Cationic polymers and their uses in biomolecule delivery

Abstract

A polymer comprising the structure of formula (1) is provided, as well as a method for preparing said polymer. A composition comprising said polymer and nucleic acids and / or peptides is also provided, as well as a method for delivering nucleic acids and / or peptides to cells.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 663,985, filed April 27, 2018, and U.S. Provisional Patent Application No. 62 / 750,097, filed October 24, 2018, the entire disclosure of which is incorporated herein by reference.

[0003] By invoking materials submitted electronically.

[0004] The computer-readable nucleotide / amino acid sequence listing submitted and identified with respect to this application by reference in its entirety is a 62,527-byte ASCII (text) file named “512879.TXT” created on April 29, 2019. Background of the Invention

[0006] Peptide, protein, and nucleic acid-based technologies have been applied countless times for the prevention, cure, and treatment of diseases. However, the safe and efficient delivery of macromolecules (e.g., peptides and nucleic acids) to their target tissues remains a challenge. Therefore, new compositions and methods for delivering therapeutic molecules are still needed. Summary of the Invention

[0007] This article provides polymers containing the structure of Formula 1:

[0008]

[0009] in:

[0010] m 1 and n 1 Each is an integer between 0 and 1000;

[0011] m 2 and n 2 Each is an integer between 0 and 1000, and the condition is m. 2 +n 2 The sum is greater than 5;

[0012] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0013] R 3a and R 3b Each is independently a methylene or ethylene group;

[0014] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0015] A 1 and A2 Each is an independent group of the following formula

[0016] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0017] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0018] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0019] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0020] B 1 and B 2 Each independently

[0021] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ;

[0022] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2;

[0023] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R4 -R 5 };

[0024] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2;

[0025] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ;

[0026] -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ;

[0027] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ;

[0028] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2;

[0029] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ;

[0030] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2;

[0031] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5};

[0032] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2;

[0033] -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or

[0034] -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2,

[0035] Where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independent integers from 1 to 5; R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups; R 4 Each occurrence of is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 Each occurrence of the group independently comprises, optionally, 1 to 8 (e.g., 2 to 8) substituted or unsubstituted alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups or combinations thereof, of a secondary or tertiary amine, or a substituent comprising a tissue-specific or cell-specific targeting moiety.

[0036] Polymers of Formula 2 are also provided:

[0037]

[0038] in:

[0039] m 1 and n 1 Each is an integer between 0 and 1000, and the condition is m. 1 and n 1 The sum is between 10 and 2000;

[0040] c is an integer from 0 to 50;

[0041] Y is optionally present and is a cleavable linker;

[0042] R 1 It is hydrogen, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, or C1-C groups optionally substituted with one or more substituents (e.g., one or more amines). 12 Straight-chain or branched alkyl groups;

[0043] R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0044] R 8 It is a tissue-specific or cell-specific targeting component; and

[0045] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

[0046] This disclosure also provides compositions comprising a structure of Formula 1 and / or a polymer of Formula 2, as well as nucleic acids and / or peptides. Methods for preparing polymers comprising a structure of Formula 1 or a polymer of Formula 2 are also provided, as well as methods for delivering nucleic acids or proteins into cells, for example, using said polymers and compositions comprising said polymers.

[0047] Brief description of the attached figures

[0048] Figure 1 The amino acid sequence of Cas9 from Streptococcus pyogene is provided (SEQ ID NO: 1).

[0049] Figure 2 The amino acid sequence of Cpf1 from the subspecies of Francisella tularensis, Novicida U112, is provided (SEQ ID NO:2).

[0050] Figure 3This is a graph illustrating the Cas9 RNP delivery level as measured by the percentage of green fluorescent protein (“GFP”) negative in GFP-HEK cells at different doses of the polymer / Cas9 composition.

[0051] Figure 4 This is a schematic diagram of the alleles reported by ai9.

[0052] Figure 5 The levels of Cre recombinase delivery to primary myoblasts, measured by RFP+ expression, are shown at different concentrations of the polymer / Cre composition.

[0053] Figure 6 This illustrates the level of Cre recombinase delivery in mouse muscle visualized using red fluorescent protein (lighter areas indicate fluorescence).

[0054] Figure 7 The indel mutation rate of human neuronal stem cells demonstrated by Cas9 delivery is shown.

[0055] Figure 8 This is a schematic diagram of the Traffic Light Reporter in HEK 293T.

[0056] Figure 9 The levels of Cre recombinase delivery to TLR-HEK 293T, measured by RFP+ expression, are shown at different concentrations of the polymer / Cre composition.

[0057] Figure 10 This is a graph illustrating the Cas9 RNP delivery level as measured by the percentage of green fluorescent protein (“GFP”) in GFP-HEK cells at different doses of the polymer / Cas9 composition.

[0058] Figure 11 This indicates the level of eGFP mRNA delivered to HEK 293T cells, as measured by GFP%.

[0059] Figure 12 This shows the incubation time dependence of the level of eGFP mRNA delivered to HEK 293T cells, as measured by GFP%.

[0060] Figure 13 This study describes the level of red fluorescent protein (“RFP”) mRNA delivered to HEK 293T cells by RFP+ expression in the presence and absence of a 1.5 kDa PEG-PAsp (DET) polymer.

[0061] Figure 14This describes the red fluorescent protein (“RFP”) mRNA levels measured by RFP+ expression in the presence and absence of 1.5 kDa PEG-PAsp (DET) polymer.

[0062] Figure 15 The illustration shows the Cas9 RNP delivery level, measured by the percentage of green fluorescent protein (“GFP”) in GFP-HEK cells, for polymer / Cas9 nanoparticles in buffer.

[0063] Figure 16 Provide the sequence of AsCpf1 (SEQ ID NO: 19).

[0064] Figure 17 The sequence of LbCpf1 is provided (SEQ ID NO: 20).

[0065] Figure 18 This indicates the average particle size of the nanoparticles, measured by dynamic light scattering at 0, 3, 5, and 7 days after preparation (n=1). Detailed Implementation

[0066] This invention provides polymers comprising the structure of Formula 1:

[0067]

[0068] in:

[0069] m 1 and n 1 Each is an integer between 0 and 1000;

[0070] m 2 and n 2 Each is an integer between 0 and 1000, and the condition is m. 2 +n 2 The sum is greater than 5;

[0071] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0072] R 3a and R 3b Each is independently a methylene or ethylene group;

[0073] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0074] A 1 and A 2 Each is an independent group of the following formula

[0075] -(CH2)p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0076] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0077] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0078] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0079] B 1 and B 2 Each independently is:

[0080] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ;

[0081] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2;

[0082] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5};

[0083] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2;

[0084] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ;

[0085] -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ;

[0086] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ;

[0087] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2;

[0088] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ;

[0089] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2;

[0090] -(CH2) p3 -N{[-(CH2)q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5};

[0091] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2;

[0092] -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or

[0093] -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2,

[0094] in

[0095] p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independent integers from 1 to 5;

[0096] R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0097] R 4 Each occurrence of this is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-;

[0098] And R 5 Each occurrence of the group independently comprises, optionally, 1 to 8 or 2 to 8 substituted or unsubstituted alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, heteroalkyl groups, heterocyclic groups or combinations thereof, or substituents containing tissue-specific or cell-specific targeting moieties.

[0099] As used herein, "alkyl" refers to a substituted or unsubstituted hydrocarbon chain. Alkyl groups can have any number of carbon atoms (e.g., C1-C1). 100 Alkyl, C1-C 50 Alkyl, C1-C 12 Alkyl, C1-C8 alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C2 alkyl, etc.). The hydrocarbon chain may be saturated, or in some embodiments may be unsaturated to any degree (e.g., providing alkenyl or alkynyl groups), and may be linear, branched, straight-chain, cyclic (e.g., cycloalkyl or cycloalkenyl) or combinations thereof. Cyclic groups may be monocyclic, fused to form bicyclic or tricyclic groups, linked by bonds, or spirocyclic. Cyclic groups contain at least three carbons (e.g., C3-C4). 12 C3-C 10 (C3-C8, C3-C6, or C5-C6). In some embodiments, the hydrocarbon chain may be interrupted by one or more heteroatoms (e.g., 1, 2, 3, 4, or 5 or more oxygen, nitrogen, and / or sulfur atoms) to provide a heteroalkyl, heteroalken, heteroynyl, or heterocyclic group (e.g., a heterocyclic group). In some embodiments, the alkyl, alken, or ynyl group is substituted with one or more substituents.

[0100] The term "heterocyclic group" or "heterocyclic group" refers to a cyclic group, such as aromatic (e.g., heteroaryl) or non-aromatic, wherein the cyclic group has one or more heteroatoms (e.g., 1, 2, 3, 4, or 5 or more atoms of oxygen, nitrogen, and / or sulfur). In some embodiments, the heterocyclic group or heterocyclic group (i.e., the cyclic group, such as aromatic (e.g., heteroaryl) or non-aromatic, wherein the cyclic group has one or more heteroatoms) is substituted with one or more substituents.

[0101] The term "aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. An aryl group may contain any suitable number of ring atoms, such as 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, fused to form a bicyclic or tricyclic group, or linked by bonds to form a biaryl group. Representative aryl groups include phenyl, naphthyl, and biphenyl. In some embodiments, the aryl group contains an alkylene linker 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, an aryl substituent contains one or more heteroatoms (e.g., 1, 2, 3, 4, or 5 or more oxygen, nitrogen, and / or sulfur atoms), thereby providing a heteroaryl group. In some implementations, the aryl group is substituted by one or more substituents.

[0102] It should be understood that any of the aforementioned groups can be monovalent, divalent, or polyvalent (e.g., alkylene, alkenylene, ynylene, arylene, heteroalkylene, alkenylene, ynylene, heterocyclic, heteroaryl, etc.).

[0103] As used herein, the term "substituted" can refer to the substitution of one or more hydrogen atoms on a specified atom or group (e.g., a substituted alkyl group) by another group, provided that the substitution does not exceed the normal valence of the specified atom. For example, when the substituent is oxo (i.e., ═O), two hydrogen atoms on the atom are replaced. Substituents can include one or more of the following: aldehydes, esters, amides, ketones, nitro groups, cyano groups, fluoroalkyl groups (e.g., trifluoromethane), halogens (e.g., fluorine), aryl groups (e.g., phenyl), heterocyclic groups or heterocyclic groups (i.e., cyclic groups, such as aromatic (e.g., heteroaryl) or non-aromatic groups, wherein said cyclic group has one or more heteroatoms), oxo groups, or combinations thereof. Combinations of substituents and / or variables are permitted, provided that the substitution does not significantly adversely affect the synthesis or use of the compound.

[0104] According to Equation 1, m 1 and n 1 Each is an integer from 0 to 1000, such as 0-500, 0-200, 0-100, or 0-50. Furthermore, m 2 and n 2 Each is an integer between 0 and 1000, for example, 0-500 or 0-100, provided that m 2 +n 2 The sum is greater than 5 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, or 5-50). In other words, the polymer contains at least some of the groups comprising B. 1 and / or B 2 The monomer units are collectively referred to herein as "B monomers". In some implementations, m 1 and n 1 A value of 0 means that the polymer does not contain A. 1 Or A 2 Groups. In other embodiments, the polymer contains some A groups. 1 and / or A 2 Groups and some B 1 and / or B 2 Group. In such an implementation, (m 1 +n 1 ) / (m 2 +n 2 The ratio is about 20 or less (e.g., about 10 or less, about 5 or less, about 2 or less, or even about 1 or less). Furthermore, in some embodiments, (m...1 +n 1 ) / (m 2 +n 2 The ratio is about 0.2 or greater, for example, about 0.5 or greater.

[0105] The polymer can exist in any suitable structural type. For example, the polymer can exist as an alternating polymer, a random polymer, a block polymer, a graft polymer, a linear polymer, a branched polymer, a cyclic polymer, or a combination thereof. In some embodiments, the polymer is a random polymer, a block polymer, a graft polymer, or a combination thereof.

[0106] Therefore, in the structure of Equation 1, the monomers (which can be separated from their respective side chains A) 1 A 2 B 1 and B 2 (Mentions) can be arranged randomly or in any order. Integer m 1 n 1 m 2 and n 2 This only indicates the total number of corresponding monomers appearing in the chain, and does not necessarily represent the blocks of those monomers; however, in some embodiments, blocks or extensions of a given monomer may exist. For example, the structure of Equation 1 may contain the sequence -A 1 -A 2 -B 1 -B 2 -、-A 2 -A 1 -B 2 -B 1 -、-A 1 -B 1 -A 2 -B 2 - and other monomers. In some embodiments, the polymer has a polypeptide (e.g., polyasparagine) backbone arranged in an α / β configuration, such that "1" and "2" monomers alternate (e.g., -A) 1 -A 2 -B 1 -B 2 -、-A 2 -A 1 -B 2 -B 1 -、-A 1 -B 2 -B 1 -A 2 -、-A 2 -B 1 -B 2 -A 1 -、-A 1 -A2 -B 1 -A 2 -etc. However, "A" and "B" monomers or side chains (e.g., A 1 / A 2 and B 1 / B 2 They can be randomly dispersed throughout the polymer backbone, or they can be arranged in the form of blocks or some combination thereof.

[0107] The polymer can be further described in a manner consistent with Formula 1.1:

[0108]

[0109] Alternatively, when the skeleton is of α or β configuration, it can be described as Equation 1.2:

[0110]

[0111] in,

[0112] m 1 and n 1 As mentioned above;

[0113] m 3 Integers between 5 and 2000 (e.g., 5-1000, 5-500, 5-100, 25-2000, 25-500, 25-100, 50-2000, 50-1000, 50-500, or 50-100);

[0114] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0115] R 3a and R 3b Each is independently a methylene or ethylene group;

[0116] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0117] Each A 3 Independently, it is a group of the following formula

[0118] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0119] -(CH2) p2 -N[-(CH2) q2 -NR2 2]2;

[0120] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2 };

[0121] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2;

[0122] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ;

[0123] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2;

[0124] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 };

[0125] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2;

[0126] -(CH2) p1 -[NR 2 -(CH2) q1-] r1 NR 2 -CH2-CHOH-R 5 ;

[0127] -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ;

[0128] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ;

[0129] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2;

[0130] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ;

[0131] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2;

[0132] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5 };

[0133] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2-(CH2) s4 -R 5 ]2}2;

[0134] -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or

[0135] -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2,

[0136] Where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independent integers from 1 to 5;

[0137] R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0138] R 4 Each occurrence of this is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and

[0139] R 5 Each occurrence of the group is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group or combination thereof that optionally contains 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety;

[0140] The condition is that A is at least about 5% (e.g., at least about 10%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even 100%). 3 Groups are selected from

[0141] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R4 -R 5 ;

[0142] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2;

[0143] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 };

[0144] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2;

[0145] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ;

[0146] -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ;

[0147] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ;

[0148] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6-NR 2 -CH2-CHOH-R 5 ]2}2;

[0149] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ;

[0150] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2;

[0151] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5};

[0152] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2;

[0153] -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or

[0154] -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2.

[0155] In other embodiments, some or even most of the polymer A 3The groups (at least about 5%, at least about 10%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%) are selected from

[0156] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0157] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0158] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0159] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2.

[0160] In any of the above polymer structures, R 3a and R 3b Each is independently a methylene or ethylene group. In some embodiments, R 3a It is ethylene, while R 3b Methylene; or R 3a It is a methylene group, while R 3b It is an ethylene group. In some embodiments, R 3a and R 3b Each is an ethylene group. In a preferred embodiment, R 3a and R 3b Each of them is a methylene group.

[0161] Group A 1 and A 2 They are chosen independently, and therefore can be the same as or different from each other. Similarly, group B... 1 and B 2 They are chosen independently and can be the same as or different from each other. Furthermore, A3 Each appearance of [a] can be combined with other A's. 3 The functional groups may be the same or different.

[0162] In group A 1 A 2 A 3 B 1 and B 2 In this context, integers p1 to p4 (i.e., p1, p2, p3, and p4), q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6), and r1 and r2 are each 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), and / or r1 and r2 are each independently integers from 1 to 3 (e.g., 1, 2, or 3). 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. In some implementations, p1 to p4 (i.e., q1, p1, p2, p3, and p4) and / or q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6) are each 2, and r1 and r2 are each 1. In some implementations, s1-s4 are each independently an integer 1, 2, 3, 4, or 5, for example, 1, 2, or 3, or 1-2.

[0163] In the polymer structure, R 4 Each occurrence of R is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-. In some implementations, R 4 Each occurrence of R is independently -C(O)O- or -C(O)NH-. In some embodiments, R 4 Each occurrence of is -C(O)O-. In some implementations, R 4 Each occurrence of is -C(O)NH-.

[0164] In some implementations of Equation 1, A 1 and A 2 Each is a formula -(CH2) p1 -[NH-(CH2) q1 -] r1 A group of NH2, such as the group -(CH2)2-NH-(CH2)2-NH2. Alternatively or optionally, B 1 and B 2 Each is a formula -(CH2) p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R5 Groups, such as -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 Or the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 In some implementations, B 1 and B 2 Each is a formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 or -(CH2)2-NR 2 -(CH2)2-NR 2 -(CH2)2-R 5 The group, optionally wherein R 2 Each is independently a C1-C3 alkyl group (e.g., methyl), and optionally R 5 It is a C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 It is -CH2-NH-CH3 or -CH2-N-(CH3)2).

[0165] In some implementations of Equation 1.1, A 3 For example, -(CH2) p1 -[NH-(CH2) q1 -] r1 A group containing NH2, such as the group -(CH2)2-NH-(CH2)2-NH2; or the formula -(CH2). p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 Groups, such as -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 ;-(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 ; or the group -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 For example, -(CH2)2-NR 2-(CH2)2-NR 2 -(CH2)2-R 5 Optional of R 2 Each is independently a C1-C3 alkyl group (e.g., methyl), and optionally R 5 It is a C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 For -CH2-NH-CH3 or -CH2-N-(CH3)2); the condition is that at least about 5% (e.g., at least about 10%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even 100%) of A 3 The functional group is -(CH2). p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 For example, the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 Or the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 ; or the group -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 For example, -(CH2)2-NR 2 -(CH2)2-NR 2 -(CH2)2-R 5 Optional of R 2 Each is independently a C1-C3 alkyl group (e.g., methyl), and optionally R 5 It is a C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 It is -CH2-NH-CH3 or -CH2-N-(CH3)2). Additionally, in some embodiments, some or even most of the polymer's A... 3The radical (at least about 5%, at least about 10%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%) is -(CH2). p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 For example, the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 Or the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 ; or the group -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 For example, -(CH2)2-NR 2 -(CH2)2-NR 2 -(CH2)2-R 5 Optional of R 2 Each is independently a C1-C3 alkyl group (e.g., methyl), and optionally R 5 It is a C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 It is -CH2-NH-CH3 or -CH2-N-(CH3)2).

[0166] Furthermore, in the polymer structure, R 5 Each occurrence of the group R independently comprises, optionally, 1 to 8 (or 2 to 8) alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroalkyl, heterocyclic, or combinations thereof, of a secondary or tertiary amine. 5 Either of them can be substituted or unsubstituted. R 5 It can also be a substituent containing tissue-specific or cell-specific targeting portions. In some embodiments, R 5It may contain about 1 to about 50 carbon atoms (e.g., about 2 to about 50 carbon atoms, such as about 2 to about 40 carbon atoms, about 2 to about 30 carbon atoms, about 2 to about 20 carbon atoms, about 1 to about 16 or about 2 to about 16 carbon atoms, about 1 to about 12 or about 2 to about 12 carbon atoms, about 1 to about 10 or about 2 to about 10 carbon atoms, or about 1 to about 8 or about 2 to about 8 carbon atoms). In some embodiments, R 5 It is a heteroalkyl group comprising 1 to 8 or 2 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7, or 8) secondary or tertiary amines. The secondary or tertiary amine can be a portion of the heteroalkyl skeleton (i.e., the longest continuous atomic chain in the heteroalkyl group) or a pendant substituent. Therefore, for example, the heteroalkyl group comprising a secondary or tertiary amine can be an alkylamino group, a dialkylamino group, an aminoalkyl group, an alkylaminoalkyl group, a dialkylaminoalkyl group, an aminoalkylamino group, etc., comprising 1 to 8 (or 2 to 8) secondary or tertiary amines.

[0167] In some implementations, each R 5 Selected independently from:

[0168]

[0169]

[0170] in

[0171] R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0172] R 7 C1-C can be optionally substituted with one or more amines. 50 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0173] z is an integer from 1 to 5;

[0174] c is an integer from 0 to 50;

[0175] Y is optionally present and is a cleavable linker;

[0176] n is an integer from 0 to 50; and

[0177] R 8 It is a tissue-specific or cell-specific targeting region, C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

[0178] R 2Each occurrence can be either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. In some embodiments, R 2 For C1-C 12 (For example, C1-C) 10 Alkyl; C1-C8 alkyl group; C1-C6 alkyl group; C1-C4 alkyl group, C1-C3 alkyl group, or C1 or C2 alkyl group) straight-chain or branched alkyl group. In some embodiments, R 2 It can be methyl or hydrogen.

[0179] R 7 It can be a C1-C 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 groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. In some embodiments, R 7 C4-C substituted with one or more amines 12 (e.g., C6-C8) alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. In some embodiments, R 7 It is replaced by one or more amines. In some embodiments, R 7 It is substituted with 1 to 8 or 2 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7 or 8) secondary or tertiary amines. The amine may be part of the alkyl group (i.e., contained in the alkyl group skeleton) or a side substituent.

[0180] R 13 It can be hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. In some embodiments, R 13 For C1-C 12 (For example, C1-C) 10 Alkyl; C1-C8 alkyl group; C1-C6 alkyl group; C1-C4 alkyl group, C1-C3 alkyl group, or C1 or C2 alkyl group) straight-chain or branched alkyl group. In some embodiments, R 13 It is methyl. In other embodiments, R 13 It can be hydrogen. Typically, within a given polymer, each R... 13 They are the same (e.g., all methyl or all hydrogen); however, each R 13 They are chosen independently and can be the same or different.

[0181] Y is optionally present in each occurrence. As used herein, the phrase "optionally present" means that a substituent, referred to as optionally present, may or may not be present, and when the substituent is absent, adjacent substituents are directly bonded to each other. When Y is present, Y is a cleavable linker. As used herein, the phrase "cleavable linker" refers to any chemical element that links two substances (species) and that element can be cleaved to separate the two substances. For example, a cleavable linker can be cleaved by hydrolysis, photochemical processes, radical processes, enzymatic processes, electrochemical processes, or combinations thereof. Exemplary cleavable linkers include, but are not limited to:

[0182]

[0183] Where R 14 Each occurrence of this is independently a C1-C4 alkyl group, R 15 Each occurrence of is independently hydrogen, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, and R 16 It is a six-membered aromatic or heteroaromatic group, which is optionally substituted by one or more -OCH3, -NHCH3, -N(CH3)2, -SCH3, -OH or combinations thereof.

[0184] The polymer can be any suitable polymer, provided that it comprises the aforementioned polymer structure as part of the polymer. In some embodiments, the polymer is a block copolymer comprising polymer blocks having a structure of formula 1, 1.1, or 1.2 and one or more other polymer blocks (e.g., ethylene oxide secondary units or propylene oxide secondary units). In other embodiments, the structure of formula 1, 1.1, or 1.2 is the sole polymeric unit, which may be "end-capped" at either end with a suitable end. In some embodiments, the polymer also comprises substituents that contain tissue-specific or cell-specific targeting portions.

[0185] In some embodiments, the polymer has the structure of Formula 1A:

[0186]

[0187] Where Q has the following formula:

[0188]

[0189] Or it may have a structure of formula 1A1:

[0190]

[0191] in

[0192] c is an integer from 0 to 50;

[0193] Y is optionally present and is a cleavable linker as previously described;

[0194] R 1 It is hydrogen, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, or C1-C groups optionally substituted with one or more substituents (e.g., one or more amines). 12 Straight-chain or branched alkyl groups;

[0195] R 6 It is hydrogen, amino group, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, optionally substituted with one or more amines, C1-C 12 Straight-chain or branched alkyl groups; or tissue-specific or cell-specific targeting regions;

[0196] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0197] And all other substituents (e.g., m) 1 n 1 m 2 n 2 、R 2 、R 3a 、R 3b 、R 4 、R 5 and R 13 As mentioned above.

[0198] Similarly, the polymer may have the structure of Formula 1.1A:

[0199]

[0200] Alternatively, when the skeleton is of α or β configuration, as shown in Equation 1.2A:

[0201]

[0202] in,

[0203] c is an integer from 0 to 50;

[0204] Y is optionally present and is a cleavable linker as previously described;

[0205] R 1 It is hydrogen, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, or C1-C groups optionally substituted with one or more substituents (e.g., one or more amines). 12 Straight-chain or branched alkyl groups;

[0206] R 6 It is hydrogen, amino group, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, optionally substituted with one or more amines, C1-C 12 Straight-chain or branched alkyl groups; or tissue-specific or cell-specific targeting regions;

[0207] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0208] And all other substituents (e.g., m) 3 、R 3a 、R 3b and A 3 As mentioned above.

[0209] In some of the aforementioned implementation schemes, R 1 and / or R 6 C1-C substituted with one or more substituents 12 (For example, C1-C) 10 Alkyl groups; C1-C8 alkyl groups; C1-C6 alkyl groups; C1-C4 alkyl groups; C1-C3 alkyl groups, or C1 or C2 alkyl groups) straight-chain or branched alkyl groups. In some embodiments, the heteroalkyl or alkyl group comprises or is substituted with one or more amines, such as 1 to 8 or 2 to 8 (i.e., 1, 2, 3, 4, 5, 6, 7 or 8) secondary or tertiary amines. The amine may be a portion of the heteroalkyl backbone chain or a side substituent. Non-limiting examples of polymers provided herein include, for example:

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223] Where a, b, c, and d are each independently an integer from 0 to 1000, such as 0-500, 0-200, 0-100, or 0-50, provided that (a+b+c+d) > approximately 5. In some embodiments, (a+b) is approximately 0 to approximately 75 (e.g., approximately 5 to approximately 75, approximately 20 to approximately 75, approximately 40 to approximately 75, approximately 40 to approximately 60, approximately 50 to approximately 75, approximately 60 to approximately 75, or approximately 70 to approximately 75), and (c+d) is approximately 5 to approximately 80 (e.g., approximately 5 to approximately 75, approximately 5 to approximately 60, approximately 5 to approximately 40, approximately 20 to approximately 40, approximately 5 to approximately 30, approximately 5 to approximately 20, or approximately 5 to approximately 10). In some embodiments, (a+b) is greater than (c+d). For example, in some embodiments, the ratio of (a+b) / (c+d) is about 1 to about 3 (e.g., (a+b) is about 55 and (c+d) is about 25), or the ratio of (a+b) / (c+d) is about 3 to about 10 (e.g., (a+b) is about 70 and (c+d) is about 10). In other embodiments, (a+b) is less than (c+d) such that the ratio of (a+b) / (c+d) is less than 1 (e.g., about 0.1 or greater, but less than 1). Similarly, the indication of the number of units (“a”, “b”, “c”, and “d”) in these exemplary polymers does not imply a block copolymer structure; rather, these numbers represent the total number of units, which may be randomly arranged as indicated by the “ / ” symbol in the formula.

[0224] The present invention also provides polymers comprising the structure of Formula 2:

[0225]

[0226] in:

[0227] a is an integer between 10 and 2000;

[0228] c is an integer from 0 to 50;

[0229] Y is optionally present and is a cleavable linker as previously described herein;

[0230] R 1 、R 2 and R 13 And all other substituent groups as previously described herein with respect to other polymer formulas (e.g., Formula 1, Formula 1A, Formula 1A1, Formula 1.1A1 and Formula 1.2A1);

[0231] And R 8 It is a tissue-specific or cell-specific targeting component.

[0232] This invention also provides polymers comprising the structure of Formula 4:

[0233]

[0234] in,

[0235] m 1 and n 1 Each is an integer between 0 and 1000; the condition is m. 1 +n 1 The sum is greater than 2;

[0236] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0237] A 1 and A 2 Each is an independent group of the following formula

[0238] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0239] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0240] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0241] -(CH2) p4 -N{-(CH2)q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0242] Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5;

[0243] R 2 Each occurrence of this substance is independently hydrogen, C1-C 12 Alkyl groups, C1-C 12 alkenyl groups, C3-C 12 Cycloalkyl groups, or C3-C 12 Cycloalkenyl groups, condition A 1 and A 2 Contains at least one (e.g., at least two, at least three, at least four, or even at least five tertiary amines (e.g., R) 2 At least some of the occurrences are C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups or cycloalkenyl groups);

[0244] R 3a and R 3b Each is independently a methylene or ethylene group; and

[0245] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

[0246] This invention also provides polymers having the structure of Formula 4A:

[0247]

[0248] in,

[0249] c is an integer from 0 to 50;

[0250] Y is optionally present and is a cleavable linker;

[0251] m 1 and n 1 Each is an integer between 0 and 1000; the condition is m. 1 +n 1 The sum is greater than 2;

[0252] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0253] A 1 and A 2 Each is an independent group of the following formula

[0254] -(CH2)p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0255] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0256] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0257] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0258] Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5;

[0259] R 1 It is hydrogen, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, or C1-C groups optionally substituted with one or more substituents. 12 Straight-chain or branched alkyl groups;

[0260] R 2 Each occurrence of this substance is independently hydrogen, C1-C 12 Alkyl groups, C1-C 12 alkenyl groups, C3-C 12 Cycloalkyl groups, or C3-C 12 Cycloalkenyl groups, condition A 1 and A 2 Contains at least one (e.g., at least two, at least three, at least four, or even at least five tertiary amines (e.g., R) 2 At least some of the occurrences are C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups or cycloalkenyl groups);

[0261] R 3a and R 3a Each is independently a methylene or ethylene group;

[0262] R 6 It is hydrogen, amino group, aryl group, heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, optionally substituted with one or more amines, C1-C 12 Straight-chain or branched alkyl groups; or tissue-specific or cell-specific targeting fractions; and

[0263] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

[0264] In embodiments of polymers comprising the structure of Formula 4 and polymers having the structure of Formula 4A, R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, C1-C 12 alkenyl groups, C3-C 12 Cycloalkyl groups or C3-C 12 Cycloalkenyl groups, condition A 1 and A 2 Each contains at least one tertiary amine (e.g., at least two, at least three, at least four, or even at least five tertiary amines). In some embodiments, R 2 Each occurrence of is independently C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. Therefore, in embodiments of polymers comprising formula 4 and polymers having formula 4A, each containing R... 2 The nitrogen in the substituent is a tertiary amine, except that the terminal amine can be a primary, secondary, or tertiary amine, preferably a secondary or tertiary amine. In some embodiments of polymers comprising the formula 4 structure and polymers having the formula 4A structure, A 1 and A 2 Each is a separate formula -(CH2)2-NR 2 -(CH2)2-NR 2 2 groups, wherein R 2 Each occurrence of is independently C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, except that the terminal amine can be a primary, secondary, or tertiary amine, preferably a secondary or tertiary amine. In certain embodiments of polymers comprising the structure of Formula 4 and polymers having the structure of Formula 4A, R 2 Each occurrence of is ethyl or methyl, except that the terminal amine may optionally be a primary, secondary, or tertiary amine, preferably a secondary or tertiary amine.

[0265] In embodiments of polymers comprising the structure of Formula 4 and polymers having the structure of Formula 4A, R13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. Typically, R... 13 Each occurrence of R is independently either a hydrogen or a methyl substituent. In some embodiments, R 13 Each occurrence of is hydrogen.

[0266] As stated above, group A 1 and A 2 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(CH 3)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).

[0267] Therefore, non-limiting examples of polymers provided herein include, for example:

[0268]

[0269]

[0270]

[0271]

[0272] Where a and b are each independently integers from 0 to 1000, such as 0-500, 0-200, 0-100, or 0-50. Therefore, in some embodiments, a or b can be 0, or the polymer can have a certain ratio of a to b. In some embodiments, (a+b) is about 5 or greater (e.g., about 5 to about 160, about 5 to about 140, about 5 to about 120, about 5 to about 100, about 5 to about 80, or about 5 to about 60) or about 25 or greater (e.g., about 25 to about 160, about 25 to about 140, about 25 to about 120, about 25 to about 100, about 25 to about 80, or about 25 to about 60) or even about 50 or greater (e.g., about 50 to about 160, about 50 to about 140, about 50 to about 120, about 50 to about 100, or about 50 to about 80). Other examples include polymers 30-33, in which the terminal tertiary amine of one or both side chains is demethylated to provide a secondary amine (e.g., -NHCH3):

[0273]

[0274]

[0275] Where a and b are as described above.

[0276] Any of the aforementioned polymers may contain a tissue-specific or cell-specific targeting portion at the location indicated in the formula, or the polymer may be further modified to contain a tissue-specific or cell-specific targeting portion. Methods for attaching the tissue-specific or cell-specific targeting portion are known in the art, some of which include Michael addition, epoxide ring-opening, displacement reactions, or “click” chemistry (e.g., reactions of CuAAC, SPAAC, SPANC, or strained olefins) using a tissue-specific or cell-specific targeting portion having appropriate functional groups. The tissue-specific or cell-specific targeting portion may be any small molecule, protein (e.g., antibody or antigen), amino acid sequence, sugar, oligonucleotide, metal-based nanoparticle, or combination thereof capable of recognizing (e.g., specifically binding) a given target tissue or cell (e.g., specifically binding to a specific ligand, receptor, or other protein or molecule that allows the targeting portion to distinguish the target tissue or cell from other non-target tissues or cells). In some embodiments, the tissue-specific or cell-specific targeting portion is a receptor for a ligand. In some embodiments, the tissue-specific or cell-specific targeting portion is a ligand for a receptor.

[0277] The tissue-specific or cell-specific targeting portion can be used to target any desired tissue or cell type. In some embodiments, the tissue-specific or cell-specific targeting portion localizes the polymer to tissues of an individual's peripheral nervous system, central nervous system, liver, muscle (e.g., cardiac muscle), lung, bone (e.g., hematopoietic cells), or eye. In some embodiments, the tissue-specific or cell-specific targeting portion localizes the polymer to tumor cells. For example, the tissue-specific or cell-specific targeting portion may be a sugar that binds to receptors on a specific tissue or cell.

[0278] In some implementations, the tissue-specific or cell-specific targeting portion is:

[0279]

[0280] Where R 9 、R 10 、R 11 and R 12 Each component is independently hydrogen, halogen, or a C1-C4 alkyl or C1-C4 alkoxy group optionally 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.

[0281] Typically, the polymer is cationic (i.e., positively charged at pH 7 and 23°C). As used herein, a "cationic" polymer means a polymer having a total net positive charge, regardless of whether the polymer contains only cationic monomer units or a combination of cationic monomer units and nonionic or anionic monomer units.

[0282] In some embodiments, the polymer has a weight-average molecular weight of about 5 kDa to about 2000 kDa, for example, about 10 kDa to about 2000 kDa. The weight-average molecular weight of the polymer can be about 2000 kDa or less, for example, about 1800 kDa or less, about 1600 kDa or less, about 1400 kDa or less, about 1200 kDa or less, about 1000 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, or even about 40 kDa or less, for example, about 30 kDa or less or 25 kDa or less. Alternatively, or additionally, the weight-average molecular weight of the polymer may be about 5 kDa or higher, or about 10 kDa or higher, for example, about 50 kDa or higher, about 100 kDa or higher, about 200 kDa or higher, about 300 kDa or higher, or about 400 kDa or higher. Therefore, the polymer may have a weight-average molecular weight defined by any two of the foregoing endpoints. For example, the weight-average molecular weight of the polymer is about 5 kDa to about 50 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 500 kDa, about 50 kDa to about 500 kDa, about 100 kDa to about 500 kDa, about 200 kDa to about 500 kDa, about 300 kDa to about 500 kDa, about 400 kDa to about 500 kDa, about 400 kDa to about 600 kDa, about 400 kDa to about 700 kDa, about... 400kDa to approximately 800kDa, approximately 400kDa to approximately 900kDa, approximately 400kDa to approximately 1000kDa, approximately 400kDa to approximately 1200kDa, approximately 400kDa to approximately 1400kDa, approximately 400kDa to approximately 1600kDa, approximately 400kDa to approximately 1800kDa, approximately 400kDa to approximately 2000kDa, approximately 200kDa to approximately 2000kDa, approximately 500kDa to approximately 2000kDa, or approximately 800kDa to approximately 2000kDa.

[0283] Weight-average molecular weight can be determined using any suitable technique. Typically, it is determined using column-packed size exclusion chromatography with columns selected from TSKgel Guard, GMPW, GMPW, G1000PW, and Waters 2414 (Waters Corporation, Milford, Massachusetts) refractive index detectors. Furthermore, it is determined using polyethylene oxide / polyethylene glycol standards ranging from 150 to 875,000 Daltons for calibration.

[0284] Preparation method

[0285] The polymers provided herein can be provided by any suitable method. In some embodiments, the polymer can be provided by including groups A of polymers comprising a structure containing formula 3. 1 and / or A 2 Prepared by at least a part of the method:

[0286]

[0287] in:

[0288] m 1 and n 1 Each is an integer between 0 and 1000, and the condition is m. 1 +n 1 Greater than 5;

[0289] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0290] R 3a and R 3b Each is independently a methylene or ethylene group;

[0291] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0292] A 1 and A 2 Each is an independent group of the following formula

[0293] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0294] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0295] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0296] -(CH2) p4 -N{-(CH2) q5-N[-(CH2) q6 -NR 2 2]2}2,

[0297] Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5; and R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups

[0298] To produce polymers containing the structure of Formula 1:

[0299]

[0300] Where m 1 and n 1 Integers from 0 to 1000;

[0301] m 2 and n 2 Each is an integer between 0 and 1000, and the condition is m. 2 +n 2 The sum is greater than 5;

[0302] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0303] R 3a and R 3b Each is independently a methylene or ethylene group;

[0304] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0305] A 1 and A 2 Each is an independent group of the following formula

[0306] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0307] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0308] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2)q4 -NR 2 -] r2 R 2};or

[0309] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0310] B 1 and B 2 Each independently is:

[0311] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ;

[0312] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2;

[0313] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5};

[0314] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2;

[0315] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R5 ;

[0316] -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ;

[0317] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ;

[0318] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2;

[0319] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ;

[0320] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2;

[0321] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5 };

[0322] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2;

[0323] -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or

[0324] -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2,

[0325] Where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independent integers from 1 to 5; R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups; R 4 Each occurrence of is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 Each occurrence of the group is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group or combination thereof that optionally contains 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

[0326] The polymer containing the structure of Formula 1 prepared by the method can be any polymer of Formula 1, including Formula 1.1, 1.2, 1A1, 1A, 1.1A, 1.2A, 4 or 4A, and any and all embodiments thereof as described with respect to the polymers of the present invention.

[0327] The polymer comprising the structure of Formula 3 can be any suitable polymer that satisfies this criterion. In some embodiments, the polymer may instead be described as Formula 3.1:

[0328]

[0329] Alternatively, when the skeleton is of α or β configuration, it can be described as Equation 3.2:

[0330]

[0331] in,

[0332] m 3Integers between 5 and 2000 (e.g., 5-1000, 5-500, 5-100, 25-2000, 25-500, 25-100, 50-2000, 50-1000, 50-500, or 50-100);

[0333] The symbol " / " indicates that the units separated by it are connected randomly or in any order;

[0334] R 3a and R 3b Each is independently a methylene or ethylene group;

[0335] R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0336] Each A 3 Independently, it is a group of the following formula

[0337] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0338] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0339] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0340] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0341] Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5; and R 2 Each occurrence of is independently hydrogen or 11C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. In this case, the method may include modification A. 3At least a portion of the group is sufficient to provide a polymer having a structure of formula 1.1 or 1.2 as described herein. Examples of polymers containing one of the following structures are pAsp(DET) (poly(2-[(2-aminoethyl)amino]ethyl)asparagine) or PEG11-pAsp(DET) (polyethylene glycol-b-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]asparagine}).

[0342] The specified name A can be modified in any suitable way. 1 and / or A 2 The group (or the group A of the polymer of formula 3.1 or 3.2) 3 To produce what is designated as B 1 and / or B 2 The group (or the group A of the polymer of formula 1.1 or 1.2) 3 For example, designated as A 1 and / or A 2 The group (or the group A of the polymer of formula 3.1 or 3.2) 3 It can be modified by Michael addition reaction, epoxide ring-opening or substitution reaction. In a preferred embodiment, it is designated as A. 1 and / or A 2 The group (or the group A of the polymer of formula 3.1 or 3.2) 3 It is modified by Michael addition reaction.

[0343] In one embodiment, the polymer comprising the structure of formula 3 has group A. 1 and / or A 2 The group A of the polymer of formula 3.1 or 3.2 3 Modification is achieved via a Michael addition reaction between a polymer comprising Formula 3 and an α,β-unsaturated carbonyl compound. As used herein, the term "Michael addition" refers to the nucleophilic addition of a nucleophile (e.g., a carbanion, oxoanion, nitroanion, oxygen atom, nitrogen atom, or a combination thereof) of the polymer to an α,β-unsaturated carbonyl compound. Therefore, the Michael addition reaction occurs between a polymer comprising Formula 3 and an α,β-unsaturated carbonyl compound. In some embodiments, the nucleophile of the polymer is a nitroanion, a nitrogen atom, or a combination thereof.

[0344] The α,β-unsaturated carbonyl compound can be any α,β-unsaturated carbonyl compound capable of accepting Michael addition from a nucleophile. In some embodiments, the α,β-unsaturated carbonyl compound is an acrylate, acrylamide, vinyl sulfone, or a combination thereof. Therefore, the Michael addition reaction can be carried out between a polymer comprising formula 3 and an acrylate, acrylamide, vinyl sulfone, or a combination thereof. Therefore, in some embodiments, the method includes contacting the polymer comprising formula 3 with an acrylate; contacting the polymer comprising formula 3 with an acrylamide; or contacting the polymer comprising formula 3 with a vinyl sulfone.

[0345] Designated as A in it 1 and / or A 2 The group (or the group A of the polymer of formula 3.1 or 3.2) 3 In embodiments modified via Michael addition reactions, they produce as described herein with respect to formulas 1, 1.1, 1.2, 1A, and 1A. 1 The name specified as B, etc. 1 and / or B 2 The group (or the group A of the polymer of formula 1.1 or formula 1.2) 3 For example, B 1 and / or B 2 It can have the following formula:

[0346] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ;

[0347] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2;

[0348] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5};

[0349] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2,

[0350] Where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independent integers from 1 to 5; R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups; R 4 Each occurrence of is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 Each occurrence of the group is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group or combination thereof that optionally comprises 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent comprising a tissue-specific or cell-specific targeting moiety; or the group and substituent may be as otherwise described herein with respect to the polymers of the present invention.

[0351] Examples of suitable acrylates, acrylamides, and vinyl sulfones include acrylates of the following formula:

[0352]

[0353] Where R 5 As described with respect to any of Equations 1, 1.1, 1.2, 1A, 1.1A, or 1.2A.

[0354] In some embodiments, the Michael addition reaction is promoted by an acid and / or a base. The acid and / or base can be any suitable acid and / or base having any suitable pKa. The acid and / or base can be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0355] In some embodiments, an acid is used to promote the Michael addition reaction. The acid can be a Brønsted acid or a Lewis acid. In embodiments where the acid is a Brønsted acid, the acid can 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 some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfonyl)imide or p-toluenesulfonic acid.

[0356] In some embodiments, the Michael addition reaction is promoted 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 can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethylpiperazine or derivatives thereof.

[0357] In some embodiments, the Michael addition reaction is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents capable of solubilizing the polymer to be reacted and the α,β-unsaturated carbonyl compound. For example, the solvent may include water, protic organic solvents, and / or aprotic organic solvents. Exemplary lists of solvents include water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0358] In one embodiment, the polymer's group A 1 and / or A 2 (or the polymer of formula 3.1 or 3.2, group A) 3 The modification is achieved through an epoxide ring-opening reaction between the polymer and the epoxide compound. As used herein, "epoxide ring-opening" refers to the nucleophilic addition of a nucleophile (e.g., a carbide ion, oxyanion, nitrogen anion, oxygen atom, nitrogen atom, or a combination thereof) of the polymer to the epoxide compound, thereby causing the epoxide to ring-open. Therefore, the epoxide ring-opening reaction occurs between the polymer and the epoxide compound. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0359] In this process, modification via an epoxide ring-opening reaction is designated as A. 1 and / or A 2 The group (or the group A of the polymer of formula 3.1 or 3.2) 3 In the implementation scheme of ), they generate what is designated as B 1 and / or B 2 The group (or the group A of the polymer of formula 1.1 or 1.2) 3 It has the following formula:

[0360] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ;

[0361] -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5;

[0362] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ;

[0363] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2,

[0364] Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5; R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups; and R 5 Each occurrence of the group is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group or combination thereof that optionally contains 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

[0365] Examples of suitable epoxides include epoxides with the following formula:

[0366]

[0367] Where R 5 As described with respect to any polymer of the present invention (e.g., Formula 1, 1.1, 1.2, 1A, 1.1A or Formula 1.2A).

[0368] In some embodiments, the ring-opening reaction of the epoxide is promoted by an acid and / or a base. The acid and / or base can be any suitable acid and / or base having any suitable pKa. The acid and / or base can be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0369] In some embodiments, the ring-opening reaction of the epoxide is promoted by an acid. The acid can be a Brønsted acid or a Lewis acid. In embodiments where the acid is a Brønsted acid, the acid can 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 some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfonyl)imide or p-toluenesulfonic acid.

[0370] In some embodiments, the ring-opening reaction of the epoxide is promoted 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 can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethylpiperazine or derivatives thereof.

[0371] In some embodiments, the ring-opening reaction of the epoxide is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents capable of solubilizing the polymer and the epoxide 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.

[0372] In one embodiment, group A of the polymer is modified by a substitution reaction between the polymer and a compound containing a leaving group (e.g., chlorine atom, bromine atom, iodine atom, toluenesulfonate, trifluoromethanesulfonate, methanesulfonate, etc.). 1 and / or A 2 (or the polymer of formula 3.1 or 3.2, group A) 3 As used herein, the term "substitution" refers to the nucleophilic addition of a nucleophile (e.g., a carbide ion, oxoanion, nitroanion, oxygen atom, nitrogen atom, or a combination thereof) of the polymer to a compound containing a leaving group. Therefore, the substitution reaction occurs between the polymer and the compound containing the leaving group. In some embodiments, the nucleophile of the polymer is a nitroanion, nitrogen atom, or a combination thereof.

[0373] In which it is modified by substitution reaction and designated as A 1 and / or A 2 The group (or the polymer of formula 3.1 or 3.2) of A 3 In embodiments of the group), they produce what is designated as B 1 and / or B 2 The group (or the polymer of formula 1.1 or 1.2) of A 3 (group), which has the following formula:

[0374] -(CH2) p1 -[NR2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ;

[0375] -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2;

[0376] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5};or

[0377] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2,

[0378] Where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independent integers from 1 to 5; R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups; and R 5 Each occurrence of the group is independently an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group or combination thereof that optionally contains 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

[0379] In some embodiments, the method includes preparing a polymer comprising a structure of Formula 5 from a compound of Formula A:

[0380]

[0381] in,

[0382] R 3b It is a methylene or ethylene group;

[0383] W is -NR 14- or -O-, where R 14 It is hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups;

[0384] Z is A 1 Or optionally comprising 1 to 8 or 2 to 8 alkyl groups, cycloalkyl groups, alkenyl groups, cycloalkenyl groups, aryl groups, arylalkyl groups, heteroalkyl groups, heterocyclic groups or combinations thereof of secondary or tertiary amines, or comprising substituents of tissue-specific or cell-specific targeting moieties;

[0385] A 1 It is a group of the following formula

[0386] -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2;

[0387] -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2;

[0388] -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 R 2};or

[0389] -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2,

[0390] Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5; and R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

[0391] Typically, methods involving the formation of polymers comprising a structure of Formula 5 from compounds of Formula A include the ring-opening polymerization of said compound A. The ring-opening polymerization of said compound A can be initiated by any suitable method (e.g., temperature, light, catalyst, compound, etc.). In some embodiments, the ring-opening polymerization of said compound A is initiated using an amine-containing compound.

[0392] In some embodiments, the ring-opening polymerization of compound A is initiated using an amine-containing compound of the following formula:

[0393]

[0394] in,

[0395] c is an integer from 0 to 50;

[0396] Y is optionally present and is a cleavable linker; and

[0397] R 6 It is hydrogen, amino group, aryl group, heterocyclic group, C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, optionally substituted with one or more amines, C1-C 12 Straight-chain or branched alkyl groups; or tissue-specific or cell-specific targeting moieties. In a preferred embodiment, the amine-containing compound is

[0398]

[0399] In some embodiments, in the compound of formula A, Y is -O-, and Z is an alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, arylalkyl group, heteroalkyl group, heterocyclic group, or combination thereof optionally comprising 1 to 8 or 2 to 8 secondary or tertiary amines. In some embodiments, Z is an arylalkyl group, such as benzyl. The method for preparing the polymer of formula 5 further includes, after ring-opening polymerization, reacting the resulting intermediate polymer with formula NHR 17 -A l The treatment of compounds, in which A l As defined with respect to Equation 1. The equation NHR... 17 -A l The compound is optionally diethyleneamine triamine. In some embodiments, the formula NHR is... 17 -A l The compound is not diethylenetriamine.

[0400] R 17 It can be hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups. In some embodiments, R 17 For C1-C 12 (For example, C1-C) 10Alkyl groups; C1-C8 alkyl groups; C1-C6 alkyl groups; C1-C4 alkyl groups, C1-C3 alkyl groups, or C1 or C2 alkyl groups) straight-chain or branched alkyl groups. In some embodiments, R 17 It is methyl. In other embodiments, R 17 It can be hydrogen. Typically, within a given polymer, each R... 17 They are the same (e.g., all methyl or all hydrogen); however, each R 17 They are chosen independently and can be the same or different.

[0401] Examples of applicable compounds containing leaving groups include compounds of the following formula:

[0402]

[0403] Where LG is a leaving group (e.g., chlorine atom, bromine atom, iodine atom, toluenesulfonate, trifluoromethanesulfonate, methanesulfonate, etc.), and R 5 As described with respect to any of Equations 1, 1.1, 1.2, 1A, or 1.2A.

[0404] In some embodiments, the displacement reaction is facilitated by an acid and / or a base. The acid and / or base can be any suitable acid and / or base having any suitable pKa. The acid and / or base can be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or a combination thereof.

[0405] In some embodiments, the acid promotes the displacement reaction. The acid can be a Brønsted acid or a Lewis acid. In embodiments where the acid is a Brønsted acid, the acid can 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 some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfonyl)imide or p-toluenesulfonic acid.

[0406] In some embodiments, the displacement reaction is promoted 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 can be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethylpiperazine or derivatives thereof.

[0407] In some embodiments, the displacement reaction is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents capable of solubilizing 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. Exemplary lists of solvents include water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0408] In some embodiments, the method further includes separating the polymer containing the structure of Formula 1. The polymer containing the structure of Formula 1 can be separated by any suitable method. For example, the polymer containing the structure of Formula 1 can be separated by extraction, crystallization, recrystallization, column chromatography, filtration, or any combination thereof.

[0409] Composition

[0410] The polymers provided herein can be used for any purpose. However, these polymers are considered particularly useful for delivering nucleic acids and / or peptides (e.g., proteins) into cells. Therefore, compositions comprising the polymers described herein and nucleic acids and / or peptides (e.g., proteins) are provided herein.

[0411] In some embodiments, the composition comprises nucleic acids. Any nucleic acid can be used. Exemplary lists of nucleic acids include guide nucleic acids and / or donor nucleic acids for CRISPR systems, siRNA, microRNA, interfering RNA or RNAi, dsRNA, mRNA, DNA vectors, ribozymes, antisense polynucleotides, and DNA expression cassettes encoding siRNA, microRNA, dsRNA, ribozymes, or antisense nucleic acids. siRNA comprises a double-stranded structure, typically containing 15-50 base pairs, preferably 19-25 base pairs, and has the same or nearly identical nucleotide sequence as the target gene or RNA expressed in the cell. siRNA can consist of annealed dipolynucleotides or hairpin-forming monopolynucleotides. MicroRNA (miRNA) is a small non-coding polynucleotide, about 22 nucleotides long, that guides the disruption or translational repression of its mRNA target. Antisense polynucleotides contain a sequence complementary to a gene or mRNA. Antisense polynucleotides include, but are not limited to: morpholino, 2'-O-methyl polynucleotides, DNA, RNA, etc. Polynucleotide-based expression inhibitors can be polymerized, recombined, and contain chimeric sequences or derivatives of these groups in vitro. Polynucleotide-based expression inhibitors can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination thereof, thereby inhibiting target RNA and / or genes. The polynucleotide can also be a sequence used as a "barcode" for purposes such as tracking delivery in vitro or in vivo.

[0412] In addition to or in place of nucleic acids, the composition may also contain any protein for delivery. 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-guided endonuclease (e.g., a Cas9 polypeptide, a Cpf1 polypeptide, or a variant thereof) or a DNA recombinase (e.g., a Cre polypeptide).

[0413] The polymers described herein are considered particularly useful for delivering one or more components of the CRISPR system. Therefore, in some embodiments, the composition comprises guide RNA, RNA-guided endonucleases or nucleic acids encoding them, and / or donor nucleic acids. The composition may comprise one, two, or all three components, as well as the polymers described herein. Furthermore, the composition may comprise multiple guide RNAs, RNA-guided endonucleases or nucleic acids encoding them, and / or donor nucleic acids. For example, it may comprise multiple different guide RNAs for different target sites, and optionally multiple different donor nucleic acids and even multiple different RNA-guided endonucleases or nucleic acids encoding them.

[0414] Furthermore, the components of the CRISPR system can be combined with each other (when multiple components are present) and the polymer in any specific manner or order. In some embodiments, the guide RNA is complexed with an RNA endonuclease prior to combination with the polymer. Alternatively, the guide RNA may be ligated (covalently or non-covalently) to the donor nucleic acid prior to combination with the polymer.

[0415] The composition is not limited to any particular CRISPR system (i.e., any particular guide RNA, RNA-guided endonuclease, or donor nucleic acid), many of which are known. However, for further illustration, the components of some such systems are described below.

[0416] donor nucleic acid

[0417] A donor nucleic acid (or “donor sequence”, “donor polynucleotide”, or “donor DNA”) is a nucleic acid sequence to be inserted at a cleavage site induced by an RNA-guided endonuclease (e.g., a Cas9 peptide or a Cpf1 peptide). The donor polynucleotide will have sufficient homology to the target genomic sequence at the cleavage site, for example, 70%, 80%, 85%, 90%, 95%, or 100% homology to a nucleotide sequence flanking the cleavage site, for example, within about 50 bases or less of the cleavage site, such as within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately adjacent to the cleavage site, to support homology-directed repair between itself and the homologous genomic sequence. Approximately 25, 50, 100, or 200 nucleotides, or more (or any integer value between 10 and 200 nucleotides, or more), of sequence homology between the donor and genomic sequences will support homology-directed repair. The donor sequence can be of any length, such as 10 or more nucleotides, 50 or more nucleotides, 100 or more nucleotides, 250 or more nucleotides, 500 or more nucleotides, 1000 or more nucleotides, 5000 or more nucleotides, etc.

[0418] The donor sequence is typically not identical to the genomic sequence it replaces. Instead, the donor sequence may contain one or more single-base alterations, insertions, deletions, inversions, or rearrangements relative to the genomic sequence, provided there is sufficient homology to support homology-directed repair. In some embodiments, the donor sequence includes non-homologous sequences flanked by two homologous regions, such that homology-directed repair between the target DNA region and the two flanking sequences results in the insertion of the non-homologous sequences at the target region. The donor sequence may also include a vector backbone containing sequences that are dislogged from the target DNA region and are not intended to be inserted into it. Typically, the homologous regions of the donor sequence will have at least 50% sequence identity with the genomic sequence to be recombined. In some embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity. Depending on the length of the donor polynucleotide, any value between 1% and 100% sequence identity may exist.

[0419] Compared to the genome sequence, the donor sequence may contain certain sequence differences, such as restriction sites, nucleotide polymorphisms, and selective markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which can be used to assess the successful insertion of the donor sequence at the cleavage site, or in some embodiments for other purposes (e.g., indicating expression at a targeted genomic locus). In some embodiments, if located in a coding region, such nucleotide sequence differences do not alter the amino acid sequence or produce silent amino acid changes (i.e., do not affect the structure or function of the protein). Alternatively, these sequence differences may include flanking recombination sequences, such as FLP, loxP sequences, etc., which can be activated at a later time to remove the marker sequence.

[0420] The donor sequence can be provided to the cell in the form of single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It can be introduced into the cell in a linear or circular form. If introduced in a linear form, the ends of the donor sequence can be protected (e.g., from exonuclease degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of a linear molecule and / or a self-complementary oligonucleotide can 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 methods such as rolling circle amplification can also be advantageously used, as exemplified herein. Other methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino groups and using modified internucleotide bonds, such as phosphate thioesters, aminophosphate esters, and O-methylribose or deoxyribose residues.

[0421] As an alternative to protecting the ends of linear donor sequences, additional sequences of length can be included outside the homologous regions that can be degraded without affecting recombination. The donor sequence can be introduced into cells as part of a vector molecule containing additional sequences, such as origins of replication, promoters, and genes encoding antibiotic resistance. Furthermore, the donor sequence can be introduced as naked nucleic acid, as nucleic acid complexed with a substance (agent) such as a liposome or polymer, or can be delivered by a virus (e.g., adenovirus, AAV), as described herein for nucleic acids encoding Cas9 guide RNA and / or Cas9 fusion polypeptides and / or donor polynucleotides.

[0422] Guided nucleic acid

[0423] In some embodiments, the composition comprises a guide nucleic acid. Guide nucleic acids suitable for inclusion in the compositions of this disclosure include single-molecule guide RNA (“single guide RNA” / “sgRNA”) and dual-molecule guide nucleic acids (“dual guide RNA” / “dgRNA”).

[0424] Guide nucleic acids (e.g., guide RNA) suitable for inclusion in the complexes disclosed herein direct the activity of RNA-guided endonucleases (e.g., CSF9 or Cpf1 peptides) to specific target sequences within target nucleic acids. The guide nucleic acid (e.g., guide RNA) comprises: a first fragment (also referred to herein as a “nucleic acid targeting fragment,” or simply a “target fragment”); and a second fragment (also referred to herein as a “protein-binding fragment”). The terms “first” and “second” do not imply the order in which the fragments appear in the guide RNA. The order of the elements relative to each other depends on the specific RNA-guided peptide to be used. For example, guide RNA for CSF9 typically has a protein-binding fragment located at the 3' end of the target fragment, while guide RNA for Cpf1 typically has a protein-binding fragment located at the 5' end of the target fragment.

[0425] Guide RNA can be introduced into the cell in a linear or circular form. If 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 methods such as rolling circle amplification can also be advantageously used, as illustrated herein.

[0426] First segment: Targeted segment

[0427] The first segment of a guide nucleic acid (e.g., guide RNA) comprises a nucleotide sequence complementary to a sequence (target site) in the target nucleic acid. In other words, the target 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). Therefore, the nucleotide sequence of the target segment can be varied, and the location within the target nucleic acid where the guide nucleic acid (e.g., guide RNA) and the target nucleic acid will interact can be determined. The target segment of the guide nucleic acid (e.g., guide RNA) can be modified (e.g., through genetic engineering) to hybridize with any desired sequence (target site) within the target nucleic acid.

[0428] Target fragments can range in length from 12 to 100 nucleotides. The nucleotide sequence of a target fragment complementary to the nucleotide sequence (target site) of the target nucleic acid (target sequence, also known as the guide sequence) can be 12 nt or longer. For example, the target sequence of a target fragment complementary to the target site of the target nucleic acid can be 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 in length.

[0429] The percentage of complementarity between the target sequence (i.e., the guide sequence) of the targeting fragment and the target site of the target nucleic acid can be 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, 97% or more, 98% or more, 99% or more, or 100%). In some embodiments, the percentage of complementarity between the target sequence of the targeting fragment and the target site of the target nucleic acid is 100% for the seven consecutive 5'-most nucleotides of the target site of the target nucleic acid. In some embodiments, the percentage of complementarity between the target sequence of the targeting fragment and the target site of the target nucleic acid is 60% or more for 20 consecutive nucleotides. In some embodiments, the percentage of complementarity between the target sequence of the targeting region and the target site of the target nucleic acid is 100% for 17, 18, 19, or 20 consecutive 5'-most nucleotides of the target site of the target nucleic acid, and as low as 0% or more for the remaining nucleotides. In this case, the length of the targeting sequence can be considered to be 17, 18, 19, or 20 nucleotides, respectively.

[0430] Second segment: Protein-binding fragment

[0431] A protein-binding fragment of a guide nucleic acid (e.g., guide RNA) interacts (binds) with an RNA-guided endonuclease. The guide nucleic acid (e.g., guide RNA) guides the bound endonuclease to a specific nucleotide sequence (target site) within the target nucleic acid via the aforementioned target fragment / target sequence / guide sequence. The protein-binding fragment of the guide nucleic acid (e.g., guide RNA) contains two complementary nucleotide segments. The complementary nucleotides of the protein-binding fragment hybridize to form a double-stranded RNA duplex (dsRNA duplex).

[0432] Single-guided nucleic acid and double-guided nucleic acid

[0433] A double-guide nucleic acid (e.g., guide RNA) comprises two separate nucleic acid molecules. Each of the two molecules of the double-guide nucleic acid (e.g., guide RNA) contains a segment of a complementary nucleotide, such that the complementary nucleotides of the two molecules hybridize to form a double-stranded RNA duplex of a protein-binding fragment.

[0434] In some embodiments, for segments of 8 or more consecutive nucleotides (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides), the double-stranded fragment of the activator has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher identity or 100% identity with one of the activator (tracrRNA) molecules or their complements described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0435] In some embodiments, for a segment of 8 or more consecutive nucleotides (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides), the double-stranded fragment of the target has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher identity or 100% identity with one of the target (crRNA) sequences or their complements shown in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0436] Dual-guide nucleic acids (e.g., guide RNA) can be designed to allow controlled (i.e., conditional) binding of target molecules to activators. Because dual-guide nucleic acids (e.g., guide RNA) are non-functional unless both the activator and the target are bound to Cas9 in a functional complex, dual-guide nucleic acids (e.g., guide RNA) can be inducible (e.g., drug-induced) by making the binding between the activator and the target inducible. As a non-limiting example, RNA aptamers can be used to regulate (i.e., control) the binding of activators to targets. Therefore, activators and / or targets can include RNA aptamer sequences.

[0437] Aptamers (e.g., RNA aptamers) are known in the art and are generally synthetic forms of riboswitches. The terms “RNA aptamer” and “riboswitch” are used interchangeably herein to refer to both synthetic and natural nucleic acid sequences that allow inducible regulation of the structure of the nucleic acid molecule to which they belong (e.g., RNA, DNA / RNA hybrids, etc.) (and thus inducible regulation of the availability of a specific sequence). RNA aptamers typically contain sequences folded into a specific structure (e.g., a hairpin) that specifically binds to a specific drug (e.g., a small molecule). Drug binding causes a structural change in the RNA fold, which alters the characteristics of the nucleic acid to which the aptamer is a part. As non-limiting examples: (i) an activator containing an aptamer may not bind to a homologous target unless the aptamer is bound by a suitable drug; (ii) a target containing an aptamer may not bind to a homologous activator unless the aptamer is bound by a suitable drug; and (iii) a target and an activator each containing different aptamers that bind different drugs may not bind to each other unless both drugs are present. As these examples illustrate, dual-guide nucleic acids (e.g., guide RNA) can be designed to be inducible.

[0438] Examples of aptamers and riboswitches can be found, 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., Wiley Interdiscip Rev RNA. 2012 May-Jun; 3(3):369-84; All of these references are incorporated herein by reference in their entirety.

[0439] Non-limiting examples of nucleotide sequences that may be included in dual guide nucleic acids (e.g., guide RNA) or complements thereof that can hybridize to form protein-binding segments, as described in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0440] The single-guide nucleic acid (e.g., guide RNA) of the present invention comprises two nucleotide segments (much like the "target" and "activator" of a dual-guide nucleic acid) that are complementary to each other, hybridize to form a double-stranded RNA duplex (dsRNA duplex) of a protein-binding segment (resulting in a stem-loop structure), and are covalently linked by intercalation nucleotides ("linker bases" or "linker base nucleotides"). Therefore, the single-guide nucleic acid (e.g., single-guide RNA) may comprise a target and an activator, each having a duplex-forming segment, wherein the duplex-forming segments of the target and activator hybridize to each other to form a dsRNA duplex. The target and activator may be covalently linked via the 3' end of the target and the 5' end of the activator. Alternatively, the target and activator may be covalently linked via the 5' end of the target and the 3' end of the activator.

[0441] The linker of a single guide nucleic acid can be from 3 nucleotides to 100 nucleotides in length. In some implementations, the linker of a single guide nucleic acid (e.g., guide RNA) is 4 nt.

[0442] An exemplary single guide nucleic acid (e.g., guide RNA) comprises two complementary nucleotide segments that hybridize to form a dsRNA double helix. In some embodiments, for segments of 8 or more consecutive nucleotides (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides), one of the two complementary nucleotide segments of the single guide nucleic acid (e.g., guide RNA) (or the DNA encoding that segment) has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher, or 100% identity with one of the activator (tracrRNA) molecules listed in International Patent Applications Nos. PCT / US 2016 / 052690 and PCT / US 2017 / 062617 or their complement.

[0443] In some embodiments, for a segment of 8 or more consecutive nucleotides (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides), one of the two complementary nucleotide segments of a single guide nucleic acid (e.g., guide RNA) (or DNA encoding the segment) has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher identity or 100% identity with one of the target (crRNA) sequences or their complements shown in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0444] In some embodiments, for a segment of 8 or more consecutive nucleotides (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides), one of the two complementary nucleotide segments of a single guide nucleic acid (e.g., guide RNA) (or DNA encoding the segment) has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher identity or 100% identity with one of the target (crRNA) sequences or activator (tracrRNA) sequences or their complements shown in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0445] By considering species name and base pairing (for dsRNA duplexes with protein-binding domains), suitable target molecules and activator homologous pairs can be routinely determined. Any activator / target pair can be used as part of a dual-guide nucleic acid (e.g., guide RNA) or as part of a single-guide nucleic acid (e.g., guide RNA).

[0446] In some embodiments, the activator (e.g., trRNA, trRNA-like molecule, etc.) of a dual-guide nucleic acid (e.g., guide RNA) or a single-guide nucleic acid (e.g., guide RNA) comprises a nucleotide segment having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or higher sequence identity or 100% sequence identity with the activator (tracrRNA) molecule or its complement shown in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0447] 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 molecules, etc.) comprises 30 or more nucleotides (nt) (e.g., 40 or more, 50 or more, 60 or more, 70 or more, 75 or more nt). In some embodiments, the activator 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 molecules, etc.) has a length of 30 to 200 nucleotides (nt).

[0448] Protein-binding fragments can range in length from 10 to 100 nucleotides.

[0449] Similarly, for both the single-guide nucleic acid (e.g., single-guide RNA) and the double-guide nucleic acid (e.g., double-guide RNA) of the present invention, the length of the dsRNA duplex of the protein-binding fragment can be from 6 base pairs (bp) to 50 bp. The percentage of complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding fragment can be 60% or higher. For example, the percentage of complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding fragment can be 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more (e.g., in some embodiments, there are some non-hybridized nucleotides that thus create bumps within the dsRNA duplex). In some embodiments, the percentage of complementarity between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding fragment is 100%.

[0450] Hybridization-guided nucleic acids

[0451] In some embodiments, the guiding nucleic acid is two RNA molecules (double-guide RNA molecule). In some embodiments, the guiding nucleic acid is one RNA molecule (single-guide RNA molecule). In some embodiments, the guiding nucleic acid is a DNA / RNA hybrid molecule. In such embodiments, the protein-binding fragment of the guiding nucleic acid is RNA and forms an RNA double helix. Therefore, the double-helix-forming fragments of both the activator and the target are RNA. However, the target fragment of the guiding nucleic acid can be DNA. Therefore, if the DNA / RNA hybrid guiding nucleic acid is a double-guide nucleic acid, then the "target" molecule is a hybrid molecule (e.g., the target fragment can be DNA, while the double-helix-forming fragment can be RNA). In such embodiments, the double-helix-forming fragment of the "activator" molecule can be RNA (e.g., to form an RNA double helix with the double-helix-forming fragment of the target molecule), while the nucleotides other than the double-helix-forming fragment of the "activator" molecule can be DNA (in this case, the activator molecule is a hybrid DNA / RNA molecule) or can be RNA (in this case, the activator molecule is RNA). If the DNA / RNA hybridization guide nucleic acid is a single guide nucleic acid, then the target fragment can be DNA, the fragment that forms the double helix (which is the protein-binding fragment that makes up the single guide nucleic acid) can be RNA, and the nucleotides other than the target and the fragment that forms the double helix can be RNA or DNA.

[0452] DNA / RNA hybridization guide nucleic acids can be used in some implementations, for example, when the target nucleic acid is RNA. Cas9 typically binds to guide RNA that hybridizes with the target DNA, thereby forming a DNA-RNA duplex at the target site. Therefore, when the target nucleic acid is RNA, it is sometimes advantageous to re-acquire the DNA-RNA duplex at the target site by using a targeting fragment that is DNA rather than RNA (the guide nucleic acid). However, because the protein-binding fragment of the guide nucleic acid is an RNA-duplex, the targeting molecule is the DNA in the targeting fragment and the RNA in the fragment that forms the duplex. Hybridization guide nucleic acids can bias the binding of Cas9 to single-stranded target nucleic acids relative to double-stranded target nucleic acids.

[0453] Exemplary Guided Nucleic Acid

[0454] 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-guided endonuclease being used). Thus, the guide nucleic acid can be, for example, a guide nucleic acid corresponding to any RNA-guided endonuclease described herein or known in the art. Guide nucleic acids and RNA-guided endonucleases are described, for example, in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US 2017 / 062617.

[0455] In some embodiments, a suitable guide nucleic acid comprises two separate RNA polynucleotide molecules. In some embodiments, the first of the two separate RNA polynucleotide molecules (the activator) contains a nucleotide sequence having 60% or more (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides) nucleotide sequence identity with any of the nucleotide sequences shown in International Patent Applications PCT / US2016 / 052690 and PCT / US2017 / 062617 or their complements. In some embodiments, the second (target) of two separate RNA polynucleotide molecules contains a nucleotide sequence having 60% or more (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides) nucleotide sequence identity with any of the nucleotide sequences listed in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US / 201062617 or their complements.

[0456] In some embodiments, a suitable guide nucleic acid is a single RNA polynucleotide and comprises a first nucleotide sequence and a second nucleotide sequence, wherein for a segment of 8 or more consecutive nucleotides (e.g., 8 or more consecutive nucleotides, 10 or more consecutive nucleotides, 12 or more consecutive nucleotides, 15 or more consecutive nucleotides, or 20 or more consecutive nucleotides), the first nucleotide sequence and the second nucleotide sequence have 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) nucleotide sequence identity with any of the nucleotide sequences shown in International Patent Applications Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 or their complements.

[0457] In some embodiments, the guide RNA is a Cpf1 and / or Cas9 guide RNA. The Cpf1 and / or Cas9 guide RNA can have a total length of 30 nucleotides (nt) to 100 nt, for example, 30 nt to 40 nt, 40 nt to 45 nt, 45 nt to 50 nt, 50 nt to 60 nt, 60 nt to 70 nt, 70 nt to 80 nt, 80 nt to 90 nt, or 90 nt to 100 nt. In some embodiments, the total length of the Cpf1 and / or Cas9 guide RNA is 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt. Cpf1 and / or Cas9 guide RNA may contain target nucleic acid binding fragments and double-strand forming fragments.

[0458] The target nucleic acid binding fragment of the Cpf1 and / or Cas9 guide RNA can have a length of 15 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 fragment is 23 nt long. In some embodiments, the target nucleic acid binding fragment is 24 nt long. In some embodiments, the target nucleic acid binding fragment is 25 nt long.

[0459] The target nucleic acid binding fragment of the Cpf1 and / or Cas9 guide RNA can be 100% complementary to a target nucleic acid sequence of appropriate length. The target fragment can also be less than 100% complementary to a target nucleic acid sequence of appropriate length. For example, the target nucleic acid binding fragment of the Cpf1 and / or Cas9 guide RNA can have 1, 2, 3, 4, or 5 nucleotides that are not complementary to the target nucleic acid sequence. For example, in some embodiments, the target nucleic acid binding fragment is 25 nucleotides long, and the target nucleic acid sequence is 25 nucleotides long; in some embodiments, the target nucleic acid binding fragment is 100% complementary to the target nucleic acid sequence. As another example, in some embodiments, the target nucleic acid binding fragment is 25 nucleotides long, and the target nucleic acid sequence is 25 nucleotides long; in some embodiments, the target nucleic acid binding fragment has 1 non-complementary nucleotide and 24 complementary nucleotides to the target nucleic acid sequence.

[0460] The double-stranded fragments of Cpf1 and / or Cas9 guide RNA can have lengths from 15 nt to 25 nt, such as 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt or 25 nt.

[0461] In some implementations, the double-stranded fragment of the Cpf1 guide RNA may contain the nucleotide sequence 5'-AAUUUCUACUGUUGUAGAU-3'.

[0462] Additional components

[0463] In some embodiments, the guide nucleic acid (e.g., guide RNA) includes one or more additional segments (in some embodiments at the 5' end, in some embodiments at the 3' end, in some embodiments at the 5' or 3' end, in some embodiments embedded within the sequence (i.e., not at the 5' and / or 3' end), in some embodiments at both the 5' and 3' ends, in some embodiments embedded and at both the 5' and / or 3' ends, etc.). For example, suitable additional fragments may include a 5' cap (e.g., a 7-methylguanylic acid cap (M... 7 G); 3' poly(A) tail (i.e., 3' poly(A) tail); ribozyme sequence (e.g., allowing self-cleavage of nucleic acid or components of nucleic acid such as targets, activators, etc.); riboswitch sequence (e.g., to allow regulated stability and / or regulated accessibility of proteins and protein complexes); sequence forming dsRNA duplexes (i.e., hairpins); sequence targeting RNA to subcellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.); modification or sequence providing tracer (e.g., labeling with fluorescent molecules (i.e., fluorescent dyes), sequences or other parts that facilitate fluorescence detection); sequence or other modification providing binding sites for proteins (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, RNA-binding proteins (e.g., RNA aptamers), labeled proteins, fluorescently labeled proteins, etc.); modification or sequence providing increased, decreased and / or controllable stability; and combinations thereof.

[0464] RNA-guided endonucleases

[0465] In addition to, or instead of, the guide nucleic acid, the composition may contain an RNA-guided endonuclease protein or a nucleic acid (e.g., mRNA or a vector) encoding it. Any RNA-guided endonuclease may be used. The choice of RNA-guided endonuclease used depends at least in part on the intended end use of the CRISPR system used.

[0466] In some embodiments, the polypeptide is a Cas9 polypeptide. Suitable Cas9 polypeptides to be included in the compositions of this disclosure include naturally occurring Cas9 polypeptides (e.g., naturally occurring in bacterial and / or archaea cells) or non-naturally occurring Cas9 polypeptides (e.g., Cas9 polypeptides are variant Cas9 polypeptides, chimeric polypeptides, etc., discussed below), as described below. In some embodiments, those skilled in the art will understand that the Cas9 polypeptides disclosed herein can be any variant derived from or isolated from any source. In other embodiments, the Cas9 peptides of this disclosure may include one or more mutations described in the literature, including but not limited to functional mutations described in the following literature: Fonfara et al. Nucleic Acids Res. 2014 Feb; 42(4):2577-90; Nishimasu H. et al. Cell. 2014 Feb 27; 156(5):935-49; Jinek M. et al. Science. 2012 337:816-21; and Jinek M. et al. Science. 2014 Mar 14; 343(6176); see also U.S. Patent Application No. 13 / 842,859, filed March 15, 2013, which is incorporated herein by reference; see also 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, the entire contents of which are incorporated herein by reference. Therefore, in some embodiments, the systems and methods disclosed herein can be used with wild-type Cas9 protein having double-stranded nuclease activity, Cas9 mutants acting as single-stranded cleavage enzymes, or other mutants having modified nuclease activity. Therefore, the Cas9 polypeptide suitable for inclusion in the compositions disclosed herein may be an enzymatic Cas9 polypeptide, for example, capable of generating single- or double-strand breaks in the target nucleic acid, or may have reduced enzymatic activity compared to the wild-type Cas9 polypeptide.

[0467] Naturally occurring Cas9 peptides bind to guide nucleic acids, thereby directing them to a specific sequence (target site) within the target nucleic acid and cleaving the target nucleic acid (e.g., cleaving dsDNA to create double-strand breaks, cleaving ssDNA, cleaving ssRNA, etc.). The Cas9 peptide comprises two parts: an RNA-binding moiety and an active moiety. The RNA-binding moiety interacts with the guide nucleic acid, and the active moiety exhibits site-directed enzymatic activity (e.g., nuclease activity, DNA and / or RNA methylation activity, DNA and / or RNA cleavage activity, histone acetylation activity, histone methylation activity, RNA modification activity, RNA binding activity, RNA splicing activity, etc.). In some embodiments, the active moiety is non-enzymatically active.

[0468] An assay to determine whether a protein has an RNA-binding moiety that interacts with a test guide nucleic acid can be any convenient binding assay that tests the binding between a protein and a nucleic acid. Exemplary binding assays include those involving the addition of a guide nucleic acid and a Cas9 peptide to the target nucleic acid (e.g., gel translocation assay).

[0469] An assay to determine whether a protein has an active moiety (e.g., to determine whether a peptide has nuclease activity to cleave target nucleic acids) can be any convenient nucleic acid lysis assay that tests for nucleic acid lysis. An exemplary lysis assay involves adding a guide nucleic acid and a Cas9 peptide to the target nucleic acid.

[0470] In some embodiments, the Cas9 peptide suitable for inclusion in the compositions disclosed herein has enzymatic activity for modifying target nucleic acids (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, superoxide 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 glycosylation activity).

[0471] In other embodiments, the Cas9 peptide suitable for inclusion in the compositions disclosed herein has enzymatic activity (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitin activity, adenylation activity, deadenylation activity, ribosylation activity, deadenylation activity, myristylation activity, or demyelination activity) of a peptide (e.g., histone) associated with the target nucleic acid.

[0472] Numerous Cas9 orthologs from multiple species have been identified, and in some embodiments, the proteins share only a few identical amino acids. All identified Cas9 orthologs share the same domain structure, which includes a central HNH endonuclease domain and a split RuvC / RNaseH domain. The Cas9 protein contains four key motifs with conserved structures. Motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif.

[0473] In some embodiments, a suitable Cas9 polypeptide comprises an amino acid sequence having four motifs, each of motifs 1-4 being... Figure 1 The Cas9 amino acid sequence described in (SEQ ID NO: 1), or motifs 1-4 of the Cas9 amino acid sequences described in Table 1 below (motifs 1-4 of SEQ ID NO: 1 are SEQ ID NO: 3-6, as described in Table 1 below), or Figure 1 The amino acid sequences 7-166 or 731-1003 of the Cas9 amino acid sequence shown (SEQ ID NO: 1) have 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.

[0474] In some implementations, the Cas9 peptide contains... Figure 1 The amino acid sequence described in and listed in SEQ ID NO: 1 has 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 98% amino acid sequence identity; and contains amino acid substitutions of N497, R661, Q695, and Q926 relative to the amino acid sequence shown in SEQ ID NO: 1; or contains amino acid substitution of K855 relative to the amino acid sequence shown in SEQ ID NO: 1; or contains amino acid substitutions of K810, K1003, and R1060 relative to the amino acid sequence shown in SEQ ID NO: 1; or contains amino acid substitutions of K848, K1003, and R1060 relative to the amino acid sequence shown in SEQ ID NO: 1.

[0475] As used herein, the term “Cas9 polypeptide” encompasses the term “variant Cas9 polypeptide”; the term “variant Cas9 polypeptide” encompasses the term “chimeric Cas9 polypeptide”.

[0476] Variant Cas9 peptide

[0477] Suitable Case9 peptides for inclusion in compositions of this disclosure include variant Case9 peptides. A variant Cas9 peptide has an amino acid sequence that differs from that of a wild-type Cas9 peptide (e.g., deletion, insertion, substitution, fusion) by one amino acid (i.e., at least one amino acid differs). In some cases, the variant Cas9 peptide has amino acid alterations (e.g., deletion, insertion, or substitution) that reduce the nuclease activity of the Cas9 peptide. For example, in some cases, the variant Cas9 peptide 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 peptide. In some embodiments, the variant Cas9 peptide has essentially no nuclease activity. When a Case9 peptide is a variant Case9 peptide that does not have substantial nuclease activity, it can be referred to as "dCas9".

[0478] In some embodiments, the variant Cas9 peptide has reduced nuclease activity. For example, variant Cas9 peptides suitable for the binding methods of this disclosure exhibit less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of wild-type Cas9 peptide (e.g., containing, for example,...). Figure 1 Endonuclease activity of the wild-type Cas9 polypeptide (the amino acid sequence depicted in SEQ ID NO: 1).

[0479] In some implementations, the variant Cas9 peptide can cleave the complementary strand of the target nucleic acid, but its ability to cleave the non-complementary strand of the double-stranded target nucleic acid is reduced. For example, the variant Cas9 peptide may have a reduced RuvC domain (e.g., Figure 1 The mutation (amino acid substitution) of the function of “domain 1”. As a non-limiting example, in some embodiments, the variant Cas9 peptide has a D10A mutation (e.g., aspartic acid is replaced with alanine at the amino acid position corresponding to position 10 of SEQ ID NO: 1), and thus can cleave the complementary strand of the double-stranded target nucleic acid, but the ability to cleave the non-complementary strand of the double-stranded target nucleic acid is reduced (therefore, when the variant Cas9 peptide cleaves the double-stranded target nucleic acid, it results in a single-strand break (SSB) instead of a double-strand break (DSB)) (see, for example, Jinek et al., Science. 2012 Aug 17; 337(6096):816-21).

[0480] In some implementations, the variant Cas9 peptide can cleave the non-complementary strand of the double-stranded target nucleic acid, but its ability to cleave the complementary strand of the target nucleic acid is reduced. For example, the variant Cas9 peptide may have a reduced HNH domain (RuvC / HNH / RuvC domain motif, Figure 1The "domain 2" of the Cas9 polypeptide may be mutated (amino acid substitution). As a non-limiting example, in some embodiments, the variant Cas9 polypeptide may have an H840A mutation (e.g., histidine at position 840 of SEQ ID NO: 1 is replaced with alanine). Figure 1 And thus it can cleave the non-complementary strand of the target nucleic acid, but its ability to cleave the complementary strand of the target nucleic acid is reduced (therefore, when the variant Cas9 peptide cleaves the double-stranded target nucleic acid, it produces SSB instead of DSB). Such Cas9 peptides have a reduced ability to cleave the target nucleic acid (e.g., single-stranded target nucleic acid), but retain the ability to bind to the target nucleic acid (e.g., single-stranded or double-stranded target nucleic acid).

[0481] In some embodiments, the variant Cas9 peptide 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 variant Cas9 peptide simultaneously contains both D10A and H840A mutations (e.g., a common mutation in both the RuvC and HNH domains), such that the peptide has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target nucleic acid. Such a Cas9 peptide has a reduced ability to cleave target nucleic acids (e.g., single-stranded or double-stranded target nucleic acids) but retains the ability to bind target nucleic acids (e.g., single-stranded or double-stranded target nucleic acids).

[0482] As another non-limiting example, in some embodiments, the variant Cas9 peptide contains W476A and W1126A mutations, resulting in a reduced ability to cleave target nucleic acids. Such a Cas9 peptide has a reduced ability to cleave target nucleic acids but retains the ability to bind to them.

[0483] As another non-limiting example, in some embodiments, the variant Cas9 peptide contains mutations of P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability to cleave target nucleic acids. Such a Cas9 peptide has a reduced ability to cleave target nucleic acids but retains the ability to bind to them.

[0484] As another non-limiting example, in some embodiments, the variant Cas9 peptide contains H840A, W476A, and W1126A mutations, resulting in a reduced ability to cleave target nucleic acids. Such Cas9 peptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to them.

[0485] As another non-limiting example, in some embodiments, the variant Cas9 peptide contains H840A, D10A, W476A, and W1126A mutations, resulting in a reduced ability to cleave target nucleic acids. Such Cas9 peptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to them.

[0486] As another non-limiting example, in some embodiments, the variant Cas9 peptide contains mutations of H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, resulting in a reduced ability to cleave target nucleic acids. Such Cas9 peptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to them.

[0487] As another non-limiting example, in some embodiments, the variant Cas9 peptide contains mutations of D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A, such that the peptide has a reduced ability to cleave target nucleic acids. Such Cas9 peptides have a reduced ability to cleave target nucleic acids but retain the ability to bind to target nucleic acids.

[0488] Other residues can be mutated to achieve the above effect (i.e., to partially inactivate one or more nucleases). As non-restrictive examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 can be altered (e.g., substituted) (see Table 1 for more information on the conservation of Cas9 amino acid residues). Furthermore, mutations other than alanine substitution are also suitable.

[0489] In some implementations, variant Cas9 peptides with reduced catalytic activity (e.g., when the Cas9 protein has mutations such as D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, such as D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A) can still bind to the target nucleic acid in a site-specific manner, as long as they retain their ability to interact with the guide nucleic acid (because they are still guided to the target nucleic acid sequence by the guide nucleic acid).

[0490] Table 1 lists four motifs present in Cas9 sequences from different species. The amino acids listed here are from Cas9 (SEQ ID NO: 1) of Streptococcus pyogenes.

[0491]

[0492] In addition to the above, variant Cas9 proteins can have the same sequence identity parameters as the Cas9 polypeptide described above. Therefore, in some embodiments, a suitable variant Cas9 polypeptide comprises an amino acid sequence having four motifs, each of motifs 1-4 being identical to... Figure 1 The Cas9 amino acid sequence shown in (SEQ ID NO: 1), or motifs 1-4 (moleculars 1-4 of SEQ ID NO: 1 are SEQ ID NO: 3-6, as shown in Table 1), or Figure 1 The amino acid sequence of Cas9 described in (SEQ ID NO: 1) has amino acid sequence identity of 60% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 99% or higher, or 100% as amino acid sequence 7-166 or 731-1003. 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.

[0493] In some implementations, suitable variants of the Cas9 peptide contain [the following]. Figure 1 The Cas9 amino acid sequence depicted in (SEQ ID NO:1) 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. Any Cas9 protein as defined above may be used as a variant Cas9 polypeptide or as part of a chimeric variant Cas9 polypeptide in the compositions disclosed herein, including those specifically referenced in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0494] Chimeric peptides (fusion peptides)

[0495] In some implementations, the variant Cas9 peptide is a chimeric Cas9 peptide (also referred to herein as a fusion peptide, e.g., "Cas9 fusion peptide"). The Cas9 fusion peptide may bind to and / or modify the target nucleic acid (e.g., cleavage, methylation, demethylation, etc.) and / or peptides associated with the target nucleic acid (e.g., methylation, acetylation, etc. of histone tails).

[0496] Cas9 fusion peptides are variant Cas9 peptides because they differ in sequence from wild-type Cas9 peptides (e.g., naturally occurring Cas9 peptides). Cas9 fusion peptides are Cas9 peptides fused with a covalently linked heteropeptide (also called a "fusion partner") (e.g., wild-type Cas9 peptides, variant Cas9 peptides, variant Cas9 peptides with reduced nuclease activity (as described above), etc.). In some embodiments, the Cas9 fusion peptide is a variant Cas9 peptide (e.g., dCas9) with reduced nuclease activity fused with a covalently linked heteropeptide. In some embodiments, the heteropeptide exhibits (and thus provides) activities (e.g., enzymatic activities) that are also exhibited by the Cas9 fusion peptide (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.). In some such embodiments, the method of binding to the target nucleic acid, for example, when the Cas9 peptide is a variant Cas9 peptide with the activity of a fusion partner (i.e., having a heteropeptide) 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, the method of binding to the target nucleic acid (e.g., a single-stranded target nucleic acid) can result in modification of the target nucleic acid. Therefore, in some implementations, the method of binding the target nucleic acid (e.g., a single-stranded target nucleic acid) can be a method of modifying the target nucleic acid.

[0497] In some embodiments, the heterologous sequence provides subcellular localization; that is, the heterologous sequence is a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting the cell nucleus, a sequence that retains the fusion protein outside the cell nucleus, such as a nuclear export sequence (NES or NES), a sequence that allows the fusion protein to remain in the cytoplasm, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). In some embodiments, the variant Cas9 does not include an NLS, such that the protein does not target the cell nucleus (this can be advantageous, for example, when the target nucleic acid is RNA present in the cytosol). In some embodiments, the heterologous sequence can provide a label (i.e., the heterologous sequence is a detectable label) to facilitate tracking and / or purification (e.g., fluorescent proteins such as green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; histidine labels, such as 6XHis labels; hemagglutinin (HA) labels; FLAG labels; Myc labels; etc.). In some embodiments, the heterologous sequence may provide increased or decreased stability (i.e., the heterologous sequence is a stability-controlling peptide, such as a degradation determinant, which in some embodiments is controllable (e.g., a temperature-sensitive or drug-controlled degradation determinant sequence, see below). In some embodiments, the heterologous sequence may provide increased or decreased transcription from the target nucleic acid (i.e., the heterologous sequence is a transcriptional regulatory sequence, such as a transcription factor / activator or a fragment thereof, a protein or a fragment thereof that recruits a transcription factor / activator, a transcriptional repressor protein or a fragment thereof, a protein or a fragment thereof that recruits a transcriptional repressor protein, a small molecule / drug-responsive transcriptional regulator, etc.). In some embodiments, the heterologous sequence may provide a binding domain (i.e., the heterologous sequence is a protein-binding sequence, e.g., providing the ability of a Cas9 fusion polypeptide to bind to another target protein, such as a DNA or histone-modified protein, a transcription factor or transcriptional repressor protein, a recruitment protein, an RNA-modifying enzyme, an RNA-binding protein, a translation initiation factor, an RNA splicing factor, etc.). The heterologous nucleic acid sequence may be linked to another nucleic acid sequence (e.g., by genetic engineering) to produce a chimeric nucleotide sequence encoding a chimeric polypeptide.

[0498] The tested Cas9 fusion polypeptide (Cas9 fusion protein) may have multiple (one or more, two or more, three or more, etc.) fusion couplers in any combination of the above. As an illustrative example, the Cas9 fusion protein may have a heterologous sequence providing activity (e.g., for transcriptional regulation, target modification, modification of proteins associated with target nucleic acids, etc.) and may also have a subcellular localization sequence. In some embodiments, such a Cas9 fusion protein may also have a marker for easy tracking and / or purification (e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; histidine markers, such as 6XHis markers; hemagglutinin (HA) markers; FLAG markers; Myc markers, etc.). As another illustrative example, the Cas9 protein may have one or more NLS (e.g., two or more, three or more, four or more, five or more, 1, 2, 3, 4, or 5 NLS). In some embodiments, the fusion partner (or multiple fusion partners) (e.g., NLS, tag, active 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, active fusion partner, etc.) is located at the N-terminus of Cas9. In some embodiments, Cas9 has a fusion partner (or multiple fusion partners) (e.g., NLS, tag, active fusion partner, etc.) at both the N-terminus and C-terminus.

[0499] Suitable fusion partners that provide increased or decreased stability include, but are not limited to, degradation determinant sequences. Degradation determinants, as readily understood by those skilled in the art, are amino acid sequences that control the stability of a protein (of which the degradation determinant is a part). For example, the stability of a protein containing a degradation determinant sequence is partially controlled by the degradation determinant sequence. In some embodiments, suitable degradation determinants are constitutive, such that they influence protein stability independently of experimental control (i.e., they are not drug-induced, temperature-induced, etc.). In some embodiments, the degradation determinant provides a variant Cas9 peptide with controllable stability, such that the variant Cas9 peptide can be “on” (i.e., stable) or “off” (i.e., unstable, degradable) depending on desired conditions. For example, if the degradation determinant is temperature-sensitive, the variant Cas9 peptide 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 non-functional (i.e., “off,” degraded) above the threshold temperature. As another example, if the degradation determinant is drug-inducible, 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 degradation determinant is derived from the FKBP12 protein. The stability of the degradation determinant is controlled by the presence or absence of a small molecule binding to the degradation determinant.

[0500] Examples of suitable degradation determinants include, but are not limited to, those controlled by Shield-1, DHFR, auxin, and / or temperature.Non-limiting examples of suitable degradation determinants are known in the art (e.g., Dohmen et al., Science, 1994, 263(5151): p. 1273-1276: Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol. 2009 Jan; 296(1): F204-11: Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med ChemLett. 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.2013Jan18;33(1).:Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase;and Greussing et al.,J VisExp.2012Nov 10;(69):Monitoring of ubiquitin-proteasome activity in living cells using aDegron(dgn)-destabilized green fluorescent protein (GFP)-based reporter protein;The entire contents are incorporated herein by reference in their entirety).

[0501] Exemplary degradation determinant sequences have been well characterized and tested in cells and animals. Therefore, fusing Cas9 (e.g., wild-type Cas9; variant Cas9; variant Cas9 with reduced nuclease activity, such as dCas9; etc.) with degradation determinant sequences yields “tunable” and “inducible” Cas9 peptides. Any fusion partner described herein can be used in any desired combination. As a non-limiting example illustrating this, a Cas9 fusion protein (i.e., a chimeric Cas9 peptide) may comprise a YFP sequence for detection, a degradation determinant sequence for stabilization, and a transcriptional activator sequence that increases transcription of the target nucleic acid. Suitable reporter proteins used as fusion partners of Cas9 peptides (e.g., wild-type Cas9, variant Cas9, variant Cas9 with reduced nucleic acid function, etc.) include, but are not limited to, the following exemplary proteins (or functional fragments thereof): his3, β-galactosidase, fluorescent proteins (e.g., GFP, RFP, YFP, Cherry, Tomato, etc., and various derivatives thereof), luciferase, β-glucuronidase, and alkaline phosphatase. Furthermore, the number of fusion partner bodies that can be used in the Cas9 fusion protein is unlimited. In some embodiments, the Cas9 fusion protein comprises one or more (e.g., two or more, three or more, four or more, or five or more) heterologous sequences.

[0502] Suitable fusion couplers include, but are not limited to, peptides providing methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, ubiquitination activity, deubiquitination activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity, any of which may be intended to modify nucleic acids (e.g., methylation of DNA or RNA) or to modify nucleic acid-related peptides (e.g., histones, DNA-binding proteins, and RNA-binding proteins). Other suitable fusion couplers include, but are not limited to, boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).

[0503] Examples of various other suitable fusion couplers (or fragments thereof) for the said variant Case9 peptide include, but are not limited to, those described in the following PCT patent applications: WO 2010 / 075303, WO 2012 / 068627 and WO 2013 / 155555, which are incorporated herein by reference in their entirety.

[0504] Suitable fusion couplers include, but are not limited to, peptides that provide activity that indirectly increases transcription by acting directly on target nucleic acids or peptides associated with target nucleic acids (e.g., histones, DNA-binding proteins, RNA-editing proteins, etc.). Suitable fusion couplers include, but are not limited to, peptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitin activity, adenylation activity, deadenylation activity, ubiquitination activity, deubiquitination activity, ribosylation activity, deadenylation activity, myristoylation activity, or deribosylation activity.

[0505] Other suitable fusion couplers include, but are not limited to, peptides that directly provide increased transcription and / or translation of target nucleic acids (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecule / drug-responsive transcription and / or translation regulators, translation regulatory proteins, etc.).

[0506] Non-limiting examples of fusion couplers that achieve increased or decreased transcription include transcriptional activators and transcriptional repressor protein domains (e.g., Krüppel associated box (KRAB or SKD); Mad mSIN3 interaction domain (SID); ERF repressor protein domain (ERD), etc.). In some such embodiments, the Cas9 fusion protein is directed by a guide nucleic acid to a specific location (i.e., sequence) in the target nucleic acid and exerts locus-specific regulation, such as blocking RNA polymerase binding to a promoter (which selectively represses transcriptional activator function), and / or modifying local chromatin states (e.g., when using a fusion sequence that modifies the target nucleic acid or a peptide associated with the target nucleic acid). In some embodiments, the alteration is temporary (e.g., transcriptional repression or activation). In some embodiments, the alteration is heritable (e.g., when epigenetic modifications are made to the target nucleic acid or proteins associated with the target nucleic acid (e.g., nucleosome histones).

[0507] Non-limiting examples of fusion couplers used 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 methyltransferases; RNA editing enzymes (e.g., RNA deaminases, such as adenosine deaminases acting on RNA (ADAR), including A-to-I and / or C-to-U editing enzymes); heliembodiments; RNA-binding proteins; and so on. It should be understood that fusion couplers may comprise whole proteins, or in some embodiments may comprise fragments of proteins (e.g., functional domains).

[0508] In some embodiments, the heterologous sequence may be fused to the C-terminus of the Cas9 peptide. In some embodiments, the heterologous sequence may be fused to the N-terminus of the Cas9 peptide. In some embodiments, the heterologous sequence may be fused to an internal portion of the Cas9 peptide (i.e., a portion other than the N-terminus or C-terminus).

[0509] Furthermore, the fusion partner of the chimeric Cas9 polypeptide can be any domain capable of interacting with ssRNA temporarily or irreversibly, directly or indirectly (for the purposes of this disclosure, this includes intramolecular and / or intermolecular secondary structures, such as double-stranded RNA duplexes like hairpins, stem-loops, etc.), including but not limited to effector domains selected from: endonucleases derived from proteins such as SMG5 and SMG6 (e.g., RNase I, CRR). 2 2. DYW domain, Dicer and PIN (PilT N-terminal) domain); proteins and protein domains responsible for stimulating RNA cleavage (e.g., CPSF, CstF, CFIm and CFI1m); exonucleases (e.g., XRN-1 or exonuclease T); deadenylate enzymes (e.g., HNT3); proteins and protein domains responsible for nonsense-mediated RNA decay (e.g., UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2 and SRm). 160); proteins and protein domains responsible for stabilizing RNA (e.g., PABP); proteins and protein domains responsible for inhibiting translation (e.g., Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (e.g., Staufen); proteins and protein domains responsible for (e.g., capable of) regulating translation (e.g., translation factors, such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (e.g., PAP). 1. GLD-2 and Star-PAP); proteins and protein domains responsible for RNA polysaccharidation (e.g., CID1 and terminal uracil transferase); proteins and protein domains responsible for RNA localization (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for RNA nuclear retention (e.g., Rrp6); proteins and protein domains responsible for RNA nuclear export (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for inhibiting RNA splicing (e.g., PTB, Sam 68, and hnRNP A1); proteins and protein domains responsible for stimulating RNA splicing (e.g., serine / arginine-rich (SR) domains); proteins and protein domains responsible for reducing transcription efficiency (e.g., FUS(TLS)); and proteins and protein domains responsible for stimulating transcription (e.g., CDK7 and HIV Tat). Alternatively, the effector domain can be selected from: endonucleases; proteins and protein domains that can stimulate RNA cleavage; exonucleases; deadenylate enzymes; proteins and protein domains with nonsense-mediated RNA decay activity; proteins and protein domains that can stabilize RNA; proteins and protein domains that can inhibit translation; proteins and protein domains that can stimulate translation; proteins and protein domains that can regulate translation (e.g., translation factors, such as initiation factors, elongation factors, release factors, etc., eIF4G, etc.); proteins and protein domains that can polyadenylate RNA; proteins and protein domains that can polysaccharide RNA; proteins and protein domains with RNA localization activity; proteins and protein domains that can retain RNA in the nucleus; proteins and protein domains with RNA nuclear export activity; proteins and protein domains that can inhibit RNA splicing; proteins and protein domains that can stimulate RNA splicing; proteins and protein domains that can reduce transcription efficiency; and proteins and protein domains that can stimulate transcription. Another suitable fusion partner is the PUF RNA-binding domain, which is described in more detail in WO 2012068627.

[0510] Some RNA splicing factors that can be used (in whole or as fragments) as fusion couplers for Cas9 peptides are modularly organized, with separate sequence-specific RNA-binding modules and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exon splicing enhancers (ESEs) in pre-mRNA and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNPA... 1 It binds to exon splice silencers (ESSs) via its RRM domain and inhibits exon retention via its C-terminal glycine-rich domain. Some splicing factors can regulate the substitution use of splice sites (SSs) by binding to regulatory sequences between two substitution sites. For example, ASF / SF2 can recognize ESEs and promote the use of intron proximal sites, while hnRNP A 1 ESSs can bind to and utilize transfer splicing at sites distal to introns. One application of these factors is the generation of ESFs that regulate alternative splicing of endogenous genes, particularly disease-related genes. For example, Bcl-x premRNA produces two splice isoforms with two optional 5' splice sites encoding proteins with opposite functions. The long splice isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is upregulated in many cancer cells, protecting cells from apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform expressed at high levels in cells with high transformation rates (e.g., developing lymphocytes). The ratio of the two Bcl-x splice isoforms is regulated by multiple cis elements located in the core exon region or exon extension region (i.e., between the two optional 5' splice sites). See WO2010075303 for more examples.

[0511] In some implementations, the Cas9 peptide (e.g., wild-type Cas9, variant Cas9, variant Cas9 with reduced nuclease activity, etc.) can be linked to the fusion partner via a peptide spacer region.

[0512] In some embodiments, the Cas9 peptide comprises a “protein transduction domain” or PTD (also known as a CPP-cell-penetrating peptide), which can refer to a peptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates passage through a lipid bilayer, micelles, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule's passage through a membrane, e.g., from the extracellular space to the intracellular space, or from the cytosol to the organelle; said other molecule can be a small polar molecule to a large polymer and / or nanoparticle. In some embodiments, a PTD linked 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 peptide comprises two or more NLSs, e.g., two or more NLSs in tandem. In some embodiments, the PTD is covalently linked to the N-terminus of the Cas9 peptide. In some embodiments, the PTD is covalently linked to the C-terminus of the Cas9 peptide. In some embodiments, the PTD is covalently linked to both the N-terminus and C-terminus of the Cas9 peptide. In some implementations, the PTD is covalently linked to a nucleic acid (e.g., a guide nucleic acid, a polynucleotide encoding a guide nucleic acid, a polynucleotide encoding a Cas9 polypeptide, etc.). Exemplary PTDs include, but are not limited to, 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 arginine residues to guide entry into the cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginine residues); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila antennal transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1256); and polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA). 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:8); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:9); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:10); and RQIKIWFQNRRMKWKK (SEQ ID NO:11).Exemplary PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 12), RKKRRQRRRR (SEQ ID NO: 13); arginine homopolymers of 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following sequences: YGRKKRRQRRR (SEQ ID NO: 14); RKKRRQRR (SEQ ID NO: 15); YARAAARQARA (SEQ ID NO: 16); THRLPRRRRRR (SEQ ID NO: 17); and GGRRARRRRRR (SEQ ID NO: 18). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPP contains 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 cell adhesion and uptake. Upon linker cleavage, the polyanion is released, locally exposing the polyarginine and its inherent adhesiveness, thereby "activating" the ACPP to traverse the membrane.

[0513] In some embodiments, the composition may comprise a Cpf1 RNA-guided endonuclease, examples of which are... Figure 2 , 16 Alternatively, it is provided in 17. Another name for the Cpf1 RNA-guided endonuclease is Cas12a. The Cpf1 CRISPR system disclosed herein comprises i) a single endonuclease protein and ii) crRNA, wherein the 3' end portion of the crRNA contains a guide sequence complementary to the target nucleic acid. In this system, the Cpf1 nuclease is directly recruited to the target DNA by the crRNA. In some embodiments, for detectable DNA cleavage, the Cpf1 guide sequence must be at least 12 nt, 13 nt, 14 nt, 15 nt, or 16 nt; for efficient DNA cleavage, the Cpf1 guide sequence must be at least 14 nt, 15 nt, 16 nt, 17 nt, or 18 nt.

[0514] The Cpf1 system disclosed herein differs from Cas9 in several ways. 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 bases long, with 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 sequences can be approximately 100 bases long.

[0515] Second, Cpf1 preferably has a “TTN” PAM motif located upstream of the 5' of its target. This is the opposite of the “NGG” PAM motif located on the 3' of the target DNA in 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” Cell 163, 759-771, which is incorporated herein by reference in its entirety for all purposes).

[0516] Third, the cleavage site of Cpf1 is offset by approximately 3-5 bases, resulting in "sticky ends" (Kim et al., 2016, "Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells," published online June 6, 2016). These sticky ends with 3-5 bp protrusions are thought to facilitate NHEJ-mediated ligation and improve gene editing of DNA fragments with matching ends. The cleavage site is located at the 3' end of the target DNA, away from the 5' end where PAM is located. The cleavage position typically follows the 18th base on the unhybridized strand and the corresponding 23rd base on the complementary strand that hybridizes to crRNA.

[0517] Fourth, in the Cpf1 complex, the "seed" region is located within the first 5 nt of the guide sequence. The Cpf1 crRNA seed region is highly sensitive to mutations; 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). Strictly speaking, unlike Cas9 CRISPR targets, the cleavage site and seed region of the Cpf1 system do not overlap. Further guidance for designing oligomers targeting Cpf1 crRNA can be obtained from (Zetsche B. et al. 2015, "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System," Cell 163, 759-771).

[0518] Those skilled in the art will understand that the Cpf1 disclosed herein can be any variant derived from or isolated from any source, many of which are known in the art. For example, in some embodiments, the Cpf1 peptide disclosed herein may include Figure 2 The FnCPF1 (e.g., SEQ ID NO: 2) and AsCpf1 (e.g.) are shown. Figure 16 ), LbCpf1 (e.g.) Figure 17 (or any other known Cpf1 protein from various other microbial species and its synthetic variants.)

[0519] In some embodiments, the composition comprises a Cpf1 peptide. In some embodiments, the Cpf1 peptide has enzymatic activity, for example, the Cpf1 peptide cleaves target nucleic acids upon binding to guide RNA. In some embodiments, the Cpf1 peptide is relative to a wild-type Cpf1 peptide (e.g., relative to a composition comprising...). Figure 2 , 16 The Cpf1 polypeptide (or the amino acid sequence described in 17) showed reduced enzyme activity while maintaining DNA-binding activity.

[0520] In some implementation schemes, for Figure 2 , 16 Or the amino acid sequence depicted in 17, 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. In some embodiments, for Figure 2, 16 The amino acid sequence described in or 17 may be a continuous segment of 100 to 200 amino acids (aa), 200 to 400 aa, 400 to 600 aa, 600 to 800 aa, 800 to 1000 aa, 1000 to 1100 aa, 1100 to 1200 aa, or 1200 to 1300 aa, and the Cpf1 polypeptide may contain 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.

[0521] In some implementation schemes, for Figure 2 , 16 The RuvCI domain of the Cpf1 polypeptide with the amino acid sequence depicted in Figure 17, wherein 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. In some embodiments, for Figure 2 , 16 The RuvCII domain of the Cpf1 polypeptide with the amino acid sequence depicted in or 17, wherein 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. In some embodiments, for Figure 2 , 16 The RuvCIII domain of the Cpf1 polypeptide with the amino acid sequence depicted in 17, wherein the Cpf1 polypeptide contains 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.

[0522] In some embodiments, the Cpf1 peptide is relative to the wild-type Cpf1 peptide (e.g., relative to a peptide comprising...). Figure 2 , 16 The Cpf1 polypeptide (or the amino acid sequence described in section 17) exhibits reduced enzyme activity while maintaining DNA-binding activity. In some embodiments, for Figure 2 , 16 Or the amino acid sequence depicted in 17, the Cpf1 polypeptide contains 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; and in accordance with Figure 2 , 16 Or, the amino acid residue corresponding to amino acid position 917 in the amino acid sequence shown in 17 contains an amino acid substitution (e.g., D→A substitution). In some embodiments, for Figure 2 , 16 Or the amino acid sequence depicted in 17, the Cpf1 polypeptide contains 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; and in accordance with Figure 2 , 16 Or, the amino acid residue corresponding to amino acid position 1006 in the amino acid sequence shown in 17 contains an amino acid substitution (e.g., E→A substitution). In some embodiments, for Figure 2 , 16 Or the amino acid sequence depicted in 17, the Cpf1 polypeptide contains 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; and in accordance with Figure 2 , 16 Or, the amino acid residue corresponding to the 1255th amino acid in the amino acid sequence shown in 17 contains an amino acid substitution (e.g., D→A substitution).

[0523] In some embodiments, the Cpf1 peptide is a fusion peptide, for example, wherein the Cpf1 fusion peptide comprises: a) a Cpf1 peptide; and b) a heterologous fusion partner. In some embodiments, the heterologous fusion partner is fused to the N-terminus of the Cpf1 peptide. In some embodiments, the heterologous fusion partner is fused to the C-terminus of the Cpf1 peptide. In some embodiments, the heterologous fusion partner is fused to both the N-terminus and C-terminus of the Cpf1 peptide. In some embodiments, the heterologous fusion partner is inserted internally into the Cpf1 peptide.

[0524] Suitable heterologous fusion couplers include NLS, epitope markers, fluorescent peptides, etc.

[0525] Connecting guide RNA and donor nucleic acid

[0526] In one aspect, the present invention provides a complex comprising a CRISPR system comprising an RNA-guided endonuclease (e.g., Cas9 or Cpf1 peptide), a guide RNA, and a donor polynucleotide, wherein the guide RNA and the donor polynucleotide are linked. As illustrated herein, the guide RNA and the donor polynucleotide may be covalently or non-covalently linked. In one embodiment, the guide RNA is chemically linked to the donor polynucleotide. In another embodiment, the guide RNA and the donor polynucleotide are enzymatically linked. In one embodiment, the guide RNA and the donor polynucleotide hybridize to each other. In another embodiment, both the guide RNA and the donor polynucleotide hybridize to a bridge sequence. Any number of such hybridization schemes are possible.

[0527] Deaminase

[0528] In some embodiments, the complex or composition further comprises a deaminase (e.g., an adenine base editor). As used herein, the term "deaminase" or "deaminase domain" refers to an enzyme that catalyzes the removal of an amine group or deamination from a molecule. In some embodiments, the deaminase is a cytidine deaminase that catalyzes the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase is a cytosine deaminase that catalyzes the hydrolysis and deamination of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA).

[0529] 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) can be derived from any organism, such as bacteria. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.

[0530] In some embodiments, the deaminase or deaminase domain does not exist in nature. For example, in some embodiments, for naturally occurring deaminases, the deaminase or deaminase domain has 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% identity. In some embodiments, the adenosine deaminase is derived from bacteria such as *Escherichia coli*, *Staphylococcus aureus*, *Salmonella typhi*, *Streptomyces putrefaciens*, *Haemophilus influenzae*, or *C. crescentus*. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is *E. coli* TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated *E. coli* TadA deaminase. For example, the truncated ecTadA may lack one or more N-terminal amino acids compared to the full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues compared to the full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues compared to 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.

[0531] The deaminase may be used in combination with any other CRISPR element described herein (i.e., as a composition), or the deaminase may be fused with any other CRISPR element described herein (e.g., Cas9 or Cpf1) (i.e., as a complex). In some embodiments, the deaminase is fused with Cas9, Cpf1, or a variant thereof.

[0532] Other components

[0533] The composition may further comprise any other components commonly used in nucleic acid or protein delivery formulations. For example, the composition may further comprise lipids, lipoproteins (e.g., cholesterol and derivatives), phospholipids, polymers, or other components of liposomes or micellar delivery carriers. The composition may also comprise a solvent or carrier suitable for administration to cells or a host (e.g., mammals or humans).

[0534] In some embodiments, the composition comprises a second polymer comprising polyethylene oxide (PEG). For example, the composition may comprise PEG-pAsp(DET), PEG-pAsp, derivatives of PEG-pAsp(DET), derivatives of PEG-pAsp, or combinations thereof. Without wishing to be bound by any particular theory, it is believed that these PEGylated polymers can control the size of the nanoparticles and their interactions with serum proteins and target cells. The polyethylene oxide polymer can be combined with other components in any manner and in any order.

[0535] In some embodiments, the composition further comprises one or more surfactants. The surfactant may be a nonionic surfactant and / or an amphoteric surfactant. The list of exemplary surfactants includes, but is not limited to: polyoxyethylene dehydrated sorbitan ester surfactants (commonly known as Tween), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO) sold under the trade name DOWFAX, such as linear EO / PO block copolymers; octylbenzyl alcohol, which may have a varying number of repeating ethoxy groups (oxy-1,2-ethylenedimethyl), of which octylbenzyl alcohol-9 (Triton X-100, or tert-octylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-6301NP-40); phospholipids, such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl alcohol, cetyl alcohol, stearyl alcohol, and oleyl alcohol (referred to as Brij surfactants), such as triethylene glycol monolaurate (Brij). 30); polyoxyethylene-9-lauryl ether, and sorbitan esters (commonly referred to 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.

[0536] In some cases, components (e.g., nucleic acid components (e.g., guide nucleic acids, etc.); protein components (e.g., CAF9 or Cpf1 peptides, variant CAF9 or Cpf1 peptides); etc.) include a labeled portion. As used herein, the terms “label,” “detectable label,” or “labeled portion” refer to any portion that allows signal detection and can vary widely depending on the specific properties of the assay. Labeled portions of interest include directly detectable labels (direct labels) (e.g., fluorescent labels) and indirectly detectable labels (e.g., binding pair members). Fluorescent labels can be any fluorescent label (e.g., fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, ALEXAFLOR labels, 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 any fluorescent derivatives thereof), etc.). Suitable detectable (direct or indirect) labeling portions for the method include any portion detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, suitable indirect labeling includes biotin (a binding pair member) that can be bound by streptoacidin (which itself can be directly or indirectly labeled). Labeling may also include: radioactive labeling (direct labeling) (e.g., 3 H, 125 I, 35 S, 14 C or 32 P); enzymes (indirect labeling) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.); fluorescent proteins (direct labeling) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivative thereof); metal labels (direct labeling); colorimetric labels; binding pair members; etc. A “partner of a binding pair” or “binding pair member” refers to one of the first and second parts, wherein the first and second parts have specific binding affinity for each other. Suitable binding pairs include, but are not limited to: antigen / antibody (e.g., digitoxin / antidigitoxin, dinitrophenyl (DNP) / antiDNP, dansyl-X-antidansyl, luciferin / antiluciferin, fluorescein / antifluorescein, and rhodamine / rhodamine), biotin / antibiotin (or biotin / streptomycin), and calmodulin-binding protein (CBP) / calmodulin. Any binding pair member may be suitable as a labeling moiety that can be detected indirectly.

[0537] Any given component or combination of components may be unlabeled or detectably labeled with a labeled portion. In some embodiments, when two or more components are labeled, they may be labeled with labeled portions that are distinguishable from each other.

[0538] Encapsulation and nanoparticles

[0539] In some embodiments of the composition, the polymer is combined with the nucleic acid and / or peptide, and the nucleic acid and / or peptide are partially or completely encapsulated. In some formulations, the composition may provide nanoparticles comprising the polymer and the nucleic acid and / or peptide.

[0540] In some embodiments, in addition to the polymers and nucleic acids or peptides described herein, the composition may also comprise a core nanoparticle. Any suitable nanoparticles may be used, including metal (e.g., gold) nanoparticles or polymer nanoparticles.

[0541] The polymers and nucleic acids (e.g., guide RNA, donor polynucleotides, or both) or peptides described herein can be directly or indirectly conjugated to the surface of nanoparticles. For example, the polymers and nucleic acids (e.g., guide RNA, donor polynucleotides, or both) or peptides described herein can be directly conjugated to the surface of nanoparticles or indirectly conjugated to the surface of nanoparticles via intercalation linkers.

[0542] Any type of molecule can be used as a linker. For example, a linker can be an aliphatic chain comprising at least two carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or more carbon atoms) and can be substituted with one or more functional groups, including ketones, ethers, esters, amides, alcohols, amines, ureas, thioureas, sulfoxides, sulfones, sulfonamides, and disulfide functional groups. In embodiments where the nanoparticles contain gold, the linker can be any thiol-containing molecule. The reaction of the thiol group with gold produces a covalent sulfide (-S-) bond. Linker design and synthesis are well known in the art.

[0543] In some embodiments, the nucleic acid conjugated to the nanoparticle is a linker nucleic acid, which is used to non-covalently bind one or more elements described herein (e.g., Cas9 peptide and guide RNA, donor polynucleotide, and Cpf1 peptide) to the nanoparticle-nucleic acid conjugate. For example, the linker nucleic acid may have a sequence that hybridizes with the guide RNA or the donor polynucleotide.

[0544] Nucleic acids conjugated to nanoparticles (e.g., colloidal metal (e.g., gold) nanoparticles; nanoparticles containing biocompatible polymers) can have any suitable length. When the nucleic acid is guide RNA or donor polynucleotide, its length is suitable for such molecules, as discussed herein and known in the art. If the nucleic acid is a linker nucleic acid, it can have any suitable length for the linker, such as from 10 nucleotides (nt) to 1000 nt, for example, about 1 nt to about 25 nt, about 25 nt to about 50 nt, about 50 nt to about 100 nt, about 100 nt to about 250 nt, about 250 nt to about 500 nt, or 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) can have a length greater than 1000 nt.

[0545] When a nucleic acid linked (e.g., covalently or non-covalently) to a nanoparticle comprises a nucleotide sequence that hybridizes with at least a portion of a guide RNA or donor polynucleotide present in the complex of this disclosure, it has a region of sequence identity sufficient to promote hybridization with a region complementary to the guide RNA or donor polynucleotide sequence. In some embodiments, the nucleic acid linked to the nanoparticle in the complex of this disclosure has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% nucleotide sequence identity with a complement of 10 to 50 nucleotides (e.g., 10 nucleotides (nt) to 15 nt, 15 nt to 20 nt, 20 nt to 25 nt, 25 nt to 30 nt, 30 nt to 40 nt, or 40 nt to 50 nt) of the guide RNA or donor polynucleotide present in the complex.

[0546] In some embodiments, the nucleic acid linked (e.g., covalently or non-covalently) to the nanoparticle is a donor polynucleotide or has the same or substantially the same nucleotide sequence as the donor polynucleotide. In some embodiments, the nucleic acid linked (e.g., covalently or non-covalently) to the nanoparticle comprises a nucleotide sequence complementary to the donor DNA template.

[0547] How to use

[0548] The polymers provided herein can be used for any purpose, but are considered particularly suitable for use with and for combining with biomolecules (e.g., nucleic acids and peptides) for various purposes, and in some embodiments, can be used to encapsulate biomolecules (e.g., nucleic acids and peptides) for various purposes. In one aspect, a method for encapsulating biomolecules (e.g., peptides or nucleic acids) is provided, said method by combining the polymers of the present invention described herein with said biomolecules, thereby partially or completely encapsulating said biomolecules with said polymers. Biomolecules partially or completely encapsulated by said polymers are sometimes referred to as nanoparticles.

[0549] This document also provides methods for delivering nucleic acids and / or peptides to cells, wherein the cells may be in vitro or in vivo. The methods include administering to the cells or to an individual containing the cells a composition comprising the polymer and nucleic acids and / or peptides as described herein. The methods can be used with any type of cell or individual, but are particularly suitable for mammalian cells (e.g., human cells). In some embodiments, the polymer comprises a targeting agent such that the nucleic acids and / or peptides are delivered primarily or entirely to target cells or tissues (e.g., cells or tissues of the peripheral nervous system, central nervous system, an individual's eye, liver, muscle, lung, bone (e.g., hematopoietic cells), or tumor cells or tissues).

[0550] When used with a composition comprising one or more components of a CRISPR system, the method can be used to induce editing of target nucleic acids or genes. In some embodiments, the method of modifying the target nucleic acid includes homology-directed repair (HDR). In some embodiments, HDR using the complexes of this disclosure 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 greater than 25%. In some embodiments, the method of modifying the target nucleic acid includes non-homologous end joining (NHEJ). In some embodiments, HDR using the complexes of this disclosure 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 greater than 25%.

[0551] The following embodiments further illustrate the present invention, but should not be construed as limiting its scope in any way.

[0552] Example 1

[0553] This embodiment provides guidance for the synthesis of the polymer described herein. The synthesis involves Michael addition with an acrylate. An exemplary method is as follows.

[0554] Route 1.

[0555]

[0556] In a glass vial, pAsp(DET) (5 mg, 0.22 mol) was suspended in dimethyl sulfoxide (“DMSO”; 700 μL). 30 μL of trimethylamine (“TEA”) was added to the suspension, and the mixture was stirred until all polymers were completely dissolved. Acrylate 1 (1.15 mg, 5.5 mol) was added to the reaction mixture, and the mixture was stirred at room temperature for 40 hours. The crude product was purified by precipitation in acetonitrile and washed three times with acetonitrile to obtain 4 mg of polymer 5, where (a+b) = 55 and (c+d) = 25. 1 H NMR (400MHz, D2O): δ4.8-4.6 (bs, 4H), 4.2 (t, 2H), 3.4-2.4 (m, 40H).

[0557] As shown in route 1, Michael addition with acrylate 1 produces amine-based bonds (see, for example, polymer 5). Thus, the original amine functional groups in pAsp(DET) remain intact.

[0558] Other polymers of Formula 1 (e.g., polymers 1-4 and 6-12) can be prepared using similar methods. Nucleophilic substitution can be used to provide polymers 28 and 29.

[0559] Furthermore, this method can be applied to different starting polymers, such as the polymers prepared as shown in Example 7, to provide other polymers of Formula 1 (e.g., starting from polymer 30 or 32, demethylation at the terminal nitrogen to provide a primary or secondary amine, the method can provide polymers 13-24).

[0560] Example 2

[0561] This embodiment provides guidance for the synthesis of the polymer described herein. The synthesis involves Michael addition with an acrylate. An exemplary method is as follows.

[0562] Route 2.

[0563]

[0564] In a glass vial, pASP(DET) (5 mg, 0.22 mol) was suspended in anhydrous methanol (300 μL), and 30 μL of triethylamine (“TEA”) was added to the suspension. The resulting solution was stirred at room temperature for 10 min to completely dissolve the polymer. The solution was diluted with 300 μL of DCM. Acrylate 2 (1.05 mg, 5.5 mol) in 30 μL of DCM was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 48 h. The crude product was purified by precipitation in a large excess of diethyl ether to give 4.3 mg of polymer 8, where (a+b) was 55 and (c+d) was 25. 1 HNMR (400MHz, D2O): δ4.8-4.6(bs,4H),4.03(t,2H),3.8-2.5(m,27H),1.5(t,2H),1.25(s,12H),0.7(t,3H).

[0565] As shown in route 2, Michael addition with acrylate 2 produces amine-based bonds (see, for example, polymer 8). Thus, the original amine functional groups in pAsp(DET) remain intact.

[0566] Example 3

[0567] This embodiment demonstrates the ability of the polymer described herein to deliver Cas9 ribonucleoprotein (“Cas9 RNP”) into cells. Cas9 RNP delivery levels were assessed using green fluorescent protein (“GFP”)-induced HEK293T (“GFP-HEK”) cells.

[0568] 10 pmol of Cas9 RNP was mixed with the following: (i) polymer 5, (ii) polymer 8, and (iii) pAsp (DET) as a control. GFP-HEK cells were treated with the resulting mixture under serum-free conditions. Polymers were added at doses of 0.375 μg, 0.75 μg, 1.25 μg, 2.5 μg, 5 μg, and 10 μg to screen for the optimal dose that yielded the highest efficiency and lowest toxicity. Results are shown in... Figure 3 middle.

[0569] Figure 3The levels of Cas9 RNP delivery, measured by the percentage of GFP(-) in GFP-HEK cells treated with the three mixtures, are shown. At polymer doses ranging from 0.375 μg to 2.5 μg, all three polymers (i.e., polymer 5, polymer 8, and pAsp(DET)) showed similar levels of gene editing. However, at polymer doses of 5 μg and 10 μg, polymer 8 and pAsp(DET) showed significant levels of cytotoxicity (cell viability less than 50%). Polymer 5, however, showed optimal delivery at a polymer dose of 5 μg, as demonstrated by the GFP% level, and exhibited no significant toxicity.

[0570] Example 4

[0571] This embodiment demonstrates the ability of the polymer described herein to deliver Cre recombinase into cells, which can alter the loxP sequence.

[0572] Using a termination sequence between loxPs from ai9 mice (see [link]). Figure 4 In primary myoblasts (gray arrow in the image), the level of delivered Cre recombinase can be measured by RFP expression. Ai9 is reported to have an allele containing a STOP box flanked by a loxP sequence, thus preventing RFP transcription. The Cre recombinase removes the STOP box flanked by the loxP sequence and allows RFP transcription.

[0573] Cre recombinase (2 μg) was mixed with (i) polymer 5 and (ii) pAsp(DET) as controls to generate nanoparticles. Polymers were added at doses of 1.25 μg, 2.5 μg, 5 μg, 10 μg, and 20 μg to screen for the optimal dose yielding the highest efficiency and lowest toxicity. Ai9 myoblasts were treated with the resulting nanoparticles, and the RFP+ population was quantified by flow cytometry 4 days post-treatment. Results are shown in… Figure 5 middle.

[0574] Figure 5 This shows the level of Cre recombinase delivery to primary myoblasts, as measured by RFP+ expression levels. Figure 5 As shown, at polymer doses of 5 μg, 10 μg, and 20 μg, polymer 5 produced higher levels of RFP+ expression than the control (pAsp(DET)), thus demonstrating that polymer 5 is more effective than pAsp(DET) at higher doses in Cre recombinase delivery.

[0575] Example 5

[0576] This embodiment demonstrates the ability of the polymer described herein to deliver Cre recombinase in mice.

[0577] Ai9 mice were injected with (i) Cre recombinase alone and (ii) PEGylated polymer nanoparticles encapsulating Cre recombinase (i.e., a mixture of Polymer 5 and PEG-polymer). Cre recombinase (35 μg) was delivered together with a mixture of Polymer 5 and PEG-PAsp (DET) (100 μg). The co-mixing of Polymer 5 and PEG-PAsp (DET) helped control the size of the polymer nanoparticles. Gastrocnemius muscle was harvested from ai9 mice two weeks after injection. Cross-sections of the harvested muscle were imaged to visualize the red fluorescent protein generated due to DNA recombination. Results are shown in... Figure 5 middle.

[0578] Figure 6 This indicates that the delivery of Cre recombinase in mouse muscle is enhanced by polymer nanoparticles. Injection of Cre recombinase alone resulted in RFP expression in a limited area of ​​muscle, as evidenced by the reduced visualization of red fluorescent protein. However, injection of Cre recombinase encapsulated in polymer nanoparticles showed RFP expression in a large area of ​​the gastrocnemius muscle.

[0579] A challenge in protein delivery is how proteins can be delivered extensively and affect large areas of tissue. Nanoparticles derived from a mixture of polymer 5 and PEG-polymers can efficiently deliver Cre recombinase and facilitate its distribution in ai9 mice.

[0580] Example 6

[0581] This embodiment demonstrates the ability of the polymer described herein to deliver Cas9 to GFP-expressing neurons.

[0582] This study investigated whether polymer 5 could deliver Cas9 RNPs into neurons using GFP-expressing neurons differentiated from neuronal progenitor cells. Using the polymer described herein as the delivery method, GFP-expressing neurons were treated with sgRNA and Cas9 protein. After 7 days of treatment, genomic DNA was extracted from the cells, and the GFP gene was amplified by PCR. TIDE analysis (TIDE software from Desktop Genetics, Netherlands Cancer Institute) was performed to measure the frequency of indel mutations resulting from gene editing by Cas9. Results are shown below. Figure 7 middle.

[0583] like Figure 7 As demonstrated, polymer 5 is able to deliver Cas9 RNPs and induce 11% indel mutations in neuronal cells.

[0584] Example 7

[0585] This embodiment provides guidance for the synthesis of the polymer described herein. The synthesis involves ring-opening polymerization, followed by further modification to produce the polymer of Formula 4. An exemplary method is as follows.

[0586] Route 3.

[0587]

[0588] Synthesis of polymer 33: (a) toluenesulfonyl chloride, TEA, DCM; (b) N,N,N-trimethylethylenediamine, K2CO3, acetonitrile, reflux; (c) LiAlH4, THF; (d) 7, K2CO3, acetonitrile, reflux; (e) LiAlH4, THF; (f) butylamine, DCM-DMF (9:1), 48 h; (g) 9, NMP, 6 h.

[0589] As shown in route 3, the ring-opening polymerization of compound 10 leads to growth to form compound 11 with n=63, which can be further modified to form polymer 33 when treated with compound 9.

[0590] Similar methods can be used to prepare other polymers of Formula 4 (e.g., polymers 30-32). Additionally, the terminal tertiary amines on the polymer side chains can be demethylated using conventional techniques to provide polymers 39-42.

[0591] Example 8

[0592] This embodiment demonstrates the ability of the polymer described herein to deliver Cre recombinase into cells, which can alter the loxP sequence.

[0593] Traffic light reporter (TLR)-HEK 293T cells were used, which were generated in HEK 293T cells via viral transduction of the traffic light reporter (see Traffic Light Reporter). Figure 8 The level of delivered Cre recombinase can be measured by the expression of red fluorescent protein (“RFP”). The traffic light reporter in HEK 293T contains a STOP box and two loxP sequences flanking the GFP sequence, thereby preventing RFP transcription and sequentially expressing GFP in the absence of Cre. The Cre recombinase removes the loxP sequences and allows RFP transcription.

[0594] Cre recombinase (1 μg) was mixed with polymer 33 prepared in Example 7 or pAsp (DET) as a control to generate nanoparticles. Polymer was added at doses of 1.25 μg or 2.5 μg to screen for the optimal dose yielding the highest efficiency and lowest toxicity. TLR-HEK293T cells were treated with the resulting nanoparticles, and the RFP+ population was quantified by flow cytometry 3 days after treatment. Results are shown in… Figure 9 middle.

[0595] Figure 9 The level of Cre recombinase delivered to (TLR)-HEK 293T cells is shown as a measure of the amount of RFP+ expression. Figure 9 As shown, polymer 33 provides enhanced delivery at polymer doses of 1.25 μg and 2.5 μg, compared to samples containing only Cre and samples containing the comparative compound pAsp (DET).

[0596] Example 9

[0597] This embodiment demonstrates the ability of the polymer described herein to deliver Cas9 ribonucleoprotein (“Cas9 RNP”) into cells. Cas9 RNP delivery levels were assessed using green fluorescent protein (“GFP”)-induced HEK 293T (“GFP-HEK”) cells.

[0598] 15 pmol of Cas9 RNP (sgRNA + Cas9 protein) was mixed with polymer 33 prepared in Example 7 or pAsp (DET) as a control to generate nanoparticles. The polymer was added at a dose of 2.5 μg. GFP-HEK cells were treated with the resulting mixture under serum-free conditions. The results are shown in… Figure 10 middle.

[0599] Figure 10 The Cas9 RNP delivery level is shown as measured by GFP% in GFP-HEK cells treated with the two mixtures. Both polymers (i.e., polymer 33 and pAsp(DET)) demonstrated the ability to deliver Cas9 RNPs at a polymer dose of 2.5 μg, relative to the control. However, at a polymer dose of 2.5 μg, polymer 33 outperformed pAsp(DET).

[0600] Example 10

[0601] This example demonstrates the ability of the polymer described herein to deliver nucleic acids. The level of eGFP mRNA delivery to HEK 293T cells was assessed using green fluorescent protein (“GFP”).

[0602] eGFP mRNA (200 ng) was mixed with Polymer 33 (600 ng) prepared in Example 7 and incubated for 5 min. The resulting polymer nanoparticles were then used to treat HEK 293T cells in an optimized culture medium. After 24 hours, the cells were isolated from the plate and analyzed by flow cytometry. Lipofectamine was used as a positive control for eGFP mRNA delivery. Results are shown in... Figure 11 middle.

[0603] Figure 11Compared to the control, polymer 33 provided increased delivery of eGFP mRNA to HEK 293T cells.

[0604] Example 11

[0605] This example demonstrates the effect of incubation time on the ability of the polymers described herein to deliver nucleic acids. The level of eGFP mRNA delivery to HEK 293T cells was assessed using green fluorescent protein (“GFP”).

[0606] eGFP mRNA (200 ng) was mixed with polymer 33 (1.2 μg) prepared as in Example 7 and incubated for 2 min, 5 min, 10 min, and 30 min. The resulting polymer nanoparticles were then used to treat HEK 293T cells in OptiMEM. After 24 hours, the cells were separated from the plate and analyzed by flow cytometry. Lipofectamine was used as a positive control for eGFP mRNA delivery. Results are shown in... Figure 12 middle.

[0607] Figure 12 The nanoparticles and polymer 33 formed within 2 minutes of incubation provide efficient delivery of eGFP mRNA throughout all incubation times.

[0608] Example 12

[0609] This example demonstrates the ability of the polymer described herein to deliver mRNA. RFP+ expression was used to assess the level of red fluorescent protein (RFP) mRNA delivery to HEK 293T cells.

[0610] RFP mRNA (200 ng) was mixed with either polymer 33 or pAsp(DET) prepared in Example 7 to prepare nanoparticles. Polymer 33 and pAsp(DET) were added at a dose of (i) 600 ng, or (ii) a combination of 480 ng and 120 ng of 1.5 kDa PEG-PAsp(DET) polymer. The 1.5 kDa PEG-PAsp(DET) polymer helped in controlling the size of the nanoparticles. TLR-HEK293T cells were treated with the resulting nanoparticles, and the RFP+ population was quantified by flow cytometry 24 hours after treatment. Results are shown in… Figure 13 middle.

[0611] Figure 13 The results showed that, compared to the control polymer (PAsp(DET)), polymer 33 improved mRNA delivery to HEK 293T cells in the absence of the 1.5 kDa PEG-PAsp(DET) polymer. Additionally, Figure 13The results showed that 600 ng of polymer 33 provided more efficient mRNA delivery to HEK 293T cells than the combination of 480 ng of polymer 33 and 120 ng of 1.5 kDa PEG-PAsp (DET) polymer.

[0612] Example 13

[0613] This example demonstrates the ability of the polymer described herein to deliver mRNA. RFP+ expression was used to assess the level of red fluorescent protein (RFP) mRNA delivery to HEK 293T cells.

[0614] RFP mRNA (200 ng) was mixed with either polymer 33 or pAsp(DET) prepared in Example 7 to prepare nanoparticles. Polymer 33 and pAsp(DET) were added at (i) a dose of 1 μg or (ii) a dose of 800 ng combined with 200 ng of a 1.5 kDa PEG-PAsp(DET) polymer. The 1.5 kDa PEG-PAsp(DET) polymer helped in controlling the size of the nanoparticles. TLR-HEK293T cells were treated with the resulting nanoparticles, and the RFP+ population was quantified by flow cytometry 24 hours after treatment. The results are shown in… Figure 14 middle.

[0615] Figure 14 The results show that, relative to the control polymer (PAsp(DET)), polymer 33 delivered comparable mRNA to HEK 293T cells in both the presence and absence of 1.5 kDa PEG-PAsp(DET) polymer, at a dose of (i) 1 μg or (ii) 800 ng combined with 200 ng of 1.5 kDa PEG-PAsp(DET) polymer.

[0616] Example 14

[0617] This embodiment uses different buffers to demonstrate the ability of the polymer described herein to deliver Cas9 ribonucleoprotein (“Cas9RNP”) into cells. Cas9 RNP delivery levels were assessed using green fluorescent protein (“GFP”)-induced HEK 293T (“GFP-HEK”) cells.

[0618] Nanoparticles were prepared by mixing 30 pmol of Cas9 RNP (sgRNA + Cas9 protein) with 4 μg of Polymer33 prepared in Example 7 or 5 μg of pAsp(DET) as a control. GFP-HEK cells (200,000 cells) were treated under three different buffer conditions: (i) (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) (“HEPES”; 20 mM), (ii) Opti-MEM. TM (Available from Thermo Fisher Scientific; Waltham, MA) or Dulbecco's Modified Eagle's Medium (“DMEM”; available from Thermo Fisher Scientific; Waltham, MA). Results are shown in Figure 15 middle.

[0619] Figure 15 The levels of Cas9 RNP delivery, measured by the percentage of GFP(-) in GFP-HEK cells treated with two mixtures (i.e., 4 μg polymer 33 or 5 μg pAsp(DET))), are shown under three different buffer conditions. Both polymers (i.e., polymer 33 and pAsp(DET)) showed the same trend across the three different buffers, with HEPES showing superiority over Opti-MEM. TM Opti-MEM TM It is superior to DMEM. Furthermore, Polymer33 outperforms pAsp(DET) in all three buffers.

[0620] Example 15

[0621] This embodiment demonstrates the ability of the polymer described herein to stably encapsulate nucleic acids.

[0622] Polymers 2, 5, and 33 of this disclosure (1 μg / μL, 10 mM HEPES) were pipetted together with oligonucleotides (1 μg / μL, 10 mM HEPES) at a 5:1 mass ratio and then left to stand at room temperature for 10 minutes to form nanoparticles. When not in use, they were stored at 4°C. The nanoparticles from the stock solution were diluted with 10 mM HEPES at a ratio of approximately 1:20 and characterized by dynamic light scattering (DLS) at 0, 3, 5, and 7 days (n=1) after preparation. Intensity-average particle size was shown in... Figure 18 middle.

[0623] Nanoparticle sizes ranging from 150 nm to 350 nm were observed. The polymer showed minimal size change over 7 days, indicating that the nanoparticles are stable. pAsp[DET], run as a control (data not shown), showed slightly larger size change by day 7 compared to other tested nanoparticles.

[0624] Example 16

[0625] This embodiment illustrates the preparation of a polymer according to the present disclosure.

[0626] 100 mg (0.0074 mmol) of poly(β-1-aspartic acid benzyl ester) (PBLA) was dissolved in 3 mL of NMP. 1.5 g m of 1,4,7,10-tetramethyl-triethylenetetramine was added to the reaction mixture, and the reaction was stirred at RT for 16 h. The crude reaction mixture was precipitated in diethyl ether, and the crude product was collected by centrifugation (5000 g, 15 min). The crude product was dissolved in 3 mL of 1 M HCl and dialyzed with water to obtain pH = 6. The resulting polymer solution was lyophilized to obtain polymer 41 as a white powder.

[0627] Example 17

[0628] This example illustrates the use of the polymer described herein for delivering mRNA to cells.

[0629] mRNA encoding green or red fluorescent proteins was mixed with the test polymer and combined with one of several different cell types as shown in Table 2. Transfection was measured as a function of fluorescence. The results are shown in Table 2, which demonstrate that almost all polymers produced a certain level of transfection in at least one cell type.

[0630] Table 2

[0631]

[0632] N / A = Untested

[0633] Simulated experiment = polymer without mRNA

[0634] Mouse primary myoblasts were tested using mRNA encoding green fluorescent protein. All other cell types were detected using mRNA encoding red fluorescent protein.

[0635] Preferred embodiments of the invention have been described herein, including the best modes known to the inventors for carrying out the invention. Variations of those preferred embodiments will be apparent to those skilled in the art from the foregoing description. The inventors intend that those skilled in the art will appropriately employ these variations, and the inventors intend that the invention be practiced in a manner different from that specifically described herein. Therefore, the invention includes all modifications and equivalents to the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, the invention covers any combination of the foregoing elements in all possible variations, unless otherwise stated herein or clearly contradicted by the context.

[0636] Where numerical ranges are provided, it should be understood that, unless the context clearly indicates otherwise, every intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other specified or intermediate value within the specified range, are included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also covered within the scope of this invention, subject to any specific exclusions within the ranges. The ranges described include one or both limits, and ranges excluding any or both of those included limits are also included in this invention.

[0637] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are described hereafter. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials relating to the methods and / or materials referenced in those publications.

[0638] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly specifies otherwise. Thus, for example, reference to “complex” includes multiple such complexes, and reference to “Cas9 polypeptide” includes reference to one or more Cas9 polypeptides and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims may be drafted to exclude any optional elements. Similarly, this statement is intended to serve as a prior basis for the use of exclusive terms such as “uniquely” in relation to the statement of a claim element, or for the use of a negative limitation.

[0639] It should be understood that, for clarity, certain features of the invention described in the context of separate embodiments 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 brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments relating to the invention are specifically included by the invention and disclosed herein, just as each and every combination is individually and explicitly disclosed. Furthermore, the invention particularly includes all sub-combinations of various embodiments and elements thereof, and discloses them herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0640] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. This document should not be construed as an admission that the invention is not entitled to any prior disclosure due to a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification.

Claims

1. A polymer having a structure comprising formula 4: in, m 1 and n 1 Each is an integer between 0 and 1000; the condition is m. 1 +n 1 The sum is greater than 2; The symbol " / " indicates that the units separated by it are connected randomly or in any order; A 1 and A 2 Each is an independent group of the following formula -(CH2) p1 -[NO 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 -] r2 R 2 };or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2, Where p1 to p4, q1 to q6, and r1 and r2 are each independent integers from 1 to 5; R 2 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups, provided that A 1 and A 2 Each contains at least one tertiary amine; R 3a and R 3b Each is independently a methylene or ethylene group; and R 13 Each occurrence of is independently either hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups, or cycloalkenyl groups.

2. The polymer according to claim 1, wherein group A 1 and A 2 Each contains at least two tertiary amines.

3. The polymer according to claim 1, wherein, In group A 1 and A 2 In this context, except for the terminal amine being a primary, secondary, or tertiary amine, each contains R. 2 The nitrogen in the substituent is a tertiary amine.

4. The polymer according to claim 1, wherein, In group A 1 and A 2 In the middle, except for A 1 and A 2 The terminal amine is either a primary, secondary, or tertiary amine, R 2 Each occurrence of is either ethyl or methyl.

5. The polymer according to claim 1, wherein in group A 1 and A 2 In this context, the terminal amine is either a secondary or tertiary amine.

6. The polymer according to claim 1, wherein R 13 It is hydrogen.

7. The polymer according to claim 1, wherein R 13 It is a methyl group.

8. The polymer according to claim 1, having the structure of formula 4A: in, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; R 6 It is hydrogen, amino group, aryl group, heterocyclic group, C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, optionally substituted with one or more amines, C1-C 12 Straight-chain or branched alkyl groups; or tissue-specific or cell-specific targeting portions.

9. The polymer according to claim 1, wherein A 1 and A 2 Each is a separate formula -(CH2)2-NR 2 -(CH2)2-NR 2 2 groups, wherein R 2 Each occurrence of is independently C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups or cycloalkenyl groups, except that the terminal amine is a primary amine, secondary amine or tertiary amine.

10. The polymer according to claim 6, wherein group A 1 and A 2 R 2 Each occurrence is either ethyl or methyl, except for group A. 1 and A 2 The terminal amine is a primary amine, a secondary amine, or a tertiary amine.

11. The polymer according to claim 1, wherein R 2 Each occurrence of is a methyl group.

12. The polymer according to claim 6, wherein R 2 Each occurrence of is a methyl group.

13. The polymer of claim 8, wherein the tissue-specific or cell-specific targeting portion is: Where R 9 、R 10 、R 11 and R 12 Each is independently hydrogen, halogen, or a C1-C4 alkyl or C1-C4 alkoxy group optionally substituted with one or more amino groups.

14. The polymer according to any one of claims 1-13, wherein the polymer is a cationic polymer.

15. The polymer according to claim 1, having the following formula: Where (a+b) ranges from 5 to 160.

16. The polymer according to claim 1, having the following formula: Where (a+b) is between 5 and 75, and (c+d) is between 5 and 80.

17. A composition comprising the polymer of any one of claims 1-16, and nucleic acids and / or polypeptides.

18. The composition of claim 17, wherein the composition comprises a guide nucleic acid and / or a donor nucleic acid.

19. The composition of claim 17, wherein the composition comprises a nuclease.

20. The composition of claim 19, wherein the composition comprises an RNA-guided endonuclease.

21. The composition of claim 20, wherein the RNA-guided endonuclease is Cas9, Cpf1, or a combination thereof.

22. The composition of claim 17, wherein the composition comprises a nucleic acid encoding an RNA-guided endonuclease.

23. The composition of claim 22, wherein the RNA-guided endonuclease is Cas9, Cpf1, or a combination thereof.

24. The composition of claim 17, wherein the composition comprises a DNA recombinase.

25. The composition of claim 24, wherein the DNA recombinase is Cre recombinase.

26. The composition of claim 17, wherein the composition comprises a zinc finger nuclease.

27. The composition of claim 17, wherein the composition comprises a transcription activator-like effector nuclease.

28. The composition of claim 17, wherein the polymer partially or completely encapsulates the nucleic acid and / or polypeptide.

29. The composition of claim 17, wherein the composition comprises a second polymer comprising polyethylene oxide.

30. Use of the composition of any one of claims 17-29 in the preparation of a pharmaceutical agent for delivering nucleic acids and / or polypeptides to cells.

31. The use according to claim 30, wherein the cell is in an individual.

32. The use according to claim 31, wherein the polymer comprises a tissue-specific targeting portion that positions the polymer to tissues of the individual’s peripheral nervous system, central nervous system, liver, muscle, lungs, bone, or eye.

33. The use according to claim 31, wherein the polymer comprises a targeting portion that preferentially binds to tumor cells.

34. The use according to claim 30, wherein the composition comprises one or more of an RNA-guided endonuclease or a nucleic acid encoding an RNA-guided endonuclease, a guide nucleic acid, and a donor nucleic acid, and the composition promotes the editing of a target gene in the cell.

35. The use according to claim 34, wherein the cell is in a host.

36. The use according to claim 35, wherein the host is a human being.

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