Cationic polymer having alkyl side chains

Abstract

This provides a novel composition useful for delivering therapeutic molecules. [Solution] A polymer is provided comprising a hydrolyzable polymer main chain, wherein the polymer main chain comprises (i) monomer units having a side chain containing a hydrophobic group, (ii) monomer units having a side chain containing an oligoamine or polyamine, and optionally (iii) monomer units having a side chain containing an ionizable group.

Description

[Technical Field]

[0001] Cross-references to related applications This patent application claims priority to U.S. Provisional Patent Application No. 62 / 837,658, filed on 23 April 2019, and U.S. Provisional Patent Application No. 62 / 853,658, filed on 28 May 2019, 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 countless applications for preventing, curing, and treating diseases. However, the safe and effective delivery of large molecules (e.g., polypeptides and nucleic acids) to their target tissues remains a challenge. Therefore, there continues to be a need for novel compositions and methods useful for delivering therapeutic molecules. [Overview of the Initiative]

[0003] Provided herein are polymers comprising a hydrolyzable polymer backbone, wherein the polymer backbone comprises (i) monomer units having side chains containing hydrophobic groups; (ii) monomer units having side chains containing oligoamines or polyamines; and optionally (iii) monomer units having side chains containing ionizable groups and optionally having a pKa of less than 7.

[0004] Also provided herein is Formula 1:

[0005] [ka]

[0006] (In the formula: m 1 , m 2 , m 3 , and m 4 Each of them is an integer from 0 to 1000, where m 1 +m2 +m 3 +m 4 The sum of is greater than 5; n 1 and n 2 each of which is an integer from 0 to 1000, provided that n 1 +n 2 the sum of is greater than 2; The symbol " / " indicates that the units separated thereby are concatenated randomly or in any order; R 3a each instance of is independently a methylene or ethylene group; R 3b each instance of is independently a methylene or ethylene group; Each X 1 is independently, -C(O)O-, -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; R 13 each instance of is independently hydrogen, an aryl group, a heterocyclic group, a C1-C 12 alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, any of which may optionally be substituted with one or more substituents; X 2 each instance of is independently a C1-C 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 one or more primary, secondary, or tertiary amines; any of which may be substituted with one or more substituents; 12 A A 1 and A 2 are each independently of the formula -(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 It is the basis of B 1 and B 2 They are independent of each other. -(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 (wherein p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R 2 Each case independently involves hydrogen or C1-C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 The second R 2 It combines with R to form a heterocyclic group; 4 Each of these cases is independently -C(O)O-, -C(O)NH-, -COC(O)-OC-, -O-, or -S(O)(O)-;R 5 Each of these examples is independently a polymer containing a structure that is an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or a combination thereof, optionally comprising 2 to 8 tertiary amines or substituents containing tissue-specific or cell-specific targeting moieties.

[0007] The polymer of formula 1 described above may further contain an ionizable group. In some embodiments, the ionizable group is R of formula 1. 5 Provided by [company name]. In other embodiments, the polymer further comprises monomers having side chains containing ionizable groups.

[0008] The disclosure also provides polymers comprising the structure of Formula 1, as well as compositions comprising nucleic acids and / or polypeptides. Further provided are methods for producing polymers comprising the structure of Formula 1, and methods for using the polymer and compositions comprising the same to deliver nucleic acids or proteins to cells, for example. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the amino acid sequence (SEQ ID NO: 1) of Cas9 derived from Streptococcus pyogenes. [Figure 2] Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of Cpf1 derived from the tularemia subspecies Novicida U112. [Figure 3] Figure 3 is a graph showing the degree of substitution of the hydrophobic moiety of polymer A as a result of the equivalent number of hydrophobic moieties added to the reaction mixture. [Figure 4] Figure 4 is a graph showing the degree of substitution of the hydrophobic moiety of polymer B as a result of the equivalent number of hydrophobic moieties added to the reaction mixture. [Figure 5] Figure 5 is a graph illustrating the transfection efficiency of polymer A nanoparticles in HEK293T cells as described in Example 3, as a function of RFP fluorescence. [Figure 6] Figure 6 is a graph illustrating the transfection efficiency of polymer A nanoparticles in HEK293T cells as a function of RFP fluorescence, as described in Example 4. [Figure 7] Figure 7 is a graph illustrating the transfection efficiency of polymer A nanoparticles in HepG2 cells as a function of RFP fluorescence, as described in Example 4. [Figure 8] Figure 8 is a graph illustrating the transfection efficiency of polymer A nanoparticles in primary myoblasts as a function of RFP fluorescence, as described in Example 4. [Figure 9]Figure 9 is a graph illustrating the transfection efficiency of polymer A nanoparticles and polymer B nanoparticles in HEK293T cells as described in Example 4, as a function of RFP fluorescence. [Figure 10] Figure 10 is a graph illustrating the transfection efficiency of Cas9-containing polymer A and polymer B nanoparticles in HEK293T cells, as described in Example 5, as a function of GFP knockout. [Figure 11] Figure 11 is a graph illustrating the transfection efficiency of polymer A and polymer B nanoparticles containing Cpf1 in HEK293T cells, as described in Example 5, as a function of GFP knockout. [Figure 12] Figure 12 is a graph illustrating the transfection efficiency of polymer A nanoparticles in HEK293T cells as a function of RFP fluorescence, as described in Example 6. [Figure 13] Figure 13 is a graph illustrating the cell viability of Hep3B cells after treatment with polymer A nanoparticles, as described in Example 7. [Figure 14] Figure 14 shows the sequence of AsCpf1 (sequence number 19). [Figure 15] Figure 15 shows the sequence of LbCpf1 (sequence number 20). [Figure 16] Figure 16 shows the dynamic light scattering of particles containing mCherry mRNA and polymer H27N as described in Example 9. [Figure 17] Figure 17 shows the dynamic light scattering of particles containing Cas9 RNP and polymer H27N as described in Example 10. [Figure 18] Figure 18 is a graph illustrating the transfection efficiency of polymer H27N nanoparticles in HEK293T cells as a function of RFP fluorescence, as described in Example 12. [Figure 19]Figure 19 is a graph illustrating the transfection efficiency of polymer H27N nanoparticles in Hep3B cells, as described in Example 13, as a function of non-homologous end joining (NHEJ) efficiency. [Figure 20] Figure 20 is a graph illustrating the transfection efficiency of polymer H27N nanoparticles in Hep3B cells, as described in Example 14, as a function of non-homologous end joining (NHEJ) efficiency. [Figure 21] Figure 21 is a schematic diagram of the mouse Loxp-luciferase reporter function. [Figure 22] Figures 22A-22C show bioluminescence imaging of luciferase-expressing mice treated with the composition described in Example 15. [Figure 23] Figure 23 is a schematic diagram of the mouse AI9 reporter function. [Figure 24] Figure 24 shows bioluminescence imaging of Cre mRNA delivery to rostral and caudal sections of the brain of luciferase-expressing mice treated with the composition described in Example 17. [Figure 25] Figure 25 is a graph illustrating the transfection efficiency of polymer nanoparticles as a function of RFP fluorescence, as described in Example 23. [Modes for carrying out the invention]

[0010] Detailed description of the invention The present invention provides a polymer comprising a hydrolyzable polymer backbone, wherein the polymer backbone comprises (i) monomer units having side chains containing hydrophobic groups; (ii) monomer units having side chains containing oligoamines or polyamines; and optionally (iii) monomer units having side chains containing ionizable groups and optionally having a pKa of less than 7.

[0011] As used herein, the term "hydrolyzable polymer backbone" refers to a polymer backbone having bonds that are easily cleaved by naturally occurring factors (e.g., enzymes) under physiological conditions (e.g., physiological pH, physiological temperature, or in a given in vivo tissue such as blood or serum). Generally, hydrolyzable polymer backbones include polyamides, poly-N-alkylamides, polyesters, polycarbonates, polycarbamates, or combinations thereof. In certain embodiments, the hydrolyzable polymer backbone includes polyamides.

[0012] A monomer unit having a side chain containing a hydrophobic group may contain any hydrophobic group. An example of a hydrophobic group is, 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 group, C2-C 12 (For example, 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-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 ,) containing a cycloalkyl group or a cycloalkenyl group. In certain embodiments, the hydrophobic group is C4-C 12The hydrophobic group comprises an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. In some embodiments, the hydrophobic group comprises fewer than eight carbon atoms or fewer than six carbon atoms. 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 embodiments described above, the hydrophobic group may be linked directly to the polymer backbone or via a linkage comprising, for example, an ester, amide, or ether group, and optionally further comprising an alkylene linker (e.g., a methylene or ethylene linker).

[0013] The polymer also comprises monomer units having side chains containing oligoamines or polyamines. As used herein, the term "oligoamine" refers to a group having two or three amine groups. The term "polyamine" refers to any chemical moiety having four or more amine groups (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc.). The amine groups may be primary amine groups, secondary amine groups, tertiary amine groups, or any combination thereof. In certain embodiments, an oligoamine or polyamine may have the formula: -(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}; -(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 -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 -NR2 -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 (In the formula, p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R 2 Each case independently involves hydrogen or C1-C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 The second R 2 It combines with R to form a heterocyclic group; 4 Each of these cases is independently -C(O)O-, -C(O)NH-, -O-, -COC(O)-CO-, or -S(O)(O)-:R 5 Each of these examples is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or combination thereof, which optionally contains 2 to 8 tertiary amines or substituents containing tissue-specific or cell-specific targeting moieties.

[0014] In some embodiments, the oligoamine or polyamine is given by formula: -(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 -NR2 2]2}2 (In the formula, p1 to p4, q1 to q6, and r1 and r2 are independently integers from 1 to 5 (e.g., 1, 2, or 3); R 2 Each case independently involves hydrogen or C1-C 12 (For example, C1-C6, C1-C3, C2, or C1) alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or R 2 The second R 2 It is a group that bonds with a carbon atom to form a heterocyclic group. The alkenyl group consists of at least two carbon atoms (for example, C2-C2). 12 The cycloalkyl and cycloalkenyl groups must have at least three carbon atoms (e.g., C2-C6), and each cycloalkyl and cycloalkenyl group must have at least three carbon atoms (e.g., C3-C6). 12 It is understood that the polyamine must have (C3-C6, etc.). In some embodiments, the polyamine is -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2, and here, arbitrarily R 2 These are independently hydrogen or a C1-C3 alkyl group (e.g., methyl or ethyl).

[0015] In some embodiments, the polymer further comprises monomer units having side chains containing ionizable groups. As used herein, the phrase “ionizable group” refers to any chemical moiety having substituents readily convertible to charged species. For example, an ionizable group can be a group that is a proton donor or a proton acceptor. The group can be protonated or deprotonated under physiological conditions. In certain embodiments, the ionizable group has a pKa of less than 7 (in water at 25°C). For example, the ionizable groups 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 further, the ionizable groups described herein may have 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. Accordingly, the ionizable groups described herein may have pKas ranging from -2 to 7, for example, -1 to 7, 0 to 7, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 0 to 6, 2 to 6, 4 to 6, 0 to 5, 2 to 5, or 4 to 5. Examples of ionizable groups include, for example, sulfonic acids, sulfonamides, carboxylic acids, thiols, phenols, amine salts, imides, and amide groups.

[0016] In some embodiments, the polymer has an overall pKa of less than 7 (in water at 25°C). 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, the polymers described herein may have 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. Accordingly, the polymers described herein may have a pKa of -2 to 7, for example, -1 to 7 pKa, 0 to 7 pKa, 1 to 7 pKa, 2 to 7 pKa, 3 to 7 pKa, 4 to 7 pKa, 5 to 7 pKa, 6 to 7 pKa, 0 to 6 pKa, 2 to 6 pKa, 4 to 6 pKa, 0 to 5 pKa, 2 to 5 pKa, or 4 to 5 pKa.

[0017] The polymer may comprise any suitable number or amount (e.g., by weight or a few percent composition) of monomer units having side chains containing hydrophobic groups, monomer units having side chains containing oligoamines or polyamines, and monomer units having side chains containing ionizable groups, if present. In some embodiments, the polymer comprises about 1 to about 80 moles. % (for example, 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 having hydrophobic groups, about 1 to about 80 mol% (for example, 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 It comprises monomer units having oligoamines or polyamines in about 40 mol%, about 1 to about 20 mol%, or about 1 to about 10 mol%, and monomer units having ionizable groups in 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%).

[0018] Provided herein is Formula 1:

[0019] [ka]

[0020] (In the formula: m 1 , m 2 , m 3 , and m 4 Each of them is an integer from 0 to 1000, where m 1 +m 2 +m 3 +m 4 The sum is greater than 5; n 1 and n 2 Each of them is an integer from 0 to 1000, where n 1 +n 2 The sum is greater than 2; The symbol " / " indicates that the units separated by it are concatenated randomly or in any order; R 3a Each of these cases is independently a methylene group or an ethylene group; R 3bEach of these cases is independently a methylene group or an ethylene group; each X 1 These are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; R 13 Each of these examples independently involves hydrogen, an aryl group, a heterocyclic group, and a C1-C group. 12 Alkyl alkyl group, C2-C 12 Alkenyl group, C3-C 12 Cycloalkyl groups, or C3-C 12 These are cycloalkenyl groups, and any of them may be optionally substituted with one or more substituents; X 2 Each of these cases independently contains, arbitrarily, one or more primary, secondary, or tertiary amines, C1-C 12 Alkyl or heteroalkyl, C3-C 12 Cycloalkyl groups, C2-C 12 Alkenyl group, C3-C 12 A cycloalkenyl group, an aryl group, a heterocyclic group, or a combination thereof; any of these being optionally substituted with one or more substituents; A 1 and A 2 These are each an independent expression -(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 It is the basis of B 1 and B 2 They are independent of each other. -(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 (wherein p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R 2 Each case independently involves hydrogen or C1-C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 The second R 2 It combines with R to form a heterocyclic group; 4 Each of these cases is independently -C(O)O-, -C(O)NH-, -O-, -COC(O)-CO-, or -S(O)(O)-;R 5 Each of these examples is independently a polymer containing a structure that is an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or a combination thereof, optionally comprising 2 to 8 tertiary amines or substituents containing tissue-specific or cell-specific targeting moieties.

[0021] As used herein, “alkyl” or “alkylene” refers to a substituted or unsubstituted hydrocarbon chain. An alkyl group is any number of carbon atoms (e.g., C1-C1). 100 Alkyl, C1-C 50 Alkyl, C1-C 12The alkyl or alkylene may have alkyl groups, C1-C8 alkyl groups, C1-C6 alkyl groups, C1-C4 alkyl groups, C1-C2 alkyl groups, etc. The alkyl or alkylene may be saturated or unsaturated (e.g., to provide alkenyl or alkynyl groups), and may be linear, branched, straight-chained, cyclic (e.g., cycloalkyl or cycloalkenyl), or a combination thereof. The cyclic group may be monocyclic, condense to form a bicyclic or tricyclic group, be linked by bonds, or be spirocyclic. In some embodiments, the alkyl substituent may have one or more heteroatoms (e.g., oxygen, nitrogen, and sulfur) interposed, thereby providing a heteroalkyl, heteroalkylene, or heterocycline (i.e., a heterocyclic group). In some embodiments, the alkyl is substituted with one or more substituents.

[0022] The term "aryl" refers to an aromatic compound having any appropriate number of ring atoms and any appropriate number of rings. This refers to a cyclic system. An aryl group may 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. An aryl group may be monocyclic, condense to form a bicyclic or tricyclic group, or be bonded to form a biaryl group. Typical aryl groups include phenyl, naphthyl, and biphenyl. In some embodiments, the aryl group contains an alkylene linking group to form 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 may have one or more heteroatoms (e.g., oxygen, nitrogen, and sulfur) interposed, thereby providing a heterocyclyl (i.e., a heterocyclic or heteroaryl group). In some embodiments, the aryl is substituted with one or more substituents.

[0023] The term “heterocyclyl” or “heterocyclic group” refers to a cyclic group having one or more heteroatoms (e.g., oxygen, nitrogen, and sulfur), such as an aromatic (e.g., heteroaryl) or non-aromatic group. In some embodiments, the heterocyclyl or heterocyclic group (i.e., a cyclic group having one or more heteroatoms, such as an aromatic (e.g., heteroaryl) or non-aromatic group) is substituted with one or more substituents.

[0024] As used herein, the term "substituted" may mean that one or more hydrogen atoms on a given atom or group are replaced by another group, provided that the valence of the given atom does not exceed the normal valence of the given atom (e.g., substituted alkyl group). For example, when the substituent is oxo (i.e., =O), then two hydrogen atoms on the atom are replaced. The substituent may be one or more hydroxyl, amino (e.g., primary, secondary, and Substituents may include tertiary compounds, aldehydes, carboxylic acids, esters, amides, ketones, nitro compounds, ureas, guanidines, cyano compounds, fluoroalkyl compounds (e.g., trifluoromethane), halo compounds (e.g., fluoro compounds), aryl compounds (e.g., phenyl compounds), heterocyclyl or heterocyclic groups (i.e., cyclic groups having one or more heteroatoms, e.g., aromatic (e.g., heteroaryl) or non-aromatic cyclic groups), oxo compounds, or combinations thereof. Combinations of substituents and / or variables are permitted on the condition that the substitution does not have a material adverse effect on the synthesis or use of the compound.

[0025] According to Equation 1, m 1 , m 2 , m 3 , and m 4 Each of them is m 1 +m 2 +m 3 +m 4 The sum of the numbers is greater than 5, such as 5 to 5000, 5 to 2000, 5 to 1000, 5 to 500, 5 to 100, or 5 to 50, and is an integer from 0 to 1000 (for example, 0 to 500, 0 to 200, 0 to 100, or 0 to 50). In some embodiments, m 1 +m2 +m 3 +m 4 The sum is greater than 10 or greater than 20 (for example, 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). Furthermore, n 1 and n 2 Each of them is n 1 +n 2 The sum of the numbers is greater than 2 (for example, 2 to 2000, 2 to 1000, 2 to 500, 2 to 200, 2 to 100, 2 to 50, or 2 to 25), and the integers are from 0 to 1000 (for example, 0 to 500, 0 to 200, 0 to 100, 0 to 50, or 0 to 25). In some embodiments, n 1 +n 2 The sum of the groups is greater than 5 or greater than 10 (for example, 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 other words, polymers are collectively referred to herein as "A monomer" and "B monomer," respectively, with group A 1 , A 2 B 1 , and / or B 2 It contains at least several monomer units, including the same. Similarly, polymers are collectively referred to as "X monomers" in this specification. The base X 1 and / or X 2 It includes at least several monomer units, including m 1 and m 2 It is zero, and the polymer is A 1 Or A 2 It contains no base. In some embodiments, m 3 and m 4 It is zero, and the polymer is B 1 or B 2 It contains no base.

[0026] The polymer may contain A and B monomers in any suitable ratio to X monomer. In some embodiments, the polymer has a ratio of A and B monomers to X monomer of about 25 or less, and optionally about 1 or more (e.g., (m 1 +m 2 +m 3 +m 4 ) / (n 1 +n 2 This includes the ratio of A and B monomers to X monomer. For example, the ratio of A and B monomers to X monomer may be about 1 to about 25, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 5 to about 25, about 10 to about 25, or about 15 to about 25.

[0027] In embodiments where the polymer contains both A monomer and B monomer, the polymer may contain A monomer in any suitable ratio to B monomer. In some embodiments, the ratio of A monomer to B monomer is (for example, (m 1 +m 2 ) / (m 3 +m 4 )) can be about 20 or less (for example, 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 The ratio of ) is approximately 0.5 or higher, or approximately 0.2 or higher.

[0028] The polymer 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 a particular embodiment, the polymer is a random polymer, a block polymer, a graft polymer, or a combination thereof.

[0029] In other words, in the structure of formula 1, the monomers (each of their side chains A) 1 , A 2 B 1 B2 , X 1 , and X 2 The integers (which can be referenced by) can be arranged randomly or in any order. 1 , m 2 , m 3 , m 4 , n 1 , and n 2 This simply indicates the number of each monomer appearing in the entire chain, and in some embodiments there may be blocks or stretches of a given monomer, but this does not necessarily mean or represent a specific order or block of those monomers. For example, the structure of formula 1 is -A 1 -A 2 -B 1 -B 2 -, -A 2 -A 1 -B 2 -B 1 -, -A 1 -B 1 -A 2 -B 2 -The polymer may contain monomers in the following order. Furthermore, the polymer may consist of blocks of A and / or B polymers (e.g., [A monomer] in any order) m1+m2 -[B monomer] m3+m4 The polymer may contain individual X monomers (e.g., -AXB-, -ABX-, -BXA, etc.) with A and B monomers interspersed, or the polymer may be "capped" at one or both ends of the polymer with one or more X monomers (e.g., blocks of X monomers). Similarly, when the polymer contains blocks of A and / or B monomers, the polymer may contain blocks of X monomers interspersed between the blocks of A and / or B monomers, or the polymer may be "capped" at one or both ends of the polymer with one or more X monomers (e.g., blocks of X monomers). In some embodiments, the polypeptide (e.g., polyaspartamide) backbone is arranged in an alpha / beta configuration with "1" and "2" monomers alternating (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 polymer is capped with X monomer or X monomer is scattered throughout). However, the "A" and "B" side chains (for example, A 1 / A 2 and B 1 / B 2 ) can be randomly dispersed throughout the polymer backbone.

[0030] In polymer structures, R 3a and R 3b These are independently methylene or e It is a ethylene group. In some embodiments, R 3a It is an ethylene group, and R 3b is a methylene group; or R 3a is a methylene group, R 3b is an ethylene group. In a particular embodiment, R 3a and R 3b These are ethylene groups, respectively. In some embodiments, R 3a and R 3b These are methylene groups, respectively.

[0031] In the polymers described herein, each X 1 The bases are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond. Each X 1 The bases can be the same or different from each other. In some embodiments, X 1 is -C(O)NR 13- is. In some embodiments, X 1 It is -C(O)O-.

[0032] R 13 Each case independently involves hydrogen or C1-C 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkenyl groups, aryl groups, or heterocyclic groups (for example, 3-12, 3-10, 3-8, or 3-6 member heterocyclic groups containing one, two, or three heteroatoms), any of which may be substituted with one or more substituents. In some embodiments, R 13 C1-C can be linear or branched. 12 Alkyl group (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 a particular embodiment, each R 13 is methyl or hydrogen. In some embodiments, R 13 is methyl; in other embodiments, R 13 It is hydrogen. Each R 13 They are selected independently and can be the same or different; however, in some embodiments, each R 13 These are the same (for example, all methyl or all hydrogen).

[0033] X 2 Each of these cases is independently C1-C 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12(For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (e.g., C3-C8, C3-C6, or C3-C5) cycloalkenyl groups, aryl groups, or heterocyclic groups (e.g., 3-12, 3-10, 3-8, or 3-6 member heterocyclic groups containing one, two, or three heteroatoms) or combinations thereof, any of which may be substituted with one or more substituents. In some embodiments, X 2 X may optionally contain one or more primary, secondary, or tertiary amines. Therefore, each X 2 They are selected independently and therefore can be the same or different from one another. In a particular embodiment, X 2 Each of these cases independently contains, arbitrarily, one or more primary, secondary, or tertiary amines, C1-C 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkenyl groups, or combinations thereof. In some embodiments, one or more (or all) X 2 The base is independently C2-C 12 (For example, C3-C 12 , C3-C8, C3-C6, C4-C 12 , C4-C6, C6-C 12 , or C8-C 12 ) alkyl group or alkenyl group, or C3-C 12 (For example, C3-C8, C3-C6, C4-C 12 , C4-C6, C6-C 12 , or C8-C 12 ) may be a cycloalkenyl group. In other embodiments, one or more (or all) X 2The group may independently be a C1-C8 (e.g., C1-C6, C1-C4, C1-C3, C2-C8, or C2-C6) alkyl group. Either of the aforementioned alkyl or alkenyl groups may be linear or branched.

[0034] Base A 1 and A 2 These are selected independently and therefore can be the same or different from one another. Similarly, base B 1 and B 2 These are selected independently and may be the same or different from one another. However, in some embodiments, A 1 and A 2 They are the same and / or B 1 and B 2 They are the same.

[0035] Base A 1 , A 2 B 1 , and B 2 In this, 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 independently integers from 1 to 5 (e.g., 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 independently integers from 1 to 3 (e.g., 1, 2, or 3). In certain embodiments, p1 to p4 (i.e., p1, p2, p3, and p4), q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6), and / or s1 to s4 (i.e., s1, s2, s3, and s4) are each 2. In some embodiments, p1 to p4 (i.e., p1, p2, p3, and p4) and / or q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6) are each 2, and r1, r2, and s1 to s4 (i.e., s1, s2, s3, and s4) are each 1.

[0036] R 2 Each of these cases involves hydrogen or C1-C 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) may be cycloalkenyl groups, or R 2 The second R 2 It combines with R to form a heterocyclic group. In some embodiments, 2 C1-C, which can be hydrogen or linear or branched. 12 Alkyl (for example, 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 a particular embodiment, R 2 is methyl. In another embodiment, R 2 It can be hydrogen. Each R 2 These are selected independently and can be the same or different. In some embodiments, each R 2 These are the same (for example, all methyl or all hydrogen).

[0037] R 4 Each of these cases is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-. In some embodiments, R 4 Each of these cases is independently -C(O)O- or -C(O)NH-. In a particular embodiment, R 4 Each of these cases is -C(O)O-. In a particular embodiment, R 4 Each of these cases is -C(O)NH-.

[0038] R 5Each of these examples is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or combination thereof, comprising optionally 2 to 8 tertiary amines or substituents containing tissue-specific or cell-specific targeting moieties. 5 This may include about 2 to about 50 carbon atoms (for example, 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). Some embodiments include R 5 This is a heteroalkyl group containing 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 (i.e., part of the longest continuous chain of atoms in the heteroalkyl group) or pendant substituents. For example, a heteroalkyl group containing tertiary amines may be an alkylamino group, aminoalkyl group, alkylaminoalkyl group, or aminoalkylamino group containing 2 to 8 tertiary amines. It can provide the basis, etc.

[0039] In some embodiments, each R 5 Independently:

[0040] [ka]

[0041] [ka]

[0042] (In the formula, R 2 The details of each case are as described above; R 7 This is a C1-C molecule that is optionally substituted with one or more amines. 50 It is 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 between 0 and 50; Y is an arbitrarily existing and severable linker; n is an integer from 0 to 50; R 8 This refers to tissue-specific or cell-specific targeting areas. C1-C 12 Selected from alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

[0043] R 7 This is a C1-C molecule that is optionally substituted with one or more amines. 50 (For example, C1-C 40 , C1-C 30 , C1-C 20 , C1-C 10 , C4-C 12 , or C6-C8) alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group. In some embodiments, R 7 This is a C4-C8 molecule, such as C6-C8, which is optionally substituted with one or more amines. 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. In some embodiments, R 7 is substituted with one or more amines. In certain embodiments, R 7 2 to 8 (i.e., 2, 3, 4, 5, It is substituted with 6, 7, or 8 tertiary amines. The tertiary amines may be part of the alkyl group (i.e., incorporated into the alkyl group backbone) or pendant substituents.

[0044] Each instance of Y is arbitrary. As used herein, the phrase "arbitrarily present" means that a substituent designated as arbitrarily present may or may not exist, and if that substituent is absent, adjacent substituents are directly bonded to each other. When Y exists, Y is a cleavable linker. As used herein, the phrase "cleavable linker" refers to any chemical element that connects two species and can be cleaved to separate them. For example, a cleavable linker can be cleaved by hydrolysis, photochemical processes, radical processes, enzymatic processes, electrochemical processes, or a combination thereof. An example of a cleavable linker is:

[0045] [ka]

[0046] (In the formula, R 14 Each of these cases is independently a C1-C4 alkyl group, and R 15 In each case, In addition, hydrogen, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 R is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. 16 This includes, but is not limited to, a six-membered aromatic or heteroaromatic group (which is optionally substituted with one or more -OCH3, -NHCH3, -N(CH3)2, -SCH3, -OH, or a combination thereof).

[0047] In some embodiments, A 1 and A 2 Each of these independently corresponds to equation -(CH2) p1 -[NH-(CH2) q1 -] r1 NH2 or -(CH2) p1 -[NH-(CH2) q1 -] r1The group is NHCH3, or the group -(CH2)2-NH-(CH2)2-NH2 or -(CH2)2-NH-(CH2)2-NHCH3 or -(CH2)2-NH-(CH2)2-NH2. Some embodiments are shown in A 1 and A 2 Each of these independently corresponds to equation -(CH2) p1 -[N(R 2 ))-(CH2) q1 -] r1 N(R 2 )2 or -(CH2) p1 -[N(R 2 )-(CH2) q1 -] r1 NH(R 2 )(wherein, R 2 The group 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.

[0048] Furthermore, or alternatively, B 1 and B 2 Each of these is the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 , or group-(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 Equations such as (CH2) p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 (In the formula, R 4 and R 5 It is the basis of (as stated above).

[0049] In some embodiments, the polymer of formula 1 does not contain any B monomer (for example, m 3 and m 4 (Both are 0). That is, what is also provided is Equation 4:

[0050] [ka]

[0051] (In the formula: m 1 and m 2 Each of them is m 1 +m 2 The polymer has a structure where the sum of the elements is greater than 5 (for example, 5 to 2000, 5 to 1000, 5 to 500, 5 to 100, or 5 to 50), and the elements are integers from 0 to 1000 (for example, 0 to 500, 0 to 200, 0 to 100, or 0 to 50). In some embodiments, m 1 +m 2 The sum is greater than 10 or greater than 20 (for example, 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). Furthermore, n 1 and n 2 Each of them is n 1 +n 2 The sum of the numbers is greater than 2 (for example, 2 to 2000, 2 to 1000, 2 to 500, 2 to 200, 2 to 100, 2 to 50, or 2 to 25), and the integers are from 0 to 1000 (for example, 0 to 500, 0 to 200, 0 to 100, 0 to 50, or 0 to 25). In some embodiments, n 1 +n 2 The sum is greater than 5 or greater than 10 (for example, 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).

[0052] Some embodiments of the polymer of formula 4 are A 1 and A 2 These are each an independent expression -(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 (In the formula, p1 to p4, q1 to q6, and r1 and r2 are independent integers from 1 to 5 (for example, integers from 1 to 3), R 2 Each case independently involves hydrogen or C1-C 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, C3-C 12 It is a group of a cycloalkenyl group (for example, C3-C8, C3-C6, or C3-C5). In some embodiments, R 2 Substituent group A 1 and A 2 Each nitrogen in 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. For further example, A 1 and A 2 Each of these is -(CH2)2-NR 2 -(CH2)2-NR 2 It can be 2, and here, R 2Each of these examples is independently a hydrogen atom, alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, particularly an alkyl group such as methyl or ethyl, where each amine is optionally a tertiary amine except that its terminal amine is a secondary or tertiary amine.

[0053] Base A 1 and A 2 Certain non-restrictive examples include, for example, -NH-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;-NH-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N (CH3)2;-NH-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);-NH-CH2-CH2-N (CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3); -N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3).

[0054] All other aspects of the polymer of formula 4, including all embodiments relating to the substituents of formula 4, are as described with respect to formula 1. That is, for example, in some embodiments of formula 4, R 13 Each case is R 13 R can be any group described with respect to Formula 1, including specific embodiments in which is hydrogen or methyl, 3a and R 3b Each case is R 3a and R 3b It can be any of the groups described with respect to Formula 1, including embodiments in which is methylene or ethylene. Similarly, X 1 and X 2 X 1 -C(O)NR 13 -or -C(O)O- and / or one or more (or all) X2 The group may be any of the groups described with respect to Formula 1, including embodiments in which the group may independently be a C1-C8 (e.g., C1-C6, C1-C4, C1-C3, C2-C8, or C2-C6) alkyl group.

[0055] In some embodiments, the polymer is of formula 1A:

[0056] [ka]

[0057] (In the formula, Q is:

[0058] [ka]

[0059] and c is an integer between 0 and 50; Y is an arbitrarily existing and severable linker; m 1 , m 2 , m 3 , and m 4 Each of them is an integer from 0 to 1000, where m 1 +m 2 +m 3 +m 4 The sum is greater than 5; n 1 and n 2 Each of them is an integer from 0 to 1000, where n 1 +n 2 The sum is greater than 2; The symbol " / " indicates that the units separated by it are concatenated randomly or in any order; R 1 This includes hydrogen, an aryl group optionally substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12(For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkenyl groups; R 6 This includes hydrogen, an amino group, an aryl group substituted with one or more amines, a heterocyclic group, and a C1-C group. 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 It has a structure that is (for example, a C3-C8, C3-C6, or C3-C5) cycloalkenyl group; or a tissue-specific or cell-specific targeting moiety.

[0060] All other aspects of Formula 1A, including any and all embodiments thereof, are as described with respect to Formula 1.

[0061] In some embodiments, the polymer is of formula 1B:

[0062] [ka]

[0063] (In the formula, c is an integer between 0 and 50; Y is an arbitrarily existing and severable linker; m 1 and m 2 Each of them is an integer from 0 to 1000, where m 1 +m 2 The sum is greater than 5; n 1 and n 2Each of them is an integer from 0 to 1000, where n 1 +n 2 The sum is greater than 2; The symbol " / " indicates that the units separated by it are concatenated randomly or in any order; R 1 This includes hydrogen, an aryl group optionally substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkenyl groups; R 6 This includes hydrogen, an amino group, an aryl group substituted with one or more amines, a heterocyclic group, and a C1-C group. 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 It has a structure that is (for example, a C3-C8, C3-C6, or C3-C5) cycloalkenyl group; or a tissue-specific or cell-specific targeting moiety.

[0064] All other aspects of Equation 1B, including any and all embodiments thereof, are as described with respect to Equations 1 and 4.

[0065] In some embodiments, the polymer is of formula 1C:

[0066] [ka]

[0067] (In the formula, m 1 and m 2 Each of them is an integer from 0 to 1000, where m 1 +m 2 The sum is greater than 5; n 1 and n 2 Each of them is an integer from 0 to 1000, where n 1 +n 2 The sum is greater than 2; The symbol " / " indicates that the units separated by it are concatenated randomly or in any order; R 1 This includes hydrogen, an aryl group optionally substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkenyl groups; R 6 This includes hydrogen, an amino group, an aryl group substituted with one or more amines, a heterocyclic group, and a C1-C group. 12 (For example, C1-C8, C1-C6, or C1-C3) alkyl or heteroalkyl groups, C2-C 12 (For example, C2-C8, C2-C6, or C2-C3) alkenyl group, C3-C 12 (For example, C3-C8, C3-C6, or C3-C5) cycloalkyl groups, or C3-C 12 It has a structure that is (for example, a C3-C8, C3-C6, or C3-C5) cycloalkenyl group; or a tissue-specific or cell-specific targeting moiety. All other aspects of Formula 1C, including any and all embodiments thereof, are as described with respect to Formulas 1 and 4.

[0068] In some embodiments, R 1 and / or R 6 It can be linear or branched, and optionally substituted with one or more substituents, C1-C 12 Alkyl (for example, C1-C 10 The alkyl group is an 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 certain 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 amine may be part of the heteroalkyl backbone or a pendant substituent.

[0069] The polymer can be any suitable polymer, provided that it contains the polymer structure described above. 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 (e.g., an ethylene oxide subunit or a propylene oxide subunit). In other embodiments, the structure of formula 1 is the sole polymer unit of the polymer, which may contain any suitable end groups. In certain embodiments, the polymer is tissue-specific or cell-specific. The molecule further comprises substituents that include a specific targeting moiety.

[0070] In some embodiments, the polymer is formula 5A:

[0071] [ka]

[0072] (In the formula, Q is:

[0073] [ka]

[0074] and c is an integer between 2 and 200 (for example, 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); Y is an arbitrarily existing and severable linker; All other substituents, including any and all embodiments thereof, have the structure described with respect to Formulas 1, 1A-1C, and 4.

[0075] In some embodiments, the polymer is formula 5B:

[0076] [ka]

[0077] (In the formula, c is an integer between 2 and 200 (for example, 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); Y is an arbitrarily existing and severable linker; All other substituents, including any and all embodiments thereof, have the structure described with respect to Formulas 1, 1A-1C, and 4.

[0078] Non-limiting examples of polymers provided herein include, for example:

[0079] [ka]

[0080] [ka]

[0081]

change

[0082]

change

[0083]

change

[0084]

change

[0085]

change

[0086]

change

[0087]

change

[0088]

change

[0089]

change

[0090]

change

[0091] (wherein (a+b) is approximately 5 to approximately 65 (e.g., approximately 5 to approximately 50, approximately 5 to approximately 40, approximately 5 to approximately 30, approximately 5 to approximately 20, or approximately 5 to approximately 10), and (c+d) is approximately 2 to approximately 60 (e.g., approximately 2 to approximately 50, approximately 2 to approximately 40, approximately 2 to approximately 30, approximately 2 to approximately 20, or approximately 2 to approximately 10)). In other embodiments, (a+b) is approximately 45 and (c+d) is approximately 20. Again, the representation of the number of units ("a", "b", "c", and "d") in these exemplary polymers does not imply a block copolymer structure; rather, these numbers indicate the number of units throughout, which can be arranged randomly as indicated by the " / " symbol in the formula.

[0092] Additional specific examples of polymers provided by this disclosure (e.g., polymers having ionic or ionizable groups) include:

[0093] [ka]

[0094] [ka]

[0095] [ka]

[0096] [ka]

[0097] [ka]

[0098] [ka]

[0099] [ka]

[0100] [ka]

[0101] [ka]

[0102] [ka]

[0103] [ka]

[0104] Further examples of polymers provided herein that contain PEG-terminated groups are as follows:

[0105] [ka]

[0106] The designation of the number of units ("a", "b", "c", and "d") in these exemplary polymers does not imply a block copolymer structure; rather, these numbers indicate the number of specific monomer units throughout, which can be arranged in any order, including blocks or monomers randomly distributed throughout the polymer. In some, but not all, instances, this is further indicated by the " / " symbol in the formula; however, the absence of " / " should not be interpreted as meaning that the polymer is bonded in a specific order. In some embodiments of polymers 1-69 described above, the monomers specified by parentheses and integers ("a", "b", "c", or "d") are randomly distributed or dispersed throughout the polymer.

[0107] In any of the polymers described above, (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 a particular embodiment, (a+b) is about 55 and (c+d) is about 10. In another embodiment, (a+b) is about 45 and (c+d) is about 20. In a particular embodiment, (a+b+c+d) is about 10 to 500, such as about 10 to 400, about 10 to 200, or about 10 to 100 (e.g., about 25 to 100 or about 50 to 75).

[0108] The polymer may contain (a+b) in any suitable ratio to (c+d). In other embodiments, (a+b) is in the range of 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) is in the range of 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 yet another embodiment, the ratio (a+b):(c+d) may be about 1 to about 25, about 1 to about 20, about 1 to about 10, about 1 to about 5, about 5 to about 25, about 10 to about 25, or about 15 to about 25.

[0109] Some of the polymers described above include monomers having ionizable side chains "e" and "f", in which case a, b, c, and d are as described above, and (e+f) is from 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). Furthermore, each case of p is an integer from 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). Furthermore, (a+b+c+d+e+f) is approximately 10-500, such as approximately 10-400, approximately 10-200, or approximately 10-100 (e.g., approximately 25-100 or approximately 50-75). Again, the representation of the number of units ("a", "b", "c", "d", "e", and "f") in these exemplary polymers does not imply a block copolymer structure; rather, these numbers indicate the number of units throughout, which may be arranged randomly.

[0110] Some of the above specific examples of polymers provided by this disclosure are shown with specific end groups (e.g., alkylamino, hydrogen, or PEG); however, any of the aforementioned specific structures may contain different end groups. For example, any of the aforementioned structures may have R groups as described herein at one or both ends of the polymer backbone. 1 , R 6 , or including the base of Q.

[0111] Any of the aforementioned polymers may contain a tissue-specific or cell-specific targeting moiety at the position indicated in the described formula, or the polymer may be modified to contain a tissue-specific or cell-specific targeting moiety. For example, the moiety may be added to the end of the polymer, or to group A 1 , A 2 B 1 , and / or B 2 The terminal amine can be modified to add a tissue-specific or cell-specific targeting moiety (e.g., by Michael addition, epoxide ring-opening, substitution, or other suitable techniques). The tissue-specific or cell-specific targeting moiety may be any small molecule, protein (e.g., antibody or antigen), amino acid sequence, sugar, oligonucleotide, metal-based nanoparticle, or combination thereof that can recognize (e.g., specifically bind to) a given target tissue or cell (e.g., specifically bind to a specific ligand, receptor, or other protein or molecule that enables the targeting moiety to distinguish between the target tissue or cell and other non-target tissues or cells). In some embodiments, the tissue-specific or cell-specific targeting moiety is a ligand receptor. In some embodiments, the tissue-specific or cell-specific targeting moiety is a receptor ligand.

[0112] 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 target tissue of the peripheral nervous system, central nervous system, liver, muscle (e.g., cardiac muscle), lung, bone (e.g., hematopoietic cells), or eye. In certain embodiments, the tissue-specific or cell-specific targeting moiety localizes the polymer to tumor cells. For example, the tissue-specific or cell-specific targeting moiety may be a sugar that binds to a receptor on a specific tissue or cell.

[0113] In some embodiments, the tissue-specific or cell-specific targeting portion is

[0114] [ka]

[0115] (In the formula, R 9 , R 10 , R 11 , and R 12 Each of these is independently a hydrogen atom, a halogen atom, or a C1-C4 alkyl or C1-C4 alkoxy atom substituted with one or more amino groups. Specific tissue-specific or cell-specific targeting moieties can be selected to localize the polymer to the tissues described herein. For example, α-d-mannose can be used to localize the polymer to the peripheral nervous system, central nervous system, or immune cells; α-d-galactose and N-acetylgalactosamine can be used to localize the polymer to hepatocytes; and folic acid can be used to localize the polymer to tumor cells.

[0116] Typically, polymers are cationic (i.e., positively charged at pH 7 and 23°C). As used herein, “cationic” polymers refer to polymers that have a net positive charge throughout, whether the polymer consists solely of cationic monomer units or a combination of cationic monomer units and nonionic or anionic monomer units.

[0117] In certain embodiments, the polymer has a weight-average molecular weight ranging from 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, 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. In other words, a polymer can have a weight-average molecular weight enclosed by any two of the aforementioned endpoints. For example, a polymer can have molecular weights ranging from approximately 10 kDa to approximately 50 kDa, approximately 10 kDa to approximately 100 kDa, approximately 10 kDa to approximately 500 kDa, approximately 50 kDa to approximately 500 kDa, and approximately 100 kDa. From approximately 500kDa, from approximately 200kDa to approximately 500kDa, from approximately 300kDa to approximately 500kDa, from approximately 400kDa to approximately 500kDa, from approximately 400kDa to approximately 600kDa, from approximately 400kDa to approximately 700kDa, from approximately 400kDa to approximately 800kDa, from approximately 400kDa to approximately 900kDa, from approximately 400kDa to approximately 1,000kDa, from approximately 400kDa to approximately It may have a weight-average molecular weight of 1,200 kDa, approximately 400 kDa to approximately 1,400 kDa, approximately 400 kDa to approximately 1,600 kDa, approximately 400 kDa to approximately 1,800 kDa, approximately 400 kDa to approximately 2,000 kDa, approximately 200 kDa to approximately 2,000 kDa, approximately 500 kDa to approximately 2,000 kDa, or approximately 800 kDa to approximately 2,000 kDa.

[0118] The weight-average molecular weight can be determined by any suitable technique. Generally, the weight-average molecular weight is determined using size exclusion chromatography with columns selected from TSKgel Guard, GMPW, GMPW, G1000PW, and Waters 2414 (Waters Corporation, Milford, Massachusetts) refractive index detectors. Furthermore, the weight-average molecular weight is determined by calibration using polyethylene oxide / polyethylene glycol standards in the range of 150 to 875,000 daltons.

[0119] Manufacturing method The present invention also provides a method for producing the polymers described herein. In some embodiments, the method is as described herein: Formula 4:

[0120] [ka]

[0121] The polymer of formula 2 or formula 3:

[0122] [ka]

[0123] (In the formula, p 1 is an integer between 1 and 2000 (for example, 1 to 1000, 1 to 500, 1 to 200, 1 to 100, 5 to 2000, 5 to 1000, 5 to 500, 5 to 200, or 5 to 100); p 2 This ranges from 1 to 2000 (for example, 1 to 1000, 1 to 500, 1 to 200, 1 or The integers are between 100, 2 to 2000, 2 to 1000, 2 to 500, 2 to 200, or 2 to 100; Each R 3 These are independently a methylene group or an ethylene group; X 1and X 2 is produced from a polymer comprising the structure as described above for Formulas 1, 1A - 1C, and 4).

[0124] That is, for example, each X 1 is independently, -C(O)O-, -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; each instance of X 2 is independently a C1 - C 12 alkyl or heteroalkyl group, a C3 - C 12 cycloalkyl group, a C2 - C 12 alkenyl group, a C3 - C 12 cycloalkenyl group, an aryl group, a heterocyclic group, or a combination thereof. All other embodiments of X 1 and X 2 for Formulas 1, 1A - 1C, and 4 also apply to X 1 and X 2 for Formulas 2 and 3.

[0125] The method involves combining the structure of Formula 2 or Formula 3 with a compound of formula HNR 13 A 1 and / or HNR 13 A 2 and optionally a compound of formula H2NX 2 or HOX 2 More specifically, the structure of Formula 2 can be combined (reacted) with (a) a compound of formula HNR 13 A 1 and / or HNR 13 A 2 and (b) a compound of formula H2NX 2 or HOX 2 simultaneously or sequentially in any order to provide a compound of Formula 4. Similarly, a compound of Formula 3 already containing an X 2 group can be combined (reacted) with a compound of formula HNR 13 A<00009​​​​​

[0126] HNR 13 A 1 and / or HNR 13 A 2 In the compounds of A, R 13 In each case, including any and all embodiments thereof, is as described above for the polymers of Formulas 1, 1A - 1C, and 4. That is, for example, R 13 In each case can independently be hydrogen, an aryl group, a heterocyclic group, an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, any of which can optionally be substituted with one or more substituents.

[0127] Similarly, A 1 and A 2 are as described above for the polymers of Formulas 1, 1A - 1C, and 4. That is, for example, A 1 and A 2 each independently is of the formula -(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 rl and r2 are each independently an integer from 1 to 5; R 2 In each case is independently hydrogen, an aryl group, a heterocyclic group, optionally substituted with one or more substituents, C1 - C 12Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C1-C 12 It is either a linear or branched alkyl group, or R 2 The second R 2 It is a group that combines with (to form a heterocyclic group). In some embodiments, A 1 and A 2 They are the same.

[0128] Formula H2NX 2 or HOX 2 Compound group X 2 This, including any and all embodiments thereof, is as described with respect to formulas 1, 1A-1C, 3, and 4.

[0129] All other substituents and faces of Formulas 2, 3, and 4, including any and all embodiments thereof, are as described herein with respect to the polymers of the present invention (e.g., Formulas 1, 1A, 1B, 1C, and 4).

[0130] HNR 13 A 1 and / or HNR 13 A 2 In the order of formula H2NX 2 or HOX 2 of The compound can be added to the compound of formula 2 or 3 in any suitable method and amount, depending on the desired degree of substitution. In some embodiments, about 1 to 400 equivalents (e.g., about 1 to 350, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 100, 2 Formula H2NX (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) 2 or HOX 2 The compound is added to the polymer of formula 2. In some embodiments, about 1 to 400 equivalents (for example, about 1 to 350, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, 1 to 50, 10 to 400, 10 to 350, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 10 to 100, 10 to 50, 20 to 400, 20 to 350, 20 to 300, 20 to 250, 20 to 200, 20 to 150, 20 to 100) Formula H (equivalent to 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) 13 A 1 and / or HNR 13 A 2 The compound is added to the polymer of formula 2 or formula 3.

[0131] The method is formula HNR 13 A 1 and / or HNR 13 A 2The compounds of and the formula H2NX 2 or HOX 2 In an embodiment that includes adding a compound of formula HNR 13 A 1 and / or HNR 13 A 2 The compounds of and the formula H2NX 2 or HOX 2 may be present in any suitable ratio in the reaction mixture. For example, the compounds of formula HNR 13 A 1 and / or HNR 13 A 2 The compounds of and the formula H2NX 2 or HOX 2 may be present in a molar ratio of from about 150:1 to about 1:150. In some embodiments, a ratio of from about 150:1 to about 1:1, such as from about 50:1 to about 1:1 (e.g., from about 25:1 to about 1:1, from about 10:1 to about 1:1, from about 5:1 to about 1:1, or from about 2.5:1 to about 1:1), etc., is used. In other embodiments, the ratio is from about 1:50 to about 1:1 (e.g., from about 1:25 to about 1:1, from about 1:10 to about 1:1, from about 1:5 to about 1:1, or from about 1:2.5 to about 1:1), etc., of from about 1:150 to about 1:1. In still other embodiments, the ratio is from about 1:10 to about 1:150, from about 1:40 to about 1:150, or from about 1:80 to about 1:150.

[0132] In some embodiments, the polymers containing the structure of formula 2 or formula 3 are, respectively, formula 2A or formula 3A:

[0133]

Chemical formula

[0134] (Wherein, c, Y, R 1 , and R 6 are as described above for the polymers of formula 1A and 1B, including any and all of their embodiments; p 1 , p 2 , R 3 , X1 , and X 2 It is a polymer of formulas 2 and 3 as described above. That is, for example: p 1 is an integer from 1 to 2000; p 2 is an integer from 1 to 2000; Each R 3 These are independently a methylene group or an ethylene group; each X 1 These are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; X 2 Each of these cases is independently a C1-C, substituted with one or more substituents. 12 Alkyl or heteroalkyl, C3-C 12 Cycloalkyl groups, C2-C 12 Alkenyl group, C3-C 12 A cycloalkenyl group, an aryl group, a heterocyclic group, or a combination thereof, or the aforementioned X with respect to formulas 1, 1A-1C, 2, 3, and 4. 1 and X 2 Any other embodiment of; The symbol " / " indicates that the units separated by it are concatenated randomly or in any order; c is an integer between 0 and 50; Y is an arbitrarily existing and severable linker; R 1 This includes hydrogen, an aryl group optionally substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C1-C 12 It is a linear or branched alkyl group; R 6 This includes hydrogen, an amino group, an aryl group substituted with one or more amines, a heterocyclic group, and a C1-C group. 12 Alkyl alkyl group, C1-C 12 Heteroalkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, C1-C 12It is a linear or branched alkyl group; or it is a tissue-specific or cell-specific targeting moiety. Formulas 2A and 3A All aspects thereof are otherwise as described herein with respect to the polymers of the present invention (e.g., formulas 1, 2, 1A, 1B, 1C, 3, and 4).

[0135] In certain embodiments, polymers containing the structure of formula 2 or formula 3 are, respectively, formula 2B or formula 3B:

[0136] [ka]

[0137] (In the formula, p 1 , p 2 , R 3 , X 1 , and X 2 (as described above with respect to formulas 2, 2A, 3, and 3A) is a polymer. That is, for example, p 1 is an integer from 1 to 2000; p 2 is an integer from 1 to 2000; Each R 3 These are independently a methylene group or an ethylene group; each X 1 These are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; X 2 Each of these cases is independently a C1-C, substituted with one or more substituents. 12 Alkyl or heteroalkyl, C3-C 12 Cycloalkyl groups, C2-C 12 Alkenyl group, C3-C 12 A cycloalkenyl group, an aryl group, a heterocyclic group, or a combination thereof, or the aforementioned X with respect to formulas 1, 1A-1C, 2, 3, and 4. 1 and X 2 Any other embodiment of; The symbol " / " indicates that the units separated by it are linked randomly or in any order. All one side of formulas 2B and 3B is otherwise the polymer of the present invention (e.g. For example, with respect to formulas 1, 1A, 1B, 1C, 2, 2A, 3, 3A, and 4), as described herein.

[0138] In some embodiments, the method also involves formula 1:

[0139] [ka]

[0140] A method for producing a polymer, wherein formula 4:

[0141] [ka]

[0142] Group A of a polymer containing the structure 1 and / or A 2 The present invention provides a method for producing a polymer containing the structure of formula 1 by modifying at least a portion of the formula.

[0143] All aspects of the polymers of formulas 1 and 4 are as described herein. That is, for example: m 1 , m 2 , m 3 , and m 4 Each of them is an integer from 0 to 1000, where m 1 +m 2 +m 3 +m 4 The sum is greater than 5; n 1 and n 2 Each of them is an integer from 0 to 1000, where n 1 +n 2 The sum is greater than 2; The symbol " / " indicates that the units separated by it are concatenated randomly or in any order; R 3a Each of these cases is independently a methylene group or an ethylene group; R 3b Each of these cases is independently a methylene group or an ethylene group; each X 1 These are independently -C(O)O- and -C(O)NR 13 -, -C(O)-, -S(O)(O)-, or a bond; R 13 Each of these examples independently involves hydrogen, an aryl group, a heterocyclic group, and a C1-C group. 12 The group is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, any of which may be optionally substituted with one or more substituents; X 2 Each of these cases is independently a C1-C, substituted with one or more substituents. 12 Alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof; A 1 and A 2 These are each an independent expression -(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 It is the basis of B 1 and B 2 They are independent of each other. -(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 (wherein p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R 2 Each case independently involves hydrogen or C1-C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R 2 The second R 2 It combines with R to form a heterocyclic group; 4 Each of these cases is independently -C(O)O-, -C(O)NH-, -OC(O)O-, or -S(O)(O)-;R 5 Each example independently comprises an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or a combination thereof, optionally containing 2 to 8 tertiary amines or substituents comprising tissue-specific or cell-specific targeting moieties. All aspects of Formulas 1, 1A-1C, and 4, and otherwise including any and all embodiments of the structures of Formulas 1, 1A-1C, and 4 as described herein, are as described herein with respect to the polymers of the present invention.

[0144] Polymers comprising the structure of Formula 1 or Formula 4 may be any polymer described herein, including Formulas 1A, 1B, and 1C, as well as any and all embodiments thereof described with respect to the polymers of the present invention.

[0145] A of polymer in formula 4 1 and / or A 2 The base specified as B 1 and / or B 2The base specified as can be modified by any appropriate means to generate it. For example, A 1 and / or A 2 The group specified is used in Michael addition reactions, epoxide ring opening, and It can be modified by a substitution reaction. In a preferred embodiment, A 1 and / or A 2 The group specified is modified by a Michael addition reaction.

[0146] One embodiment involves a polymer group A containing the structure of formula 4. 1 and / or A 2 The polymer is modified by a Michael addition reaction between a polymer containing the structure of formula 4 and an α,β-unsaturated carbonyl compound. As used herein, the term "Michael addition" refers to the nucleophilic addition of a polymer nucleophile (e.g., a carbanion, oxygen anion, nitrogen anion, oxygen atom, nitrogen atom, or a combination thereof) to an α,β-unsaturated carbonyl compound. Thus, the Michael addition reaction is between a polymer containing the structure of formula 4 and an α,β-unsaturated carbonyl compound. In some embodiments, the polymer nucleophile is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0147] The α,β-unsaturated carbonyl compound can be any α,β-unsaturated carbonyl compound that can accept Michael addition from a nucleophile. In some embodiments, the α,β-unsaturated carbonyl compound is an acrylate, acrylamide, vinyl sulfone, or a combination thereof. Thus, the Michael addition reaction may occur between a polymer containing the structure of formula 4 and an acrylate, acrylamide, vinyl sulfone, or a combination thereof. That is, in some embodiments, the method includes contacting a polymer containing the structure of formula 4 with an acrylate; contacting a polymer containing the structure of formula 4 with an acrylamide; or contacting a polymer containing the structure of formula 4 with a vinyl sulfone.

[0148] A 1 and / or A2 In an embodiment in which the groups specified are modified by a Michael addition reaction, they are given by formula: -(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; or -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2 (In the formula, p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R 2 Each example independently includes hydrogen, an aryl group substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C1-C 12 It is either a linear or branched alkyl group, or R 2The second R 2 It combines with R to form a heterocyclic group; 4 Each of these cases is independently -C(O)O-, -C(O)NH-, -OC(O)O-, or -S(O)(O)-;R 5 Each of these examples independently contains optionally 2 to 8 tertiary amines, or substituents including tissue-specific or cell-specific targeting moieties, such as alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof. 1 and / or B 2 It generates a base specified as follows.

[0149] Examples of suitable acrylates, acrylamides, and vinyl sulfones are given by formula:

[0150] [ka]

[0151] (In the formula, R 5 This includes an acrylate (as described with respect to formula 1, 1A, 1B, or 1C).

[0152] In some embodiments, the Michael addition reaction is facilitated by an acid and / or 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.

[0153] In some embodiments, the Michael addition reaction is facilitated by an acid. The acid may be a Brønsted acid or a Lewis acid. In embodiments where the acid is a Brønsted acid, the acid may 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 certain embodiments, the acid is a Lewis acid. For example, the acid may be bis(trifluoromethanesulfone)imide or p-toluenesulfonic acid.

[0154] In some embodiments, the Michael addition reaction is facilitated by a base. The base can be a weak base (i.e., with a pKa of about 7 to about 12) or a strong base (i.e., with a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base may be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethylpiperazine, or derivatives thereof.

[0155] 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 dissolve the polymers to be reacted and the α,β-unsaturated carbonyl compounds. For example, the solvent may include water, protic organic solvents, and / or aprotic organic solvents. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0156] In one embodiment, polymer group A 1 and / or A 2The polymer is modified by an epoxide ring-opening reaction between the polymer and the epoxide compound. As used herein, the term "epoxide ring-opening" refers to the nucleophilic addition of a polymer nucleophile (e.g., a carbanion, oxygen anion, nitrogen anion, oxygen atom, nitrogen atom, or a combination thereof) to the epoxide compound, thereby causing the epoxide to open its ring. Thus, the epoxide ring-opening reaction is between the polymer and the epoxide compound. In some embodiments, the polymer nucleophile is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0157] A 1 and / or A 2 In an embodiment in which the groups specified as are modified by an epoxide ring-opening reaction, they are given by formula: -(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 (In the formula, p1 to p4, q1 to q6, and r1 and r2 are independent integers from 1 to 5; R 2 Each example independently includes hydrogen, an aryl group substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C1-C 12 It is either a linear or branched alkyl group, or R 2 The second R 2 It combines with R to form a heterocyclic group; 5 Each of these examples independently contains optionally 2 to 8 tertiary amines, or substituents including tissue-specific or cell-specific targeting moieties, such as alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof. 1 and / or B 2 It generates a base specified as follows.

[0158] An example of an epoxide suitable for use is formula:

[0159] [ka]

[0160] (In the formula, R 5 This includes an epoxide of formula 1, 1A, 1B, or 1C as described.

[0161] In some embodiments, the epoxide ring-opening reaction is facilitated by an acid and / or 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.

[0162] In some embodiments, the epoxide ring-opening reaction is facilitated by an acid. The acid may be a Brønsted acid or a Lewis acid. In embodiments where the acid is a Brønsted acid, the acid may be a weak acid (i.e., pKa of about 4 to about 7) or a strong acid (i.e., pKa of about -2 to about 4). Typically, the acid is a weak acid. In certain embodiments, the acid is a Lewis acid. For example, the acid may be bis(trifluoromethanesulfone)imide or p-toluenesulfonic acid.

[0163] In some embodiments, the epoxide ring-opening reaction is facilitated by a base. The base can be a weak base (i.e., pKa of about 7 to about 12) or a strong base (i.e., pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base may be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethylpiperazine, or derivatives thereof.

[0164] 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 dissolve the polymer and epoxide compound to be reacted. For example, the solvent may include water, protic organic solvents, and / or aprotic organic solvents. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0165] In one embodiment, polymer group A 1 and / or A 2 The polymer and desorption The polymer is modified by a substitution reaction with a compound containing a leaving group (e.g., chloride atom, bromide atom, iodide atom, tosylate, triflate, mesylate, etc.). As used herein, "displacement" refers to the nucleophilic addition of a polymer nucleophile (e.g., a carbanion, oxygen anion, nitrogen anion, oxygen atom, nitrogen atom, or a combination thereof) to a compound containing a leaving group. Thus, the substitution reaction is between the polymer and the compound containing the leaving group. In some embodiments, the polymer nucleophile is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0166] A 1 and / or A 2 In embodiments in which the groups specified as are modified by a substitution reaction, they are given by formula: -(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; or -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR2 2 (In the formula, p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R 2 Each example independently includes hydrogen, an aryl group substituted with one or more substituents, a heterocyclic group, and a C1-C group. 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C1-C 12 It is either a linear or branched alkyl group, or R 2 The second R 2 It combines with R to form a heterocyclic group; 5 Each of these examples independently contains optionally 2 to 8 tertiary amines, or substituents including tissue-specific or cell-specific targeting moieties, such as alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof. 1 and / or B 2 It generates a base specified as follows.

[0167] Examples of compounds containing a suitable leaving group for use are given by formula:

[0168] [ka]

[0169] (In the formula, LG is a leaving group (e.g., chloride atom, bromide atom, iodide atom, tosylate, triflate, mesylate, etc.), R 5 This includes compounds of formula 1, 1A, 1B, or 1C as described.

[0170] In some embodiments, the substitution reaction is facilitated by an acid and / or 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.

[0171] In some embodiments, the substitution reaction is facilitated by an acid. The acid may be a Brønsted acid or a Lewis acid. In embodiments where the acid is a Brønsted acid, the acid may 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 certain embodiments, the acid is a Lewis acid. For example, the acid may be bis(trifluoromethanesulfone)imide or p-toluenesulfonic acid.

[0172] In some embodiments, the substitution reaction is facilitated by a base. The base can be a weak base (i.e., with a pKa of about 7 to about 12) or a strong base (i.e., with a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base may be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethylpiperazine, or derivatives thereof.

[0173] 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 dissolve the polymer to be reacted and the compound containing the leaving group. For example, the solvent may include water, protic organic solvents, and / or aprotic organic solvents. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0174] In some embodiments, the method further includes isolating a polymer containing the structure of Formula 1. The polymer containing the structure of Formula 1 can be isolated by any suitable means. For example, the polymer containing the structure of Formula 1 can be isolated by extraction, crystallization, recrystallization, column chromatography, filtration, or any combination thereof.

[0175] composition The polymers provided herein can be used for any purpose. However, the polymers are considered particularly useful for delivering nucleic acids and / or polypeptides (e.g., proteins) to cells. That is, provided herein are compositions comprising the polymers described herein and nucleic acids and / or polypeptides (e.g., proteins).

[0176] In some embodiments, the composition includes nucleic acids. Any nucleic acid can be used. An exemplary list of nucleic acids includes guide and / or donor nucleic acids for the CRISPR system, 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. siRNA typically contains 15–50 base pairs, preferably 19–25 base pairs, and comprises a double-stranded structure having a nucleotide sequence identical or nearly identical to the target gene or RNA expressed in the cell. siRNA may consist of two annealed polynucleotides or a single polynucleotide forming a hairpin structure. MicroRNA (miRNA) is a small non-coding polynucleotide about 22 nucleotides in length that directs the disruption or translational repression of its mRNA target. Antisense polynucleotides contain sequences complementary to the gene or mRNA. Antisense polynucleotides include, but are not limited to, morpholino, 2'-O-methyl polynucleotide, DNA, RNA, etc. Polynucleotide-based expression inhibitors may be polymerized in vitro, may be recombinant, may contain chimeric sequences, or may be derivatives of these. Polynucleotide-based expression inhibitors may contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and / or gene is inhibited.

[0177] The composition may also include any protein for delivery, in addition to or instead of the nucleic acid. The polypeptide can be any suitable polypeptide. For example, the polypeptide may 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-induced endonuclease. These are either creases (e.g., Cas9 polypeptide, Cpf1 polypeptide, or their variants) or DNA recombinases (e.g., Cre polypeptide).

[0178] The polymers provided herein are considered particularly useful for delivering one or more components of a CRISPR system. Specifically, in some embodiments, the composition comprises a guide RNA, an RNA-inducing endonuclease or nucleic acid encoding it, and / or a donor nucleic acid. The composition may comprise one, two, or all three components, along with the polymers described herein. Furthermore, the composition may comprise multiple guide RNAs, RNA-inducing endonucleases or nucleic acids encoding them, and / or donor nucleic acids. For example, multiple different guide RNAs for different target sites may optionally comprise multiple different donor nucleic acids and even multiple different RNA-inducing endonucleases or nucleic acids encoding them.

[0179] Furthermore, the components of the CRISPR system can be combined with each other and with the polymer in any particular way or order (when multiple components are present). In some embodiments, the guide RNA is complexed with an RNA endonuclease before being combined with the polymer. Alternatively, or instead, the guide RNA may be ligated (covalently or noncovalently) to a donor nucleic acid before being combined with the polymer.

[0180] The compositions are not limited to any particular CRISPR system (i.e., any particular guide RNA, RNA-inducing endonuclease, or donor nucleic acid), many of which are known. Nevertheless, for further explanation, some components of such systems are described below.

[0181] Donor nucleic acid A donor nucleic acid (or “donor sequence,” “donor polynucleotide,” or “donor DNA”) is a nucleic acid sequence inserted into a cleavage site caused by an RNA-directed endonuclease (e.g., Cas9 polypeptide or Cpf1 polypeptide). A donor polynucleotide may contain sufficient homology to the target genomic sequence at the cleavage site, e.g., within approximately 50 bases or less from the cleavage site, e.g., within approximately 30 bases, 15 bases, 10 bases, 5 bases, or directly adjacent to the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequence adjacent to the cleavage site, so as to support homologous recombination repair between it and the genomic sequence with which it has homology. Sequence homology between the donor and the genomic sequence of approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides (or any integer value of nucleotides between 10 and 200, or more), will support homologous recombination repair. The donor sequence can be of any length, for example, 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0182] A donor sequence is typically not identical to the genomic sequence it replaces. Rather, a donor sequence may contain one or more single-nucleotide changes, insertions, deletions, inversions, or rearrangements with respect to the genomic sequence, as long as sufficient homology exists to support homologous recombination repair. In some embodiments, the donor sequence includes non-homologous sequences adjacent to two homologous regions so that homologous recombination repair between the target DNA region and the two adjacent sequences results in the insertion of the non-homologous sequence in the target region. The donor sequence may also include a vector backbone containing sequences that are not homologous to the DNA region of interest and are not intended for insertion into the DNA region of interest. Generally, homologous regions of a donor sequence will have at least 50% sequence identity with respect to the genomic sequence to which recombination is desired. In certain embodiments, sequence identity of 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% may be present. Depending on the length of the polynucleotide, sequence identity can exist at any value between 1% and 100%.

[0183] The donor sequence may include specific sequence differences, such as restriction sites, nucleotide polymorphisms, or selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which can be used to assess the success of insertion of the donor sequence at a cleavage site compared to the genome sequence, or, in some embodiments, for other purposes (e.g., to indicate expression at a target genomic locus). In some embodiments, if located in a coding region, such nucleotide sequence differences will not alter the amino acid sequence or will perform silent amino acid changes (i.e., changes that do not affect the structure or function of the protein). Alternatively, these sequence differences may include adjacent recombinant sequences such as FLP, loxP sequences, which can be activated later for the removal of the marker sequence.

[0184] The donor sequence may be supplied to the cell as single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It may be introduced into the cell in linear or circular form. When introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues may be added to the 3' end of the linear molecule and / or a self-complementary oligonucleotide may be attached to one or both ends. See, for example, 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, as illustrated herein, may also be advantageously used. 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 nucleotide linkages such as phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues.

[0185] As an alternative to protecting the ends of linear donor sequences, sequences of additional length may be included outside the homology region, which can be degraded without affecting recombination. Donor sequences can be introduced into cells as part of a vector molecule having additional sequences, such as genes encoding the origin of replication, promoter, and antibiotic resistance. Furthermore, donor sequences can be introduced as naked nucleic acids, as nucleic acids complexed with drugs such as liposomes or polymers, or by viruses (e.g., adenoviruses, AAVs) as described herein for nucleic acids encoding Cas9 guide RNA and / or Cas9 fusion polynucleotides and / or donor polynucleotides.

[0186] Guide nucleic acids In some embodiments, the composition includes a guide nucleic acid. Suitable guide nucleic acids to be included in the compositions of this disclosure include single-molecule guide RNA ("single guide RNA" / "sgRNA") and double-molecule guide nucleic acid ("double guide RNA" / "dgRNA").

[0187] A guide nucleic acid (e.g., guide RNA) suitable for inclusion in the complex of this disclosure directs the activity of an RNA-induced endonuclease (e.g., Cas9 or Cpf1 polypeptide) to a specific target sequence within the target nucleic acid. The guide nucleic acid (e.g., guide RNA) comprises: a first segment (hereinafter also referred to herein as the “nucleic acid targeting segment” or simply the “targeting segment”); and a second segment (hereinafter also referred to herein as the “protein-binding segment”). The terms “first” and “second” do not imply the order in which the segments occur in the guide RNA. The order of the elements depends on the specific RNA-inducing polypeptide used. For example, the guide RNA for Cas9 typically has a protein-binding segment located at 3' of the targeting segment, while the guide RNA for Cpf1 typically has a protein-binding segment located at 5' of the targeting segment.

[0188] Guide RNA can be introduced into cells in a linear or circular form. When introduced in a linear form, the ends of the guide RNA can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. Amplification procedures, such as rolling circle amplification as illustrated herein, can also be advantageously used.

[0189] Segment 1: Targeting Segment The first segment of a guide nucleic acid (e.g., guide RNA) contains a nucleotide sequence complementary to the sequence (target site) in the target nucleic acid. In other words, the targeting segment 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 via hybridization (i.e., base pairing). Thus, the nucleotide sequence of the targeting segment can be modified to 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 segment 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) in the target nucleic acid.

[0190] The targeting segment may have a length of 12 to 100 nucleotides. The nucleotide sequence of the targeting segment (also called the targeting sequence or guide sequence) 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 a targeting segment 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.

[0191] The complementarity percentage between the targeting sequence of the targeting segment (i.e., the guide sequence) and the target site of the target nucleic acid may 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 complementarity percentage between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over seven adjacent 5'-terminal nucleotides of the target site of the target nucleic acid. In some embodiments, the complementarity percentage between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or greater over 20 adjacent nucleotides. In some embodiments, the complementarity percentage between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% over 17, 18, 19 or 20 consecutive 5'-terminal nucleotides of the target site of the target nucleic acid, and low at 0% or greater for the remainder. In such cases, the targeting sequences can be considered to have lengths of 17, 18, 19, or 20 nucleotides, respectively.

[0192] Second segment: Protein-binding segment The protein-binding segment of the guide nucleic acid (e.g., guide RNA) interacts with (binds to) the RNA-induced 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 aforementioned targeting segment / targeting sequence / guide sequence. The protein-binding segments of the guide nucleic acid (e.g., guide RNA) are complementary to each other. It contains two stretches of nucleotides. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA).

[0193] Single and dual guide nucleic acids A dual guide nucleic acid (e.g., guide RNA) comprises two distinct nucleic acid molecules. Each of the two molecules of the dual guide nucleic acid (e.g., guide RNA) in question contains stretches of nucleotides that are complementary to each other, such that the complementary nucleotides of the two molecules hybridize to form a double-stranded RNA double helix of a protein-binding segment.

[0194] In some embodiments, the double-stranding segment of the activator is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% identical to one of the activator (tracrRNA) molecules described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or its complement, across eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides).

[0195] In some embodiments, the double-stranding segment of the targeter is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% identical to one of the targeter (crRNA) sequences described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or its complement, across eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides).

[0196] Dual guide nucleic acids (e.g., guide RNA) can be designed to enable controlled (i.e., conditional) binding of a targeter to an activator. Since dual guide nucleic acids (e.g., guide RNA) only function if both the activator and targeter are bound in a functional complex with Cas9, they can be inducible (e.g., drug-inducible) by making binding between the activator and targeter induceable. As one non-limiting example, RNA aptamers can be used to modulate (i.e., control) the binding of an activator to a targeter. Thus, the activator and / or targeter may contain RNA aptamer sequences.

[0197] 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 to encompass both synthetic and native nucleic acid sequences that provide inducible regulation (and thus the availability of specific sequences) of the structure of the nucleic acid molecule (e.g., RNA, DNA / RNA hybrids, etc.) of which they are part; and thus, the availability of specific sequences. RNA aptamers typically contain sequences that fold into a specific structure (e.g., a hairpin) that specifically binds to a particular drug (e.g., a small molecule). Drug binding causes a structural change in RNA folding, which alters the characteristics of the nucleic acid of which the aptamer is part. As non-limiting examples: (i) an activator having an aptamer may not be able to bind to a congener targeter unless the aptamer is bound by a suitable drug; (ii) a targeter having an aptamer may not be able to bind to a congener activator unless the aptamer is bound by a suitable drug; (iii) Targeters and activators, each containing different aptamers that bind to different drugs, may not be able to bind to each other without both drugs present. As illustrated by these examples, dual guide nucleic acids (e.g., guide RNA) can be designed to be reducible.

[0198] Examples of aptamers and riboswitches include, for example, 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. These can be found in al., Wiley Interdiscip Rev RNA. 2012 May-Jun;3(3):369-84, all of which are incorporated herein by reference in their entirety.

[0199] Nucleotide sequences that can be included in dual guide nucleic acids (e.g., guide RNA) contained in international patent applications PCT / US2016 / 052690 and PCT / US2017 / 062617, or non-limiting examples thereof, that can hybridize to form protein-binding segments, or their complements.

[0200] The target single guide nucleic acid (e.g., guide RNA) contains two stretches of nucleotides (similar to the "targeter" and "activator" of a dual guide nucleic acid) that are complementary to each other and hybridize to form a double-stranded RNA double helix (dsRNA double helix) of a protein-binding segment (i.e., resulting in a stem-loop structure), covalently linked by an intervening nucleotide ("linker" or "linker nucleotide"). That is, the target single guide nucleic acid (e.g., single guide RNA) may contain a targeter and an activator, each having a double-stranding segment, where the double-stranding segments of the targeter and activator hybridize to each other to form a dsRNA double helix. The targeter and activator can be covalently linked via the 3' end of the targeter and the 5' end of the activator. Alternatively, the targeter and activator can be covalently linked via the 5' end of the targeter and the 3' end of the activator.

[0201] The linker of a single guide nucleic acid can have a length of 3 to 100 nucleotides. In some embodiments, the linker of a single guide nucleic acid (e.g., guide RNA) is 4 nt.

[0202] An exemplary single guide nucleic acid (e.g., guide RNA) includes two complementary stretches of nucleotides that hybridize to form a dsRNA double helix. In some embodiments, one of the two complementary stretches of nucleotides of a 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, or 100% identical to one of the activator (tracrRNA) molecules described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or its complement, over eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides).

[0203] In some embodiments, one of the two complementary stretches of nucleotides in a single guide nucleic acid (e.g., guide RNA) (or DNA encoding the stretch) is: It is identical by 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% identical to one of the targeter (crRNA) sequences described in international patent applications PCT / US2016 / 052690 and PCT / US2017 / 062617, or its complement, across eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides).

[0204] In some embodiments, one of two complementary stretches of nucleotides of a single guide nucleic acid (e.g., guide RNA) (or DNA encoding a stretch) is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% identical to one of the targeter (crRNA) sequences or activator (tracrRNA) sequences, or their complements, described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, over eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides).

[0205] Appropriate congeneral pairs of targeters and activators can be routinely determined by considering species name and base pairing (for dsRNA double helix of the protein-binding domain). Any activator / targeter 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).

[0206] In some embodiments, the activator (e.g., trRNA, trRNA-like molecule, etc.) of a dual guide nucleic acid (e.g., guide RNA) (e.g., dual guide RNA) or a single guide nucleic acid (e.g., guide RNA) (e.g., single guide RNA) comprises a stretch of a molecule having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% sequence identity with an activator (tracrRNA) molecule or its complement described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0207] In some embodiments, the activator of a dual-guide nucleic acid (e.g., dual-guide RNA) or a single-guide nucleic acid (e.g., single-guide RNA) (e.g., trRNA, trRNA-like molecule, etc.) contains 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 of a dual-guide nucleic acid (e.g., dual-guide RNA) or a single-guide nucleic acid (e.g., single-guide RNA) (e.g., trRNA, trRNA-like molecule, etc.) has a length in the range of 30 to 200 nucleotides (nt).

[0208] The protein-binding segment can have a length of 10 to 100 nucleotides.

[0209] Furthermore, for both the target single guide nucleic acid (e.g., single guide RNA) and the target dual guide nucleic acid (e.g., dual guide RNA), the dsRNA double helix of the protein-binding segment may have a length of 6 base pairs (bp) to 50 bp. The complementarity percentage between nucleotide sequences that hybridize to form the dsRNA double helix of the protein-binding segment may be 60% or more. For example, the complementarity percentage between nucleotide sequences that hybridize to form the dsRNA double helix of the protein-binding segment may be 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more. % or more, 98% or more, or 99% or more (for example, in some embodiments, there may be some nucleotides that do not hybridize and thus form a bulge within the dsRNA double helix. In some embodiments, the percentage of complementarity between nucleotide sequences that hybridize to form the dsRNA double helix of the protein-binding segment is 100%).

[0210] 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 segment of the guide nucleic acid is RNA and forms an RNA double helix. That is, the double-stranding segments of the activator and targeter are RNA. However, the targeting segment of the guide nucleic acid can be DNA. That is, if the DNA / RNA hybrid guide nucleic acid is a dual guide nucleic acid, the "targeter" molecule is a hybrid molecule (e.g., the targeting segment can be DNA and the double-stranding segment can be RNA). In such embodiments, the double-stranding segment of the "activator" molecule can be RNA (e.g., to form an RNA double helix with the double-stranding segment of the targeter molecule), while the nucleotides of the "activator" molecule outside the double-stranding segment can be DNA (in this case, the activator molecule is a hybrid DNA / RNA molecule) or RNA (in this case, the activator molecule is RNA). If the DNA / RNA hybrid guide nucleic acid is a single guide nucleic acid, then the targeting segment may be DNA, the double-stranding segment (which constitutes the protein-binding segment of the single guide nucleic acid) may be RNA, and the nucleotides outside the targeting and double-stranding segments may be RNA or DNA.

[0211] DNA / RNA hybrid guide nucleic acids can be useful in some embodiments, for example, when the target nucleic acid is RNA. Cas9 typically works in conjunction with a guide RNA that hybridizes with the target DNA, i.e., forms a DNA-RNA double helix at the target site. Therefore, when the target nucleic acid is RNA, it is sometimes advantageous to repeat the DNA-RNA double helix at the target site by using a targeting segment (of the guide nucleic acid) that is DNA instead of RNA. However, since the protein-binding segment of the guide nucleic acid is an RNA double helix, the targeter molecule is DNA in the targeting segment and RNA in the double-helix-forming segment. Hybrid guide nucleic acids can bias Cas9 binding to single-stranded target nucleic acids compared to double-stranded target nucleic acids.

[0212] 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 will be appropriately paired with the specific CRISPR system being used (e.g., the specific RNA-inducing endonuclease being used). That is, the guide nucleic acid may be, for example, a guide nucleic acid corresponding to any RNA-inducing endonuclease described herein or known in the art. Guide nucleic acids and RNA-inducing endonucleases are described, for example, in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0213] In some embodiments, the appropriate guide nucleic acid comprises two distinct RNA polynucleotide molecules. The first molecule (activator) contains 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 over a stretch of eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides) with respect to any one of the nucleotide sequences described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 or its complement. In some embodiments, the second of two distinct RNA polynucleotide molecules (targeters) comprises a nucleotide sequence having 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) nucleotide sequence identity over a stretch of eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides) with respect to any one of the nucleotide sequences described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 or its complement.

[0214] In some embodiments, a suitable guide nucleic acid is a single RNA polynucleotide comprising first and second nucleotide sequences having 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) nucleotide sequence identity over a stretch of eight or more consecutive nucleotides (e.g., eight or more consecutive nucleotides, ten or more consecutive nucleotides, twelve or more consecutive nucleotides, fifteen or more consecutive nucleotides, or twenty or more consecutive nucleotides) with respect to any one of the nucleotide sequences described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 or its complement.

[0215] In some embodiments, the guide RNA is Cpf1 and / or Cas9 guide RNA. Cpf1 and / or Cas9 guide RNA may have a total length of 30 nucleotides (nt) to 100 nt, for example, 30nt to 40nt, 40nt to 45nt, 45nt to 50nt, 50nt to 60nt, 60nt to 70nt, 70nt to 80nt, 80nt to 90nt, or 90nt to 100nt. In some embodiments, Cpf1 and / or Cas9 guide RNA may have a total length of 35nt, 36nt, 37nt, 38nt, 39nt, 40nt, 41nt, 42nt, 43nt, 44nt, 45nt, 46nt, 47nt, 48nt, 49nt, or 50nt. Cpf1 and / or Cas9 guide RNA may include a target nucleic acid binding segment and a double-strand formation segment.

[0216] The target nucleic acid binding segment of the Cpf1 and / or Cas9 guide RNA may have a length of 15 nt to 30 nt, 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 some embodiments, the target nucleic acid binding segment has a length of 23 nt. In some embodiments, the target nucleic acid binding segment has a length of 24 nt. In some embodiments, the target nucleic acid binding segment has a length of 25 nt.

[0217] The target nucleic acid binding segment of Cpf1 and / or Cas9 guide RNA may have 100% complementarity with the target nucleic acid sequence of the corresponding length. The targeting segment may have less than 100% complementarity with the target nucleic acid sequence of the corresponding length. For example, Cpf1 and The target nucleic acid binding segment of the 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 where the target nucleic acid binding segment is 25 nucleotides long and the target nucleic acid sequence is 25 nucleotides long, the target nucleic acid binding segment has 100% complementarity to the target nucleic acid sequence. As another example, in some embodiments where the target nucleic acid binding segment is 25 nucleotides long and the target nucleic acid sequence is 25 nucleotides long, the target nucleic acid binding segment has 1 non-complementary nucleotide and 24 complementary nucleotides that have the target nucleic acid sequence.

[0218] The double-stranding segments of Cpf1 and / or Cas9 guide RNA may have lengths ranging from 15nt to 25nt, for example, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, or 25nt.

[0219] In some embodiments, the double-stranding segment of the Cpf1 guide RNA may include the nucleotide sequence 5'-AAUUUCUACUGUUGUAGAU-3'.

[0220] Additional elements In some embodiments, the guide nucleic acid (e.g., guide RNA) includes additional segments or multiple segments (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' ends), 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 segment is a 5' cap (e.g., a 7-methylguanylate cap (m 7 G)); 3' polyadenylated tails (i.e., 3' poly(A) tails); ribozyme sequences (e.g., guide nucleic acids and components of guide nucleic acids, e.g., for enabling self-cleavage of targeters, activators, etc.); riboswitch sequences (e.g., for enabling regulated stability and / or regulated accessibility by proteins and protein complexes); sequences that form dsRNA double helix (i.e., hairpins); sequences that target RNA to intracellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.); modifications or sequences that provide tracking (e.g., labeling such as fluorescent molecules (i.e., fluorescent dyes), sequences or other parts that facilitate fluorescence detection); sequences or modifications that provide protein binding sites (e.g., proteins acting on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, RNA-binding proteins (e.g., RNA aptamers), labeling proteins, fluorescently labeled proteins, etc.); modifications or sequences that provide increased, decreased, and / or controllable stability; and combinations thereof.

[0221] RNA-induced endonuclease In addition to or instead of the guide nucleic acid, the composition may include an RNA-inducing endonuclease protein or the nucleic acid encoding it (e.g., mRNA or a vector). Any RNA-inducing endonuclease can be used. The choice of RNA-inducing endonuclease used will depend, at least in part, on the intended end use of the CRISPR system being used.

[0222] In some embodiments, the polypeptide is a Cas9 polypeptide. Suitable Cas9 polypeptides for inclusion in the compositions of this disclosure include naturally occurring Cas9 polypeptides (e.g., naturally occurring in bacterial and / or archaeal cells), or The following non-naturally occurring Cas9 polypeptides are included (e.g., Cas9 polypeptides are mutant Cas9 polypeptides, chimeric polypeptides as discussed below). In some embodiments, those skilled in the art will understand that the Cas9 polypeptides disclosed herein may be any variant derived from or isolated from any source. In other embodiments, the Cas9 peptides of this disclosure are 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 This may include, but is not limited to, the functional mutations described in 14;343(6176), one or more mutations described in the literature; see also U.S. Patent Application No. 13 / 842,859, filed March 15, 2013, which is incorporated herein by reference; and further see U.S. Patents 8,697,359;8,771,945;8,795,965;8,865,406;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. In other words, in some embodiments, the systems and methods disclosed herein can be used with wild-type Cas9 protein having double-strand nuclease activity, Cas9 mutants acting as single-strand nickases, or other mutants having modified nuclease activity. Thus, a Cas9 polypeptide suitable for inclusion in the compositions of this disclosure may be an enzymatically active Cas9 polypeptide that, for example, can perform single- or double-strand breaks in target nucleic acids, or may have reduced enzymatic activity compared to a wild-type Cas9 polypeptide.

[0223] Naturally occurring Cas9 polypeptides bind to a guide nucleic acid, thereby being directed to a specific sequence within the target nucleic acid (target site) and cleaving the target nucleic acid (e.g., cleaving dsDNA to produce double-strand breaks, cleaving ssDNA, cleaving ssRNA, etc.). The Cas9 polypeptide in question comprises two parts: an RNA-binding part and an active part. The RNA-binding part interacts with the guide nucleic acid in question, while the active part exhibits site-specific 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 part exhibits reduced nuclease activity compared to the corresponding part of the wild-type Cas9 polypeptide. In some embodiments, the active part is enzymatically inactive.

[0224] Assays for determining whether a protein has an RNA-binding moiety that interacts with a target guide nucleic acid can be any convenient binding assay for testing 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 the target nucleic acid.

[0225] Assays for determining whether a protein has an active site (for example, whether a polypeptide has nuclease activity to cleave a target nucleic acid) can be any convenient nucleic acid cleavage assay for testing nucleic acid cleavage. An exemplary cleavage assay involves adding a guide nucleic acid and a Cas9 polypeptide to the target nucleic acid.

[0226] In some embodiments, a Cas9 polypeptide suitable for inclusion in the composition of this disclosure may have enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity). It possesses 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.

[0227] In other embodiments, a Cas9 polypeptide suitable for inclusion in the composition of the Disclosure has enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity) that modifies a polypeptide related to a target nucleic acid (e.g., histone).

[0228] Numerous Cas9 orthologues have been identified from a wide variety of species, and in some embodiments, the proteins share only a few identical amino acids. All identified Cas9 orthologues have the same domain architecture, possessing a central HNH endonuclease domain and a divided RuvC / RNaseH domain. The Cas9 protein shares four key motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif.

[0229] In some embodiments, a suitable Cas9 polypeptide comprises an amino acid sequence having four motifs, each of motifs 1-4 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 with respect to the Cas9 amino acid sequence (SEQ ID NO: 1) shown in Figure 1; or to motifs 1-4 of the Cas9 amino acid sequence shown in Table 1 below; or to respect to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence (SEQ ID NO: 1) shown in Figure 1.

[0230] In some embodiments, the Cas9 polypeptide includes an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% amino acid sequence identity with the amino acid sequence shown in Figure 1 and described in SEQ ID NO: 1; includes amino acid substitutions N497, R661, Q695, and Q926 related to the amino acid sequence described in SEQ ID NO: 1; includes amino acid substitution K855 related to the amino acid sequence described in SEQ ID NO: 1; includes amino acid substitutions K810, K1003, and R1060 related to the amino acid sequence described in SEQ ID NO: 1; or includes amino acid substitutions K848, K1003, and R1060 related to the amino acid sequence described in SEQ ID NO: 1.

[0231] As used herein, the term "Cas9 polypeptide" encompasses the term "mutant Cas9 polypeptide"; and the term "mutant Cas9 polypeptide" encompasses the term "chimeric Cas9 polypeptide."

[0232] Mutant Cas9 polypeptide Suitable Cas9 polypeptides for inclusion in the compositions of this disclosure include mutant Cas9 polypeptides. Mutant Cas9 polypeptides have an amino acid sequence that differs by one amino acid (e.g., having deletions, insertions, substitutions, or fusions) (i.e., differing by at least one amino acid) when compared to the amino acid sequence of wild-type Cas9 polypeptide (e.g., naturally occurring Cas9 polypeptides as described above). In some cases, mutant Cas9 polypeptides have an amino acid sequence that differs from the nuclease activity of Cas9 polypeptides. They have amino acid changes (e.g., deletions, insertions, or substitutions) that reduce nuclease activity. For example, in some cases, the mutant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide. In some embodiments, the mutant Cas9 polypeptide has virtually no nuclease activity. When a Cas9 polypeptide is a mutant Cas9 polypeptide that has virtually no nuclease activity, it can be called "dCas9".

[0233] In some embodiments, the mutant Cas9 polypeptide has reduced nuclease activity. For example, mutant Cas9 polypeptides suitable for use in the conjugation method of this disclosure exhibit endonuclease activity of less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.1% of that of a wild-type Cas9 polypeptide, e.g., a wild-type Cas9 polypeptide containing the amino acid sequence shown in Figure 1 (SEQ ID NO: 1).

[0234] In some embodiments, mutant Cas9 polypeptides can cleave the complementary strand of a target nucleic acid but have a reduced ability to cleave the non-complementary strand of a double-stranded target nucleic acid. For example, mutant Cas9 polypeptides may have mutations (amino acid substitutions) that reduce the function of the RuvC domain (e.g., "domain 1" in Figure 1). As a non-limiting example, in some embodiments, mutant Cas9 polypeptides have a D10A mutation (e.g., from aspartic acid to alanine at the amino acid position corresponding to position 10 in SEQ ID NO: 1), and thus can cleave the complementary strand of a double-stranded target nucleic acid but have a reduced ability to cleave the non-complementary strand of a double-stranded target nucleic acid (i.e., when mutant Cas9 polypeptides cleave a double-stranded target nucleic acid, they result in single-strand breaks (SSBs) instead of double-strand breaks (DSBs)) (see, e.g., Jinek et al., Science. 2012 Aug 17;337(6096):816-21).

[0235] In some embodiments, a mutant Cas9 polypeptide can cleave the non-complementary strand of a double-stranded target nucleic acid but has a reduced ability to cleave the complementary strand of the target nucleic acid. For example, a mutant Cas9 polypeptide may 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 may have an H840A mutation (e.g., histidine to alanine at the amino acid position corresponding to position 840 of SEQ ID NO: 1) (Figure 1), and thus can cleave the non-complementary strand of the target nucleic acid but has a reduced ability to cleave the complementary strand of the target nucleic acid (i.e., when the mutant Cas9 polypeptide cleaves a double-stranded target nucleic acid, it results in an SSB instead of a DSB). Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid (e.g., a single-stranded target nucleic acid) but retains the ability to bind a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid).

[0236] In some embodiments, the mutant Cas9 polypeptide has a reduced ability to cleave both the 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 D10A and H840A mutations (e.g., mutations in both the RuvC domain and the HNH domain) such that the polypeptide has a reduced ability to cleave both the complementary and non-complementary strands of 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 or double-stranded target nucleic acid) but retains the ability to bind to the target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid).

[0237] As another non-limiting example, in some embodiments, mutant Cas9 polypeptide The Cas9 polypeptide has W476A and W1126A mutations, resulting in a reduced ability for the polypeptide to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to them.

[0238] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to target nucleic acids.

[0239] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has mutations H840A, W476A, and W1126A, such that the polypeptide has a reduced ability to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to target nucleic acids.

[0240] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has mutations H840A, D10A, W476A, and W1126A, such that the polypeptide has a reduced ability to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to target nucleic acids.

[0241] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to target nucleic acids.

[0242] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to target nucleic acids.

[0243] To achieve the above effects (i.e., to inactivate one or the other nuclease moiety), other residues can be mutated. Non-limiting examples include modifying (i.e., substituting) residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (see Table 1 for further information regarding the conservation of Cas9 amino acid residues). Mutations other than alanine substitutions are also appropriate.

[0244] In some embodiments, when a mutant Cas9 polypeptide has reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A), the mutant Cas9 polypeptide can still site-specifically bind to the target nucleic acid, as long as it retains the ability to interact with the guide nucleic acid (because it is still guided to the target nucleic acid sequence by the guide nucleic acid).

[0245] [Table 1]

[0246] In addition to the above, mutant Cas9 proteins may have the same sequence identity parameters as those described above for Cas9 polypeptides. That is, in some embodiments, a suitable mutant Cas9 polypeptide comprises an amino acid sequence having four motifs, where each of motifs 1-4 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 with respect to the Cas9 amino acid sequence (SEQ ID NO: 1) shown in Figure 1, or motifs 1-4 (as shown in Table 1, motifs 1-4 of SEQ ID NO: 1 are SEQ ID NOs: 3-6, respectively); or respect to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence (SEQ ID NO: 1) shown in Figure 1. Any Cas9 protein as defined above may be used in the compositions of this disclosure as a Cas9 polypeptide or as part of a chimeric Cas9 polypeptide, including those specifically referenced in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0247] 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 with respect to the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO: 1). Any Cas9 protein as defined above can be used in the compositions of this disclosure as part of a mutant Cas9 polypeptide or a chimeric mutant Cas9 polypeptide, including those specifically referenced in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0248] Chimeric polypeptide (fusion polypeptide) In some embodiments, the mutant Cas9 polypeptide is a chimeric Cas9 polypeptide (also referred herein as a fusion polypeptide, e.g., "Cas9 fusion polypeptide"). The Cas9 fusion polypeptide can conjugate and / or modify target nucleic acids (e.g., cleavage, methylation, demethylation, etc.) and / or polypeptides related to the target nucleic acid (e.g., methylation, acetylation, etc. of histone tails).

[0249] Cas9 fusion polypeptides are mutant Cas9 polypeptides because they differ in sequence from wild-type Cas9 polypeptides (e.g., naturally occurring Cas9 polypeptides). Cas9 fusion polypeptides are Cas9 polypeptides fused to a covalently bonded heterologous polypeptide (also called a "fusion partner") (e.g., wild-type Cas9 polypeptide). These include plutidosides, mutant Cas9 polypeptides, and mutant Cas9 polypeptides having reduced nuclease activity (as described above). In some embodiments, the Cas9 fusion polypeptide is a mutant Cas9 polypeptide (e.g., dCas9) having reduced nuclease activity, fused to a covalently bonded heterologous polypeptide. In some embodiments, the heterologous polypeptide exhibits (and thus provides) activity (e.g., enzymatic activity) (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that would also be exhibited by the Cas9 fusion polypeptide. In some such embodiments, for example, a method of binding when the Cas9 polypeptide is a mutant Cas9 polypeptide having a fusion partner having activity (e.g., a heterologous polypeptide) that modifies the target nucleic acid (e.g., enzymatic activity), can also be considered a method of modifying the target nucleic acid. In some embodiments, a method of binding a target nucleic acid (e.g., a single-stranded target nucleic acid) can result in modification of the target nucleic acid. In other words, in some embodiments, the method for attaching a target nucleic acid (e.g., a single-stranded target nucleic acid) may be a method for modifying the target nucleic acid.

[0250] In some embodiments, heterologous sequences provide intracellular localization, i.e., the heterologous sequences are intracellular localization sequences (e.g., nuclear localization signals (NLS) targeting the nucleus, sequences that move the fusion protein away from the nucleus, e.g., nuclear export sequences (NES), sequences that retain the fusion protein in the cytoplasm, mitochondrial localization signals targeting mitochondria, chloroplast localization signals targeting chloroplasts, endoplasmic reticulum (ER) retention signals, etc.). In some embodiments, the mutant Cas9 does not contain an NLS so that the protein does not target the nucleus (which may be advantageous, for example, when the target nucleic acid is RNA present in the cytosol). In some embodiments, heterologous sequences can provide tags for facilitating tracking and / or purification (e.g., fluorescent proteins, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; histidine tags, e.g., 6XHis tags; hemagglutinin (HA) tags; FLAG tags; Myc tags, etc.) (i.e., heterologous sequences are detectable labels). In some embodiments, heterologous sequences can provide increased or decreased stability (i.e., heterologous sequences are controllable in some embodiments (e.g., temperature-sensitive or drug-controllable degron sequences, see below; stability-controlling peptides, e.g., degron)). In some embodiments, heterologous sequences can provide increased or decreased transcription from target nucleic acids (i.e., heterologous sequences are transcription regulatory sequences, e.g., transcription factors / activators or their fragments; proteins or their fragments that recruit transcription factors / activators; transcriptional repressors or their fragments; proteins or their fragments that recruit transcriptional repressors; small molecule / drug-responsive transcription regulators, etc.).In some embodiments, heterologous sequences can provide a binding domain (i.e., the heterologous sequence is a protein-binding sequence that provides the ability of a Cas9 fusion polypeptide to bind to another protein of interest, such as a DNA or histone-modifying protein, a transcription factor or transcriptional repressor, a recruiting protein, an RNA-modifying enzyme, an RNA-binding protein, a translation initiation factor, an RNA splicing factor, etc.). Heterologous nucleic acid sequences can be ligated to another nucleic acid sequence (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide.

[0251] The target Cas9 fusion polypeptide (Cas9 fusion protein) may have multiple (one or more, two or more, three or more, etc.) fusion partners in any combination as described above. Exemplary examples include a Cas9 fusion protein having heterologous sequences that provide activity (e.g., transcriptional regulation, targeted modification, modification of proteins related to target nucleic acids, etc.) and also having intracellular localization sequences. In some embodiments, such a Cas9 fusion protein may also have a tag (e.g., green fluorescent protein) to facilitate tracking and / or purification. The Cas9 protein may have fusion partners (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; histidine tags, e.g., 6XHis tag, hemagglutinin (HA) tag; FLAG tag; Myc tag, etc.). As another exemplary example, the Cas9 protein may 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 multiple fusion partners) (e.g., NLS, tag, activity-providing fusion partner, etc.) is located at or near the C-terminus of Cas9. In some embodiments, the fusion partner (or multiple fusion partners) (e.g., NLS, tag, activity-providing fusion partner, etc.) is located at the N-terminus of Cas9. In some embodiments, Cas9 has fusion partners (or multiple fusion partners) (e.g., NLS, tag, activity-providing fusion partner, etc.) at both the N-terminus and the C-terminus.

[0252] Suitable fusion partners that provide increased or decreased stability include, but are not limited to, degron sequences. It will be readily apparent to those skilled in the art that degrons are amino acid sequences that control the stability of the protein they are part of. For example, the stability of a protein containing degron sequences is partially controlled by the degron sequences. In some embodiments, a suitable degron is constitutive such that the degron affects the stability of the protein independently of experimental control (i.e., the degron is not drug-inducible, temperature-inducible, etc.). In some embodiments, the degron provides a mutant Cas9 polypeptide with controllable stability so that the mutant Cas9 polypeptide can be “on” (i.e., stable) or “off” (i.e., unstable, degraded) depending on desired conditions. For example, if a degron is a temperature-sensitive degron, a mutant Cas9 polypeptide may be functional (i.e., "on," stable) 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 not functional (i.e., "off," degraded) above the threshold temperature. As another example, if a degron is a drug-inducible degron, the presence or absence of a drug can 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 a degron is controlled by the presence or absence of small molecules that bind to the degron.

[0253] Examples of suitable degrons include, but are not limited to, degrons controlled by Shield-1, DHFR, auxin, and / or temperature. Non-limiting examples of suitable degrons are known in the art (e.g., all of them, which are incorporated herein by reference in their entirety; 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 1 8;33(1).:Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase;および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)。

[0254] Exemplary degron sequences have been well-characterized and tested in both cells and animals. Specifically, fusion of Cas9 (e.g., wild-type Cas9; mutant Cas9; mutant Cas9 with reduced nuclease activity, e.g., dCas9) with a degron sequence generates "adjustable" and "inducible" Cas9 polypeptides. 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) may include a YFP sequence for detection, a degron sequence for stability, and a transcription activator sequence for increasing the transcription of the target nucleic acid. Reporter proteins suitable 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 their functional fragments): his3, β-galactosidase, fluorescent proteins (e.g., GFP, RFP, YFP, cherry, tomato, etc., and various derivatives thereof), luciferase, β-glucuronidase, and alkaline phosphatase. Furthermore, the number of fusion partners that can be used in a Cas9 fusion protein is unlimited. In some embodiments, the Cas9 fusion protein contains one or more (e.g., two or more, three or more, four or more, or five or more) heterologous sequences.

[0255] 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, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity, any of which can be directed to directly modify nucleic acids (e.g., methylation of DNA or RNA) or modify nucleic acid-related 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 their fragments that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0256] Examples of various additional suitable fusion partners (or their fragments) for the target mutant Cas9 polypeptide include, but are not limited to, those described in PCT patent applications: WO2010 / 075303, WO2012 / 068627, and WO2013 / 155555, which are incorporated herein by reference in their entirety.

[0257] Suitable fusion partners include, but are not limited to, polypeptides that provide activity to indirectly increase transcription by acting directly on or on polypeptides related to target nucleic acids (e.g., histones, DNA-binding proteins, RNA-binding proteins, RNA-editing proteins, etc.). Suitable fusion partners include polypeptides with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, and adenyl This includes, but is not limited to, polypeptides that provide rosinylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.

[0258] Additional suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription and / or translation of the target nucleic acid (e.g., transcription activators or their fragments, proteins or their fragments that recruit transcription activators, small molecule / drug-responsive transcription and / or translation regulators, translational regulatory proteins, etc.).

[0259] Non-limiting examples of fusion partners for achieving increased or decreased transcription include transcription activators and transcriptional repressor domains (e.g., Krueppel-associated box (KRAB or SKD); Mad mSIN3 interaction domain (SID); ERF repressor domain (ERD), etc.). In some such embodiments, the Cas9 fusion protein is targeted to a specific site (i.e., sequence) in the target nucleic acid by a guide nucleic acid and exerts locus-specific regulation such as blocking RNA polymerase binding to the promoter (which selectively inhibits transcription 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 modification is performed on the target nucleic acid or a protein associated with the target nucleic acid, e.g., nucleosome histone).

[0260] 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 release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, including adenosine deaminases (ADARs) that act on RNA, e.g., RNA deaminases, e.g., A to I and / or C to U editing enzymes); heliembodiments; RNA-binding proteins; etc. It is understood that fusion partners may include an entire protein or, in some embodiments, a fragment of a protein (e.g., a functional domain).

[0261] 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).

[0262] Furthermore, the fusion partners of the chimeric Cas9 polypeptide, whether transient or irreversible, directly or indirectly, include: endonucleases (e.g., RNase I, CRR22 DYW domain, Dicer, and PIN (PilT N-terminal) domain derived 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 mutation-dependent RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and protein domains involved in stabilizing RNA (e.g., PABP); proteins and protein domains involved in repressing translation (e.g., Ago2 and Ago4); and proteins that stimulate translation. Proteins and protein domains involved in (e.g., Staufen); proteins and protein domains involved in (e.g., can) regulating translation (e.g., translation factors such as initiation factors, elongation factors, release 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., IMP1, ZBP1, She2p, She3p, and from Bicaudal-D); proteins and protein domains involved in the nuclear retention of RNA (e.g., Rrp6); proteins and protein domains involved in the nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains involved in the repression of RNA splicing (e.g., PTB, Sam68, and hnRNP) A1); an effector domain 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 transcription efficiency (e.g., FUS(TLS)); and proteins and protein domains involved in stimulating transcription (e.g., CDK7 and HIV Tat), which may be any domain capable of interacting with intramolecular and / or intermolecular secondary structures, such as double-stranded RNA double helixes, hairpins, stem-loops, etc., for the purposes of this disclosure.Alternatively, the effector domain may be selected from the group including endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense mutation-dependent RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing 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, release factors, 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 nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repressing RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of reducing transcription efficiency; and proteins and protein domains capable of stimulating transcription. Another suitable fusion partner is the PUF RNA-binding domain, which is described in more detail in WO2012068627.

[0263] Some RNA splicing factors that can be used as fusion partners (either as a whole or as fragments) for the Cas9 polypeptide have a modular configuration with separate sequence-specific RNA-binding modules 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 the exon splicing enhancer (ESE) in premRNA and a C-terminal RS domain that promotes exon incorporation. As another example, the hnRNP protein hnRNP Al binds to the exon splicing silencer (ESS) through its RRM domain and inhibits exon incorporation through its C-terminal glycine-rich domain. Some splicing factors can modulate the alternative use of splice sites (ss) by binding to regulatory sequences between two alternative sites. For example, ASF / SF2 can recognize ESEs and promote the use of proximal intron sites, while hnRNP Al can bind to ESSs and shift splicing to the use of distal intron sites. One application of such factors is the generation of ESFs that regulate the alternative splicing of endogenous genes, particularly disease-related genes. For example, Bcl-x pre-mRNA generates two splicing isoforms, each containing two alternative 5' splice sites encoding proteins with opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmittal cells, upregulated in many cancer cells, and protects cells from apoptotic signaling. The short isoform Bcl-xS is a pro-apoptotic isoform, 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 cis-elements (cω-elements) located within either the core-exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For further examples, see WO2010075303.

[0264] In some embodiments, Cas9 polypeptides (e.g., wild-type Cas9, mutant Cas9, mutant Cas9 with reduced nuclease activity, etc.) can be linked to a fusion partner via a peptide spacer.

[0265] In some embodiments, the Cas9 polypeptide includes a “protein delivery domain” or PTD (also known as CPP—a cell-permeable peptide) which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates passage through lipid bilayers, micelles, cell membranes, organelle membranes, or vesicular membranes. A PTD attached to another molecule, which can range from small polar molecules to large macromolecules and / or nanoparticles, facilitates the molecule’s passage through membranes, e.g., from extracellular space to intracellular space, or from cytosol to organelles. In some embodiments, a PTD attached to another molecule facilitates the molecule’s entry into the nucleus (e.g., in some embodiments, the PTD includes a nuclear localization signal (NLS)). In some embodiments, the Cas9 polypeptide includes two or more NLSs, e.g., two or more NLSs in tandem. In some embodiments, the PTD is covalently bonded to the amino terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently bonded to the carboxyl terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently bonded to the amino and carboxyl terms of the Cas9 polypeptide. In some embodiments, the PTD is covalently bonded to a nucleic acid (e.g., a guide nucleic acid, a polynucleotide encoding the guide nucleic acid, a polynucleotide encoding the Cas9 polypeptide, etc.). Exemplary PTDs include: a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT containing YGRKKRRQRRR; SEQ ID NO: 7); a polyarginine sequence containing a sufficient number of arginines to direct cell entry (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); and a Drosophia Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); truncated human calcitonin peptide (Trehin Polylysine (Wender et al. (2004) Pharm. Research 21:1248-1256); RRQRRTSKLMKR (SEQ ID NO: 8); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 9); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 10); and RQIKIWFQNRRMKWKK (SEQ ID NO: 11), but not limited to these. Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 12), RKKRRQRRR (SEQ ID NO: 13); arginine homopolymers ranging from 3 to 50 arginine residues; exemplary. Typical 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); andGGRRARRRRRR(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). The ACPP comprises a polycationic CPP (e.g., Arg9 or "R9") linked to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero, thereby inhibiting adhesion and uptake into the cell. Cleavage of the linker releases the polyanion, locally revealing polyarginine and its intrinsic adhesiveness, i.e., "activating" the ACPP to pass through the membrane.

[0266] In some embodiments, the composition may include a Cpf1 RNA-induced endonuclease, an example of which is provided in Figure 2, 16, or 17. The Cpf1 RNA-induced endonuclease is also known as Cas12a. The Cpf1 CRISPR system of this disclosure comprises i) a single endonuclease protein and ii) a crRNA, where a portion of the 3' end 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 of Cpf1 must be at least 12nt, 13nt, 14nt, 15nt, or 16nt to achieve detectable DNA cleavage, and at least 14nt, 15nt, 16nt, 17nt, or 18nt to achieve efficient DNA cleavage.

[0267] The Cpf1 system of this disclosure differs from Cas9 in several respects. First, unlike Cas9, Cpf1 does not require a separate tracrRNA for cleavage. In some embodiments, the Cpf1 crRNA can be as short as approximately 42–44 nucleotides in length—23–25 nt being the guide sequence and 19 nt being the constitutive direct repeat sequence. In contrast, the combined Cas9 tracrRNA and crRNA synthetic sequence can be approximately 100 nucleotides in length.

[0268] Secondly, Cpf1 prefers a “TTN” PAM motif located 5' upstream of its target. This is in contrast to the “NGG” PAM motif located 3' above the target DNA for the Cas9 system. In some embodiments, the uracil base immediately preceding the guide sequence cannot be substituted (Zetsche, B. et al. 2015. “Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” is incorporated herein by reference in its entirety for all purposes).

[0269] Thirdly, the cleavage sites of Cpf1 are shifted by approximately 3-5 base pairs that create "sticky ends" (Kim et al., 2016. “Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells” published online). (June 06, 2016). These sticky ends, with overhangs of 3–5 bp, are thought to facilitate NHEJ-mediated ligation and improve gene editing of DNA fragments with matching ends. The cleavage site is located in the 3' end of the target DNA, distal to the 5' end where the PAM is located. The cleavage site typically follows the 18th base on the non-hybridized strand and the corresponding 23rd base on the complementary strand hybridized to crRNA.

[0270] Fourth, in the Cpf1 complex, the "seed" region is located within the first 5nt of the guide sequence. The Cpf1 crRNA seed region is highly susceptible to mutations, and even a single base substitution in this region can significantly 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). Importantly, unlike Cas9 CRISPR targets, the cleavage sites and seed regions of the Cpf1 system do not overlap. Additional guidance on designing Cpf1 crRNA targeting oligos can be found in (Zetsche B. et al. 2015 “Cpf1 Is It is available in "A Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System" (Cell 163, 759-771).

[0271] Those skilled in the art will understand that the Cpf1s disclosed herein may be any variants derived or isolated from any source known in the art, many of which may be known in the art. For example, in some embodiments, the Cpf1 peptides of this disclosure may include FnCPF1 (e.g., SEQ ID NO: 2), AsCpf1 (e.g., Figure 14), LbCpf1 (e.g., Figure 15), or any other of the many known Cpf1 proteins from various other microbial species, as described in Figure 2, and synthetic variants thereof.

[0272] In some embodiments, the composition comprises a Cpf1 polypeptide. In some embodiments, the Cpf1 polypeptide is enzymatically active, for example, cleaving a target nucleic acid when it binds to a guide RNA. In some embodiments, the Cpf1 polypeptide exhibits reduced enzymatic activity compared to a wild-type Cpf1 polypeptide (for example, compared to a Cpf1 polypeptide containing the amino acid sequence shown in Figure 2, 16, or 17), while retaining DNA binding activity.

[0273] 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 with respect to the amino acid sequence shown in Figure 2, 16, or 17. In some embodiments, the Cpf1 polypeptide contains an amino acid sequence having amino acid sequence identity of 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%, at least 90%, or 100% of a continuous stretch of amino acid sequence from 100 to 200 amino acids (aa), 200aa to 400aa, 400aa to 600aa, 600aa to 800aa, 800aa to 1000aa, 1000aa to 1100aa, 1100aa to 1200aa, or 1200aa to 1300aa as shown in Figures 2, 16, or 17.

[0274] 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 with respect to the RuvCI domain of the Cpf1 polypeptide of the amino acid sequence shown in Figure 2, 16, or 17. The Cpf1 polypeptide contains an amino acid sequence having amino acid sequence identity with respect to the RuvCII domain of the Cpf1 polypeptide of the amino acid sequence shown in Figure 17, in amounts of 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%. In some embodiments, the Cpf1 polypeptide contains an amino acid sequence having amino acid sequence identity with respect to the RuvCIII domain of the Cpf1 polypeptide of the amino acid sequence shown in Figure 2, 16, or 17, in amounts of 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%.

[0275] In some embodiments, the Cpf1 polypeptide exhibits reduced enzymatic activity compared to the wild-type Cpf1 polypeptide (e.g., compared to a Cpf1 polypeptide containing the amino acid sequence shown in Figure 2, 16, or 17) while retaining DNA binding activity. 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 with respect to the amino acid sequence shown in Figure 2, 16, or 17; and comprises an amino acid substitution (e.g., D→A substitution) at the amino acid residue corresponding to amino acid 917 of the amino acid sequence shown in Figure 2, 16, or 17. 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, 16, or 17; and comprises an amino acid substitution (e.g., E→A substitution) at the amino acid residue corresponding to amino acid 1006 of the amino acid sequence shown in Figure 2, 16, or 17. 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 with respect to the amino acid sequence shown in Figure 2, 16, or 17; and comprises amino acid substitutions (e.g., D→A substitutions) at amino acid residues corresponding to amino acid 1255 of the amino acid sequence shown in Figure 2, 16, or 17.

[0276] In some embodiments, the Cpf1 polypeptide is a fusion polypeptide, for example, here, the Cpf1 fusion polypeptide comprises: a) the Cpf1 polypeptide; and b) a heterogeneous fusion partner. In some embodiments, the heterogeneous fusion partner fuses to the N-terminus of the Cpf1 polypeptide. In some embodiments, the heterogeneous fusion partner fuses to the C-terminus of the Cpf1 polypeptide. In some embodiments, the heterogeneous fusion partner fuses to both the N-terminus and the C-terminus of the Cpf1 polypeptide. In some embodiments, the heterogeneous fusion partner is internally inserted into the Cpf1 polypeptide.

[0277] Suitable heterogeneous fusion partners include NLS, epitope tags, and fluorescent polypeptides.

[0278] Linked guide RNA and donor nucleic acids In one aspect, the present invention provides a complex comprising a CRISPR system including an RNA-induced endonuclease (e.g., Cas9 or Cpf1 polynucleotide), a guide RNA, and a donor polynucleotide, wherein the guide RNA and donor polynucleotide are ligated. As illustrated herein, the guide RNA and donor polynucleotide may be ligated by either covalent or noncovalent bonds. In one embodiment, the guide RNA and donor polynucleotide are chemically linked. In another embodiment, the guide RNA and donor polynucleotide are enzymatically linked. In one embodiment, the guide RNA and donor polynucleotide hybridize with each other. In another embodiment, both the guide RNA and donor polynucleotide hybridize into a bridge sequence. Any number of such hybridization schemes are possible.

[0279] Deaminase In some embodiments, the complex or composition further comprises a deaminase (e.g., an adenine base editor). As used herein, the terms “deaminase” or “deaminase domain” refer to an enzyme that catalyzes the removal of an amine group from a molecule, or deamination. In some embodiments, the deaminase is a cytidine deaminase that catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase is a cytosine deaminase that catalyzes the hydrolytic deamination of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA).

[0280] In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that 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 deaminases provided herein (e.g., engineered adenosine deaminases, evolved adenosine deaminases) may be derived from any organism, such as bacteria. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase derived from an organism such as a human, chimpanzee, gorilla, monkey, cattle, dog, rat, or mouse.

[0281] In some embodiments, the deaminase or deaminase domain does not occur in nature. 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 naturally occurring deaminase. In some embodiments, the adenosine deaminase is derived from bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhi, Lactobacillus putlefaciens, Haemophilus influenzae, or Caulobacter crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is Escherichia coli TadA deaminase (ecTadA). In some embodiments, TadA deaminase is a shortened E. coli TadA deaminase. For example, shortened ecTadA may lack one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, shortened ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 compared to full-length ecTadA. , 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues may be missing. In some embodiments, the shortened ecTadA may be missing 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 the 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 variant thereof.

[0282] Deaminase can be used in combination with any of the other CRISPR elements described herein (i.e., as a composition), or deaminase can be fused with any of the other CRISPR elements described herein (e.g., Cas9 or Cpf1) (i.e., as a complex). In certain embodiments, deaminase is fused with Cas9, Cpf1, or their variants.

[0283] 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 liposomes or micelle delivery vehicles. The composition may also contain a solvent or carrier suitable for administration to cells or hosts such as mammals or humans.

[0284] In some embodiments, the composition further comprises one or more surfactants. The surfactants may be nonionic surfactants and / or zwitterionic surfactants. In some embodiments, the surfactants are polymers or copolymers of ethylene oxide (EO), propylene oxide (PO), butylene oxide (BO), glycolic acid (GA), lactic acid (LA), or combinations thereof. For example, the surfactants may be polyethylene glycol (PEG), polypropylene glycol, polyglycolic acid (PGA), polylactic acid, or mixtures thereof. The list of exemplary surfactants includes polyoxyethylene sorbitan ester surfactants (commonly called tweens), particularly 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; octoxynol, which may differ in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being particularly important; (octylphenoxy)polyethoxyethanol (IGEPAL CA-6301NP-40); phospholipids such as phosphatidylcholine (lecithin); and triethylene glycol monolauryl ether (Brij The composition includes, but is not limited to, polyoxyethylene fatty 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). In some embodiments, the composition further comprises one or more pharmaceutically acceptable carriers and / or excipients.

[0285] In some cases, the components (e.g., nucleic acid components (e.g., guide nucleic acids); protein components (e.g., Cas9 or Cpf1 polypeptide, mutant Cas9 or Cpf1 polypeptide)) include a labeled portion. As used herein, the terms “label,” “detectable label,” or “labeled portion” refer to any portion that provides signal detection and may vary significantly depending on the specific nature of the assay. The labeled portion of interest is directly detectable. The method includes both direct labels (e.g., fluorescent labels) and indirectly detectable labels (indirect labels) (e.g., binding pair members). Fluorescent labels may be any fluorescent labels (e.g., fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, ALEXAFLUOR® label, etc.), fluorescent proteins (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 their fluorescent derivatives), etc.). A suitable detectable (directly or indirectly) labeled portion for use in the method includes any portion detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, a suitable indirect label includes biotin (binding pair member) that can be bound by streptavidin (which itself can be directly or indirectly labeled). The label may also be a radioactive label (direct label) (e.g., 3 H, 125 I, 35 S, 14 C, or 32P); enzymes (indirectly labeled) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.); fluorescent proteins (directly labeled) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivative thereof); metal labels (directly labeled); colorimetric labels; binding pair members, etc. By “binding pair partner” or “binding pair member,” one of the first and second parts is meant, where the first and second parts have a specific binding affinity to each other. Suitable binding pairs include, but are not limited to, antigens / antibodies (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 coupled pair member may be suitable for use as an indirectly detectable label portion.

[0286] Any given component, or combination of components, may be left unlabeled or may be detected by labeling portions. In some embodiments, when two or more components are labeled, they may be labeled with labeling portions that are distinguishable from each other.

[0287] Encapsulation and nanoparticles In some embodiments of the composition, the polymer combines with nucleic acids and / or polypeptides to partially or completely encapsulate the nucleic acids and / or polypeptides. In some formulations, the composition can provide nanoparticles comprising the polymer and nucleic acids and / or polypeptides.

[0288] In some embodiments, the composition may include core nanoparticles in addition to the polymers and nucleic acids or polypeptides described herein. Any suitable nanoparticles, including metal (e.g., gold) nanoparticles or polymer nanoparticles, can be used.

[0289] The polymers and nucleic acids (e.g., guide RNA, donor polynucleotides, or both) or polypeptides described herein can be conjugated directly or indirectly to the surface of nanoparticles. For example, the polymers and nucleic acids (e.g., guide RNA, donor polynucleotides, or both) or polypeptides described herein can be conjugated directly to the surface of nanoparticles or indirectly through interposed linkers.

[0290] Any type of molecule can be used as a linker. For example, the linker may 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 may be substituted with one or more functional groups, including ketone, ether, ester, amide, alcohol, amine, urea, thiourea, sulfoxide, sulfone, sulfonamide, and disulfide functional groups. In embodiments in which nanoparticles contain gold... In this case, the linker can be any thiol-containing molecule. The reaction of the thiol group with gold results in a sulfide (-S-) covalent bond. The design and synthesis of linkers are well known in the art.

[0291] In some embodiments, the nucleic acid conjugated to the nanoparticle is a linker nucleic acid that helps to non-covalently bond one or more elements described herein (e.g., Cas9 polypeptide, and guide RNA, donor polynucleotide, and Cpf1 polynucleotide) to the nanoparticle-nucleic acid conjugate. For example, the linker nucleic acid may have a sequence that hybridizes to the guide RNA or donor polynucleotide.

[0292] Nucleic acids conjugated to nanoparticles (e.g., colloidal metal (e.g., gold) nanoparticles; nanoparticles containing biocompatible polymers) may have any suitable length. When the nucleic acid is a guide RNA or a donor polynucleotide, the length will be suitable for such a molecule, as discussed herein and known in the art. When the nucleic acid is a linker nucleic acid, it may have any suitable length for the linker, e.g., 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 containing biocompatible polymers) may have lengths exceeding 1000 nt.

[0293] When nucleic acids linked to nanoparticles (e.g., covalently linked; non-covalently linked) contain a nucleotide sequence that hybridizes to at least a portion of the guide RNA or donor polynucleotide present in the complex of the Disclosure, they have a region that has sufficient sequence identity to the complementary region of the guide RNA or donor polynucleotide sequence to facilitate hybridization. In some embodiments, the nucleic acids linked to nanoparticles in the complex of the Disclosure have 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 to the complement of the guide RNA or donor polynucleotide present in the complex from 10 to 50 nucleotides (e.g., 10 nucleotides (nt) to 15 nucleotides, 15 nucleotides to 20 nucleotides, 20 nucleotides to 25 nucleotides, 25 nucleotides to 30 nucleotides, 30 nucleotides to 40 nucleotides, or 40 nucleotides to 50 nucleotides).

[0294] In some embodiments, the nucleic acids linked to the nanoparticles (e.g., covalently linked; noncovalently linked) are either donor polynucleotides or have the same or substantially the same nucleotide sequence as the donor polynucleotide. In some embodiments, the nucleic acids linked to the nanoparticles (e.g., covalently linked; noncovalently linked) contain a nucleotide sequence complementary to the donor DNA template.

[0295] How to use Also provided herein are methods for delivering nucleic acids and / or polypeptides to cells, where the cells may be in vitro or in vivo. The method comprises administering a composition comprising the polymers and nucleic acids and / or polypeptides described herein to cells or to a subject containing cells. The method can be used with respect to any type of cell or subject, but is particularly useful for mammalian cells (e.g., human cells). In some embodiments, the polymer delivers nucleic acids and / or polypeptides primarily or exclusively to target cells or tissues (e.g., peripheral nervous system, central nervous system, eyes, liver, muscles, lungs, bone (e.g., hematopoietic cells), or tumor cells or tissues) of the subject. It contains a targeting agent to ensure delivery.

[0296] When used to deliver proteins or nucleic acids to cells in a target (i.e., in vivo), it is desirable that the polymer be stable in serum. Stability in serum can be evaluated as a function of the efficiency with which the polymer delivers the protein or nucleic acid payload to cells in serum (e.g., in vitro or in vivo). That is, in some embodiments, the polymer delivers a given protein or nucleic acid to cells in serum with higher efficiency than pAsp[DET] under the same conditions.

[0297] When used with a composition comprising 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 for modifying a target nucleic acid includes homologous recombination repair (HDR). In some embodiments, the use of the complex of the present disclosure for performing 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 25% or more. In some embodiments, the method for modifying a target nucleic acid includes non-homologous end joining (NHEJ). In some embodiments, the use of the complex of the present disclosure for performing 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 25% or more.

[0298] The following embodiments further illustrate the present invention, but of course, should not be construed as limiting its scope. [Examples]

[0299] Example 1 This example provides guidance for the synthesis of the polymers described herein. The synthesis involves modifying PBLA with an amine and N-(2-aminoethyl)ethane-1,2-diamine ("DET"). An exemplary procedure is as follows:

[0300] [ka]

[0301] 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 equivalents) 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 equivalents relative to the benzyl group of the PBLA segment, 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 (10-12 X 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 with excess deionized water through a 3.5-5 kD cutoff membrane. When the pH of the solution was between 5 and 6, dialyzing was stopped, and the solution was lyophilized to obtain approximately 60 mg of polymer product. A similar procedure was carried out using different ratios of n-hexylamine relative to PBLA to provide polymers A1-A6. The concentration ratios and the polymers obtained therefrom are listed in Table 2, with the values ​​x and y reported as averages. The degree of substitution is: 1 This was confirmed by 1H NMR spectroscopy. The results are also plotted in Figure 3.

[0302] [Table 2]

[0303] As demonstrated by Table 2 and Figure 3, the degree of substitution of the hydrophobic moiety can be controlled by the equivalent amount of hydrophobic moiety added to the reaction mixture.

[0304] Example 2 This example provides guidance for the synthesis of the polymers described herein. The synthesis involves modifying PBLA with an amine and N-(2-aminoethyl)ethane-1,2-diamine ("DET"). An exemplary procedure is as follows:

[0305] [ka]

[0306] Lyophilized PBLA (50 mg, 0.0037 mMol) was placed in a flask and dissolved in tetrahydrofuran / N-methyl-2-pyrrolidine (1 mL each). 1-(4-butylcyclohexyl)methaneamine (75 mg, 0.44 mMol, 120 equivalents) 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 equivalents relative to the benzyl group of the PBLA segment) 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 (10-12 X 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 with excess deionized water through a 3.5-5 kD cutoff membrane. Dialysis was stopped when the pH of the solution was between 5 and 6, and the solution was lyophilized to obtain the polymer product. A similar procedure was carried out using 1-(4-butylcyclohexyl)methaneamine in different ratios relative to PBLA to provide polymers B1-B3. The concentration ratios and the polymers obtained therefrom are listed in Table 3, with the values ​​x and y reported as averages. The degree of substitution is: 1 This was confirmed by 1H NMR spectroscopy. The results are also plotted in Figure 4.

[0307] [Table 3]

[0308] As demonstrated by Table 3 and Figure 4, the degree of substitution of the hydrophobic moiety can be controlled by the equivalent amount of hydrophobic moiety added to the reaction mixture.

[0309] Example 3 The following examples illustrate the effect of incremental hydrophobic side chain substitution on the polymers described herein on mRNA delivery to cells.

[0310] Polymers A1, A2, and A3 from Example 1 were formulated with mRNA encoding red fluorescent protein (RFP) and cultured with HEK293T cells under both serum and serum-free conditions. pAsp[DET] was used as a positive control. The results (Figure 5) showed some transfection in all serum-free samples, and the use of polymers with a higher degree of hexylamine substitution increased transfection efficiency. Much higher transfection was observed when polymer A3 was used.

[0311] Example 4 The following examples illustrate the use of the polymer of the present invention for delivering mRNA to various cells.

[0312] Polymers A4 and A5 from Example 1 were formulated with mRNA encoding mCherry and cultured with HEK293T and HepG2 under both serum and serum-free conditions, as well as with primary myoblasts from Mdx mice under serum conditions. The results, showing good transfection in all samples, are shown in Figures 6-8. The highest transfection level was obtained using polymer A5, which has a higher level of hexylamine substitution.

[0313] Polymer A5 (hexylamine substituted) from Example 1 and polymer B3 ((4-butylcyclohexyl)methanamine substituted) from Example 2 were formulated into mCherry mRNA and cultured with HEK293T cells. The results are shown in Figure 9. Both polymers showed excellent transfection efficiency.

[0314] Example 5 The following examples illustrate the use of the polymers of the present invention for delivering CRISPR ribonucleoprotein and single guide RNA (sgRNA) to cells.

[0315] Polymers A4 and A5 (hexylamine substituted) from Example 1 and polymer B3 ((4-butylcyclohexyl)methaneamine substituted) from Example 2 were used in this experiment. Each polymer was mixed with either (a) GFP-targeting sgRNA (600 ng) and Cas9 (3 μg), or (b) GFP-targeting crRNA (300 ng) and Cpf1 (3 μg) to provide loaded polymer nanoparticles. GFP-expressing HEK293T cells (GFP-HEK cells) seeded in serum at a cell density of 20,000 were treated with the loaded polymer nanoparticles, and doxycycline induction was performed 2 days after transfection. Flow cytometry was performed 48 hours after induction to quantify the percentage of cells that were GFP-. The results are shown in Figure 10 (polymers loaded with Cas9; n=3 and error bars = SEM) and Figure 11 (polymers loaded with Cpf1; n=3 and error bars = SEM). All three polymers showed significant GFP knockout using either Cas9 or Cpf1.

[0316] Example 6 The following examples illustrate the stability of nanoparticles containing the polymer of the present invention.

[0317] mCherry mRNA was mixed with polymer A5 from Example 1 to provide loaded nanoparticles. One sample of nanoparticles was incubated at room temperature for 1 minute to 2 hours. Another sample was stored at 4 degrees Celsius for 2 hours. A third sample was incubated at -80 degrees Celsius for 2 hours. The cells were frozen. HEK293T cells were treated with nanoparticles, and mCherry expression was quantified by flow cytometry. The results are shown in Figure 12 (n=2, error bars=SEM). As shown, the nanoparticles retained almost all transfection efficiency, demonstrating that the nanoparticles were stable under the test conditions.

[0318] Example 7 The following examples illustrate the use of the polymers provided herein, co-mixed with PEGylated polymers, for delivering mRNA to cells.

[0319] Polymer A5 from Example 1 was mixed with gradually increasing amounts of GalNAc-PEG-PAsp or GalNAc-PEG-PAsp-C6 (i.e., 10% by weight, 20% by weight, 40% by weight, and 60% by weight of the total composition) and used to deliver mCherry mRNA to Hep3B cells.

[0320] [ka]

[0321] Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay, and RFP+ percent was measured by flow cytometry. The results are shown in Figure 13 (n=2, error bars=SEM). Polymer A5 of Example 1, mixed with either GalNAc-PEG-PAsp or GalNAc-PEG-PAsp-C6, showed very efficient delivery for compositions containing 10%, 20%, 40%, and 60% by weight of GalNAc-PEG-PAsp or GalNAc-PEG-PAsp-C6 of the total composition. GalNAc-PEG-PAsp and GalNAc-PEG-PAsp-C6 showed a slight decrease in transfection efficiency when moving from 40% to 60% by weight of the total composition, which was expected. It is known that a high PEG composition may hinder cell uptake. The CCK-8 assay showed that the polymers tested did not cause cytotoxicity at the doses used. At doses that resulted in mRNA transfection of over 60%, no cytotoxicity was observed.

[0322] The results for both transfection efficiency and cell viability indicate that the polymers provided herein can be co-formulated with PEG-polymers.

[0323] Example 8 Polymer H27N was prepared and used in Examples 9, 10, 12-14, 16-21, and 23 provided herein.

[0324] [ka]

[0325] H27N uses PBLA with N 1 -(2-aminoethyl)-N 1 ,N 2 ,N 2 It can be produced by modification with -trimethylethane-1,2-diamine and hexylamine. An exemplary procedure is as follows:

[0326] [ka]

[0327] 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). n-hexylamine (160 equivalents) was added to the clear solution, and the clear reaction mixture was stirred at room temperature for 24 hours. After approximately 24 hours, under mild anhydrous conditions, N 1 -(2-aminoethyl)-N 1 ,N 2 ,N 2 -Trimethylethane-1,2-diamine (50 equivalents relative to the benzyl group of the PBLA segment) was added to the clear mixture. After approximately 18 hours at room temperature, the reaction mixture was precipitated in 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 with excess deionized water through a 3.5-5 kD cutoff membrane. Dialysis was stopped when the pH of the solution was between 5 and 6, and the solution was freeze-dried to obtain the polymer product.

[0328] Example 9 The following examples demonstrate the ability of polymer H27N from Example 8 to form nanoparticles when combined with mCherry mRNA.

[0329] Polymer H27N was combined with mCherry mRNA, and the resulting mixture was analyzed using dynamic light scattering. The results are plotted in Figure 16.

[0330] As demonstrated by the dynamic light scattering plot in Figure 16, the combination of H27N and mCherry mRNA results in clear nanoparticle formation. The resulting nanoparticles had an average diameter of 195 nm and a polydispersity index of 0.16.

[0331] Example 10 The following examples demonstrate the ability of the polymer H27N from Example 8 to form nanoparticles when combined with Cas9 RNP.

[0332] The polymer H27N was combined with Cas9 RNP, and the resulting mixture was subjected to dynamic light scattering. The analysis was performed using [the specified method]. The results are plotted in Figure 17.

[0333] As demonstrated by the dynamic light scattering plot in Figure 17, the combination of H27N and Cas9 RNP results in clear nanoparticle formation. The resulting nanoparticles had an average diameter of 92 nm and a polydispersity index of 0.21.

[0334] Example 11 Four polymers were prepared using the same synthesis procedure as described in Example 8. The resulting polymers were used in Examples 12-14 provided herein. As is evident from the preparation methods, the parenthetical designations in the following structures do not indicate block copolymer structures.

[0335] [ka]

[0336] Example 12 The following examples illustrate the use of polymer H27N and PEG-polymers 2-4 for delivering mRNA to HEK293T cells.

[0337] RFP mRNA was delivered in H27N and a comixture of H27N and PEG-polymers 2-4, in a ratio of 20:80 or 40:60 wt% of PEG-polymers to H27N. The comixture of H27N and PEG-polymers 2-4 (in a ratio of 20:80 or 40:60 wt% of PEG-polymers to H27N) was prepared before the addition of RFP mRNA. The resulting nanoparticles were treated in HEK293T cells, and RFP+ cells were quantified 24 hours after transfection using flow cytometry. The results are plotted in Figure 18.

[0338] As demonstrated by Figure 18, polymer H27N, both alone and in combination with PEG-polymers 2-4, is efficient at delivering mRNA into HEK293T cells, and these combinations yielded equivalent or slightly reduced trans-trans The infection efficiency was demonstrated.

[0339] Example 13 The following examples illustrate the effects of PEG-polymers 1-3 on the ability of polymer H27N to deliver Cas9 RNP to Hep3B cells.

[0340] Hep3B cells were seeded at 50,000 cells / well in a culture medium consisting of Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS) to form 40ρmol of Cas9 RNPs. sgRNA targeting the SERPINA1 gene was prepared, and Cas9 protein was slowly added and thoroughly mixed by pipetting. Separately, compositions containing H27N polymer and PEG-polymers 1-3 were prepared using a 1:1 ratio. Nanoparticles were formed by mixing the resulting compositions with sgRNA using a 4:1 mass ratio of polymer to sgRNA. The resulting nanoparticles were treated in Hep3B cells, and genomic DNA (gDNA) was extracted 72 hours after transfection using the Qiagen DNeasy Blood and Tissue Protocol. Experiments were performed using biological and assay replication, and non-homologous end joining (NHEJ) efficiency was quantified using ddPCR. The results are plotted in Figure 19.

[0341] As demonstrated in Figure 19, only polymer H27N was effective for gene editing in Hep3B cells. PEG-polymers 1-3 showed comparable or slightly reduced gene editing efficiency in Hep3B cells.

[0342] Example 14 The following examples illustrate the effects of PEG-polymers 1-3 on the ability of polymer H27N to deliver Cas9 RNP to Hep3B cells.

[0343] Hep3B cells were seeded at 50,000 cells / well in a culture medium consisting of Dulbecco's Modified Eagle Medium (DMEM) and 10% fetal bovine serum (FBS) to form 40ρmol of Cas9 RNPs. sgRNA targeting the SERPINA1 gene was prepared, and Cas9 protein was slowly added and thoroughly mixed by pipetting. Separately, compositions containing H27N polymer and PEG-polymers 1-3 were prepared using a 1:1 ratio. Nanoparticles were formed by mixing the resulting compositions with sgRNA using an 8:1 mass ratio of polymer to sgRNA. The resulting nanoparticles were treated in Hep3B cells, and genomic DNA (gDNA) was extracted 72 hours after transfection using the Qiagen DNeasy Blood and Tissue Protocol. Experiments were performed with biological and assay replication, and non-homologous end joining (NHEJ) efficiency was quantified using ddPCR. The results are plotted in Figure 20.

[0344] As demonstrated in Figure 20, only polymer H27N was effective for gene editing in Hep3B cells. PEG-polymers 1-3 showed comparable or slightly reduced gene editing efficiency in Hep3B cells.

[0345] Example 15 The following examples illustrate the use of the polymer of the present invention for delivering Cre mRNA to mice, as demonstrated by Loxp-luciferase mice.

[0346] Loxp-luciferase mice possessing the reporter sequence shown in Figure 21 were treated with nanoparticles formulated with H27N and Cre mRNA. The control group remained untreated. This represents the mice. Administration was via intrathecal (IT) injection. Cre mRNA delivery was evaluated via bioluminescence, and the resulting images are shown in Figures 22A-22C.

[0347] Mice treated with the nanoparticle composition showed significant delivery of Cre mRNA to mice.

[0348] Example 16 Polymer C is PBLA N 1 -(2-aminoethyl)-N 1 ,N 2 ,N 2 It can be produced by modification with -trimethylethane-1,2-diamine and 4-methylpentane-1-amine. An exemplary procedure is as follows: Scheme 4.

[0349] [ka]

[0350] Lyophilized PBLA (50 mg, 0.0037 mMol) was placed in a flask and dissolved in tetrahydrofuran / N-methyl-2-pyrrolidine (1 mL each). n-4-methylpentan-1-amine (160 equivalents) was added to the clear solution, and the clear reaction mixture was stirred at room temperature for 24 hours. After approximately 24 hours, under mild anhydrous conditions, N 1 -(2-aminoethyl)-N 1 ,N 2 ,N 2 -Trimethylethane-1,2-diamine (50 equivalents relative to the benzyl group of the PBLA segment) was added to the clear mixture. After approximately 18 hours at room temperature, the reaction mixture was precipitated in 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 with excess deionized water through a 3.5-5 kD cutoff membrane. Dialysis was stopped when the pH of the solution was between 5 and 6, and the solution was freeze-dried to obtain the polymer product.

[0351] Example 17 The following examples illustrate the use of the polymer of the present invention for delivering Cre mRNA to mice, as demonstrated by ai9 mice.

[0352] ai9 mice having the same reporter construct illustrated in Figure 23 were subjected to two nanoparticle compositions: (i) H27N and P with a 4:1 ratio of PGA-PEG:mRNA. (ii) Nanoparticles formulated with a mixture of GA-PEG and Cre mRNA ("H27N+PGA-PEG ("4:1 PGA-PEG:mRNA ratio")) and (ii) one of the nanoparticles formulated with a mixture of H27N, PGA-PEG, and Cre mRNA having a 6:1 PGA-PEG:mRNA ratio ("H27N+PGA-PEG ("6:1 PGA-PEG:mRNA ratio")). Controls represent untreated mice. The properties of the nanoparticles are summarized in Table 4.

[0353] [Table 4]

[0354] The resulting nanoparticle formulation was administered to mice via intrathecal (IT) injection. Ten days after treatment, the mice were sacrificed by CO2 asphyxiation, and blood was removed by perfusing the left ventricle with 1% heparinized saline followed by PBS. The brain and spinal cord were then collected. The mouse brains were sectioned in the coronal plane to a thickness of 100 μm, and every other section was collected and imaged.

[0355] In vivo Cre mRNA delivery was evaluated in the rostral and caudal regions of the brain via bioluminescence, and the resulting images are shown in Figure 24.

[0356] In Figure 24, nanoparticle compositions (i) and (ii), respectively, showed increased delivery of Cre mRNA to the caudal portions of the brainstem and cerebellum (i.e., the brain region surrounded by cerebrospinal fluid (CSF)) compared to untreated mice (negative control). Furthermore, nanoparticle compositions (i) and (ii) containing PGA-PEG qualitatively showed significant visible RFP expression, suggesting that PGA-PEG nanoparticles have an enhanced ability to transfect around the brainstem in the caudal region of the brain.

[0357] Example 18 This example provides guidance for the synthesis of polymer D as described herein. The synthesis involves modifying PBLA with hexylamine and amine compound 10. An exemplary procedure is as follows:

[0358] [ka]

[0359] Amine compound 10 was synthesized using the protocol described in Scheme 5.

[0360] [ka]

[0361] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Hexylamine (21.85 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Next, amine compound 10 (607 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer D (17 mg) as a white solid. 1 H NMR(D2O):4.53(65H), 3.63-2.29(m), 2.17-2.00(s), 1.41-0.52(m).

[0362] Example 19 This example provides guidance for the synthesis of polymer E as described herein. The synthesis involves modifying PBLA with hexylamine and amine compound 13. An exemplary procedure is as follows:

[0363] [ka]

[0364] Amine compound 13 was synthesized using the protocol described in Scheme 7.

[0365] [ka]

[0366] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Hexylamine (21.85 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Amine compound 13 (371.71 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was then added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer E (20 mg) as a white solid. 1H NMR(D2O): 4.53(br s), 3.63-2.29(m), 2.17-2.00(s), 1.54(m), 1.41-0.52(m).

[0367] Example 20 This example provides guidance for the synthesis of polymer F described herein. The synthesis involves modifying PBLA with cyclohexylethylamine and amine compound 13. An exemplary procedure is as follows:

[0368] Amine compound 13 was synthesized using the protocol described in Scheme 7 of Example 19.

[0369] [ka]

[0370] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Cyclohexylethylamine (27.48 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Next, amine compound 13 (371.71 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer F (20 mg) as a white solid. 1H NMR(D2O): 4.53(br s), 3.63-2.29(m), 2.17-2.00(s), 1.55-1.15(m).

[0371] Example 21 This example provides guidance for the synthesis of polymer G described herein. The synthesis involves modifying PBLA with hexylamine and amine compound 20. An exemplary procedure is as follows:

[0372] [ka]

[0373] Amine compound 20 was synthesized using the protocol described in Scheme 10.

[0374] [ka]

[0375] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Hexylamine (21.85 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Next, amine compound 20 (407.8 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer G (20 mg) as a white solid. 1H NMR(D2O): 4.53(br s), 3.63-2.29(m), 2.17-2.00(s), 1.54(m), 1.41-0.52(m).

[0376] Example 22 This example provides guidance for the synthesis of polymer H described herein. The synthesis involves modifying PBLA with hexylamine and amine compound 22. An exemplary procedure is as follows:

[0377] [ka]

[0378] Amine compound 22 was synthesized using the protocol described in Scheme 12.

[0379] [ka]

[0380] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Hexylamine (21.85 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Next, amine compound 22 (541.5 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer H (20 mg) as a white solid. 1H NMR(D2O): 4.53(br s), 3.63-2.29(m), 2.17-2.00(s), 1.54(m), 1.41-0.52(m).

[0381] Example 23 The following examples illustrate the ability of nanoparticles containing the polymer of the present invention to deliver mCherry mRNA to HEK293T cells and Hep3B cells.

[0382] mCherry mRNA was mixed with polymers D-H, respectively, to provide loaded nanoparticles. HEK293T cells and Hep3B cells were treated with the resulting nanoparticles, and mCherry expression was quantified by flow cytometry. The results are shown in Figure 25 (n=2, error bars = SEM).

[0383] As demonstrated in Figure 25, treatment of HEK293T cells and Hep3B cells with nanoparticles formed from polymers E, F, and G resulted in relatively higher transfection efficiencies compared to nanoparticles formed from polymers D and H.

[0384] Example 24 This example provides guidance for the synthesis of polymer I described herein. The synthesis involves modifying PBLA with hexylamine and amine compound 15. An exemplary procedure is as follows:

[0385] [ka]

[0386] Amine compound 15 was synthesized using the protocol described in Scheme 14.

[0387] [ka]

[0388] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Hexylamine (21.85 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Amine compound 15 (473.5 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was then added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer I.

[0389] Example 25 This example provides guidance for the synthesis of polymer J described herein. The synthesis involves modifying PBLA with hexylamine and amine compound 18. An exemplary procedure is as follows:

[0390] [ka]

[0391] Amine compound 18 was synthesized using the protocol described in Scheme 16.

[0392] [ka]

[0393] PBLA (25 mg, 0.0018 mmol) was dissolved in a mixture of 500 μL of NMP and 500 μL of THF. Hexylamine (21.85 mg, 0.216 mmol) was then added to this reaction mixture, and the reaction mixture was stirred at room temperature for 23 hours. Next, amine compound 18 (607 mg, 2.34 mmol) in free amine form, dissolved in 500 μL of NMP, 500 μL of THF, and 500 μL of triethylamine, was added to this solution. The resulting reaction mixture was stirred at room temperature for 24 hours, and the crude reaction mixture was precipitated in diethyl ether (40 mL) to obtain the crude polymer. The crude polymer was dissolved in 2 mL of 1N HCl solution and dialyzed at 4°C for 48 hours using a 3.5-5 kD cutoff membrane dialysis bag. The purified polymer was lyophilized to obtain polymer J.

[0394] Preferred embodiments of the present invention, including the best modes known to the inventors for carrying out the invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend to carry out the invention in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradictory by context.

[0395] Where a range of values ​​is provided, each value in between is understood to be included in the invention up to one-tenth of the lower limit unless the context otherwise explicitly indicates, as are values ​​between the upper and lower limits of that range and other values ​​described or within the described range. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in the invention, subject to any particularly excluded restrictions in the described range. Where a described range includes one or both of the restrictions, a range excluding one or both of those included restrictions is also included in the invention.

[0396] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Any methods and materials similar to or equivalent to those described herein are also part of the present invention. Methods and materials that may be used in the implementation or testing of the method, but are preferred, are described herein. All publications referenced herein are incorporated herein by reference to disclose and explain the methods and / or materials to which the publications are cited in connection therewith.

[0397] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include multiple references unless the context otherwise explicitly indicates otherwise. That is, for example, a reference to “(a) complex” includes multiple such complexes, and a reference to “(the) Cas9 polypeptide” includes one or more Cas9 polypeptides and their equivalents known to those skilled in the art, etc. It should be further noted that the claims may be drafted to exclude any element whatsoever. As such, this statement is intended to serve as a prior basis for the use of exclusive terms such as “alone,” “only,” or “negative” restrictions in relation to the enumeration of elements of the claims.

[0398] It is understood that certain features of the Invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for simplification may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if each combination were individually and expressly disclosed. Furthermore, all subcombinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if each such subcombination were individually and expressly disclosed herein.

[0399] The publications discussed herein are provided solely for disclosure prior to the filing date of this application. Nothing herein should be construed as acknowledging that the present invention has no prior rights to such publications for the sake of prior art. Furthermore, the publication dates provided may differ from the actual publication dates which may need to be independently verified.

Claims

1. A polymer comprising a hydrolyzable polymer backbone, wherein the polymer backbone is: (i) Monomer units having a side chain containing a hydrophobic group; (ii) Monomer units having a side chain containing an oligoamine or polyamine; and optionally (iii) Monomer units having a side chain containing an ionizable group and optionally having a pKa of less than 7 A polymer containing [this component].

2. The polymer according to claim 1, wherein the hydrophobic group comprises an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group.

3. Hydrophobic group is C ３ -C １２ Linear or branched alkyl group, optionally C ３ -C ６ The polymer according to claim 1, comprising a linear or branched alkyl group.

4. Oligoamines or polyamines are given by the formula: -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ ２ ] ２ ; -(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -]r ２ R ２ }; -(CH ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ ２ ] ２ } ２ ,-(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -(CH ２ ) ｓ１ -R ４ -R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -(CH ２ ) ｓ２ -R ４ -R ５ ] ２ ;-(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ (CH ２ ) ｓ３ -R ４ -R ５ }; -(CH ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -(CH ２ ) ｓ４ -R ４ -R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -CH ２ -CHOH-R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -CH ２ -CHOH-R ５ ; -(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ -CH ２ -CHOH-R ５ ; -(CH) ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -CH ２ -CHOH-R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -(CH ２ ) ｓ１ -R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -(CH ２ ) ｓ２ -R ５ ] ２ ; -(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ (CH ２ ) ｓ３ -R ５ }; -(CH ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -(CH ２ ) ｓ４ -R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[N{(CH ２ ) ｓ１ -R ４ -R ５ }-(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH) ２ ) ｐ１ -[N{(CH ２ ) ｓ１ -R ５ }-(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ 、-(CH ２ ) ｐ１ -[NR ２ -(CH) ２ ) ｑ１ -] ｒ１ NR ２ -CH(CONG) ２ ()-(CH ２ ) ｓ１ -R ５ ; or -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -CH(CONH ２ )-(CH ２ ) ｓ１ -R ４ -R ５ (wherein p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R ２ Each of these cases independently involves hydrogen or C １ -C １２ a It is a lukyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R ２ The second R ２ It combines with to form a heterocyclic group; each instance is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-: R ５ The polymer of any one of claims 1 to 3, wherein each example independently comprises optionally 2 to 8 tertiary amines or substituents comprising tissue-specific or cell-specific targeting moieties, and is an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or a combination thereof.

5. Polyamines -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ ２ ] ２ ; - (CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ] [- (CH ２ ) ｑ４ -NR ２ -]r ２ R ２ };or -(CH) ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ ２ ] ２ } ２ Including; Each R ２ hydrogen or C １ -C ３ It is an alkyl group; Polyamines can be used arbitrarily. -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ A polymer according to any one of claims 1 to 4, comprising:

6. The polymer according to any one of claims 1 to 5, wherein the hydrolyzable polymer main chain comprises monomer units having about 1 to about 80 mol% of hydrophobic groups, monomer units having about 1 to about 80 mol% of oligoamines or polyamines, and monomer units having 0 to about 80 mol% of ionizable groups.

7. A polymer according to any one of claims 1 to 6, comprising a monomer unit having a side chain containing an ionizable group having a pKa of less than 7 in the hydrolyzable polymer main chain.

8. A polymer according to any one of claims 1 to 7, wherein the hydrolyzable polymer main chain comprises a polyamide, a poly-N-alkylamide, a polyester, a polycarbonate, a polycarbamate, or a combination thereof.

9. The polymer according to claim 8, wherein the hydrolyzable polymer main chain contains a polyamide.

10. Formula 1: 【Chemistry 1】 (In the formula: I understand １ , m ２ , m ３ , and m ４ Each of them is an integer from 0 to 1000, where m １ +m ２ +m ３ +m ４ The sum is greater than 5; n １ and n ２ Each of them is an integer from 0 to 1000, where n １ +n ２ The sum is greater than 2; The symbol " / " indicates that the units separated by it are connected randomly or in any order; R ３ａ Each of these cases is independently a methylene group or an ethylene group; R ３ｂ Each of these cases is independently a methylene group or an ethylene group; Each X １ These are independently -C(O)O- and -C(O)NR １３ -, -C(O)-, -S(O)(O)-, or bond; R １３ Each of these examples independently involves hydrogen, an aryl group, a heterocyclic group, and C. １ -C １２ alkyl group, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ These are cycloalkenyl groups, and any of them may be substituted with one or more substituents; X ２ Each case is independently C １ -C １２ Alkyl or heteroalkyl, C ３ -C １２ Cycloalkyl groups, C ２ -C １２ Alkenyl group, C ３ -C １２ A cycloalkenyl group, an aryl group, a heterocyclic group, or a combination thereof, any of which may be substituted with one or more substituents; A １ and A ２ These are each an independent expression -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ ２ ] ２ ; - (CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ] [- (CH ２ ) ｑ４ -NR ２ -]r ２ R ２ };or -(CH) ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ ２ ] ２ } ２ It is the basis of B １ and B ２ They are independent of each other. -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -(CH ２ ) ｓ１ -R ４ -R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -(CH ２ ) ｓ２ -R ４ -R ５ ] ２ ;-(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ (CH ２ ) ｓ３ -R ４ -R ５ }; -(CH ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -(CH ２ ) ｓ４ -R ４ -R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -CH ２ -CHOH-R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -CH ２ -CHOH-R ５ ; -(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ -CH ２ -CHOH-R ５ ; -(CH) ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -CH ２ -CHOH-R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -(CH ２ ) ｓ１ -R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -(CH ２ ) ｓ２ -R ５ ] ２ ; -(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ (CH ２ ) ｓ３ -R ５ }; -(CH ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -(CH ２ ) ｓ４ -R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[N{(CH ２ ) ｓ１ -R ４ -R ５ }-(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH) ２ ) ｐ１ -[N{(CH ２ ) ｓ１ -R ５ }-(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ 、-(CH ２ ) ｐ１ -[NR ２ -(CH) ２ ) ｑ１ -] ｒ１ NR ２ -CH(CONG) ２ ()-(CH ２ ) ｓ１ -R ５ ; or -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -CH(CONH ２ )-(CH ２ ) ｓ１ -R ４ -R ５ (wherein p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are independent integers from 1 to 5; R ２ Each of these cases independently involves hydrogen or C １ -C １２ alkyl group, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ It is a cycloalkenyl group, or R ２ The second R ２ It combines with R to form a heterocyclic group; ４ Each of these cases is independently -C(O)O-, -C(O)NH-, -O-C( O)O- or -S(O)(O)-; R ５ The polymer of claim 1, wherein each example independently comprises a structure that is an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or a combination thereof, comprising optionally 2 to 8 tertiary amines or substituents comprising tissue-specific or cell-specific targeting moieties.

11. Formula 1A: 【Chemistry 2】 (In the formula, Q is: 【Transformation 3】 and c is an integer between 0 and 50; Y is an arbitrarily existing and severable linker; R １ C is a hydrogen atom, an aryl group, a heterocyclic group, or a hydrogen atom substituted with one or more substituents. １ -C １２ Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C １ -C １２ It is a linear or branched alkyl group; R ６ This includes hydrogen, an amino group, an aryl group, a heterocyclic group, and C, which are optionally substituted with one or more amines. １ -C １２ alkyl group, C １ -C １２ Heteroalkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, C １ -C １２ It is a linear or branched alkyl group; or it is a tissue-specific or cell-specific targeting moiety; I understand １ , m ２ , m ３ , m ４ , n １ , n ２ , R ３ａ , R ３ｂ , R １３ , X １ , X ２ A １ A ２ , B １ , and B ２ The polymer of claim 10, having the structure as defined in claim 10.

12. B １ and B ２ Each of them is given by equation - (CH ２ ) ２ -NH-(CH ２ ) ２ -NH-(CH ２ ) ２ -R ４ -R ５ The polymer of claim 10 or 11, which is the base of the polymer.

13. Each R ５ They became independent 【Chemistry 4】 【Transformation 5】 (In the formula, R ２ Each of these cases independently involves hydrogen or C １ -C １２ It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or R ２ The second R ２ It combines with to form a heterocyclic group; R ７ C is optionally substituted with one or more amines. １ -C ５０ It is 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 between 0 and 50; Y is an arbitrarily existing and severable linker; n is an integer between 0 and 50; R ８ The polymer according to any one of claims 10 to 12, wherein the part is a tissue-specific or cell-specific targeting portion.

14. R ４ A polymer according to any one of claims 10 to 13, wherein is -C(O)-O-.

15. R ５ but 【Transformation 6】 A polymer according to any one of claims 10 to 14.

16. (m １ +m ２ +m ３ +m ４ ) / (n １ +n ２ A polymer according to any one of claims 10 to 15, wherein the ratio of ) is approximately 25 or less, and optionally approximately 1 or more.

17. The tissue-specific or cell-specific targeting portion is: 【Transformation 7】 (In the formula, R ９ , R １０ , R １１ , and R １２ Each of them is independently substituted with hydrogen, halogen, or optionally one or more amino groups, C １ -C ４ Alkyl, or C １ -C ４ A polymer according to any one of claims 10 to 16, wherein it is an alkoxy polymer.

18. Formula 4: 【Transformation 8】 (In the formula, m １ , m ２ , n １ , n ２ , R ３ａ , R ３ｂ , R １３ , X １ , X ２ A １ , and A ２ The polymer of claim 10 having the structure as defined in claim 10.

19. Formula 1B: 【Chemistry 9】 (In the formula, c is an integer between 0 and 50; Y is an arbitrarily existing and severable linker; R １ These are hydrogen, aryl groups, heterocyclic groups, and C １ -C １２ alkyl group, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ These are cycloalkenyl groups, each of which is optionally substituted with one or more substituents; R ２ Each of these cases independently involves hydrogen or C １ -C １２ alkyl group, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ It is a cycloalkenyl group; R ６ These include hydrogen, an amino group, an aryl group optionally substituted with one or more amines, a heterocyclic group, and C １ -C １２ alkyl group, C １ -C １２ Heteroalkyl groups, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ It is a cycloalkenyl group; or it is a tissue-specific or cell-specific targeting moiety; I understand １ , m ２ , n １ , n ２ , R ３ａ , R ３ｂ , R １３ , X １ , X ２ A １ , and A ２ The polymer of claim 10, having the structure as defined in claim 10.

20. Formula 1C: 【Chemistry 10】 (In the formula, R １ These are hydrogen, aryl groups, heterocyclic groups, and C １ -C １２ alkyl group, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ These are cycloalkenyl groups, each of which is optionally substituted with one or more substituents; R ６ These are hydrogen, amino groups, aryl groups, heterocyclic groups, and C １ -C １２ alkyl group, C １ -C １２ Heteroalkyl groups, C ２ -C １２ Alkenyl group, C ３ -C １２ Cycloalkyl groups, or C ３ -C １２ A cycloalkenyl group, each of which is optionally substituted with one or more amines; or a tissue-specific or cell-specific targeting moiety; I understand １ , m ２ , n １ , n ２ , R ３ａ , R ３ｂ , R １３ , X １ , X ２ A １ , and A ２ The polymer of claim 10 having the structure (as defined in 8).

21. formula: 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 [Chemistry 18] 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemistry 27】 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 【Chemistry 31】 【Chemistry 32】 The polymer of claim 10, having (wherein (a+b) is about 5 to about 65, (c+d) is about 2 to about 60, and (e+f) is about 2 to about 60).

22. formula: 【Transformation 33】 The polymer of claim 10, having (wherein (a+b) is from about 5 to about 65, (c+d) is from about 2 to about 60, and each instance of p is an integer from 2 to 200 independently).

23. A polymer according to any one of claims 1 to 22, wherein the polymer is a cationic polymer.

24. A method for producing a polymer of formula 1 according to claim 10, wherein the method (a) Formula 4: 【Transformation 34】 To provide polymers and (b) Polymer group A of formula 4 １ and / or A ２ Modify a part of it to form equation 1: 【Chemistry 35】 A method comprising providing a polymer (wherein formulas 1 and 4 of the formulas, m １ , m ２ , m ３ , m ４ , n １ , n ２ , R ３ａ , R ３ｂ , R １３ , X １ , X ２ A １ A ２ , B １ , and B ２ This is as defined in claim 10).

25. Polymer group A of formula 4 １ and / or A ２ Modifying a part of the base structure: 【Transformation 36】 The method of claim 21, comprising reacting a compound having the １ and A ２ These are each an independent expression -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ ２ ] ２ ; - (CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ] [- (CH ２ ) ｑ４ -NR ２ -]r ２ R ２ };or -(CH) ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ ２ ] ２ } ２ It is the basis of B １ and B ２ teeth, -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -(CH ２ ) ｓ１ -R ４ -R ５ ; -(CH ２ ) ｐ２ -N[-(CH ２ ) ｑ２ -NR ２ -(CH ２ ) ｓ２ -R ４ -R ５ ] ２ ;-(CH ２ ) ｐ３ -N{[-(CH ２ ) ｑ３ -NR ２ ２ ][-(CH ２ ) ｑ４ -NR ２ -] ｒ２ (CH ２ ) ｓ３ -R ４ -R ５ }; -(CH ２ ) ｐ４ -N{-(CH ２ ) ｑ５ -N[-(CH ２ ) ｑ６ -NR ２ -(CH ２ ) ｓ４ -R ４ -R ５ ] ２ } ２ ; -(CH ２ ) ｐ１ -[N{(CH ２ ) ｓ１ -R ４ -R ５ }-(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ ; -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -CH(CONH ２ )-(CH ２ ) ｓ１ -R ４ -R ５ (That is the case.)

26. A １ and A ２ Both: -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ ２ And; B １ and B ２ Both: -(CH ２ ) ｐ１ -[NR ２ -(CH ２ ) ｑ１ -] ｒ１ NR ２ -(CH ２ ) ｓ１ -R ４ -R ５ The method of claim 25.

27. Formula 4 according to claim 18: 【Chemistry 37】 A method for producing a polymer, wherein the method (I) Equation 2: 【Transformation 38】 The polymer of formula (a) HNR １３ A １ and / or HNR １３ A ２ Compounds of formula H ２ NX ２ Or HOX ２ Reacting with the compound simultaneously or in any order; or (II) Formula 3 【Chemistry 39】 The polymer of formula HNR １３ A １ and / or HNR １３ A ２ A method comprising reacting with a compound (in the formula, p １ is an integer from 1 to 2000; p ２ is an integer from 1 to 2000; Each R ３ These are independently a methylene group or an ethylene group; I understand １ , m ２ , n １ , n ２ , R ３ａ , R ３ｂ , R １３ , X １ , X ２ A １ , and A ２ This is as defined in claim 10).

28. Polymers containing the structure of formula 2 or formula 3 are, respectively, formula 2A or formula 3A: 【Chemistry 40】 (In the formula, p １ is an integer from 1 to 2000; p ２ is an integer from 1 to 2000; Each R ３ These are independently a methylene group or an ethylene group; Each X １ These are independently -C(O)O- and -C(O)NR １３ -, -C(O)-, -S(O)(O)-, or bond; X ２ Each of these cases independently contains, arbitrarily, one or more primary, secondary, or tertiary amines, C １ -C １２ Alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups, or combinations thereof; any of these may be substituted with one or more substituents; c is an integer between 0 and 50; Y is an arbitrarily existing and severable linker; R １ C is a hydrogen atom, an aryl group, a heterocyclic group, or a hydrogen atom substituted with one or more substituents. １ -C １２ Alkyl group, alkenyl group, cycloalkyl group, or cycloalkenyl group, or C １ -C １２ It is a linear or branched alkyl group; R ６ This includes hydrogen, an amino group, an aryl group, a heterocyclic group, and C, which are optionally substituted with one or more amines. １ -C １２ alkyl group, C １ -C １２ Heteroalkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, C １ -C １２ It is a linear or branched alkyl group; or it is a tissue-specific or cell-specific targeting moiety; p １ , p ２ , R ３ , X １ , and X ２ The method of claim 27, wherein the polymer is as defined in claim 27.

29. Polymers containing the structure of formula 2 or formula 3 are, respectively, formula 2B or formula 3B: 【Chemistry 41】 (In the formula, and p １ , p ２ , R ３ , X １ , and X ２ The method of claim 27, wherein the polymer is as defined in claim 27.

30. The compound in formula 2 is (a) HNR １３ A １ and / or HNR １３ A ２ The compound of (b) formula H ２ NX ２ or HOX ２ The method of claim 27, comprising combining with a compound, wherein (a) and (b) are present in a molar ratio of about 1:10 to about 1:150, optionally about 1:40 to about 1:150, or about 1:80 to about 1:

150.

31. A composition comprising a polymer according to any one of claims 1 to 22 and nucleic acids and / or polypeptides.

32. The composition according to claim 31, wherein the composition comprises a guide nucleic acid and / or a donor nucleic acid.

33. The composition according to claim 31 or 32, wherein the composition comprises an endonuclease.

34. The composition according to claim 33, wherein the composition comprises an RNA-induced endonuclease or a nucleic acid encoding it.

35. The RNA-induced endonuclease is Cas9, Cpf1, or a combination thereof. The composition according to claim 34.

36. A composition according to any one of claims 31 to 35, wherein the composition comprises a DNA recombinase.

37. The composition of claim 36, wherein the DNA recombinase is Cre recombinase.

38. A composition according to any one of claims 31 to 37, wherein the composition comprises a zinc finger nuclease.

39. A composition according to any one of claims 31 to 38, wherein the composition comprises a transcription activator-like effector nuclease.

40. The composition according to any one of claims 31 to 39, wherein the composition comprises a polymer of any one of claims 1 to 20 and nanoparticles comprising nucleic acids or polypeptides.

41. The composition according to any one of claims 31 to 40, wherein the composition comprises a second polymer containing polyethylene oxide.

42. A method for delivering nucleic acids and / or polypeptides to cells, the method comprising administering to the cells a composition according to any one of claims 31 to 41.

43. The method of claim 42, wherein cells are present in the subject, and the composition of any one of claims 30 to 40 is administered to the subject.

44. The method according to claim 42 or 43, wherein the polymer includes a tissue-specific targeting portion that localizes the polymer to the target tissue of the peripheral nervous system, central nervous system, liver, muscle, lung, bone, or eye.

45. The method of claim 44, wherein the polymer includes a targeting portion that preferentially binds to tumor cells.

46. A method according to any one of claims 42 to 45, wherein the composition comprises one or more RNA-inducible endonucleases or nucleic acids encoding them, guide nucleic acids, and donor nucleic acids, and the composition promotes the editing of a target gene in a cell.

47. A method according to any one of claims 42 to 46, wherein the cells are located in a host, optionally a human, and the composition is delivered to the cells by administering the composition to the host.

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