 units, 25 to 50 units, 50 to 200 units, 50 to 150 units, or 50 to 100 units). In some embodiments, the side chains comprising a polyamine group and a polyalkylene oxide group comprise at least one polyethylene glycol / polypropylene oxide group having a total of 2 to 200 ethylene glycol and / or propylene oxide units. The side chains comprising the polyamine and polyalkylene oxide groups can comprise ethylene glycol and propylene oxide units in any suitable configuration, such as alternating, random, block, graft, linear, branched, cyclic, or combinations thereof. For example, at least some of the side chains of the monomers can be derived from Pluronic® F65 or Pluronic® F127.

[0018] In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 wherein each of p1-p3 is independently an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s2 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R in each instance 2 are independently C1-C 12 (e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; Z is optionally present and, if present, is —C(O)—, —C(O)O—, —S(O)(O)—, —C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 -, or optionally substituted aryl or heteroaryl; 4 are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, —S(O)(O)—, or a bond; and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof). In some embodiments, p1, p3, or both are greater than p2; and / or p1, p3, or both are integers from 3 to 5 (e.g., 3, 4, or 5), and optionally, p2 is an integer of 1 or 2. In some embodiments, p1, p2, and p3 are the same. In some embodiments, Z is present and is -C(O)-. In other embodiments, Z is absent.

[0019] In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1-[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 wherein each of p1 to p3 is independently an integer of 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 1 to 5; R in each case 2 are independently hydrogen or a C1-C6 alkyl group, a C2-C6 alkenyl group, a C3-C6 cycloalkyl group, or a C3-C6 cycloalkenyl group; R in each case 4are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —O—, or —S(O)(O)—; and R 5 is a group containing polyalkylene oxide.

[0020] In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-CH2-CH2-C(O)OR 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-CH2-CH2-C(O)NR2-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(O)-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(O)-OR 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(O)-NH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(S)-NH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2)p3 -NH-C(NH)-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-S(O)(O)-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-CH2-C(O)NH-R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-Ar-C(O)OR 5 It has.

[0021] In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 and optionally wherein R 2 is hydrogen. In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 and optionally wherein R 2is a C1-C6 alkyl group (e.g., methyl or ethyl). In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 wherein R 4 is -C(O)O-, -C(O)-, or -C(O)NH-, and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).

[0022] In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 4 -R 5 and optionally wherein R 2 is hydrogen. In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 4 -R 5 and optionally wherein R 2 is a C1-C6 alkyl group (e.g., methyl or ethyl). In some embodiments, the side chains comprising the polyamine group and the polyalkylene oxide group have the formula: -(CH2)2-NH-(CH2)2-NH-C(O)-(CH2)2-R 4 -R 5 wherein R4 is -C(O)O-, -C(O)-, or -C(O)NH-, and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).

[0023] In some embodiments, the polymer further comprises a monomer unit comprising a side chain that comprises a polyamine group and no polyalkylene oxide group, the group having the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 wherein each of p1-p3 is independently an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R 2 are independently C1-C 12 (e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group, or R 2 is the second R 2 and combine to form a heterocyclic group. In some embodiments, p1, p3, or both are greater than p2; and / or p1, p3, or both are integers from 3 to 5 (e.g., 3, 4, or 5), and optionally, p2 is an integer of 1 or 2. In some embodiments, p1, p2, and p3 are the same. In some embodiments, the side chain containing the polyamine group and not the polyalkylene oxide group has the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 wherein R 2 is hydrogen. In some embodiments, the side chain containing the polyamine group and no polyalkylene oxide group has the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 wherein R in one or both instances 2 is a C1-C6 alkyl group (e.g., methyl or ethyl). In some embodiments, the side chain containing the polyamine group and not the polyalkylene oxide group has the formula: -(CH2)2-NH-(CH2)2-NH2 or -(CH2)2-NH-(CH2)2-NHCH2 It has.

[0024] The polymer can contain any suitable number or amount (e.g., by weight or percentage) of monomer units containing hydrophobic side chains, monomer units containing side chains containing polyamine groups and polyalkylene oxide groups, and, when present, monomer units containing side chains containing polyamine groups but not polyalkylene oxide groups. In some embodiments, the polymer contains about 1 to about 80 mol % (e.g., about 5 to about 80 mol %, about 10 to about 80 mol %, about 20 to about 80 mol %, about 40 to about 80 mol %, about 1 to about 60 mol %, about 1 to about 40 mol %, about 1 to about 20 mol %, or about 1 to about 10 mol %) of monomer units containing hydrophobic groups, about 1 to about 50 mol % (e.g., about 5 to about 50 mol %, about 10 to about 50 mol %, about 20 to about 50 mol %, about 40 to about 50 mol %, about 1 to about 40 mol %, about 1 to about 20 mol %) of monomer units containing hydrophobic groups. 0 mol%, about 1 to about 10 mol%, or about 1 to about 5 mol%) of monomer units containing a side chain containing a polyamine group and a polyalkylene oxide group; and 0 to about 80 mol% (e.g., about 5 to about 80 mol%, about 10 to about 80 mol%, about 20 to about 80 mol%, about 40 to about 80 mol%, about 1 to about 60 mol%, about 1 to about 40 mol%, about 1 to about 20 mol%, or about 1 to about 10 mol%) of monomer units containing a side chain containing a polyamine group but not a polyalkylene oxide group.

[0025] Individual polymer side chains can have any suitable pKa profile. For example, a polymer side chain can have one or more protic hydrogens with a pKa (in water at 25°C) greater than -2 and less than 14. For example, an individual polymer side chain described herein can have one or more protic hydrogens with a pKa less than 12, less than 10, less than 7, less than 5, less than 3, or less than 1. Alternatively, or in addition, an individual polymer side chain described herein can have one or more protic hydrogens with a pKa greater than -2, greater than -1, greater than 0, greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 6. Thus, an individual polymer side chain described herein can have one or more protic hydrogens with a pKa of -2 to 14, e.g., a pKa of -1 to 12, a pKa of 0 to 12, a pKa of 1 to 12, a pKa of 2 to 12, a pKa of 3 to 12, a pKa of 4 to 12, a pKa of 5 to 12, a pKa of 6 to 12, a pKa of -1 to 10, a pKa of 0 to 10, a pKa of 1 to 10, a pKa of 2 to 10, a pKa of 3 to 10, a pKa of 4 to 10, a pKa of 5 to 10, a pKa of 6 to 10, a pKa of -1 to 7, a pKa of 0 to 7, a pKa of 1 to 7, a pKa of 2 to 7, a pKa of 3 to 7, a pKa of 4 to 7, a pKa of 5 to 7, or a pKa of 6 to 7.

[0026] In some embodiments, the polymers have an overall pKa (in water at 25° C.) of less than about 10 or less than about 7. For example, the polymers described herein may have a pKa of less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. Alternatively, or in addition, the polymers described herein may have a pKa of greater than −2, greater than −1, greater than 0, greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, or greater than 6. Alternatively, the polymers described herein can have a pKa of -2 to 7, e.g., a pKa of -1 to 7, a pKa of 0 to 7, a pKa of 1 to 7, a pKa of 2 to 7, a pKa of 3 to 7, a pKa of 4 to 7, a pKa of 5 to 7, a pKa of 6 to 7, a pKa of 0 to 6, a pKa of 2 to 6, a pKa of 4 to 6, a pKa of 0 to 5, a pKa of 2 to 5, or a pKa of 4 to 5.

[0027] As used herein, "alkyl" or "alkylene" refers to a substituted or unsubstituted hydrocarbon chain. An alkyl group can have any number of carbon atoms (e.g., C-C 100 Alkyl, C1-C 50 Alkyl, C1-C 12 Alkyl, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, etc. The alkyl or alkylene can be saturated or unsaturated (e.g., to provide alkenyl or alkynyl) and can be linear, branched, straight-chain, cyclic (e.g., cycloalkyl or cycloalkenyl), or a combination thereof. The cyclic groups can be monocyclic, fused to form bicyclic or tricyclic groups, linked by bonds, or spirocyclic. In some embodiments, the alkyl substituents can be interrupted by one or more heteroatoms (e.g., oxygen, nitrogen, and sulfur), thereby providing heteroalkyl, heteroalkylene, or heterocyclyl (i.e., heterocyclic groups). In some embodiments, the alkyl is substituted with one or more substituents.

[0028] The term "aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can contain, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms and 6 to 10, 6 to 12, or 6 to 14 ring members. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. In some embodiments, aryl groups contain an alkylene linking group, thereby forming an arylalkyl group (e.g., a benzyl group). Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl. In some embodiments, the aryl substituent can be interrupted by one or more heteroatoms (e.g., oxygen, nitrogen, and sulfur), thereby providing a heterocyclyl (i.e., heterocyclic or heteroaryl group). In some embodiments, the aryl is substituted with one or more substituents.

[0029] The term "heterocyclyl" or "heterocyclic group" refers to a cyclic group, e.g., aromatic (e.g., heteroaryl) or non-aromatic, where the cyclic group has one or more heteroatoms (e.g., oxygen, nitrogen, and sulfur). In some embodiments, the heterocyclyl or heterocyclic group (i.e., a cyclic group, e.g., aromatic (e.g., heteroaryl) or non-aromatic, where the cyclic group has one or more heteroatoms) is substituted with one or more substituents.

[0030] As used herein, the term "substituted" may mean that one or more hydrogens on the specified atom or group (e.g., a substituted alkyl group) are replaced with another group, provided that the normal valence of the specified atom is not exceeded. For example, if the substituent is oxo (i.e., =0), two hydrogens on the atom are replaced. Substituents may include one or more hydroxyl, amino (e.g., primary, secondary, or tertiary), aldehyde, carboxylic acid, ester, amide, ketone, nitro, urea, guanidine, cyano, fluoroalkyl (e.g., trifluoromethane), halo (e.g., fluoro), aryl (e.g., phenyl), heterocyclyl, or heterocyclic groups (i.e., cyclic groups, e.g., aromatic (e.g., heteroaryl) or non-aromatic, where the cyclic group has one or more heteroatoms), oxo, or combinations thereof. Combinations of substituents and / or variables are permissible provided that the substituents do not significantly adversely affect the synthesis or use of the compound.

[0031] In some embodiments, the polymers provided herein have Formula 1:

[0032] [ka]

[0033] During the ceremony: m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000, 1 + m 2 + m 3 + m 4 The sum of is greater than 2, and m 3 + m 4 The sum of is at least 1; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is at least 1, The symbol " / " indicates that the units separated by it are combined randomly or in any order; R in each case 3a are independently a methylene or ethylene group; R in each case 3b are independently a methylene or ethylene group; R in each case 11 are independently hydrogen or a C1-C4 alkyl group or a C2-C4 alkenyl group, any of which may be substituted with one or more substituents; each X 1 are independently -C(O)O- and -C(O)NR 11 -, -C(O)-, -S(O)(O)- or a bond; X in each case 2 is a hydrophobic side chain; A in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 and B in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2-Z-(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and During the ceremony, Each of p1 to p3 independently represents an integer of 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; s2 is an integer from 0 to 5; R in each case 2 are independently hydrogen or C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 a cycloalkenyl group, or R 2 If applicable, the second R 2 in combination with to form a heterocyclic group; Z may be present, and when present, is -C(O)-, -C(O)O-, -S(O)(O)-, or -C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or optionally substituted aryl or heteroaryl; R in each case 4are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, —S(O)(O)— or a bond; and R 5 is a group containing polyalkylene oxide It is a polymer containing the structure:

[0034] In some embodiments, each occurrence of A 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 is a group of the formula each of p1 to p3 is independently an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R in each case 2 are independently hydrogen, C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl groups. In some embodiments, p1, p3, or both are greater than p2; and / or p1, p3, or both are integers from 3 to 5 (e.g., 3, 4, or 5), and optionally, p2 is an integer of 1 or 2. In some embodiments, p1, p2, and p3 are the same.

[0035] In some embodiments, each occurrence of A 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1NH-(CH2) p3 -NR 2 2 In the formula, R 2 is hydrogen. In some embodiments, each occurrence of A 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 where R in one or both cases 2 is a C1-C6 alkyl group (e.g., methyl or ethyl).

[0036] A 1 Specific non-limiting examples of groups include, for example: -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH2; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NHCH3; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH2; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NHCH3; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH2; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NHCH3; -CH2-CH2-CH2-NH-CH2-CH2-CH2-NH2; -CH2-CH2-CH2-NH-CH2-CH2-CH2-NHCH3; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH2; CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NHCH3; CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH2; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NHCH3; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH2; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NHCH3; -CH2-CH2-NH-CH2-CH2-NH2; or -CH2-CH2-NH-CH2-CH2-NHCH3 Includes.

[0037] B in each case 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2-Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 is a group of the formula: each of p1 to p3 is independently an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s2 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); R in each case 2 are independently C1-C 12 (e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; Z may be present, and when present, is -C(O)-, -C(O)O-, -S(O)(O)-, or -C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or optionally substituted aryl or heteroaryl; R in each case 4 are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, —S(O)(O)—, or a bond; and R5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof). In some embodiments, p1, p3, or both are greater than p2; and / or p1, p3, or both are integers from 3 to 5 (e.g., 3, 4, or 5), and optionally, p2 is an integer of 1 or 2. In some embodiments, p1, p2, and p3 are the same. In some embodiments, Z is present and is -C(O)-. In other embodiments, Z is absent.

[0038] In some embodiments, each B 1 teeth: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2)p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 wherein p1 to p3 each independently represent an integer of 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; R in each case 2 are independently hydrogen or a C1-C6 alkyl group, a C2-C6 alkenyl group, a C3-C6 cycloalkyl group, or a C3-C6 cycloalkenyl group; R in each case 4 are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, —S(O)(O)—, or a bond; and R 5 is a group containing polyalkylene oxide.

[0039] In some embodiments, in each instance of B 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-CH2-CH2-C(O)OR 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-CH2-CH2-C(O)NR2-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(O)-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(O)-OR5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(O)-NH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(S)-NH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-C(NH)-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-S(O)(O)-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-CH2-C(O)NH-R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NH-Ar-C(O)OR 5 It is based on.

[0040] In some embodiments, in each instance of B 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 In the formula, R2 is hydrogen. In some embodiments, each occurrence of B 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 In the formula, R 2 is a C1-C6 alkyl group (e.g., methyl or ethyl) group.

[0041] In some embodiments, in each instance of B 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 4 -R 5 In the formula, R 2 is hydrogen. In some embodiments, each occurrence of B 1 are independently expressed as: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 4 -R 5 In the formula, R 2 is a C1-C6 alkyl group (e.g., methyl or ethyl) group.

[0042] B 1 Specific non-limiting examples of groups include, for example: -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH-(CH2) s1 -R4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-N(CH3)-(CH2) s1-R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-N(CH3)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-CH2-NH-CH2-CH2-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-N(CH3)-C(O)-(CH2)s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; -CH2-CH2-NH-CH2-CH2-NH-C(O)-(CH2) s1 -R 4 -R 5 ;or -CH2-CH2-NH-CH2-CH2-N(CH3)-C(O)-(CH2) s1 -R 4 -R 5 ; where s1 is an integer from 0 to 5, 1 to 5, or 1 to 3. Includes.

[0043] In some embodiments, each occurrence of A 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 and in each case B 1 teeth: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 is.

[0044] In other embodiments, in each instance of A 1 are independently: -(CH2)2-[NH-(CH2)2-]r1 NH-(CH2)2-NR 2 2 and in each case B 1 teeth: -(CH2)2-[NH-(CH2)2-] r1 NH-(CH2)2-NR 2 -(CH2)2-R 4 -R 5 is.

[0045] In some embodiments, each occurrence of A 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 and in each case B 1 teeth: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 4 -R 5 is.

[0046] In other embodiments, in each instance of A 1 are independently: -(CH2)2-[NH-(CH2)2-] r1 NH-(CH2)2-NR 2 2 and in each case B 1 teeth: -(CH2)2-[NH-(CH2)2-] r1 NH-(CH2)2-NR 2 -C(O)-(CH2)2-R 4 -R 5 is.

[0047] Base A 1 and B 1In any of the foregoing embodiments, variables p1-p3 (i.e., p1, p2, and p3) are each independently an integer between 1 and 5 (e.g., 1, 2, 3, 4, or 5). In some embodiments, p1, p3, or both are an integer greater than the integer p2. Additionally, or alternatively, p1 or p3, or both, are an integer between 3 and 5 (e.g., 3, 4, or 5). In some embodiments, both p1 and p3 are an integer greater than p2, or both are integers between 3 and 5 (e.g., 3, 4, or 5). In still other embodiments, p1, p2 (if present), and p3 are the same.

[0048] Base A 1 and B 1 In any of the foregoing embodiments, the variable r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5). In some embodiments, r1 is 0; in other embodiments, the variable r1 is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5).

[0049] Base A 1 and B 1 In any of the foregoing embodiments, the variable s1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5). In some embodiments, the variable s1 is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5). In some embodiments, the variable s1 is an integer from 1 to 3 (e.g., 1, 2, or 3). In some embodiments, the variable s1 is 2. In some embodiments, r1 and s1 are each independently an integer from 1 to 3 (e.g., 1, 2, or 3).

[0050] Base B 1 In any of the foregoing embodiments, variable s2 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5). In some embodiments, variable s2 is an integer from 0 to 3 (e.g., 0, 1, 2, or 3). In some embodiments, variable s2 is 0. In some embodiments, variable s2 is 2.

[0051] In any of the foregoing embodiments, each occurrence of R 2 is hydrogen or C1-C 12 (e.g., C1-C6, C1-C4, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C5, C2-C4, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C5 or C3-C4) cycloalkyl groups, or C3-C 12 (e.g., C3-C5 or C3-C4) cycloalkenyl group, or, where applicable, R 2 is the second R 2 and may combine with the nitrogen to which they are attached to form a heterocyclic group. In some embodiments, each occurrence of R 2 is hydrogen. In some embodiments, one or more (or all) instances of R 2 is a C1-C6 (e.g., C1-C6, C1-C4, or C1-C3) alkyl group, such as an ethyl or methyl group.

[0052] According to any of the foregoing embodiments, Z at each occurrence, if present, is —C(O)—, —C(O)O—, —S(O)(O)—, —C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 -, or optionally substituted aryl or heteroaryl. In some embodiments, Z is present and is -C(O)-. In other embodiments, Z is absent (i.e., a bond).

[0053] According to any of the foregoing embodiments, in each instance R 4 is independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —COC(O)—O—, —O—, —S(O)(O)—, or a bond. 4 is independently —C(O)O—, —C(O)—, or —C(O)NH—. In some embodiments, each occurrence of R 4is —C(O)O—. In some embodiments, each occurrence of R 4 is -C(O)NH-.

[0054] According to any of the foregoing embodiments, in each instance R 5 is a group containing a polyalkylene oxide, and optionally contains a linking group. 5 is a group containing two or three or more alkylene oxide groups (e.g., oligoalkylene oxides) or larger polyalkylene oxides containing four or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 25, 50, 75, 100 or more) alkylene oxide groups. Examples of polyalkylene oxide groups include polyethylene oxide (PEG), polypropylene oxide (PPO), or mixtures thereof (EO / PO).

[0055] In some embodiments, R 5 contains at least one polyethylene glycol group (also known as a polyethylene oxide group) having a total of 2 to 200 ethylene oxide units (e.g., 2 to 150 units, 2 to 100 units, 2 to 50 units, 10 to 200 units, 10 to 150 units, 10 to 100 units, 10 to 50 units, 25 to 200 units, 25 to 150 units, 25 to 100 units, 25 to 50 units, 50 to 200 units, 50 to 150 units, or 50 to 100 units). In some embodiments, R 5 R contains at least one polypropylene oxide group having a total of 2 to 200 propylene oxide units (e.g., 2 to 150 units, 2 to 100 units, 2 to 50 units, 10 to 200 units, 10 to 150 units, 10 to 100 units, 10 to 50 units, 25 to 200 units, 25 to 150 units, 25 to 100 units, 25 to 50 units, 50 to 200 units, 50 to 150 units, or 50 to 100 units). 5In an embodiment, R comprises at least one polyethylene glycol / polypropylene oxide group having a total of 2 to 200 ethylene glycol and propylene oxide units (e.g., 2 to 150 units, 2 to 100 units, 2 to 50 units, 10 to 200 units, 10 to 150 units, 10 to 100 units, 10 to 50 units, 25 to 200 units, 25 to 150 units, 25 to 100 units, 25 to 50 units, 50 to 200 units, 50 to 150 units, or 50 to 100 units). 5 can exist as any suitable structural type. For example, R 5 may contain ethylene glycol and / or propylene oxide units as alternating, random, block, graft, linear, branched, cyclic, or combinations thereof. For example, at least some of the R 5 may be derived from Pluronic® F65 or Pluronic® F127. It will be understood by those skilled in the art that the number of alkylene oxide groups may exist as a range of species having different amounts of alkylene oxide groups, so that the reported number of alkylene oxide groups is an average.

[0056] In some embodiments, each occurrence of R 5 are independently expressed as:

[0057] [ka]

[0058] wherein: R 12 is a bond or a methylene, ethylene, or propylene group; R 13 is hydrogen, an aryl group (e.g., C6-C 12 alkyl groups), heterocyclic groups (e.g., C2-C 12 heterocyclic groups), alkyl groups (e.g., C1-C 12 alkyl groups), alkenyl groups (e.g., C2-C 12 alkenyl groups), cycloalkyl groups (e.g., C3-C 12cycloalkyl groups), or cycloalkenyl groups (e.g., C3-C 12 cycloalkenyl groups), any of which may be substituted with one or more substituents, optionally hydrogen or a C1-C3 alkyl group; and t1 is an integer of 2 to 200 (e.g., 2 to 150, 2 to 100, 2 to 50, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 25 to 200, 25 to 150, 25 to 100, 25 to 50, 50 to 200, 50 to 150, or 50 to 100).

[0059] In any of the foregoing embodiments, R 3a and R 3b are each independently a methylene or ethylene group. In some embodiments, R 3a is an ethylene group, and R 3b is a methylene group; or R 3a is a methylene group, and R 3b is an ethylene group. In some embodiments, R 3a and R 3b are both ethylene groups. In some embodiments, R 3a and R 3b are both methylene groups.

[0060] In any of the foregoing embodiments, each X in formula 1 1 The groups are independently -C(O)O- and -C(O)NR 11 -, -C(O)-, -S(O)(O)-, or a bond. 1 The groups may be the same or different from each other. In some embodiments, X 1 is -C(O)NR 11 In some embodiments, X 1 is -C(O)O-.

[0061] In any of the foregoing embodiments, each occurrence of R 11are independently hydrogen or a C1-C4 (e.g., C1-C3 or C1-C2) alkyl group or a C2-C4 (e.g., C2, C3, or C4) alkenyl group, any of which can be substituted with one or more substituents. In some embodiments, each R 11 is independently a C1-C4 alkyl group or a C2-C4 alkenyl group. In some embodiments, R 11 is methyl; in other embodiments, R 11 is hydrogen. Each R 11 are independently selected and may be the same or different; however, in some embodiments, each R 11 are identical (e.g., all methyl or all hydrogen).

[0062] In any of the foregoing embodiments, each occurrence of X 2 is a hydrophobic side chain. X 2 can be any hydrophobic side chain such as an aryl or an aliphatic group, including cyclic, straight-chain, or branched aliphatic groups. For example, X in each instance 2 are independently C1-C 12 (e.g., C1-C 10 , C1-C8, C1-C6, C1-C3;C2-C 12 , C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , or C8-C 10 ) alkyl groups, C2-C 12 (For example, C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 ,C6-C8, C8-C 12, C8-C 10 ,) alkenyl group, or C3-C 12 (C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , C8-C 10 ) may be a cycloalkyl or cycloalkenyl group. Any of the foregoing may further include one or more heteroatoms to provide a heteroalkyl, heteroalkenyl, or heterocyclic group. Any of the foregoing may be substituted with one or more substituents. In some embodiments, X 2 is an aliphatic group. In some embodiments, one or more (or all) X 2 The group is C2-C 12 (e.g., C3-C 12 , C3-C8, C3-C6, C4-C 12 , C4-C6, C6-C 12 , or C8-C 12 ) alkyl or alkenyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, C4-C 12 , C4-C6, C6-C 12 , or C8-C 12 ) cycloalkyl or cycloalkenyl groups. In other embodiments, one or more (or all) X 2 The group may be C1-C8 (e.g., C1-C6, C1-C4, C1-C 3、 Each X is a C2-C8, or C2-C6) alkyl group. Any of the foregoing alkyl or alkenyl groups may be linear or branched. 2 are independently selected and therefore may be the same or different from one another; in some embodiments, all instances of X2 are the same.

[0063] The polymers described herein can have any suitable end groups. In some embodiments, the polymer has Formula 1A:

[0064] [ka]

[0065] During the ceremony Q is the formula:

[0066] [ka]

[0067] and c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; R 1 is hydrogen, an aryl group, a heterocyclic group, C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 cycloalkenyl groups, any of which may be substituted with one or more substituents; and R 6 is hydrogen, amino group, aryl group, heterocyclic group, C1-C 12 Alkyl groups, C1-C 12 Heteroalkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 cycloalkenyl groups, any of which may be substituted with one or more substituents or tissue- or cell-specific targeting moieties. It has the following structure. All other aspects of Formula 1A are as described above for Formula 1, including any and all embodiments thereof.

[0068] In some embodiments, the polymer has Formula 1B:

[0069] [ka]

[0070] During the ceremony R 1 is hydrogen, an aryl group, a heterocyclic group, C1-C 12 (e.g., C1-C 10 , C1-C8, C1-C6, C1-C3;C2-C 12 , C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , or C8-C 10 ) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 ,C6-C8, C8-C 12 , C8-C 10 ) an alkenyl or heteroalkenyl group, or a C3-C 12 (C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , C8-C 10 ) a cycloalkyl, heterocycloalkyl, cycloalkenyl, or heterocycloalkenyl group, any of which may be substituted with one or more substituents; and R 6 is hydrogen, amino group, aryl group, heterocyclic group, C1-C 12 (e.g., C1-C 10 , C1-C8, C1-C6, C1-C3;C2-C 12 , C2-C 10, C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , or C8-C 10 ) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 ,C6-C8, C8-C 12 , C8-C 10 ) an alkenyl or heteroalkenyl group, or a C3-C 12 (C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C 10 , C6-C8, C8-C 12 , C8-C 10 ) a cycloalkyl, heterocycloalkyl, cycloalkenyl, or heterocycloalkenyl group, any of which may be substituted with one or more substituents or tissue- or cell-specific targeting moieties. In some embodiments of Formula 1B and 1C, R 1 is hydrogen, and R 6 is C1-C8 or C1-C6 alkyl.

[0071] All other aspects of Formula 1B are as described for Formulas 1 and 1A, including any and all embodiments thereof.

[0072] In some embodiments, the polymer has Formula 1C:

[0073] [ka]

[0074] It has the following structure.

[0075] All other aspects of Formula 1C are as described for Formulas 1, 1A, and 1B, including any and all embodiments thereof.

[0076] According to Equation 1 (or Equations 1A-1C), m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000 (e.g., 0 to 500, 0 to 200, 0 to 100, or 0 to 50), 1 + m 2 + m 3 + m 4 The sum of is greater than 2, for example, 2-5000, 2-2000, 2-1000, 2-500, 2-100, or 2-50, and m 3 + m 4 is at least 1 (e.g., 1-2000, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-25, 2-2000, 2-1000, 2-500, 2-200, 2-100, 2-50, or 2-25). 1 + m 2 + m 3 + m 4 is greater than 5, or greater than 10 (e.g., 5-5000, 5-2000, 5-1000, 5-500, 5-100, or 5-50; or 10-5000, 10-2000, 10-1000, 10-500, 10-100, or 10-50). 1 + m 2 + m 3 + m 4 is approximately 10-50 or 20-40. 1 and n 2Each of n is an integer from 0 to 1000 (e.g., 0 to 500, 0 to 200, 0 to 100, 0 to 50, or 0 to 25), provided that 1 + n 2 is at least 1 (e.g., 1-2000, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-25, 2-2000, 2-1000, 2-500, 2-200, 2-100, 2-50, or 2-25). 1 + n 2 is greater than 5, or greater than 10 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, 5-50, or 5-25; or 10-2000, 10-1000, 10-500, 10-200, 10-100, 10-50, or 10-25). 1 + n 2 The sum of m is about 20-50 or 20-40. 1 + m 2 + m 3 + m 4 is about 5-65, for example about 20-50 or 20-40, and n 1 +n 2 The sum is about 10-50 or 20-40.

[0077] In other words, the polymer contains the group B 1 and optionally a group A 1 and several monomer units comprising the group X, collectively referred to herein as "A monomers" and "B monomers," respectively. In some embodiments, the polymer comprises at least some A monomers and B monomers. Similarly, the polymer may comprise at least some A monomers and B monomers. 1 and / or X 2 In some embodiments, the monomeric units comprise at least some of the monomeric units comprising m 1 and m 2 is zero, and the polymer is A 1 Does not contain groups.

[0078] Polymers according to Formula 1 (or Formulas 1A-1C) may be prepared using any suitable degree of polymerization (e.g., m 1 + m 2 + m 3 + m 4 + n 1 + n 2 In some embodiments, the polymer has a degree of polymerization of about 10 or more, or about 20 or more (e.g., about 20 to about 1000, or about 20 to about 100, or about 20 to about 65, or about 20 to 64). In some embodiments, the polymer has a degree of polymerization of about 50 or more (e.g., about 50 to about 1000, or about 50 to about 500, or 66 to about 200). In some embodiments, the degree of polymerization is about 20-100 (e.g., about 20-80, or about 20-75, or about 20-50). In some embodiments, the degree of polymerization is about 40-100 (e.g., about 40-80, about 40-75, or about 50-75).

[0079] The polymer can comprise any suitable ratio of A and B monomers to X monomers. In some embodiments, the polymer has a ratio of A and B monomers to X monomers (e.g., (m 1 +m 2 +m 3 +m 4 ) / (n 1 +n 2 )). For example, the ratio of A and B monomers to X monomer can be about 0.3 to about 25, about 0.3 to about 20, about 0.3 to about 10, about 0.3 to about 5, about 0.3 to about 3, or about 0.3 to about 2. In some embodiments, the ratio of (m1+m2+m3+m4) / (n1+n2) is about 0.5-2 or about 1-2.

[0080] In embodiments where the polymer comprises both A and B monomers, the polymer can comprise any suitable ratio of A monomers to B monomers. In some embodiments, the ratio of A monomers to B monomers (e.g., (m 1 +m 2 ) / (m 3 +m 4 )) can be about 20 or less (e.g., about 10 or less, about 5 or less, about 2 or less, or even about 1 or less, e.g., about 0.5 or less or 0.2 or less). In some embodiments, (m 1 +m 2 ) / (m 3 +m 4 The ratio of (m1+m2) / (m3+m4) is about 0.1 or greater, or about 0.2 or greater, e.g., about 0.5 or greater. In some embodiments, the ratio of A monomers to B monomers is higher, e.g., about 5 or greater (e.g., about 5, 6, 7, 8, 9, or 10 or greater). In some embodiments, the ratio of (m1+m2) / (m3+m4) is about 0.5-5, e.g., about 0.5-3 or about 1-4.

[0081] The tissue- or cell-specific targeting moiety referred to in connection with any of the foregoing polymers can be any small molecule, protein (e.g., antibody or antigen), amino acid sequence, sugar, oligonucleotide, metal-based nanoparticle, or combination thereof, capable of recognizing (e.g., specifically binding to) a predetermined target tissue or cell (e.g., specifically binding to a particular ligand, receptor, or other protein or molecule that allows the targeting moiety to distinguish the target tissue or cell from other non-target tissues or cells). In some embodiments, the tissue- or cell-specific targeting moiety is a small molecule (e.g., a small molecule drug or other small molecule moiety having less than about 900 daltons or less than about 500 daltons and / or fewer than about 100 atoms, e.g., about 10-100 or about 20-100 atoms). In other embodiments, the tissue- or cell-specific targeting moiety is a receptor for a ligand or a ligand for a receptor.

[0082] Tissue-specific or cell-specific targeting moieties can be used to target any desired tissue or cell type. In some embodiments, the tissue-specific or cell-specific targeting moiety localizes the polymer to the peripheral nervous system, central nervous system, liver, muscle (e.g., cardiac muscle), lung, bone (e.g., hematopoietic cells), or ocular tissue of a subject. In some embodiments, the tissue-specific or cell-specific targeting moiety localizes the polymer to tumor cells. In certain embodiments, the tissue-specific or cell-specific targeting moiety targets lung tissue. For example, the tissue-specific or cell-specific targeting moiety can be a sugar that binds to a receptor on a specific tissue or cell.

[0083] By way of illustration, non-limiting examples of tissue-specific or cell-specific targeting moieties include:

[0084] [ka]

[0085] In the formula, R 7 , R 8 , R 9 , and R 10 each independently represents hydrogen, halogen, C1-C4 alkyl, or C1-C4 alkoxy, optionally substituted with one or more amino groups. Specific tissue- or cell-specific targeting moieties can be selected to localize the polymer to the tissues described herein. For example, alpha-d-mannose can be used to localize the polymer to the peripheral nervous system, central nervous system, or immune cells; alpha-d-galactose and N-acetylgalactosamine can be used to localize the polymer to liver cells; and folic acid can be used to localize the polymer to tumor cells.

[0086] The polymers described herein (e.g., Formulas 1 and 1A-1C) can exist as any suitable structural type. For example, the polymer can exist as an alternating polymer, a random polymer, a block polymer, a graft polymer, a linear polymer, a branched polymer, a cyclic polymer, or a combination thereof. In some embodiments, the polymer is a random polymer, a block polymer, a graft polymer, or a combination thereof.

[0087] Thus, in the structure of Formula 1 (or Formulas 1A-1C), the monomers (their respective side chains A 1 , B 1 , and X(X 1 and / or X 2 ) may be arranged randomly or in any order. 1 , m 2 , m 3 , m 4 , n 1 , and n 2 The (if applicable) simply indicate the number of each monomer that appears in the overall chain, and do not necessarily imply or represent any particular order or block of these monomers, although blocks or stretches of a given monomer may be present in some embodiments. For example, the structure of Formula 1 may be represented by -A 1 -A 1 -B 1 -B 1 -, -B 1 -A 1 -B 1 -A 1 -, -A 1 -B 1 -B 1 -B 1 - etc. Additionally, the polymer may comprise blocks of A and / or B polymers (e.g., [A monomer] m1+m2 -[B monomer] m3+m4) in any order. The polymer may contain individual X monomers (e.g., -AXB-, -ABX-, -BXA, etc.) interspersed with A and B monomers, or the polymer may be "capped" with one or more X monomers (e.g., blocks of X monomers) at one or both ends of the polymer. Similarly, if the polymer contains blocks of A and / or B monomers, the polymer may contain blocks of X monomers interspersed between blocks of A and / or B monomers, or the polymer may be "capped" with one or more X monomers (e.g., blocks of X monomers) at one or both ends of the polymer. In some embodiments, the polypeptide (e.g., polyaspartamide) backbone is arranged in an alpha / beta configuration, resulting in alternating "m1-like" or "m3-like" and "m2-like" or "m4-like" monomers, where the polymer is capped with X monomers or where X monomers are interspersed throughout. However, the "A" and "B" side chains (e.g., A 1 and B 1 ) may be randomly dispersed throughout the polymer backbone.

[0088] Each A 1 The groups (if present) are independently selected and therefore may be the same or different from one another. Similarly, each B 1 The groups are independently selected and may be the same or different from one another, and each X 1 and / or X 2 The groups are independently selected and may be the same or different from one another. However, in some embodiments, all A 1 The groups are identical and all B 1 The groups are identical and / or all X 1 and / or X 2 The groups are identical.

[0089] The polymer can be any suitable polymer, provided that the polymer comprises the above-mentioned polymer structure.In some embodiments, the polymer is a block copolymer comprising a polymer block having the structure of Formula 1 and one or more other polymer blocks, which can comprise any suitable end group.In some embodiments, the polymer can further comprise a substituent comprising tissue-specific or cell-specific targeting moiety.

[0090] Non-limiting examples of polymers provided herein include, for example:

[0091] [ka]

[0092] [ka]

[0093] [ka]

[0094] [ka]

[0095] [ka]

[0096] [ka]

[0097] [ka]

[0098] [ka]

[0099]

change

[0100]

change

[0101]

change

[0102]

change

[0103]

change

[0104]

change

[0105]

change

[0106]

change

[0107]

change

[0108]

change

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] [ka]

[0113] In the formula, R 12 is a bond or a methylene, ethylene, or propylene group (e.g., ethylene); R 13 is hydrogen, an aryl group (e.g., C6-C 12 alkyl groups), heterocyclic groups (e.g., C2-C 12 heterocyclic groups), alkyl groups (e.g., C1-C 12 alkyl groups), alkenyl groups (e.g., C2-C 12 alkenyl groups), cycloalkyl groups (e.g., C3-C 12 cycloalkyl groups), or cycloalkenyl groups (e.g., C3-C 12 cycloalkenyl groups), any of which may be substituted with one or more substituents, optionally hydrogen or a C1-C3 alkyl group; and t1 is an integer from 2 to 200 (e.g., from 2 to 150, from 2 to 100, from 2 to 50, from 10 to 200, from 10 to 150, from 10 to 100, from 10 to 50, from 25 to 200, from 25 to 150, from 25 to 100, from 25 to 50, from 50 to 200, from 50 to 150, or from 50 to 100). 12 is a C1-C3 alkyl, such as an ethylene group. In some embodiments, R 13 is methyl. In some embodiments, t1 is 20-80 (e.g., 30-60 or 25-50).

[0114] The unit number designations ("a," "b," "c," "d," "e," and "f") in these exemplary polymers do not imply a block copolymer structure; rather, these numbers indicate the total number of specific monomer units, which may be arranged in any order, including blocks of monomers or monomers randomly arranged throughout the polymer. In some, but not all, cases, this is further indicated by a " / " symbol in the formula; however, the absence of a " / " should not be construed as indicating that the polymer is joined in a particular order. In some embodiments of the foregoing polymers 1-64, the monomers designated by the parentheses and integer ("a," "b," "c," "d," "e," or "f") are randomly arranged or dispersed throughout the polymer.

[0115] In the foregoing exemplary polymers, in Formulas 1 and 1A-1C, "a," "b," "e," and "f" are as defined for m1, m2, m3, and m4, respectively; and "c" and "d" are as defined for n1 and n2, respectively. Thus, each of a, b, e, and f is an integer between 0 and 1000 (e.g., between 0 and 500, between 0 and 200, between 0 and 100, or between 0 and 50), provided that the sum of a + b + e + f is greater than 2, e.g., 2-5000, 2-2000, 2-1000, 2-500, 2-100, or 2-50, and the sum of c + d is at least 1 (e.g., 1-2000, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-25, 2-2000, 2-1000, 2-500, 2-200, 2-100, 2-50, or 2-25). In some embodiments, the sum of a + b + e + f is greater than 5 or greater than 10 (e.g., 5-5000, 5-2000, 5-1000, 5-500, 5-100, or 5-50; or 10-5000, 10-2000, 10-1000, 10-500, 10-100, or 10-50). In some embodiments, a + b + e + f is about 10-50 or 20-40. Further, each of c and d is an integer between 0 and 1000 (e.g., between 0 and 500, between 0 and 200, between 0 and 100, between 0 and 50, or between 0 and 25), provided that the sum of c + d is at least 1 (e.g., between 1 and 2000, between 1 and 1000, between 1 and 500, between 1 and 200, between 1 and 100, between 1 and 50, between 1 and 25, between 2 and 2000, between 2 and 1000, between 2 and 500, between 2 and 200, between 2 and 100, between 2 and 50, or between 2 and 25). In some embodiments, the sum of c + d is greater than 5 or greater than 10 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, 5-50, or 5-25; or 10-2000, 10-1000, 10-500, 10-200, 10-100, 10-50, or 10-25). In some embodiments, the sum of c + d is about 20-50 or 20-40. In certain embodiments, a + b + e + f is about 20-50 or 20-40, and the sum of c + d is about 10-50 or 20-40.

[0116] The polymer can have any suitable degree of polymerization (e.g., the sum of m1 + m2 + m3 + m4 + n1 + n2, or the sum of a+b+c+d+e+f, as applicable) as described above with respect to Formulas 1 and 1A-1C. In some cases, the polymer has a degree of polymerization (a+b+c+d+e+f) of about 10 or greater, or about 20 or greater (e.g., about 20 to about 1000, or about 20 to about 100, or about 20 to about 65, or about 20 to 64). In some cases, the polymer has a degree of polymerization of about 50 or greater (e.g., about 50 to about 1000, or about 50 to about 500, or 66 to about 200). In some embodiments, (a+b+c+d+e+f) is about 10-500, eg, about 10-400, about 10-200, or about 10-100 (eg, about 25-100 or about 50-75).

[0117] In some embodiments, (a+b+e+f) is greater than 5 or greater than 10 (e.g., 5-5000, 5-2000, 5-1000, 5-500, 5-100, or 5-50; or 10-5000, 10-2000, 10-1000, 10-500, 10-100, or 10-50). In some embodiments, (a+b+e+f) is about 10-50, 10-30, or 20-40.

[0118] In some embodiments, (c + d) is at least 1 (e.g., 1-2000, 1-1000, 1-500, 1-200, 1-100, 1-50, 1-25, 2-2000, 2-1000, 2-500, 2-200, 2-100, 2-50, or 2-25). In some embodiments, (c + d) is greater than 5 or greater than 10 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, 5-50, or 5-25; or 10-2000, 10-1000, 10-500, 10-200, 10-100, 10-50, or 10-25). In some embodiments, (c + d) is about 20-50 or 20-40.

[0119] In some embodiments, (a+b+e+f) is about 5 to about 65 (e.g., about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, or about 5 to about 10), and (c+d) is about 2 to about 60 (e.g., about 2 to about 50, about 2 to about 40, about 2 to about 30, about 2 to about 20, or about 2 to about 10). In some embodiments, (a+b+e+f) is about 15-30, e.g., about 15-20 or 20-30, and (c+d) is about 30-60, e.g., about 30-50 or 35-50). In some embodiments, (a+b+e+f) is about 55, and (c+d) is about 10. In other embodiments, (a+b+e+f) is about 45, and (c+d) is about 20.

[0120] A polymer can contain any suitable proportions of (a+b), (e+f), and (c+d). In some embodiments, (a+b+e+f) ranges from 10-95% (e.g., 10-75%, 10-65%, 10-50%, 20-95%, 20-75%, 20-65%, 20-50%, 30-95%, 30-75%, 30-65%, or 30-50%) of the total number of polymer units (a+b+c+d+e+f). In other embodiments, (c+d) ranges from 5-90% (e.g., 5-75%, 5-65%, 5-50%, 5-40%, 5-30%, 10-90%, 10-75%, 10-65%, 10-50%, 10-40%, or 10-30%) of the total number of polymer units (a+b+c+d+e+f).

[0121] In some embodiments, the ratio of (a+b+e+f):(c+d) can be about 25 or less (e.g., about 10 or less, about 5 or less, about 3 or less, or about 2 or less), and, optionally, about 0.1 or more, or about 0.2 or more (e.g., about 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, or about 1 or more). For example, in some embodiments, the ratio of (a+b+e+f):(c+d) can be about 0.1 to about 1, about 0.1 to about 0.5, or about 0.5 to about 1. In other embodiments, the ratio of (a+b+e+f):(c+d) can be about 0.3 to about 25, about 0.3 to about 20, about 0.3 to about 10, about 0.3 to about 5, about 0.3 to about 3, or about 0.3 to about 2. In still other embodiments, the ratio of (a+b+e+f):(c+d) can be from about 1 to about 25, from about 1 to about 20, from about 1 to about 10, from about 1 to about 5, from about 5 to about 25, from about 10 to about 25, or from about 15 to about 25. In certain embodiments, the ratio of (a+b+e+f):(c+d) is about 0.5-2 or about 1-2.

[0122] With any of the aforementioned polymer configurations, the polymers can have any suitable ratio of (a+b):(e+f). In some embodiments, the ratio of (a+b):(e+f) is about 0.1 or greater, or about 0.2 or greater, such as about 0.5 or greater, or even 1 or greater (e.g., 1.5 or greater, 2 or greater, 2.5 or greater, 3 or greater, 3.5 or greater, 4 or greater, or 5 or greater). In some embodiments, the ratio of (a+b):(e+f) is about 20 or less (e.g., about 10 or less, about 5 or less, about 2 or less, or even about 1 or less, such as about 0.5 or less or 0.2 or less). In other embodiments, the ratio of (a+b):(e+f) is less than 1 (e.g., 0.75 or less, 0.5 or less, or 0.25 or less). In certain embodiments, the ratio of (a+b):(e+f) is about 0.5 to 5, such as about 0.5-3 or about 1-4.

[0123] The amount of PEG-containing side chains (e+f) can also be expressed as a percentage of the total number of monomers ((e+f) / (a+b+c+d+e+f) x 100). In some embodiments, (e+f) is about 5% or more (e.g., about 10% or more, or about 15% or more) of the total number of monomers. In some embodiments, (e+f) is about 50% or less (e.g., about 40% or less, or 30% or less) of the total number of monomers. In some embodiments, (e+f) is about 5-50%, 5-40%, 5-30%, 5-20%, 10-50%, 10-40%, 10-30%, 10-20%, 15-50%, 15-40%, or 15-30%. The degree of polymerization can be as described above, but in some embodiments, the degree of polymerization is about 25-100, such as about 25-50 (e.g., about 20-45) or about 50-100 (e.g., about 55-75). The relative amounts of other monomers can be as described above.

[0124] Some of the above specific examples of polymers provided by the present disclosure are depicted with specific end groups (e.g., alkylamino or hydrogen); however, any of the above specific structures may include different end groups. For example, any of the above structures may include the R 1 , R 6 or Q groups at either or both ends of the polymer backbone. Thus, provided herein are polymers comprising the structure of any of Polymers 1-64, wherein the terminal hydrogen is an R group as defined for any of Formulas 1A-1C. 1 and / or the terminal alkylamino is substituted with group Q of formula 1A or group -NHR of formula 1B 6 is replaced by

[0125] In some embodiments, the polymer is cationic (i.e., positively charged at pH 7 and 23° C.). As used herein, a "cationic" polymer refers to a polymer that has an overall net positive charge, regardless of whether the polymer contains only cationic monomer units or a combination of cationic and nonionic or anionic monomer units.

[0126] In some embodiments, the polymer has a weight-average molecular weight of about 5 kDa to about 2,000 kDa. The polymer may have a weight-average molecular weight of about 2,000 kDa or less, for example, about 1,800 kDa or less, about 1,600 kDa or less, about 1,400 kDa or less, about 1,200 kDa or less, about 1,000 kDa or less, about 900 kDa or less, about 800 kDa or less, about 700 kDa or less, about 600 kDa or less, about 500 kDa or less, about 100 kDa or less, or about 50 kDa or less. Alternatively, or in addition, the polymer may have a weight-average molecular weight of about 10 kDa or more, for example, about 50 kDa or more, about 100 kDa or more, about 200 kDa or more, about 300 kDa or more, or about 400 kDa or more. Thus, a polymer can have a weight average molecular weight bounded by any two of the aforementioned endpoints. For example, the polymer may be from about 10 kDa to about 50 kDa, from about 10 kDa to about 100 kDa, from about 10 kDa to about 500 kDa, from about 50 kDa to about 500 kDa, from about 100 kDa to about 500 kDa, from about 200 kDa to about 500 kDa, from about 300 kDa to about 500 kDa, from about 400 kDa to about 500 kDa, from about 400 kDa to about 600 kDa, from about 400 kDa to about 700 kDa, from about 400 kDa to about 800 kDa, from about 400 kDa to about 900 kDa, from about 400 kDa to about 1,000 kDa, from about 400 kDa to about 1,200 kDa, or from about 400 kDa to about 1,400 kDa. kDa, about 400 kDa to about 1,600 kDa, about 400 kDa to about 1,800 kDa, about 400 kDa to about 2,000 kDa, about 200 kDa to about 2,000 kDa, about 500 kDa to about 2,000 kDa, or about 800 kDa to about 2,000 kDa.

[0127] The weight average molecular weight can be determined by any suitable technique. Typically, the weight average molecular weight is determined using size exclusion chromatography equipped with a column selected from TSKgel Guard, GMPW, GMPW, G1000PW, and a Waters 2414 (Waters Corporation, Milford, Massachusetts) refractive index detector. Additionally, the weight average molecular weight is determined by calibration using polyethylene oxide / polyethylene glycol standards ranging from 150 to 875,000 daltons.

[0128] In certain aspects, the polymers provided herein have the formula of polymers (a)-(t):

[0129] [ka]

[0130] [ka]

[0131] [ka]

[0132] [ka]

[0133] [ka]

[0134] [ka]

[0135] [ka]

[0136] [ka]

[0137] [ka]

[0138] wherein x and y can be present in any amounts and in any suitable ratio as previously defined for (a+b) and (e+f), respectively.

[0139] The designations of the number of units in these exemplary polymers do not imply a block copolymer structure; rather, these numbers indicate the total number of units, which may be arranged in any order, including blocks of monomers or monomers randomly arranged throughout the polymer. Furthermore, the grouping of monomers with similar side chains in the above formula with a single designation for the total number of units does not imply that both units are present or that they are present in any particular order. For example, the representation:

[0140] [ka]

[0141] is the expression:

[0142] [ka]

[0143] where "x" is equal to the sum of a+b, with the understanding that either a or b can be zero, and that the units corresponding to "a" and "b" can be arranged in any order or randomly dispersed throughout the polymer backbone. The same is true for the number of units containing hydrophobic side chains (equivalent to c+d) and the number of units containing polyalkylene oxide-containing side chains (equivalent to e+f) shown in the formula above.

[0144] Preparation method The present invention also provides methods for preparing the polymers described herein. In some embodiments, the methods include preparing a polymer of Formula 1, the method comprising: (a) Formula 2:

[0145] [ka]

[0146] providing a polymer of (b) Group A of the polymer of formula 2 1 Modify the part to get formula 1:

[0147] [ka]

[0148] All aspects of the polymers of Formula 1 and 2 are as previously disclosed herein. Thus, for example: m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000, 1 + m 2 + m 3 + m 4 The sum of is greater than 2, and m 3 + m 4 The sum of is at least 1; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is at least 1, The symbol " / " indicates that the units separated by it are combined randomly or in any order; R in each case 3a are independently a methylene or ethylene group; R in each case 3bare independently a methylene or ethylene group; R in each case 11 are independently hydrogen or a C1-C4 alkyl or alkenyl group, any of which may be optionally substituted with one or more substituents, optionally hydrogen or a C1-C3 alkyl group; each X 1 are independently -C(O)O- and -C(O)NR 11 -, -C(O)-, -S(O)(O)- or a bond; X in each case 2 is a hydrophobic side chain; A in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 2 and B in each case 1 are independently: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2)p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -Z-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and In the formula, each of p1 to p3 is independently an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer of 0 to 5 (e.g., 1, 2, 3, 4, or 5); s1 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s2 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R in each instance 2 are independently C1-C 12 (e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; Z may be present, and when present, is —C(O)—, —C(O)O—, —S(O)(O)—, —C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or optionally substituted aryl or heteroaryl; 4 are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, —S(O)(O)— or a bond; and R 5is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof). In some embodiments, p1, p3, or both are greater than p2, and / or p1, p3, or both are integers from 3 to 5 (e.g., 3, 4, or 5), and optionally, p2 is an integer of 1 or 2. In some embodiments, Z is present and is -C(O)-. In other embodiments, Z is absent. All aspects of Formulas 1 and 2 are as otherwise described herein for the polymers of the invention, including any and all embodiments of the structures of Formulas 1 and 1A-1C described herein.

[0149] The polymer comprising the structure of Formula 1 can be any polymer described herein, including Formulas 1A, 1B, and 1C, and any and all embodiments thereof described with respect to the polymers of the invention.

[0150] The polymer comprising the structure of Formula 2 can be any polymer described herein, including those of Formulas 2A, 2B, 2C:

[0151] [ka]

[0152] During the ceremony, Q is the formula:

[0153] [ka]

[0154] and c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; R 1 is hydrogen, an aryl group, a heterocyclic group, C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C12 cycloalkenyl groups, any of which may be substituted with one or more substituents; and R 6 is hydrogen, amino group, aryl group, heterocyclic group, C1-C 12 Alkyl groups, C1-C 12 Heteroalkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 cycloalkenyl groups, any of which may be substituted with one or more substituents or tissue- or cell-specific targeting moieties;

[0155] [ka]

[0156] During the ceremony, R 1 is hydrogen, an aryl group, a heterocyclic group, C1-C 12 Alkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 cycloalkenyl groups, any of which may be substituted with one or more substituents; and R 6 is hydrogen, amino group, aryl group, heterocyclic group, C1-C 12 Alkyl groups, C1-C 12 Heteroalkyl groups, C2-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 cycloalkenyl groups, any of which may be substituted with one or more substituents or tissue- or cell-specific targeting moieties; and

[0157] [ka]

[0158] wherein all other aspects of Formulas 2A, 2B, and 2C are as described for Formulas 1 and 1A-1C, including any and all embodiments thereof, and any and all embodiments thereof described for the polymers of the invention.

[0159] A of the polymer of formula 2 1 The group designated as B is modified by any suitable means to form B of the polymer of Formula 1. 1 For example, A 1 A group designated as may be modified by Michael addition reaction, epoxide ring opening, amide formation with an ester (e.g., an activated ester), nitrogen-halogen exchange reaction, activated carbonate or carbamate reaction, urea formation, thiourea formation, guanidine formation, sulfonamide formation, reductive amination, nucleophilic alkylation, nucleophilic aromatic substitution, or substitution reaction. 1 In a preferred embodiment, the group designated A is modified by amide formation with an activated ester. 1 Groups designated as are modified by Michael addition reactions.

[0160] In one embodiment, A of the polymer comprising the structure of Formula 2 1 The group is modified by a Michael addition reaction between a polymer comprising the structure of Formula 2 and an α,β-unsaturated carbonyl compound. As used herein, the term "Michael addition" refers to the nucleophilic addition of a nucleophile (e.g., a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) of the polymer to an α,β-unsaturated carbonyl compound. Thus, the Michael addition reaction is between a polymer comprising the structure of Formula 2 and an α,β-unsaturated carbonyl compound. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0161] The α,β-unsaturated carbonyl compound can be any α,β-unsaturated carbonyl compound that can undergo Michael addition from a nucleophile. In some embodiments, the α,β-unsaturated carbonyl compound is an acrylate, an acrylamide, a vinyl sulfone, or a combination thereof. Thus, the Michael addition reaction can be between a polymer comprising the structure of Formula 2 and an acrylate, an acrylamide, a vinyl sulfone, or a combination thereof. Thus, in some embodiments, the method comprises contacting a polymer comprising the structure of Formula 2 with an acrylate; contacting a polymer comprising the structure of Formula 2 with an acrylamide; or contacting a polymer comprising the structure of Formula 2 with a vinyl sulfone.

[0162] A 1 In embodiments where groups designated as: are modified by a Michael addition reaction, they have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 B 1 and generating a group designated as: In the formula, each of p1 to p3 is independently an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer of 0 to 5 (e.g., 1, 2, 3, 4, or 5); s1 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R in each instance 2 are independently C1-C 12 (e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; 4are independently —C(O)O—, —C(O)—, —C(O)NH—, —O—, or —S(O)(O)—; and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).

[0163] Examples of acrylates, acrylamides, and vinyl sulfones suitable for use are those of the formula:

[0164] [ka]

[0165] In the formula, R 5 is as described for any of Formulas 1 and 1A-1C Contains acrylates.

[0166] In some embodiments, the Michael addition reaction is promoted by an acid and / or a base. The acid and / or base can be any suitable acid and / or base having any suitable pKa. The acid and / or base can be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0167] In some embodiments, the Michael addition reaction is promoted by an acid. The acid can be a Bronsted acid or a Lewis acid. In embodiments where the acid is a Bronsted acid, the acid can be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfon)imide or p-toluenesulfonic acid.

[0168] In some embodiments, the Michael addition reaction is promoted by a base. The base can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, or a derivative thereof.

[0169] In some embodiments, the Michael addition reaction is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents that can solubilize the polymer and the α,β-unsaturated carbonyl compound to be reacted. For example, the solvent can include water, a protic organic solvent, and / or an aprotic organic solvent. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0170] In some embodiments, A of the polymer comprising the structure of Formula 2 1 The group is modified by amide formation between a polymer comprising the structure of Formula 2 and an ester (e.g., an activated ester). As used herein, the term "activated ester" refers to any electron-deficient ester suitable for amide bond formation, many of which are known in the art. For example, the activated ester can be an N-hydroxysuccinimide (NHS) ester or a fluorophenol ester (e.g., a tetrafluorophenol ester or a pentafluorophenol ester). In some embodiments, the amide formation is between a nitrogen anion, a nitrogen atom, or a combination thereof of a polymer comprising the structure of Formula 2 and an activated ester.

[0171] A 1 In embodiments where groups designated as: are modified by amide formation with an ester (e.g., an activated ester), they have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s1 -R 5 ; -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -C(O)-(CH2) s2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 B 1 Generate a group designated as In the formula, each of p1 to p3 is independently an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s1 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s2 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R in each instance 2 are independently C1-C 12(e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; 4 are independently —C(O)O—, —C(O)—, —C(O)NH—, —CH—OC(O)—O—CH—, —OC(O)—O—, —O—, or —S(O)(O)—; and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).

[0172] Examples of activated esters suitable for use include those of the formula:

[0173] [ka]

[0174] In the formula, E A is an activated ester, and s1 and R 5 is as described with respect to any of Formulas 1 and 1A-1C. For example, activated esters for use include esters of the formula:

[0175] [ka]

[0176] In the formula, s1 and R 5 may include an NHS ester of, wherein: is as described with respect to any of Formulas 1 and 1A-1C. In some embodiments, the active ester for use is of the formula:

[0177] [ka]

[0178] is.

[0179] In some embodiments, the amide-forming reaction is promoted by an acid and / or a base. The acid and / or base can be any suitable acid and / or base having any suitable pKa. The acid and / or base can be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0180] In some embodiments, the amide-forming reaction is promoted by an acid. The acid can be a Bronsted acid or a Lewis acid. In embodiments where the acid is a Bronsted acid, the acid can be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfon)imide or p-toluenesulfonic acid.

[0181] In some embodiments, the amide-forming reaction is promoted by a base. The base can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, or a derivative thereof.

[0182] In some embodiments, the amide-forming reaction is carried out in a solvent.The solvent can be any suitable solvent or mixture of solvents that can solubilize the polymer and ester (e.g., activated ester) compound to be reacted.For example, the solvent can include water, a protic organic solvent, and / or an aprotic organic solvent.An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0183] In one embodiment, A of the polymer comprising the structure of Formula 2 1 The group is modified by an epoxide ring-opening reaction between the polymer and an epoxide compound. As used herein, the term "epoxide ring-opening" refers to the nucleophilic addition of a nucleophile (e.g., a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) of the polymer to the epoxide compound, thereby opening the epoxide ring. Thus, the epoxide ring-opening reaction is between the polymer and the epoxide compound. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0184] A 1 In embodiments where groups designated as: are modified by an epoxide ring-opening reaction, they may be represented by the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -CH2-CHOH-R 5 B 1 and generating a group designated as: In the formula, each of p1 to p3 is independently an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); R 2 are independently C1-C 12 (e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).

[0185] Examples of epoxides suitable for use include those of the formula:

[0186] [ka]

[0187] In the formula, R 5 is as described with respect to any of Formulas 1 and 1A-1C.

[0188] In some embodiments, the epoxide ring-opening reaction is promoted by an acid and / or a base. The acid and / or base can be any suitable acid and / or base having any suitable pKa. The acid and / or base can be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0189] In some embodiments, the epoxide ring-opening reaction is promoted by an acid. The acid can be a Bronsted acid or a Lewis acid. In embodiments where the acid is a Bronsted acid, the acid can be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfon)imide or p-toluenesulfonic acid.

[0190] In some embodiments, the epoxide ring-opening reaction is promoted by a base. The base can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, or a derivative thereof.

[0191] In some embodiments, the epoxide ring-opening reaction is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents that can solubilize the polymer and epoxide compound to be reacted. For example, the solvent can include water, a protic organic solvent, and / or an aprotic organic solvent. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0192] In one embodiment, A of the polymer comprising the structure of Formula 2 1 The group is modified by a substitution reaction between the polymer and a compound containing a leaving group (e.g., chlorine atom, bromine atom, iodine atom, tosylate, triflate, mesylate, etc.). As used herein, the term "substitution" refers to the nucleophilic addition of a nucleophile (e.g., a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) of the polymer to a compound containing a leaving group. Thus, the substitution reaction is between the polymer and a compound containing a leaving group. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0193] A 1 In embodiments where groups designated as: are modified by substitution reactions, they may be modified to have the formula: -(CH2) p1 -[NH-(CH2) p2 -] r1 NH-(CH2) p3 -NR 2 -(CH2) s1 -R 5 B 1 and generating a group designated as: In the formula, each of p1 to p3 is independently an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s1 is an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); and R in each instance 2 are independently C1-C 12(e.g., C1-C6, C1-C3, C2, or C1) alkyl groups, C2-C 12 (e.g., C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkyl groups, or C3-C 12 (e.g., C3-C8 or C3-C6) cycloalkenyl group; and R 5 is a polyalkylene oxide-containing group (e.g., polyethylene oxide, polypropylene oxide, or a combination thereof).

[0194] Examples of compounds containing leaving groups suitable for use include those of the formula:

[0195] [ka]

[0196] In the formula, LG is a leaving group (e.g., chlorine atom, bromine atom, iodine atom, tosylate, triflate, mesylate, etc.), and R 5 is as described with respect to any of Formulas 1 and 1A-1C.

[0197] In some embodiments, the substitution reaction is promoted by an acid and / or a base. The acid and / or base may be any suitable acid and / or base having any suitable pKa. The acid and / or base may be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0198] In some embodiments, the substitution reaction is promoted by an acid. The acid can be a Bronsted acid or a Lewis acid. In embodiments where the acid is a Bronsted acid, the acid can be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfon)imide or p-toluenesulfonic acid.

[0199] In some embodiments, the substitution reaction is promoted by a base. The base can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, or a derivative thereof.

[0200] In some embodiments, the substitution reaction is carried out in a solvent.The solvent can be any suitable solvent or mixture of solvents that can solubilize the polymer to be reacted and the compound that comprises a leaving group.For example, the solvent can include water, a protic organic solvent, and / or an aprotic organic solvent.An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0201] In some embodiments, the method further comprises isolating the polymer comprising the structure of Formula 1. The polymer comprising the structure of Formula 1 can be isolated by any suitable method. For example, the polymer comprising the structure of Formula 1 can be isolated by extraction, crystallization, recrystallization, column chromatography, filtration, or any combination thereof.

[0202] Polymers comprising the structure of Formula 2 can be made by any suitable method. For example, polymers comprising the structure of Formula 2:

[0203] [ka]

[0204] Polymers containing the structure: (I) Formula 3:

[0205] [ka]

[0206] The polymer (a) is 11 A 1 and (b) a compound of formula HNX 2 or HOX 2 simultaneously or in any order; or (II) Formula 4

[0207] [ka]

[0208] The polymer of formula HNR 11 A 1 reacting with a compound of; may be prepared by a method comprising During the ceremony, p 1 is an integer between 1 and 2000 (e.g., between 1 and 1000, between 1 and 500, between 1 and 200, between 1 and 100, between 5 and 2000, between 5 and 1000, between 5 and 500, between 5 and 200, or between 5 and 100); p 2 is an integer between 1 and 2000 (e.g., between 1 and 1000, between 1 and 500, between 1 and 200, between 1 and 100, between 2 and 2000, between 2 and 1000, between 2 and 500, between 2 and 200, or between 2 and 100); Each R 3 are independently a methylene or ethylene group; and m 1 , m 2 , n 1 , n 2 , R 3a , R 3b , R 11 , X 1 , X2 , and A 1 is as described above with respect to Equations 1 and 1A-1C.

[0209] The method comprises reacting a structure of Formula 3 or Formula 4 with a compound of Formula HNR 11 A 1 and optionally a compound of formula HNX 2 or HOX 2 More specifically, the structure of Formula 3 can include combining with a compound of formula (a) HNR 11 A 1 and (b) a compound of formula HNX 2 or HOX 2 can be combined (reacted) simultaneously or in any order to provide a compound of formula 2. 2 Compounds of formula 4 that already contain a group have the formula HNR 11 A 1 to provide a compound of formula 2.

[0210] All other substituents and aspects of Formulas 3 and 4 are as described herein for the polymers of the invention (eg, Formulas 1 and 1A-1C), including any and all embodiments thereof.

[0211] HNR 11 A 1 and the formula H2NX 2 or HOX 2can be added to the compound of Formula 3 or 4 in any suitable manner and amount depending on the desired degree of substitution. In some embodiments, about 1-400 equivalents (e.g., about 1-350, 1-300, 1-250, 1-200, 1-150, 1-100, 1-50, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10-50, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100, 2 0-50, 30-400, 30-350, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 40-400, 40-350, 40-300, 40-250, 40-200, 40-150, 40-100, 40-50, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, or 50-100 equivalents) of formula HNX 2 or HOX 2 is added to the polymer of Formula 3. Also, in some embodiments, about 1-400 equivalents (e.g., about 1-350, 1-300, 1-250, 1-200, 1-150, 1-100, 1-50, 10-400, 10-350, 10-300, 10-250, 10-200, 10-150, 10-100, 10-50, 20-400, 20-350, 20-300, 20-250, 20-200, 20-150, 20-100 , 20-50, 30-400, 30-350, 30-300, 30-250, 30-200, 30-150, 30-100, 30-50, 40-400, 40-350, 40-300, 40-250, 40-200, 40-150, 40-100, 40-50, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, or 50-100 equivalents) of the formula HNR 11 A 1 is added to a polymer of formula 3 or formula 4.

[0212] This method is based on the formula HNR 11 A 1 and compounds of formula HNX 2 or HOX 2to a polymer of Formula 3. 11 A 1 and compounds of formula HNX 2 or HOX 2 Compounds of formula HNR can be present in the reaction mixture in any suitable ratio. 11 A 1 and compounds of formula HNX 2 or HOX 2 The compounds may be present in a molar ratio of about 150:1 to about 1:150. In some embodiments, a ratio of about 150:1 to about 1:1, e.g., about 50:1 to about 1:1 (e.g., about 25:1 to about 1:1, about 10:1 to about 1:1, about 5:1 to about 1:1, or about 2.5:1 to about 1:1) is used. In other embodiments, the ratio is about 1:150 to about 1:1, e.g., about 1:50 to about 1:1 (e.g., about 1:25 to about 1:1, about 1:10 to about 1:1, about 1:5 to about 1:1, or about 1:2.5 to about 1:1). In still other embodiments, the ratio is about 1:10 to about 1:150, about 1:40 to about 1:150, or about 1:80 to about 1:150.

[0213] In some embodiments, the polymer comprising the structure of Formula 3 or Formula 4 has Formula 3A or Formula 4A, respectively:

[0214] [ka]

[0215] wherein c, Y, R 1 , and R 6 is as described above for the polymers of Formula 1A and 2A, including any and all embodiments thereof; and p 1 , p 2 , R 3 , X 1 , and X 2 is as described above for Equations 3 and 4.

[0216] In some embodiments, the polymer comprising the structure of Formula 3 or Formula 4 has Formula 3B or Formula 4B, respectively:

[0217] [ka]

[0218] is a polymer of the formula: 1 , p 2 , R 3 , X 1 , and X 2 is as described above for Equations 3 and 4.

[0219] composition The polymers provided herein can be used for any application. However, it is believed that the polymers are particularly useful for delivering nucleic acids and / or polypeptides (e.g., proteins) to cells. The polymers provided herein can provide such delivery vehicles in the form of nanoparticles. Without wishing to be bound by any particular theory or mechanism of action, it is believed that the polymers described herein, in some embodiments, can provide nanoparticles with improved stability, particularly in physiological environments, such as body fluids (e.g., blood, serum, or CSF). Stability can be measured as a factor of change in particle size over time, where a larger change in particle size over a given period indicates less stability than a smaller change in particle size over a given period.

[0220] Thus, provided herein are compositions comprising the polymers described herein and nucleic acids and / or polypeptides (e.g., proteins). In some embodiments, the compositions contain nucleic acids. Any nucleic acid may be used. An exemplary list of nucleic acids includes guide and / or donor nucleic acids for CRISPR systems, siRNA, microRNA, interfering RNA or RNAi, dsRNA, mRNA, DNA vectors, ribozymes, antisense polynucleotides, and DNA expression cassettes encoding siRNA, microRNA, dsRNA, ribozymes, or antisense nucleic acids. SiRNAs typically contain 15-50 base pairs, and preferably 19-25 base pairs, and comprise a double-stranded structure with a nucleotide sequence identical or nearly identical to a target gene or RNA expressed in a cell. siRNAs can consist of two annealed polynucleotides or one polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small, non-coding polynucleotides, approximately 22 nucleotides in length, that direct the destruction or translational repression of their mRNA targets. Antisense polynucleotides contain sequences that preferentially target genes or mRNAs. Antisense polynucleotides include, but are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA, RNA, etc. Polynucleotide-based expression inhibitors may be polymerized in vitro and may contain recombinant or chimeric sequences, or derivatives of these groups. Polynucleotide-based expression inhibitors may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and / or gene is inhibited.

[0221] The composition can also contain any protein for delivery in addition to or instead of nucleic acid. The polypeptide can be any suitable polypeptide. For example, the polypeptide can be a zinc finger nuclease, a transcription activator-like effector nuclease ("TALEN"), a recombinase, a deaminase, an endonuclease, or a combination thereof. In some embodiments, the polypeptide is an RNA-guided endonuclease (e.g., a Cas9 polypeptide, a Cpf1 polypeptide, or a variant thereof) or a DNA recombinase (e.g., a Cre polypeptide).

[0222] The polymers provided herein are believed to be particularly useful for delivering one or more components of the CRISPR system.Therefore, in some embodiments, the composition contains guide RNA, RNA-guided endonuclease or the nucleic acid encoding it, and / or donor nucleic acid.The composition can contain one, two, or all three components together with the polymers described herein.In addition, the composition can contain multiple guide RNAs, RNA-guided endonuclease or the nucleic acid encoding it, and / or donor nucleic acid.For example, multiple different guide RNAs for different target sites can be included, optionally with multiple different donor nucleic acids and even multiple different RNA-guided endonuclease or the nucleic acid encoding it.

[0223] Furthermore, the components of CRISPR system can be combined with each other (when there are multiple components) and polymer in any specific manner or order.In some embodiments, guide RNA is combined with RNA endonuclease before being combined with polymer.In addition, or instead, guide RNA can be linked (covalently or non-covalently) with donor nucleic acid before being combined with polymer.

[0224] The compositions are not limited with respect to any particular CRISPR system (i.e., any particular guide RNA, RNA-guided endonuclease, or donor nucleic acid), many of which are known. Nevertheless, for further explanation, components of some such systems are described below.

[0225] The polymers provided herein can be used with additional polymers. The polymers can be combined in any suitable manner (e.g., blended) to provide polymeric nanoparticles. The composition can include any suitable amount of a first polymer (provided herein) and a second polymer. For example, the composition can include a weight ratio of the first polymer to the second polymer of about 1:99 to 99:1. In some embodiments, the composition includes a weight ratio of the first polymer to the second polymer of about 1:1 to about 1:20 (e.g., about 1:1 to about 1:15, or about 1:1 to about 1:10). Relative amounts can also be expressed as a weight percent composition. In some embodiments, the composition includes about 1 wt.% or more (e.g., about 5 wt.% or more, about 10 wt.% or more, about 20 wt.% or more, about 30 wt.% or more, or about 40 wt.% or more) of the first polymer based on the total weight of the combined first and second polymers. Also, in some embodiments, the composition comprises about 60 wt.% or less (e.g., about 50 wt.% or less) of the first polymer based on the total weight of the first and second polymers combined. The foregoing percent compositions can also be stated as ranges. Thus, for example, in some embodiments, the composition comprises about 1 wt.% to about 60 wt.% of the first polymer based on the total weight of the first and second polymers (e.g., about 5 wt.% to about 60 wt.%, about 10 wt.% to about 60 wt.%, about 20 wt.% to about 60 wt.%, about 30 wt.% to about 60 wt.%, about 5 wt.% to about 50 wt.%, about 10 wt.% to about 50 wt.%, about 20 wt.% to about 50 wt.%, etc.).

[0226] The composition comprising the first and optionally second polymers can further comprise any carrier, typically an aqueous carrier, suitable for administration to a cell or host, e.g., a mammal or human. The polymer(s) in the carrier, if present, form nanoparticles that partially or completely encapsulate the compound to be delivered.

[0227] The various components of the polymer composition, including examples of second polymers, nucleic acids, and polypeptide compounds, are described in more detail below.

[0228] Additional Polymers In one embodiment, a composition is provided that includes a first polymer and a second polymer. The first polymer is as described herein, for example, a polymer that includes (i) a monomer unit that includes a hydrophobic side chain; (ii) a monomer unit that includes a side chain that includes a polyamine group and a polyalkylene oxide group; and optionally (iii) a monomer unit that includes a side chain that includes a polyamine group but does not include a polyalkylene oxide group. All aspects and embodiments of the first polymer of the composition are as described above.

[0229] The second polymer can be any polymer suitable for forming nanoparticles for nucleic acid or polypeptide delivery. In some embodiments, the second polymer comprises (a) a monomer unit having a side chain comprising a hydrophobic group, and (b) a monomer unit having a side chain comprising an oligoamine or polyamine. In some embodiments, the second polymer also comprises other monomers, such as a monomer unit having a side chain comprising an ionic group, optionally with a pKa of less than 7. In some embodiments, the second polymer comprises a hydrolyzable polymer backbone, such as a polyamide, a poly-N-alkylamide, a polyester, a polycarbonate, a polycarbamate, or a combination thereof. In some embodiments, the hydrolyzable polymer backbone comprises a polyamide. In some embodiments, the second polymer does not comprise a polyalkylene oxide moiety. In some embodiments, the second polymer is a polymer of any of WO2021 / 217082, WO2020219776, WO2020086910A1, WO2020243370, or WO2019210326A2.

[0230] The monomer unit having a side chain containing a hydrophobic group can contain any hydrophobic group. Examples of hydrophobic groups include, for example, C1-C 12 (For example, C2-C 12 , C2-C 10 , C2-C8, C2-C6, C3-C 12 , C3-C 10 , C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C8, C8-C 12 , C8-C 10 ,) alkyl groups, C2-C 12 (e.g., C2-C6, C3-C 12 , C3-C10, C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C8, C8-C 12 , C8-C 10 ) alkenyl group, or C3-C12 (C3-C10, C3-C8, C3-C6, C4-C 12 , C4-C 10 , C4-C8, C4-C6, C6-C 12 , C6-C8, C8-C 12 , C8-C 10 ) cycloalkyl or cycloalkenyl groups. In some embodiments, the hydrophobic group is a C4-C 12 The hydrophobic group may comprise an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. The hydrophobic group may also comprise a heteroatom (e.g., a heteroalkyl, heteroalkenyl, or heterocyclyl group) or an aryl group. In some embodiments, the hydrophobic group comprises fewer than 8 carbons or fewer than 6 carbons. For example, the hydrophobic group may comprise a C2-C8 or C2-C6 (e.g., C3-C8 or C3-C6) alkyl group. The alkyl or alkenyl group may be branched or linear. In any of the foregoing embodiments, the hydrophobic group may be linked to the polymer backbone directly or via a linkage comprising, for example, an ester, amide, or ether group, optionally further comprising an alkylene linker (e.g., a methylene or ethylene linker).

[0231] The second polymer also comprises a monomer unit having a side chain comprising an oligoamine or polyamine. As used herein, the term "oligoamine" refers to any chemical moiety having two or three amine groups, and the term "polyamine" refers to any chemical moiety having four or more (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc.) amine groups. The amine groups can be primary amine groups, secondary amine groups, tertiary amine groups, or any combination thereof. In some embodiments, the oligoamine or polyamine has the structure: -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2 or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2、 -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR2 -CH2-CHOH-R 5 ; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5 }-(CH2) q1 -] r1 NR 2 2; -(CH2) p1-[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2. -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and In the formula, p1 to p4, q1 to q6, r1 and r2, and s1 to s4 each independently represent an integer of 1 to 5; 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 is the second R 2 and R combine to form a heterocyclic group; 4 is independently —C(O)O—, —C(O)NH—, —CH—OC(O)—O—CH—, —O—, or —S(O)(O)—; and R 5 are independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroalkyl, heterocyclic, or combinations thereof, optionally containing 2 to 8 tertiary amines, or a substituent containing a tissue- or cell-specific targeting moiety. In some embodiments, the oligoamine or polyamine has the structure: -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NHR 2 , -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -(CH2) s1 -R 5 ; -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and In the formula, each of p1 to p3 is independently an integer of 1 to 5; r1 is an integer of 0 to 5; s1 is an integer of 1 to 5; and R 2 are independently hydrogen or C1-C12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 is the second R 2 and R combine to form a heterocyclic group; 4 is independently —C(O)O—, —C(O)NH—, —CH—OC(O)—O—CH—, —O—, or —S(O)(O)—; and R 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 2 to 8 tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

[0232] For example, in some embodiments, the composition comprises a compound of Formula 5:

[0233] [ka]

[0234] and further comprising a polymer of During the ceremony: m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000, 1 + m 2 + m 3 + m 4 The sum of is greater than 5; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is greater than 2; The symbol " / " indicates that the units separated by it are combined randomly or in any order; R in each case 3a are independently a methylene or ethylene group; R in each case 3b are independently a methylene or ethylene group; each X 1 are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; R in each case 13 are independently hydrogen, an aryl group, a heterocyclic group, or a C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, any of which may be substituted with one or more substituents; X in each case 2 are independently C1-C, optionally containing one or more primary, secondary, or tertiary amines. 12 alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof; any of which may be substituted with one or more substituents; A 1 and A 2 are each independently expressed as -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2 ;or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 is the basis of B 1 and B 2 are each independently -(CH2) p1 -[NR 2 -(CH2) q1 -]r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2、 -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH(CONH2)-(CH2) s1 -R 5 or -(CH2) p1 -[NR 2 -(CH2)q1 -] r1 NR 2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and In the formula, p1 to p4, q1 to q6, r1 and r2, and s1 to s4 each independently represent an integer of 1 to 5; 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 is the second R 2 and R combine to form a heterocyclic group; 4 is independently —C(O)O—, —C(O)NH—, —CH—OC(O)—O—CH—, —O—, or —S(O)(O)—; and R 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 2 to 8 tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

[0235] Alternatively, or in addition, in some embodiments, the composition comprises a compound of Formula 5':

[0236] [ka]

[0237] and further comprising a polymer of During the ceremony: m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000, 1 + m 2 + m 3 + m 4 The sum of is greater than 5; n 1 and n 2Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is greater than 2; The symbol " / " indicates that the units separated by it are combined randomly or in any order; R in each case 3a are independently a methylene or ethylene group; R in each case 3b are independently a methylene or ethylene group; each X 1 are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; R in each case 13 are independently hydrogen, an aryl group, a heterocyclic group, or a C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, any of which may be substituted with one or more substituents; X in each case 2 are independently C1-C, optionally containing one or more primary, secondary, or tertiary amines. 12 alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof; any of which may be substituted with one or more substituents; A 1 and A 2 are each independently expressed as -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NHR 2 is the basis of B 1 and B 2 are each independently -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3-NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -(CH2) s1 -R 5 ; -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 5 ;or -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NR 2 -CH(CONH2)-(CH2) s1 -R 4 -R 5 and In the formula, each of p1 to p3 is independently an integer of 1 to 5; r1 is an integer of 0 to 5; s1 is an integer of 1 to 5; and R 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 is the second R 2 and R combine to form a heterocyclic group;4 is independently —C(O)O—, —C(O)NH—, —CH—OC(O)—O—CH—, —O—, or —S(O)(O)—; and R 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 2 to 8 tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

[0238] According to formula 5 or 5', m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000 (e.g., 0 to 500, 0 to 200, 0 to 100, or 0 to 50), 1 + m 2 + m 3 + m 4 The sum of m is greater than 5, e.g., 5-5000, 5-2000, 5-1000, 5-500, 5-100, or 5-50. 1 + m 2 + m 3 + m 4 The sum of n is greater than 10 or greater than 20 (e.g., 10-5000, 10-2000, 10-1000, 10-500, 10-100, or 10-50; or 20-5000, 20-2000, 20-1000, 20-500, 20-100, or 20-50). 1 and n 2 Each of n is an integer from 0 to 1000 (e.g., 0 to 500, 0 to 200, 0 to 100, 0 to 50, or 0 to 25), provided that 1 + n 2 The sum of n is greater than 2 (e.g., 2-2000, 2-1000, 2-500, 2-200, 2-100, 2-50, or 2-25). 1 + n 2is greater than 5, or greater than 10 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, 5-50, or 5-25; or 10-2000, 10-1000, 10-500, 10-200, 10-100, 10-50, or 10-25). 1 , A 2 , B 1 and / or B. 2 and the second polymer comprises at least some monomer units comprising the group X 1 and / or X 2 In some embodiments, the monomeric units comprise at least some of the monomeric units comprising m 1 and m 2 is zero, so that the second polymer is A 1 or A 2 In some embodiments, m 3 and m 4 is zero, so that the second polymer is B 1 or B 2 Does not contain groups.

[0239] The second polymer can include any suitable ratio of A and B monomers to X monomers. In some embodiments, the second polymer has a ratio of A and B monomers to X monomers (e.g., m 1 +m 2 +m 3 +m 4 ) / (n 1 +n 2 For example, the ratio of A and B monomers to X monomer can be from about 1 to about 25, from about 1 to about 20, from about 1 to about 10, from about 1 to about 5, from about 5 to about 25, from about 10 to about 25, or from about 15 to about 25.

[0240] In embodiments where the second polymer comprises both A and B monomers, the second polymer can comprise any suitable ratio of A monomers to B monomers. In some embodiments, the ratio of A monomers to B monomers (e.g., (m 1 +m 2 ) / (m 3 +m 4 )) can be about 20 or less (e.g., about 10 or less, about 5 or less, about 2 or less, or even about 1 or less). In some embodiments, (m 1 +m 2 ) / (m 3 +m 4 ) is about 0.2 or greater, for example, about 0.5 or greater.

[0241] The second polymer can be any suitable structural type.For example, the second polymer can be an alternating polymer, a random polymer, a block polymer, a graft polymer, a linear polymer, a branched polymer, a cyclic polymer, or a combination thereof.In some embodiments, the second polymer is a random polymer, a block polymer, a graft polymer, or a combination thereof.

[0242] Thus, in the structures of formulas 5 and 5′, the monomers (their respective side chains A 1 , A 2 , B 1 , B 2 , X 1 , and X 2 The integers m may be arranged randomly or in any order. 1 , m 2 , m 3 , m 4 , n 1 , and n 2 The -A simply indicates the number of each monomer that appears in the overall chain, and does not necessarily imply or represent any particular order or block of these monomers, although blocks or stretches of certain monomers may be present in some embodiments. For example, a structure of formula 5 or 5' may have -A 1 -A 2 -B1 -B 2 -, -A 2 -A 1 -B 2 -B 1 -, -A 1 -B 1 -A 2 -B 2 - etc. Additionally, the second polymer may comprise blocks of A and / or B polymers (e.g., [A monomer] m1+m2 -[B monomer] m3+m4 ) in any order. The second polymer may comprise individual X monomers interspersed with A and B monomers (e.g., -AXB-, -ABX-, -BXA, etc.), or the second polymer may be "capped" with one or more X monomers (e.g., blocks of X monomers) at one or both ends of the polymer. Similarly, if the second polymer comprises blocks of A and / or B monomers, the second polymer may comprise blocks of X monomers interspersed between blocks of A and / or B monomers, or the second polymer may be "capped" with one or more X monomers (e.g., blocks of X monomers) at one or both ends of the polymer. In some embodiments, the polypeptide (e.g., polyaspartamide) backbone is arranged in an alpha / beta configuration, such that "1" and "2" monomers alternate (e.g., -A 1 -A 2 -B 1 -B 2 -, -A 2 -A 1 -B 2 -B 1 -, -A 1 -B 2 -B 1 -A 2 -, -A 2 -B 1 -B 2 -A 1 -, -B 1 -A 2 -B 1 -A 2 -etc.), where the second polymer is capped with X monomers or X monomers are interspersed throughout. However, the "A" and "B" side chains (e.g., A 1 / A2 and B 1 / B 2 ) may be randomly dispersed throughout the polymer backbone.

[0243] In the polymer structure, R 3a and R 3b are each independently a methylene or ethylene group. In some embodiments, R 3a is an ethylene group, and R 3b is a methylene group; or R 3a is a methylene group, and R 3b is an ethylene group. In some embodiments, R 3a and R 3b are each an ethylene group. In some embodiments, R 3a and R 3b are each methylene groups.

[0244] In the polymers described herein, each X 1 The groups are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond. 1 The groups may be the same or different from each other. In some embodiments, X 1 is -C(O)NR 13 In some embodiments, X 1 is -C(O)O-.

[0245] R in each case 13 are independently hydrogen or C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12(e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group, aryl group, or heterocyclic group (e.g., 3-12, 3-10, 3-8, or 3-6 membered heterocyclic group containing 1, 2, or 3 heteroatoms), any of which can be substituted with one or more substituents. 13 may be linear or branched, C1-C 12 Alkyl groups (e.g., C1-C 10 alkyl group; C1-C8 alkyl group; C1-C6 alkyl group; C1-C4 alkyl group, C1-C3 alkyl group, or C1 or C2 alkyl group). In some embodiments, each R 13 is methyl or hydrogen. In some embodiments, R 13 is methyl; in other embodiments, R 13 is hydrogen. Each R 13 are independently selected and may be the same or different; however, in some embodiments, each R 13 are identical (e.g., all methyl or all hydrogen).

[0246] X in each case 2 are independently C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl, aryl, or heterocyclic groups (e.g., 3-12, 3-10, 3-8, or 3-6 membered heterocyclic groups containing 1, 2, or 3 heteroatoms), or combinations thereof, any of which may be substituted with one or more substituents. In some embodiments, X 2 may optionally contain one or more primary, secondary, or tertiary amines. Thus, each X 2are independently selected and therefore may be the same or different from one another. In some embodiments, each occurrence of X 2 are independently C1-C, optionally containing one or more primary, secondary, or tertiary amines. 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl groups, or combinations thereof. In some embodiments, one or more (or all) X 2 The groups are independently C2-C 12 (e.g., C3-C 12 , C3-C8, C3-C6, C4-C 12 , C4-C6, C6-C 12 , or C8-C 12 ) alkyl or alkenyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, C4-C 12 , C4-C6, C6-C 12 , or C8-C 12 ) cycloalkenyl groups. In other embodiments, one or more (or all) X 2 The groups are independently C1-C8 (e.g., C1-C6, C1-C4, C1-C 3、 It can be a C2-C8, or C2-C6) alkyl group. Any of the foregoing alkyl or alkenyl groups can be straight-chain or branched.

[0247] Base A 1 and A 2 are independently selected and therefore may be the same or different from one another. 1 and B 2 are independently selected and therefore may be the same or different from one another. However, in some embodiments, A 1 and A 2are identical and / or B 1 and B 2 are identical.

[0248] Base A 1 , A 2 , B 1 , and B 2 In the formula (I), integers p1 to p4 (i.e., p1, p2, p3, and p4), q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6), r1, r2, and s1 to s4 (i.e., s1, s2, s3, and s4) are each independently an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5). However, in some embodiments, r1 can be an integer of 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5). In some embodiments, p1 to p4 (i.e., p1, p2, p3, and p4), q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6), r1, r2, and / or s1 to s4 are each independently an integer of 1 to 3 (e.g., 1, 2, or 3). In some embodiments, p1 through p4 (i.e., p1, p2, p3, and p4), q1 through q6 (i.e., q1, q2, q3, q4, q5, and q6), and / or s1 through s4 (i.e., s1, s2, s3, and s4) are each 2. In some embodiments, p1 through p4 (i.e., p1, p2, p3, and p4) and / or q1 through q6 (i.e., q1, q2, q3, q4, q5, and q6) are each 2, and r1, r2, and s1 through s4 (i.e., s1, s2, s3, and s4) are each 1.

[0249] R in each case 2 is hydrogen or C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12(e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group, or R 2 is the second R 2 and form a heterocyclic group. In some embodiments, R 2 is hydrogen or C1-C which can be straight or branched 12 Alkyl (e.g., C1-C 10 alkyl group; C1-C8 alkyl group; C1-C6 alkyl group; C1-C4 alkyl group, C1-C3 alkyl group, or C1 or C2 alkyl group). In some embodiments, R 2 is methyl. In other embodiments, R 2 can be hydrogen. 2 are independently selected and may be the same or different. In some embodiments, each R 2 are identical (e.g., all methyl or all hydrogen).

[0250] R in each case 4 is independently —C(O)O—, —C(O)NH—, or —S(O)(O)—. In some embodiments, each occurrence of R 4 is independently —C(O)O— or —C(O)NH—. In some embodiments, each occurrence of R 4 is —C(O)O—. In some embodiments, each occurrence of R 4 is -C(O)NH-.

[0251] R in each case 5 R is independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 2 to 8 tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety. 5 can contain about 2 to about 50 carbon atoms (e.g., about 2 to about 40 carbon atoms, about 2 to about 30 carbon atoms, about 2 to about 20 carbon atoms, about 2 to about 16 carbon atoms, about 2 to about 12 carbon atoms, about 2 to about 10 carbon atoms, or about 2 to about 8 carbon atoms). In some embodiments, R5 is a heteroalkyl group containing 2 to 8 (i.e., 2, 3, 4, 5, 6, 7, or 8) tertiary amines. The tertiary amines can be part of the heteroalkyl backbone (i.e., the longest continuous chain of atoms in the heteroalkyl group), or a pendant substituent. Thus, for example, a heteroalkyl group containing a tertiary amine can provide an alkylamino group, aminoalkyl group, alkylaminoalkyl group, aminoalkylamino group, etc., containing 2 to 8 tertiary amines.

[0252] In some embodiments, each R 5 are independently:

[0253] [ka]

[0254] [ka]

[0255] [ka]

[0256] Selected from During the ceremony, R in each case 2 is as above; R 7 is optionally substituted with one or more amines, C1-C 50 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; z is an integer from 1 to 5; c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; N is an integer from 0 to 50; and R 8 are tissue- or cell-specific targeting moieties, C1-C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group.

[0257] R 7 is optionally substituted with one or more amines, C1-C 50 (e.g., C1-C 40 , C1-C 30 , C1-C 20 , C1-C 10 , C4-C 12 , or C-C) alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups. In some embodiments, R 7 is optionally substituted with one or more amines, C4-C 12 , e.g., C6-C8, alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups. In some embodiments, R 7 is substituted with one or more amines. In some embodiments, R 7 is substituted with 2 to 8 (i.e., 2, 3, 4, 5, 6, 7, or 8) tertiary amines. The tertiary amines can be part of the alkyl group (i.e., contained within the alkyl group backbone) or as pendant substituents.

[0258] Y in each instance is optionally present. As used herein, the phrase "optionally present" means that the substituent designated as being optionally present may or may not be present, and that if the substituent is absent, adjacent substituents are directly bonded to each other. When Y is present, Y is a cleavable linker. As used herein, the phrase "cleavable linker" refers to any chemical moiety connecting two species that can be cleaved to separate the two species. For example, the cleavable linker can be cleaved by a hydrolysis process, a photochemical process, a radical process, an enzymatic process, an electrochemical process, or a combination thereof. Exemplary cleavable linkers are:

[0259] [ka]

[0260] where R 14 are independently a C1-C4 alkyl group, and each occurrence of R 15 are independently hydrogen, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), C-C 12 is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, and R 16 is a six-membered aromatic or heteroaromatic group optionally substituted with one or more -OCH3, -NHCH3, -N(CH3)2, -SCH3, -OH, or combinations thereof.

[0261] In some embodiments, A 1 and A 2 each independently having the formula -(CH2) p1 -[NH-(CH2) q1 -] r1 NH2 or -(CH2) p1 -[NH-(CH2) q1 -] r1 NHCH3, or the group -(CH2)2-NH-(CH2)2-NH2 or -(CH2)2-NH-(CH2)2-NHCH3 or -(CH2)2-NH-(CH2)2-NH2. In some embodiments, A 1 and A 2 each independently having the formula -(CH2) p1 -[N(R 2 ))-(CH2) q1 -] r1 N(R 2 )2 or -(CH2) p1 -[N(R 2 )-(CH2) q1 -] r1 NH(R 2 ), where R 2 is methyl or ethyl; or the group -(CH2)2-N(CH3)-(CH2)2-NH2 or -(CH2)2-N(CH3)-(CH2)2-NHCH3 or -(CH2)2-N(CH3)-(CH2)2-N(CH3)2.

[0262] Additionally or alternatively, B 1and B 2 Each of the formula -(CH2) p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 groups such as the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 or the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 , where R 4 and R 5 is as above, .

[0263] In some embodiments, the polymer comprising the structure of formula 5 or 5′ does not have any B monomers (e.g., m 3 and m 4 are both 0). Therefore, the second polymer has the formula 6:

[0264] [ka]

[0265] may include the structure During the ceremony: m 1 and m 2 Each of m is an integer from 0 to 1000 (e.g., 0 to 500, 0 to 200, 0 to 100, or 0 to 50), 1 + m 2 The sum of m is greater than 5 (e.g., 5-2000, 5-1000, 5-500, 5-100, or 5-50). 1 + m 2 The sum of n is greater than 10 or greater than 20 (e.g., 10-5000, 10-2000, 10-1000, 10-500, 10-100, or 10-50; or 20-5000, 20-2000, 20-1000, 20-500, 20-100, or 20-50). 1 and n 2Each of n is an integer between 0 and 1000 (e.g., between 0 and 500, between 0 and 200, between 0 and 100, between 0 and 50, or between 0 and 25), provided that 1 + n 2 The sum of n is greater than 2 (e.g., 2-2000, 2-1000, 2-500, 2-200, 2-100, 2-50, or 2-25). 1 + n 2 The sum is greater than 5 or greater than 10 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, 5-50, or 5-25; or 10-2000, 10-1000, 10-500, 10-200, 10-100, 10-50, or 10-25).

[0266] In some embodiments of the polymer comprising the structure of Formula 6, A 1 and A 2 are each independently expressed as -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 wherein p1 to p4, q1 to q6, and r1 and r2 are each independently an integer of 1 to 5 (e.g., an integer of 1 to 3); and R 2 are independently hydrogen or C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group. In some embodiments of the polymer comprising the structure of Formula 6, A 1 and A 2 are each independently expressed as -(CH2) p1 -[NR 2 -(CH2) p2 -] r1 NR 2 -(CH2) p3 -NHR 2 wherein each of p1-p3 is independently an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); r1 is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4, or 5); s1 is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and R 2 are independently hydrogen or C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group. In some embodiments, R 2 Group A containing substituents 1 and A 2 Each nitrogen in A is a tertiary amine, except that the terminal amine may be a primary, secondary, or tertiary amine, or, in some embodiments, a secondary or tertiary amine. 1 and A 2 Each of these is -(CH2)2-NR 2 -(CH2)2-NR 2 2, where R in each case 2are independently hydrogen, alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, especially alkyl such as methyl or ethyl, as defined above, and optionally wherein each amine is a tertiary amine, except that the terminal amine is a secondary or tertiary amine.

[0267] Base A 1 and A 2 Specific non-limiting examples of are, for example, -CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2; -CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2; -CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2; -CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2; -CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N( CH3)2;-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);-CH2-CH2-N(CH 3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3); Contains -CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3).

[0268] All other aspects of the polymer containing the structure of Formula 6 are as described for Formulas 5 and 5', including all embodiments thereof, for the structure of Formula 6. Thus, for example, in some embodiments of Formula 4, each occurrence of R 13 can be any group as described for formulas 5 and 5′, and R 13 and each occurrence of R 3a and R 3b can be any group as described for formulas 5 and 5′, and R 3a and R 3 is methylene or ethylene. 1 and X 2 can be any group as described for formulas 5 and 5′, and X 1 -C(O)NR 13 - or -C(O)O-, and / or one or more (or all) X 2The groups are independently C1-C8 (e.g., C1-C6, C1-C4, C1-C 3、 It can be a C2-C8, or C2-C6) alkyl group.

[0269] In some embodiments, the second polymer has Formula 5A:

[0270] [ka]

[0271] having the structure During the ceremony, Q is the formula:

[0272] [ka]

[0273] and c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 , m 2 , m 3 and m 4 Each of m is an integer from 0 to 1000, 1 + m 2 + m 3 + m 4 The sum of is greater than 5; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is greater than 2; The symbol " / " indicates that the units separated by it are combined randomly or in any order; R 1 is hydrogen, an aryl group, a heterocyclic group, a C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12(e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group; and R 6 is hydrogen, optionally substituted with one or more amines, an amino group, an aryl group, a heterocyclic group, a C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group; or a tissue- or cell-specific targeting moiety. All other aspects of Formula 5A are as described above for Formulas 5 and 5', including any and all embodiments thereof.

[0274] In some embodiments, the second polymer has Formula 5B:

[0275] [ka]

[0276] having the structure During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 and m 2 Each of m is an integer from 0 to 1000, 1 + m 2 The sum of is greater than 5; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n2 The sum of is greater than 2; The symbol " / " indicates that the units separated by it are combined randomly or in any order; R 1 is hydrogen, an aryl group, a heterocyclic group, a C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group; and R 6 is hydrogen, optionally substituted with one or more amines, an amino group, an aryl group, a heterocyclic group, a C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group; or a tissue- or cell-specific targeting moiety. All other aspects of Formula 5B are as described above for Formula 5, Formula 5', and Formula 6, including any and all embodiments thereof.

[0277] In some embodiments, the second polymer has Formula 5C:

[0278] [ka]

[0279] having the structure During the ceremony, m 1 and m 2Each of m is an integer from 0 to 1000, 1 + m 2 The sum of is greater than 5; n 1 and n 2 Each of n is an integer from 0 to 1000, 1 + n 2 The sum of is greater than 2; The symbol " / " indicates that the units separated by it are combined randomly or in any order; R 1 is hydrogen, an aryl group, a heterocyclic group, a C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group; and R 6 is hydrogen, optionally substituted with one or more amines, an amino group, an aryl group, a heterocyclic group, a C1-C 12 (e.g., C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (e.g., C2-C8, C2-C6, or C2-C3) alkenyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl group; or a tissue- or cell-specific targeting moiety. All other aspects of Formula 5C are as described for Formula 5, Formula 5', and Formula 6, including any and all embodiments thereof.

[0280] In some embodiments, R 1 and / or R 6 is C1-C 12Alkyl (e.g., C1-C 10 Heteroalkyl or alkyl groups include alkyl groups; C1-C8 alkyl groups; C1-C6 alkyl groups; C1-C4 alkyl groups, C1-C3 alkyl groups, or C1 or C2 alkyl groups, which may be linear or branched and optionally substituted with one or more substituents. In some embodiments, the heteroalkyl or alkyl group comprises or is substituted with one or more amines, for example, 2 to 8 (i.e., 2, 3, 4, 5, 6, 7, or 8) tertiary amines. The tertiary amines may be part of the heteroalkyl backbone chain or pendant substituents.

[0281] The second polymer can be any suitable polymer, provided that the polymer comprises the polymer structure described above. In some embodiments, the second polymer is a block copolymer comprising a polymer block having the structure of Formula 5 or 5' and one or more other polymer blocks, such as a polyalkylene oxide, polylactic acid, or polyglycolic acid block. However, the second polymer of the composition does not necessarily comprise such additional polymer blocks. In some embodiments, the second polymer does not comprise polyalkylene oxide, polylactic acid, or polyglycolic acid in the side chain of the polymer. In some embodiments, the second polymer does not comprise polyalkylene oxide, polylactic acid, or polyglycolic acid in the backbone or at either end of the polymer. In some embodiments, the second polymer does not comprise any polyalkylene oxide, polylactic acid, or polyglycolic acid. In still other embodiments, the second polymer does not comprise any additional polymer units other than those shown in the structure of Formula 5 or 5' and may comprise any suitable terminal group. In some embodiments, the polymer further comprises a substituent comprising a tissue-specific or cell-specific targeting moiety.

[0282] In some embodiments, the second polymer has Formula 5A':

[0283] [ka]

[0284] having the structure During the ceremony, Q is the formula:

[0285] [ka]

[0286] and c is an integer between 2 and 200 (e.g., between 2 and 150, between 2 and 100, between 2 and 50, between 10 and 200, between 10 and 150, between 10 and 100, between 10 and 50, between 25 and 200, between 25 and 150, between 25 and 100, between 25 and 50, between 50 and 200, between 50 and 150, or between 50 and 100); Y is optionally present and is a cleavable linker; and all other substituents are as described for Formulas 5, 5', and 6, including any and all embodiments thereof.

[0287] In some embodiments, the second polymer has Formula 5B':

[0288] [ka]

[0289] having the structure During the ceremony, c is an integer between 2 and 200 (e.g., between 2 and 150, between 2 and 100, between 2 and 50, between 10 and 200, between 10 and 150, between 10 and 100, between 10 and 50, between 25 and 200, between 25 and 150, between 25 and 100, between 25 and 50, between 50 and 200, between 50 and 150, or between 50 and 100); Y is optionally present and is a cleavable linker; and all other substituents are as described for Formulas 5, 5', and 6, including any and all embodiments thereof.

[0290] Non-limiting examples of second polymers provided herein include, for example:

[0291]

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[0292]

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[0293]

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[0294]

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[0295]

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[0296]

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[0297]

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[0298]

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[0299]

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[0300]

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[0301]

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[0302] [ka]

[0303] wherein (a+b) is about 5 to about 65 (e.g., about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 15, about 5 to about 10, about 10-50, about 10-40, about 10-30, or about 10-20), and (c+d) is about 2 to about 60 (e.g., about 2 to about 50, about 2 to about 40, about 2 to about 30, about 2 to about 20, about 2 to about 10), or about 10-60 (e.g., about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20). In some embodiments, (a+b) is about 10-50 or 10-30, and (c+d) is about 20-50 or 20-40. In other embodiments, (a+b) is about 45 and (c+d) is about 20. Again, the designations of the numbers of units in these exemplary polymers ("a," "b," "c," and "d") do not imply a block copolymer structure; rather, these numbers indicate the total number of units, which may be randomly arranged as indicated by the " / " symbol in the formula.

[0304] Further specific examples of second polymers provided by the present disclosure include:

[0305] [ka]

[0306] [ka]

[0307] [ka]

[0308] [ka]

[0309]

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[0310]

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[0311]

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[0312]

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[0313]

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[0314]

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[0315]

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[0316]

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[0317]

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[0318]

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[0319] [ka]

[0320] [ka]

[0321] [ka]

[0322] [ka]

[0323] Includes.

[0324] Further examples of polymers comprising PEG end groups provided herein are as follows:

[0325] [ka]

[0326] [ka]

[0327] The unit number designations ("a," "b," "c," and "d") in these exemplary polymers do not imply a block copolymer structure; rather, these numbers indicate the total number of specific monomer units, which units may be arranged in any order, including blocks of monomers or monomers randomly arranged throughout the polymer. In some cases, but not all, this is further indicated by a " / " symbol in the formula; however, the absence of a " / " should not be construed as indicating that the polymer is joined in a particular order. In some embodiments of the foregoing polymers 1-95, the monomers designated by the parentheses and integer ("a," "b," "c," or "d") are randomly arranged or dispersed throughout the polymer.

[0328] In any of the aforementioned second polymers, (a+b) is about 5 to about 65 (e.g., about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, or about 5 to about 10), and (c+d) is about 2 to about 60 (e.g., about 2 to about 50, about 2 to about 40, about 2 to about 30, about 2 to about 20, or about 2 to about 10). In some embodiments, (a+b) is about 55, and (c+d) is about 10. In other embodiments, (a+b) is about 45, and (c+d) is about 20. In some embodiments, (a+b+c+d) is about 10-500, e.g., about 10-400, about 10-200, or about 10-100 (e.g., about 25-100 or about 50-75).

[0329] The second polymer can include any suitable ratio of (a+b) to (c+d). In other embodiments, (a+b) ranges from 10-95% (e.g., 10-75%, 10-65%, 10-50%, 20-95%, 20-75%, 20-65%, 20-50%, 30-95%, 30-75%, 30-65%, or 30-50%) of the total number of polymer units (a+b+c+d). In other embodiments, (c+d) ranges from 5-90% (e.g., 5-75%, 5-65%, 5-50%, 5-40%, 5-30%, 10-90%, 10-75%, 10-65%, 10-50%, 10-40%, or 10-30%) of the total number of polymer units (a+b+c+d). In still other embodiments, the ratio of (a+b):(c+d) can be from about 1 to about 25, from about 1 to about 20, from about 1 to about 10, from about 1 to about 5, from about 5 to about 25, from about 10 to about 25, or from about 15 to about 25.

[0330] Some of the second polymers include monomers having ionic side chains "e" and "f," where a, b, c, and d are as defined above, and (e+f) is from about 2 to about 60 (e.g., from about 2 to about 50, from about 2 to about 40, from about 2 to about 30, from about 2 to about 20, or from about 2 to about 10). Further, p in each instance is independently an integer from 2 to 200 (e.g., from 2 to 150, from 2 to 100, from 2 to 50, from 6 to 36, from 6 to 30, from 6 to 24, from 6 to 18, from 10 to 200, from 10 to 150, from 10 to 100, from 10 to 50, from 25 to 200, from 25 to 150, from 25 to 100, from 25 to 50, from 50 to 200, from 50 to 150, or from 50 to 100). Further, (a+b+c+d+e+f) is about 10-500, e.g., about 10-400, about 10-200, or about 10-100 (e.g., about 25-100 or about 50-75). Again, the designations of the numbers of units in these exemplary second polymers ("a," "b," "c," "d," "e," and "f") do not imply a block copolymer structure; rather, these numbers refer to the total number of units, which may be randomly arranged. In some embodiments, the second polymer has the structure of polymer 29, 30, 35, 36, 37, 39, or 40 above, where a, b, c, and d are as described herein. In some embodiments, the second polymer is polymer 29, 30, 35, 36, 37, 39, or 40, where (a+b) is about 10-50 or 10-30, and (c+d) is about 20-50 or 20-40.

[0331] Some of the above specific examples of second polymers provided by the present disclosure are depicted with specific end groups (e.g., alkylamino, hydrogen, or PEG); however, any of the above specific structures may include different end groups. For example, any of the above structures may include the R 1 , R 6 or Q groups at either or both ends of the polymer backbone.

[0332] Typically, the second polymer is cationic (i.e., positively charged at pH 7 and 23° C.). As used herein, a "cationic" polymer refers to a polymer that has an overall net positive charge, regardless of whether the polymer contains only cationic monomer units or a combination of cationic and nonionic or anionic monomer units.

[0333] In some embodiments, the second polymer has a weight-average molecular weight of about 5 kDa to about 2,000 kDa. The second polymer can have a weight-average molecular weight of about 2,000 kDa or less, e.g., about 1,800 kDa or less, about 1,600 kDa or less, about 1,400 kDa or less, about 1,200 kDa or less, about 1,000 kDa or less, about 900 kDa or less, about 800 kDa or less, about 700 kDa or less, about 600 kDa or less, about 500 kDa or less, about 100 kDa or less, or about 50 kDa or less. Alternatively, or in addition, the second polymer can have a weight-average molecular weight of about 10 kDa or more, e.g., about 50 kDa or more, about 100 kDa or more, about 200 kDa or more, about 300 kDa or more, or about 400 kDa or more. Thus, the second polymer can have a weight average molecular weight bounded by any two of the aforementioned endpoints. For example, the second polymer may be from about 10 kDa to about 50 kDa, from about 10 kDa to about 100 kDa, from about 10 kDa to about 500 kDa, from about 50 kDa to about 500 kDa, from about 100 kDa to about 500 kDa, from about 200 kDa to about 500 kDa, from about 300 kDa to about 500 kDa, from about 400 kDa to about 500 kDa, from about 400 kDa to about 600 kDa, from about 400 kDa to about 700 kDa, from about 400 kDa to about 800 kDa, from about 400 kDa to about 900 kDa, from about 400 kDa to about 1,000 kDa, from about 400 kDa to about 1,200 kDa, or from about 400 kDa to about 1,400 kDa. The weight-average molecular weight may be about 1,600 kDa, about 400 kDa to about 1,800 kDa, about 400 kDa to about 2,000 kDa, about 200 kDa to about 2,000 kDa, about 500 kDa to about 2,000 kDa, or about 800 kDa to about 2,000 kDa. The weight-average molecular weight may be determined by any suitable technique. Typically, the weight-average molecular weight is determined using size exclusion chromatography equipped with a column selected from a TSKgel Guard, GMPW, GMPW, G1000PW, and Waters 2414 (Waters Corporation, Milford, Massachusetts) refractive index detector.Additionally, weight average molecular weights are determined from calibration using polyethylene oxide / polyethylene glycol standards ranging from 150-875,000 daltons.

[0334] Without wishing to be bound by any particular theory or mechanism of action, it is believed that compositions comprising a first and a second polymer described herein can provide nanoparticles that are more stable than polymeric nanoparticles comprising only the second polymer, particularly in physiological environments, such as body fluids (e.g., blood, serum, or CSF). Stability can be measured as a factor of particle size over time, where a larger change in particle size over a given period indicates less stability than a smaller change in particle size over a given period.

[0335] Donor nucleic acid A donor nucleic acid (or "donor sequence" or "donor polynucleotide" or "donor DNA") is a nucleic acid sequence to be inserted at the cleavage site induced by an RNA-dependent endonuclease (e.g., a Cas9 polypeptide or a Cpfl polypeptide). The donor polynucleotide contains sufficient homology to the target genomic sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100%, to support homology-directed repair between the genomic sequence flanking the cleavage site or immediately flanking the cleavage site and having homology thereto, e.g., within about 50 bases or less, e.g., about 30 bases, about 15 bases, about 10 bases, about 5 bases, of the cleavage site. About 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between the donor and the genomic sequence (or any integer value between 10 and 200 nucleotides, or more) support homology-directed repair. The donor sequence can be any length, for example, 10 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 250 or more nucleotides, 500 or more nucleotides, 1000 or more nucleotides, 5000 or more nucleotides, etc.

[0336] The donor sequence is typically not identical to the genomic sequence it replaces. Rather, the donor sequence may contain one or more single base changes, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, as long as sufficient homology exists to support homology-directed repair. In some embodiments, the donor sequence contains a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequence into the target region. The donor sequence may also contain a vector backbone that is not homologous to the DNA region of interest and contains a sequence that is not intended for insertion into the DNA region of interest. Generally, the homologous region of the donor sequence has at least 50% sequence identity with the genomic sequence into which recombination is desired. In some embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity. Depending on the length of the donor polynucleotide, there can be any value between 1% sequence identity and 100% sequence identity.

[0337] The donor sequence may contain sequence differences compared to the genomic sequence, such as restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which can be used to evaluate successful insertion of the donor sequence at the cleavage site, or in some embodiments, for other purposes (e.g., to indicate expression at the targeted genomic locus). In some embodiments, when located in a coding region, such nucleotide sequence differences do not change the amino acid sequence or create silent amino acid changes (i.e., changes that do not affect the structure or function of the protein). Alternatively, these sequence differences may include flanking recombination sequences, such as FLP and loxP sequences, which can be subsequently activated to remove the marker sequence.

[0338] The donor sequence can be provided to cells as single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It can be introduced into cells in linear or circular form. When introduced in linear form, the ends of the donor sequence can be protected (e.g., from extranuclear degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides can be attached to one or both ends. For example, see Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Amplification procedures such as rolling circle amplification can also be used advantageously, as exemplified herein. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups and the use of modified internucleotide linkages such as phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues.

[0339] As an alternative to protecting the ends of linear donor sequences, additional lengths of sequence can be included outside the regions of homology that can be degraded without affecting recombination. Donor sequences can be introduced into cells as part of a vector molecule with additional sequences, such as an origin of replication, a promoter, and a gene encoding antibiotic resistance. Furthermore, donor sequences can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or polymer, or delivered by a virus (e.g., adenovirus, AAV), as described herein for nucleic acids encoding Cas9 guide RNAs and / or Cas9 fusion polypeptides and / or donor polynucleotides.

[0340] Guide nucleic acid In some embodiments, the composition contains a guide nucleic acid. Suitable guide nucleic acids for inclusion in the compositions of the present disclosure include single-guide RNAs ("single-guide RNAs" / "sgRNAs") and dual-guide nucleic acids ("dual-guide RNAs" / "dgRNAs").

[0341] A guide nucleic acid (e.g., guide RNA) suitable for inclusion in a complex of the present disclosure directs the activity of an RNA-guided endonuclease (e.g., a Cas9 or Cpfl polypeptide) against a specific target sequence within a target nucleic acid. The guide nucleic acid (e.g., guide RNA) includes: a first fragment (also referred to herein as a "nucleic acid targeting fragment" or simply "targeting fragment"); and a second fragment (also referred to herein as a "protein-binding fragment"). The terms "first" and "second" do not refer to the order in which the fragments appear in the guide RNA. The order of the elements relative to each other depends on the particular RNA-guided polypeptide used. For example, a guide RNA for Cas9 typically has the protein-binding fragment located 3' of the targeting fragment, while a guide RNA for Cpfl typically has the protein-binding fragment located 5' of the targeting fragment.

[0342] Guide RNA can be introduced into cells in linear or circular form.When introduced in linear form, the end of guide RNA can be protected (for example, from extranuclear degradation) by methods known to those skilled in the art.Amplification procedures such as rolling circle amplification can also be used advantageously, as exemplified herein.

[0343] First fragment: targeting fragment The first fragment of the guide nucleic acid (e.g., guide RNA) comprises a nucleotide sequence that is complementary to a sequence (target site) in the target nucleic acid. In other words, the targeting fragment of the guide nucleic acid (e.g., guide RNA) can interact with the target nucleic acid (e.g., RNA, DNA, double-stranded DNA) in a sequence-specific manner through hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the targeting fragment can be varied and can determine the position within the target nucleic acid where the guide nucleic acid (e.g., guide RNA) and the target nucleic acid will interact. The targeting fragment of the guide nucleic acid (e.g., guide RNA) can be modified (e.g., by genetic engineering) to hybridize to any desired sequence (target site) within the target nucleic acid.

[0344] The targeting fragment may have a length of 12 to 100 nucleotides. The nucleotide sequence (targeting sequence, also referred to as guide sequence) of the targeting fragment that is complementary to the nucleotide sequence (target site) of the target nucleic acid may have a length of 12 nt or more. For example, the targeting sequence of the targeting fragment that is complementary to the target site of the target nucleic acid may have a length of 12 nt or more, 15 nt or more, 17 nt or more, 18 nt or more, 19 nt or more, 20 nt or more, 25 nt or more, 30 nt or more, 35 nt or more, or 40 nt.

[0345] The percent complementarity between the targeting sequence of the targeting fragment (i.e., guide sequence) and the target site of the target nucleic acid can be 60% or greater (e.g., 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%). In some embodiments, the percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 100% for the 7 contiguous 5'-most nucleotides of the target site of the target nucleic acid. In some embodiments, the percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 60% or greater for 20 contiguous nucleotides. In some embodiments, the percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 100% for the 17, 18, 19, or 20 contiguous 5'-most nucleotides of the target site of the target nucleic acid, and as low as 0% or greater for the remainder. In such cases, the targeting sequence may be considered to be 17, 18, 19 or 20 nucleotides in length, respectively.

[0346] Second fragment: protein-binding fragment The protein-binding fragment of the guide nucleic acid (e.g., guide RNA) interacts with (binds to) an RNA-guided endonuclease. The guide nucleic acid (e.g., guide RNA) guides the bound endonuclease to a specific nucleotide sequence within the target nucleic acid (target site) via the targeting fragment / targeting sequence / guide sequence described above. The protein-binding fragment of the guide nucleic acid (e.g., guide RNA) contains two stretches of nucleotides that are complementary to each other. The complementary nucleotides of the protein-binding fragment hybridize to form a double-stranded RNA duplex (dsRNA).

[0347] Single and dual guide nucleic acids A dual guide nucleic acid (e.g., guide RNA) comprises two separate nucleic acid molecules, each of the two molecules of the analyte dual guide nucleic acid (e.g., guide RNA) comprising a stretch of nucleotides that are complementary to each other, such that the complementary nucleotides of the two molecules hybridize to form the double-stranded RNA duplex of the protein-binding fragment.

[0348] In some embodiments, the duplex-forming fragment of the activator is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical, or 100% identical, over a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) to one of the activators (tracrRNAs) described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements.

[0349] In some embodiments, the duplex-forming fragment of the target is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical, or 100% identical, over a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) to one of the target (crRNA) sequences set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements.

[0350] Dual guide nucleic acids (e.g., guide RNAs) can be designed to allow controlled (i.e., conditional) binding of a target with an activator. Because dual guide nucleic acids (e.g., guide RNAs) are not functional unless both the activator and the target are bound in a functional complex with Cas9, dual guide nucleic acids (e.g., guide RNAs) can be inducible (e.g., drug-inducible) by providing an inducible binding between the activator and the target. As one non-limiting example, RNA aptamers can be used to regulate (i.e., control) the binding of an activator with a target. Thus, the activator and / or the target can comprise an RNA aptamer sequence.

[0351] Aptamers (e.g., RNA aptamers) are known in the art and are generally synthetic versions of riboswitches. The terms "RNA aptamer" and "riboswitch" are used interchangeably herein and include both synthetic and natural nucleic acid sequences that provide inducible modulation of the structure (and thus the availability of specific sequences) of the nucleic acid molecule (e.g., RNA, DNA / RNA hybrids, etc.) of which they are a part. RNA aptamers usually contain a sequence that folds into a specific structure (e.g., a hairpin), which specifically binds a specific drug (e.g., a small molecule). Binding of the drug causes a structural change in the folding of the RNA, which alters the characteristics of the nucleic acid of which the aptamer is a part. Non-limiting examples include: (i) an activator bearing an aptamer cannot bind to its cognate target unless the aptamer is bound by an appropriate drug; (ii) a target bearing an aptamer cannot bind to its cognate activator unless the aptamer is bound by an appropriate drug; and (iii) a target and an activator containing different aptamers, each binding a different drug, cannot bind to each other unless both drugs are present. As illustrated by these examples, dual guide nucleic acids (e.g., guide RNAs) can be designed to be inducible.

[0352] Examples of aptamers and riboswitches can be found, for example, in: Nakamura et al., Genes Cells. 2012 May;17(5):344-64; Vavalle et al., Future Cardiol. 2012 May;8(3):371-82; Citartan et al., Biosens Bioelectron. 2012 Apr 15;34(1):1-11; and Liberman et al., Wiley Interdiscip Rev RNA. 2012 May-Jun;3(3):369-84; all of which are incorporated by reference in their entirety.

[0353] Non-limiting examples of nucleotide sequences that may be contained in dual guide nucleic acids (e.g., guide RNAs) contained in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements, that may hybridize to form protein-binding fragments.

[0354] An analyte single guide nucleic acid (e.g., a guide RNA) hybridizes to form a double-stranded RNA duplex (dsRNA duplex) with a protein-binding fragment (thus creating a stem-loop structure) and contains two complementary stretches of nucleotides (analogous to the "target" and "activator" of a dual guide nucleic acid) covalently linked by an intervening nucleotide (a "linker" or "linker nucleotide"). Thus, an analyte single guide nucleic acid (e.g., a single guide RNA) can contain a target and an activator, each having a duplex-forming fragment, where the duplex-forming fragments of the target and activator hybridize to each other to form a dsRNA duplex. The target and activator can be covalently linked via the 3' end of the target and the 5' end of the activator. Alternatively, the target and activator can be covalently linked via the 5' end of the target and the 3' end of the activator.

[0355] The linker of a single guide nucleic acid can have a length of 3 nucleotides to 100 nucleotides. In some embodiments, the linker of a single guide nucleic acid (e.g., a guide RNA) is 4 nt.

[0356] Exemplary single guide nucleic acids (e.g., guide RNAs) comprise two complementary stretches of nucleotides that hybridize to form a dsRNA duplex. In some embodiments, one of the two complementary stretches of nucleotides of the single guide nucleic acid (e.g., guide RNA) (or DNA encoding the stretch) is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical, or 100% identical, over a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) to one of the activator (tracrRNA) molecules described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements.

[0357] In some embodiments, one of the two complementary stretches of nucleotides of the single guide nucleic acid (e.g., guide RNA) (or DNA encoding the extension) is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical, or 100% identical, for a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) to one of the target (crRNA) sequences set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements.

[0358] In some embodiments, one of the two complementary stretches of nucleotides of the single guide nucleic acid (e.g., guide RNA) (or DNA encoding the extension) is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical, or 100% identical, for a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) to one of the target (crRNA) or activator (tracrRNA) sequences, or their complements, set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0359] Suitable cognate pairs of target and activator can be routinely determined by considering the species name and base pairing (for the dsRNA duplex of the protein-binding domain). Any activator / target pair can be used as part of a dual guide nucleic acid (e.g., guide RNA) or as part of a single guide nucleic acid (e.g., guide RNA).

[0360] In some embodiments, the activator (e.g., trRNA, trRNA-like molecule, etc.) of the dual guide nucleic acid (e.g., guide RNA) (e.g., dual guide RNA) or single guide nucleic acid (e.g., guide RNA) (e.g., single guide RNA) comprises a stretch of nucleotides having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% sequence identity to an activator (tracrRNA) molecule described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or a complement thereof.

[0361] In some embodiments, the activator (e.g., trRNA, trRNA-like molecule, etc.) of a dual guide nucleic acid (e.g., dual guide RNA) or single guide nucleic acid (e.g., single guide RNA) comprises 30 or more nucleotides (nt) (e.g., 40 or more, 50 or more, 60 or more, 70 or more, 75 or more nt). In some embodiments, the activator (e.g., trRNA, trRNA-like molecule, etc.) of a dual guide nucleic acid (e.g., dual guide RNA) or single guide nucleic acid (e.g., single guide RNA) has a length ranging from 30 to 200 nucleotides (nt).

[0362] The protein-binding fragment can have a length of 10 to 100 nucleotides.

[0363] Also, for both analyte single guide nucleic acids (e.g., single guide RNAs) and analyte dual guide nucleic acids (e.g., dual guide RNAs), the dsRNA duplexes of the protein-binding fragments can have lengths between 6 base pairs (bp) and 50 bp. The percent homology between the nucleotide sequences that hybridize to form the dsRNA duplexes of the protein-binding fragments can be 60% or greater. For example, the percent homology between the nucleotide sequences that hybridize to form the dsRNA duplexes of the protein-binding fragments can be 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater (e.g., in some embodiments, there are some nucleotides that do not hybridize and thus create a bulge in the dsRNA duplex). In some embodiments, the percent homology between the nucleotide sequences that hybridize to form the dsRNA duplexes of the protein-binding fragments is 100%.

[0364] Hybrid guide nucleic acid In some embodiments, the guide nucleic acid is two RNA molecules (dual guide RNA). In some embodiments, the guide nucleic acid is one RNA molecule (single guide RNA). In some embodiments, the guide nucleic acid is a DNA / RNA hybrid molecule. In such embodiments, the protein-binding fragment of the guide nucleic acid is RNA and forms an RNA duplex. Thus, the activator and target duplex-forming fragments are RNA. However, the targeting fragment of the guide nucleic acid can be DNA. Thus, when a DNA / RNA hybrid guide nucleic acid is a dual guide nucleic acid, the "target" molecule is a hybrid molecule (e.g., the targeting fragment can be DNA and the duplex-forming fragment can be RNA). In such embodiments, the duplex-forming fragment of the "activator" molecule can be RNA (e.g., to form an RNA duplex with the duplex-forming fragment of the target molecule), while the nucleotides of the "activator" molecule outside the duplex-forming fragment can be DNA (in which case the activator molecule is a hybrid DNA / RNA molecule) or RNA (in which case the activator molecule is RNA). When the DNA / RNA hybrid guide nucleic acid is a single guide nucleic acid, the targeting fragment can be DNA, the duplex-forming fragment (which makes up the protein-binding fragment of the single guide nucleic acid) can be RNA, and the nucleotides outside the targeting and duplex-forming fragments can be RNA or DNA.

[0365] DNA / RNA hybrid guide nucleic acids can be useful in some embodiments, for example, when the target nucleic acid is RNA. Cas9 typically associates with a guide RNA that hybridizes with the target DNA and thus forms a DNA-RNA duplex at the target site. Therefore, when the target nucleic acid is RNA, it is sometimes advantageous to replicate the DNA-RNA duplex at the target site by using a targeting fragment (of the guide nucleic acid) that is DNA instead of RNA. However, because the protein-binding fragment of the guide nucleic acid is RNA-duplexed, the target molecule is DNA in the targeting fragment and RNA in the duplex-forming fragment. Hybrid guide nucleic acids can bias Cas9 binding to single-stranded target nucleic acids relative to double-stranded target nucleic acids.

[0366] Exemplary Guide Nucleic Acids Any guide nucleic acid can be used.Many different types of guide nucleic acids are known in the art.The selected guide nucleic acid is appropriately paired with the specific CRISPR system used (for example, the specific RNA-guided endonuclease used).Therefore, the guide nucleic acid can be, for example, the guide nucleic acid corresponding to any RNA-guided endonuclease described herein or known in the art.Guide nucleic acids and RNA-guided endonucleases are described, for example, in International Patent Application No. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0367] In some embodiments, a suitable guide nucleic acid comprises two separate RNA polynucleotide molecules. In some embodiments, the first two separate RNA polynucleotide molecules (activators) comprise a nucleotide sequence having 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) nucleotide sequence identity with any one of the nucleotide sequences set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements, over a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides). In some embodiments, the second two distinct RNA polynucleotide molecules (targets) comprise a nucleotide sequence having 60% or greater (e.g., 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100%) nucleotide sequence identity to any one of the nucleotide sequences set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements, over a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides).

[0368] In some embodiments, a suitable guide nucleic acid is a single RNA polynucleotide and comprises first and second nucleotide sequences having 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) nucleotide sequence identity to any one of the nucleotide sequences set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements, over a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides).

[0369] In some embodiments, the guide RNA is a Cpf1 and / or Cas9 guide RNA. The Cpf1 and / or Cas9 guide RNA can have a total length of 30 nucleotides (nt) to 100 nt, for example, 30 nt to 40 nt, 40 nt to 45 nt, 45 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, or 90 nt to 100 nt. In some embodiments, the Cpf1 and / or Cas9 guide RNA has a total length of 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt. The Cpf1 and / or Cas9 guide RNA can comprise a target nucleic acid-binding fragment and a duplex-forming fragment.

[0370] The target nucleic acid-binding fragment of the Cpf1 and / or Cas9 guide RNA can be 15 nt to 30 nt in length, for example, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, or 30 nt in length. In some embodiments, the target nucleic acid-binding fragment is 23 nt in length. In some embodiments, the target nucleic acid-binding fragment is 24 nt in length. In some embodiments, the target nucleic acid-binding fragment is 25 nt in length.

[0371] The target nucleic acid-binding fragment of a Cpf1 and / or Cas9 guide RNA may have 100% complementarity with the corresponding length of the target nucleic acid sequence. The targeting fragment may have less than 100% complementarity with the corresponding length of the target nucleic acid sequence. For example, the target nucleic acid-binding fragment of a Cpf1 and / or Cas9 guide RNA may have 1, 2, 3, 4, or 5 nucleotides that are not complementary to the target nucleic acid sequence. For example, in some embodiments, the target nucleic acid-binding fragment is 25 nucleotides in length and the target nucleic acid sequence is 25 nucleotides in length, and in some embodiments, the target nucleic acid-binding fragment is 100% complementary to the target nucleic acid sequence. As another example, in some embodiments, the target nucleic acid-binding fragment is 25 nucleotides in length and the target nucleic acid sequence is 25 nucleotides in length, and in some embodiments, the target nucleic acid-binding fragment has one non-complementary nucleotide and 24 nucleotides complementary to the target nucleic acid sequence.

[0372] The duplex-forming fragment of the Cpf1 and / or Cas9 guide RNA can have a length of 15 nt to 25 nt, e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt.

[0373] In some embodiments, the duplex-forming fragment of the Cpf1 guide RNA may comprise the nucleotide sequence 5'-AAUUUCUACUGUUGUAGAU-3'.

[0374] Further elements In some embodiments, the guide nucleic acid (e.g., guide RNA) comprises an additional fragment(s) (in some embodiments, at the 5' end, in some embodiments, at the 3' end, in some embodiments, at either the 5' or 3' end, in some embodiments, embedded within the sequence (i.e., not at the 5' and / or 3' end), in some embodiments, at both the 5' and 3' ends, in some embodiments, embedded and at the 5' and / or 3' ends, etc.). For example, a suitable additional fragment may be a 5' cap (e.g., a 7-methylguanylic acid cap (m 7 3' polyadenylation tail (i.e., 3' poly(A) tail); ribozyme sequence (e.g., to allow self-cleavage of the guide nucleic acid or a component of the guide nucleic acid, e.g., a target, an activator, etc.); riboswitch sequence (e.g., to allow regulated stability and / or regulated accessibility by proteins and protein complexes); sequence that forms a dsRNA duplex (i.e., a hairpin); sequence that targets the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplast, etc.); modification or sequence that provides tracking (e.g., a fluorescent molecule (i.e., a fluorescent dye) sequences or other modifications that provide binding sites for proteins (e.g., including proteins that act on DNA, transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, proteins that bind RNA (e.g., RNA aptamers), labeled proteins, fluorescently labeled proteins, etc.); modifications or sequences that provide increased, decreased, and / or controllable stability; and combinations thereof.

[0375] RNA-guided endonucleases In addition to or instead of the guide nucleic acid, the composition may contain an RNA-guided endonuclease protein or a nucleic acid (e.g., mRNA or vector) encoding the same. Any RNA-guided endonuclease may be used. The choice of the RNA-guided endonuclease to be used depends, at least in part, on the intended end use of the CRISPR system used.

[0376] In some embodiments, the polypeptide is a Cas9 polypeptide. Cas9 polypeptides suitable for inclusion in the compositions of the present disclosure include naturally occurring Cas9 polypeptides (e.g., naturally occurring in bacterial and / or archaeal cells), as described below, or non-naturally occurring Cas9 polypeptides (e.g., Cas9 polypeptides include mutant Cas9 polypeptides, chimeric polypeptides, etc., as discussed below). In some embodiments, one of skill in the art will understand that the Cas9 polypeptides disclosed herein can be any variant derived from or isolated from any source. In other embodiments, the Cas9 peptides of the present disclosure may include one or more mutations described in the literature, including, but not limited to, the functional mutations described in Fonfara et al. Nucleic Acids Res. 2014 Feb;42(4):2577-90; Nishimasu H. et al. Cell. 2014 Feb 27;156(5):935-49; Jinek M. et al. Science. 2012 337:816-21; and Jinek M. et al. Science. 2014 Mar 14;343(6176); see also U.S. Patent Application No. 13 / 842,859, filed March 15, 2013, which is incorporated herein by reference; and further, U.S. Patent Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; See, e.g., 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, all of which are incorporated herein by reference. Thus, in some embodiments, the systems and methods disclosed herein can be used with wild-type Cas9 proteins that have double-stranded nuclease activity, Cas9 mutants that act as single-stranded nickases, or other mutants with modified nuclease activity.Thus, a Cas9 polypeptide suitable for inclusion in a composition of the present disclosure can be, for example, an enzymatically active Cas9 polypeptide that can make a single- or double-stranded cleavage in a target nucleic acid or that can have reduced enzymatic activity compared to a wild-type Cas9 polypeptide.

[0377] Naturally occurring Cas9 polypeptides bind guide nucleic acids, thereby directing them to specific sequences within a target nucleic acid (target site), and cleave the target nucleic acid (e.g., cleaving dsDNA to generate double-strand breaks, cleaving ssDNA, cleaving ssRNA, etc.). The analyte Cas9 polypeptide comprises two portions: an RNA-binding portion and an active portion. The RNA-binding portion interacts with the analyte guide nucleic acid, and the active portion exhibits site-directed enzymatic activity (e.g., nuclease activity, activity for DNA and / or RNA methylation, activity for DNA and / or RNA cleavage, activity for histone acetylation, activity for histone methylation, activity for RNA modification, activity for RNA-binding, activity for RNA splicing, etc.). In some embodiments, the active portion exhibits reduced nuclease activity relative to the corresponding portion of a wild-type Cas9 polypeptide. In some embodiments, the active portion is enzymatically inactive.

[0378] The assay for determining whether a protein has an RNA-binding moiety that interacts with the analyte guide nucleic acid can be any convenient binding assay that tests for binding between a protein and a nucleic acid. Exemplary binding assays include binding assays (e.g., gel shift assays) that involve adding a guide nucleic acid and a Cas9 polypeptide to a target nucleic acid.

[0379] The assay to determine whether a protein has an active portion (e.g., to determine whether a polypeptide has nuclease activity that cleaves a target nucleic acid) can be any convenient nucleic acid cleavage assay that tests for nucleic acid cleavage. An exemplary cleavage assay includes adding a guide nucleic acid and a Cas9 polypeptide to a target nucleic acid.

[0380] In some embodiments, a Cas9 polypeptide suitable for inclusion in a composition of the present disclosure has an enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0381] In other embodiments, a Cas9 polypeptide suitable for inclusion in a composition of the present disclosure has an enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid.

[0382] Many Cas9 orthologs from a wide variety of species have been identified, and in some embodiments, the proteins share only a few identical amino acids. All identified Cas9 orthologs have the same domain structure: a central HNH endonuclease domain and a split RuvC / RNaseH domain. Cas9 proteins share four key motifs with conserved structures. Motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif.

[0383] In some embodiments, a suitable Cas9 polypeptide comprises an amino acid sequence having four motifs, each of which has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% amino acid sequence identity to the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO:1); or to motifs 1-4 of the Cas9 amino acid sequence shown in Table 1 below; or to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO:1).

[0384] In some embodiments, the Cas9 polypeptide comprises an amino acid sequence set forth in FIG. 1 and having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and comprises amino acid substitutions of N497, R661, Q695, and Q926 with respect to the amino acid sequence set forth in SEQ ID NO:1; or comprises amino acid substitutions of K855 with respect to the amino acid sequence set forth in SEQ ID NO:1; or comprises amino acid substitutions of K810, K1003, and R1060 with respect to the amino acid sequence set forth in SEQ ID NO:1; or comprises amino acid substitutions of K848, K1003, and R1060 with respect to the amino acid sequence set forth in SEQ ID NO:1.

[0385] As used herein, the term "Cas9 polypeptide" includes the term "mutant Cas9 polypeptide"; and the term "mutant Cas9 polypeptide" includes the term "chimeric Cas9 polypeptide."

[0386] Mutant Cas9 polypeptides Cas9 polypeptides suitable for inclusion in the compositions of the present disclosure include mutant Cas9 polypeptides. A mutant Cas9 polypeptide has an amino acid sequence that differs by one amino acid (e.g., has a deletion, insertion, substitution, or fusion) (i.e., at least one amino acid differs) when compared to the amino acid sequence of a wild-type Cas9 polypeptide (e.g., a naturally occurring Cas9 polypeptide as described above). In some cases, the mutant Cas9 polypeptide has an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some cases, the mutant Cas9 polypeptide has less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide. In some embodiments, the mutant Cas9 polypeptide does not have substantial nuclease activity. When a Cas9 polypeptide is a mutant Cas9 polypeptide that does not have substantial nuclease activity, it may be referred to as "dCas9."

[0387] In some embodiments, mutant Cas9 polypeptides have reduced nuclease activity. For example, mutant Cas9 polypeptides suitable for use in the conjugation methods of the present disclosure exhibit less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 polypeptide, e.g., a wild-type Cas9 polypeptide comprising the amino acid sequence depicted in Figure 1 (SEQ ID NO: 1).

[0388] In some embodiments, a mutant Cas9 polypeptide can cleave the complementary strand of a target nucleic acid but has a reduced ability to cleave the non-complementary strand of a double-stranded target nucleic acid. For example, a mutant Cas9 polypeptide can have a mutation (amino acid substitution) that reduces the function of the RuvC domain (e.g., "Domain 1" in FIG. 1). As a non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a D10A mutation (e.g., aspartate to alanine at the amino acid position corresponding to position 10 of SEQ ID NO: 1) and therefore can cleave the complementary strand of a double-stranded target nucleic acid but has a reduced ability to cleave the non-complementary strand of a double-stranded target nucleic acid (thus, when the mutant Cas9 polypeptide cleaves a double-stranded target nucleic acid, it generates a single-strand break (SSB) instead of a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug 17;337(6096):816-21).

[0389] In some embodiments, a mutant Cas9 polypeptide can cleave a non-complementary strand of a double-stranded target nucleic acid but has a reduced ability to cleave a complementary strand of the target nucleic acid. For example, a mutant Cas9 polypeptide can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif, "Domain 2" in Figure 1). As a non-limiting example, in some embodiments, a mutant Cas9 polypeptide can have an H840A mutation (e.g., a histidine to alanine at an amino acid position corresponding to position 840 of SEQ ID NO: 1) (Figure 1), and thus can cleave a non-complementary strand of a target nucleic acid but has a reduced ability to cleave a complementary strand of the target nucleic acid (thus generating an SSB instead of a DSB when the mutant Cas9 polypeptide cleaves a double-stranded target nucleic acid). Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid (e.g., a single-stranded target nucleic acid) but maintains the ability to bind a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid).

[0390] In some embodiments, the mutant Cas9 polypeptide has a reduced ability to cleave both complementary and non-complementary strands of a double-stranded target nucleic acid. As a non-limiting example, in some embodiments, the mutant Cas9 polypeptide has both a D10A mutation and an H840A mutation (e.g., mutations in both the RuvC domain and the HNH domain), such that the polypeptide has a reduced ability to cleave both complementary and non-complementary strands of a double-stranded target nucleic acid. Such Cas9 polypeptides have a reduced ability to cleave a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid), but retain the ability to bind a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid).

[0391] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a W476A mutation and a W1126A mutation such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but maintains the ability to bind to a target nucleic acid.

[0392] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a P475A mutation, a W476A mutation, an N477A mutation, a D1125A mutation, a W1126A mutation, and a D1127A mutation, such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but retains the ability to bind to a target nucleic acid.

[0393] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a H840A mutation, a W476A mutation, and a W1126A mutation such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but maintains the ability to bind to a target nucleic acid.

[0394] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a H840A mutation, a D10A mutation, a W476A mutation, and a W1126A mutation, such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but retains the ability to bind to a target nucleic acid.

[0395] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a H840A mutation, a P475A mutation, a W476A mutation, a N477A mutation, a D1125A mutation, a W1126A mutation, and a D1127A mutation, such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but retains the ability to bind to a target nucleic acid.

[0396] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a D10A mutation, a H840A mutation, a P475A mutation, a W476A mutation, a N477A mutation, a D1125A mutation, a W1126A mutation, and a D1127A mutation, such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but retains the ability to bind to a target nucleic acid.

[0397] Other residues may be mutated to achieve the above effects (i.e., to inactivate one or other nuclease moieties). By way of non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted) (see Table 1 for further information regarding conservation of Cas9 amino acid residues). Mutations other than alanine substitutions are also suitable.

[0398] In some embodiments, a mutant Cas9 polypeptide with reduced catalytic activity (e.g., when the Cas9 protein has a D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutation, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and / or D986A), the mutant Cas9 polypeptide can still bind to a target nucleic acid in a site-specific manner (as it is still guided to the target nucleic acid sequence by the guide nucleic acid), so long as it maintains the ability to interact with a guide nucleic acid.

[0399] [Table 1]

[0400] In addition, mutant Cas9 proteins can have the same parameters for sequence identity as those described above for Cas9 polypeptides. Thus, in some embodiments, suitable mutant Cas9 polypeptides comprise an amino acid sequence having four motifs, each of which has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% amino acid sequence identity to the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO: 1), or alternatively to motifs 1-4 (motifs 1-4 of SEQ ID NO: 1 are SEQ ID NOs: 3-6, respectively, as shown in Table 1); or alternatively to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO: 1). Any of the Cas9 proteins defined above, including those specifically referenced in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, may be used as a Cas9 polypeptide or as part of a chimeric Cas9 polypeptide in the compositions of the disclosure.

[0401] In some embodiments, a suitable mutant Cas9 polypeptide comprises an amino acid sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% amino acid sequence identity to the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO: 1). Any of the Cas9 proteins defined above, including those specifically referenced in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, can be used as a mutant Cas9 polypeptide or as part of a chimeric mutant Cas9 polypeptide in the compositions of the disclosure.

[0402] Chimeric polypeptides (fusion polypeptides) In some embodiments, the mutant Cas9 polypeptide is a chimeric Cas9 polypeptide (also referred to herein as a fusion polypeptide, e.g., a "Cas9 fusion polypeptide"). A Cas9 fusion polypeptide can bind to and / or modify (e.g., cleave, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with the target nucleic acid (e.g., methylate, acetylate, etc., of histone tails).

[0403] A Cas9 fusion polypeptide is a mutant Cas9 polypeptide due to sequence differences from a wild-type Cas9 polypeptide (e.g., a naturally occurring Cas9 polypeptide). A Cas9 fusion polypeptide is a Cas9 polypeptide (e.g., a wild-type Cas9 polypeptide, a mutant Cas9 polypeptide, a mutant Cas9 polypeptide with reduced nuclease activity (as described above), etc.) fused to a covalently linked heterologous polypeptide (also referred to as a "fusion partner"). In some embodiments, a Cas9 fusion polypeptide is a mutant Cas9 polypeptide with reduced nuclease activity (e.g., dCas9) fused to a covalently linked heterologous polypeptide. In some embodiments, the heterologous polypeptide exhibits (and thus provides) an activity (e.g., an enzymatic activity) that is also exhibited by the Cas9 fusion polypeptide (e.g., a methyltransferase activity, an acetyltransferase activity, a kinase activity, a ubiquitination activity, etc.). In some such embodiments, for example, when the Cas9 polypeptide is a mutant Cas9 polypeptide having a fusion partner (i.e., having a heterologous polypeptide) that has an activity (e.g., enzymatic activity) that modifies the target nucleic acid, the method can also be considered a method of modifying the target nucleic acid. In some embodiments, the method of binding a target nucleic acid (e.g., a single-stranded target nucleic acid) can result in modification of the target nucleic acid. Thus, in some embodiments, the method of binding a target nucleic acid (e.g., a single-stranded target nucleic acid) can be a method of modifying the target nucleic acid.

[0404] In some embodiments, the heterologous sequence provides for subcellular localization, i.e., the heterologous sequence is a subcellular localization sequence for targeting to the nucleus (e.g., a nuclear localization signal (NLS), a sequence for maintaining the fusion protein outside the nucleus, e.g., a nuclear export sequence (NES), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). In some embodiments, the mutant Cas9 does not contain an NLS, and as a result, the protein is not targeted to the nucleus (this can be advantageous, for example, when the target nucleic acid is RNA present in the cytosol). In some embodiments, the heterologous sequence may provide a tag (i.e., the heterologous sequence is a detectable label) for ease of tracking and / or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; etc.). In some embodiments, the heterologous sequence may provide increased or decreased stability (i.e., the heterologous sequence is a stability control peptide, e.g., a degron, which in some embodiments is controllable (e.g., a temperature-sensitive or drug-controllable degron sequence, see below). In some embodiments, the heterologous sequence may provide increased or decreased transcription from the target nucleic acid (i.e., the heterologous sequence is a transcription regulatory sequence, e.g., a transcription factor / activator or fragment thereof, a protein or fragment thereof that recruits a transcription factor / activator, a transcription repressor or fragment thereof, a protein or fragment thereof that recruits a transcription repressor, a small molecule / drug-responsive transcriptional regulator gene, etc.). In some embodiments, the heterologous sequence may provide a binding domain (i.e., the heterologous sequence is a protein binding sequence, e.g., a transcription factor or transcription repressor, a recruiting protein, an RNA-modifying enzyme, an RNA-binding protein, a translation initiation factor, an RNA splicing factor, etc., to provide the ability of the Cas9 fusion polypeptide to bind to another protein of interest, e.g., a DNA- or histone-modifying protein).A heterologous nucleic acid sequence can be linked (eg, by genetic engineering) to another nucleic acid sequence to produce a chimeric nucleotide sequence that encodes a chimeric polypeptide.

[0405] A subject Cas9 fusion polypeptide (Cas9 fusion protein) can have multiple (one or more, two or more, three or more, etc.) fusion partners in any combination of the above. As an illustrative example, a Cas9 fusion protein can have a heterologous sequence that provides an activity (e.g., for transcriptional regulation, target modification, modification of a protein associated with a target nucleic acid, etc.) and can also have a subcellular localization sequence. In some embodiments, such Cas9 fusion proteins can also have a tag for ease of tracking and / or purification (e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; etc.). As another illustrative example, a Cas9 protein can have one or more NLSs (e.g., two or more, three or more, four or more, five or more, one, two, three, four, or five NLSs). In some embodiments, the fusion partner (or fusion partners) (e.g., an NLS, a tag, a fusion partner that provides an activity, etc.) is located at or near the C-terminus of Cas9. In some embodiments, the fusion partner (or fusion partners) (e.g., an NLS, a tag, a fusion partner that provides an activity, etc.) is located at the N-terminus of Cas9. In some embodiments, Cas9 has a fusion partner (or fusion partners) (e.g., an NLS, a tag, a fusion partner that provides an activity, etc.) at both the N-terminus and the C-terminus.

[0406] Suitable fusion partners that provide increased or decreased stability include, but are not limited to, degron sequences. Those skilled in the art will readily appreciate that degrons are amino acid sequences that control the stability of the protein of which they are a part. For example, the stability of a protein containing a degron sequence is controlled, in part, by the degron sequence. In some embodiments, suitable degrons are constitutive, such that the degron exerts its effect on protein stability independently of experimental control (i.e., the degron is not drug-inducible, temperature-inducible, etc.). In some embodiments, degrons provide mutant Cas9 polypeptides with controllable stability, such that the mutant Cas9 polypeptide can be directed "on" (i.e., stable) or "off" (i.e., unstable, degraded) depending on the desired conditions. For example, if the degron is a temperature-sensitive degron, the mutant Cas9 polypeptide may be functional (i.e., "on," stable) at or below a threshold temperature (e.g., 42°C, 41°C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, etc.), but may be non-functional (i.e., "off," degraded) above the threshold temperature. As another example, if the degron is a drug-inducible degron, the presence or absence of a drug may switch the protein from an "off" (i.e., unstable) state to an "on" (i.e., stable) state, or vice versa. An exemplary drug-inducible degron is derived from the FKBP12 protein. The stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron.

[0407] Examples of suitable degrons include, but are not limited to, degrons regulated by Shield-1, DHFR, auxin, and / or temperature. Non-limiting examples of suitable degrons are known in the art (e.g., Dohmen et al., Science, 1994. 263(5151): p. 1273-1276: Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol. 2009 Jan;296(1):F204-11: Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett. 2008 Nov 15;18(22):5941-4: Recent progress with FKBP-derived destabilizing domains; Kanemaki, Pflugers Arch. 2012 Dec 28: Frontiers of protein expression control with conditional degrons; Yang et al., Mol Cell. 2012 Nov 30;48(4):487-8: Titivated for destruction: the methyl degron; Barbour et al., Biosci Rep. 2013 Jan 18;33(1).: Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase; and Greussing et al., J Vis Exp.2012 Nov 10;(69): Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein; all of which are incorporated herein by reference in their entireties.

[0408] Exemplary degron sequences have been well characterized and tested in both cells and animals. Thus, fusing Cas9 (e.g., wild-type Cas9; mutant Cas9; mutant Cas9 with reduced nuclease activity, e.g., dCas9; etc.) to a degron sequence produces a "regulatable" and "inducible" Cas9 polypeptide. Any of the fusion partners described herein can be used in any desired combination. As one non-limiting example illustrating this point, a Cas9 fusion protein (i.e., a chimeric Cas9 polypeptide) can contain a YFP sequence for detection, a degron sequence for stability, and a transcriptional activator sequence to increase transcription of the target nucleic acid. Suitable reporter proteins for use as fusion partners for Cas9 polypeptides (e.g., wild-type Cas9, mutant Cas9, mutant Cas9 with reduced nuclease function, etc.) include, but are not limited to, the following exemplary proteins (or functional fragments thereof): his3, β-galactosidase, fluorescent proteins (e.g., GFP, RFP, YFP, cherry, tomato, etc., and various derivatives thereof), luciferase, β-glucuronidase, and alkaline phosphatase. Furthermore, there is no limit to the number of fusion partners that can be used in a Cas9 fusion protein. In some embodiments, a Cas9 fusion protein comprises one or more (e.g., two or more, three or more, four or more, or five or more) heterologous sequences.

[0409] Suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity, any of which can be directed to directly modifying nucleic acids (e.g., methylating DNA or RNA) or modifying nucleic acid-associated polypeptides (e.g., histones, DNA-binding proteins, and RNA-binding proteins, etc.). Further suitable fusion partners include, but are not limited to, boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0410] Examples of various additional suitable fusion partners (or fragments thereof) for the subject mutant Cas9 polypeptides include, but are not limited to, those described in PCT patent applications WO2010 / 075303, WO2012 / 068627, and WO2013 / 155555, which are incorporated by reference herein in their entireties.

[0411] Suitable fusion partners include, but are not limited to, polypeptides that provide an activity that indirectly increases transcription by acting directly on the target nucleic acid or a polypeptide associated with the target nucleic acid (e.g., histones, DNA-binding proteins, RNA-binding proteins, RNA-editing proteins, etc.) Suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity.

[0412] Additional suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription and / or translation of a target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that recruit transcriptional activators, small molecule / drug-responsive transcriptional and / or translational regulatory genes, translation-regulatory proteins, etc.).

[0413] Non-limiting examples of fusion partners for achieving increased or decreased transcription include transcriptional activator and transcriptional repressor domains (e.g., Kruppel-associated box (KRAB or SKD); Mad mSIN3-interacting domain (SID); ERF repressor domain (ERD), etc.). In some such embodiments, the Cas9 fusion protein is targeted to a specific location (i.e., sequence) in the target nucleic acid by the guide nucleic acid and exerts locus-specific modulation, such as blocking RNA polymerase binding to the promoter (which selectively inhibits transcriptional activator function) and / or modifying the local chromatin state (e.g., when a fusion sequence is used that modifies the target nucleic acid or a polypeptide associated with the target nucleic acid). In some embodiments, the change is transient (e.g., transcriptional repression or activation). In some embodiments, the change is heritable (e.g., when epigenetic modifications are made to the target nucleic acid or to proteins associated with the target nucleic acid, e.g., nucleosomal histones).

[0414] Non-limiting examples of fusion partners for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or termination factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, including e.g., adenosine deaminases acting on RNA (ADARs), AI and / or CU editing enzymes); heliembodiments; RNA-binding proteins; and the like. It is understood that fusion partners can include entire proteins, or in some embodiments, can include fragments of proteins (e.g., functional domains).

[0415] In some embodiments, the heterologous sequence can be fused to the C-terminus of the Cas9 polypeptide. In some embodiments, the heterologous sequence can be fused to the N-terminus of the Cas9 polypeptide. In some embodiments, the heterologous sequence can be fused to an internal portion of the Cas9 polypeptide (i.e., a portion other than the N- or C-terminus).

[0416] Additionally, the fusion partner of a chimeric Cas9 polypeptide can be any domain (which, for the purposes of this disclosure, includes intramolecular and / or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.) that can interact with ssRNA, whether primary or irreversible, directly or indirectly; endonucleases (e.g., RNase I, CRR22 DYW domain, Dicer, and the PIN (PilT N-terminal) domain from proteins such as SMG5 and SMG6); proteins and protein domains involved in stimulating RNA cleavage (e.g., CPSF, CstF, CFIm, and CFIIm); exonucleases (e.g., XRN-1 or exonuclease T); deadenylases (e.g., HNT3); proteins and protein domains involved in nonsense-mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and protein domains involved in RNA stabilization (e.g., PABP); proteins and protein domains involved in translational repression (e.g., Ago2 and Ago4); proteins and protein domains involved in translational stimulation (e.g., Staufen); proteins and protein domains involved in (e.g., capable of regulating) translation (e.g., translation factors such as initiation factors, elongation factors, and termination factors, e.g., eIF4G); proteins and protein domains involved in RNA polyadenylation (e.g., PAP1, GLD-2, and Star-PAP); proteins and protein domains involved in RNA polyuridinylation (e.g., CI D1 and terminal uridylate transferase); proteins and protein domains involved in RNA localization (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains involved in nuclear retention of RNA (e.g., Rrp6); proteins and protein domains involved in nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains involved in repression of RNA splicing (e.g., PTB, Sam68, and hnRNP A1);These include, but are not limited to, effector domains selected from the group including proteins and protein domains involved in stimulating RNA splicing (e.g., serine / arginine-rich (SR) domains); proteins and protein domains involved in reducing the efficiency of transcription (e.g., FUS (TLS)); and proteins and protein domains involved in stimulating transcription (e.g., CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group consisting of endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense-mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of suppressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of regulating translation (e.g., translation factors such as initiation factors, elongation factors, and release factors, e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of retaining RNA in the nucleus; proteins and protein domains having RNA export activity; proteins and protein domains capable of suppressing RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable fusion partner is a PUF RNA-binding domain, which is described in more detail in WO2012068627.

[0417] Some RNA splicing factors that can be used as fusion partners for Cas9 polypeptides (either in whole or as fragments thereof) have a modular structure with distinct sequence-specific RNA-binding and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain an N-terminal RNA recognition motif (RRM) that binds to exon splicing enhancers (ESEs) in pre-mRNAs and a C-terminal RS domain that promotes exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to exon splicing silencers (ESSs) via its RRM domain and inhibits exon inclusion via its C-terminal glycine-rich domain. Some splicing factors can regulate the alternative use of splice sites (SSs) by binding to regulatory sequences between two alternative sites. For example, ASF / SF2 can recognize ESEs and promote the use of intron-proximal sites, while hnRNP A1 can bind to ESSs and shift splicing to the use of intron-distal sites. One use of such factors is to generate ESFs that regulate alternative splicing of endogenous genes, particularly those associated with gene-associated diseases. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites, encoding proteins with opposing functions. The long splicing isoform, Bcl-xL, is a potent apoptosis inhibitor expressed in long-lived, postmitotic cells and is upregulated in many cancer cells, protecting cells against apoptotic signals. The short isoform, Bcl-xS, is a pro-apoptotic isoform and is expressed at high levels in cells with high turnover rates (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cω-elements located either in the core exon region or in the exon extension region (i.e., between the two alternative 5' splice sites). For further examples, see WO2010075303.

[0418] In some embodiments, the Cas9 polypeptide (e.g., wild-type Cas9, mutant Cas9, mutant Cas9 with reduced nuclease activity, etc.) can be linked to the fusion partner via a peptide spacer.

[0419] In some embodiments, the Cas9 polypeptide comprises a "protein transduction domain" or PTD (also known as a CPP - cell penetrating peptide), which can refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from small polar molecules to large macromolecules and / or nanoparticles, facilitates the molecule's crossing of a membrane, e.g., from the extracellular space to the intracellular space, or from the cytosol into an organelle. In some embodiments, a PTD attached to another molecule facilitates the molecule's entry into the nucleus (e.g., in some embodiments, the PTD comprises a nuclear localization signal (NLS)). In some embodiments, the Cas9 polypeptide comprises two or more NLSs, e.g., two or more NLSs in tandem. In some embodiments, the PTD is covalently attached to the amino terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently attached to the carboxyl terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently linked to the amino and carboxyl termini of the Cas9 polypeptide. In some embodiments, the PTD is covalently linked to a nucleic acid (e.g., a guide nucleic acid, a polynucleotide encoding a guide nucleic acid, a polynucleotide encoding a Cas9 polypeptide, etc.).Exemplary PTDs include a minimal undecapeptide protein transduction domain (YGRKKRRQRRR; corresponding to residues 47-57 of HIV-1 TAT, comprising SEQ ID NO: 7); a polyarginine sequence containing a sufficient number of arginines to directly enter cells (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:8); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:9); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:10); and RQIKIWFQNRRMKWKK (SEQ ID NO:11). Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO:12), RKKRRQRRR (SEQ ID NO:13); arginine homopolymers of 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO:14); RKKRRQRR (SEQ ID NO:15); YARAAARQARA (SEQ ID NO:16); THRLPRRRRRR (SEQ ID NO:17); and GGRRARRRRRR (SEQ ID NO:18). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381).ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero and thereby inhibits cellular adhesion and uptake. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thus "activating" the ACPP to cross membranes.

[0420] In some embodiments, the composition may contain a Cpf1 RNA-guided endonuclease, examples of which are provided in Figures 2, 3, or 4. Another name for the Cpf1 RNA-guided endonuclease is Cas12a. The Cpf1 CRISPR system of the present disclosure includes i) a single endonuclease protein and ii) a crRNA, where the 3'-end portion of the crRNA contains a guide sequence complementary to the target nucleic acid. In this system, the Cpf1 nuclease is directly recruited to the target DNA by the crRNA. In some embodiments, the guide sequence for Cpf1 must be at least 12 nt, 13 nt, 14 nt, 15 nt, or 16 nt to achieve detectable DNA cleavage, and is at least 14 nt, 15 nt, 16 nt, 17 nt, or 18 nt to achieve sufficient DNA cleavage.

[0421] The Cpfl system of the present disclosure differs from Cas9 in several ways. First, unlike Cas9, Cpfl does not require a separate tracrRNA for cleavage. In some embodiments, the Cpfl crRNA can be as short as about 42-44 bases in length—of which 23-25 ​​nt are guide sequences and 19 nt are constitutive direct repeats. In contrast, the combined Cas9 tracrRNA and crRNA synthetic sequence can be about 100 bases in length.

[0422] Second, Cpf1 prefers a "TTN" PAM motif located 5' upstream of its target. This contrasts with the "NGG" PAM motif located 3' of the target DNA for the Cas9 system. In some embodiments, the uracil base immediately preceding the guide sequence cannot be replaced (Zetsche, B. et al. 2015. "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System" Cell 163, 759-771, which is incorporated herein by reference in its entirety for all purposes).

[0423] Third, the cleavage sites for Cpf1 are staggered by approximately 3-5 bases, creating "sticky ends" (Kim et al., 2016. "Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells," published online June 6, 2016). These sticky ends with 3-5 bp overhangs are thought to facilitate NHEJ-mediated ligation and improve gene editing of DNA fragments with matching ends. The cleavage site is at the 3' end of the target DNA, distal to the 5' end of the PAM. The cleavage position usually follows the 18th base on the unhybridized strand and the corresponding 23rd base on the complementary strand hybridized to the crRNA.

[0424] Fourth, in the Cpf1 complex, a "seed" region is located within the first 5 nt of the guide sequence. The Cpf1 crRNA seed region is highly sensitive to mutations, and even a single base substitution in this region can dramatically reduce cleavage activity (see Zetsche B. et al. 2015 "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System" Cell 163, 759-771). Notably, unlike Cas9 CRISPR targets, the cleavage site and seed region of the Cpf1 system do not overlap. Further guidance for designing Cpf1 crRNA-targeting oligos is available (Zetsche B. et al. 2015 "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System" Cell 163, 759-771).

[0425] Those skilled in the art will understand that the Cpf1 disclosed herein can be any variant derived or isolated from any source, many of which are known in the art. For example, in some embodiments, the Cpf1 peptides of the present disclosure can include FnCPF1 (e.g., SEQ ID NO: 2) shown in Figure 2, AsCpf1 (e.g., Figure 3), LbCpf1 (e.g., Figure 4), or any of many other known Cpf1 proteins from various other microbial species and synthetic variants thereof.

[0426] In some embodiments, the composition contains a Cpf1 polypeptide. In some embodiments, the Cpf1 polypeptide is enzymatically active, e.g., the Cpf1 polypeptide cleaves a target nucleic acid when bound to a guide RNA. In some embodiments, the Cpf1 polypeptide exhibits reduced enzymatic activity relative to a wild-type Cpf1 polypeptide (e.g., relative to a Cpf1 polypeptide comprising the amino acid sequence shown in Figure 2, 3, or 4) and maintains DNA binding activity.

[0427] In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 2, 3, or 4. In some embodiments, the Cpfl polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to a contiguous stretch of 100 amino acids to 200 amino acids (aa), 200 aa to 400 aa, 400 aa to 600 aa, 600 aa to 800 aa, 800 aa to 1000 aa, 1000 aa to 1100 aa, 1100 aa to 1200 aa, or 1200 aa to 1300 aa of the amino acid sequence shown in Figures 2, 3, or 4.

[0428] In some embodiments, the Cpfl polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCI domain of a Cpfl polypeptide having the amino acid sequence depicted in Figure 2, 3, or 4. In some embodiments, the Cpfl polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCII domain of a Cpfl polypeptide having the amino acid sequence depicted in Figure 2, 3, or 4. In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the RuvCIII domain of a Cpf1 polypeptide having an amino acid sequence shown in Figure 2, 3, or 4.

[0429] In some embodiments, the Cpfl polypeptide exhibits reduced enzymatic activity relative to a wild-type Cpfl polypeptide (e.g., relative to a Cpfl polypeptide comprising the amino acid sequence shown in Figure 2, 3, or 4) and maintains DNA binding activity. In some embodiments, the Cpfl polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 2, 3, or 4; and comprises an amino acid substitution (e.g., a D-to-A substitution) at the amino acid residue corresponding to amino acid 917 of the amino acid sequence shown in Figure 2, 3, or 4. In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 2, 3, or 4; and comprises an amino acid substitution (e.g., an E→A substitution) at the amino acid residue corresponding to amino acid 1006 of the amino acid sequence shown in Figure 2, 3, or 4. In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 2, 3, or 4; and comprises an amino acid substitution (e.g., a D to A substitution) at the amino acid residue corresponding to amino acid 1255 of the amino acid sequence shown in Figure 2, 3, or 4.

[0430] In some embodiments, the Cpf1 polypeptide is a fusion polypeptide, for example, wherein the Cpf1 fusion polypeptide comprises: a) a Cpf1 polypeptide; and b) a heterologous fusion partner. In some embodiments, the heterologous fusion partner is fused to the N-terminus of the Cpf1 polypeptide. In some embodiments, the heterologous fusion partner is fused to the C-terminus of the Cpf1 polypeptide. In some embodiments, the heterologous fusion partner is fused to both the N-terminus and C-terminus of the Cpf1 polypeptide. In some embodiments, the heterologous fusion partner is inserted internally within the Cpf1 polypeptide.

[0431] Suitable heterologous fusion partners include NLSs, epitope tags, fluorescent polypeptides, and the like.

[0432] Combined guide RNA and donor nucleic acid In one aspect, the present invention provides a complex comprising a CRISPR system, comprising an RNA-guided endonuclease (e.g., Cas9 or Cpf1 polypeptide), a guide RNA and a donor polynucleotide, wherein the guide RNA and the donor polynucleotide are linked.As exemplified herein, the guide RNA and the donor polynucleotide can be either covalently linked or non-covalently linked.In one embodiment, the guide RNA and the donor polynucleotide are chemically linked.In another embodiment, the guide RNA and the donor polynucleotide are enzymatically linked.In one embodiment, the guide RNA and the donor polynucleotide are hybridized with each other.In another embodiment, the guide RNA and the donor polynucleotide are both hybridized with a bridge sequence.Any number of such hybridization schemes are possible.

[0433] Deaminase In some embodiments, the complex or composition further comprises a deaminase (e.g., an adenine base editor). As used herein, the term "deaminase" or "deaminase domain" refers to an enzyme that catalyzes the removal of an amine group from a molecule, i.e., deamination. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase is a cytosine deaminase, which catalyzes the hydrolytic deamination of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA).

[0434] In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., modified adenosine deaminase, developed adenosine deaminase) can be from any organism, such as bacteria. In some embodiments, the deaminase or deaminase domain is a mutant of a naturally occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.

[0435] In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may lack one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length ecTadA. In some embodiments, the ecTadA deaminase does not contain an N-terminal methionine. In some embodiments, the deaminase is APOBEC1 or a mutant thereof.

[0436] The deaminase can be used in conjunction with any other CRISPR element described herein (i.e., as a composition), or the deaminase can be fused to any other CRISPR element described herein (e.g., Cas9 or Cpfl) (i.e., as a complex). In some embodiments, the deaminase is fused to Cas9, Cpfl, or a mutant thereof.

[0437] Other ingredients The composition may further contain any other components typically used in nucleic acid or protein delivery formulations. For example, the composition may further contain lipids, lipoproteins (e.g., cholesterol and derivatives), phospholipids, polymers, or other components of a liposome or micelle delivery vehicle. The composition may also contain a solvent or carrier suitable for administration to a cell or host, such as a mammal or human.

[0438] In some embodiments, the composition further comprises one or more surfactants.The surfactant can be a nonionic surfactant and / or an amphoteric surfactant.In some embodiments, the surfactant is a polymer or copolymer of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO), glycolic acid (GA), lactic acid (LA), or a combination thereof.For example, the surfactant can be polyethylene glycol (PEG), polypropylene glycol, polyglycolic acid (PGA), polylactic acid, or a mixture thereof. A list of exemplary surfactants is: polyoxyethylene sorbitan ester surfactants (commonly called Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), sold under the trade name DOWFAX®, such as linear EO / PO block copolymers; octoxynols, which may vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, of which octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-6301NP-40); phospholipids such as phosphatidylcholines (lecithins); triethylene glycol monolauryl ether (Brij Examples of surfactants include, but are not limited to, polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants), such as 30; polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known as Span), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. In some embodiments, the surfactant is an anticoagulant (e.g., heparin, etc.). In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0439] In some cases, a component (e.g., a nucleic acid component (e.g., a guide nucleic acid, etc.); a protein component (e.g., a Cas9 or Cpfl polypeptide, a mutant Cas9 or Cpfl polypeptide, etc.) comprises a labeling moiety. As used herein, the terms "label," "detectable label," or "labeling moiety" refer to any moiety that provides signal detection and can vary widely depending on the particular nature of the assay. Labeling moieties of interest include both directly detectable labels (direct labels) (e.g., fluorescent labels) and indirectly detectable labels (indirect labels) (e.g., binding pair members). The fluorescent label can be any fluorescent label (e.g., a fluorescent dye (e.g., fluorescein, Texas red, rhodamine, ALEXAFLUOR® label, etc.), a fluorescent protein (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, tangerine, and any fluorescent derivatives thereof), etc. Detectable (direct or indirect) label moieties suitable for use in the present methods include any moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, a suitable indirect label includes biotin (a binding pair member), which can be bound by streptavidin (which itself can be directly or indirectly labeled). Labels also include: radiolabels (direct labels) (e.g., 3 H, 125 I, 35 S, 14 C, or 32P); enzymes (indirect labels) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.); fluorescent proteins (direct labels) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivatives thereof); metal labels (direct labels); colorimetric labels; binding pair members; etc. "Partner of a binding pair" or "binding pair member" means one of a first and second moiety, wherein the first and second moieties have specific binding affinity for each other. Suitable binding pairs include, but are not limited to: antigen / antibody (e.g., digoxigenin / anti-digoxigenin, dinitrophenyl (DNP) / anti-DNP, dansyl-X-anti-dansyl, fluorescein / anti-fluorescein, lucifer yellow / anti-lucifer yellow, and rhodamine anti-rhodamine), biotin / avidin (or biotin / streptavidin), and calmodulin-binding protein (CBP) / calmodulin. Any binding pair member may be suitable for use as an indirectly detectable labeling moiety.

[0440] Any given component, or combination of components, can be unlabeled or detectably labeled with a labeling moiety. In some embodiments, when two or more components are labeled, they can be labeled with labeling moieties that are distinguishable from one another.

[0441] Encapsulation and Nanoparticles In some embodiments of the composition, the polymer is combined with the nucleic acid and / or polypeptide and partially or completely encapsulates the nucleic acid and / or polypeptide. The composition may, in some formulations, provide nanoparticles comprising the polymer and the nucleic acid and / or polypeptide.

[0442] In some embodiments, the composition may contain, in addition to the polymer and nucleic acid or polypeptide described herein, a core nanoparticle. Any suitable nanoparticle may be used, including metal (e.g., gold) nanoparticles or polymeric nanoparticles.

[0443] The polymers and nucleic acids (e.g., guide RNA, donor polynucleotide, or both) or polypeptides described herein can be directly or indirectly conjugated to the surface of the nanoparticle. For example, the polymers and nucleic acids (e.g., guide RNA, donor polynucleotide, or both) or polypeptides described herein can be directly or indirectly conjugated to the surface of the nanoparticle via an intervening linker.

[0444] Any type of molecule can be used as a linker. For example, the linker can be an aliphatic chain containing at least two carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more carbon atoms) and can be substituted with one or more functional groups, including ketone, ether, ester, amide, alcohol, amine, urea, thiourea, sulfoxide, sulfone, sulfonamide, and disulfide functionalities. In embodiments in which the nanoparticles contain gold, the linker can be any thiol-containing molecule. Reaction of a thiol group with gold results in a covalent sulfide (-S-) bond. The design and synthesis of linkers are well known in the art.

[0445] In some embodiments, the nucleic acid conjugated to the nanoparticle is a linker nucleic acid that serves to non-covalently link one or more elements described herein (e.g., a Cas9 polypeptide, and a guide RNA, a donor polynucleotide, and a Cpfl polypeptide) to the nanoparticle-nucleic acid conjugate. For example, the linker nucleic acid can have a sequence that hybridizes to the guide RNA or the donor polynucleotide.

[0446] Nucleic acids conjugated to nanoparticles (e.g., colloidal metal (e.g., gold) nanoparticles; nanoparticles comprising biocompatible polymers) can have any suitable length. When the nucleic acid is a guide RNA or a donor polynucleotide, the length is appropriate for such molecules as discussed herein and known in the art. When the nucleic acid is a linker nucleic acid, it can have any suitable length for the linker, for example, from 10 nucleotides (nt) to 1000 nt, e.g., from about 1 nt to about 25 nt, from about 25 nt to about 50 nt, from about 50 nt to about 100 nt, from about 100 nt to about 250 nt, from about 250 nt to about 500 nt, or from about 500 nt to about 1000 nt. In some cases, nucleic acids conjugated to nanoparticles (e.g., colloidal metal (e.g., gold) nanoparticles; nanoparticles comprising biocompatible polymers) can have a length greater than 1000 nt.

[0447] When a nucleic acid bound to a nanoparticle (e.g., covalently or non-covalently) comprises a nucleotide sequence that hybridizes to at least a portion of a guide RNA or donor polynucleotide present in a complex of the present disclosure, it has a region having sufficient sequence identity with a region of the complement of the guide RNA or donor polynucleotide sequence to facilitate hybridization. In some embodiments, the nucleic acid bound to a nanoparticle in a complex of the present disclosure has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% nucleotide sequence identity with a 10-50 nucleotide (e.g., 10 nucleotides (nt) to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 40 nt, or 40 nt to 50 nt) complement of a guide RNA or donor polynucleotide present in the complex.

[0448] In some embodiments, the nucleic acid bound (e.g., covalently bound; non-covalently bound) to the nanoparticle is a donor polynucleotide or has the same or substantially the same nucleotide sequence as a donor polynucleotide. In some embodiments, the nucleic acid bound (e.g., covalently bound; non-covalently bound) to the nanoparticle comprises a nucleotide sequence that is complementary to a donor DNA template.

[0449] The nanoparticles can have any suitable size as long as they are stable. For example, the nanoparticles can have an average diameter of about 20 nm to about 500 nm, about 50 nm to about 500 nm, about 20 nm to about 400 nm, about 20 nm to about 300 nm, about 20 nm to about 250 nm, about 50 nm to about 400 nm, about 50 nm to about 300 nm, or about 50 nm to about 250 nm. In some embodiments, the nanoparticles have an average diameter of about 50 nm to about 25 nm.

[0450] The nanoparticles can have any suitable polydispersity index (PDI). For example, the nanoparticles can have a polydispersity index of 0.05 to about 0.5, about 0.1 to about 0.5, about 0.2 to about 0.5, about 0.05 to about 0.4, about 0.1 to about 0.4, about 0.2 to about 0.4, about 0.05 to about 0.3, about 0.1 to about 0.3, or about 0.2 to about 0.3. In some embodiments, the nanoparticles have an average polydispersity index of about 0.1 to about 0.3.

[0451] How to use Also provided herein are methods for delivering nucleic acids and / or polypeptides to cells, where the cells can be in vitro or in vivo. The methods include administering a composition containing the polymers provided herein and nucleic acids and / or polypeptides described herein to cells or a subject containing cells. The methods can be used with any type of cell or subject, but are particularly useful for mammalian cells (e.g., human cells). In some embodiments, the polymers include a targeting agent, such that the nucleic acids and / or polypeptides are delivered primarily or exclusively to target cells or tissues (e.g., cells or tissues of the peripheral nervous system, central nervous system (e.g., brain or spinal cord), a subject's eye, liver, muscle, lung, bone (e.g., hematopoietic cells), or tumor cells or tissues).

[0452] When used to deliver proteins or nucleic acids to cells in a subject (i.e., in vivo), it is desirable for the polymer to be stable in serum. Serum stability can be evaluated as a function of the efficiency with which the polymer delivers a protein or nucleic acid payload to cells in serum (e.g., in vitro or in vivo). Thus, in some embodiments, the polymer delivers a given protein or nucleic acid to cells in serum with greater efficiency or directionality than occurs with pAsp[DET] under the same conditions for a particular tissue type.

[0453] In some embodiments, the polymers disclosed herein exhibit specific tropism for certain cell or tissue types. In some embodiments, the polymers disclosed herein are tropic for tissues of the peripheral or central nervous system (i.e., upon administration to a host, the polymer is delivered in the greatest amount or concentration to tissues of the peripheral or central nervous system relative to other tissues) and are used to deliver proteins or nucleic acids to tissues of the peripheral or central nervous system (e.g., to deliver proteins or nucleic acids to cells or tissues of the peripheral or central nervous system of a mammal).

[0454] The polymer can be any polymer described herein (e.g., a polymer comprising a hydrolyzable polymer backbone, such as a polymer of Formula 1 or 1A-1C, or otherwise as described herein, including any and all features and embodiments thereof, the polymer backbone comprising (i) monomer units comprising hydrophobic side chains; and (ii) monomer units comprising side chains comprising polyamine groups and polyalkylene oxide groups, and optionally (iii) monomer units comprising side chains comprising polyamine groups and no polyalkylene oxide groups).

[0455] In some embodiments, the composition contains an "additional polymer" or "second polymer" as described herein, e.g., a polymer comprising (a) a monomeric unit having a side chain comprising a hydrophobic group, and (b) a monomeric unit having a side chain comprising an oligoamine or polyamine. In some embodiments, the second polymer also comprises another monomer, e.g., a monomeric unit having a side chain comprising an ionic group, optionally having a pKa of less than 7. In some embodiments, the second polymer comprises a hydrolyzable polymer backbone such as a polyamide, a poly-N-alkylamide, a polyester, a polycarbonate, a polycarbamate, or a combination thereof. In some embodiments, the hydrolyzable polymer backbone comprises a polyamide. In some embodiments, the second polymer does not comprise a polyalkylene oxide moiety. In some embodiments, the second polymer is a polymer of any of WO2021 / 217082, WO2020219776, WO2020086910A1, WO2020243370, or WO2019210326A2. All other aspects and embodiments of the additional or "second" polymer are as previously described herein and may be used in accordance with the present method of use.

[0456] All other aspects of the composition for use in the method are as previously described herein.

[0457] When used with a composition containing one or more components of a CRISPR system, the method can be used to edit a target nucleic acid or gene. In some embodiments, the method of modifying a target nucleic acid comprises homology-directed repair (HDR). In some embodiments, the use of the complex of the present disclosure to perform HDR provides an HDR efficiency of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or more than 25%. In some embodiments, the method of modifying a target nucleic acid comprises non-homologous end joining (NHEJ). In some embodiments, the use of the complex of the present disclosure to perform HDR provides an NHEJ efficiency of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or more than 25%.

[0458] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0459] Example 1 Comparative polymer 1 ("H27N") and PBLA were 1 -(2-aminoethyl)-N 1 ,N 2 ,N 2 The procedure is illustrated in Scheme 1. Scheme 1: Synthesis of polymer H27N

[0460] [ka]

[0461] (The brackets in H27N do not mean a block copolymer structure.)

[0462] Lyophilized PBLA (50 mg, 0.0037 mmol; degree of polymerization ("DP") 65) was placed in a flask and dissolved in tetrahydrofuran / N-methyl-2-pyrrolidine (1 mL each). To the clear solution, n-hexylamine (160 equivalents) was added, and the clear reaction mixture was stirred at room temperature for 24 hours. After approximately 24 hours, N 1 -(2-aminoethyl)-N 1 ,N 2 ,N 2 1,2-trimethylethane-1,2-diamine (50 equivalents relative to the benzyl groups of the PBLA moiety) was added to the clear mixture under mild, anhydrous conditions. After approximately 18 hours at room temperature, the reaction mixture was precipitated into diethyl ether (10-12X volume, 35 mL). The precipitate was then centrifuged and washed twice with diethyl ether. The polymer was dissolved in 1 M HCl (3 mL) and dialyzed against excess deionized water using a 3.5-5 KD cutoff membrane. When the pH of the solution was between 5 and 6, dialysis was stopped, and the solution was lyophilized to obtain comparative polymer 1 (H27N). As can be seen from the procedure described, the resulting polymer contained polyamine and hydrophobic side chains randomly dispersed throughout the polymer. Therefore, the bracketed groups in the above structure merely represent the approximate number of side chain species generated and do not imply any specific arrangement of side chains in the polymer.

[0463] Example 2 Polymer Precursor A was prepared by modifying PBLA with amines and N-(2-aminoethyl)ethane-1,2-diamine ("DET"). The procedure is illustrated in Scheme 2.

[0464] [ka]

[0465] Lyophilized PBLA (50 mg, 0.0037 mMol) was placed in a flask and dissolved in tetrahydrofuran / N-methyl-2-pyrrolidine (1 mL each). n-Hexylamine (58.8 μL, 0.44 mMol, 120 equiv.) was added to the clear solution, and the clear reaction mixture was stirred at room temperature for 24 hours. After approximately 24 hours, diethylenetriamine (50 equiv. relative to the benzyl group of the PBLA moiety, 1.0 g) was added to the clear mixture under mild anhydrous conditions. After approximately 18 hours at room temperature, the reaction mixture was precipitated in diethyl ether (10-12X volume, 35 mL). The white precipitate was then centrifuged and washed twice with diethyl ether. The white polymer was dissolved in 1 M HCl (3 mL) and dialyzed against excess deionized water using a 3.5-5 KD cutoff membrane. When the pH of the solution was between 5-6, dialysis was stopped, and the solution was lyophilized to obtain approximately 60 mg of polymer precursor A. As shown in Table 2 (x and y represent average values), polymer precursors A1, A2, and A3 were obtained using different amounts of n-hexylamine and PBLA. 1 Confirmed by H NMR spectroscopy. The bracketed groups shown in the Polymer A structure merely represent the approximate number of side chain species that occurred and do not imply any particular arrangement of the side chains in the polymer.

[0466] [Table 2]

[0467] Example 3 Polymers (a)-(l) were prepared by modifying Polymer Precursor A1, Polymer Precursor A2, or Polymer Precursor A3 with pegylated acrylamide or pegylated acrylate. The procedure is illustrated in Scheme 3. Scheme 3: Synthesis of polymers (a)-(l)

[0468] [ka]

[0469] General Procedure A. Triethylamine was added to a methanol solution of Polymer Precursor A1, Polymer Precursor A2, or Polymer Precursor A3. PEGylated acrylamide or PEGylated acrylate (~2000 Da; n = ~45) was added to the resulting solution and stirred at 25 °C. The crude PEG-conjugated product was precipitated in ether and purified to yield the final product.

[0470] General Procedure B. Polymer Precursor A1, Polymer Precursor A2, or Polymer Precursor A3 was added to an aqueous solution (pH = 9). Varying amounts of PEGylated acrylamide or PEGylated acrylate (~2000 Da; n = ~45) were added to the resulting solution and stirred at 50 °C. The crude PEG-conjugated product was precipitated in ether and purified to yield the final product.

[0471] General Procedure C. Triethylamine was added to a methanol solution of Polymer Precursor A1, Polymer Precursor A2, or Polymer Precursor A3. PEGylated acrylamide or PEGylated acrylate (~400 Da; n = ~9) was added to the resulting solution and stirred at 25 °C. The crude PEG-conjugated product was precipitated in ether and purified to yield the final product.

[0472] Polymers (a)-(l) were prepared according to General Procedure A, General Procedure B, or General Procedure C, as shown in Table 3.

[0473] [Table 3]

[0474] Example 4 The following example describes the use of the polymers of the invention to deliver Cre mRNA to mice, as represented by Ai9 mice.

[0475] Two polymer nanoparticle compositions were prepared by combining Cre mRNA with either (i) Comparative Polymer 1 alone (composition (i)), or (ii) a mixture of Comparative Polymer 1 and polymer (c). The mRNA and polymer solutions were combined by microfluidic mixing followed by lyophilization. Both preparations showed good encapsulation efficiency.

[0476] To test the ability of the nanoparticle compositions to deliver mRNA in vivo, Ai9 mice carrying the red fluorescent reporter construct illustrated in Figure 5 were injected (intrathecally) with 0.5 mg / ml of composition (i) or composition (ii). Termination sequences inserted 5' of the reporter gene are flanked by LoxP elements and prevent expression of the reporter genes until they are excited by Cre recombinase protein. Untreated mice served as controls.

[0477] After treatment, brains and spinal cords were harvested and imaged by ex vivo epifluorescence, with the results shown in Figure 6. Brains were also sagittally sectioned and reimaged to more clearly display the epifluorescence, with the results shown in Figure 7.

[0478] The results show increased delivery of Cre mRNA to the brain, particularly to the posterior regions of the brain, with composition (ii) compared to composition (i).

[0479] Example 5 The following examples illustrate the preparation of additional polymers in accordance with the present disclosure.

[0480] The synthesis scheme was as follows:

[0481] [ka]

[0482] Synthesis of BLA-NCA (1): β-Benzyl aspartic acid (10 gm, 0.44 mol) was added to an oven-dried 250 mL round-bottom flask and suspended in anhydrous THF (90 mL). To this solution was added triphosgene (7.97 gm, 0.026 mol) in one portion. The reaction mixture was heated at 55°C for 1 hour. THF was removed under reduced pressure while maintaining the water bath temperature at 28°C to give the crude BLA-NCA product (13.75 gm). The crude BLA-NCA product was dissolved in 50 mL THF, and the crude product was precipitated into 750 mL of hexane to give crude BLA-NCA (9.87 g). The crude BLA-NCA was suspended in anhydrous DCM (250 mL) and stirred under argon for 30–40 minutes. The BLA-NCA solution was filtered through a 25 g bed of Celite® S (the Celite was dried in an oven at 130°C for 2 days). The Celite bed was further washed with 200 mL of DCM. The combined DCM (approximately 350 mL) washes of BLA-NCA were filtered twice through the same Celite® S bed and washed with DCM (200 mL). The combined DCM solution was evaporated under reduced pressure while maintaining the water bath temperature at 28°C. The purified BLA-NCA was dissolved in THF (30 mL), and the product was precipitated into hexane (450 mL) to give BLA-NCA (1) as a white solid, 2.87 g, in 25.7% yield. The solid was dried under high vacuum for 2-3 hours and stored at -80°C. 1H NMR (DMSO-d6; 800 MHz): 9.0 (1H), 7.35 (m, 5H), 5.15 (s, 2H), 4.62 (m, 1H), 3.20-2.85 (dd, 2H).

[0483] Synthesis of PBLA (2): BLA-NCA (1) (2.0 g, 8.025 mmol) was added to an oven-dried 1000 mL round-bottom flask and dissolved in a mixture of 25 mL of dichloromethane (DCM) and 2.5 mL of N,N-dimethylformamide (DMF). To this solution was added n-butylamine (13.22 μL, 0.1337 mmol). The reaction mixture was stirred at room temperature for 96 hours to give PBLA 2. The reaction mixture was precipitated into diethyl ether (420 mL). The PBLA-containing diethyl ether solution was collected in a Falcon tube (50 mL) and centrifuged (3500 x g, 7 min, 25 °C) to give intermediate 2 (1.7 gm) as a white solid. 1H NMR (DMSO-d6; 800 MHz): 8.25 (m, 60H), 7.40-7.10 (m, 332 H), 5.10-4.85 (m, 134H), 4.63 (63H), 2.75 (m, 65H), 2.50 (m, 65H), 1.25 (m, 4H), 0.82 (t, 3H).

[0484] Synthesis of intermediate (3) (H41D24): To an oven-dried 250 mL round-bottom flask was added PBLA 2 (1.0 g, 0.0074 mmol) dissolved in 30 mL of DMSO. The solution was then diluted with 10 mL of THF. n-Hexylamine (1183 μL, 8.95 mmol) was added to the solution and stirred at room temperature for 28 h. Diethylenetriamine (DET) (7.7 gm, 74.6 mmol) was then added to the reaction mixture and stirred for 20 h. Crude intermediate 3 was precipitated in 750 mL of diethyl ether in an Erlenmeyer flask to give an oily precipitate. The precipitate was collected by centrifugation at 3500 × g for 7 min at 25 °C using 16 x 50 mL Falcon tubes (8 x 2). The ether was decanted, and the sample was dried at RT for 2 h. The precipitate was suspended in 0.5 M HCl (3 mL) and allowed to dissolve overnight at 4 °C. The crude intermediate 3 was further purified using an 8-10 kD cutoff dialysis bag with 0.01 M HCl as the dialysate at 4 °C for 18 h. The dialysate was then replaced with freshly prepared 0.01 M HCl, and dialysis continued at 4 °C for 12 h. Subsequently, the dialysate was replaced with molecular biology-grade water every 4 h, and dialysis continued at 4 °C. A 70 mL solution of pure intermediate 3 was lyophilized to give intermediate 3 as a fluffy white solid (840 mg). H NMR (DO, 800 MHz): 3.86-2.20 (0.9H, m), 1.90-0.34 (1H, m).

[0485] Synthesis of Polymer A (4): To a 20 mL scintillation vial was added intermediate (3) (150 mg, 0.01 mmol) dissolved in 7 mL of water (molecular biology grade). To this solution was added 2000 kDa PEG acrylate (800 mg, 0.4 mmol) dissolved in 5 mL of molecular biology grade water. The reaction mixture was heated at 50 °C for 48 h. The reaction mixture was cooled to RT and lyophilized to give crude Polymer A (4) as a light brown, fluffy solid. The approximate crude yield was 950 mg. Crude Polymer A (4) was purified using a Pierce™ strong cation exchange spin column (ThermoFisher Scientific, Catalog No.: 90009). Finally, the polymer solution was lyophilized to give pure Polymer A (4) as a light yellow powder (230 mg). 1H NMR (D2O, 800 MHz): 4.45-4.12 (m, 19H), 4.00-3.60 (m, 1171H), 3.57-2.12 (m, 427H), 1.72-0.46 (m, 451H).

[0486] Additional polymers were made using the above procedure, but with the same structure as intermediate 3 but different ratios of hydrophobic (H) to amine (D) side chains, as intermediate (3), and others modified by using compounds as shown in Tables 4 and 5:

[0487] [ka]

[0488] [Table 4]

[0489] [Table 5]

[0490] Example 6 The following examples illustrate the use of polymers according to the present disclosure to deliver nucleic acids.

[0491] The polymer blends shown below were prepared to a final concentration of 1 mg / ml, and luciferase mRNA was prepared to a final concentration of 0.2 mg / ml using 20 mM HEPES. Polymer nanoparticles (PNPs) were formulated by adding equal volumes of luciferase mRNA (0.2 mg / ml) and polymer (1 mg / ml). This resulted in a PNP formulation with an mRNA:polymer ratio of 1:5. The PNPs were incubated for 10 minutes, followed by the addition of sucrose to a final concentration of 10 mg / mL. The resulting PNPs were flash-frozen in liquid nitrogen and lyophilized.

[0492] PNP was resuspended to a final concentration of 1 mg / ml mRNA by slowly pipetting and dropping into 20 mM HEPES buffer and incubated for 10 minutes. Male FVB mice (6-8 weeks) were anesthetized with 1.5-2% isoflurane in an induction chamber, and 10 μl PNP was administered by intrathecal lumbar puncture between the L5 and L6 spinous processes. After PNP administration, mice were kept under isoflurane anesthesia for 30 minutes in the Trendelenburg position with their heads tilted 30 degrees on their backs before being returned to their cages to recover.

[0493] Transfection and biodistribution were analyzed by bioluminescence imaging. Animals were anesthetized and maintained with 2-2.5% isoflurane in oxygen. D-luciferin was administered intraperitoneally at 150 mg / kg. Animals were continuously imaged using an in vivo imaging system (IVIS). Photons were collected and integrated for 1 min. Total flux intensity was measured from the region of interest across the FUS-targeted area.

[0494] Immediately after the final in vivo bioluminescence imaging period, animals were euthanized. The brains and spinal cords were quickly removed, immersed in 10 mg / ml D-luciferin, and imaged with IVIS. Photons were integrated for 2 minutes.

[0495] The results are shown in Figures 8-10. The polymers identified in Figures 8-10 correlate to the polymer of Example 5 shown in Table 6.

[0496] [Table 6]

[0497] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to adopt such variations as necessary, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0498] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value in that stated range is included within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0499] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0500] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a complex" includes a plurality of such complexes, and reference to "the Cas9 polypeptide" includes reference to one or more Cas9 polypeptides and equivalents thereof known to those of skill in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of such exclusive terminology, such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

[0501] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments of the invention are specifically embraced by the present invention and disclosed herein, just as if each and every combination were individually and explicitly disclosed herein. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and disclosed herein, just as if each and every subcombination were individually and explicitly disclosed herein.

[0502] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

Claims

1. A polymer containing a hydrolyzable polymer backbone, wherein the polymer backbone is: (i) a monomer unit containing a hydrophobic side chain; (ii) a monomer unit containing a side chain containing a polyamine group and a polyalkylene oxide group, wherein the polyamine group and the polyalkylene oxide group together form a structure: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH 2 -CHOH-R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH(CONH 2 )-(CH 2 ) s1 -R 5 ; or -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH(CONH 2 )-(CH 2 ) s1 -R 4 -R 5 , wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; s2 is an integer from 0 to 5; In each case, R 2 is independently hydrogen or a C 1 -C 12 alkyl group, a C 2 -C 12 alkenyl group, a C 3 -C 12 cycloalkyl group or a C 3 -C 12 cycloalkenyl group; Z may be present, and when present, is -C(O)-, -C(O)O-, -S(O)(O)-, -C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or an optionally substituted aryl or heteroaryl; In each case, R 4 is independently -C(O)O-, -C(O)-, -C(O)NR 2 -, -CH 2 -O-C(O)-O-CH 2 -,-O-C(O)-O-,-O-,-S(O)(O)- or a bond; and R 5 is a group containing a polyalkylene oxide; having a monomer unit; and, optionally, (iii) the formula: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 2 , wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; and in each case R 2 is independently hydrogen or a C 1 -C 12 alkyl group, a C 2 -C 12 alkenyl group, a C 3 -C 12 cycloalkyl group or a C 3 -C 12 cycloalkenyl group, or R 2 forms a heterocyclic group in combination with the second R 2 and the nitrogen to which it is attached; a monomer unit containing a polyamine group having and not containing a polyalkylene oxide group a side chain containing containing a polymer.

2. The polymer according to claim 1, wherein the hydrophobic group is an aliphatic group or an aryl group, optionally containing an alkyl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, a heteroalkyl group or a heterocyclic group.

3. The polymer according to claim 1, wherein the hydrophobic group contains a C 3 -C 12 linear or branched alkyl group.

4. The polymer according to claim 1, wherein the polyalkylene oxide group contains a polyethylene oxide group, a polypropylene oxide group, or a combination thereof.

5. A side chain containing a polyamine group and a polyalkylene oxide group has the formula: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH 2 -CHOH-R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH(CONH 2 )-(CH 2 ) s1 -R 5 ; or -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH(CONH 2 )-(CH 2 ) s1 -R 4 -R 5 、 wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; and in each case R 2 is independently hydrogen or C 1 -C 6 alkyl group, C 2 -C 6 alkenyl group, C 3 -C 6 cycloalkyl group or C 3 -C 6 cycloalkenyl group; and in each case R 4 is independently -C(O)O-, -C(O)-, -C(O)NH-, -CH 2 -O-C(O)-O-CH 2 -, -O-, -S(O)(O)- or a bond; and R 5 is a group containing a polyalkylene oxide having the polymer of claim 1.

6. The formula: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 2 、 wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; and in each case R 2 is independently hydrogen or C 1 -C 6 alkyl group, C 2 -C 6 alkenyl group, C 3 -C 6 cycloalkyl group or C 3 -C 62is a cycloalkenyl group, or R 2 is the second R 2 and combines with the nitrogen to which it is attached to form a heterocyclic group A polymer according to claim 1, comprising a monomer unit containing a polyamine group having and not containing a polyalkylene oxide group, and having a side chain not containing a polyalkylene oxide group.

7. A polymer according to claim 1, comprising monomer units containing hydrophobic side chains in an amount of about 1 to about 80 mol%, monomer units containing side chains containing polyamine groups and polyalkylene oxide groups in an amount of about 1 to about 50 mol%, and, if present, monomer units containing side chains containing polyamine groups and not containing polyalkylene oxide groups in an amount of about 1 to about 80 mol%.

8. A polymer according to claim 1, wherein the hydrolyzable polymer backbone comprises a polyamide, a poly-N-alkylamide, a polyester, a polycarbonate, a polyurethane, or a combination thereof.

9. A polymer according to claim 8, wherein the hydrolyzable polymer backbone comprises a polyamide.

10. Formula 1: 【Chemical Formula 1】 wherein: m 1 、m 2 、m 3 and m 4 each of which is an integer from 0 to 1000, provided that the sum of m 1 + m 2 + m 3 + m 4 is greater than 2, and the sum of m 3 + m 4 is at least 1; n 1 and n 2 each of which is an integer from 0 to 1000, provided that the sum of n 1 + n 2 is at least 1, The symbol " / " indicates that the units separated thereby are joined randomly or in any order; R in each case 3a is independently a methylene or ethylene group; R in each case 3b is independently a methylene or ethylene group; R in each case 11 is independently hydrogen or a C 1 -C 4 alkyl group or a C 2 -C 4 alkenyl group, and any of these may be substituted with one or more substituents; Each X 1 is independently -C(O)O-, -C(O)NR 11 -, -C(O)-, -S(O)(O)- or a bond; X in each case 2 is a hydrophobic side chain; A in each case 1 is independently: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 2 and B in each case 1 is independently: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH 2 -CHOH-R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH(CONH 2 )-(CH 2 ) s1 -R 5 ; or -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH(CONH 2 )-(CH 2 ) s1 -R 4 -R 5 and wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; s2 is an integer from 0 to 5; in each case R 2 is independently hydrogen or a C 1 -C 12 alkyl group, C 2 -C 12An alkenyl group, C 3 -C 12 a cycloalkyl group or C 3 -C 12 a cycloalkenyl group, or R 2 is, when applicable, combined with a second R 2 and the nitrogen to which they are attached to form a heterocyclic group; Z may be present, and when present, is -C(O)-, -C(O)O-, -S(O)(O)-, -C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or an optionally substituted aryl or heteroaryl; In each case, R 4 is independently -C(O)O-, -C(O)-, -C(O)NH-, -CH 2 -O-C(O)-O-CH 2 -, -O-C(O)-O-, -O-, -S(O)(O)- or a bond; and R 5 is a group containing a polyalkylene oxide The polymer according to claim 1, comprising the structure of

11. Formula 1A: 【Chemical Formula 2】 Wherein: Q is of the formula: 【Chemical Formula 3】 and c is an integer from 0 to 50; Y may be present and is a cleavable linker; R 1 is hydrogen, an aryl group, a heterocyclic group, C 1 -C 12 an alkyl group, C 2 -C 12 an alkenyl group, C 3 -C 12 a cycloalkyl group or C 3 -C 12 a cycloalkenyl group, any of which may be optionally substituted with one or more substituents; and R 6 is hydrogen, an amino group, an aryl group, a heterocyclic group, C 1 -C 12 an alkyl group, C 1 -C 12 a heteroalkyl group, C 2 -C 12 an alkenyl group, C 3 -C 12 a cycloalkyl group or C 3 -C 12 a cycloalkenyl group, and any of these may be substituted with one or more substituents or tissue-specific or cell-specific targeting sites having the structure of; or Formula 1B: [Chemical Formula 4] wherein: R 1 is hydrogen, an aryl group, a heterocyclic group, C 1 -C 12 an alkyl group, C 2 -C 12 an alkenyl group, C 3 -C 12 a cycloalkyl group or C 3 -C 12 a cycloalkenyl group, and any of these may be substituted with one or more substituents; and R 6 is hydrogen, an amino group, an aryl group, a heterocyclic group, C 1 -C 12 an alkyl group, C 1 -C 12 a heteroalkyl group, C 2 -C 12 an alkenyl group, C 3 -C 12 a cycloalkyl group or C 3 -C 12 a cycloalkenyl group, and any of these may be substituted with one or more substituents or tissue-specific or cell-specific targeting sites having the structure of; or Formula 1C: [Chemical Formula 5] The polymer of claim 10 having the structure.

12. X in each case 2 is independently C 1 -C 12 an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof; wherein any of these may be substituted with one or more substituents, the polymer of claim 10.

13. B in each case 1 is: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH 2 -CHOH-R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH(CONH 2 )-(CH 2 ) s1 -R 5 ; or -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH(CONH 2 )-(CH 2 ) s1 -R 4 -R 5 、 wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; in each case R 2 is independently hydrogen or a C 1 -C 6 alkyl group, a C 2 -C 6 alkenyl group, a C 3 -C 6 cycloalkyl group or a C 3 -C 6 cycloalkenyl group; in each case R 4 is independently -C(O)O-, -C(O)-, -C(O)NH-, -CH 2 -O-C(O)-O-CH 2 -, -O-, -S(O)(O)- or a bond; and R 5 is a group containing a polyalkylene oxide is, the polymer of claim 10.

14. In each case A 1 is -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 2 and B in each case 1 is: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 4 -R 5 The polymer of claim 10, wherein

15. (i) A in each case 1 is -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 2 and B in each case 1 is: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -C(O)-(CH 2 ) s1 -R 4 -R 5 or (ii) A1 in each case is -(CH2)2-[NH-(CH2)2-]r1NH-(CH2)2-NR22 and B1 in each case is: -(CH2)2-[NH-(CH2)2-]r1NH-(CH2)2-NR2-C(O)-(CH2)2-R4-R5 The polymer of claim 10, wherein

16. (m 1 +m 2 +m 3 +m 4 ) / (n 1 +n 2 ) has a ratio of about 0.3 to 3, the polymer of claim 10.

17. In each case, R 5 is independently of the formula: 【Chemical Formula 6】 In the formula: R 12 is a bonding or methylene, ethylene or propylene group; R 13 is hydrogen, an aryl group, a heterocyclic group, C 1 -C 12 alkyl group, C 2 -C 12 alkenyl group, C 3 -C 12 cycloalkyl group or C 3 -C 12 cycloalkenyl group, any of which may be substituted with one or more substituents, optionally hydrogen or C 1 -C 3 alkyl group; and t1 is an integer from 2 to 200 is the polymer of claim 10.

18. (a) providing a polymer of formula 2: 【Chemical Formula 7】 and (b) modifying the moiety of group A of the polymer of formula 2 to formula 1: 1 to give formula 1: 【Chemical Formula 8】 In the formula: m 1 、m 2 、m 3 and m 4 are each an integer from 0 to 1000, provided that m 1 + m 2 + m 3 + m 4 The sum of which is greater than 2, and m 3 + m 4 The sum of which is at least 1; n 1 and n 2 each of which is an integer from 0 to 1000, provided that n 1 + n 2 The sum of which is at least 1, The symbol " / " indicates that the units separated thereby are combined randomly or in any order; Each R in each case 3a is independently a methylene or ethylene group; Each R in each case 3b is independently a methylene or ethylene group; Each R in each case 11 is independently hydrogen or a C 1 -C 4 alkyl or alkenyl group, any of which may be substituted with one or more substituents, optionally hydrogen or a C 1 -C 3 alkyl group; Each X 1 is independently -C(O)O-, -C(O)NR 11 -, -C(O)-, -S(O)(O)- or a bond; Each X in each case 2 is a hydrophobic side chain; Each A in each case 1 is independently: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 2 and Each B in each case 1 is independently: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH 2 -CHOH-R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH(CONH 2 )-(CH 2 ) s1 -R 5 ; or -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s2 -CH(CONH 2 )-(CH 2 ) s1 -R 4 -R 5 and is, wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; s2 is an integer from 0 to 5; R in each case 2 is independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, or in the applicable case, R 2 is, the second R 2 combines to form a heterocyclic group; Z may be present, and if present, -C(O)-, -C(O)O-, -S(O)(O)-, -C(NH)NR 2 -, -C(S)O-, -C(S)NR 2 -, -C(O)NR 2 - or an optionally substituted aryl or heteroaryl; R in each case 4 is independently -C(O)O-, -C(O)-, -C(O)NH-, -CH 2 -O-C(O)-O-CH 2 -, -O-C(O)-O-, -O-, -S(O)(O)- or a bond; and R 5 is a group containing a polyalkylene oxide to provide a polymer of A process for preparing a polymer of formula 1 comprising.

19. B in each case 1 is: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH 2 -CHOH-R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 5 ; -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH(CONH 2 )-(CH 2 ) s1 -R 5 ; or -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -CH(CONH 2 )-(CH 2 ) s1 -R 4 -R 5 wherein each of p1 to p3 is independently an integer from 1 to 5; r1 is an integer from 0 to 5; s1 is an integer from 0 to 5; In each case, R 2 is independently hydrogen or C 1 -C 6 an alkyl group, C 2 -C 6 an alkenyl group, C 3 -C 6 a cycloalkyl group or C 3 -C 6 a cycloalkenyl group; In each case, R 4 is independently -C(O)O-, -C(O)-, -C(O)NH-, -CH 2 -O-C(O)-O-CH 2 -, -O-, -S(O)(O)- or a bond; and R 5 is a group containing a polyalkylene oxide The method of claim 18.

20. (i) Modifying the moiety of group A 1 of the polymer of formula 2 to provide a polymer of formula 1 by reacting with a compound having the structure: 1 【Formula 9】 【Formula 9】 including; Here, in each case, B is: 1 is: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -(CH 2 ) s1 -R 4 -R 5 or (ii) Modifying the moiety of group A1 of the polymer of formula 2 to provide a polymer of formula 1 by reacting with a compound having the structure: 【Formula 10】 wherein EA is an activated ester; including; Here, in each case, B1 is: Here, in each case, B1 is: -(CH 2 ) p1 -[NH-(CH 2 ) p2 -] r1 NH-(CH 2 ) p3 -NR 2 -Z-(CH 2 ) s1 -R 4 -R 5 The method according to claim 18.

21. R in each case 5 is independently of the formula: 【Chemical Formula 11】 In the formula: R 12 is a bond or a methylene, ethylene or propylene group; R 13 is hydrogen, an aryl group, a heterocyclic group, C 1 -C 12 alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group, any of which may be substituted by one or more substituents, optionally hydrogen or C 1 -C 3 alkyl group; and t1 is an integer from 2 to 200 The polymer according to claim 18.

22. A composition comprising a polymer according to any one of claims 1-17 and a nucleic acid and / or a polypeptide, and optionally nanoparticles comprising a polymer according to any one of claims 1-17 and a nucleic acid or a polypeptide.

23. A composition according to claim 22, comprising a guide nucleic acid and / or a donor nucleic acid; an RNA-guided endonuclease or a nucleic acid encoding the same, optionally the RNA-guided endonuclease is Cas9 or Cpf1; a DNA recombinase; a zinc finger nuclease; or a transcription activator-like effector nuclease.

24. A composition according to claim 22, comprising a second polymer comprising (a) a monomer unit having a side chain containing a hydrophobic group and (b) a monomer unit having a side chain containing an oligoamine or a polyamine, wherein the second polymer does not contain a polyalkylene oxide group. A composition according to claim 22 for use in delivering a nucleic acid and / or polypeptide to a cell, optionally wherein the cell is in a host. Claim 26 A method for delivering a nucleic acid and / or polypeptide to a cell in vitro, comprising administering the composition of claim 22 to the cell.

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