Cationic polymer and its use for biomolecule delivery

Abstract

To provide a composition that enables safe and efficient delivery of large molecules (e.g., polypeptides and nucleic acids) to their target tissues.SOLUTION: Provided are a cationic polymer comprising a structure of Formula (1), a method of preparing the polymer, and a composition comprising the polymer and a nucleic acid and / or polypeptide.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture of U.S. Provisional Patent Application No. 62 / 663,985, filed April 27, 2018, and U.S. Provisional Patent Application No. 62 / 663,985, filed October 27, 2018. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 750,097, filed on May 24, 2013, the entire disclosure of which is incorporated herein by reference.

[0002] INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIAL The computer readable nucleotide / amino acid sequence disclosed herein is The string is incorporated herein by reference and is identified as follows: One 62527 Byte ASCII (Text) file named "512879.TXT," created on April 29, 2019. [Background technology]

[0003] 2. Background of the Invention Peptide, protein, and nucleic acid-based technologies have a myriad of applications for preventing, curing, and treating disease. However, the safe and efficient delivery of large molecules (e.g., polypeptides and nucleic acids) to their target tissues remains problematic. Thus, there is a continuing need for new compositions and methods useful for the delivery of therapeutic molecules. Summary of the Invention [Means for solving the problem]

[0004] Brief Summary of the Invention As used herein, formula 1:

[0005] [ka]

[0006] During the ceremony: m 1 and n 1Each of is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on B 1 and B. 2 are each independently -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 }; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2 and where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independently an integer of 1 to 5; 2 are independently hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkenyl groups R is an alkyl group, or a cycloalkenyl group; 4is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 are independently a substituted or unsubstituted alkyl group, cycloalkyl group, alkenyl group, cycloalkenyl group, aryl group, heteroalkyl group, heterocyclic group, or combinations thereof, optionally containing 1 to 8 (e.g., 2 to 8) secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety; A polymer comprising the structure:

[0007] Also, Equation 2:

[0008] [ka]

[0009] During the ceremony: m 1 and n 1 Each of m is an integer from 0 to 1000, 1 and n 1 The total is between 10 and 2000. the law of nature; c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; R 1 is hydrogen, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, or C1-C 12 Linear or branched alkyl groups the law of nature; R in each case 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 8 is a tissue- or cell-specific targeting moiety; and R in each case 13 are independently hydrogen or C1-C12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A polymer of the formula is provided.

[0010] The present disclosure also provides compositions containing the polymers of the structure of Formula 1 and / or the structure of Formula 2, as well as nucleic acids and / or polypeptides. Methods of preparing the polymers comprising the structure of Formula 1 or the polymers of Formula 2, as well as methods of using the polymers and compositions containing the polymers, for example, to deliver nucleic acids or proteins to cells, are further provided. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 provides the amino acid sequence of Cas9 from Streptococcus pyogene (SEQ ID NO:1). [Diagram 2] FIG. 2 provides the amino acid sequence of Cpf1 from Francisella tularensis subsp. Novicida U112 (SEQ ID NO:2). [Diagram 3] FIG. 3 is a graph of the level of Cas9 RNP delivery as measured by % green fluorescent protein ("GFP") negative in GFP-HEK cells at different dosages of polymer / Cas9 composition. [Figure 4] FIG. 4 is a schematic representation of the ai9 reporter allele. [Diagram 5] FIG. 5 shows the level of Cre recombinase delivery to primary myoblasts as measured by the amount of RFP+ expression at different concentrations of polymer / Cre compositions. [Figure 6] FIG. 6 illustrates the levels of Cre recombinase delivery in mouse muscle visualized by red fluorescent protein (lighter areas indicate fluorescence). [Figure 7] FIG. 7 shows indel mutation rates in human neural stem cells demonstrated by Cas9 delivery. [Figure 8]FIG. 8 is a schematic representation of the traffic light reporter in HEK 293T. [Figure 9] FIG. 9 shows the level of Cre recombinase delivery to TLR-HEK 293T as measured by the amount of RFP+ expression at different concentrations of polymer / Cre compositions. [Figure 10] FIG. 10 is a graph of the level of Cas9 RNP delivery as measured by % green fluorescent protein ("GFP") in GFP-HEK cells at different dosages of polymer / Cas9 composition. [Figure 11] FIG. 11 illustrates the level of eGFP mRNA delivery into HEK 293T cells as measured by GFP %. [Figure 12] FIG. 12 shows the incubation time dependence of the level of eGFP mRNA delivery into HEK 293T cells as measured by GFP %. [Figure 13] FIG. 13 illustrates the level of red fluorescent protein ("RFP") mRNA delivery into HEK 293T cells as measured by RFP+ expression in both the presence and absence of 1.5 kDa PEG-PAsp(DET) polymer. [Figure 14] FIG. 14 illustrates the levels of red fluorescent protein ("RFP") mRNA as measured by RFP+ expression into HEK 293T cells both in the presence and absence of 1.5 kDa PEG-PAsp(DET) polymer. [Figure 15] FIG. 15 is a graph of the level of Cas9 RNP delivery as measured by % green fluorescent protein ("GFP") in GFP-HEK cells for polymer / Cas9 nanoparticles in buffer. [Figure 16] FIG. 16 provides the sequence of AsCpf1 (SEQ ID NO:19). [Figure 17] FIG. 17 provides the sequence of LbCpf1 (SEQ ID NO:20). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention The present invention provides a polymer comprising a structure of Formula 1:

[0013] [ka]

[0014] During the ceremony: m 1 and n 1 Each of is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2)q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on B 1 and B. 2 are each independently -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p3 -N{[-(CH2) q3 -NR2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2 and Where: p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independently an integer of 1 to 5; R 2 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group group, or a cycloalkenyl group; R in each case 4 are independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; And R in each case 5 are independently arranged as needed, with 1-8 or 2-8 second or third class nets. The substituents are substituted or unsubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, aryl, heteroalkyl, heterocyclic groups, or combinations thereof, including those containing an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or combinations thereof, or those containing a tissue- or cell-specific targeting moiety.

[0015] As used herein, "alkyl" refers to a substituted or unsubstituted hydrocarbon chain. An alkyl group can have any number of carbon atoms (e.g., C1-C6 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 can be saturated, or in some embodiments, unsaturated to any degree (e.g., providing an alkenyl or alkynyl group), and can be linear, branched, straight-chain, cyclic (e.g., cycloalkyl or cycloalkenyl), or combinations thereof. Cyclic groups can be monocyclic, fused to form bicyclic or tricyclic groups, linked by bonds, or spirocyclic. Cyclic groups are groups that have at least three carbons (e.g., , C3-C 12 , C3-C 10, C3-C8, C3-C6, or C5-C6). In some embodiments, the hydrocarbon chain can be interrupted by one or more heteroatoms (e.g., 1, 2, 3, 4, or 5 or more oxygen, nitrogen, and / or sulfur atoms), thereby providing a heteroalkyl, heteroalkenyl, heteroalkynyl, or heterocyclyl (i.e., a heterocyclic group). In some embodiments, the alkyl, alkenyl, alkynyl is substituted with one or more substituents.

[0016] The term "heterocyclyl," or "heterocyclic group," refers to a cyclic group, e.g., an aromatic (e.g., aryl) or cyclic group having one or more heteroatoms (e.g., 1, 2, 3, 4, or 5 or more acids In some embodiments, a heterocyclyl or heterocyclic group (i.e., a cyclic group, e.g., an aromatic (e.g., heteroaryl) group) is a non-aromatic group having carbon, nitrogen and / or sulfur atoms. or a non-aromatic ring having one or more heteroatoms) is substituted with one or more substituents. do.

[0017] The term "aryl" refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups can contain any suitable number of ring atoms, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 ring atoms, as well as 6 to 10, 6 to 12, or 6 to 14 ring members. The aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or linked by bonds to form biaryl groups. Representative aryl groups include phenyl, naphthyl, and biphenyl. In some embodiments, the aryl groups include an alkylene linking group, thereby forming an arylalkyl group (e.g., a benzyl group). Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl. In some embodiments, the aryl substituents can include one or more heteroatoms (e.g., 1, 2, 3, 4, or or five or more oxygen, nitrogen and / or sulfur atoms), thereby forming a heteroaryl In some embodiments, the aryl is substituted with one or more substituents.

[0018] Any of the foregoing groups may be monovalent, divalent, or polyvalent (e.g., alkylene, alkenylene, alkynyl, It is understood that the heteroaryl group can be heteroaryl, aryl, aryl, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocyclylene, heteroarylene, etc.

[0019] As used herein, the term "substituted" may mean that one or more hydrogens on the specified atom or group (e.g., a substituted alkyl group) are replaced with another group, provided that the normal valence of the specified atom is not exceeded. For example, if the substituent is oxo (i.e., -O), then two hydrogens on the atom are replaced. Substituents may be one or more of aldehydes, esters, amides, ketones, nitro, cyano, fluoroalkyl (e.g., trifluoromethyl), methyl, ethyl ... halo (e.g., fluoro), aryl (e.g., phenyl), heterocyclyl or heterocyclic group (e.g., That is, it may include cyclic groups, for example, aromatic (e.g., heteroaryl) or non-aromatic, where the cyclic group has one or more heteroatoms), oxo, or combinations thereof. Combinations of substituents and / or variables are permissible provided that the substituents do not significantly adversely affect the synthesis or use of the compound.

[0020] According to Equation 1, m 1 and n 1 Each of m is an integer from 0 to 1000, for example, 0-500, 0-200, 0-100, or 0-50. 2 and n 2 Each of m 2 + n 2 If the sum of is greater than 5 In other words, the polymer is an integer from 0 to 1000, e.g., 0-500, or 0-100 (e.g., 5-2000, 5-1000, 5-500, 5-200, 5-100, or 5-50), provided that 1 and / or B. 2 Includes At least some of the monomer units are collectively referred to herein as "B monomers." In some embodiments, m 1 and n 1 is 0, so that the polymer is A 1 Or A 2 In another embodiment, the polymer does not contain any A groups. 1 and / or A 2 Base and And some B 1 and / or B. 2 In such embodiments, (m 1 +n 1 ) / (m 2 +n 2 ) 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). In some embodiments, (m 1 +n 1 ) / (m 2 +n 2 ) ratio is about 0.2 or more, for example about 0.5 or more. do.

[0021] The polymer may be of any suitable structural type. For example, the polymer may be an alternating polymer, a random polymer, a block polymer, a graft polymer, a linear polymer, a branched polymer, a cyclic polymer, or a combination thereof. In some embodiments, the polymer is a random polymer, a block polymer, a graft polymer, or a combination thereof.

[0022] Thus, in the structure of Formula 1, the monomers (with their respective side chains A 1 , A 2 , B 1 , and B. 2The integers m may be arranged randomly or in any order. 1 , n 1 , m 2 , and n 2 simply indicates the number of each monomer that occurs in the entire chain, and this Although not necessarily representing a block of monomers from each other, a block or stretch of a given monomer may be depicted in some embodiments. For example, the structure of Formula 1 may be represented by -A 1 -A 2 -B 1 -B 2 -, -A 2 -A 1 -B 2 -B 1 -, -A 1 -B 1 -A 2 -B 2 - etc. In some embodiments, the polymer may comprise a polypeptide arranged in an alpha / beta configuration (e.g., polyaspartamide). backbone, resulting in alternating "1" and "2" monomers (e.g., -A 1 -A 2 -B 1 -B 2 -, -A 2 -A 1 -B 2 -B 1 -, -A 1 -B 2 -B 1 -A 2 -, -A 2 -B 1 -B 2 -A 1 -, -B 1 -A 2 -B 1 -A 2 -, etc.), but the "A" and "B" monomers or side chains (e.g., A 1 / A 2 and B. 1 / B 2 ) are randomly distributed throughout the polymer backbone They may be distributed or arranged in blocks, or some combination thereof.

[0023] By way of further explanation, the polymer may alternatively be represented by formula 1.1:

[0024] [ka]

[0025] or, if the backbone is in the alpha, beta configuration, formula 1.2:

[0026] [ka]

[0027] It can be written as During the ceremony, m 1 and n 1 is as in the previous period; m 3 is an integer between 5-2000 (e.g., 5-1000, 5-500, 5-100, 25-2000, 25-500, 25-100, 50-2000, 50-1000, 50-500, or 50-100); The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; Each A 3 is an independent formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 22]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2 }; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 }; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR2 -CH2-CHOH-R 5 ; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or -(CH2)p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2 Based on wherein p1 to p4, q1 to q6, r1 and r2, and s1 to s4 each independently represent an integer of 1 to 5; R in each case 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R in each case 4 is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R in each case 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 1-8 or 2-8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety; However, 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 at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even 100%) A 3 The basis is -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R4 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5 }; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2-N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2 Selected from.

[0028] In yet another embodiment, the polymer comprises some or even a majority of A 3 Base (at least 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%), 3 The basis is -(CH2) p1 -[NR 2-(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Selected from.

[0029] In any of the foregoing polymer structures, R 3a and R 3b are each independently a methylene or ethylene group. 3a is an ethylene group, and R 3b is a methylene group; or R 3a is a methylene group, and R 3b is an ethylene group In one embodiment, R 3a and R 3b are each an ethylene group. In a preferred embodiment, R 3a and R 3b are each a methylene group.

[0030] Base A 1 and A 2 are independently selected and therefore may be the same or different from one another. Similarly, the groups B 1 and B. 2 are independently selected and therefore may be the same or different from each other. 3 is another A 3 It may be the same as or different from the group.

[0031] Base A 1 , A 2 , A 3 , B 1 , and B. 2 In the above, 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 an integer of 1 to 5 (e.g., 1, 2, 3, 4, or 5). In some embodiments, integers 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 an integer of 1 to 3 (e.g., 1, 2, or 3). In some embodiments, p1 to p4 (i.e., p1, p2, p3, and p4) and / or In some cases, q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6) are 2. In an embodiment, p1 to p4 (i.e., p1, p2, p3, and p4) and / or q1 to q6 (i.e., q1, q2, q3, q4, q5, and q6) are each 2, and r1 and r2 are each 1. In some embodiments, s1-s4 are each independently an integer of 1, 2, 3, 4, or 5, e.g., 1, 2, or 3, or 1-2.

[0032] In the polymer structure, R in each case 4 is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-. In some embodiments, each occurrence of R 4 are independently -C(O)O- or -C(O)NH- In one embodiment, each occurrence of R 4 is -C(O)O-. In some embodiments, R in combination 4 is -C(O)NH-.

[0033] In some embodiments of Formula 1, A 1 and A2 Each of the formula -(CH2) p1 -[NH-(CH2) q1 -] r1 NH2, for example the group -(CH2)2-NH-(CH2)2-NH2. Additionally or alternatively, B 1 and B. 2 Each of the formula -(CH2) p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 groups such as the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-R 4 -R 5 or the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR 5 It is. In some embodiments, B 1 and B. 2 Each of the 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 where each R 2 are independently C1-C3 alkyl (e.g., methyl), and optionally where R 5 is C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 is -CH2-NH-CH3 or -CH2-N-(CH3)2).

[0034] In some embodiments of Formula 1.1, A 3 is the formula -(CH2) p1 -[NH-(CH2) q1 -] r1 a group of NH2, such as the group -(CH2)2-NH-(CH2)2-NH2; or a group of the formula -(CH2)p1 -[NH-(CH2) q1 -] r1 NH-(CH2)2-R 4 -R 5 Based on For example, the group -(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 where R is the number of Rs as needed. 2 are independently C1-C3 alkyl (e.g., methyl), and optionally where R 5 is C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 is -CH2-NH-CH3 or -CH2-N-(CH3)2); provided 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 At least about 90%, at least about 95%, or even 100%) of A 3 The 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)-OR5 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 where R is the number of Rs as needed. 2 are C1-C3 al alkyl (e.g., methyl), and where R is an integer of 1 to 5, 5 is C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 is -CH2-NH-CH3 or -CH2-N-(CH3)2). Further, in some embodiments, the polymer may have some or even a majority of A 3 Base (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%); 3 The 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 where R is the number of Rs as needed. 2 are independently C1-C3 alkyl (e.g., methyl), and optionally where R 5 is C1-C3 alkylamino or C1-C3 dialkylamino (e.g., R 5 is -CH2-NH-CH3 or -CH2-N-(CH3)2).

[0035] Furthermore, in the polymer structure, R in each case 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 1 to 8 (or 2 to 8) secondary or tertiary amines. 5 R can be substituted or unsubstituted. 5 Also , a substituent that includes a tissue-specific or cell-specific targeting moiety. In some embodiments, R 5 may contain about 1 to about 50 carbon atoms (e.g., about 2 to about 50 carbon atoms, e.g., about 2 to about 40 carbon atoms, about 2 to about 30 carbon atoms, about 2 to about 20 carbon atoms, about 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 1-8 or 2-8 (i.e., 1, 2, 3, 4, 5, 6, 7, 8 The secondary or tertiary amine is a heteroalkyl group containing one or more secondary or tertiary amines. It may be part of a heteroalkyl skeleton (i.e., the longest sequence of atoms in a heteroalkyl group). Thus, for example, a heteroalkyl group containing a secondary or tertiary amine can be used with an alkylamino group, a diamino group, or a heteroaryl group containing 1-8 (or 2-8) secondary or tertiary amines. The alkyl group may be an alkylamino group, an aminoalkyl group, an alkylaminoalkyl group, a dialkylaminoalkyl group, an aminoalkylamino group, or the like.

[0036] In some embodiments, each R 5 are independently selected from:

[0037] [ka]

[0038] [ka]

[0039] [ka]

[0040] During the ceremony R in each case 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 7 is optionally substituted with one or more amines, C1-C 50 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; z is an integer from 1 to 5; c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; n is an integer from 0 to 50; and R 8 are tissue- or cell-specific targeting sites, C1-C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group.

[0041] R in each case 2 is hydrogen or C1-C 12It can be an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. In some embodiments, R 2 is C1-C 12 (Example, C1-C 10 an alkyl group; a C1-C8 alkyl group; a C1-C6 alkyl group; a C1-C4 alkyl group, a C1-C3 alkyl group, or a C1 or C2 alkyl group) linear or branched alkyl group. , R 2 is methyl or hydrogen.

[0042] R 7 is optionally substituted with one or more amines, C1-C 50 (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 can be a cycloalkenyl group. In some embodiments, R 7 may be substituted with one or more amines, C4-C 12 , for example C6-C8, alkyl groups, alkenyl groups, cyclo In some embodiments, R 7 is substituted with one or more amines. In certain embodiments, R 7 1 to 8 or 2 to 8 ( 1, 2, 3, 4, 5, 6, 7, or 8) secondary or tertiary amines. The amine is either part of an alkyl group (i.e., contained within the alkyl group backbone) or a pendant substituent. could be.

[0043] R 13 is hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkyl group In some embodiments, R 13 is C1-C12 (Example, C1-C 10 Alkyl group; C1-C8 alkyl group; C1-C6 alkyl group; C1-C4 alkyl group, C1-C3 alkyl group, or is a C1 or C2 alkyl group) linear or branched alkyl group. In some embodiments, R 13 is methyl. In another embodiment, R 13 can be hydrogen. Typically, Within each R 13 are the same (e.g., all methyl or all hydrogen); however, each R 13 is independent The nucleotides are selected as desired and may be the same or different.

[0044] In each instance, Y is optionally present. As used herein, the phrase "optionally present" means "optionally" means that the substituent specified as being present may or may not be present, and if the substituent is absent, the 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 connecting two species that can be cleaved to separate the two species. For example, the cleavable linker can be cleaved by a hydrolytic process, a photochemical process, a radical process, an enzymatic process, an electrochemical process, or a combination thereof. Exemplary cleavable linkers include, but are not limited to:

[0045] [ka]

[0046] where R in each case 14 is independently a C1-C4 alkyl group, and each occurrence of R 15 are independently hydrogen, Aryl groups, heterocyclic groups (e.g., aromatic or non-aromatic), C1-C 12 is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, and R 16is a six-membered aromatic or heteroaromatic group optionally substituted with one or more -OCH3, -NHCH3, -N(CH3)2, -SCH3, -OH, or combinations thereof.

[0047] The polymer can be any suitable polymer, provided that the polymer comprises the aforementioned polymer structure as part of the polymer. In some embodiments, the polymer comprises a polymer block having a structure of Formula 1, 1.1, or 1.2 and one or more other polymer blocks (e.g., ethylene oxide subunits, or propylene oxide subunits). In other embodiments, the structure of formula 1, 1.1, or 1.2 is a poly(block copolymer). The mer is the only polymer unit, which can be "capped" at either end at a suitable terminus. In some embodiments, the polymer further comprises a substituent that comprises a tissue-specific or cell-specific targeting moiety.

[0048] In some embodiments, the polymer has Formula 1A:

[0049] [ka]

[0050] wherein Q has the formula:

[0051] [ka]

[0052] is; Or formula 1A1:

[0053] [ka]

[0054] During the ceremony c is an integer from 0 to 50; Y is optionally present and is a cleavable linker as described above; R 1 is hydrogen, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), optionally substituted with one or more substituents, such as one or more amines, C-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, or C1-C 12 is a linear or branched alkyl group; R 6 is hydrogen, an amino group, optionally substituted with one or more amines, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), a C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, C1-C 12 Linear or branched alkyl groups; or tissue-specific or is a cell-specific targeting moiety; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; 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 )teeth As stated above It has the structure:

[0055] Similarly, the polymer may have the formula 1.1A:

[0056] [ka]

[0057] Or, if the backbone is in the alpha, beta configuration, formula 1.2A:

[0058] [ka]

[0059] During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker as described above; R 1 is hydrogen, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), optionally substituted with one or more substituents, such as one or more amines, C-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, or C1-C 12 is a linear or branched alkyl group; R 6 is hydrogen, an amino group, optionally substituted with one or more amines, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), a C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, C1-C 12 Linear or branched alkyl groups; or tissue-specific or is a cell-specific targeting moiety; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; and all other substituents (e.g., m 3 , R 3a , R 3b , and A 3 ) is as above The structure may be:

[0060] In some of the foregoing embodiments, R 1 and / or R 6 is substituted with one or more substituents C1-C 12 (Example, C1-C 10 Alkyl group;C1-C8 alkyl group;C1-C6 alkyl group;C1-C4 alkyl group The heteroalkyl or alkyl group is a linear or branched alkyl group, such as a C1-C3 alkyl group, or a C1 or C2 alkyl group. In certain embodiments, the heteroalkyl or alkyl group contains 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 amines can be part of the heteroalkyl backbone chain or as pendant substituents.

[0061] Non-limiting examples of polymers provided herein include, for example, the following:

[0062] [ka]

[0063] [ka]

[0064] [ka]

[0065] [ka]

[0066] [ka]

[0067] [ka]

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] [ka]

[0075] wherein a, b, c, and d are each independently 0 to 1000, with the proviso that (a+b+c+d)>about 5; For example, it is an integer of 0-500, 0-200, 0-100, or 0-50. In some embodiments, (a+b) is about 0 to about 75 (e.g., about 5 to about 75, about 20 to about 75, about 40 to about 75, about 40 to about 60, about 50 to about 75, (c+d) is about 5 to about 80 (e.g., about 5 to about 75, about 5 to about 60, about 5 to about 40, about 20 to about 40, about 5 to about 30, about 5 to about 20, or about 5 to about 10). In 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 (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 (a+b) / (c+d) is greater than 1. less than about 0.1 (e.g., greater than or equal to about 0.1, but less than 1). Again, the designations of numbers of units ("a", "b", "c", and "d") in these exemplary polymers do not imply a block copolymer structure; rather, these numbers indicate the total number of units, which may be randomly arranged as indicated by the symbol " / " in the formula.

[0076] The present invention also provides a polymer comprising the structure of Formula 2:

[0077] [ka]

[0078] During the ceremony: a is an integer from 10 to 2,000; c is an integer from 0 to 50; Y is optionally present and is a cleavable linker as described herein; R 1 , R 2 , and R 13 and all other substituents are as defined herein above for other polymer formulas (e.g., Formulas 1, 1A, 1A1, 1.1A, and 1.2A; and R 8 is a tissue- or cell-specific targeting moiety.

[0079] The present invention also provides a polymer comprising a structure of Formula 4:

[0080] [ka]

[0081] During the ceremony, m 1 and n 1 Each of m is an integer from 0 to 1000; 1 + n 1 the sum of is greater than 2; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; A 1 and A 2 are each independently represented by the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on wherein p1 to p4, q1 to q6, and r1 and r2 each independently represent an integer of 1 to 5; R in each case 2 are independently hydrogen, C1-C 12 Alkyl groups, C1-C 12 Alkenyl groups, C3-C 12 Cycloa Alkyl group, or C3-C 12 A cycloalkenyl group, 1 and A 2 At least one ( For example, at least two, at least three, at least four, or even at least five tertiary amines (e.g., at least some of R 2 is C1-C 12 Alkyl groups, alkenyl groups, cyclo groups a cycloalkenyl group; R 3a and R3b are each independently a methylene or ethylene group; and R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group It is a cycloalkenyl group, or a cycloalkenyl group.

[0082] The present invention further provides a polymer having the structure of Formula 4A:

[0083] [ka]

[0084] During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 and n 1 Each of m is an integer from 0 to 1000; 1 + n 1 the sum of is greater than 2; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; A 1 and A 2 are each independently represented by the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4-N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on wherein p1 to p4, q1 to q6, and r1 and r2 each independently represent an integer of 1 to 5; R 1 is a hydrogen atom, an aryl group, a heterocyclic group (e.g., an aromatic aromatic or non-aromatic), C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkyl groups Chloroalkenyl group, or C1-C 12 is a linear or branched alkyl group; R in each case 2 are independently hydrogen, C1-C 12 Alkyl groups, C1-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups, or C3-C 12 A cycloalkenyl group, 1 and A 2 At least one ( For example, at least two, at least three, at least four, or even at least five tertiary amines (e.g., at least some of R 2 is C1-C 12 Alkyl groups, alkenyl groups, cyclo groups a cycloalkenyl group; R 3a and R 3a are each independently a methylene or ethylene group; R 6 is hydrogen, an amino group, optionally substituted with one or more amines, an aryl group, a heterocyclic group (e.g., aromatic or non-aromatic), a C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, C1-C 12 Linear or branched alkyl groups; or tissue-specific or is a cell-specific targeting moiety; and R in each case 13are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group It is a cycloalkenyl group, or a cycloalkenyl group.

[0085] In embodiments of the polymer comprising the structure of Formula 4 and the polymer having the structure of Formula 4A, each In the case of R 2 are independently hydrogen or C1-C 12 Alkyl groups, C1-C 12 Alkenyl groups, C3-C 12 Cycloalkyl groups, or C3-C 12 A cycloalkenyl group, 1 and A 2 each of which contains at least one tertiary amine (e.g., at least two, at least three, at least four, or In some embodiments, each occurrence of R 2 teeth Independently C1-C 12 The alkyl group is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. Thus, the implementation of a polymer having a structure of Formula 4 and a polymer having a structure of Formula 4A is In an embodiment, each nitrogen-containing R 2 The substituents are tertiary amines, except that the terminal amine can be a primary, secondary, or tertiary amine, preferably a secondary or tertiary amine. In some embodiments of the polymer comprising the polymer having the structure of Formula 4A, 1 and A 2 each of which has the formula -(CH)-NR, except that the terminal amine may be a primary, secondary, or tertiary amine, preferably a secondary or tertiary amine. 2 -(CH2)2-NR 2 2 group, where R in each case 2 are independent, C1-C 12 The alkyl group, the alkenyl group, the cycloalkyl group, or the cycloalkenyl group. Polymers Having the Structure of Formula 4 and Polymers Having the Structure of Formula 4A In certain embodiments, each occurrence of R 2 is ethyl or methyl, except that the terminal amine may be a primary, secondary, or tertiary amine, preferably a secondary or tertiary amine, as appropriate.

[0086] In embodiments of the polymer comprising the structure of Formula 4 and the polymer having the structure of Formula 4A, each In the case of R 13 are independently hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkyl groups , or a cycloalkenyl group. In general, R in each case 13 are independently hydrogen or methyl In certain embodiments, each occurrence of R 13 is hydrogen.

[0087] Examples of groups A1 and A2 in accordance with any of the foregoing are, 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)-CH2-CH2-N(CH3)2; -N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2; -CH2-N(CH3)2;-NH-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);-NH-CH2-CH Contains 2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3); -N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3).

[0088] Thus, non-limiting examples of polymers provided herein include, for example, the following:

[0089] [ka]

[0090] [ka]

[0091] [ka]

[0092] [ka]

[0093] wherein each of a and b is independently an integer from 0 to 1000, e.g., 0-500, 0-200, 0-100, or 0-50. Thus, either a or b can be 0 in some embodiments; Or the polymer can have some ratio of a and b. In some embodiments, (a+b) is about 5 or more (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 more (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 more (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). Further examples include polymers 30-33, in which the terminal tertiary amines of one or both side chains are demethylated to secondary amines. Provide an anion (e.g., -NHCH3):

[0094] [ka]

[0095] [ka]

[0096] [ka]

[0097] In the formula, a and b are as defined above.

[0098] Any of the aforementioned polymers may contain a tissue- or cell-specific targeting moiety at the position indicated in the written formula, or the polymer may be otherwise modified to contain a tissue- or cell-specific targeting moiety. Methods for attaching tissue- or cell-specific targeting moieties are known in the art, some of which include Michael addition, epoxide ring-opening, substitution reactions, or the like, with a tissue- or cell-specific targeting moiety bearing an appropriate functional group. involves "click" chemistry (e.g., CuAAC, SPAAC, SPANC, or reactions of strained alkenes). Tissue-specific or cell-specific targeting moieties are those that target a given target tissue or cell (e.g., a particular ligase). The targeting moiety specifically binds to a target protein or molecule that allows the target tissue or cell to be distinguished from other non-target tissues or cells. Any small molecule, protein (e.g., an antibody or an antibody) that can bind (e.g., specifically bind) to a The nucleotide sequence, amino acid sequence, sugar, oligonucleotide, metal-based nanoparticle, or their It may be a combination. In some embodiments, the tissue-specific or cell-specific targeting moiety is a receptor for a ligand. In some embodiments, the tissue-specific or cell-specific targeting moiety is a ligand for a receptor.

[0099] Tissue-specific or cell-specific targeting moieties can be used to target any desired tissue or cell type. In some embodiments, tissue-specific or cell-specific targeting moieties localize the polymer to the peripheral nervous system, central nervous system, liver, muscle (e.g., cardiac muscle), lung, bone (e.g., hematopoietic cells), or eye tissue of a subject. In some embodiments, tissue-specific or cell-specific targeting moieties localize the polymer to tumor cells. For example, tissue-specific or cell-specific targeting moieties can be sugars that bind to receptors on specific tissues or cells.

[0100] In some embodiments, the tissue-specific or cell-specific targeting moiety is:

[0101] [ka]

[0102] In the formula, R 9 , R 10 , R 11 , and R 12 Each of the formulas is independently hydrogen, halogen, one or more amines. The polymer may be C1-C4 alkyl, C1-C4 alkoxy, or C1-C4 alkoxy, optionally substituted with a cyclic alkyl group. Specific tissue-specific or cell-specific targeting moieties may be selected to localize the polymer to the tissues described herein. For example, alpha-d-mannose targets the polymer to the peripheral nervous system, central nervous system, and / or nervous system. It can be used to localize to the central nervous system or immune cells, and can be used to localize to alpha-d-galactose and and N-acetylgalactosamine can be used to localize the polymer to liver cells, and folic acid can be used to localize the polymer to tumor cells.

[0103] Typically, the polymers are cationic (i.e., positively charged at pH 7 and 23° C.). As used herein, a "cationic" polymer refers to a polymer that has an overall net positive charge, regardless of whether the polymer contains only cationic monomer units or a combination of cationic and nonionic or anionic monomer units.

[0104] In some embodiments, the polymer has a weight average molecular weight of about 5 kDa to about 2000 kDa, e.g., about 10 kDa to about 2,000 kDa. The polymer may be about 2,000 kDa or less, e.g., about 1,800 kDa or less, about 1,600 kDa or less, about 1,400 kDa or less, about 1,200 kDa or less, about 1,000 kDa or less, about 900 kDa or less. Below, 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 Alternatively, or in addition, the polymer may have a weight average molecular weight of about 50 kDa or less, or even about 40 kDa or less, such as about 30 kDa or less or 25 kDa or less. about 10 kDa or more, for example, about 50 kDa or more, about 100 kDa or more, about 200 kDa or more, about 300 kDa or more The polymer may have a weight average molecular weight of about 400 kDa or more. For example, the polymer may have a weight average molecular weight bounded by any two of the following points: 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 500 kDa to about 500 kDa, about 600 kDa to about 600 kDa, about 700 kDa to about 700 kDa, about 800 kDa to about 800 kDa, about 900 kDa to about 900 kDa, about 1000 kDa to about 10 ... Approximately 500 kDa, approximately 400 kDa to approximately 500 kDa, approximately 400 kDa to approximately 600 kDa, approximately 400 kDa to approximately 700 kDa, approximately 400 kDa to approximately 800 kDa, approximately 400 kDa to approximately 900 kDa, approximately 400 kDa to approximately 1,000 kDa, approximately 400 kDa to approximately 1,200 kDa, approximately 400 kDa to approximately 1,400 kDa, approximately 400 kDa to approximately 1,600 kDa, approximately 400 kDa to approximately 1,800 kDa , about 400 kDa to about 2,000 kDa, about 200 kDa to about 2,000 kDa, about 500 kDa to about 2,000 kDa, or about 800 kDa to about 2,000 kDa.

[0105] The weight average molecular weight can be determined by any suitable technique. Generally, the weight average molecular weight is determined using size exclusion chromatography equipped with a column selected from TSKgel Guard, GMPW, GMPW, G1000PW, and a Waters 2414 (Waters Corporation, Milford, Massachusetts) refractive index detector. Additionally, the weight average molecular weight is in the range of 150-875,000 Daltons. The concentration is determined from calibration with polyethylene oxide / polyethylene glycol standards.

[0106] Preparation method The polymers provided herein can be provided by any suitable method. In some embodiments, the polymer has Formula 3:

[0107] [ka]

[0108] During the ceremony: m 1 and n 1 Each of m is an integer from 0 to 1000, 1 + n 1 is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on wherein p1 to p4, q1 to q6, and r1 and r2 each independently represent an integer of 1 to 5; and R in each case 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; The polymer group A includes the structure 1 and / or A 2 by modifying at least a portion of the formula 1:

[0109] [ka]

[0110] In the formula, m 1 and n 1 Each of is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on B 1and B. 2 are each independently -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2; -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2; -(CH2) p1 -[N{(CH2) s1 -R 4 -R 5}-(CH2) q1 -] r1 NR 2 2; or -(CH2) p1 -[N{(CH2) s1 -R 5}-(CH2) q1 -] r1 NR 2 2 where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independently an integer from 1 to 5; 2 are independently hydrogen or C1-C12 Alkyl groups, alkenyl groups, silyl groups R is a cycloalkyl group, or a cycloalkenyl group; 4 is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, which may contain 1-8 or 2-8 secondary or tertiary amines, or a substituent which contains a tissue-specific or cell-specific targeting moiety; The compound may be prepared by a process comprising producing a polymer comprising the structure:

[0111] The polymer comprising the structure of Formula 1 produced by this method can be any polymer of Formula 1, including formula 1.1, 1.2, 1A1, 1A, 1.1A, 1.2A, 4, or 4A, as well as any and all of the embodiments thereof described with respect to the polymers of the invention.

[0112] The polymer containing the structure of formula 3 can be any suitable polymer that meets this criteria. In some embodiments, the polymer may instead be represented by formula 3.1:

[0113] [ka]

[0114] Or, if the backbone is in the alpha, beta configuration, formula 3.2:

[0115] [ka]

[0116] During the ceremony, m 3is an integer between 5-2000 (e.g., 5-1000, 5-500, 5-100, 25-2000, 25-500, 25-100, 50-2000, 50-1000, 50-500, or 50-100); The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; Each A 3 is an independent formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 where p1 through p4, q1 through q6, and r1 and r2 are each independently an integer from 1 to 5; and each occurrence of R 2 are independently hydrogen or C1-C 12 Alkyl group, alkenyl group , a cycloalkyl group, or a cycloalkenyl group; In this case, the method comprises reacting sufficient A to provide a polymer having the structure of formula 1.1 or 1.2 as described herein. 3 An example of a polymer that includes one of the following structures is pAsp(DET)(poly(2-[(2-aminoethyl) (phenyl)amino]ethyl aspartamide) or PEG-pAsp(DET) (polyethylene glycol-b- Poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}).

[0117] A 1 and / or A 2 (or group A of the polymer of formula 3.1 or 3.2) 3 ) is optional. B is modified by any appropriate method. 1 and / or B. 2 Groups designated as (or formula 1.1 or or 1.2 of the polymer group A 3 ) can occur. For example, A 1 and / or A 2 (or group A of the polymer of formula 3.1 or 3.2) 3 ) can be modified by Michael addition reactions, epoxide ring opening, or substitution reactions. In a preferred embodiment, A 1 and / or A 2 It is specified (or group A of the polymer of formula 3.1 or 3.2) 3 ) is modified by Michael addition reaction can be.

[0118] In one embodiment, the group A of the polymer comprising the structure of formula 3 1 and / or A 2 or group A of the polymer of formula 3.1 or 3.2 3 The polymers containing the structure of formula 3 and α,β-unsaturated carbonyls are The term "Michael addition" as used herein refers to the reaction of a nucleophile (e.g., a carbanion, an oxygen anion, a nitrogen anion, etc.) with a polymer. , oxygen atom, nitrogen atom, or a combination thereof) to an α,β-unsaturated carbonyl compound Thus, the Michael addition reaction is between a polymer comprising the structure of Formula 3 and an α,β-unsaturated carbonyl compound. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0119] α,β-Unsaturated carbonyl compounds can accept Michael addition from nucleophiles. In some embodiments, the α,β-unsaturated carbonyl compound may be any α,β-unsaturated carbonyl compound. β-Unsaturated carbonyl compounds include acrylates, acrylamides, vinyl sulfones, and and combinations thereof. Thus, the Michael addition reaction can be between a polymer comprising the structure of formula 3 and an acrylate, an acrylamide, a vinyl sulfone, or combinations thereof. Thus, in some embodiments, the method comprises the step of reacting a polymer comprising the structure of formula 3 with contacting a polymer comprising the structure of Formula 3 with an acrylamide; or contacting a polymer comprising the structure of Formula 3 with a vinyl sulfone.

[0120] A 1 and / or A 2 (or group A of the polymer of formula 3.1 or 3.2) 3 In embodiments in which ) is modified by Michael addition reaction, they are represented by formulas 1, 1.1., 1.2, 1A, 1A1, etc. B as described herein with respect to 1 and / or B. 2 Groups designated as (or formula 1.1 or or 1.2 of the polymer group A 3 ) is produced. For example, B 1and / or B. 2 can be the following formula: -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 4 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 4 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 4 -R 5}; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 4 -R 5 ]2}2, where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independently an integer of 1 to 5; 2 are independently hydrogen or C1-C 12 Alkyl groups, alkenyl groups, cycloalkenyl groups R is an alkyl group, or a cycloalkenyl group; 4 is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety; or groups and The substituents may be otherwise as described herein for the polymers of the invention.

[0121] Examples of acrylates, acrylamides, and vinyl sulfones suitable for use include acrylates of the formula:

[0122] [ka]

[0123] Here, R 5 is as described for any of Equations 1, 1.1, 1.2, 1A, 1.1A, or 1.2A. do.

[0124] In some embodiments, the Michael addition reaction is promoted by an acid and / or a base. The acid and / or base may be any suitable acid having any suitable pKa and / or The acid and / or base may be an organic acid (e.g., p-toluenesulfonic acid), an organic salt, or the like. The reactant may be a group (eg, triethylamine), an inorganic acid (eg, titanium tetrachloride), an inorganic base (eg, potassium carbonate), or a combination thereof.

[0125] In some embodiments, the Michael addition reaction is promoted by an acid. In embodiments where the acid is a Bronsted acid, the acid may be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfonyl)imide or p-toluenesulfonic acid.

[0126] In some embodiments, the Michael addition reaction is promoted by a base. It can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base may be triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, or or derivatives thereof.

[0127] In some embodiments, the Michael addition reaction is carried out in a solvent. The solvent may be any suitable solvent or mixture of solvents capable of solubilizing the polymer and the α,β-unsaturated carbonyl compound to be polymerized. For example, the solvent may include water, a protic organic solvent, and / or an aprotic organic solvent. An exemplary list of solvents includes water, dichloromethane, ethyl acetate ... These include methane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0128] In one embodiment, the group A of the polymer 1 and / or A 2 (or the group A of the polymer of formula 3.1 or 3.2) 3 ) is formed by the epoxide ring-opening reaction between the polymer and the epoxide compound. As used herein, the term "epoxide ring opening" refers to the nucleophilic (e.g. The term refers to the nucleophilic addition of an anion (such as a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) to an epoxide compound, thereby opening the epoxide. Thus, the epoxide ring-opening reaction is between the polymer and the epoxide compound. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0129] A 1 and / or A 2 (or group A of the polymer of formula 3.1 or 3.2) 3 ) is Epo In embodiments modified by an oxide ring-opening reaction, they are represented by the following formula B 1 and / or Or B 2 (or group A of the polymer of formula 1.1 or 1.2) 3 ) results in: -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -CH2-CHOH-R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -CH2-CHOH-R 5 ; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 -CH2-CHOH-R 5 ; -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -CH2-CHOH-R 5 ]2}2, wherein p1 to p4, q1 to q6, and r1 and r2 each independently represent an integer of 1 to 5; R in each case 2 are independently hydrogen or C1-C 12an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; and 5 are independently 1 to 8 or 2 to 8 or a combination thereof, optionally containing a secondary or tertiary amine of the formula:

[0130] Examples of epoxides suitable for use include epoxides of the formula:

[0131] [ka]

[0132] Here, R 5 for any polymer of the invention (e.g., formula 1, 1.1, 1.2, 1A, 1.1A, or 1.2A), This is as stated above.

[0133] In some embodiments, the epoxide ring-opening reaction is carried out with an acid and / or a base. The acid and / or base may be any suitable acid and / or base having any suitable pKa. or a base. The acid and / or base may be an organic acid (e.g., p-toluenesulfonic acid), Organic bases (e.g., triethylamine), inorganic acids (e.g., titanium tetrachloride), inorganic bases (e.g., potassium carbonate um), or a combination thereof.

[0134] In some embodiments, the epoxide ring-opening reaction is promoted by an acid. The acid can be a Bronsted acid or a Lewis acid. In embodiments where the acid is a Bronsted acid, the acid can be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfonyl)imide or p-toluenesulfonic acid.

[0135] In some embodiments, the epoxide ring-opening reaction is promoted by a base. The base can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base is triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, Or it may be a derivative thereof.

[0136] In some embodiments, the epoxide ring-opening reaction is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents that can solubilize the polymer and epoxide compound to be reacted. For example, the solvent can be water, a protic organic solvent, and / or may include an aprotic organic solvent. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0137] In one embodiment, the group A of the polymer 1 and / or A 2 (or Equation 3.1 or 3.2 Polymer Group A 3 ) can be modified by substitution reactions between the polymer and compounds containing leaving groups (e.g., chlorine, bromine, iodine, tosylate, triflate, mesylate, etc.) As used herein, the term "substitution" refers to the substitution of a nucleophile (e.g., a carbanion, acid) of a polymer. leaving group of an anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof The substitution reaction is therefore between a polymer and a compound containing a leaving group. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

[0138] A 1 and / or A 2 (or group A of the polymer of formula 3.1 or 3.2) 3 ) is replaced In the embodiment modified by the reaction, they are represented by the following formula B 1 and / or B. 2 and finger The group (or group A of the polymer of formula 1.1 or 1.2) 3 ) results in: -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 -(CH2) s1 -R 5 ; -(CH2) p2 -N[-(CH2) q2 -NR 2 -(CH2) s2 -R 5 ]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -] r2 (CH2) s3 -R 5};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 -(CH2) s4 -R 5 ]2}2, where p1 to p4, q1 to q6, r1 and r2, and s1 to s4 are each independently an integer of 1 to 5; 2 are independently hydrogen or C1-C12 Alkyl groups, alkenyl groups, cycloalkenyl groups and R in each occurrence is an alkyl group, or a cycloalkenyl group; 5 are independent, 1 to 8 or is an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, which may contain 2-8 secondary or tertiary amines, or a substituent which contains a tissue-specific or cell-specific targeting moiety.

[0139] In some embodiments, the method comprises the step of preparing a compound of formula A:

[0140] [ka]

[0141] During the ceremony, R 3b is a methylene or ethylene group; W is -NR 14 - or -O-, where R 14 is hydrogen or C1-C 12 Alkyl group, alkenyl group a cycloalkyl group, or a cycloalkenyl group; Z is A 1 or 1 to 8 or 2 to 8 secondary or tertiary amines. a substituent that includes an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, an arylalkyl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, or a tissue- or cell-specific targeting moiety; A 1 is the formula -(CH2) p1 -[NR 2 -(CH2) q1 -] r1 NR 2 2; -(CH2) p2 -N[-(CH2) q2 -NR 2 2]2; -(CH2) p3 -N{[-(CH2) q3 -NR 2 2][-(CH2) q4 -NR 2 -]r2R 2};or -(CH2) p4 -N{-(CH2) q5 -N[-(CH2) q6 -NR 2 2]2}2 Based on wherein p1 to p4, q1 to q6, and r1 and r2 each independently represent an integer of 1 to 5; and R in each case 2 are independently hydrogen or C1-C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; The method includes preparing a polymer comprising the structure of formula 5 from a compound of formula

[0142] Generally, the method comprising forming a polymer comprising the structure of formula 5 from a compound of formula A comprises ring-opening polymerization of the compound of formula A. The ring-opening polymerization of the compound of formula A can be carried out by any suitable method (e.g., temperature, light, The ring-opening polymerization of the compound of formula A can be initiated by an amine-containing compound, such as a catalyst, compound, etc. In some embodiments, the ring-opening polymerization of the compound of formula A is initiated with an amine-containing compound.

[0143] In one embodiment, the ring-opening polymerization of a compound of formula A is carried out using an amine-containing compound of the formula: Starts with:

[0144] [ka]

[0145] During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; and R 6is hydrogen, an amino group, an aryl group, a heterocyclic group, a C1-C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl groups, C1-C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. In a preferred embodiment, the amine-containing compound is

[0146] [ka]

[0147] It is.

[0148] In some embodiments, in the compound of formula A, Y is -O- and Z is an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, an arylalkyl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, which may contain 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 then includes, after ring-opening polymerization, converting the resulting intermediate polymer to a compound of formula NHR. 17 -A 1 , where A 1 is defined in terms of Eq. The method further comprises treating the compound of formula NHR 17 -A 1 The compound of formula NHR is optionally a diethyleneamine triamine. 17 -A 1 The compound is not a diethyleneaminetriamine.

[0149] R 17 is hydrogen or C1-C 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkyl group In some embodiments, R 17 is C1-C 12 (Example, C1-C10 Al alkyl group; C1-C8 alkyl group; C1-C6 alkyl group; C1-C4 alkyl group, C1-C3 alkyl group, or is a C1 or C2 alkyl group) linear or branched alkyl group. In some embodiments, R 17 is methyl. In another embodiment, R 17 can be hydrogen. Typically, Within each R 17 are the same (e.g., all methyl or all hydrogen); however, each R 17 is independent The nucleotides are selected as desired and may be the same or different.

[0150] Examples of compounds containing leaving groups suitable for use include compounds of the formula:

[0151] [ka]

[0152] Here, LG is a leaving group (e.g., chlorine atom, bromine atom, iodine atom, tosylate, triflate, mesylate, etc.), and R 5 is as described for any of Formulas 1, 1.1, 1.2, 1A, 1.1A, or 1.2A.

[0153] In some embodiments, the substitution reaction is promoted by acid and / or base. The acid and / or base may be any suitable acid and / or base having any suitable pKa. The acid and / or base may be an organic acid (e.g., p-toluenesulfonic acid), an organic base (e.g., triethylamine), an inorganic acid (e.g., titanium tetrachloride), an inorganic base (e.g., potassium carbonate), or may be a combination thereof.

[0154] In some embodiments, the substitution reaction is promoted by an acid. The acid can be a Bronsted acid or a Lewis acid. In embodiments where the acid is a Bronsted acid, the acid can be a weak acid (i.e., a pKa of about 4 to about 7) or a strong acid (i.e., a pKa of about -2 to about 4). Typically, the acid is a weak acid. In some embodiments, the acid is a Lewis acid. For example, the acid can be bis(trifluoromethanesulfonyl)imide or p-toluenesulfonic acid.

[0155] In some embodiments, the substitution reaction is promoted by a base. The base can be a weak base (i.e., a pKa of about 7 to about 12) or a strong base (i.e., a pKa of about 12 to about 50). Typically, the base is a weak base. For example, the base is triethylamine, diisopropylethylamine, pyridine, N-methylmorpholine, or N,N-dimethyl-piperazine, or It may be a derivative thereof.

[0156] In some embodiments, the substitution reaction is carried out in a solvent. The solvent can be any suitable solvent or mixture of solvents that can solubilize the polymer to be reacted and the compound containing the leaving group. For example, the solvent can be water, a protic organic solvent, and / or a non-protic organic solvent. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol, and ethanol.

[0157] In some embodiments, the method further comprises isolating a polymer comprising the structure of Formula 1. The polymer comprising the structure of Formula 1 can be isolated by any suitable method. For example, polymers containing the structure of Formula 1 can be prepared by extraction, crystallization, recrystallization, column chromatography, etc. The extract may be isolated by extraction, filtration, or any combination thereof.

[0158] composition The polymers may be used for any application. However, the polymers may be used in combination with nucleic acids and / or are believed to be particularly useful for delivering polypeptides (e.g., proteins) to cells. Accordingly, provided herein are compositions that contain the polymers described herein and nucleic acids and / or polypeptides (e.g., proteins).

[0159] In some embodiments, the composition contains a nucleic acid. Any nucleic acid can be used. An exemplary list of nucleic acids includes guide and / or donor nucleic acids for CRISPR systems, siRNA, microRNA, interfering RNA or RNAi, dsRNA, mRNA, DNA vectors, ribozymes, antisense polynucleotides, and the like. The present invention includes a nucleic acid sequence encoding a target gene or RNA, and a DNA expression cassette encoding a siRNA, microRNA, dsRNA, ribozyme, or antisense nucleic acid. SiRNAs typically contain 15-50 base pairs, and preferably 19-25 base pairs, and are identical or nearly identical to a target gene or RNA expressed in a cell. siRNAs are composed of two annealed polynucleotides that have a double-stranded structure with a nucleotide sequence. MicroRNAs (miRNAs) are small non-coding polynucleotides that direct the destruction or translational repression of their mRNA targets. Antisense polynucleotides are: morpholino, 2'-O-methyl, about 22 nucleotides in length. Antisense polynucleotides contain sequences that target genes or mRNA. Antisense polynucleotides include: morpholino, 2'-O-methyl Polynucleotides include, but are not limited to, recombinant polynucleotides, DNA, RNA, etc. Polynucleotide-based expression inhibitors can be polymerized in vitro and can be recombinant, chimeric sequences, or combinations thereof. Polynucleotide-based expression inhibitors may include derivatives of ribonucleic acid, The polynucleotide may comprise a sequence that is a nucleotide, a deoxyribonucleotide, a synthetic nucleotide, or any suitable combination such that the target RNA and / or gene is inhibited. The polynucleotide may also be a sequence that serves as a "bar code" for purposes such as tracking delivery in vitro or in vivo.

[0160] The composition may also contain any protein for delivery in addition to or instead of the nucleic acid. The polypeptide may 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 can be an RNA-guided endonuclease (e.g., a Cas9 polypeptide, a Cpf1 polypeptide, or a variant thereof) or a DNA recombinase. enzyme (e.g., a Cre polypeptide).

[0161] The polymers provided herein are believed to be particularly useful for delivering one or more components of a CRISPR system. Thus, in some embodiments, the composition contains a guide RNA, an RNA-guided endonuclease or a nucleic acid encoding the same, and / or a donor nucleic acid. The composition may contain one, two, or all three components together with the polymers described herein. Furthermore, the composition may contain multiple guide RNAs, an RNA-guided endonuclease or a nucleic acid encoding the same, and / or a donor nucleic acid. For example, the composition may contain multiple guide RNAs, an RNA-guided endonuclease or a nucleic acid encoding the same, and / or a donor nucleic acid. Depending on the donor nucleic acid and the RNA-guided endonucleases, In addition to the enzyme or a nucleic acid encoding same, multiple different guide RNAs for different target sites can be included.

[0162] Furthermore, the components of a CRISPR system can be linked together in any particular manner or order (if multiple components are present). In some embodiments, the guide RNA is complexed with an RNA endonuclease prior to combining with the polymer. Alternatively, the guide RNA can be linked (covalently or non-covalently) to the donor nucleic acid prior to combining with the polymer.

[0163] The compositions are not limited with respect to any particular CRISPR system (i.e., any particular guide RNA, RNA-guided endonuclease, or donor nucleic acid), many of which are known. Nevertheless, for the sake of further explanation, the components of some such systems are described below.

[0164] Donor Nucleic Acid Donor nucleic acid (or "donor sequence" or "donor polynucleotide" or "donor The "DNA" ("DNA") is an RNA-dependent endonuclease (e.g., Cas9 polypeptide or Cpf1 polypeptide) The donor polynucleotide is the nucleic acid sequence to be inserted at the cleavage site induced by the donor polynucleotide. The donor comprises sufficient homology to the target genomic sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100%, for example, homology with a nucleotide sequence flanking the cleavage site or immediately flanking the cleavage site and having homology thereto, within about 50 bases or less, e.g., about 30 bases, about 15 bases, about 10 bases, about 5 bases, of the cleavage site, to support homologous recombination repair between the genomic sequence flanking the cleavage site or immediately flanking the cleavage site and having homology thereto, within about 25, 50, 100, or 200 nucleotides of sequence homology between the donor and the genomic sequence (or any integer value between 10 and 200 nucleotides, or more). The donor sequence is a sequence of 10 nucleotides, or more than 200 nucleotides, that supports homology-directed repair. For example, any of 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc. It can be of any length.

[0165] The donor sequence is typically not identical to the genomic sequence it replaces. Rather, the donor sequence may contain one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, as long as there is sufficient homology to support homology-directed repair. In some embodiments, the donor sequence contains a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences is not affected by the target. This results in the insertion of non-homologous sequences at the target region. The donor sequence must also be homologous to the DNA region of interest. and may contain vector backbones that contain sequences that are not intended for insertion into the DNA region of interest. Generally, the region of homology of the donor sequence has at least 50% sequence identity to the genomic sequence with which recombination is desired. In certain embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity. Depending on the length of the donor polynucleotide, there may be up to 1% sequence identity. Any value between sequence identity and 100% sequence identity can exist.

[0166] The donor sequence may contain certain sequence differences compared to the genomic sequence, such as restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which may be used to assess successful insertion of the donor sequence at the cleavage site, or in some embodiments may be used for other purposes (e.g., to indicate expression at the targeted genomic locus). In some embodiments, when located in a coding region, such nucleotide sequence differences do not change the amino acid sequence, or result in silent amino acid changes ( i.e., changes that do not affect the structure or function of the protein. The differences in these sequences are due to the presence of FLP, loxP sequences, etc., which can then be activated to remove the marker sequence. Any flanking recombination sequence may be included.

[0167] The donor sequence can be provided to the cell as single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It can be introduced into the cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from extranuclear degradation) by methods known to those skilled in the art. For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides can be attached to one or both ends. See, e.g., Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; See, et al. (1996) Science 272:886-889. Amplification procedures such as rolling circle amplification may also be advantageously used, as exemplified herein. Additional methods for protecting exogenous polynucleotides from degradation include the addition of terminal amino groups and the addition of nucleotides such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. These include, but are not limited to, the use of modified internucleotide linkages such as

[0168] As an alternative to protecting the ends of the linear donor sequence, additional lengths of sequence that can be degraded without affecting recombination can be included outside the regions of homology. The donor sequence can be introduced into cells as part of a vector molecule with additional sequences, such as, for example, a replication origin, a promoter, and a gene encoding antibiotic resistance. In addition, the donor sequence can be a vector molecule that encodes a Cas9 guide RNA and / or a Cas9 fusion polypeptide and / or a donor polynucleotide. As described herein for nucleic acids that can be delivered, the nucleic acid can be delivered as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or polymer, or can be delivered via a virus (e.g., adenovirus). The antibody can be delivered by an AAV (antibody, AAV).

[0169] Guide Nucleic Acid In some embodiments, the composition contains a guide nucleic acid. Guide nucleic acids suitable for inclusion in the compositions of the present disclosure include single-molecule guide RNAs ("single-guide RNAs" / "sgRNAs") and and dual guide nucleic acids ("dual-guide RNAs" / "dgRNAs").

[0170] Suitable guide nucleic acids (e.g., guide RNAs) for inclusion in the complexes of the present disclosure include RNA-guided endonucleases. The guide nucleic acid (e.g., guide RNA) directs the activity of a nucleic acid cleavage enzyme (e.g., Cas9 or Cpf1 polypeptide) against a specific target sequence within the target nucleic acid. a "targeting fragment" or simply a "targeting fragment"); and a second fragment (also referred to herein as a "protein targeting fragment"). The terms "first" and "second" refer to a fragment that is a guide RNA. The order of elements relative to each other depends on the particular RNA-guided polypeptide used. For example, guide RNAs for Cas9 typically have a protein-binding fragment located 3' to the targeting fragment, while guide RNAs for Cpf1 typically have a protein-binding fragment located 5' to the targeting fragment.

[0171] Guide RNAs can be introduced into cells in linear or circular form. When introduced in linear form The ends of the guide RNA are protected (e.g., from extranuclear degradation) by methods known to those of skill in the art. Amplification procedures such as rolling circle amplification may also be used to advantage, as exemplified herein.

[0172] First fragment: targeting fragment A first fragment of a guide nucleic acid (e.g., a guide RNA) is complementary to a sequence (target site) in a target nucleic acid. In other words, the targeting of the guide nucleic acid (e.g., guide RNA) The fragments bind to the target in a sequence-specific manner via hybridization (i.e., base pairing). It can interact with nucleic acids (e.g., RNA, DNA, double-stranded DNA). The sequence of the guide nucleic acid (e.g., guide RNA) and the target nucleic acid may vary and may be modified to interact with each other. The targeting fragment of the guide nucleic acid (e.g., guide RNA) can be modified (e.g., by genetic engineering) to hybridize to any desired sequence (target site) within the target nucleic acid. It can be hybridized.

[0173] The targeting fragment may have a length of 12 nucleotides to 100 nucleotides. The nucleotide sequence of the targeting fragment (targeting sequence, nucleotide sequence) that is complementary to the nucleotide sequence (target site) of the targeting fragment. For example, the targeting sequence of the targeting fragment that is 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, or 20 nt or more. It may have a length of nt or more, 20 nt or more, 25 nt or more, 30 nt or more, 35 nt or more, or 40 nt.

[0174] The percent complementarity between the targeting sequence (i.e., 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 percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 100% over the seven contiguous 5'-most nucleotides of the target site of the target nucleic acid. In some embodiments, the percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 60% or greater over the 20 contiguous nucleotides. In some embodiments, the percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 60% or greater over the 20 contiguous nucleotides. In some cases, the percent complementarity between the targeting sequence of the targeting fragment and the target site of the target nucleic acid is 100% for the 17, 18, 19, or 20 contiguous 5'-most nucleotides of the target site of the target nucleic acid, and is as low as 0% or less for the remainder. In such cases, the targeting sequence may be considered to be 17, 18, 19, or 20 nucleotides in length, respectively.

[0175] Second fragment: Protein-binding fragment The protein-binding fragment of the guide nucleic acid (e.g., guide RNA) interacts with (binds to) the RNA-guided endonuclease. The guide nucleic acid (e.g., guide RNA) can be linked to the targeting fragment / targeting sequence / targeting fragment described above. The guide sequence guides the bound endonuclease to a specific nucleotide sequence within the target nucleic acid (target site). The protein-binding fragment of the guide nucleic acid (e.g., guide RNA) contains two stretches of nucleotides that are complementary to each other. The complementary nucleotides of the protein-binding fragment The tides hybridize to form double-stranded RNA duplexes (dsRNA).

[0176] Single and dual guide nucleic acids A dual guide nucleic acid (e.g., a guide RNA) comprises two separate nucleic acid molecules. Each of the two molecules of a guide nucleic acid (e.g., a guide RNA) is a nucleoside that is complementary to the other. Each molecule contains a stretch of tides such that complementary nucleotides in the two molecules hybridize to form the double-stranded RNA duplex of the protein-binding fragment.

[0177] In some embodiments, the duplex-forming fragment of the activator is 60%, 65%, 70% identical to one of the activators (tracrRNA) described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements, for a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides). , 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical or 100% identical.

[0178] In some embodiments, the duplex-forming fragment of the target is a stretch of 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides), as described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617. The nucleic acid sequence of the target gene (crRNA) may be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical or 100% identical to one of the target (crRNA) sequences described herein, or their complements.

[0179] Dual guide nucleic acids (e.g., guide RNAs) can be designed to allow for controlled (i.e., conditional) binding of a target to an activator. Dual guide nucleic acids (e.g., guide RNAs) are not functional unless both the activator and the target are bound in a functional complex with Cas9, so dual guide nucleic acids (e.g., guide RNAs) can be designed to allow for controlled (i.e., conditional) binding of an activator and an inducer. The activator may be inducible (e.g., drug inducible) by providing an inducible bond between the activator and the target. As one non-limiting example, an RNA aptamer may be used to induce the binding of an activator to a target. Thus, the activator and / or the target may comprise an RNA aptamer sequence.

[0180] Aptamers (e.g., RNA aptamers) are known in the art and generally refer to ribosomal switching The terms "RNA aptamer" and "riboswitch" are used interchangeably herein. are used interchangeably to refer to the nucleic acid molecules of which they are a part (e.g., RNA, DNA / RNA hybrids, etc.) Synthetic and experimental methods that provide inducible control of the structure (and thus the availability of specific sequences) of RNA aptamers include both RNA and natural nucleic acid sequences. RNA aptamers are usually designed to bind to specific structures (e.g., hairpins). The aptamer contains a folding sequence that specifically binds a particular drug (e.g., a small molecule). Binding of the drug induces a conformational change in the folding of the RNA, which then binds to the nucleus of which the aptamer is a part. As non-limiting examples: (i) an activator with an aptamer cannot bind to a cognate target unless the aptamer is bound by an appropriate drug; (ii) a target with an aptamer cannot bind to a cognate target unless the aptamer is bound by an appropriate drug. , cannot bind to the cognate activator; and (iii) a target and an activator that each contain different aptamers that bind different drugs cannot bind to each other unless both drugs are present. As illustrated by these examples, dual guide nucleic acids (e.g., guide RNAs) can be designed to be inducible.

[0181] Examples of aptamers and riboswitches can be found in, 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 which are incorporated by reference in their entireties.

[0182] Dual guide nucleic acids (e.g., guide RNAs) contained in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 that can hybridize to form protein-binding fragments, or Non-limiting examples of nucleotide sequences that may be included in these complements.

[0183] An analyte single guide nucleic acid (e.g., guide RNA) comprises two complementary stretches of nucleotides (analogous to the "target" and "activator" of a dual guide nucleic acid) that hybridize to form a double-stranded RNA duplex (dsRNA duplex) that hybridizes to a protein-binding fragment (thus generating a stem-loop structure) and are covalently linked by an intervening nucleotide (a "linker" or "linker nucleotide"). Thus, an analyte single guide nucleic acid (e.g., syn The guide RNA (G-RNA) contains a target and an activator with double-stranded fragments. where the duplex-forming fragments of the target and activator hybridize to each other. The target and activator can be covalently linked via the 3' end of the target and the 5' end of the activator. The activator can be covalently linked via the 5' end of the target and the 3' end of the activator.

[0184] The linker of a single guide nucleic acid can have a length of 3 nucleotides to 100 nucleotides. In some embodiments, the linker of a single guide nucleic acid (e.g., a guide RNA) is 4 nt or less. It is.

[0185] Exemplary single guide nucleic acids (e.g., guide RNAs) comprise two complementary stretches of nucleotides that hybridize to form a dsRNA duplex. In some embodiments, one of the two complementary stretches of nucleotides of the single guide nucleic acid (e.g., guide RNA) (or DNA encoding the stretch) comprises 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides). nucleotide, or 20 or more adjacent nucleotides) one of the activator (tracrRNA) molecules described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617. , or their complements that are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical are one or 100% identical.

[0186] In some embodiments, one of the two complementary stretches of nucleotides of the single guide nucleic acid (e.g., guide RNA) (or DNA encoding the stretch) has 8 or more adjacent nucleotides. nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) ) as described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 or their complements.

[0187] In some embodiments, one of the two complementary stretches of nucleotides of the single guide nucleic acid (e.g., guide RNA) (or DNA encoding the stretch) has 8 or more adjacent nucleotides. For extensions of nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides), the methods described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617 one of the target (crRNA) or activator (tracrRNA) sequences of, or their complements 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical to the body or 100% identical do.

[0188] Suitable cognate pairs of targets and activators can be routinely determined taking into account the species name and base pairing (for the dsRNA duplex of the protein-binding domain). Any activator / target pair can be used as part of a dual guide nucleic acid (e.g., guide RNA) or as part of a single guide nucleic acid (e.g., guide RNA).

[0189] In some embodiments, the activator (e.g., trRNA, trRNA-like molecule, etc.) of a dual guide nucleic acid (e.g., guide RNA) (e.g., dual guide RNA) or single guide nucleic acid (e.g., guide RNA) (e.g., single guide RNA) comprises a stretch of nucleotides having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, or 100% sequence identity to an activator (tracrRNA) molecule described in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or a complement thereof.

[0190] In some embodiments, a dual guide nucleic acid (e.g., a dual guide RNA) or Activators of single-guide nucleic acids (e.g., single-guide RNAs) (e.g., trRNAs, trRNA-like molecules) 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- Such nucleic acid-like molecules have lengths ranging from 30 to 200 nucleotides (nt).

[0191] The protein-binding fragment may have a length of from 10 nucleotides to 100 nucleotides.

[0192] In addition, the subject single-guide nucleic acid (e.g., single-guide RNA) and the subject dual-guide For both the dsRNA duplex and the dsRNA duplex of the protein-binding fragment, the dsRNA duplex can have a length of 6 base pairs (bp) to 50 bp. The percent homology between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding fragment can be 60% or more. For example, The percent homology 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. The hybridization rate may be 98% or more, or 99% or more (e.g., in some embodiments, There are some nucleotides that do not size and therefore generate bulges within the dsRNA duplex. In some embodiments, the percent homology between the nucleotide sequences that hybridize to form the dsRNA duplex of the protein-binding fragment is 100%.

[0193] Hybrid Guide Nucleic Acid In some embodiments, the guide nucleic acid is two RNA molecules (dual guide RNA). In some embodiments, the guide nucleic acid is one RNA molecule (single guide RNA). In some embodiments, the guide nucleic acid is a DNA / RNA hybrid molecule. In such an embodiment, the protein-binding fragment of the guide nucleic acid is RNA, and the RNA duplex Thus, the duplex-forming fragments of the activator and target are RNA. However, the targeting fragment of the guide nucleic acid can be DNA. Thus, when a DNA / RNA hybrid guide nucleic acid is a dual guide nucleic acid, the "target" molecule is a hybrid molecule (e.g., a target In such an embodiment, the duplex-forming fragment of the "activator" molecule can be RNA (e.g., the duplex-forming fragment of the target molecule can be DNA and the duplex-forming fragment can be RNA). to form an RNA-duplex with the double-stranded fragment, while The nucleotides of an "activator" molecule can be DNA (in which case the activator molecule is a hybrid DNA / RNA molecule) or RNA (in which case the activator molecule is RNA). A DNA / RNA hybrid guide nucleic acid is a single guide nucleic acid. In the case of a nucleic acid, the targeting fragment can be DNA, and the double-stranded-forming fragment (the target of a single guide nucleic acid) can be a nucleic acid. The targeting and duplex-forming fragments (which create the protein-binding fragment) can be RNA, and the outer nucleotides of the targeting and duplex-forming fragments can be RNA or DNA.

[0194] A DNA / RNA hybrid guide nucleic acid may be useful in some embodiments, for example when the target nucleic acid is RNA. Cas9 normally hybridizes to the target DNA and thus the target The target nucleic acid is then associated with a guide RNA that forms a DNA-RNA duplex at the target site. In this case, it is sometimes advantageous to repeat the DNA-RNA duplex at the target site by using a targeting fragment (of the guide nucleic acid) that is DNA instead of RNA. However, since the protein-binding fragment of the guide nucleic acid is an RNA-duplex, the target molecule is DNA in the targeting fragment, and In the double-stranded-forming fragment, the hybrid guide nucleic acid is RNA. The hybrid guide nucleic acid can bias Cas9 binding to single-stranded target nucleic acid versus double-stranded target nucleic acid.

[0195] Exemplary Guide Nucleic Acids Any guide nucleic acid can be used. Many different types of guide nucleic acids are known in the art. The guide nucleic acid selected will be appropriately paired with the particular CRISPR system being used (e.g., the particular RNA-guided endonuclease being used). Thus, the guide nucleic acid can be, for example, any of the nucleic acids described herein. Any RNA-guided endonuclease described herein or known in the art may be used. The guide nucleic acid and the RNA-guided endonuclease can be, for example, Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617.

[0196] In some embodiments, a suitable guide nucleic acid is a nucleic acid sequence comprising two separate RNA polynucleotides. In some embodiments, the first two distinct RNA polynucleotides The molecule (activator) binds eight or more contiguous nucleotides (e.g., tide, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more) identical to any one of the nucleotide sequences set forth in PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements. , 90% or more, 95% or more, 98% or more, 99% or more, or 100%) of nucleotide sequence identity with In some embodiments, the second two separate RNA polypeptides comprise a nucleotide sequence The nucleotide molecule (target) is a sequence of eight or more contiguous nucleotides (e.g., eight or more contiguous nucleotides). nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides) Any one of the nucleotide sequences set forth in application numbers PCT / US2016 / 052690 and PCT / US2017 / 062617, or their complements, has a sequence identity of 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more). or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100%) nucleotide sequence identity The present invention includes a nucleotide sequence having the following structure:

[0197] In some embodiments, a suitable guide nucleic acid is a single RNA polynucleotide. and 8 or more contiguous nucleotides (e.g., 8 or more contiguous nucleotides, 10 or more adjacent nucleotides, 12 or more adjacent nucleotides, 15 or more adjacent nucleotides or any one of the nucleotide sequences set forth in International Patent Application Nos. PCT / US2016 / 052690 and PCT / US2017 / 062617, or any one of the nucleotide sequences set forth therein, for a stretch of 20 or more contiguous nucleotides. The nucleic acid sequence includes a first and a second nucleotide sequence having 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) nucleotide sequence identity to their complement.

[0198] In some embodiments, the guide RNA is Cpf1 and / or Cas9 guide RNA.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, Cpf1 and / or Cas9 guide RNA has a total length of 35 nt, 36 nt, 37 nt, 38 nt, 39 nt, 40 nt, 41 nt, 42 nt, 43 nt, 44 nt, 45 nt, 46 nt, 47 nt, 48 nt, 49 nt, or 50 nt. The Cpf1 and / or Cas9 guide RNA may comprise a target nucleic acid-binding fragment and a duplex-forming fragment.

[0199] The target nucleic acid-binding fragment of the Cpf1 and / or Cas9 guide RNA may be 15 nt to 30 nt, e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, In some embodiments, the target nucleic acid-binding fragment has a length of 23 nt. In some embodiments, the target nucleic acid-binding fragment has a length of 24 nt. The binding fragment has a length of 24 nt. In some embodiments, the target nucleic acid-binding fragment has a length of 25 nt.

[0200] The target nucleic acid-binding fragment of the Cpf1 and / or Cas9 guide RNA is a fragment of the corresponding length of the target nucleic acid sequence. The targeting fragment may have 100% complementarity to the corresponding length of the target nucleic acid sequence. The targeting fragment may have less than 100% complementarity to the corresponding length of the target nucleic acid sequence. For example, the target nucleic acid binding fragment of the Cpf1 and / or Cas9 guide RNA may have 1, 2, 3, 4, or 5 nucleotides that are not complementary to the target nucleic acid sequence. For example, some In some embodiments, the target nucleic acid-binding fragment has a length of 25 nucleotides and The target nucleic acid sequence has a length of 25 nucleotides, and in some embodiments, the target nucleic acid-binding As another example, in some embodiments, the target nucleic acid-binding fragment has a length of 25 nucleotides and the target nucleic acid sequence is 25 nucleotides long. In some embodiments, the target nucleic acid-binding fragment has a length of 24 nucleotides and in some embodiments, the target nucleic acid-binding fragment has one non-complementary nucleotide and 24 complementary nucleotides with the target nucleic acid sequence.

[0201] The Cpf1 and / or Cas9 guide RNA duplex-forming fragment can have a length of 15 nt to 25 nt, e.g., 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, or 25 nt.

[0202] In some embodiments, the double-stranded-forming fragment of the Cpf1 guide RNA may comprise the nucleotide sequence 5'-AAUUUCUACUGUUGUAGAU-3'.

[0203] Further elements In some embodiments, the guide nucleic acid (e.g., guide RNA) can include an additional fragment (single or multiple fragments). or more) (in some embodiments, at the 5' end, in some embodiments, at the 3' end at the 5' end, in some embodiments, at either the 5' or 3' end, and in some embodiments, embedded within the sequence (i.e., not at the 5' and / or 3' end). In some embodiments, the fragment contains a 5' cap (e.g., a 7-methylguanylic acid cap (m), in some embodiments, both the 5' and 3' ends, in some embodiments, embedded and at the 5' and / or 3' ends, etc.). For example, suitable additional fragments include a 5' cap (e.g., a 7-methylguanylic acid cap (m), 73' polyadenylation tail (i.e., 3' poly(A) tail); ribozyme sequence (e.g., to enable self-cleavage of the guide nucleic acid or a component of the guide nucleic acid, e.g., target, activator, etc. ... switch sequences (e.g., to allow for regulated stability and / or regulated accessibility by proteins and protein complexes); sequences that target the RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.); modifications or sequences that provide tracking (e.g., labels such as fluorescent molecules (i.e., fluorescent dyes), fluorescent detection a sequence or other moiety that facilitates the synthesis of a protein (e.g., a protein that acts on DNA, a transcriptional binding proteins for proteins that bind RNA (e.g., RNA aptamers), labeled proteins, fluorescently labeled proteins, etc.) These may include sequences or other modifications that provide binding sites; modifications or sequences that provide increased, decreased and / or controllable stability; and combinations thereof.

[0204] RNA-guided endonucleases In addition to or instead of a guide nucleic acid, a composition may contain an RNA-guided endonuclease protein or a nucleic acid (e.g., an mRNA or vector) encoding the same. Any RNA-guided endonuclease may be used. Selection of the RNA-guided endonuclease to be used will depend, at least in part, on the intended end use of the CRISPR system used.

[0205] In some embodiments, the polypeptide is a Cas9 polypeptide. Cas9 polypeptides suitable for inclusion in the compositions of the present disclosure include naturally occurring Cas9 polypeptides (e.g., naturally occurring in bacterial and / or archaeal cells), or non-naturally occurring Cas9 polypeptides, as described below. A live Cas9 polypeptide (e.g., a Cas9 polypeptide can be a mutant Cas9 polypeptide, as discussed below). In some embodiments, those skilled in the art can use the present disclosure to It will be understood that the Cas9 polypeptides disclosed herein can be any variant derived from or isolated from any source. In other embodiments, the Cas9 peptides of the present disclosure can include one or more mutations described in the literature, including but not limited to the functional mutations described in: Fonfara et al. Nucleic Acids Res. 2014 Feb;42(4):2577-90; Nishimasu et al. 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); filed March 15, 2013 See also U.S. Patent Application No. 13 / 842,859, incorporated herein by reference; see also U.S. Patent Nos. 8,697,359; 8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, all of which are incorporated herein by reference. Thus, in some embodiments, the systems and methods disclosed herein can be used with wild-type Cas9 proteins that have double-stranded nuclease activity, Cas9 mutants that act as single-stranded nickases, or other mutants that have modified nuclease activity. Thus, a Cas9 polypeptide suitable for inclusion in a composition of the disclosure can be, for example, an enzymatically active Cas9 polypeptide that can effect a single- or double-stranded cleavage in a target nucleic acid or that can have reduced enzymatic activity compared to a wild-type Cas9 polypeptide.

[0206] Naturally occurring Cas9 polypeptides bind the guide nucleic acid, thereby targeting the target nucleic acid (target site) and cleaves the target nucleic acid (e.g., cleaves dsDNA to form double stranded nucleic acid). (e.g., generating strand breaks, cleaving ssDNA, cleaving ssRNA, etc.) The subject Cas9 polypeptide is The RNA-binding portion interacts with the analyte guide nucleic acid, and the active portion exhibits site-directed enzymatic activity (e.g., nuclease activity, activity for DNA and / or RNA methylation, activity for DNA and / or RNA cleavage, activity for histone acetylation, activity for histone methylation, activity for RNA modification, activity for RNA-binding). In some embodiments, the nucleic acid molecule exhibits an activity for the synthesis of a nucleic acid molecule, an activity for RNA splicing, etc. In some embodiments, an active portion exhibits reduced nuclease activity relative to a corresponding portion of a wild-type Cas9 polypeptide. In some embodiments, an active portion is enzymatically inactive.

[0207] The assay to determine whether a protein has an RNA-binding moiety that interacts with the analyte guide nucleic acid can be any convenient binding assay that tests for binding between a protein and a nucleic acid. Exemplary binding assays include binding assays (e.g., gel shift assays) that include adding a guide nucleic acid and a Cas9 polypeptide to a target nucleic acid.

[0208] Determine whether the protein has an active portion (e.g., whether the polypeptide cleaves a target nucleic acid) The assay for determining whether a nucleic acid has nuclease activity that cleaves nucleic acids is performed The assay can be any convenient nucleic acid cleavage assay to be tested. An exemplary cleavage assay includes adding a guide nucleic acid and a Cas9 polypeptide to a target nucleic acid.

[0209] In some embodiments, a Cas9 polypeptide suitable for inclusion in a composition of the present disclosure has an enzymatic activity that modifies a target nucleic acid (e.g., a nuclease activity, a methyltransferase activity, , demethylase activity, DNA repair activity, DNA damaging activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity).

[0210] In other embodiments, a Cas9 polypeptide suitable for inclusion in a composition of the present disclosure has an enzymatic activity (e.g., a methyltransferase) that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid. The enzyme has ubiquitin ligase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).

[0211] Many Cas9 orthologs from a wide variety of species have been identified, and in some embodiments, the proteins share only a few identical amino acids. All identified Cas9 orthologs have the same domain structure with a central HNH endonuclease domain and a split RuvC / RNaseH domain. The Cas9 protein contains four key domains with a conserved structure. Motifs 1, 2, and 4 are RuvC-like motifs, whereas -3 is the HNH motif.

[0212] In some embodiments, a suitable Cas9 polypeptide has an amino acid sequence having four motifs. Each of motifs 1-4 comprises the amino acid sequence of the Cas9 amino acid sequence shown in FIG. 1 (SEQ ID NO: 1); or motifs 1-4 of the Cas9 amino acid sequence shown in Table 1 below (motifs 1-4 of SEQ ID NO: 1 are 3-6, respectively, as shown in Table 1 below); or having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% amino acid sequence identity to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence shown in Figure 1 (SEQ ID NO:1).

[0213] In some embodiments, the Cas9 polypeptide comprises an amino acid sequence set forth in FIG. 1 and having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and comprises amino acid substitutions of N497, R661, Q695, and Q926 with respect to the amino acid sequence set forth in SEQ ID NO:1; or comprises amino acid substitutions of K855 with respect to the amino acid sequence set forth in SEQ ID NO:1; or comprises amino acid substitutions of K810, K1003, and R1060 with respect to the amino acid sequence set forth in SEQ ID NO:1; or comprises amino acid substitutions of K848, K1003, and R1060 with respect to the amino acid sequence set forth in SEQ ID NO:1.

[0214] As used herein, the term "Cas9 polypeptide" includes the term "mutant Cas9 polypeptide"; and the term "mutant Cas9 polypeptide" includes the term "chimeric Cas9 polypeptide." include.

[0215] Mutant Cas9 Polypeptides Suitable Cas9 polypeptides for inclusion in the compositions of the present disclosure include mutant Cas9 polypeptides. Mutant Cas9 polypeptides can be any combination of wild-type Cas9 polypeptides (e.g., naturally occurring Cas9 polypeptides as described above). The amino acid sequence of the Cas9 polypeptide (which corresponds to the Cas9 polypeptide of the present invention) is different from that of the Cas9 polypeptide of the present invention (i.e., has a deletion, insertion, substitution, fusion) (i.e., has at least one amino acid difference). In some cases, the mutant Cas9 polypeptide has an amino acid sequence that is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% of the nuclease activity of a corresponding wild-type Cas9 polypeptide. In some embodiments, the mutant Cas9 polypeptide has no substantial nuclease activity. When a Cas9 polypeptide is a mutant Cas9 polypeptide that has no substantial nuclease activity, it can be referred to as "dCas9."

[0216] In some embodiments, the mutant Cas9 polypeptide has reduced nuclease activity. For example, a mutant Cas9 polypeptide suitable for use in the conjugation methods of the present disclosure is a wild-type Cas9 polypeptide, e.g., a wild-type Cas9 polypeptide comprising the amino acid sequence shown in Figure 1 (SEQ ID NO: 1). Less than about 20%, less than about 15%, less than about 10%, less than about 5% of the endonuclease activity of the polypeptide , less than about 1%, or less than about 0.1%.

[0217] In some embodiments, a mutant Cas9 polypeptide can cleave a complementary strand of a target nucleic acid, but has a reduced ability to cleave a non-complementary strand of a double-stranded target nucleic acid. For example, a mutant Cas9 polypeptide can have a mutation (amino acid substitution) that reduces the function of the RuvC domain (e.g., "Domain 1" in FIG. 1). As a non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a D10A mutation (e.g., an aspartate substitution at an amino acid position corresponding to position 10 of SEQ ID NO:1). thate to alanine) and therefore capable of cleaving the complementary strand of a double-stranded target nucleic acid. However, when the mutant Cas9 polypeptide cleaves a double-stranded target nucleic acid, , generating a single-strand break (SSB) instead of a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug 17;337(6096):816-21).

[0218] In some embodiments, the mutant Cas9 polypeptide can cleave the non-complementary strand of a double-stranded target nucleic acid, but has a reduced ability to cleave the complementary strand of the target nucleic acid. For example, the mutant Cas9 polypeptide can have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC / HNH / RuvC domain motif, "Domain 2" in FIG. 1). As a non-limiting example, some In embodiments, a mutant Cas9 polypeptide can have an H840A mutation (e.g., histidine to alanine at an amino acid position corresponding to position 840 of SEQ ID NO:1) ( FIG. 1 ) and thus can cleave a non-complementary strand of a target nucleic acid, but not a double-stranded target nucleic acid (thus, a mutant Cas9 polypeptide can cleave a double-stranded target nucleic acid). When cleaving nucleic acids, it creates single strand breaks (SSBs) instead of double strand breaks (DSBs) and reduces the ability of the complementary strand to cleave. Such Cas9 polypeptides have a reduced ability to cleave a target nucleic acid (e.g., a single-stranded target nucleic acid) but retain the ability to bind a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid).

[0219] In some embodiments, the mutant Cas9 polypeptide has a reduced ability to cleave both complementary and non-complementary strands of a double-stranded target nucleic acid. As a non-limiting example, in some embodiments, the mutant Cas9 polypeptide has both a D10A mutation and a H840A mutation (e.g., mutations in both the RuvC domain and the HNH domain), such that the polypeptide has a reduced ability to cleave both complementary and non-complementary strands of a double-stranded target nucleic acid. Such Cas9 polypeptides have a reduced ability to cleave a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid), but maintain the ability to bind a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid).

[0220] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has a W476A and a W1126A mutation, such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids, but retain the ability to bind target nucleic acids.

[0221] As another non-limiting example, in some embodiments, the mutant Cas9 polypeptide has P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations, such that the polypeptide Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids. Such Cas9 polypeptides have a reduced ability to cleave target nucleic acids, but retain the ability to bind target nucleic acids.

[0222] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has H840A, W476A, and W1126A mutations such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but maintains the ability to bind to a target nucleic acid.

[0223] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has H840A, D10A, W476A, and W1126A mutations such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but maintains the ability to bind to a target nucleic acid.

[0224] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutations such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such Cas9 polypeptides have a reduced ability to cleave a target nucleic acid, but retain the ability to bind to a target nucleic acid.

[0225] As another non-limiting example, in some embodiments, a mutant Cas9 polypeptide has a D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A, and D1127A mutation such that the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid, but maintains the ability to bind to a target nucleic acid.

[0226] Other residues may be mutated to achieve the above effects (i.e., to alter one or other nuclease As a non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 may be altered (i.e., substituted) (see Table 1 for further information regarding conservation of Cas9 amino acid residues). In addition, alanine substitutions may be used. Mutations other than ribozyme mutations are appropriate.

[0227] In some embodiments, a mutant Cas9 polypeptide having reduced catalytic activity (e.g., a Cas9 protein having D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, D10B, D12C, D17D, D17E, D17F ... In the case of a mutant Cas9 polypeptide having the nucleotide sequence H982A, H983A, A984A and / or D986A, the mutant Cas9 polypeptide can still be expressed in a site-specific manner, so long as it maintains its ability to interact with the guide nucleic acid. capable of binding to the target nucleic acid (still being guided to the target nucleic acid sequence by the guide nucleic acid) so).

[0228] [Table 1]

[0229] In addition to the above, mutant Cas9 proteins can have the same parameters for sequence identity as those described above for Cas9 polypeptides. Thus, in some embodiments, suitable mutant Cas9 polypeptides include an amino acid sequence having four motifs, Each of F1-4 has an amino acid sequence identity of 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% to the Cas9 amino acid sequence (SEQ ID NO: 1) shown in FIG. or alternatively to motifs 1-4 (motifs 1-4 of SEQ ID NO:1 are SEQ ID NOs:3-6, respectively, as shown in Table 1); or alternatively to amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence shown in FIG. 1 (to SEQ ID NO:1, see International Patent Application Nos. PCT / US2016 / 052690 and Any Cas9 protein as defined above, including those specifically referenced in PCT / US2017 / 062617, may be used as a Cas9 polypeptide or chimeric Cas9 polypeptide in the compositions of the disclosure. It may be used as part of a polypeptide.

[0230] In some embodiments, a suitable mutant Cas9 polypeptide is a Cas9 amine shown in FIG. The amino acid sequence has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more or 100% amino acid sequence identity to the amino acid sequence (SEQ ID NO: 1). Any of the Cas9 proteins defined above, including those specifically referenced in application numbers PCT / US2016 / 052690 and PCT / US2017 / 062617, may be used as a mutant Cas9 polypeptide or as part of a chimeric mutant Cas9 polypeptide in the compositions of the disclosure.

[0231] Chimeric Polypeptides (Fusion Polypeptides) In some embodiments, the mutant Cas9 polypeptide is a chimeric Cas9 polypeptide (such as the present As used herein, a fusion polypeptide, e.g., also referred to as a "Cas9 fusion polypeptide," may bind and / or modify (e.g., cleave, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with the target nucleic acid (e.g., methylate, acetylate, etc., of histone tails).

[0232] A Cas9 fusion polypeptide is a polypeptide that is a wild-type Cas9 polypeptide (e.g., a naturally occurring Cas9 polypeptide). The Cas9 fusion polypeptide is a mutant Cas9 polypeptide that differs in sequence from the Cas9 polypeptide of the present invention. A Cas9 polypeptide (e.g., a wild-type Cas9 polypeptide, a mutant Cas9 polypeptide, a polypeptide with reduced nuclease activity, or a polypeptide with reduced nuclease activity) fused to a covalently linked heterologous polypeptide (also referred to as a "fusion partner"). In some embodiments, the Cas9 polypeptide is a mutant Cas9 polypeptide having the properties described above. In some embodiments, the Cas9 fusion polypeptide is a mutant Cas9 polypeptide with reduced nuclease activity (e.g., dCas9) fused to a covalently linked heterologous polypeptide. In the present invention, the heterologous polypeptide is a Cas9 fusion polypeptide (e.g., a polypeptide having a methyltransferase activity). activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc. In some embodiments, the method of binding a target nucleic acid (e.g., a single stranded target nucleic acid) can result in modification of the target nucleic acid. Thus, in some embodiments, the method of binding a target nucleic acid (e.g., a single stranded target nucleic acid) can result in modification of the target nucleic acid. The method of binding the nucleic acid (anion) can be a method of modifying the target nucleic acid.

[0233] In some embodiments, the heterologous sequence provides for subcellular localization, i.e., the heterologous sequence includes a subcellular localization sequence for targeting to the nucleus (e.g., a nuclear localization signal (NLS)), a sequence for maintaining the fusion protein outside the nucleus, e.g., a nuclear export sequence (NES), a sequence for transporting the fusion protein to ... (e.g., sequences for retention in the endoplasmic reticulum, mitochondrial localization signals for targeting to mitochondria, chloroplast localization signals for targeting to chloroplasts, and endoplasmic reticulum (ER) retention signals) In some embodiments, the mutant Cas9 does not contain an NLS, and thus the protein The heterologous sequence is not targeted to the nucleus (which may be advantageous, for example, if the target nucleic acid is an RNA that resides in the cytosol). In some embodiments, the heterologous sequence is tagged (i.e., the heterologous sequence is a detectable label) (e.g., a fluorescent protein) for ease of tracking and / or purification. proteins, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; histidine tags, e.g., 6XHis tags; hemagglutinin (HA) tags; FLAG tags; Myc tags; etc. In some embodiments, the heterologous sequence may provide decreased or increased stability (i.e., the heterologous sequence is a stability control peptide, e.g., a degron, which may be used to regulate some In embodiments, the degron sequence is regulatable (e.g., temperature-sensitive or drug-controllable). In some embodiments, the heterologous sequence is an increased or decreased fragment of the target nucleic acid. (i.e., the heterologous sequence may be a transcriptional regulatory sequence, e.g., a transcription factor / activator or fragment thereof, a protein that employs a transcription factor / activator or a fragment thereof, (e.g., a transcriptional repressor or fragment thereof, a protein or fragment thereof that employs a transcriptional repressor, a small molecule / drug-responsive transcriptional regulator, etc.). In embodiments, the heterologous sequence may provide a binding domain (i.e., the heterologous sequence may be a protein Binding sequences, e.g., of a Cas9 fusion polypeptide to another protein of interest, e.g., DNA or histones. (e.g., transcription factors or transcription repressors, recruitment proteins, RNA-modifying enzymes, RNA-binding proteins, translation initiation factors, RNA splicing factors, etc., to provide the ability to bind to a modifying protein.) A heterologous nucleic acid sequence is one that is linked (e.g., by genetic engineering) to another nucleic acid sequence and is expressed as a gene. A chimeric nucleotide sequence encoding a mer polypeptide can be generated.

[0234] A subject Cas9 fusion polypeptide (Cas9 fusion protein) can have multiple (one or more, two or more, three or more, etc.) fusion partners in any combination of the above. As an illustrative example, a Cas9 fusion protein can have a heterologous sequence that provides an activity (e.g., for transcriptional regulation, target modification, modification of a protein associated with a target nucleic acid, etc.) and can also have a subcellular localization sequence. In some embodiments, such Cas9 fusion proteins also provide tracking and / or It may have a tag for ease of purification (e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; etc.). As another illustrative example, the Cas9 protein may have one or more NLSs (e.g., two or more, three or more, four or more, five or more, 1, 2, 3, 4, or 5 NLSs). In some embodiments, the fusion partner (or fusion partners) (e.g., a fusion partner that provides an NLS, a tag, an activity, etc.) is located at or near the C-terminus of Cas9. In some embodiments, the fusion partner (or fusion partners) (e.g., a fusion partner that provides an NLS, a tag, an activity, etc.) is located at the N-terminus of Cas9. In embodiments, Cas9 is fused to a fusion partner (or fusion partners) at both the N-terminus and C-terminus. The polypeptide may have a target polypeptide (e.g., an NLS, a tag, a fusion partner that provides activity, etc.).

[0235] Suitable fusion partners that provide increased or decreased stability include, but are not limited to, degron sequences. It will be readily understood by those of skill in the art that degrons are amino acid sequences that control the stability of the protein of which they are a part. For example, the stability of a protein that contains a degron sequence is controlled, in part, by the degron sequence. In some embodiments, suitable degrons are constitutive, such that the degron exerts its effect on protein stability independent of experimental control (i.e., the degron is not drug inducible). In some embodiments, the degron is regulatable. In one embodiment, the present invention provides a mutant Cas9 polypeptide having a stable and stable state, such that the mutant Cas9 polypeptide can be "on" (i.e., stable) or "off" (i.e., unstable) depending on the desired conditions. For example, if the degron is a temperature-sensitive degron, the mutation The Cas9 polypeptide is capable of being transformed into a polypeptide that ... , 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, etc.) and are functional (i.e., "on" and stable) at The degron may be functional (i.e., "off," decomposing) above a threshold temperature. As another example, if the degron is a drug-inducible degron, the presence or absence of a drug may switch the protein from an "off" (i.e., unstable) state to an "on" (i.e., stable) state, or vice versa. An exemplary drug-inducible degron is derived from the FKBP12 protein. The stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron.

[0236] Examples of suitable degrons include those regulated by Shield-1, DHFR, auxin, and / or temperature. Non-limiting examples of suitable degrons are known in the art (e.g., Dohmen et al., Science, 1994. 263(5151): p. 1273-1276: Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol. 2009 Jan;296(1):F204-11 : Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett. 2008 Nov 15;18(22):5941-4: Recent progress with FKBP-derived destabilizing domains; Kanemaki, Pflugers Arch. 2012 Dec 28: Frontiers of protein expression control with conditional degrons; Yang et al., Mol Cell. 2012 Nov 30;48(4):487-8: Titivated for destruction: the methyl degron; Barbour et al., Biosci Rep. 2013 Jan 18;33(1).: Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase; and Greussing et al., J Vis Exp. 2012 Nov 10;(69): Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized flurescent protein (GFP)-based reporter protein; all of which are incorporated by reference in their entireties. (Incorporated herein).

[0237] Exemplary degron sequences have been well characterized and tested in both cells and animals. Thus, Cas9 (e.g., wild-type Cas9; mutant Cas9; reduced nuclease activity; Fusion of a mutant Cas9 (e.g., dCas9) with a degron sequence would be "tunable" Any of the fusion partners described herein may be used in any desired combination. As one non-limiting example illustrating this point, a Cas9 fusion protein (i.e., a chimeric Cas9 polypeptide) may be used that contains a YFP sequence for detection. The Cas9 polypeptide (e.g., wild-type Cas9, mutant Cas9, reduced nucleic acid sequence, degron sequence for stability, and transcription activator sequence for increasing transcription of the target nucleic acid) may be used. Suitable for use as a fusion partner for a mutant Cas9 with cleavage function, etc. Suitable reporter proteins 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. Additionally, reporter proteins that can be used with Cas9 fusion proteins 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. There is no limit to the number of fusion partners that can be used. 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.

[0238] Suitable fusion partners include methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity, any of which directly modifies nucleic acids (e.g., DNA or RNA methylation) or modifies nucleic acid-associated polypeptides (e.g., histones, DNA-binding proteins, and Further suitable fusion partners include, but are not limited to, boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.). do not have.

[0239] A variety of additional suitable fusion partners (or Examples of polypeptides (and fragments thereof) include, but are not limited to, those described in PCT Patent Applications: WO2010 / 075303, WO2012 / 068627, and WO2013 / 155555, which are incorporated by reference in their entireties.

[0240] Suitable fusion partners include a target nucleic acid or a polypeptide related to the target nucleic acid (e.g., His Suitable fusion partners include, but are not limited to, polypeptides that provide an activity that indirectly increases transcription by acting directly on a target protein (such as a fusion protein, a DNA-binding protein, an RNA-binding protein, an RNA-editing protein, etc.). Suitable fusion partners include, but are not limited to, polypeptides that provide a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, or a demyristoylating activity.

[0241] Further suitable fusion partners may be used to direct increased transcription and / or translation of a target nucleic acid. The provided polypeptides (e.g., transcriptional activators or fragments thereof, transcriptional activators Proteins or fragments thereof that recruit small molecules / drug-responsive transcription and / or translation These include, but are not limited to, translational regulatory genes, translational regulatory proteins, etc.

[0242] Non-limiting examples of fusion partners for achieving increased or decreased transcription include transcriptional activator and transcriptional repressor domains (e.g., Kruppel-associated box (KRAB or SKD); Mad mSIN3-interacting domain (SID); ERF repressor domain (ERD), etc.). In some embodiments, the Cas9 fusion protein is targeted to a specific location (i.e., sequence) in the target nucleic acid by a guide nucleic acid and a promoter (which may be a transcriptional activator Blocks RNA polymerase binding to and / or localizes the modifying a target nucleic acid or modifying a polypeptide associated with a target nucleic acid In some cases, fusion sequences that modify the nucleotide sequence of the target gene are used to modulate the locus-specific regulation of the target gene. In some embodiments, the change is transient (e.g., transcriptional repression or activation). In some embodiments, the change is heritable (e.g., an epigenetic modification of the target nucleic acid or the target It is a protein that is associated with nucleic acids, e.g., nucleosomes (as done with histones).

[0243] Non-limiting examples of fusion partners for use when targeting ssRNA target nucleic acids include: splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or transcription factors); or release factors; e.g., eIF4G; RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminases acting on RNA (ADAR), including A-I and / or C-U editing enzymes); These include, but are not limited to, fusion partners; heliembodiments; RNA-binding proteins; and the like. It is understood that a fusion partner can include an entire protein, or in some embodiments, a fragment of a protein (e.g., a functional domain).

[0244] In some embodiments, the heterologous sequence can be fused to the C-terminus of the Cas9 polypeptide. In some embodiments, the heterologous sequence can be fused to the N-terminus of the Cas9 polypeptide. In some embodiments, the heterologous sequence can be fused to an internal portion (i.e., the N- or can be fused to a portion other than the C-terminus).

[0245] Additionally, the fusion partner of the chimeric Cas9 polypeptide may be any domain capable of interacting with ssRNA, whether primary or irreversible, directly or indirectly (which for the purposes of this disclosure is understood to include intra- and / or intermolecular secondary structures, e.g., hairpins, stem-loops, etc.). endonucleases (e.g., RNase I, CRR22 DYW domain, Dicer, and the PIN (PilT N-terminal) domain from proteins such as SMG5 and SMG6); proteins and protein domains involved in stimulating RNA cleavage (e.g., CPSF, CstF, CFIm, and CFIIm); exonucleases (e.g., XRN-1 or exonuclease T); deadenylases (e.g., HNT3); proteins and domains involved in nonsense-mediated RNA decay. proteins and protein domains involved in RNA stabilization (e.g., PABP); proteins and protein domains involved in translational repression (e.g., Ago2 and Ago4); Proteins and protein domains involved in stimulating translation (e.g., Staufen); proteins and protein domains involved in (e.g., capable of regulating) translation (e.g., translation factors such as initiation factors, elongation factors, and termination factors, e.g., eIF4G); proteins and protein domains involved in RNA polyadenylation (e.g., PAP1, GLD-2, and Star-PAP); RNA proteins and protein domains involved in polyuridinylation of RNA (e.g., CID1 and terminal uridylate transferase); proteins and protein domains involved in RNA localization protein domains (e.g., from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains involved in the nuclear retention of RNA (e.g., Rrp6); proteins and protein domains involved in the nuclear export of RNA (e.g., TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains involved in the repression of RNA splicing (e.g., , PTB, Sam68, and hnRNP A1); proteins involved in stimulating RNA splicing and Protein domains (e.g., serine / arginine-rich (SR) domains); decreasing the efficiency of transcription Proteins and protein domains involved in promoting the expression of TLS (e.g., FUS (TLS)); effector domains selected from the group including, but not limited to, proteins and protein domains involved in stimulating transcription (e.g., CDK7 and HIV Tat); or Effector domains include endonucleases; proteins and protein domains that can stimulate RNA cleavage; exonucleases; deadenylases; proteins and protein domains that have nonsense-mediated RNA decay activity; proteins and protein domains that can stabilize RNA; proteins and protein domains that can repress translation; proteins and protein domains that can stimulate translation; proteins and protein domains that can regulate translation. domains (e.g., translation factors such as initiation factors, elongation factors, and termination factors, e.g., eIF4G); RNA polyadenylation The fusion protein may be selected from the group consisting of proteins and protein domains capable of denylation; proteins and protein domains capable of polyuridinylating RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of retaining RNA in the nucleus; proteins and protein domains having RNA export activity; proteins and protein domains capable of inhibiting RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of decreasing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. The binding partner is a PUF RNA-binding domain, which is described in more detail in WO2012068627.

[0246] Several RNA splicing factors that can be used (in whole or as fragments thereof) as fusion partners for the Cas9 polypeptide contain separate sequence-specific RNA binding modules and They have a modular structure with splicing effector domains and splicing enhancer domains. For example, members of the serine / arginine-rich (SR) protein family contain an N-terminal RNA recognition motif (RRM) that binds to exon splicing enhancers (ESEs) in pre-mRNA and a C-terminal RS domain that promotes exon inclusion. As another example, the hnRNP protein hnRNP Al It binds to exon splicing silencers (ESSs) via its RRM domain and inhibits exon inclusion via its C-terminal glycine-rich domain. Splicing factors can regulate the alternative use of splice sites (ss) by binding to regulatory sequences between two alternative sites. For example, ASF / SF2 can recognize an ESE and promote the use of an intron-proximal site, whereas hnRNP A1 can bind to an ESS and inhibit intron splicing. One use of such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly those associated with diseases. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites, encoding proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is upregulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a proapoptotic isoform and is expressed at high levels in cells with high turnover (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms can be determined by the nuclear exon region or exon. It is regulated by multiple cω-elements located either in the extension region (i.e., between the two alternative 5' splice sites). For further examples, see WO2010075303.

[0247] In some embodiments, the Cas9 polypeptide (e.g., wild-type Cas9, mutant Cas9, reduced Mutant Cas9s with enhanced nuclease activity (e.g., mutant Cas9s with enhanced nuclease activity) are linked to fusion proteins via peptide spacers. The toner may be coupled to the

[0248] In some embodiments, the Cas9 polypeptide comprises a "protein transduction domain" or PTD (also known as CPP - cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates crossing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. The PTD attached to another molecule, which can range from a chromosome and / or a nanoparticle, facilitates the molecule crossing a membrane, for example going from the extracellular space to the intracellular space, or from the cytosol into an organelle. In some embodiments, the PTD attached to another molecule In some embodiments, the Cas9 polypeptide comprises two or more NLSs, facilitating entry of the molecule into the nucleus (e.g., in some embodiments, the PTD comprises a nuclear localization signal (NLS)). For example, it comprises two or more NLSs in tandem. In some embodiments, the PTD is covalently linked to the amino terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently linked to the amino terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently linked to the carboxyl terminus of the Cas9 peptide. The PTD is covalently attached to the amino and carboxyl termini of the polypeptide. In some embodiments, the PTD is covalently attached to a nucleic acid (e.g., a guide nucleic acid, a polynucleotide encoding a guide nucleic acid, a polynucleotide encoding a Cas9 polypeptide, etc.). Exemplary PTDs include a minimal undecapeptide protein transduction domain (YGRKKRRQRRR; corresponding to residues 47-57 of HIV-1 TAT, comprising SEQ ID NO:7); a polyarginine sequence containing a sufficient number of arginines to directly enter a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein form transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); truncated human calcitonin Exemplary PTDs include, but are not limited to, arginine peptides (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:8); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:9); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:10); and RQIKIWFQNRRMKWKK (SEQ ID NO:11). Exemplary PTDs include YGRKKRRQRRR (SEQ ID NO:12), RKKRRQRRR (SEQ ID NO:13); 3 arginine Arginine homopolymers of from 1 to 50 arginine residues; e.g. Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 14); RKKRRQRR (SEQ ID NO: 15); YARAAARQARA (SEQ ID NO: 16); THRLPRRRRRR ( SEQ ID NO:17); and GGRRARRRRRR (SEQ ID NO:18). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs contain a polycationic CPP (e.g., Arg9 or "R9") connected to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, which reduces the net charge to near zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesive properties, thus "activating" the ACPP to cross the membrane.

[0249] In some embodiments, the composition may contain Cpf1 RNA-guided endonuclease. , examples of which are provided in Figures 2, 16, or 17. Another name for the Cpf1 RNA-guided endonuclease is Cas12a. The Cpf1 CRISPR system of the present disclosure includes: i) a single endonuclease ii) a crRNA, wherein the 3'-end portion of the crRNA comprises a guide sequence complementary to the target nucleic acid. In this system, the Cpf1 nuclease is directly recruited to the target DNA by the crRNA. In some embodiments, the guide sequence for Cpf1 is capable of inducing detectable DNA cleavage. must be at least 12 nt, 13 nt, 14 nt, 15 nt, or 16 nt to achieve cleavage and be a minimum of 14 nt, 15 nt, 16 nt, 17 nt, or 18 nt to achieve sufficient DNA cleavage. .

[0250] The Cpf1 system of the present disclosure differs from Cas9 in a number of ways. First, unlike Cas9, Cpf1 does not require a separate tracrRNA for cleavage. In some embodiments, the Cpf1 crRNA can be as short as about 42-44 bases long - of which 23-25 ​​nt are guide sequences and 19 nt are non-stranded. is a constitutive direct repeat. In contrast, the combined Cas9 tracrRNA and crRNA synthetic sequences can be approximately 100 bases long.

[0251] Second, Cpf1 prefers a "TTN" PAM motif located 5' upstream of its target. This is essential for Cas9 This is in contrast to the "NGG" PAM motif located 3' of the target DNA for the system. In some embodiments, the uracil base immediately preceding the guide sequence may not be replaced (Zetsche, B. et al. 2015. "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System" Cell 163, 759-771, which is incorporated herein by reference in its entirety for all purposes). (Incorporated herein by reference).

[0252] Third, the cleavage sites for Cpf1 are staggered by about 3-5 bases, which is (Kim et al., 2016. Genome-wide analysis reveals specificities of Cpf1 endonuclease s in human cells, published online June 6, 2016.) These sticky ends with overhangs are thought to facilitate NHEJ-mediated ligation and improve gene editing of DNA fragments with matching ends. The cleavage site is at the 3' end of the target DNA distal to the 5' end, which is the PAM. The cleavage site is usually located at the 3' end of the target DNA, distal to the 5' end, which is the hybridized The 18th base on the unlinked strand and the corresponding 23rd base on the complementary strand hybridized to the crRNA It continues to the th base.

[0253] Fourth, in the Cpf1 complex, a "seed" region is located within the first 5 nt of the guide sequence. The Cpf1 crRNA seed region is highly sensitive to mutations, and mononucleotides in this region are highly likely to be involved in the regulation of the Cpf1 gene. Even group substitution can dramatically reduce cleavage activity (Zetsche B. et al. 2015 “Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” Cell 163, 759-771). (see ). Remarkably, unlike Cas9 CRISPR targets, the cleavage sites and seed regions of the Cpf1 system do not overlap. Further guidance for the design of Cpf1 crRNA targeting oligos is available (Zetsche B. et al. 2015. "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System" Cell 163, 759-771).

[0254] Those skilled in the art will understand that the Cpf1 disclosed herein can be any variant derived or isolated from any source, many of which are known in the art. For example, in some embodiments, the Cpf1 peptide of the present disclosure can include FnCPF1 (e.g., SEQ ID NO: 2) shown in Figure 2, AsCpf1 (e.g., Figure 16), LbCpf1 (e.g., Figure 17), or any of many other known Cpf1 proteins from various other microbial species and their synthetic variants.

[0255] In some embodiments, the composition contains a Cpf1 polypeptide. In some embodiments, the Cpf1 polypeptide is enzymatically active, e.g., the Cpf1 polypeptide cleaves the target nucleic acid when bound to a guide RNA. In some embodiments, the Cpf1 polypeptide The polypeptides exhibit reduced enzymatic activity relative to a wild-type Cpf1 polypeptide (eg, relative to a Cpf1 polypeptide comprising the amino acid sequence shown in Figure 2, 16, or 17) and retain DNA binding activity.

[0256] In some embodiments, the Cpf1 polypeptide has the amino acid sequence shown in FIG. The amino acid sequence includes an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence. In some embodiments, the Cpf1 polypeptide has at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least a contiguous stretch of 100 amino acids to 200 amino acids (aa), 200 aa to 400 aa, 400 aa to 600 aa, 600 aa to 800 aa, 800 aa to 1000 aa, 1000 aa to 1100 aa, 1100 aa to 1200 aa, or 1200 aa to 1300 aa of the amino acid sequence shown in 2, 16, or 17. 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. The amino acid sequence includes:

[0257] In some embodiments, the Cpf1 polypeptide comprises amino acid sequence shown at 2, 16, or 17. At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, of the RuvCI domain of the Cpf1 polypeptide of the sequence At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence having at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence shown at 2, 16, or 17. 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 In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence having at least 95%, at least 90%, or 100% amino acid sequence identity to an amino acid sequence shown in FIG. The amino acid sequence of the RuvCIII domain of the Cpf1 polypeptide is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least Any of these amino acid sequences may have 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.

[0258] In some embodiments, the Cpf1 polypeptide has a decreased amino acid sequence relative to a wild-type Cpf1 polypeptide (e.g., a Cpf1 polypeptide comprising an amino acid sequence shown in FIG. 2, 16, or 17). In some embodiments, the Cpf1 polypeptide exhibits enzymatic activity and maintains DNA binding activity. The peptide has at least 30%, at least 35%, or both of the amino acid sequences shown in FIG. , 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 and the like, which have 90%, at least 95%, at least 90%, or 100% amino acid sequence identity with 2, 16, or 17; and In some embodiments, the Cpf1 polypeptide contains an amino acid substitution (e.g., a D to A substitution) at an amino acid residue that is 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 ... have at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity and comprises an amino acid substitution (e.g., an E to A substitution) at an amino acid residue corresponding to amino acid 1006 of the amino acid sequence shown in Figure 2, 16, or 17. In some embodiments, the Cpf1 polypeptide has an identical identity to the amino acid sequence shown in Figure 2, 16, or 17 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 have at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity and comprises an amino acid substitution (eg, a D to A substitution) at an amino acid residue corresponding to amino acid 1255 of the amino acid sequence shown in Figure 2, 16, or 17.

[0259] In some embodiments, the Cpf1 polypeptide is a fusion polypeptide, for example, where the Cpf1 fusion polypeptide comprises: a) a Cpf1 polypeptide; and b) a heterologous fusion partner. In some embodiments, the heterologous fusion partner is fused to the N-terminus of the Cpf1 polypeptide. In some embodiments, the heterologous fusion partner is fused to the C-terminus of the Cpf1 polypeptide. In some embodiments, the heterologous fusion partner is fused to both the N-terminus and the C-terminus of the Cpf1 polypeptide. In some embodiments, the heterologous fusion partner is inserted internally within the Cpf1 polypeptide.

[0260] Suitable heterologous fusion partners include NLSs, epitope tags, fluorescent polypeptides, and the like.

[0261] Combined guide RNA and donor nucleic acid In one aspect, the present invention provides a complex comprising a CRISPR system comprising an RNA-guided endonuclease (e.g., a Cas9 or Cpf1 polypeptide), a guide RNA, and a donor polynucleotide. wherein the guide RNA and the donor polynucleotide are linked. The guide RNA and the donor polynucleotide can be covalently linked, as exemplified in In one embodiment, the guide RNA is either covalently or non-covalently linked. In another embodiment, the guide RNA and the donor polynucleotide are enzymatically linked. The donor RNA and the donor polynucleotide hybridize to each other. Thus, both the guide RNA and the donor polynucleotide hybridize to the bridge sequence. Any number of such hybridization schemes are possible.

[0262] Deaminase In some embodiments, the complex or composition comprises a deaminase (e.g., an adenine base As used herein, the term "deaminase" or "deaminase" refers to a deaminase that is a transcription factor. "Aminase domain" refers to an enzyme that catalyzes the removal of an amine group from a molecule, i.e., deamination. In some embodiments, the deaminase is a cytidine deaminase, which hydrolyzes cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase is a cytosine deaminase, which catalyzes the hydrolytic deamination of cytosine to uracil (e.g., in RNA) or thymine (e.g., in DNA).

[0263] In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminases provided herein (e.g., modified adenosine deaminases) are The enzyme adenosine deaminase (adenosine deaminase) can be derived from any organism, including 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.

[0264] In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, or at least 70% identical to a naturally occurring deaminase. At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least In some embodiments, the adenosine deaminase is from a bacterium, such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. The deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an 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 relative to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA. In some embodiments, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues may be deleted. In some embodiments, the ecTadA deaminase does not include an N-terminal methionine. In some embodiments, the deaminase is APOBEC1 or a mutant thereof.

[0265] The deaminase may be conjugated (i.e., assembled) with any other CRISPR element described herein. In some embodiments, the deaminase may be used as a CRISPR component (i.e., as a complex) or the deaminase may be fused to any other CRISPR element described herein (e.g., Cas9 or Cpf1). In this method, the deaminase is fused to Cas9, Cpf1, or a mutant thereof.

[0266] Other Ingredients The composition may further contain any other components typically used in nucleic acid or protein delivery formulations. For example, the composition may contain lipids, lipoproteins (e.g., cholesterol, and derivatives), phospholipids, polymers or liposomal or micellar delivery vehicles, etc. The composition may also contain a suitable solvent or carrier for administration to a cell or a host, such as a mammal or human.

[0267] In some embodiments, the composition further comprises a second polymer comprising polyethylene oxide (PEG). For example, the composition may contain PEG-pAsp(DET), PEG-pAsp, a derivative of PEG-pAsp(DET), a derivative of PEG-pAsp, or a combination thereof. Not wishing to be bound by any of the above, these PEGylated polymers have been developed to be suitable for the size of nanoparticles as well as their It is believed that the polyethylene oxide polymer may be capable of controlling the interaction of the glycerol with serum proteins and target cells. The polyethylene oxide polymer may be combined with the other components in any manner and in any order.

[0268] In some embodiments, the composition further comprises one or more surfactants. The surfactants can be non-ionic surfactants and / or amphoteric surfactants. Exemplary surfactants include: The list of active agents is: polyoxyethylene sorbitan ester surfactants (commonly called Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and / or butylene oxide (BO), DOWFAX® ) trade names, such as linear EO / PO block copolymers; Octoxynol, which may vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, of which Octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) is of particular interest; phenoxy)polyethoxyethanol (IGEPAL CA-6301NP-40); phospholipids such as phosphatidylcholine (lecithin); triethylene glycol monolauryl ether (Brij 30), Polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl, and oleyl alcohols (known as Brij surfactants); polyoxyethylene-9-lauryl ether, as well as sorbitan trioleate (Span 85) and sorbitan monolaurate. In some embodiments, the surfactant is an anticoagulant (e.g., heparin, etc.). In some embodiments, the composition is provided with one or more pharma- ceutically acceptable carriers and / or excipients. It further contains a vehicle.

[0269] In some cases, the moiety (e.g., a nucleic acid moiety (e.g., a guide nucleic acid, etc.); a protein moiety (e.g., e.g., Cas9 or Cpf1 polypeptide, mutant Cas9 or Cpf1 polypeptide); etc.) is a labeling moiety. As used herein, the terms "label," "detectable label," or "labeling moiety" refer to any moiety that provides signal detection and can vary widely depending on the particular nature of the assay. Labeling moieties of interest include directly detectable labels (direct labels) (e.g., fluorescent Labels include both indirectly detectable labels (indirect labels) (e.g., binding pair members). The photolabel can be any fluorescent label (e.g., fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, ALEXAFLUOR® labels, etc.), fluorescent proteins (e.g., green fluorescent tags, GFP, enhanced GFP (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), cherry, tomato, tangerine, and any of the above. The compound may be a suitable detectable (direct or fluorescent derivative thereof, etc.) compound for use in the present method. The labeling moiety may be spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, The indirect label includes any moiety that is detectable by fluorescent, chemical, or other means. For example, suitable indirect labels include biotin (a member of a binding pair), which binds to streptavidin (which binds to Labels can also be attached by: radiolabels (direct labels) (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P; Enzymes (indirect labels) (e.g., peroxidase enzymes, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.); fluorescent proteins (direct labeling) (e.g., green fluorescent protein, red fluorescent protein, Yellow fluorescent proteins, and any convenient derivatives thereof; metal labels (direct labels); colorimetric and the like. "Partner of a binding pair" or "member of a binding pair" means one of a first and a second moiety, where the first and second moieties have a specific binding affinity for one another. Suitable binding pairs include: antigen / antibody (e.g., digoxigenin, erythrocyte / macrocellular matrix (MCM) and / or erythrocyte / macrocellular matrix (MCM)). Examples of indirectly detectable labeling moieties include, but are not limited to, dansyl / anti-digoxigenin, dinitrophenyl (DNP) / anti-DNP, dansyl-X-anti-dansyl, fluorescein / anti-fluorescein, lucifer yellow / anti-lucifer yellow, and rhodamine (anti-rhodamine), biotin / avidin (or biotin / streptavidin) and calmodulin binding protein (CBP) / calmodulin. Any binding pair member may be suitable for use as an indirectly detectable labeling moiety.

[0270] Any given component, or combination of components, may be unlabeled or detectably labeled with a labeling moiety. In some embodiments, when more than one component is labeled, they may be labeled with labeling moieties that are distinguishable from one another.

[0271] Encapsulation and Nanoparticles In some embodiments of the composition, the polymer is linked to a nucleic acid and / or a polypeptide. Combined and partially or completely encapsulated nucleic acids and / or polypeptides The composition, in some formulations, comprises a polymer and a nucleic acid and / or a polymer. Nanoparticles comprising the polypeptide may be provided.

[0272] In some embodiments, the composition may contain a core nanoparticle in addition to the polymer and nucleic acid or polypeptide described herein. Any suitable nanoparticle may be used, including metal (e.g., gold) nanoparticles or polymeric nanoparticles.

[0273] The polymers and nucleic acids (e.g., guide RNA, donor polynucleotide, or both) or polypeptides described herein can be directly or indirectly conjugated to the nanoparticle surface. For example, the polymers and nucleic acids (e.g., guide RNA, donor polynucleotide, or both) or polypeptides described herein can be attached directly to the surface of the nanoparticle or via an intervening linker. The bond can be indirectly bonded via a bond.

[0274] Any type of molecule can be used as a linker. For example, the linker can be an aliphatic chain containing at least 2 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more carbon atoms). The linker may be any thiol-containing molecule, and may be substituted with one or more functional groups, including ketone, ether, ester, amide, alcohol, amine, urea, thiourea, sulfoxide, sulfone, sulfonamide, and disulfide functionalities. In embodiments in which the nanoparticles comprise gold, the linker may be any thiol-containing molecule. The reaction of a thiol group with gold results in a covalent sulfide (-S-) bond. The design and synthesis of linkers are well known in the art.

[0275] In some embodiments, the nucleic acid conjugated to the nanoparticle is a linker nucleic acid that serves to non-covalently link one or more elements described herein (e.g., Cas9 polypeptide, and guide RNA, donor polynucleotide, and Cpf1 polypeptide) to the nanoparticle-nucleic acid conjugate. For example, the linker nucleic acid can be a nucleic acid that hybridizes to the guide RNA or donor polynucleotide. It may have a sequence corresponding to

[0276] Nanoparticles (e.g., colloidal metal (e.g., gold) nanoparticles; nanoparticles containing biocompatible polymers) The nucleic acid attached to the guide RNA or donor polynucleotide may be of any suitable length. If it is a nucleotide, the length is appropriate for such molecules as discussed herein and known in the art. If the nucleic acid is a linker nucleic acid, it can have any appropriate length for the linker, for example, 10 nucleotides (nt) to 1000 nt, e.g., 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, nanoparticles (e.g., colloids) can be used. Nucleic acids conjugated to nanoparticles (eg, nanoparticles comprising biocompatible polymers; nanoparticles comprising nanoparticles such as nanoparticles of metal (eg, gold) nanoparticles) can have a length greater than 1000 nt.

[0277] The nucleic acid bound (e.g., covalently or non-covalently) to the nanoparticles is present in the complexes of the present disclosure. hybridizes to at least a portion of the guide RNA or donor polynucleotide present in the If it contains a nucleotide sequence, it has a region that has sufficient sequence identity with a region of the complement of the guide RNA or donor polynucleotide sequence to facilitate hybridization. In some embodiments, the core bound to the nanoparticle in the complex of the present disclosure has The acid is a fragment 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) of the guide RNA or donor polynucleotide present in the complex. The nucleic acid sequence 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 to the complement of a nucleic acid sequence (e.g., 10 nt to 40 nt, or 10 nt to 50 nt).

[0278] In some embodiments, the nucleic acid attached (e.g., covalently attached; non-covalently attached) to the nanoparticle is the donor polynucleotide or is the same as the donor polynucleotide. or has substantially the same nucleotide sequence. In some embodiments, the nucleic acid attached (e.g., covalently attached; non-covalently attached) to the nanoparticle comprises a nucleotide sequence that is complementary to the donor DNA template.

[0279] How to use The polymers provided herein may be used for any purpose, but are believed to be particularly useful for combining with biomolecules (e.g., nucleic acids and polypeptides) for various purposes, and in some embodiments, for encapsulation.In one aspect, a method is provided for encapsulating biomolecules, such as polypeptides or nucleic acids, by combining the polymers of the present invention described herein with the biomolecule, whereby the polymer partially or completely encapsulates the biomolecule.The biomolecule that is partially or completely encapsulated by the polymer is sometimes called nanoparticle.

[0280] Also provided herein is a method for delivering a nucleic acid and / or a polypeptide to a cell, comprising: The cells herein can be in vitro or in vivo. The methods involve administering compositions containing the polymers and nucleic acids and / or polypeptides described herein to cells or cells containing The method can be used with any type of cell or subject, but is particularly useful for mammalian cells (e.g., human cells). In some embodiments, the polymer includes a targeting agent, such that the nucleic acid and / or polypeptide is The delivery is primarily or exclusively to a target cell or tissue (eg, cells or tissue of the peripheral nervous system, the central nervous system, the subject's eye, liver, muscle, lung, bone (eg, hematopoietic cells), or tumor cells or tissue).

[0281] When used with a composition containing one or more components of the CRISPR system, the method can be used to induce editing of a target nucleic acid or gene. In some embodiments, the method of modifying a target nucleic acid comprises homology-directed repair (HDR). In some embodiments, the use of the complex of the present disclosure to perform HDR provides an HDR efficiency of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or more than 25%. In some embodiments, the method of modifying a target nucleic acid comprises non-homologous end joining (NHEJ). In some embodiments, the use of the complex of the present disclosure to perform HDR provides an HDR efficiency of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or more than 25%. Use of the complexes of the present 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%.

[0282] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0283] Example 1 This example provides guidance for the synthesis of the polymers described herein. The synthesis involves a Michael addition with an acrylate. An exemplary procedure is as follows:

[0284] [ka]

[0285] In a glass vial, pAsp(DET) (5 mg, 0.22 μmol) was suspended in dimethyl sulfoxide ("DMSO"; 700 μL). 30 μL of trimethylamine ("TEA") was added to the suspension, and the resulting The resulting suspension was stirred until all the polymer was completely dissolved. Acrylate 1 (1.15 mg, 5.5 μmol) was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 40 h. The material was purified by precipitation in acetonitrile and washing three times with acetonitrile to give 4 mg of polymer 5, where (a+b) is 55 and (c+d) is 25. 1 H NMR (400 MHz, D2O): δ 4.8-4.6 (bs, 4H), 4.2 (t, 2H), 3.4-2.4 (m, 40H).

[0286] As demonstrated by Scheme 1, Michael addition with acrylate 1 results in an amine-based linkage (see, for example, polymer 5). Thus, the original amine- The sensuality remains intact.

[0287] Similar procedures can be used to prepare additional polymers of formula 1 (e.g., polymers 1-4 and 6-12). Nucleophilic substitution can be used to provide polymers 28 and 29.

[0288] Additionally, this procedure can be used to prepare polymers with different starting polymers, such as the polymer prepared as shown in Example 7. to provide other polymers of formula 1 (e.g., starting with polymers 30 or 32 and demethylating at the terminal nitrogen to provide a primary or secondary amine, this method can provide polymers 13-24).

[0289] Example 2 This example provides guidance for the synthesis of the polymers described herein. The synthesis involves a Michael addition with an acrylate. An exemplary procedure is as follows:

[0290] [ka]

[0291] In a glass vial, pASP(DET) (5 mg, 0.22 μmol) was suspended in anhydrous methanol (300 μL), and 30 μL of triethylamine ("TEA") was added to the suspension. The resulting solution was incubated at room temperature. The reaction mixture was stirred at rt for 10 min to completely dissolve the polymer. The solution was diluted with 300 μL of DCM. To the reaction mixture was added acrylate 2 (1.05 mg, 5.5 μmol) in 30 μL of DCM, and the reaction mixture was stirred at room temperature for 48 h. The crude product was purified by precipitation into a large excess of diethyl ether to give 4.3 mg of polymer 8, where (a+b) is 55 and (c+d) is 25. . 1 H NMR (400 MHz, D2O): δ 4.8-4.6 (bs, 4H), 4.03 (t, 2H), 3.8-2.5 (m, 27H), 1.5 (t, 2H), 1.25 (s, 12H), 0.7 (t, 3H).

[0292] As demonstrated in Scheme 2, Michael addition with acrylate 2 afforded amine-based This results in the attachment of the amine to the pAsp(DET) moiety (see, for example, polymer 8). Thus, the original amine-functionality in pAsp(DET) remains intact.

[0293] Example 3 This example demonstrates the ability of the polymers described herein to deliver Cas9 ribonucleoprotein ("Cas9 RNP") to cells. The level of Cas9 RNP delivery was measured using the green fluorescent protein ( "GFP" inducible HEK293T ("GFP-HEK") cells were used to evaluate.

[0294] 10 pmoles of Cas9 RNP were mixed with (i) polymer 5, (ii) polymer 8, and (iii) pAsp(DET) as a control. GFP-HEK cells were treated with the resulting mixture in serum-free conditions. Polymers were added at dosages of 0.375 μg, 0.75 μg, 1.25 μg, 2.5 μg, 5 μg, and 10 μg. The optimal dose that provides maximum efficacy and minimum toxicity was screened. The results are shown in Figure 3. .

[0295] Figure 3 shows the Cas9 expression level as measured by the percent GFP(-) of GFP-HEK cells treated with the three mixtures. The levels of RNP delivery are shown. All three polymers (i.e., polymer 5, polymer 8, and Polymers 1 and 2 showed similar levels of gene editing at polymer doses ranging from 0.375 μg to 2.5 μg. However, polymers 8 and 2 showed similar levels of gene editing at polymer doses of 5 μg and 10 μg. showed significant levels of cytotoxicity (less than 50% cell viability), whereas polymer 5 showed optimal delivery at a polymer dosage of 5 μg as demonstrated by the GFP % levels without significant toxicity.

[0296] Example 4 This example illustrates the use of Cre recombinases that can alter the loxP sequences of the polymers described herein. The present invention demonstrates the ability to deliver the enzyme to cells.

[0297] Using primary myoblasts from ai9 mice, which have a termination sequence between the loxPs (see grey arrows in Figure 4), the level of delivered Cre recombinase can be measured by RFP expression. The target allele contains a STOP cassette flanked by loxP sequences, thereby preventing RFP transcription. Cre recombinase removes the STOP cassette flanked by loxP sequences and allows transcription of RFP.

[0298] Cre recombinase (2 μg) was mixed with (i) polymer 5 and (ii) pAsp(DET) as a control to produce nanoparticles. The polymer was added at 1.25 μg, 2.5 μg, 5 μg, 10 μg, and 20 μg. In addition to the μg dosage, the optimal dose that gave the maximum efficacy and the minimum toxicity was screened. Ai9 myoblasts were treated with the resulting nanoparticles, and the RFP+ population was quantified 4 days after treatment using flow cytometry. The results are shown in Figure 5.

[0299] FIG. 5 shows the level of Cre recombinase delivery into primary myoblasts as measured by the amount of RFP+ expression. As shown in FIG. 5, polymer 5 was The dose produced higher levels of RFP+ expression than the control (pAsp(DET)), thereby Rimer 5 was more efficient at delivering Cre recombinase than pAsp(DET) at higher dosages. This demonstrated that

[0300] Example 5 This example demonstrates in vivo Cre recombination in mice of the polymers described herein. The ability to deliver enzymes is demonstrated.

[0301] 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 ug) was delivered with a mixture of polymer 5 and PEG-PAsp(DET) (100 ug). Co-mixture of polymer 5 and PEG-PAsp(DET) helps control the size of the polymer nanoparticles. Gastrocnemius muscles were harvested from ai9 mice 2 weeks after injection. Cross sections of muscle were imaged to detect the red fluorescent protein that had been generated as a result of DNA modification. The results are shown in Figure 6.

[0302] Figure 6 shows that Cre recombinase delivery in mouse muscle was enhanced by polymer nanoparticles. Injection of Cre recombinase alone resulted in visible red fluorescent protein. This resulted in RFP expression in limited regions of muscle, as demonstrated by less On the other hand, injection of Cre recombinase encapsulated in polymer nanoparticles resulted in Most areas in abdominal muscle show RFP expression.

[0303] The challenge of protein delivery is how widely proteins can be delivered and affect many areas of tissue. Nanoparticles derived from the IL-11 gene can efficiently deliver and aid in the distribution of Cre recombinase in ai9 mice.

[0304] Example 6 This example demonstrates the ability of the polymers described herein to deliver Cas9 to GFP-expressing neural cells. Demonstrate the following.

[0305] We investigated whether polymer 5 can deliver Cas9 RNP to neural cells using GFP-expressing neural cells differentiated from neural progenitor cells. GFP-expressing neural cells were treated with the polymers described herein. The cells were treated with sgRNA and Cas9 protein as a delivery method. Seven days after treatment, genomic DNA was extracted from the cells and PCR amplification of the GFP gene was performed. TIDE analysis (Desktop Genetics, The frequency of indel mutations due to gene editing with Cas9 was measured using the TIDE software provided by the Netherlands Cancer Institute. The results are shown in Figure 7.

[0306] As demonstrated by FIG. 7, polymer 5 was able to deliver Cas9 RNP and induce 11% indel mutations in neuronal cells.

[0307] Example 7 This example provides guidance for the synthesis of the polymers described herein. The synthesis involves ring-opening polymerization followed by further modification to produce a polymer of formula 4. An exemplary procedure is as follows: As stated above.

[0308] [ka]

[0309] Synthesis of polymer 33: (a) tosyl 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.

[0310] As demonstrated by Scheme 3, the ring-opening polymerization of compound 10 leads to the propagation of n = 63 cations. Compound 11 is formed, which can be further modified by treatment with compound 9 to form polymer 33.

[0311] Similar procedures can be used to prepare other polymers of formula 4 (e.g., polymers 30-32). Additionally, the terminal tertiary amines on the side chains of the polymers can be demethylated by conventional techniques to provide polymers 39-42.

[0312] Example 8 This example illustrates the use of Cre recombinases that can alter the loxP sequences of the polymers described herein. The present invention demonstrates the ability to deliver the enzyme to cells.

[0313] Trough generation following viral transduction of the traffic light reporter in HEK 293T cells Using a TLR-HEK 293T cell line (see FIG. 8), the level of delivered Cre recombinase can be measured by red fluorescent protein ("RFP") expression. The traffic light reporter contains a STOP cassette and flanking GFP sequences. The gene contains two loxP sequences that bind to the GFP gene, thereby preventing RFP transcription and then expressing GFP in the absence of Cre. Cre recombinase removes the loxP sequences and allows transcription of RFP. .

[0314] Cre recombinase (1 ug) was mixed with polymer 33 prepared in Example 7, or pAsp(DET) as a comparison, to produce nanoparticles. The polymer was added at a dosage of 1.25 μg or 2.5 μg to screen the optimal dose that gave the maximum efficiency and the minimum toxicity. TLR-HEK293T cells were treated with the resulting nanoparticles, and the RFP+ population was analyzed using flow cytometry. The results are shown in Figure 9.

[0315] Figure 9 shows the level of Cre recombinase delivery to (TLR)-HEK 293T cells as measured by the amount of RFP+ expression. As shown in Figure 9, polymer 33 provided enhanced delivery relative to samples containing Cre only and comparison pAsp(DET) at polymer dosages of 1.25 μg and 2.5 μg.

[0316] Example 9 This example demonstrates the ability of the polymers described herein to deliver Cas9 ribonucleoprotein ("Cas9 RNP") to cells. The level of Cas9 RNP delivery was measured using the green fluorescent protein ( "GFP" inducible HEK293T ("GFP-HEK") cells were used to evaluate.

[0317] 15 pmole of Cas9 RNP (sgRNA + Cas9 protein) was mixed with polymer 33 prepared in Example 7, or pAsp(DET) as a comparison, to produce nanoparticles. Polymer was added at a dosage of 2.5 μg. GFP-HEK cells were treated with the resulting mixture in serum-free conditions. The results are shown in Figure 10.

[0318] Figure 10 shows the level of Cas9 RNP delivery as measured by GFP % in GFP-HEK cells treated with the two mixtures. Both polymers (i.e., polymer 33 and pAsp(DET)) were significantly higher than the control. We demonstrated the ability to deliver Cas9 RNP to mice at a polymer dosage of 2.5 μg. Mer33 was superior to pAsp(DET) at a polymer dosage of 2.5 μg.

[0319] Example 10 This example demonstrates the ability of the polymers described herein to deliver nucleic acids. The level of eGFP mRNA delivery into HEK 293T cells was assessed using green fluorescent protein ("GFP").

[0320] eGFP mRNA (200 ng) was mixed with polymer 33 (600 ng) prepared in Example 7, and 5 The resulting polymer nanoparticles were then incubated with HEK 293T cells in OptiMEM medium. After 24 hours, cells were detached from the plate and analyzed by flow cytometry. Lipofectamine was used as a positive control for eGFP mRNA delivery. The results are shown in Figure 11.

[0321] FIG. 11 shows that polymer 33 increased eGFP mRNA expression in HEK 293T cells compared to the control. It indicates that delivery is provided.

[0322] Example 11 This example demonstrates the effect incubation time on the ability of the polymers described herein to deliver nucleic acids. The level of eGFP mRNA delivery into HEK 293T cells was measured using green fluorescent tag. The expression was assessed using green fluorescent protein (GFP).

[0323] eGFP mRNA (200 ng) was mixed with polymer 33 (1.2 μg) prepared in Example 7 and incubated for periods of 2 minutes, 5 minutes, 10 minutes, and 30 minutes. The resulting polymer nanoparticles were applied to HEK 293T cells in OptiMEM medium. After 24 hours, the cells were detached from the plate. The cells were then analyzed by flow cytometry. Lipofectamine was used to deliver eGFP mRNA. This was used as a positive control for the study. The results are shown in Figure 12.

[0324] FIG. 12 shows that nanoparticles formed within 2 minutes of incubation and that polymer 33 was completely 10 provided efficient delivery of eGFP mRNA within an incubation time of 10 min.

[0325] Example 12 This example demonstrates the ability of the polymers described herein to deliver mRNA. The level of red fluorescent protein ("RFP") mRNA delivery into HEK 293T cells was assessed using RFP+ expression. Ta.

[0326] RFP mRNA (200 ng) was mixed with polymer 33 prepared in Example 7, or with pAsp(DET) as a comparison, to produce nanoparticles. Polymer 33 and pAsp(DET) were combined with 120 ng of 1.5 kDa PEG-PAsp(DET) polymer to give a dosage of (i) 600 ng or (ii) 480 ng. The PEG-PAsp(DET) polymer may help play a role in controlling the size of the nanoparticles. TLR-HEK293T cells were treated with the resulting nanoparticles, and the RFP+ population was isolated by flow cytometry. The results were quantified 24 hours after treatment using cytometry, and the results are shown in Figure 13.

[0327] FIG. 13 shows that polymer 33 improved the delivery of mRNAs to HEK 293T cells in the absence of the 1.5 kDa PEG-PAsp(DET) polymer relative to the comparative polymer (PAsp(DET)). show that 600 ng of polymer 33, in combination with 120 ng of 1.5 kDa PEG-PAsp(DET) polymer, provides more efficient delivery of mRNAs into HEK 293T cells than 480 ng of polymer 33.

[0328] Example 13 This example demonstrates the ability of the polymers described herein to deliver mRNAs. The level of red fluorescent protein ("RFP") mRNA delivery into HEK 293T cells was assessed using RFP+ expression.

[0329] RFP mRNA (200 ng) was mixed with polymer 33 prepared in Example 7, or with pAsp(DET) as a comparison, to produce nanoparticles. Polymer 33 and pAsp(DET) were added in combination with 200 ng of 1.5 kDa PEG-PAsp(DET) polymer at a dosage of (i) 1 μg or (ii) 800 ng. The 1.5 kDa PEG-PAsp(DET) polymer was found to play a role in controlling the size of the nanoparticles. TLR-HEK293T cells were treated with the resulting nanoparticles, and the RFP+ population was isolated by flow cytometry. The results were quantified 24 hours after treatment using cytometry, and the results are shown in Figure 14.

[0330] FIG. 14 shows that polymer 33 provides equivalent delivery of mRNA to HEK 293T cells in combination with 200 ng of 1.5 kDa PEG-PAsp(DET) polymer relative to the comparative polymer (PAsp(DET)) at a dosage of (i) 1 μg or (ii) 800 ng in both the presence and absence of 1.5 kDa PEG-PAsp(DET) polymer. This indicates that the

[0331] Example 14 This example demonstrates the ability of the polymers described herein to deliver Cas9 ribonucleoprotein ("Cas9 RNP") to cells using different buffers. The level of Cas9 RNP delivery was assessed using green fluorescent protein ("GFP")-inducible HEK293T ("GFP-HEK") cells.

[0332] 30 pmole of Cas9 RNP (sgRNA + Cas9 protein) was mixed with 4 μg of polymer 33 prepared in Example 7 or 5 μg of pAsp(DET) for comparison to produce nanoparticles. GFP-HEK cells (200,000 cells) were incubated in three different buffer conditions: (i) (4-(2-hydroxyethyl)-1-piperazine; diethyl ethanesulfonic acid (“HEPES”; 20 mM), (ii) Opti-MEM TM(Thermo Fisher Scientific; Waltham, Mass.), or Dulbecco's Modified Eagle's Medium ("DMEM"; commercially available from Thermo Fisher Scientific; Waltham, Mass.). The results are shown in FIG.

[0333] Figure 15 shows the level of Cas9 RNP delivery as measured by percent GFP(-) in GFP-HEK cells treated with two mixtures (i.e., 4 μg of polymer 33 or 5 μg of pAsp(DET)) under three different buffer conditions. Both polymers (i.e., polymer 33 and pAsp(DET)) significantly increased the delivery rate of Cas9 RNPs over a 3-h period. The same trend for the two buffers, HEPES and Opti-MEM TM Better than Opti-MEM TM showed superiority over DMEM. Furthermore, polymer 33 was superior to pAsp(DET) in all three buffers.

[0334] Example 15 This example demonstrates the ability of the polymers described herein to stably encapsulate nucleic acids.

[0335] Polymers 2, 5, and 33 of the present disclosure (1 ug / uL, 10 mM HEPES) were mixed with oligonucleotdies (1 ug / uL, 10 mM HEPES) in a 5:1 mass ratio by pipette and then left at room temperature for 10 minutes to form nanoparticles. They were stored at 4C when not in use. Nanoparticles from the stock were diluted in 10 mM HEPES in a ~1:20 ratio and characterized by dynamic light scattering (DLS) at 0, 3, 5, and 7 days (n=1) after preparation. The intensity-averaged particle size is shown in FIG. 18.

[0336] Nanoparticle sizes between 150 nm and 350 nm were observed. The polymer showed minimal The nanoparticles showed minimal size change, indicating that they were stable. roll (data not shown), and by day 7, it was significantly higher than the other nanoparticles tested. Somewhat larger size changes were observed.

[0337] Example 16 This example demonstrates the preparation of a polymer according to the present disclosure.

[0338] 100 mg (0.0074 mmol) of poly(β-benzyl l-aspartate) (PBLA) was dissolved in 3 mL of NMP. To this reaction mixture was added 1.5 gm of 1,4,7,10-tetramethyl-triethylenetetraamine and the reaction was stirred at RT for 16 h. The crude reaction mixture was precipitated into diethyl ether and The crude product was then collected by centrifugation (5000 g for 15 min). The crude product was dissolved in 3 mL of 1 M HCl. The solution was dissolved in 10 ml of water and dialyzed against water to achieve a pH of 6. The resulting polymer solution was lyophilized to give polymer 41 as a white powder.

[0339] Example 17 This example demonstrates the use of the polymers described herein to deliver mRNA to cells.

[0340] The mRNA encoding green or red fluorescent protein was mixed with the test polymer and the results shown in Table 2 The polymers were combined with one of several different cell types shown in Table 1. Transfection was measured as a function of fluorescence. The results are shown in Table 2 and show that nearly all polymers expressed at least one 4 shows that the same levels of transfection were achieved in all cell types.

[0341] [Table 2]

[0342] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those of skill in the art, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0343] Where a range of values ​​is provided, it is understood that each intervening value, every tenth of a lower limit, between the upper and lower limits of that range and any other stated or intervening value in that stated range is included within the invention unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0344] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are expressly incorporated by reference in their entirety to disclose and describe the methods and / or materials in connection with which the publications are cited. For the purposes of disclosure, it is incorporated herein by reference.

[0345] As used herein and in the appended claims, the singular forms "a" and "an" are intended to be used interchangeably. and "the" includes plural references unless the context clearly dictates otherwise. Thus, for example, a reference to "a complex" The present invention includes any number of such complexes, and reference to "the Cas9 polypeptide" includes reference to one or more Cas9 polypeptides known to those of skill in the art and their equivalents, and so forth. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of such exclusive terminology, such as "solely," "only," and the like, or for use of a "negative" limitation in connection with the recitation of claim elements.

[0346] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments of the invention are specifically embraced by the invention and disclosed herein as if each and every combination was individually and expressly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the invention and disclosed herein as if each and every subcombination was individually and expressly disclosed herein.

[0347] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

Claims

1. A polymer comprising the structure of Formula 1: 【Chemistry 1】 During the ceremony: m 1 and n 1 each of which is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 Based on B 1 and B. 2 are each independently -(CH 2 ) p1 -[NR 2 -(CH 2 ) q1 -] r1 NR 2 -(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 -(CH 2 ) s2 -R 4 -R 5 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -] r2 (CH 2 ) s3 -R 4 -R 5 }; -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 -(CH 2 ) s4 -R 4 -R 5 ] 2 } 2 ; -(CH 2 , p1 -[NR 2 -(CH 2 , q1 , r1 NR 2 -CH 2 -CHOH-R 5 , -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 -CH 2 -CHOH-R 5 ; -(CH 2 , p3 -N{[-(CH 2 , q3 -NR 2 2 ][-(CH 2 , q4 -NR 2 , r2 -CH 2 -CHOH-R 5 , -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 -CH 2 -CHOH-R 5 ] 2 } 2 ; -(CH 2 ) p1 -[NR 2 -(CH 2 ) q1 -] r1 NR 2 -(CH 2 ) s1 -R 5 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 -(CH 2 ) s2 -R 5 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -] r2 (CH 2 ) s3 -R 5 }; -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 -(CH 2 ) s4 -R 5 ] 2 } 2 ; -(CH 2 ) p1 -[N{(CH 2 ) s1 -R 4 -R 5 }-(CH 2 ) q1 -] r1 NR 2 2 ;or -(CH 2 ) p1 -[N{(CH 2 ) s1 -R 5 }-(CH 2 ) q1 -] r1 NR 2 2 and where p1 through p4, q1 through q6, r1 and r2, and s1 through s4 are each independently an integer from 1 to 5; 2 are independently hydrogen or C 1 -C 12 Alkyl groups, alkenyl groups, cycloalkenyl groups R is an alkyl group, or a cycloalkenyl group; 4 is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 1 to 8 secondary or tertiary amines, or a substituent containing a tissue- or cell-specific targeting moiety.

2. The polymer of claim 1 having the structure of Formula 1A: 【Chemistry 2】 During the ceremony c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 and n 1 each of which is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 1 is a hydrogen atom, an aryl group, a heterocyclic group, which may be substituted with one or more substituents, 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or C 1 -C 12 is a linear or branched alkyl group; R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 3a and R 3a are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; R in each case 4 is -C(O)O-, -C(O)NH-, or -S(O)(O)-; R in each case 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 1 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety; and R 6 is a hydrogen atom, an amino group, an aryl group, a heterocyclic group, which may be substituted with one or more amines, 1 -C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl group, C 1 -C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. do.

3. Each R 5 But independently: 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 and where R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 7 may be substituted with one or more amines, C 1 -C 50 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; C 1 -C 12 Alkyl groups, alkenyl groups, cyclo groups cycloalkyl or cycloalkenyl group. z is an integer from 1 to 5; c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; n is an integer from 0 to 50; and R 8 is a tissue- or cell-specific targeting moiety; 3. The polymer of claim 1 or 2.

4. A 1 and A 2 Each of the formula -(CH 2 ) 2 -NH-(CH 2 ) 2 -NH 2 and B 1 and B. 2 Noso Each of them has the formula -(CH 2 ) 2 -NH-(CH 2 ) 2 -NH-(CH 2 ) 2 -R 4 -R 5 The polymer of any one of claims 1 to 3, wherein the group is

5. R 4 The polymer of any of claims 1-4, wherein is -C(O)-O-.

6. R 5 but 【Chemistry 6】 or 【Chemistry 7】 6. The polymer of any of claims 1-5,

7. (m 1 +n 1 ) / (m 2 +n 2 7. The polymer of claim 1, wherein the ratio of

8. Polymer of Formula 2: 【Chemistry 8】 During the ceremony m 1 and n 1 Each of m is an integer from 0 to 1000, 1 and n 1 The total is between 10 and 2000. the law of nature; c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; R 1 is a hydrogen atom, an aryl group, a heterocyclic group, which may be substituted with one or more substituents, 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or C 1 -C 12 is a linear or branched alkyl group; R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 8 is a tissue- or cell-specific targeting moiety; and R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group It is a cycloalkenyl group, or a cycloalkenyl group.

9. Tissue- or cell-specific targeting moieties: 【Chemistry 9】 and Here, R 9 , R 10 , R 11 , and R 12 are independently substituted with hydrogen, halogen, or one or more amino groups C 1 -C 4 Alkyl, or C 1 -C 4 is an alkoxy; 9. The polymer of any one of claims 1-5, 7, or 8.

10. 10. The polymer of any of claims 1-9, wherein the polymer is a cationic polymer.

11. formula: 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemical 22】 【Chemistry 23】 wherein (a+b) is from about 0 to about 75, and (c+d) is from about 5 to about 80. Rimmer.

12. A polymer comprising the structure of formula 4: 【Chemistry 24】 During the ceremony, m 1 and n 1 Each of m is an integer from 0 to 1000; 1 + n 1 The sum of is greater than 2; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; A 1 and A 2 are each independently represented by the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 wherein p1 to p4, q1 to q6, and r1 and r2 are each independently an integer from 1 to 5; R in each case 2 are independently hydrogen or C 1 -C 12 An alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, provided that A 1 and A 2 each contains at least one tertiary amine; R 3a and R 3b are each independently a methylene or ethylene group; and R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group It is a cycloalkenyl group, or a cycloalkenyl group.

13. The polymer of claim 12 having the structure of Formula 4A: 【Chemistry 25】 During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 and n 1 Each of m is an integer from 0 to 1000; 1 + n 1 The sum of is greater than 2; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; A 1 and A 2 are each independently represented by the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 where p1 through p4, q1 through q6, and r1 and r2 are each independently an integer from 1 to 5; and R in each case 2 is independently C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 1 is a hydrogen atom, an aryl group, a heterocyclic group, which may be substituted with one or more substituents, 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or C 1 -C 12 is a linear or branched alkyl group; R in each case 2 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group A is a cycloalkenyl group, or a cycloalkenyl group, 1 and A 2 each contains at least one tertiary amine; R 3a and R 3a are each independently a methylene or ethylene group; R 6 is a hydrogen atom, an amino group, an aryl group, a heterocyclic group, which may be substituted with one or more amines, 1 -C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl group, C 1 -C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. and R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group It is a cycloalkenyl group, or a cycloalkenyl group.

14. A 1 and A 2 Each of the formula -(CH 2 ) 2 -NR 2 -(CH 2 ) 2 -NR 2 2 where R in each case is 2 but Independently C 1 -C 12 14. The polymer of claim 12 or 13, wherein the terminal amine is an alkyl, alkenyl, cycloalkyl, or cycloalkenyl group and is not a primary, secondary, or tertiary amine.

15. R in each case 2 is methyl.

16. Tissue- or cell-specific targeting moieties: 【Chemistry 26】 where R 9 , R 10 , R 11 , and R 12 each independently represents hydrogen, halogen, one or more C, which may be substituted with the above amino group; 1 -C 4 Alkyl, or C 1 -C 4 16. The polymer of any one of claims 13-15, which is alkoxy.

17. 17. The polymer of any of claims 12-16, wherein the polymer is a cationic polymer.

18. formula: 【Chemistry 27】 【Chemistry 28】 【Chemical Formula 29】 【Chemistry 30】 wherein (a+b) is from about 5 to about 160.

19. A method for preparing the polymer of any one of claims 1 to 11, comprising the step of: 【Chemistry 31】 During the ceremony: m 1 and n 1 Each of m is an integer from 0 to 1000, 1 + n 1 is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 Based on wherein p1 through p4, q1 through q6, and r1 and r2 are each independently an integer from 1 to 5; and R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; The polymer group A includes the structure 1 and / or A 2 by modifying at least a portion of the formula 1: 【Chemistry 32】 During the ceremony: m 1 and n 1 each of which is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 3a and R 3b are each independently a methylene or ethylene group; R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; A 1 and A 2 are each independently represented by the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 Based on B 1 and B. 2 are each independently -(CH 2 ) p1 -[NR 2 -(CH 2 ) q1 -] r1 NR 2 -(CH 2 ) s1 -R 4 -R 5 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 -(CH 2 ) s2 -R 4 -R 5 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -] r2 (CH 2 ) s3 -R 4 -R 5 }; -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 -(CH 2 ) s4 -R 4 -R 5 ] 2 } 2 ; -(CH 2 , p1 -[NR 2 -(CH 2 , q1 , r1 NR 2 -CH 2 -CHOH-R 5 , -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 -CH 2 -CHOH-R 5 ; -(CH 2 , p3 -N{[-(CH 2 , q3 -NR 2 2 ][-(CH 2 , q4 -NR 2 , r2 -CH 2 -CHOH-R 5 , -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 -CH 2 -CHOH-R 5 ] 2 } 2 ; -(CH 2 ) p1 -[NR 2 -(CH 2 ) q1 -] r1 NR 2 -(CH 2 ) s1 -R 5 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 -(CH 2 ) s2 -R 5 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -] r2 (CH 2 ) s3 -R 5 }; -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 -(CH 2 ) s4 -R 5 ] 2 } 2 ; -(CH 2 ) p1 -[N{(CH 2 ) s1 -R 4 -R 5 }-(CH 2 ) q1 -] r1 NR 2 2 ;or -(CH 2 ) p1 -[N{(CH 2 ) s1 -R 5 }-(CH 2 ) q1 -] r1 NR 2 2 where p1 through p4, q1 through q6, r1 and r2, and s1 through s4 are each independently an integer from 1 to 5; 2 are independently hydrogen or C 1 -C 12 Alkyl groups, alkenyl groups, silyl groups R is a cycloalkyl group, or a cycloalkenyl group; 4 is independently -C(O)O-, -C(O)NH-, or -S(O)(O)-; and R 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, which may contain 1 to 8 secondary or tertiary amines, or a substituent that contains a tissue-specific or cell-specific targeting moiety; The method includes producing a polymer comprising the structure:

20. Group A of the polymer containing the structure of formula 3 1 and / or A 2 However, the polymer and the acrylic resin having the structure of formula 3 are 20. The method of claim 19, wherein the compound is modified by a Michael addition reaction between a acrylate, an acrylamide, a vinyl sulfone, or a combination thereof.

21. The acrylate, acrylamide, or vinyl sulfone has the formula: 【Chemical 33】 where R 5 is an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, optionally containing 1 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

22. A polymer comprising the structure of formula 3 is represented by formula 3A: 【Chemical Formula 34】 During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 and n 1 Each of m is an integer from 0 to 1000; 1 + n 1 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 1 is a hydrogen atom, an aryl group, a heterocyclic group, which may be substituted with one or more substituents, 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or C 1 -C 12 is a linear or branched alkyl group; R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 3a and R 3a are each independently a methylene or ethylene group; and R 6 is a hydrogen atom, an amino group, an aryl group, a heterocyclic group, which may be substituted with one or more amines, 1 -C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl group, C 1 -C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. the law of nature; R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; is a polymer of A polymer comprising the structure of formula 1 is represented by formula 1A: 【Chemistry 35】 During the ceremony c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 and n 1 each of which is an integer between 0 and 1000; m 2 and n 2 Each of m is an integer from 0 to 1000, 2 + n 2 the sum of is greater than 5; The symbol " / " indicates that the units it separates may be combined randomly or in any order. indicates that; R 1 is a hydrogen atom, an aryl group, a heterocyclic group, which may be substituted with one or more substituents, 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or C 1 -C 12 is a linear or branched alkyl group; R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; R 3a and R 3a are each independently a methylene or ethylene group; R in each case 4 is -C(O)O-, -C(O)NH-, or -S(O)(O)-; R in each case 5 are independently an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, which may contain 1 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety; R 6 is a hydrogen atom, an amino group, an aryl group, a heterocyclic group, which may be substituted with one or more amines, 1 -C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl group, C 1 -C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. and R in each case 13 are independently hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group a cycloalkenyl group, or a cycloalkenyl group; 22. The method of any one of claims 19-21, wherein the polymer is

23. A 1 and A 2 Each of the formula -(CH 2 ) 2 -NH-(CH 2 ) 2 -NH 2 and B 1 and B. 2 Noso Each of them has the formula -(CH 2 ) 2 -NH-(CH 2 ) 2 -NH-(CH 2 ) 2 -R 4 -R 5 23. The method of any of claims 19-22, wherein the group is

24. R 4 The method of any one of claims 19-23, wherein is -C(O)-O-.

25. R 5 but 【Chemical Formula 36】 or 【Chemical Formula 37】 25. The method of claim 23 or 24,

26. A of the polymer containing the structure of formula 3 1 and / or A 2 A sufficient number of groups are modified (m 1 +n 1 ) / (m 2 +n 2 ) is about 20 or less, and optionally about 0.2 or more. The method of any of claims 19-25, further comprising providing

27. A method comprising the steps of: (a) administering to a subject a polymer according to any one of claims 1 to 18 and a nucleic acid and / or a polypeptide; A composition comprising:

28. 28. The composition of claim 27, comprising a guide nucleic acid and / or a donor nucleic acid.

29. 29. The composition of claim 27 or 28, comprising an endonuclease.

30. 30. The composition of claim 29, comprising an RNA-guided endonuclease or a nucleic acid encoding same.

31. The composition of claim 30, wherein the RNA-guided endonuclease is Cas9, Cpf1, or a combination thereof.

32. 32. The composition of any one of claims 27-31, comprising a DNA recombinase.

33. The composition of claim 32, wherein the DNA recombinase is Cre recombinase.

34. 34. The composition of any one of claims 27-33, comprising a zinc finger nuclease.

35. 35. Any one of claims 27-34, comprising a transcription activator-like effector nuclease. Composition of.

36. A nanoparticle comprising a polymer according to any one of claims 1 to 9 and a nucleic acid or a polypeptide.

36. The composition of any one of claims 27-35, comprising:

37. 37. The method of claim 27, further comprising the step of: Composition of.

38. 38. A method for delivering a nucleic acid and / or a polypeptide to a cell comprising administering to the cell a composition according to any one of claims 27-37.

39. The cell is in a subject, and the composition of any one of claims 27-37 is administered to the subject. The method of claim 38.

40. 40. The method of claim 38 or 39, wherein the polymer comprises a tissue-specific targeting moiety that localizes the polymer to a tissue of the peripheral nervous system, central nervous system, liver, muscle, lung, bone, or eye of a subject.

41. 41. The method of claim 40, wherein the polymer comprises a targeting moiety that preferentially binds to tumor cells.

42. The composition comprises an RNA-guided endonuclease or a nucleic acid encoding the same, a guide nucleic acid, and and one or more of a donor nucleic acid and facilitates editing of a target gene in a cell.

43. 43. The method of any of claims 38-42, wherein the cell is in a host, optionally a human, and the composition is delivered to the cell by administering the composition to the host.

44. Formula A: 【Chemical 38】 During the ceremony, R 3b is a methylene or ethylene group; W is -NR 14 - or -O-, where R 14 is hydrogen or C 1 -C 12 Alkyl group, alkenyl group a cycloalkyl group, or a cycloalkenyl group; Z is A 1 or 1 to 8 secondary or tertiary amines, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, an arylalkyl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, or a substituent that includes a tissue-specific or cell-specific targeting moiety; A 1 is the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 Based on wherein p1 through p4, q1 through q6, and r1 and r2 are each independently an integer from 1 to 5; and R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; From the compound of formula 5: 【Chemical Formula 39】 During the ceremony, m 1 is an integer between 1 and 2000; R 3b is a methylene or ethylene group; R 17 is hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkyl group is an alkenyl group; A 1 is the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 Based on wherein p1 through p4, q1 through q6, and r1 and r2 are each independently an integer from 1 to 5; and R in each case 2 are independently hydrogen or C 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group; A method for preparing a polymer comprising the structure of:

45. The method of claim 44, wherein the polymer comprising the structure of Formula 5 is a polymer of Formula 5A: 【Chemistry 40】 During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; m 1 is an integer between 1 and 2000; R 3a is a methylene or ethylene group; R 6 is a hydrogen atom, an amino group, an aryl group, a heterocyclic group, which may be substituted with one or more amines, 1 -C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl group, C 1 -C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. the law of nature; R 1 is a hydrogen atom, an aryl group, a heterocyclic group, which may be substituted with one or more substituents, 1 -C 12 an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, or C 1 -C 12 is a linear or branched alkyl group; R 17 is hydrogen or C 1 -C 12 Alkyl group, alkenyl group, cycloalkyl group, or cycloalkyl group is an alkenyl group; A 1 is the formula -(CH 2 ) p1 -[NO 2 -(CH 2 ) q1 -] r1 NR 2 2 ; -(CH 2 ) p2 -N[-(CH 2 ) q2 -NR 2 2 ] 2 ; -(CH 2 ) p3 -N{[-(CH 2 ) q3 -NR 2 2 ][-(CH 2 ) q4 -NR 2 -]r 2 R 2 };or -(CH 2 ) p4 -N{-(CH 2 ) q5 -N[-(CH 2 ) q6 -NR 2 2 ] 2 } 2 Based on wherein p1 through p4, q1 through q6, and r1 and r2 are each independently an integer from 1 to 5; and R in each case 2 are independently hydrogen or C 1 -C 12 It is an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group.

46. A 1 is the formula -(CH 2 ) 2 -NR 2 -(CH 2 ) 2 -NR 2 2 46. ​​The method of claim 44 or 45, wherein the group is

47. 47. The method of any one of claims 44-46, wherein the ring-opening polymerization of compound Formula A is initiated with an amine-containing compound.

48. 5. The amine-containing compound of the formula: 【Chemistry 41】 During the ceremony, c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; and R 6 is a hydrogen atom, an amino group, an aryl group, a heterocyclic group, which may be substituted with one or more amines, 1 -C 12 Alkyl, alkenyl, cycloalkyl, or cycloalkenyl group, C 1 -C 12 a linear or branched alkyl group; or a tissue- or cell-specific targeting moiety. The method of claim 47.

49. Amine-containing compounds 【Chemistry 42】 49. The method of claim 47 or 48,

50. In the compound of formula A, W is -O- and Z is an alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an aryl group, an arylalkyl group, a heteroalkyl group, a heterocyclic group, or a combination thereof, which optionally contains 1 to 8 secondary or tertiary amines; and the method comprises, after ring-opening polymerization, forming the resulting intermediate polymer into a compound of the formula NHR 14 -A 1 , where A 1 50. The method of any of claims 44-49, further comprising treating with a compound of the formula:

Images