EA046334B1 - CATIONIC POLYMER AND APPLICATION FOR BIOMOLECULE DELIVERY - Google Patents

Description

Materials presented in electronic form, included by reference

Included herein is a machine-readable list of nucleotide/amino acid sequences, in its entirety by reference, provided concurrently with this document in a single 62527-byte ASCII (text) file named 512879.TXT, which was created on April 29, 2019.

State of the art

Technologies based on peptides, proteins and nucleic acids have numerous applications in the prevention, treatment and therapy of diseases. However, safe and effective delivery of large molecules (e.g., polypeptides and nucleic acids) to target tissues remains challenging. Accordingly, there continues to be a need for new compositions and methods useful for the delivery of therapeutic molecules.

The essence of the invention

The present invention relates to a polymer containing the structure of formula 4:

A' where each of m ¹ and n ¹ is an integer from 0 to 1000, provided that the sum of m ¹ +n ¹ is greater than 2;

the symbol / indicates that the links separated in this way are connected randomly or in any order;

each of A ¹ and A ² independently represents a group of the formula

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ , where each of p1-p4, q1-q6 and r1 and r2 independently represents integer from 1 to 5;

each R ² independently represents hydrogen or an γ-alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, with the proviso that A ¹ and A ² each contain at least one tertiary amine;

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or an γ-alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group.

Also presented is a composition for delivering a nucleic acid and/or polypeptide into a cell, containing a structure of formula 4 and a nucleic acid and/or polypeptide.

Also presented is a method for delivering a nucleic acid and/or a polypeptide into a cell, including introducing the composition into the cell.

Brief description of the drawings

In fig. 1 shows the amino acid sequence of Cas9 from Streptococcus pyogen (SEQ ID NO: 1).

In fig. Figure 2 shows the amino acid sequence of Cpf1 from Francisella tularensis subsp. Novicida U112 (SEQ ID NO: 2).

In fig. 3 is a graph of the level of Cas9 RNP delivery measured as the percentage of negative green fluorescent protein (GFP) in GFP-HEK cells at various doses of polymer/Cas9 formulations.

In fig. Figure 4 shows a schematic illustration of the ai9 reporter allele.

In fig. Figure 5 shows the level of delivery of Cre recombinase to primary myoblasts, measured by the magnitude of RFP+ expression at various concentrations of polymer/Cre compositions

In fig. Figure 6 shows the level of Cre recombinase delivery into mouse muscle visualized with red fluorescent protein (lighter areas indicate fluorescence).

In fig. Figure 7 shows the frequency of indel mutations in human neuronal stem cells, determined by the magnitude of Cas9 delivery.

In fig. Figure 8 schematically shows the genetic design of the Traffic light reporter in HEK 293T.

In fig. 9 shows the level of Cre recombinase delivery to TLR-HEK 293T as measured by the level of RFP+ expression at various concentrations of polymer/Cre compositions.

In fig. 10 is a graph of Cas9 RNP delivery levels measured by % green fluorescent protein (GFP) in GFP-HEK cells at various doses of polymer/Cas9 compositions.

In fig. Figure 11 shows the level of eGFP mRNA delivery into HEK 293T cells, measured by % containing

- 1 046334 niyu GFP.

In fig. Figure 12 shows the dependence of the level of eGFP mRNA delivery into HEK 293T cells on incubation time, measured by the % GFP content.

In fig. 13 shows the level of red fluorescent protein (RFP) mRNA delivery into HEK 293T cells as measured by the level of RFP+ expression in both the presence and absence of the 1.5 kDa PEG-PAsp (DET) polymer.

In fig. 14 shows the level of red fluorescent protein (RFP) mRNA in HEK 293T cells as measured by the level of RFP+ expression in both the presence and absence of the 1.5 kDa PEG-PAsp (DET) polymer.

In fig. 15 is a graph of the level of Cas9 RNP delivery measured by % green fluorescent protein (GFP) content in GFP-HEK cells for polymer/Cas9 nanoparticles in buffer.

In fig. 16 shows the sequence of AsCpf1 (SEQ ID NO: 19).

In fig. 17 shows the sequence of LbCpf1 (SEQ ID NO: 20).

Detailed Description of the Invention

The invention relates to a polymer containing the structure of formula 1:

where each of m ¹ and n ¹ is an integer from 0 to 1000;

each of m2 and ⁿ² is an integer from 0 to 1000, provided that the sum of ^m2 + ⁿ² is greater than 5;

the symbol / indicates that the links separated in this way are connected randomly or in any order;

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each of A ¹ and A ² independently represents a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ , each of B ¹ and B ² independently represents:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁴ -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁴ -R ⁵ ]2;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁴ -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁴ -R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -CH2-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] [-(CH2)q4-NR ² -]r2-CH ₂ -CHOH-R ⁵ ;

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -CH2-CHOH-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁵ ]2;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁴ -R ⁵ }-(CH2)ql-]rlNR ² 2; or

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁵ }-(CH2)ql-]rlNR ² 2, where each of p1-p4, q1-q6, r1 and r2, and s1-s4 independently represents an integer from 1 to 5;

each R ² independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each R ⁴ independently represents -C(O)O-, -C(O)NH-, or -S(O)(O)-;

each R ⁵ independently represents a substituted or unsubstituted 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 from 1 to 8 or from 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety.

As used herein, alkyl refers to substituted or unsubstituted

- 2 046334 hydrocarbon chain. An alkyl group can have any number of carbon atoms (for example, C1-C ₁₀₀ -alkyl, C1-C ₅₀ -alkyl, C1-C ₁₂ -alkyl, C1-C ₈ -alkyl, C1-C ₆ -alkyl, C1-C ₄ -alkyl, C1-C ₂ -alkyl, etc.). The hydrocarbon chain may be substituted or, in some embodiments, may be unsubstituted to any extent (eg, in the case of alkenyl or alkynyl groups) and may be linear, branched, straight chain, cyclic (eg, cycloalkyl or cycloalkenyl), or a combination thereof. The cyclic groups may be monocyclic, fused to form bicyclic or tricyclic groups linked by a bond, or spirocyclic. Cyclic groups contain at least three carbons (eg C3-C12, _C3 - _C10 , C3-C8, _C3 - _C6 or _C5 - _C6 ). In some embodiments, the hydrocarbon chain may be interrupted by one or more heteroatoms (e.g., 1, 2, 3, 4, or 5 or more oxygen, nitrogen, and/or sulfur atoms), thereby providing a heteroalkyl, heteroalkenyl, heteroalkynyl, or heterocyclyl (i.e., e. heterocyclic group). In some embodiments, the alkyl, alkenyl, alkynyl is substituted with one or more substituents.

The term heterocyclyl or heterocyclic group refers to a cyclic group, such as an aromatic (eg heteroaryl) or non-aromatic group, where the cyclic group has one or more heteroatoms (eg 1, 2, 3, 4 or 5 or more oxygen, nitrogen and/or sulfur). In some embodiments, a heterocyclyl or heterocyclyl group (ie, a cyclic group, such as aromatic (eg, heteroaryl) or non-aromatic, where the cyclic group has one or more heteroatoms) is substituted with one or more substituents.

The term aryl refers to an aromatic ring system having any suitable number of ring atoms and any suitable number of rings. Aryl groups may include 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. Aryl groups can be monocyclic, fused to form bicyclic or tricyclic groups, or bonded to form a biaryl group. Representative aryl groups include phenyl, naphthyl and biphenyl. In some embodiments, the aryl group contains an alkylene linking group to provide an arylalkyl group (eg, a benzyl group). Some aryl groups have 6 to 12 ring members, such as phenyl, naphthyl, or biphenyl. Other aryl groups have 6 to 10 ring members, such as phenyl or naphthyl. In some embodiments, the aryl substituent contains one or more heteroatoms (eg, 1, 2, 3, 4, or 5 or more oxygen, nitrogen, and/or sulfur atoms), thereby providing a heteroaryl group. In some embodiments, aryl is substituted with one or more substituents.

It is understood that any of the above groups may be monovalent, divalent or polyvalent (eg, alkylene, alkenylene, alkynylene, arylene, heteroalkylene, heteroalkenylene, heteroalkynylene, heterocyclylene, heteroarylene, etc.).

As used herein, the term substituted may mean that one or more hydrogen atoms on a specified atom or group (eg, a substituted alkyl group) is replaced by another group, provided that the normal valence of said atom is not exceeded. For example, when the substituent is oxo (i.e. =O), then two hydrogens on the atom are replaced. Substituent groups may include one or more of: aldehyde, ether, amide, ketone, nitro, cyano, fluoroalkyl (e.g., trifluoromethane), halogen (e.g., fluorine), aryl (e.g., phenyl), heterocyclyl, or heterocyclic group (i.e. a cyclic group, for example, aromatic (eg heteroaryl) or non-aromatic, wherein the cyclic group has one or more heteroatoms), oxo or substituents thereof. Combinations of substituents and/or variables are permitted provided that the substituents do not have a significant adverse effect on the synthesis or use of the compound.

According to Formula 1, each of m ¹ and n ¹ is an integer from 0 to 1000, such as 0-500, 0-200, 0-100 or 0-50. Moreover, each of m2 and ⁿ² is an integer from 0 to 1000, such as 0-500 or 0-100, provided that the sum of ^m2 + ⁿ² is greater than 5 (for example, 5-2000, 5-1000 , 5-500, 5-200, 5-100 or 5-50). In other words, the polymer contains at least a number of monomer units containing B ¹ and/or B ² groups, which are collectively referred to herein as B monomers. In some embodiments, m ¹ and n ¹ are zero, i.e. such a polymer contains neither the A ¹ group nor the A ² group. In other embodiments, the polymer contains a number of A ¹ and/or A ² groups and a number of B ¹ and/or B ² groups. In such embodiments, the ratio (m1+n1)/( ^m2 + ⁿ² ) is about 20 or less (eg, about 10 or less, about 5 or less, about 2 or less, or even about 1 or less). Also in some embodiments, the ratio (m1+n1)/( ^m2 + ⁿ² ) is about 0.2 or more, such as about 0.5 or more.

The polymer may exist in any suitable type of structure. For example, the polymer may exist as a regular 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.

Thus, in the structure of formula 1, the monomers (which can be designated accordingly)

- 3 046334 their associated side chains A ¹ , A ² , B ¹ and B ² ) can be arranged randomly or in any order. The integers m ¹ , n ¹ , m ² and n ² simply denote the number of corresponding monomers that appear in the chain as a whole, and do not necessarily represent blocks of these monomers, although in some embodiments blocks or regions of a given monomer may be present. For example, the structure of Formula 1 may include monomers in the order -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 has a polypeptide (eg, polyaspartamide) backbone arranged in an alpha/beta configuration in which monomers 1 and 2 alternate (eg, -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.). However, the monomers or side chains of A and B (eg, A1/A ² and B ¹ /B ² ) may be distributed randomly throughout the polymer backbone or may be arranged in blocks or some combination thereof.

For further illustration, the polymer could alternatively be described by Formula 1.1:

or, if the core is in alpha, beta configuration, formula 1.2:

where m ¹ and n ¹ are as described above;

m ³ is an integer from 5-2000 (for example, 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 links separated in this way are connected randomly or in any order;

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each A ³ independently represents a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁴ -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁴ -R ⁵ ]2;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁴ -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁴ -R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -CH2-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] [-(CH2)q4-NR ² -]r2-CH ₂ -CHOH-R ⁵ ;

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -CH2-CHOH-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁵ ]2;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁴ -R ⁵ }-(CH2)ql-]rlNR ² 2; or

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁵ }-(CH2)ql-]rlNR ² 2, where each of p1-p4, q1-q6, r1 and r2 and s1-s4 independently represents is an integer from 1 to 5;

each R ² independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each R ⁴ independently represents -C(O)O-, -C(O)NH- or -S(O)(O)-;

- 4 046334 each R ⁵ independently represents 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 from 1 to 8 or from 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific targeting moiety;

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 at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or even 100%) of the A ³ groups are selected from:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁴ -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁴ -R ⁵ ]2;

-(CH ₂ ) _P 3-N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _S 3-R ⁴ -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁴ -R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -CH2-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] [-(CH2)q4-NR ² -]r2-CH ₂ -CHOH-R ⁵ ;

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -CH2-CHOH-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁵ ]2;

-(CH ₂ ) _P 3-N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁴ -R ⁵ }-(CH2)ql-]rlNR ² 2; or -(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁵ }-(CH2)ql-]rlNR ² 2.

In another embodiment, the polymer contains some or even a majority of A ³ groups (at least about 5%, at least about 10%, at least about 25%, at least about 30%, at least about 35% , at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%), selected from:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2 ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or -(CH2)p4-N{-(CH2)q5-N[-(CH2)q6-NR ² 2] ₂ } ₂ .

In any of the above polymer structures, R ^3a and R ^3b independently represent a methylene or ethylene group. In some embodiments, R ^3a is an ethylene group and R ^3b is a methylene group, or R ^3a is a methylene group and R ^3b is an ethylene group. In some embodiments, R ^3a and R ^3b are each an ethylene group. In preferred embodiments, R ^3a and R ^3b are each a methylene group.

Groups A ¹ and A ² are selected independently, and therefore they may be the same or different from each other. Likewise, groups B ¹ and B ² are independently selected and may be the same or different from each other. In addition, the A ³ groups present may be the same or each A ³ group may be different from other A ³ groups.

In groups A ¹ , A ² , A ³ , B ¹ and B ^2, each of the integers p1-p4 (i.e. p1, p2, p3 and p4), q1-q6 (i.e. q1, q2, q3 , q4, q5 and q6) and r1 and r2 independently represent an integer from 1 to 5 (for example, 1, 2, 3, 4 or 5). In some embodiments, each of p1-p4 (i.e., p1, p2, p3, and p4), q1-q6 (i.e., q1, q2, q3, q4, q5, and q6), and/or r1 and r2 are independently represents an integer from 1 to 3 (for example, 1, 2, or 3). In some embodiments, each of p1-p4 (i.e., p1, p2, p3, and p4) and/or q1-q6 (i.e., q1, q2, q3, q4, q5, and q6) is 2. In some In embodiments, each of p1-p4 (i.e. p1, p2, p3 and p4) and/or q1-q6 (i.e. q1, q2, q3, q4, q5 and q6) is 2, and each of r1 and r2 is 1. In some embodiments, each of s1-s4 is independently an integer from 1, 2, 3, 4, or 5, such as 1, 2 or 3, or 1-2.

In the polymer structure, each R ⁴ independently represents -C(O)O-, -C(O)NH- or -S(O)(O)-. In some embodiments, each R ⁴ is independently -C(O)O or -C(O)NH-. In some embodiments, each R ⁴ is -C(O)O-. In some embodiments, each R ⁴ is -C(O)NH-.

- 5 046334

In some embodiments of Formula 1, A ¹ and A ² are each a group of the formula -(CH2)p1-[NH-(CH2)q1-]r1NH2, such as a -(CH2)2-NH-(CH2)2- group NH2. Additionally or alternatively, each of B ¹ and B ² represents a group of the formula -(CH2) _p1 -[NH-(CH ₂ ) _ql -] _r1 NH-(CH ₂ ) ₂ -R ⁴ -R ⁵ such as the group - (CH2)2-NH-(CH2)2-NH-(CH2)2-R ⁴ -R ⁵ , or the group -(CH2) ₂ -NH-(CH ₂ ) ₂ -NH-(CH ₂ ) ₂ -C (O)-OR ⁵ . In some embodiments, B ¹ and B ² are each a group 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 , wherein optionally each of R ² independently represents Q^-alkyl (for example, methyl), and optionally R ⁵ represents ^-^-alkylamino or C1-C3 dialkylamino (eg R ⁵ is -CH2-NH-CH3 or -CH2-N-(CH ₃ ) ₂ ).

In some embodiments of Formula 1.1, A ³ is a group of the formula -(CH2)p1-[NH-(CH2)q1-]r1NH2, such as a -(CH2)2-NH-(CH2)2-NH2 group; or a group of the formula -(CH2)p1-[NH-(CH2)q1-]r1NH-(CH2)2-R ⁴ -R ⁵ such as the -(CH2)2-NH-(CH2)2-NH-( group CH2)2-R ⁴ -R ⁵ ; -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR ⁵ ; or a -(CH2)p1-[NR ² -(CH2)q1-]r1NR ² -(CH2)s1-R ⁵ group such as -(CH2)2-NR ² -(CH2)2-NR ² -(CH2 )2-R ⁵ , optionally each of R ² independently represents Ci^-alkyl (eg, methyl), and optionally R ⁵ represents Ci-C'3-alkylamino or C1-C ₃ -dialkylamino (eg , R ⁵ represents -CH2-NH-CH3 or -CH ₂ -N-(CH ₃ ) 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 at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or even 100%) of the A ³ groups are -(CH ₂ ) _p1 -[NH-(CH ₂ ) _q1 - ] _r1 ]S[H-(CH ₂ ) ₂ -R ⁴ -R ⁵ , such as a -(CH2)2-NH-(CH2)2-NH-(CH2)2-R ⁴ -R ⁵ group or a - group (CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR ⁵ ; or a -(CH2)p1-[NR ² -(CH2)q1-]r1NR ² -(CH2)s1-R ⁵ group such as -(CH2)2-NR ² -(CH2)2-NR ² -(CH2 )2-R ⁵ , optionally each of R ² independently represents Q^-alkyl (e.g., methyl), and optionally R ⁵ represents ^-^-alkylamino or ^-^-dialkylamino (e.g., R ⁵ represents -CH2-NH-CH3 or -CH2-N-(CH3)2).

Additionally, in some embodiments, the polymer has some or even a majority of A ³ groups (at least about 5%, at least about 10%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70 %, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%), which is -(CH ₂ ) _p1 -[NH -(CH ₂ ) _q1 -] _r1 NH-(CH ₂ ) ₂ -R ⁴ -R ⁵ , such as the group -(CH2)2-NH-(CH2)2-NH-(CH2)2-R ⁴ -R ⁵ or group -(CH2)2-NH-(CH2)2-NH-(CH2)2-C(O)-OR ⁵ ; or a -(CH2)p1-[NR ² -(CH2) _q1 -] _r1 NR ² -(CH2)s1-R ⁵ group such as -(CH2) ₂ -NR ² -(CH2)2-NR ² -( CH2) ₂ -R ⁵ , optionally each of R ² being independently Q^-alkyl (e.g., methyl), and optionally R ⁵ being ^-^-alkylamino or ^-^-dialkylamino (e.g., R ⁵ is -CH2-NH-CH3 or -CH2-N-(CH3)2).

Moreover, in polymer structures, each of R ⁵ independently represents 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 from 1 to 8 (or 2 to 8) secondary or tertiary amines. Any of the above R ⁵ groups may be substituted or unsubstituted. R ⁵ may also be a substituent containing a tissue-specific or cell-specific targeting moiety. In some embodiments, R ⁵ may contain from about 1 to about 50 carbon atoms (e.g., from about 2 to about 50 carbon atoms, e.g., from about 2 to about 40 carbon atoms, from about 2 to about 30 carbon atoms, from about 2 to about 20 carbon atoms, from about 1 to about 16 or from about 2 to about 16 carbon atoms, from about 1 to about 12 or from about 2 to about 12 carbon atoms, from about 1 to about 10 or from about 2 to about 10 carbon atoms or from about 1 to about 8 or from about 2 to about 8 carbon atoms). In some embodiments, R ⁵ is a heteroalkyl group containing 1 to 8 or 2 to 8 (ie, 1, 2, 3, 4, 5, 6, 7, or 8) secondary or tertiary amines. Secondary or tertiary amines may be part of a heteroalkyl backbone (i.e., the longest continuous chain of atoms in a heteroalkyl group) or a pendant substituent. Thus, for example, the heteroalkyl group containing secondary or tertiary amines may be an alkylamino group, a dialkylamino group, an aminoalkyl group, an alkylaminoalkyl group, a dialkylaminoalkyl group, an aminoalkylamino group, or the like, containing from 1 to 8 (or 2-8) secondary or tertiary amines.

- 6 046334

In some embodiments, each of R ⁵ is independently selected from:

where each R ² independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

R ⁷ represents a C ₁ -C ₅₀ -alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group, optionally substituted by one or more amines;

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;

R ⁸ represents a tissue-specific or cell-specific targeting moiety, a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group.

Each R ² may represent hydrogen or a C ₁ -C ₁₂ -alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group. In some embodiments, R ² is

- 7 046334 is a linear or branched C1-C ₁₂ -alkyl group (for example, C1-C ₁₀ -alkyl group; ^-^-alkyl group; ^-^-alkyl group; ^-^-alkyl group, ^-^ -alkyl group or C ₁ or C ₂ alkyl group). In some embodiments, R ² is methyl or hydrogen.

R ⁷ may represent a C1-C50 (for example, C1-C40, C1-C30, C1-C20, C1-C10, C4-C12 or C6-C8) alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group, optionally substituted by one or more amines. In some embodiments, R ⁷ is a C ₄ -C ₁₂ , for example a C ₆ -C ₈ , an alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group, optionally substituted with one or more amines. In some embodiments, R ⁷ is substituted with one or more amines. In some embodiments, R ⁷ is substituted with 1-8 or 2-8 (ie, 1, 2, 3, 4, 5, 6, 7, or 8) secondary or tertiary amines. Amines can be part of an alkyl group (i.e. included in a backbone consisting of an alkyl group) or a pendant substituent.

R ¹³ may represent hydrogen or a C1-C12 alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group. In some embodiments, R ¹³ is a straight or branched C1-C ₁₂ alkyl group (e.g., C ₁ -C ₁₀ alkyl group; ^-^-alkyl group; ^-^-alkyl group; ^-^-alkyl group , ^-^-alkyl group or C1 or C ₂ alkyl group). In some embodiments, R ¹³ is methyl. In other embodiments, R ¹³ may be hydrogen. Typically, in a given polymer, all R ¹³ are the same (eg, all are methyl or all are hydrogen); however, each of the R ¹³ is independently selected and may be the same or different.

Each of the Y's is optional. As used herein, the phrase present does not necessarily mean that a substituent indicated as optionally present may or may not be present, and if the substituent is not present, then adjacent substituents are directly related to each other. If Y is present, Y is a cleavable linker. As used herein, the phrase cleavable linker refers to any chemical element connecting two parts that can be cleaved into two separate parts. For example, the cleavable linker may be cleaved by a hydrolytic process, a photochemical process, a free radical process, an enzymatic process, an electrochemical process, or a combination thereof. Examples of cleavable linkers include, but are not limited to:

wherein each R ¹⁴ independently represents a ^-^-alkyl group, each R ¹⁵ independently represents hydrogen, an aryl group, a heterocyclic group (eg, aromatic or non-aromatic), a C ₁ -C ₁₂ -alkyl group, an alkenyl group, a cycloalkyl group, or cycloalkenyl group,

R ¹⁶ represents a six-membered aromatic or heteroaromatic group, optionally substituted by one or more of: -OCH3, -NHCH3, -N(CH3)2, -SCH3, -OH, or a combination thereof.

The polymer may be any suitable polymer, provided that the polymer contains the above polymer structure as part of the polymer. In some embodiments, the polymer is a block copolymer comprising a polymer block having a structure of Formula 1, 1.1, or 1.2, and one or more other polymer blocks (eg, an ethylene oxide subunit or a propylene oxide subunit). In other embodiments, the structure of formula 1, 1.1 or 1.2 is only a polymer polymer unit that can be capped at either end with a suitable terminal end. In some embodiments, the polymer further comprises a substituent containing a tissue-specific or cell-specific targeting moiety.

- 8 046334

In some embodiments, the polymer has a structure of Formula 1A:

where Q is the formula:

or formula 1A1:

where c is an integer from 0 to 50;

Y is optionally present and is the cleavable linker described above;

R ¹ represents hydrogen, an aryl group, a heterocyclic group (eg, aromatic or non-aromatic), a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, or a linear or branched C ₁ -C ₁₂ alkyl group, optional substituted with one or more substituents, such as one or more amines;

R ⁶ represents hydrogen, an amino group, an aryl group, a heterocyclic group (eg, aromatic or non-aromatic), a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, a linear or branched C ₁ -C ₁₂ alkyl group optionally substituted with one or more amines; or a tissue-specific or cell-specific targeting moiety;

each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

and all other substituents (eg m ¹ , n ¹ , m ² , n ² , R ² , R ^3a , R ^3b , R ⁴ , R ⁵ and R ¹³ ) are as described above.

Likewise, the polymer may have the structure of Formula 1.1A:

or, if the skeleton is in alpha, beta configuration, the structure of formula 1.2A:

where c is an integer from 0 to 50;

Y is optionally present and is the cleavable linker described above;

R ¹ represents hydrogen, an aryl group, a heterocyclic group (eg, aromatic or non-aromatic), a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, or a linear or branched C ₁ -C ₁₂ alkyl group, optional

- 9 046334 specifically substituted with one or more substituents, such as one or more amines;

R ⁶ represents hydrogen, an amino group, an aryl group, a heterocyclic group (for example, aromatic or non-aromatic), a C ₁ -C ₁₂ -alkyl group, an alkenyl group, a cycloalkyl group or cycloalkenyl group, a linear or branched QC^-alkyl group, optionally substituted one or more amines; or a tissue-specific or cell-specific targeting moiety;

each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

and all other substituents (eg m ³ , R ^3a , R ^3b and A ³ ) are as described above.

In some embodiments of the above polymer, R ¹ and/or R ⁶ is a linear or branched C ₁ -C ₁₂ -alkyl group (e.g., a C ₁ -C ₁₀ -alkyl group; a C ₁ -C ₈ -alkyl group; ^-^ -alkyl group; ^-^-alkyl group, ^-^-alkyl group or C1 or _C2 alkyl group) substituted with one or more substituents. In some embodiments, the heteroalkyl or alkyl group contains or is substituted with one or more amines, such as 1-8 or 2-8 (ie, 1, 2, 3, 4, 5, 6, 7, or 8) secondary or tertiary amines. Amines can be part of a heteroalkyl backbone or pendant substituents.

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

- 10 046334

- 11 046334

- 12 046334

- 13 046334

- 14 046334

- 15 046334

- 16 046334

Polymer 15

- 17 046334

- 18 046334

- 19 046334

- 20 046334

- 21 046334

or

where each of a, b, c and d is independently an integer from 0 to 1000, such as 0-500, 0-200, 0-100 or 0-50, provided that (a+b+c+d ) > about 5. In some embodiments, (a+b) is in the range from about 0 to about 75 (e.g., from about 5 to about 75, from about 20 to about 75, from about 40 to about 75, from about 40 up to about 60,

- 22 046334 from about 50 to about 75, from about 60 to about 75, or from about 70 to about 75), and (c+d) is in the range from about 5 to about 80 (for example, from about 5 to about 75, from about 5 to about 60, from about 5 to about 40, from about 20 to about 40, from about 5 to about 30, from about 5 to about 20, or from about 5 to about 10).

In some embodiments, (a+b) is greater than (c+d). For example, in some embodiments, the ratio (a+b)/(c+d) is between about 1 and about 3 (e.g., (a+b) is about 55, and (c+d) is about 25), or the ratio (a+b)/(c+d) ranges from about 3 to about 10 (eg, (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 less than 1 (eg, about 0.1 or more, but less than 1). In this case also, the indication of the number of units (a, b, c and d) in these exemplary polymers does not imply the structure of the block copolymer; on the contrary, these quantities indicate the total number of links, and the links can be arranged in a random order, as indicated by the symbol / in these formulas.

The invention also relates to a polymer containing the structure of formula 2:

where 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 above in the present description;

R ¹ , R ² , and R ¹³ and all other substituent groups are as described above for polymers having other formulas (eg, formulas 1, 1A, 1A1, 1.1A and 1.2A); And

R ⁸ is a tissue-specific or cell-specific targeting moiety.

The invention also relates to a polymer containing the structure of formula 4:

GO OΊ > JI n n, / ,11. n n, |---4-с—с— n4 / C-R3a^C— n)-£ > I ΓΪΊ / t Π ^Ί J ^R yAo

NR ¹³

NR ¹³ A \ ¹ where each of m ¹ and n ¹ is an integer from 0 to 1000, provided that the sum of m1+n1 is greater than 2;

the symbol / indicates that the links separated in this way are connected randomly or in any order;

each of A ¹ and A ² is independently a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ , where each of p1-p4, q1-q6, and r1 and r2 independently represents is an integer from 1 to 5;

each R ² independently represents hydrogen, a C1-C12-alkyl group, a C1-C12 alkenyl group, a C3-C12 cycloalkyl group, or a C3-C12 cycloalkenyl group, provided that A ¹ and A ² contain at least one (for example, at least two, at least three, at least four or even at least five) a tertiary amine (for example, at least some of R ² are a C1-C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group);

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group.

- 23 046334

The invention also relates to a polymer having the structure of formula 4A:

oh oh

where c is an integer from 0 to 50;

Y is optionally present and is a cleavable linker;

each of m ¹ and n ¹ is an integer from 0 to 1000, provided that the sum of m1+n1 is greater than 2;

the symbol / indicates that the links separated in this way are connected randomly or in any order;

each of A ¹ and A ² is independently a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2 ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _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-p4, q1-q6 and r1 and r2 are each independently an integer from 1 to 5;

R ¹ represents hydrogen, an aryl group, a heterocyclic group (eg, aromatic or non-aromatic), a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, or a linear or branched C ₁ -C ₁₂ alkyl group, optional substituted with one or more substituents;

each R ² independently represents hydrogen, a C1-C12 alkyl group, a C1-C12 alkenyl group, a C3-C12 cycloalkyl group, or a C3-C12 cycloalkenyl group, provided that A ¹ and A ² contain at least one (for example, at least two, at least three, at least four or even at least five) a tertiary amine (for example, at least some of R ² are a C1-C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group);

each of R ^3a and R ^3a independently represents a methylene or ethylene group;

R ⁶ represents hydrogen, an amino group, an aryl group, a heterocyclic group (eg, aromatic or non-aromatic), a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, a linear or branched C ₁ -C ₁₂ alkyl group optionally substituted with one or more amines; or a tissue-specific or cell-specific targeting moiety; and each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group.

In embodiments of polymers containing the structure of Formula 4 and polymers containing the structure of Formula 4A, each R ² independently represents hydrogen or a C1-C12 alkyl group, a C1-C12 alkenyl group, a C3-C12 cycloalkyl group, or a C3-C12 -cycloalkenyl group, provided that each of A ¹ and A ² contains at least one tertiary amine (for example, at least two, at least three, at least four or even at least five tertiary amines). In some embodiments, each R ² independently represents a C1-C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. Accordingly, in embodiments of polymers containing the structure of Formula 4 and polymers containing the structure of Formula 4A, each nitrogen containing R ² substituents is a tertiary amine, except that the terminal amine may be a primary, secondary or tertiary amine, preferably a secondary amine or a tertiary amine.

In some embodiments of polymers containing the structure of formula 4, polymers containing the structure of formula 4A, each of A ¹ and A ² represents a group of formula -(CH2)2-NR ² -(CH2) ₂ -NR ² 2, where each R ² independently represents a C1- _C12 alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, except that the terminal amine may be a primary, secondary or tertiary amine, preferably a secondary or tertiary amine. In some embodiments of polymers containing the structure of Formula 4 and polymers containing the structure of Formula 4A, each R ² is ethyl or methyl, optionally except that the terminal amine may be a primary, secondary or tertiary amine, preferably a secondary or tertiary amine .

In embodiments of the polymers containing the structure of Formula 4 and the polymers containing the structure of Formula 4A, each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group, or a cycloalkenyl group. As a rule,

- 24 046334 Each R ¹³ independently represents hydrogen or a methyl substituent. In some embodiments, each R ¹³ is hydrogen.

According to any of the above polymer embodiments, examples of groups A ¹ and

And ² include, for example:

- NH-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;

- N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;

- NH-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;

- N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)2;

- NH-CH ₂ -CH ₂ -N(CH ₃ )-CH ₂ -CH ₂ -NH(CH ₃ );

- N(CH ₃ )-CH ₂ -CH ₂ -N(CH ₃ )-CH ₂ -CH ₂ -NH(CH ₃ );

- NH-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3);

- N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-N(CH3)-CH2-CH2-NH(CH3).

Non-limiting examples of polymers provided herein thus include, for example:

Polymer 31

- 25 046334

- 26 046334

where each of a and b is independently an integer from 0 to 1000, such as 0-500, 0-200, 0-100, or 0-50.

Thus, in some embodiments, either a or b may be 0, or the polymer may have some ratio of a to 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 from about 25 to about 60) or even about 50 or more (for example, from about 50 to about 160, from about 50 to about 140, from about 50 to about 120, from about 50 to about 100, or from about 50 to approximately 80). Additional examples include polymers 30-33 in which the terminal tertiary amine of one or both side chains is demethylated to form a secondary amine (e.g., -NHCH3):

- 27 046334

where a and b are as described above.

Any of the foregoing polymers may include a tissue-specific or cell-specific targeting moiety at the position indicated in the described formulas, or the polymers may be otherwise modified to include a tissue-specific or cell-specific targeting moiety. Methods for attaching a tissue-specific or cell-specific targeting moiety are known in the art, some of which include Michael addition, epoxy ring opening, displacement reactions, or click chemistry (e.g., CuAAC, SPAAC, SPANC, or strained alkene reactions) that utilize tissue-specific or cell-specific targeting moiety with appropriate functional groups. A tissue-specific or cell-specific targeting moiety can be any small molecule, protein (eg, antibody or antigen), amino acid sequence, sugar, oligonucleotide, metal-based nanoparticle, or combination thereof capable of recognizing (eg, specifically binding to) a given target tissue or cell (eg, specifically bind to a particular ligand, receptor, or other protein or molecule, allowing the target moiety to distinguish the target tissue or cell from another tissue or cell that is not the target). In some embodiments, the tissue-specific or cell-specific targeting moiety is a ligand receptor. In some embodiments, the tissue-specific or cell-specific targeting moiety is a receptor ligand.

A tissue-specific or cell-specific targeting moiety can be used to

- 28 046334 targeting any desired tissue or cell type. In some embodiments, the tissue-specific or cell-specific targeting moiety localizes the polymer to tissues of the peripheral nervous system, central nervous system, liver, muscle (eg, cardiac muscle), lung, bone (eg, hematopoietic cells), or eye of the subject. In some embodiments, the tissue-specific or cell-specific targeting moiety localizes the polymer to tumor cells. For example, a tissue-specific or cell-specific targeting moiety may be a sugar that binds to a receptor on a specific tissue or cell.

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

wherein each of R ⁹ , R ¹⁰ , R ¹¹ and R ¹² independently represents hydrogen, halogen, CrC^-alkyl or ^-^-alkoxy, independently substituted with one or more amino groups.

These tissue-specific or cell-specific targeting moieties can be selected to ensure localization of the polymer in tissue, as disclosed herein. For example, alphaG-mannose can be used to localize the polymer to the peripheral nervous system, central nervous system or immune cells, alphaG-galactose and N-acetylgalactosamine can be used to localize the polymer to liver hepatocytes, and folic acid can be used to localize the polymer to tumor cells.

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

In some embodiments, the polymer has a weight average molecular weight of from about 5 to about 2000 kDa, such as from about 10 to about 2000 kDa. The polymer may have a weight average molecular weight of about 2000 kDa or less, for example, about 1800 kDa or less, about 1600 kDa or less, about 1400 kDa or less, about 1200 kDa or less, about 1000 kDa or less, about 900 kDa, or less,

- 29 046334 about 800 kDa or less, about 700 kDa or less, about 600 kDa or less, about 500 kDa or less, about 100 kDa or less, or about 50 kDa or less, or even about 40 kDa or less, for example, about 30 kDa or less or 25 kDa or less. Alternatively or additionally, the polymer may have a weight average molecular weight of about 5 kDa or more, or about 10 kDa or more, such as about 50 kDa or more, about 100 kDa or more, about 200 kDa or more, about 300 kDa or more, or approximately 400 kDa or more. Thus, the polymer may have a weight average molecular weight limited to any two of the above endpoints. For example, the polymer may have a weight average molecular weight of 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, from about 100 to about 500 kDa, from about 200 to about 500 kDa, from about 300 to about 500 kDa, from about 400 to about 500 kDa, from about 400 to about 600 kDa, from about 400 to about 700 kDa, from about 400 to about 800 kDa, from about 400 to about 900 kDa, from about 400 to about 1000 kDa, from about 400 to about 1200 kDa, from about 400 to about 1400 kDa, from about 400 to about 1600 kDa, from about 400 to about 1800 kDa, about 400 to about 2000 kDa, about 200 to about 2000 kDa, about 500 to about 2000 kDa, or about 800 to about 2000 kDa.

The weight average molecular weight can be determined by any suitable method. Typically, weight average molecular weight is determined by size exclusion chromatography on a column selected from TSKgel Guard, GMPW, GMPW, G1000PW using a Waters 2414 refractive index detector (Waters Corporation, Milford, Massachusetts). In addition, the weight average molecular weight is determined by calibration using polyethylene oxide/polyethylene glycol with a molecular weight ranging from 150 to 875,000 Da as a standard.

Cooking methods

The proposed polymers can be obtained by any suitable method. In some embodiments, polymers can be prepared by a process that involves modifying at least a portion of the A ¹ and/or A ² groups of a polymer containing the structure of Formula 3:

oh oh

where each of m ¹ and n ¹ is an integer from 0 to 1000, provided that m ¹ +n ¹ is greater than 5;

the symbol / indicates that the links separated in this way are connected randomly or in any order;

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ -alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each of A ¹ and A ² independently represents a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _P 3-N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ , where each of p1-p4, q1-q6 and r1 and r2 independently represents integer from 1 to 5;

each R ² independently represents hydrogen or a Q-Cn-alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group to form a polymer containing the structure of formula 1:

_g OO OO,

where m ¹ and n ¹ represents an integer from 0 to 1000;

each of m2 and ⁿ² is an integer from 0 to 1000, provided that the sum of ^m2 + ⁿ² is greater than 5;

the symbol / indicates that the links separated in this way are connected randomly or in any way

- 30 046334 good order;

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or a Q-Cn-alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each of A ¹ and A ² independently represents a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ , each of B ¹ and B ² independently represents:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁴ -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁴ -R ⁵ ]2;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁴ -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁴ -R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -CH2-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][-(CH2)q4-NR ² -]r2-CH ₂ -CHOH-R ⁵ ;

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -CH2-CHOH-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁵ ]2;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁵ ]2} ₂ ;

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁴ -R ⁵ }-(CH2)ql-]rlNR ² 2; or

-(CH ₂ ) _pl -[N{(CH ₂ ) _sl -R ⁵ }-(CH2)ql-]rlNR ² 2, where each of p1-p4, q1-q6, r1 and r2 and s1-s4 independently represents is an integer from 1 to 5;

each R ² independently represents hydrogen or a Q-Cn-alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each R ⁴ independently represents -C(O)O-, -C(O)NH- or -S(O)(O)-;

each R ⁵ independently represents 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 tissue-specific or cell-specific targeting moiety.

The polymer containing the structure of formula 1 obtained by this method can be any polymer of formula 1, including formulas 1.1, 1.2, 1A1, 1A, 1.1A, 1.2A, 4 or 4A, and any embodiments thereof, as described with respect to the polymer according to the invention.

The polymer containing the structure of formula 3 can be any suitable polymer meeting these criteria. In some embodiments, the polymer may alternatively be described by Formula 3.1:

or, if the core is in alpha, beta configuration, by formula 3.2:

where m ³ represents an integer from 5 to 2000 (for example, 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 links separated in this way are connected randomly or in any order;

each of R ^3a and R ^3b independently represents a methylene or ethylene group;

each R ¹³ independently represents hydrogen or a Q-Cn-alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each A ³ independently represents a group of the formula:

- 31 046334

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _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-p4, q1-q6 and rl and r2 are each independently an integer from 1 to 5;

each R ² independently represents hydrogen or a CrCn-alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group.

In this case, the method may include modifying at least a portion of the A ³ groups sufficient to produce a polymer with a structure of formula 1.1 or 1.2, as disclosed herein. An example of a polymer containing one of the following structures is pAsp(DET) (poly(2-[(2aminoethyl)amino]ethyl aspartamide)) or PEG-pAsp(DET) (polyethylene glycol-b-poly{N'-[N-(2 -aminoethyl)-2-aminoethyl] aspartamide}).

The groups designated A ¹ and/or A ² (or the A ³ group of a polymer of formula 3.1 or 3.2) can be modified by any suitable means to produce the groups designated B ¹ and/or B ² (or the A ³ group of a polymer of formula 1.1 or 1.2 ).

For example, groups designated A ¹ and/or A ² (or group A ³ of a polymer of formula 3.1 or 3.2) can be modified by Michael addition, epoxy ring opening, or substitution reactions. In preferred embodiments, the groups designated A ¹ and/or A ² (or the A ³ group of a polymer of Formula 3.1 or 3.2) are modified by a Michael addition reaction.

In one embodiment, groups A ¹ and/or A ² of a polymer containing a structure of formula 3, or group A ³ of a polymer of formula 3.1 or 3.2, are modified by a Michael addition reaction carried out between the polymer containing a structure of formula 3 and α, β-unsaturated carbonyl compound. As used herein, the term Michael addition refers to the nucleophilic addition of a polymer nucleophile (eg, a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) to an α,β-unsaturated carbonyl compound. Accordingly, a Michael addition reaction occurs between a polymer containing 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.

The α,β-unsaturated carbonyl compound can be any α,β-unsaturated carbonyl compound capable of accepting a nucleophile via Michael addition reaction. In some embodiments, the α,β-unsaturated carbonyl compound is an acrylate, an acrylamide, a vinyl sulfone, or a combination thereof. Accordingly, a Michael addition reaction can occur between a polymer containing the structure of Formula 3 and an acrylate, acrylamide, vinyl sulfone, or a combination thereof. Thus, in some embodiments, the method comprises contacting a polymer containing the structure of Formula 3 and an acrylate; bringing into contact a polymer containing the structure of formula 3 and acrylamide; or contacting a polymer containing the structure of Formula 3 and a vinyl sulfone.

In embodiments in which the groups designated A ¹ and/or A ² (or the A ³ group of a polymer of formula 3.1 or 3.2) are modified by a Michael addition reaction, these groups form groups designated B ¹ and/or B ² . defined herein with respect to formulas 1, 1.1, 1.2, 1A, 1A1, etc. (or group A ³ polymer of formula 1.1 or 1.2). For example, groups B ¹ and/or B ² can be represented by the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁴ -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁴ -R ⁵ ]2;

-(CH ₂ ) _P 3-N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _s3 -R ⁴ -R ⁵ };

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁴ -R ⁵ ]2} ₂ , where each of p1-p4, q1-q6, r1 and r2 and s1-s4 independently represent an integer from 1 to 5;

each R ² independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group; each R ⁴ independently represents -C(O)O-, -C(O)NH- or -S(O)(O)-;

each R ⁵ independently represents 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 tissue-specific or cell-specific targeting moiety; or in other cases, the groups and substituents may be as defined in relation to the polymers of the invention.

- 32 046334

Examples of suitable acrylates, acrylamides and vinyl sulfones include an acrylate of the formula:

where R ⁵ is as defined by any of formulas 1, 1.1, 1.2, 1A, 1.1A or 1.2A.

In some embodiments, an acid and/or a base is used to facilitate the Michael addition reaction. The acid and/or base can be any suitable acid and/or base with any suitable pKa value. The acid and/or base may be an organic acid (eg, p-toluenesulfonic acid), an organic base (eg, triethylamine), an inorganic acid (eg, titanium tetrachloride), an inorganic base (eg, potassium carbonate), or a combination thereof.

In some embodiments, an acid is used to facilitate the Michael addition reaction. The acid may be a Brønsted acid or a Lewis acid. In embodiments in which the acid is a Brønsted acid, the acid can be a weak acid (ie, with a pKa of about 4 to about 7) or a strong acid (ie, with 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 may be bis(trifluoromethanesulfone)imide or p-toluenesulfonic acid.

In some embodiments, a base is used to facilitate the Michael addition reaction. The base may be a weak base (ie, a pKa of about 7 to about 12) or a strong base (ie, 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-methyl morpholine or NA-dimethylinerazine or derivatives thereof.

In some embodiments, the Michael addition reaction is performed 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 effect the reaction. For example, the solvent may include water, protic organic solvents and/or aprotic organic solvents. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol and ethanol.

In one embodiment, the A ¹ and/or A ² groups (or the A ³ group of a polymer of Formula 3.1 or 3.2) of the polymer are modified by an epoxy ring opening reaction between the polymer and the epoxy compound. As used herein, the term epoxy ring opening refers to the nucleophilic addition of a polymer nucleophile (eg, a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) to an epoxy compound, thereby opening the epoxy ring. Accordingly, the epoxy ring opening reaction occurs between the polymer and the epoxy compound. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

In embodiments in which groups designated A ¹ and/or A ² (or group A ³ of a polymer of formula 3.1 or 3.2) are modified by an epoxy ring opening reaction, these groups form groups designated B ¹ and/or B ² ( or group A ³ polymer of formula 1.1 or 1.2) formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -CH2-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][-(CH2)q4-NR ² -]r2-CH ₂ -CHOH-R ⁵ ;

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -CH2-CHOH-R ⁵ ]2} ₂ , where each of p1-p4, q1-q6 and r1 and r2 independently represent an integer from 1 to 5;

each R ² independently represents hydrogen or a C1-C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each R ⁵ independently represents 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 tissue-specific or cell-specific targeting moiety.

Examples of epoxides suitable for use include epoxides of the formula:

where R ⁵ is as defined in relation to any polymer according to the invention (for example, formulas 1, 1.1, 1.2, 1A, 1.1A or 1.2A).

In some embodiments, an acid and/or a base is used to facilitate the epoxy ring opening reaction. The acid and/or base may be any suitable

- 33 046334 acid and/or base with any suitable pKa value. The acid and/or base may be an organic acid (eg, p-toluenesulfonic acid), an organic base (eg, triethylamine), an inorganic acid (eg, titanium tetrachloride), an inorganic base (eg, potassium carbonate), or a combination thereof.

In some embodiments, an acid is used to facilitate the epoxy ring opening reaction. The acid may be a Brønsted acid or a Lewis acid. In embodiments in which the acid is a Brønsted acid, the acid can be a weak acid (ie, with a pKa of about 4 to about 7) or a strong acid (ie, with 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 may be bis(trifluoromethanesulfone)imide or p-toluenesulfonic acid.

In some embodiments, a base is used to facilitate the epoxy ring opening reaction. The base may be a weak base (ie, a pKa of about 7 to about 12) or a strong base (ie, 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-dimethylpiperazine or derivatives thereof.

In some embodiments, the epoxy ring opening reaction is performed in a solvent. The solvent may be any suitable solvent or mixture of solvents capable of solubilizing the polymer and the epoxy compound to effect the reaction. For example, the solvent may include water, protic organic solvents and/or aprotic organic solvents. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol and ethanol.

In one embodiment, the groups A ¹ and/or A ² (or group A ³ of a polymer of formula 3.1 or 3.2) of the polymer are modified by a substitution reaction carried out between the polymer and a compound containing a leaving group (eg, chloride, bromide, iodide, tosylate , triflate, mesylate, etc.). As used herein, the term substitution refers to the nucleophilic addition of a polymer nucleophile (eg, a carbanion, an oxygen anion, a nitrogen anion, an oxygen atom, a nitrogen atom, or a combination thereof) to a compound containing a leaving group. Accordingly, the substitution reaction occurs between the polymer and the compound containing the leaving group. In some embodiments, the nucleophile of the polymer is a nitrogen anion, a nitrogen atom, or a combination thereof.

In embodiments in which the groups designated A ¹ and/or A ² (or group A ³ of the polymer of formula 3.1 or 3.2) are modified by a substitution reaction, these groups form groups designated B ¹ and/or B ² (or groups A ³ polymers of formula 1.1 or 1.2) formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² -(CH2) _sl -R ⁵ ;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² -(CH2)s2-R ⁵ ]2;

-(CH ₂ ) _P 3-N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2(CH ₂ ) _S 3-R ⁵ }; or -(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² -(CH2)s4-R ⁵ ]2} ₂ , where each of p1-p4, q1- q6, r1 and r2 and s1-s4 independently represent an integer from 1 to 5;

each R ² independently represents hydrogen or a C ₁ -C ₁₂ -alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

each R ⁵ independently represents 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 tissue-specific or cell-specific targeting moiety.

In some embodiments, the method comprises preparing a polymer containing the structure of Formula 5 from a compound of Formula A:

where R ^3b represents a methylene or ethylene group;

W represents -NR ¹⁴ - or -O-, where R ¹⁴ represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group;

Z represents A ¹ or 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, optionally containing 1 to 8 or 2 to 8 secondary or tertiary amines, or a substituent containing a tissue-specific or cell-specific

- 34 046334 targeting fragment;

And ¹ represents a group of the formula:

-(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2;

-(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ;

-(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or

-(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR2 ₂ ] ₂ } ₂ , where each of p1-p4, q1-q6 and r1 and r2 independently represents an integer number from 1 to 5;

each R ² independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group.

Typically, the process comprising forming a polymer containing the structure of formula 5 from a compound of formula A involves ring opening polymerization of a compound of formula A. Ring opening polymerization of a compound of formula A can be initiated by any suitable method (eg, temperature, light, catalyst, compound, etc.). In some embodiments, ring-opening polymerization of a compound of Formula A is initiated with an amine-containing compound.

In some embodiments, ring-opening polymerization of a compound of formula A is initiated with an amine-containing compound of formula:

RMcH ₂ CH ₂ 0prC ^H ^Y— νη _ξ ^с 0-6 where с represents an integer from 0 to 50;

Y is optionally present and is a cleavable linker;

R ⁶ represents hydrogen, an amino group, an aryl group, a heterocyclic group, a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, a linear or branched C ₁ -C ₁₂ alkyl group, optionally substituted by one or more amines ; or a tissue-specific or cell-specific targeting moiety.

In preferred embodiments, the amine-containing compound is

In some embodiments, in a compound of formula, AY 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, optionally containing from 1 to 8 or 2 to 8 secondary or tertiary amines. In some embodiments, Z is an arylalkyl group, such as benzyl. The method for preparing the polymer of formula 5 further comprises, after ring opening polymerization has occurred, treating the resulting intermediate polymer with a compound of formula NHR17-A ¹ , wherein A ¹ is as defined in relation to formula 1. The compound of formula NI IR ^r -A' is optionally diethylenamine triamine. In some embodiments, the compound of formula NHR17-A ¹ is not a diethyleneamine triamine.

R ¹⁷ may represent hydrogen or a C1-C12 alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group. In some embodiments, R ¹⁷ is a straight or branched C1-C12 alkyl group (e.g., a C ₁ -C ₁₀ alkyl group; a C1-C ₈ alkyl group; a C1-C ₆ alkyl group; a C1-C ₄ - alkyl group, C1- _C3 alkyl group or C1 or _C2 alkyl group). In some embodiments, R ¹⁷ is methyl. In other embodiments, R ¹⁷ may be hydrogen. Typically, in a given polymer, all R ¹⁷ are the same (eg, all are methyl or hydrogen); however, each R ¹⁷ is independently selected and the R ¹⁷ may be the same or different.

Examples of compounds containing leaving groups suitable for use include a compound of the formula:

where LG represents a leaving group (eg, chloride, bromide, iodide, tosylate, triflate, mesylate, etc.);

R ⁵ is as described in relation to any of formulas 1, 1.1, 1.2, 1A, 1.1A or 1.2A.

In some embodiments, an acid and/or a base is used to facilitate the displacement reaction. The acid and/or base can be any acid and/or base with any suitable pKa value. The acid and/or base may be an organic acid (eg, p-toluenesulfonic acid), an organic base (eg, triethylamine), inorganic

- 35 046334 chelic acid (for example titanium tetrachloride), an inorganic base (for example potassium carbonate) or a combination thereof.

In some embodiments, an acid is used to facilitate the displacement reaction. The acid may be a Brønsted acid or a Lewis acid. In embodiments in which the acid is a Brønsted acid, the acid can be a weak acid (ie, with a pKa of about 4 to about 7) or a strong acid (ie, with 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 may be bis(trifluoromethanesulfone)imide or p-toluenesulfonic acid.

In some embodiments, a base is used to facilitate the displacement reaction. The base may be a weak base (ie, a pKa of about 7 to about 12) or a strong base (ie, 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-methyl morpholine or N,N-dimethylpiperazine or derivatives thereof.

In some embodiments, the displacement reaction is performed in a solvent. The solvent may be any suitable solvent or mixture of solvents capable of solubilizing the polymer and the leaving group-containing compound for the reaction. For example, the solvent may include water, protic organic solvents and/or aprotic organic solvents. An exemplary list of solvents includes water, dichloromethane, diethyl ether, dimethyl sulfoxide, acetonitrile, methanol and ethanol.

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

Compositions

The polymers presented herein can be used for any purpose. However, polymers are believed to be particularly useful for delivering nucleic acids and/or polypeptides (eg, protein) into cells. Thus, a composition is provided containing a polymer and a nucleic acid and/or polypeptide (eg, protein) as disclosed herein.

In some embodiments, the composition contains a nucleic acid. Any nucleic acid can be used. An exemplary list of nucleic acids includes CRISPR guide and/or donor nucleic acid systems, siRNA, microRNA, interfering RNA or RNAi, dsRNA, mRNA, vector DNA, ribozymes, antisense polynucleotides, and DNA expression cassettes encoding siRNA, microRNA, dsRNA, ribozymes or antisense nucleic acids. siRNA contains a double-stranded structure, typically containing 15-50 base pairs and preferably 19-25 base pairs, and having a nucleotide sequence identical or nearly identical to the expressed target gene or RNA within the cell. siRNA can consist of two hybridized polynucleotides or one polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small noncoding polynucleotides, approximately 22 nucleotides in length, that direct the degradation or translational repression of their target mRNAs. Antisense polynucleotides contain a sequence complementary to a gene or mRNA. Antisense polynucleotides include, but are not limited to, morpholines, 2'-O-methyl polynucleotides, DNA, RNA, and the like.

The polynucleotide-based expression inhibitor may be in vitro polymerized, recombinant, contain chimeric sequences, or derivatives of these groups. The polynucleotide-based expression inhibitor may comprise ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination that inhibits the target RNA and/or gene. The polynucleotide may also be a sequence that serves as a barcode for purposes such as tracking delivery in vitro or in vivo.

The composition may also contain any protein to be delivered 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 (TAKEN), a recombinase, a deaminase, an endonuclease, or a combination thereof. In some embodiments, the polypeptide is an RNA-guided endonuclease (eg, a Cas9 polypeptide, a Cpfl polypeptide, or variants thereof) or a DNA recombinase (eg, a Cre polypeptide).

The polymers provided herein are believed to be particularly useful for delivering one or more components of the CRISPR system. Thus, in some embodiments, the composition comprises 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 polymer disclosed herein. In addition, the composition can

- 36 046334 hold several guide RNAs, RNA-guided endonucleases or nucleic acids encoding them, and/or donor nucleic acids. For example, several different guide RNAs for different target sites may be included, optionally with several different donor nucleic acids and even with several different RNA-guided endonucleases or nucleic acids encoding them.

In addition, the components of the CRISPR system can be combined with each other (if multiple components are present) and the polymer in any particular way or in any order. In some embodiments, the guide RNA is complexed with an RNA endonuclease prior to combining with the polymer. Additionally or alternatively, the guide RNA can be linked (covalently or non-covalently) to a donor nucleic acid prior to association with the polymer.

The compositions are not limited to any particular CRISPR system (ie, any particular guide RNA, RNA-guided endonuclease, or donor nucleic acid), many of which are known. However, for further illustration, the components of some of these systems are described below.

Donor nucleic acid.

A donor nucleic acid (or donor sequence, or donor polynucleotide, or donor DNA) is a nucleic acid sequence that is intended to be introduced into a cleavage site induced by an RNA-directed endonuclease (eg, a Cas9 polypeptide or a Cpf1 polypeptide). The donor polynucleotide may have sufficient homology to the target genomic sequence at the cleavage site, e.g., 70, 80, 85, 90, 95, or 100% homology to nucleotide sequences flanking the cleavage site, e.g., within about 50 bases or less of the cleavage site, e.g. , within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases from the cleavage site, or immediately flanking the cleavage site, to support homology-driven repair between it and the genomic sequence to which it is homologous. Homology of approximately 25, 50, 100 or 200 nucleotides or more than 200 nucleotides of sequence between the donor and the genomic sequence (or any integral value of 10 to 200 nucleotides or more) will support homology-driven repair. The donor sequences can be of any length, such as 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.

The donor sequence is usually not identical to the genomic sequence it replaces. Rather, the donor sequence may contain one or more substitutions, insertions, deletions, inversions, or single base rearrangements relative to the genomic sequence as long as sufficient homology is maintained to support homology-driven repair. In some embodiments, the donor sequence includes a non-homologous sequence flanked by two regions of homology such that homology-driven repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence into the target region. Donor sequences may also include a vector backbone containing sequences that are not homologous to the DNA region of interest and which are not intended to be inserted into the DNA region of interest. Typically, the homologous region(s) of the donor sequence will have at least 50% sequence identity with the genomic sequence with which recombination is desired. In some embodiments, there is 60, 70, 80, 90, 95, 98, 99, or 99.9% sequence identity. Sequence identity can have any value from 1 to 100% depending on the length of the donor polynucleotide.

The donor sequence may differ to some extent from the genomic sequence, such as restriction sites, nucleotide polymorphisms, selectable markers (eg, drug resistance genes, fluorescent proteins, enzymes, etc.), etc., which can be used for evaluation the success of insertion of a donor sequence into a cleavage site or which, in some embodiments, can be used for other purposes (eg, to indicate expression at a target genomic locus). In some embodiments, if such nucleotide sequence differences are in the coding region, they will not change the amino acid sequence or will result in silent amino acid substitutions (ie, substitutions that do not affect the structure or function of the protein). Alternatively, these sequence differences may involve sequences flanking the recombination, such as FLP, loxP, etc. sequences, which may be activated at a later time to remove the marker sequence.

The donor sequence can be introduced into the cell as single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. It can be introduced into the cell in a linear or ring form. When introduced in linear form, the ends of the donor sequence can be protected (for example, from exonucleolytic degradation) by methods known in the art.

- 37 046334 stam in this area. For example, one or more dideoxynucleotide residues are added to the 3' end of a linear molecule, and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987), Proc. Natl. Acad. Sci. USA, 84:4959-4963; Nehls et al. (1996), Science, 272:886-889. Amplification procedures such as rolling circle amplification as shown herein can also be successfully used. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of amino terminal(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methylribose or deoxyribose residues.

Alternatively, additional sequences of a certain length may be included to protect the ends of the linear donor sequence outside the regions of homology, which can be disrupted without affecting recombination. The donor sequence can be introduced into the cell as part of a vector molecule containing additional sequences, such as, for example, origins of replication, promoters and genes encoding antibiotic resistance. Moreover, donor sequences may be administered as naked nucleic acid, as a complex of the nucleic acid with an agent such as a liposome or polymer, or may be delivered by viruses (e.g., adenovirus, AAV) as disclosed herein for nucleic acids. encoding a Cas9 guide RNA and/or a Cas9 fusion polypeptide and/or a donor polynucleotide.

Gide nucleic acid.

In some embodiments, the composition contains a guide nucleic acid. Guide nucleic acids suitable for inclusion in the composition of the present invention include single-molecule guide RNAs (single guide RNA/sgRNA) and double-molecule guide nucleic acids (double guide RNA/dgRNA).

A guide nucleic acid (eg, guide RNA) suitable for inclusion in the complex of the present invention directs the activity of an RNA-guided endonuclease (eg, a Cas9 or Cpf1 polypeptide) to a specific target sequence in the target nucleic acid. A guide nucleic acid (eg, guide RNA) contains a first segment (also referred to herein as a targeting nucleic acid segment or simply a targeting segment) and a second segment (also referred to herein as a protein binding segment). The terms first and second do not imply the order in which the segments occur in the guide RNA. The order of the elements relative to each other depends on the particular RNA-guided polypeptide used. For example, a guide RNA for Cas9 typically has a protein-binding segment located at the 3' end of the targeting segment, whereas a guide RNA for Cpf1 typically has a protein-binding segment located at the 5' end of the targeting segment.

Guide RNA can be introduced into the cell in linear or circular form. When administered in linear form, the ends of the guide RNA can be protected (eg, from exonucleolytic degradation) by methods known to those skilled in the art. Amplification procedures such as rolling circle amplification can also be used successfully, as illustrated herein.

First segment: targeting segment.

The first segment of the guide nucleic acid (eg, guide RNA) includes a nucleotide sequence complementary to the sequence (target site) of the target nucleic acid. In other words, a targeting segment of a guide nucleic acid (eg, guide RNA) can interact with a target nucleic acid (eg, RNA, DNA, dsDNA) in a sequence-specific manner by hybridization (ie, base pairing). Thus, the nucleotide sequence of the targeting segment may vary and may determine the location in the target nucleic acid at which the guide nucleic acid (eg, guide RNA) and the target nucleic acid will interact. A targeting segment of a guide nucleic acid (eg, guide RNA) can be modified (eg, by genetic engineering) to hybridize to any desired sequence (target site) in the target nucleic acid.

The targeting segment can range from 12 to 100 nucleotides in length. The nucleotide sequence (targeting sequence, also called guide sequence) of the targeting segment, which is complementary to the nucleotide sequence (target site) of the target nucleic acid, may be 12 nucleotides (nt) or more in length. For example, the targeting sequence of a targeting segment that is complementary to the target site of the target nucleic acid may be 12 nt or greater, 15 nt or greater, 17 nt or greater, 18 nt or greater, 19 nt or greater, 20 nt or greater, 25 nt or more, 30 nt or more, 35 nt or more, or 40 nt.

The percentage of complementarity between the targeting sequence (i.e., guide sequence) of the targeting segment and the target site of the target nucleic acid may 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 neko

- 38 046334 In other embodiments, the percentage of complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% within the seven contiguous nucleotides closest to the 5' end of the target site of the target nucleic acid. In some embodiments, the percentage of complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 60% or greater within 20 contiguous nucleotides. In some embodiments, the percentage of complementarity between the targeting sequence of the targeting segment and the target site of the target nucleic acid is 100% within the 17, 18, 19, or 20 contiguous nucleotides closest to the 5' terminal nucleotides of the target site of the target nucleic acid and 0% or more throughout the remainder . In this case, the targeting sequence can be considered to be 17, 18, 19 or 20 nucleotides in length, respectively.

Second segment: protein-binding segment.

The protein-binding segment of a guide nucleic acid (eg, guide RNA) interacts (binds) with an RNA-guided endonuclease. The guide nucleic acid (eg, guide RNA) directs the bound endonuclease to a specific nucleotide sequence in the target nucleic acid (target site) via the aforementioned targeting segment/targeting sequence/guide sequence. The protein-binding segment of a guide nucleic acid (eg, guide RNA) contains two stretches of nucleotides that are complementary to each other. The complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA (dsRNA) duplex.

Single and double guide nucleic acids.

A double guide nucleic acid (eg, guide RNA) consists of two separate nucleic acid molecules. Each of the two molecules of a subject double guide nucleic acid (eg, guide RNA) contains a region of nucleotides complementary to each other such that the complementary nucleotides of the two molecules hybridize to form a double-stranded RNA protein-binding segment duplex.

In some embodiments, the duplex-forming segment of the activator is 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% or more identical or 100% identical to one of the activator molecules (tracrRNA) listed in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement at a site consisting of 8 or more contiguous nucleotides (for example, 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides).

In some embodiments, the duplex-forming segment of the target (targrter) is 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% or more identical or 100% identical to one of the target sequences (crRNA ), shown in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement at a site consisting of 8 or more contiguous nucleotides (for example, 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).

A dual guide nucleic acid (eg, guide RNA) can be designed to provide controlled (ie, condition-dependent) binding of the target to the activator. Because a dual guide nucleic acid (eg, guide RNA) does not function unless the activator and target are bound into a functional complex with Cas9, the dual guide nucleic acid (eg, guide RNA) can be inducible (eg, drug-inducible) by generating an inducible binding between activator and targeter. As one non-limiting example, RNA aptamers can be used to regulate (ie, control) the binding of an activator to a target. Accordingly, the activator and/or targeter may include an RNA aptamer sequence.

Aptamers (eg, RNA aptamers) are known in the art and are typically a synthetic version of a riboswitch. The terms RNA aptamer and riboswitch are used interchangeably herein to cover both synthetic and naturally occurring nucleic acid sequences that provide inducible regulation of the structure (and hence the availability of specific sequences) of a nucleic acid molecule (e.g., RNA, DNA/RNA hybrid, and etc.) of which they are a part. RNA aptamers typically contain a sequence that folds into a specific structure (eg, a hairpin) that specifically binds to a specific drug (eg, a small molecule). Drug binding causes a structural change in the folding of the RNA, which alters the properties of the nucleic acid of which the aptamer is a part. As non-limiting examples, (i) an aptamer activator is unable to bind to a cognate target unless the aptamer is bound to a corresponding drug; (ii) the aptamer target is unable to bind to its cognate activator unless the aptamer is bound to the corresponding drug; and (iii) a targeter and an activator, each containing different aptamers that bind different drugs,

- 39 046334 are unable to bind to each other unless both drugs are present. As shown in these examples, a double guide nucleic acid (eg, guide RNA) can be designed to be inducible.

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

Non-limiting examples of nucleotide sequences that may be included in a dual guide nucleic acid (e.g., guide RNA) are provided in International Patent Application Nos. PCT/US2016/052690 and PCT/US2017/062617, and also include complements thereof that may hybridize with formation of a protein-binding segment.

The single guide nucleic acid in question (e.g., guide RNA) contains two stretches of nucleotides (like the targeter and activator of the double guide nucleic acid), which are complementary to each other, hybridize to form a double-stranded RNA duplex (dsRNA duplex) protein-binding segment (resulting in the formation stem-loop structures) and are covalently linked through intermediate nucleotides (linkers or linker nucleotides). Thus, a subject single guide nucleic acid (eg, single guide RNA) may comprise a targeter and an activator, each of which has a duplex-forming segment, wherein the duplex-forming segments of the targeter and activator hybridize with each other to form a dsRNA duplex. The targeter and activator can be covalently linked through the 3' end of the targeter and the 5' end of the activator. Alternatively, the targeter and activator can be covalently linked through the 5' end of the target and the 3' end of the activator.

The single guide nucleic acid linker can be from 3 to 100 nucleotides in length. In some embodiments, the single guide nucleic acid (eg, guide RNA) linker is 4 nucleotides.

A typical single guide nucleic acid (eg, guide RNA) contains two complementary stretches of nucleotides that hybridize to form a dsRNA duplex. In some embodiments, one of the two complementary nucleotide regions of a single guide nucleic acid (e.g., guide RNA) (or the DNA encoding that region) is 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% or more identical or 100% identical to one of the activator molecules (tracrRNA) listed in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement, within a region consisting of 8 or more contiguous nucleotides (eg, 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides).

In some embodiments, one of the two complementary nucleotide regions of a single guide nucleic acid (e.g., guide RNA) (or the DNA encoding that region) is 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% or more identical or 100% identical to one of the target sequences (crRNA) given in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement, within a region consisting of 8 or more contiguous nucleotides (eg, 8 or more contiguous nucleotides, 10 or more contiguous nucleotides, 12 or more contiguous nucleotides, 15 or more contiguous nucleotides, or 20 or more contiguous nucleotides).

In some embodiments, one of the two complementary nucleotide regions of a single guide nucleic acid (e.g., guide RNA) (or the DNA encoding that region) is 60, 65, 70, 75, 80, 85, 90, 95, 98, 99% or more identical or 100% identical to one of the targeter sequences (crRNA) or activator sequences (tracrRNA) given in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or their complement, within the region consisting of 8 or more contiguous nucleotides (eg, 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).

Appropriate cognate pairs of targeters and activators can be determined using routine procedures, taking into account the species name and base pairing (for the protein-binding domain dsRNA duplex). Any activator/targeter pair may be used as part of a double guide nucleic acid (eg, guide RNA) or as part of a single guide nucleic acid (eg, guide RNA).

In some embodiments, an activator (e.g., trRNA, trRNA-like molecule, etc.) of a double guide nucleic acid (e.g., guide RNA) (e.g., double guide RNA) or a single guide nucleic acid (e.g., guide RNA) (e.g. , single guide RNA) includes a region of nucleotides with 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or more or 100% sequence identity with the activator molecule (tracrRNA) described in international patent applications No. PCT /US2016/052690 and PCT/US2017/062617, or its complement.

In some embodiments, an activator (e.g., tRNA, a tRNA-like molecule, and

- 40 046334

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

The protein-binding segment can be from 10 to 100 nucleotides in length.

Also, for both the contemplated single guide nucleic acid (e.g., single guide RNA) and the contemplated double guide nucleic acid (e.g., double guide RNA), the protein-binding segment dsRNA duplex may be from 6 to 50 base pairs in length (bp). O.). The percentage of complementarity between the nucleotide sequences that hybridize to form a dsRNA protein-binding segment duplex can be 60% or more. For example, the percentage of complementarity between nucleotide sequences that hybridize to form a dsRNA protein-binding segment duplex may be 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more , 95% or more, 98% or more, or 99% or more (eg, in some embodiments, there are some nucleotides that do not hybridize and therefore create a bulge within the dsRNA duplex). In some embodiments, the percentage of complementarity between nucleotide sequences that hybridize to form a protein-binding segment dsRNA duplex is 100%.

Hybrid guide nucleic acids.

In some embodiments, the guide nucleic acid is two RNA molecules (double guide RNA). In some embodiments, the guide nucleic acid is a single RNA molecule (single guide RNA). In some embodiments, the guide nucleic acid is a DNA/RNA hybrid molecule. In such embodiments, the protein-binding segment of the guide nucleic acid is RNA and forms an RNA duplex. Thus, the duplex-forming segments of the activator and target are RNA. However, the targeting segment of the guide nucleic acid may be DNA. Thus, if the DNA/RNA hybrid guide nucleic acid is a double guide nucleic acid, the target molecule is a hybrid molecule (eg, the targeting segment may be DNA and the duplexing segment may be RNA). In such embodiments, the duplex-forming segment of the activator molecule may be RNA (e.g., to form an RNA duplex with the duplex-forming segment of the target molecule), while the nucleotides of the activator molecule that are outside the duplex-forming segment may be DNA ( in this case the activator molecule is a hybrid DNA/RNA molecule) or may be RNA (in this case the activator molecule is RNA). If the DNA/RNA hybrid guide nucleic acid is a single guide nucleic acid, then the targeting segment may be DNA, the duplex-forming segments (which constitute the protein-binding segment of the single guide nucleic acid) may be RNA, and nucleotides outside the targeting and duplex the forming segments can be RNA or DNA.

In some embodiments, a DNA/RNA hybrid guide nucleic acid may be used, for example when the target nucleic acid is RNA. Cas9 typically binds to a guide RNA, which hybridizes to the target DNA, thereby forming a DNA-RNA duplex at the target site. Therefore, when the target nucleic acid is RNA, it is sometimes advantageous to reproduce a DNA-RNA duplex at the target site using a targeting segment (guide nucleic acid) that is DNA rather than RNA. However, since the protein-binding segment of the guide nucleic acid is an RNA duplex, in the targeting segment the target molecule is DNA, and in the duplex-forming segment it is RNA. Hybrid guide nucleic acids can bias Cas9 binding towards binding to single-stranded nucleic acid targets versus double-stranded nucleic acid targets.

Examples of guide nucleic acids.

Any guide nucleic acid can be used. There are many different types of guide nucleic acids known in the art. The selected guide nucleic acid can be suitably paired with the particular CRISPR system used (eg, the particular RNA-guided endonuclease used). Thus, the guide nucleic acid can be, for example, a guide nucleic acid corresponding to any RNA-guided endonuclease disclosed herein or known in the art. Guide nucleic acids and RNA-guided endonucleases are described, for example, in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617.

In some embodiments, a suitable guide nucleic acid includes two separate RNA polynucleotide molecules. In some embodiments, the first of two from

- 41 046334 distinct RNA polynucleotide molecules (activator) contains a nucleotide sequence having 60% or more (for example, 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 within a region 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), with any of the nucleotide sequences disclosed in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement. In some embodiments, the second of two separate RNA polynucleotide molecules (the targeter) contains a nucleotide sequence having 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85% or more , 90% or more, 95% or more, 98% or more, 99% or more, or 100%) nucleotide sequence identity within a region 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), with any of the nucleotide sequences disclosed in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement .

In some embodiments, a suitable guide nucleic acid is a single RNA polynucleotide and contains first and second nucleotide sequences having 60% or more (e.g., 65% or more, 70% or more, 75% or more, 80% or more, 85 % or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) nucleotide sequence identity within a region 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), with any of the nucleotide sequences shown in international patent applications No. PCT/US2016/052690 and PCT/US2017/062617, or its complement.

In some embodiments, the guide RNA is a Cpf1 and/or Cas9 guide RNA. The Cpf1 and/or Cas9 guide RNA may have a total length of from 30 nucleotides (nt) to 100 nt, e.g., 30 to 40 nt, 40 to 45 nt, 45 to 50 nt, 50 to 60 nt, 60 to 70 nt, 70 to 80 nt, 80 to 90 nt, or 90 to 100 nt. In some embodiments, the Cpf1 and/or Cas9 guide RNA has a total length of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nt. The Cpf1 and/or Cas9 guide RNA may include a target nucleic acid binding segment and a duplex-forming segment.

The target nucleic acid binding segment of the Cpf1 and/or Cas9 guide RNA may be 15 to 30 nt in length, e.g., 15, 16, 17, 18, 19, 20 nt, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nt. In some embodiments, the target nucleic acid binding segment is 23 nt in length. In some embodiments, the target nucleic acid binding segment is 24 nt in length. In some embodiments, the target nucleic acid binding segment is 25 nt in length.

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

The duplex-forming segment of the Cpf1 and/or Cas9 guide RNA may be 15 to 25 nt in length, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nt.

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

Additional elements.

In some embodiments, the guide nucleic acid (e.g., guide RNA) includes an additional segment or segments (in some embodiments at the 5' end, in some embodiments at the 3' end, in some embodiments at either the 5' or at the 3' end, in some embodiments, embedded within the sequence (i.e., not at the 5' and/or

- 42 046334

3' end), in some embodiments both at the 5' end and at the 3' end, in some embodiments embedded at both the 5' end and/or 3' end, etc.). For example, a suitable additional segment may include a 5' cap (eg, a 7-methylguanylate cap (m7G)); 3'-polyadenylated tail (ie, 3'-poly(A) tail); a ribozyme sequence (eg, to cause self-cleavage of a guide nucleic acid or a component of a guide nucleic acid, eg, targeter, activator, etc.); a riboswitch sequence (eg, to provide controlled stability and/or controlled accessibility to proteins and protein complexes); sequence that forms a dsRNA duplex (i.e. hairpin)); a sequence that targets RNA at a subcellular site (eg, nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides tracking (e.g., a tag such as a fluorescent molecule (i.e., a fluorescent dye), sequence or other fragment that facilitates detection of fluorescence; a sequence or other modification that provides a binding site for proteins (e.g., proteins that act on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, RNA binding proteins (e.g., RNA aptamers), tagged proteins, fluorescently tagged proteins, etc.) ; a modification or sequence that provides increased, decreased and/or controlled stability, and combinations thereof.

RNA-guided endonuclease.

The composition may contain, in addition to or instead of a guide nucleic acid, an RNA-guided endonuclease protein or a nucleic acid encoding it (eg, mRNA or vector). Any RNA-guided endonuclease can be used. The choice of RNA-guided endonuclease used will depend, at least in part, on the intended end use of the CRISPR system being used.

In some embodiments, the polypeptide is a Cas9 polypeptide. Cas9 polypeptides suitable for inclusion in the composition of the present invention include a naturally occurring Cas9 polypeptide (e.g., naturally occurring in bacterial and/or archaeal cells) or a non-naturally occurring Cas9 polypeptide (e.g., a Cas9 polypeptide is a variant of a Cas9 polypeptide chimeric polypeptide, as discussed below, etc.) as described below. In some embodiments, one of ordinary skill in the art will appreciate that a Cas9 polypeptide disclosed herein can be any variant obtained or isolated from any source. In other embodiments, the Cas9 peptide of the present invention may include one or more mutations described in the literature, including, without limitation, functional mutations described in Fonfara et al. Nucleic Acids Res. Feb 2014; 42(4):2577-90; Nishimasu H. et al. Cell. 2014 Feb 27; 156(5):935-49; Jinek M. et al. Science. 2012, 337:816-21; and Jinek M. et al. Science. 2014 Mar 14; 343(6176); see also US Patent Application No. 13/842,859, filed March 15, 2013, which is incorporated herein by reference; also see US Pat. No. 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, incorporated herein by reference. Thus, in some embodiments, the systems and methods disclosed herein can be used with wild-type Cas9 protein having double-stranded nuclease activity, Cas9 mutants that act as single-stranded nickases, or other mutants with modified nuclease activity. Thus, a Cas9 polypeptide suitable for inclusion in a composition of the present invention may be an enzymatically active Cas9 polypeptide, for example, may produce single- or double-strand breaks in the target nucleic acid, or, alternatively, may have reduced enzymatic activity compared to a wild-type Cas9 polypeptide type.

Naturally occurring Cas9 polypeptides bind to a guide nucleic acid, thereby directing it to a specific sequence in the target nucleic acid (target site) and cleave the target nucleic acid (cleave dsDNA creating a double-strand break, cleave ssRNA, cleave ssRNA, etc.) . The Cas9 polypeptide in question has two parts: an RNA-binding part and an activity part. The RNA binding moiety interacts with the nucleic acid in question, and the activity moiety has site-directed enzymatic activity (e.g., nuclease activity, DNA and/or RNA methylation activity, DNA and/or RNA cleavage activity, histone acetylation activity, histone methylation activity, RNA modification activity, RNA binding activity, RNA splicing activity, etc. In some embodiments, the activity portion has reduced nuclease activity compared to the corresponding portion of the wild type Cas9 polypeptide.In some embodiments, the activity portion is enzymatically inactive.

Assays for determining whether a protein has an RNA-binding moiety that interacts with the nucleic acid in question can be any convenient binding assay that assesses the binding between the protein and the nucleic acid. Typical binding assays include binding assays (eg, gel mobility shift assays) that involve adding a guide nucleic acid and a Cas9 polypeptide to a target nucleic acid.

- 43 046334 leic acid.

Assays to determine whether a protein has a moiety with activity (eg, to determine whether a polypeptide has nuclease activity that cleaves a target nucleic acid) can be any convenient nucleic acid cleavage assay that assesses nucleic acid cleavage. Typical cleavage assays involve adding a guide nucleic acid and a Cas9 polypeptide to a target nucleic acid.

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

In other embodiments, a Cas9 polypeptide suitable for inclusion in a composition of the present invention has enzymatic activity that modifies a polypeptide (e.g., histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOlylation activity, de-SUMOlylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity).

Numerous Cas9 orthologs have been identified, originating from a wide variety of species, and in some embodiments, the proteins have only a few identical amino acids. All identified Cas9 orthologues share the same domain architecture with a central HNH endonuclease domain and a RuvC/RNaseH cleavage domain. Cas9 proteins share four core motifs with conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, whereas motif 3 is an HNH motif.

In some embodiments, a suitable Cas9 polypeptide comprises an amino acid sequence having 4 motifs, each of motifs 1-4 having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or greater than, 95% or greater, 99% or greater, or 100% amino acid sequence identity with the Cas9 amino acid sequence depicted in FIG. 1 (SEQ ID NO: 1); or, alternatively, with motifs 1-4 of the Cas9 amino acid sequence presented in table. 1 below (motifs 1-4 of SEQ ID NO: 1 are SEQ ID NO: 3-6, respectively, as shown in Table 1 below); or, alternatively, with amino acids 7-166 or 731-1003 of the Cas9 amino acid sequence depicted in FIG. 1 (SEQ ID NO: 1).

In some embodiments, a Cas9 polypeptide comprises an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 98% amino acid sequence identity with the amino acid sequence depicted in FIG. 1 and given in SEQ ID NO: 1; and includes amino acid substitutions N497, R661, Q695 and Q926 relative to the amino acid sequence shown in SEQ ID NO: 1; or contains an amino acid substitution K855 relative to the amino acid sequence shown in SEQ ID NO: 1; or contains amino acid substitutions K810, K1003 and R1060 relative to the amino acid sequence shown in SEQ ID NO: 1; or contains amino acid substitutions K848, K1003 and R1060 relative to the amino acid sequence shown in SEQ ID NO: 1.

As used herein, the term Cas9 polypeptide includes the term Cas9 polypeptide variant; and the term Cas9 polypeptide variant includes the term chimeric Cas9 polypeptide.

Cas9 polypeptide variants.

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

In some embodiments, the variant Cas9 polypeptide has reduced nuclease activity. For example, a variant of a Cas9 polypeptide suitable for use in a binding method

- 44 046334 of the present invention has less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity exhibited by a wild-type Cas9 polypeptide, e.g. Wild-type Cas9 containing the amino acid sequence shown in FIG. 1 (SEQ ID NO: 1).

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

In some embodiments, a variant Cas9 polypeptide can cleave the non-complementary strand of a double-stranded target nucleic acid, but has a reduced ability to cleave the complementary strand of the target nucleic acid. For example, a variant Cas9 polypeptide may have a mutation (amino acid substitution) that reduces the function of the HNH domain (RuvC/HNH/RuvC domain motifs, domain 2 in Fig. 1). As a non-limiting example, in some embodiments, a variant Cas9 polypeptide may have an H840A mutation (e.g., a replacement of histidine by alanine at the amino acid position corresponding to position 840 of SEQ ID NO: 1) (Fig. 1) and, therefore, may cleave a non-complementary strand of the target nucleic acid , but has a reduced ability to cleave the complementary strand of the target nucleic acid (resulting in an SSB instead of a DSB when the Cas9 polypeptide variant cleaves the double-stranded target nucleic acid). Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid (eg, a single-stranded target nucleic acid) but retains the ability to bind a target nucleic acid (eg, a single- or double-stranded target nucleic acid).

In some embodiments, the variant 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, a variant Cas9 polypeptide contains both D10A and H840A mutations (e.g., a mutation in both the RuvC domain and the HNH domain), resulting in the polypeptide having a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave a target nucleic acid (eg, a single-stranded target nucleic acid or a double-stranded target nucleic acid) but retains the ability to bind a target nucleic acid (eg, a single-stranded target nucleic acid or a double-stranded target nucleic acid).

As another non-limiting example, in some embodiments, a variant Cas9 polypeptide contains the W476A and W1126A mutations, whereby the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave the target nucleic acid, but retains the ability to bind the target nucleic acid.

As another non-limiting example, in some embodiments, a variant Cas9 polypeptide contains mutations P475A, W476A, N477A, D1125A, W1126A, and D1127A, whereby the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave the target nucleic acid, but retains the ability to bind the target nucleic acid.

As another non-limiting example, in some embodiments, a variant Cas9 polypeptide contains mutations H840A, W476A, and W1126A, whereby the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave the target nucleic acid, but retains the ability to bind the target nucleic acid.

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

As another non-limiting example, in some embodiments, the Cas9 polypeptide variant contains mutations H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A,

- 45 046334 as a result of which the polypeptide has a reduced ability to cleave the target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave the target nucleic acid, but retains the ability to bind the target nucleic acid.

As another non-limiting example, in some embodiments, a variant Cas9 polypeptide contains mutations D10A, H840A, P475A, W476A, N477A, D1125A, W1126A, and D1127A, whereby the polypeptide has a reduced ability to cleave a target nucleic acid. Such a Cas9 polypeptide has a reduced ability to cleave the target nucleic acid, but retains the ability to bind the target nucleic acid.

To achieve the above effects (i.e., inactivation of one or another part of the nuclease), other residues can be modified. By way of non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 may be altered (i.e., replaced) (see Table 1 for more information regarding the conservation of amino acid residues of Cas9). Mutations other than alanine substitution are also suitable.

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

In table Figure 1 lists four motifs that are present in Cas9 sequences of various species. The amino acids listed in this table are derived from S. pyogenes Cas9 (SEO ID NO: 1).

Table 1

Mochi in Motive Amino acids (no. of residue(s)) Strongly conservative 1 RuvC IGLDIGTNSVGWAVI (7-21) (SEQ ID NO:3) D10, G12, G17 2 RuvC IVIEMARE (759-766) (SEQ ID NO:4) E762 j HNH-motif DVDHIVPQSFLKDDSIDNKVLTRS DKN (837-863) (SEQ ID NO:5) H840, N854, N863 4 RuvC HHAHDAYL (982-989) (SEQ ID NO:6) H982, H983, A984, D986, A987

In addition to the above, a variant Cas9 protein may have the same sequence identity parameters as described above for Cas9 polypeptides. Thus, in some embodiments, a suitable Cas9 polypeptide variant comprises an amino acid sequence having 4 motifs, with each of motifs 1-4 having 60% or more, 70% or more, 75% or more, 80% or more, 85% or greater than, 90% or greater, 95% or greater, 99% or greater, or 100% amino acid sequence identity with the Cas9 amino acid sequence depicted in FIG. 1 (SEQ ID NO: 1), or alternatively with motifs 1-4 (motifs 1-4 of SEQ ID NO: 1 are SEQ ID NO: 3-6, respectively, as shown in Table 1); or, alternatively, with amino acids 7166 or 731-1003 of the Cas9 amino acid sequence depicted in FIG. 1 (SEQ ID NO: 1). Any Cas9 protein defined above can be used as a Cas9 polypeptide or as part of a chimeric Cas9 polypeptide in the composition of the present invention, including polypeptides that are particularly disclosed in International Patent Application Nos. PCT/US2016/052690 and PCT/US2017/ 062617.

In some embodiments, a suitable Cas9 polypeptide variant comprises an amino acid sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more or 100% amino acid sequence identity with the Cas9 amino acid sequence shown in FIG. 1 (SEQ ID NO: 1). Any Cas9 protein defined above may be used as a variant Cas9 polypeptide or as part of a chimeric variant Cas9 polypeptide in a composition of the present disclosure, including, but not limited to, the polypeptides set forth in International Patent Application Nos. PCT/US2016/052690 and PCT /US2017/062617.

Chimeric polypeptides (fusion polypeptides).

In some embodiments, the variant Cas9 polypeptide is a chimeric Cas9 polypeptide (also referred to herein as a fusion polypeptide, e.g., a Cas9 fusion polypeptide). A Cas9 fusion polypeptide can bind and/or modify a target nucleic acid (e.g., cleave, methylate, demethylate, etc.) and/or polypeptide as

- 46 046334 associated with the target nucleic acid (eg methylation, acetylation, etc., eg histone tail).

A Cas9 fusion polypeptide is a variant of a Cas9 polypeptide in that the sequence is different from a wild-type Cas9 polypeptide (eg, a naturally occurring Cas9 polypeptide). A Cas9 fusion polypeptide is a Cas9 polypeptide (e.g., a wild-type Cas9 polypeptide, a variant Cas9 polypeptide, a variant Cas9 polypeptide with reduced nuclease activity (as described above), etc.) fused to a covalently linked heterologous polypeptide (also called a fusion partner ). In some embodiments, the Cas9 fusion polypeptide is a variant of a Cas9 polypeptide with reduced nuclease activity (eg, dCas9) fused to a covalently linked heterologous polypeptide. In some embodiments, the heterologous polypeptide exhibits (and therefore provides) activities (e.g., enzymatic activity) that can also be observed in the Cas9 fusion polypeptide (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) . In some such embodiments, the binding method, for example, where the Cas9 polypeptide is a variant Cas9 polypeptide having a fusion partner (i.e., having a heterologous polypeptide) with activity (e.g., enzymatic activity) that modifies the target nucleic acid, this method also can be considered as a method for modifying a target nucleic acid. In some embodiments, the method of binding a target nucleic acid (eg, a single-stranded target nucleic acid) may result in modification of the target nucleic acid. Thus, in some embodiments, a method of binding a target nucleic acid (eg, a single-stranded target nucleic acid) may be a method of modifying the target nucleic acid.

In some embodiments, the heterologous sequence may have a subcellular localization, i.e. the heterologous sequence is a subcellular localization sequence (eg, a nuclear localization signal (NLS) to target the nucleus, a sequence that keeps the fusion protein outside the nucleus, eg, a nuclear export sequence (NES), a sequence to keep the fusion protein in the cytoplasm, a mitochondrial localization signal for mitochondria targeting, chloroplast localization signal for chloroplast targeting, endoplasmic reticulum (ER) retention signal, etc.). In some embodiments, the Cas9 variant does not include an NLS, hence the protein is not targeted to the nucleus (which may provide an advantage, for example, when the target nucleic acid is RNA present in the cytosol). In some embodiments, the heterologous sequence may provide a tag (i.e., the heterologous sequence is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; histidine tag, for example 6XHis tag; hemagglutinin (HA) tag; FLAG tag; Myc tag, etc.). In some embodiments, the heterologous sequence may provide increased or decreased stability (i.e., the heterologous sequence is a stability control peptide, such as a degron, which in some embodiments is controlled (e.g., a thermosensitive degron sequence or a drug controlled degron sequence, see below) In some embodiments, the heterologous sequence may provide increased or decreased transcription from a target nucleic acid (i.e., the heterologous sequence is a transcription modulation sequence, e.g., a transcription factor/activator or fragment thereof, a protein or fragment thereof that recruits transcription factor/activator, transcription repressor or fragment thereof, protein or fragment thereof that recruits the transcriptional repressor, small molecule/drug-responsive transcriptional regulator, etc.). In some embodiments, the heterologous sequence may provide a binding domain (i.e., the heterologous sequence is a binding protein sequence, e.g., to provide the ability of a Cas9 fusion polypeptide to bind another protein of interest, e.g., a DNA or histone modifying protein, transcription factor, or repressor transcription, recruitment protein, RNA modification enzyme, RNA-binding protein, translation initiation factor, RNA splicing factor, etc.). A heterologous nucleic acid sequence can be linked to another nucleic acid sequence (eg, by genetic engineering) to create a chimeric nucleotide sequence encoding a chimeric polypeptide.

A subject Cas9 fusion polypeptide (Cas9 fusion protein) may have multiple (1 or more, 2 or more, 3 or more, etc.) fusion partners in any combination of the above. As an illustrative example, a Cas9 fusion protein may have a heterologous sequence that mediates activity (eg, transcriptional modulation, target modification, modification of a protein associated with a target nucleic acid, etc.) and may also have a subcellular localization sequence. In some embodiments, such a Cas9 fusion protein may also be labeled to facilitate tracking and/or purification (e.g., green fluorescent

- 47 046334 protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; a histidine tag, for example a 6XHis tag; hemagglutinin (HA) tag; FLAG tag; tag Mus, etc.). As another illustrative example, a Cas9 protein may have one or more NLSs (eg, two or more, three or more, four or more, five or more, 1, 2, 3, 4 or 5 NLSs). In some embodiments, a fusion partner (or multiple fusion partners) (eg, an NLS, a tag, an activity-providing fusion partner, etc.) is located at or adjacent to the C terminus of Cas9. In some embodiments, a fusion partner (or multiple fusion partners) (eg, an NLS, a tag, an activity-providing fusion partner, etc.) is located at the N terminus of Cas9. In some embodiments, Cas9 has a fusion partner (or multiple fusion partners) (eg, an NLS, a tag, an activity-providing fusion partner, etc.) at both the N-terminus and the C-terminus.

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

Examples of suitable degrons include, but are not limited to, degrons that are controlled by Shield-1, DHFR, auxins and/or temperature. Non-limiting examples of suitable degrons are known in the art (see, for example, Dohmen et al., Science, 1994. 263(5151):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 FKBPderived destabilizing domains; Kanemaki, Pflugers Arch. 2012 Dec 28: Frontiers of protein expression control with conditional degrons; Yang et al., Mol. Cell. 2012 Nov 30 ;48(4):487-8: Titivated for destruction: the methyl degron;Barbour et al., Biosci Rep. 2013 Jan 18;33(1): Characterization of the bipartite degron that regulates ubiquitin-independent degradation of thymidylate synthase; and Greussing et al., J. Vis. Exp. 2012 Nov 10;(69): Monitoring of ubiquitin-proteasome activity in living cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein; all are incorporated herein by reference).

Typical degron sequences are well characterized and tested in both cells and animals. Thus, a fusion protein of Cas9 (eg, wild-type Cas9; variant Cas9; variant of Cas9 with reduced nuclease activity, eg dCas9, etc.) with a degron sequence produces a tunable and inducible Cas9 polypeptide. Any of the fusion partners disclosed herein may be used in any desired combination. As one non-limiting illustrative example, a Cas9 fusion protein (ie, a chimeric Cas9 polypeptide) may comprise a YFP sequence to facilitate detection, a degron sequence to provide stability, and a transcription activator sequence to enhance transcription of the target nucleic acid. Suitable reporter protein for use as a fusion partner for a Cas9 polypeptide (e.g., wild-type Cas9, variant Cas9, reduced nuclease function variant of Cas9, etc.) includes, but is not limited to, the following exemplary proteins (or functional fragments thereof): his3, β-galactosidase, fluorescent protein (eg, GFP, RFP, YFP, cherry, tomato, etc. and various derivatives thereof), luciferase, β-glucuronidase and alkaline phosphatase. In addition, the number of fusion partners that can be used in a Cas9 fusion protein is unlimited. In some embodiments, the Cas9 fusion protein contains one or more (eg, two or more, three or more, four or more, or five or more) heterologous sequences.

Suitable fusion partners include, but are not limited to, a polypeptide that provides methyltransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, ubiquitin ligase, deubiquitination, adenylation, deadenylation, SUMO-lylation, de-SUMO activity

- 48 046334 lylation, ribosylation, deribosylation, myristoylation or demyristoylation, any of which may be aimed at directly modifying the nucleic acid (methylation of DNA or RNA) or modifying the polypeptide associated with the nucleic acid (for example, histone, DNA binding protein and RNA -binding protein, etc.). Additional suitable fusion partners include, but are not limited to, boundary elements (eg, CTCF), proteins and fragments thereof that mediate peripheral recruitment (eg, lamin A, lamin B, etc.), and protein docking elements (eg, FKBP /FRB, Pil1/Aby1, etc.).

Examples of various additional suitable fusion partners (or fragments thereof) for a subject Cas9 polypeptide variant include, without limitation, those described in PCT patent applications: WO 2010/075303, WO 2012/068627 and WO 2013/155555, which are incorporated herein description by reference in its entirety.

Suitable fusion partners include, but are not limited to, a polypeptide that provides transcription-enhancing activity indirectly by acting directly on a target nucleic acid or polypeptide (e.g., histone, DNA binding protein, RNA binding protein, RNA editing protein, etc. ) associated with the target nucleic acid. Suitable fusion partners include, but are not limited to, a polypeptide that provides methyltransferase, demethylase, acetyltransferase, deacetylase, kinase, phosphatase, ubiquitin ligase, deubiquitination, adenylation, deadenylation, SUMOlylation, de-SUMOlylation, ribosylation, deribosylation, myristoylation, or demyristoylation activity.

Additional suitable fusion partners include, but are not limited to, a polypeptide that directly mediates increased transcription and/or translation of a target nucleic acid (e.g., a transcriptional activator or fragment thereof, a protein or fragment thereof that recruits a transcriptional activator, a small molecule/transcriptional regulator, and/or or translation, drug-sensing protein, translation regulatory protein, etc.).

Non-limiting examples of fusion partners to achieve increased or decreased transcription include transcriptional activator and transcriptional repressor domains (e.g., Kruppell associated box (KRAB or SKD); Mad mSIN3 interaction domain (SID); ERF repressor domain (ERD), etc. ). In some such embodiments, targeting the Cas9 fusion protein to a specific location (i.e., sequence) in a target nucleic acid is accomplished by a guide nucleic acid, wherein it exerts locus-specific regulation, such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function) and/or modifies local chromatin status (eg, when a fusion sequence is used that modifies the target nucleic acid or modifies a polypeptide associated with the target nucleic acid). In some embodiments, the changes are transient (eg, transcriptional repression or activation). In some embodiments, the changes are heritable (eg, when epigenetic modifications occur in the target nucleic acid or in proteins associated with the target nucleic acid, such as nucleosomal histones).

Non-limiting examples of fusion partners for use in targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (eg, RS domains); protein translation components (eg, translation initiation, elongation and/or release factors; eg, eIF4G); RNA methylases; RNA editing enzymes (eg, RNA deaminases, such as adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicase-dependent embodiments; RNA-binding proteins, etc. It is understood that the fusion partner may include the entire protein or, in some embodiments, may include a portion of the protein (eg, a functional domain).

In some embodiments, the heterologous sequence may be fused to the C-terminus of a Cas9 polypeptide. In some embodiments, the heterologous sequence may be fused to the N-terminus of a Cas9 polypeptide. In some embodiments, the heterologous sequence may be fused to an internal portion (ie, a portion other than the N- or C-terminus) of a Cas9 polypeptide.

Additionally, the fusion partner of a chimeric Cas9 polypeptide may be any domain capable of interacting with ssRNA (which, for the purposes of the present invention, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc. ), temporarily or irreversibly, directly or indirectly, including, without limitation, an effector domain selected from the group consisting of endonucleases (e.g., RNase I, CRR22 DYW domain, Dicer, and PEST (N-terminal PUT) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (eg, CPSF, CstF, CFIm and CFIIm); exonucleases (eg, XRN-1 or exonuclease T); deadenylase (eg HNT3); proteins and protein domains responsible for nonsense-mediated RNA decay (eg, UPF1, UPF2, UPF3, UPF3b, RNP S1, Y14, DEK, REF2, and SRm160); proteins and protein domains responsible for stabilizing RNA (for example, PABP); proteins and protein domains responsible for

- 49 046334 translational repression (for example, Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (eg Staufen); proteins and protein domains responsible for (eg, capable of) modulating translation (eg, translation factors such as initiation factors, elongation factors, release factors, etc., eg eIF4G); proteins and protein domains responsible for RNA polyadenylation (for example, PAP1, GLD-2 and Star-PAP); proteins and protein domains responsible for RNA polyuridinylation (for example, CID1 and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (for example, from IMP1, ZBP1, She2p, She3p and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example, Rrp6); proteins and protein domains responsible for RNA export into the nucleus (for example, TAP, NXF1, THO, TREX, REF and Aly); proteins and protein domains responsible for the repression of RNA splicing (for example, PTB, Sam68 and hnRNP A1); proteins and protein domains responsible for stimulating RNA splicing (eg, serine/arginine-rich (SR) domains); proteins and protein domains responsible for reducing transcription efficiency (for example, FUS (TLS)); and proteins and protein domains responsible for transcriptional stimulation (eg, CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group consisting of endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylase; proteins and protein domains having nonsense-mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (eg, translation factors such as initiation factors, elongation factors, release factors, etc., such as eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains with RNA localization activity; proteins and protein domains capable of retaining RNA in the nucleus; proteins and protein domains with RNA export activity into the nucleus; proteins and protein domains capable of inhibiting RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains that can reduce transcription efficiency; and proteins and protein domains capable of stimulating transcription. Another suitable fusion partner is the RNA binding domain PUF, which is described in more detail in WO 2012/068627.

Some RNA splicing factors that can be used (whole or fragmented) as fusion partners for a Cas9 polypeptide have a modular organization of individual modules that bind specific RNA sequences and effector domains responsible for splicing. For example, members of the serine/arginine-rich (SR) family of proteins contain N-terminal RNA recognition motifs (RRMs) that bind to exon splicing enhancers (ESEs) in the pre-mRNA and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP A1 protein binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through its C-terminal glycine-rich domain. Some splicing factors can regulate alternative splice site (ss) usage by binding to regulatory sequences between two alternative sites. For example, ASF/SF2 can recognize the ESE and promote the use of proximal intronic regions, whereas hnRNP A1 can bind to the ESS and shift splicing toward the use of distal intronic regions. One application of such factors is the creation of ESFs that modulate alternative splicing of endogenous genes, especially disease-associated genes. For example, Bcl-x pre-mRNA produces two splice isoforms with two alternative 5' splice sites to encode proteins with opposing functions. The long splice isoform Bcl-xL is a potent inhibitor of apoptosis expressed in long-lived postmitotic cells and is upregulated in many cancer cells, protecting cells from apoptotic signals. The short isoform Bcl-xS is a proapoptotic isoform and is highly expressed in cells with a high turnover rate (eg, developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cώ elements, which are located either in the core region of the exon or in the exon extension region (i.e., between two alternative 5' splice sites). For further examples see WO 2010/075303.

In some embodiments, a Cas9 polypeptide (eg, wild-type Cas9, variant Cas9, reduced nuclease activity variant of Cas9, etc.) may be linked to a fusion partner through a peptide spacer.

In some embodiments, a Cas9 polypeptide comprises a protein transduction domain or PTD (also known as a cell penetrating peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates passage of a lipid bilayer, micelle, cell membrane, membrane organelles or vesicle membranes. Attached to another molecule, a PTD, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule's passage across a membrane, such as from extracellular to intracellular or from cytosol to organelle. In some embodiments, a PTD attached to another molecule facilitates

- 50 046334 penetration of the molecule into the nucleus (eg, in some embodiments, the PTD includes a nuclear localization signal (NLS)). In some embodiments, the Cas9 polypeptide contains two or more NLSs, such as 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 carboxyl terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently linked to the amino terminus and the carboxyl terminus of the Cas9 polypeptide. In some embodiments, the PTD is covalently linked to a nucleic acid (eg, a guide nucleic acid, a polynucleotide encoding a guide nucleic acid, a polynucleotide encoding a Cas9 polypeptide, etc.). Exemplary PTDs include, but are not limited to, a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of the HIV-1 TAT containing YGRKKRRQRRR; SEQ ID NO: 7); a polyarginine sequence containing a number of arginines sufficient to directly enter the cell (for example, 3, 4, 5, 6, 7, 8, 9, 10 or 10-50 arginines); VP22 domain (Zender et al. (2002), Cancer Gene Ther. 9(6):489-96); Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003), Diabetes, 52(7):1732-1737); truncated human calcitonin peptide (Trehin et al. (2004), Pharm. Research, 21:1248-1256); polylysine (Wender et al. (2000), Proc. Natl. Acad. Sci. USA, 97:13003-13008); RRQRRTSKLMKR(SEQ ID NO: 8); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 9); KALAWEAKLAKALAKHLACALAKALKCEA (SEQ ID NO: 10); and RQIKIWFQNRRMKWKK (SEQ ID NO: 11). Examples of PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 12), RKKRRQRRR (SEQ ID NO: 13); an arginine homopolymer containing from 3 to 50 arginine residues; exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 14); RKKRRQRR (SEQ ID NO: 15); YARAAARQARA (SEQ ID NO: 16); THRLPRRRRRR (SEQ ID NO: 17) and GGRRARRRRRR (SEQ ID NO: 18).

In some embodiments, the PTD is an activated 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) linked via a cleavable linker to a suitable polyanion (e.g., Glu9 or E9), which reduces the net charge to almost zero and thereby inhibits adhesion and cellular uptake. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thereby activating ACPP to cross the membrane.

In some embodiments, the composition may comprise the RNA-guided endonuclease Cpf1, an example of which is shown in FIG. 2, 16 or 17. Another name for the RNA-guided endonuclease Cpf1 is Cas12a. The Cpf1 CRISPR systems of the present disclosure comprise i) a single protein with endonuclease activity and ii) a crRNA in which a portion of the 3' end contains a guide sequence complementary to the target nucleic acid. In this system, the Cpf1 nuclease is recruited directly to the target DNA via crRNA. In some embodiments, guide sequences for Cpf1 must be at least 12, 13, 14, 15, or 16 nt for detectable DNA cleavage and at least 14, 15, 16, 17, or 18 nt for efficient DNA cleavage.

The Cpf1 systems of the present invention differ from Cas9 in many aspects. First, unlike Cas9, Cpf1 does not require a separate tracrRNA for cleavage. In some embodiments, the Cpf1 crRNA may be approximately 42-44 bases in length, of which 23-25 nucleotides are guide sequence and 19 nucleotides are constitutive direct repeat sequence. In contrast, combined synthetic tracrRNA and Cas9 crRNA sequences can be approximately 100 bases in length.

Second, the preferred motif of Cpf1 is TTN PAM, located 5′ upstream of its target. This is in contrast to the NGG PAM motifs located at the 3' end of the target DNA of the Cas9 system. In some embodiments, the uracil base immediately preceding the guide sequence is replaceable (Zetsche, B. et al. 2015. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, 759-771, entire incorporated herein by reference for all purposes).

Third, the cutting sites for Cpf1 are offset by approximately 3-5 bases, which creates sticky ends (Kim et al., 2016. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells, published online June, 2016). These sticky ends are thought to have 3-5 bp overhangs. promote NHEJ-mediated ligation and improve gene editing of DNA fragments with matching ends. The cutting sites are at the 3' end of the target DNA, distal to the 5' end, where PAM is located. The cut positions are typically located below the 18th base on the unhybridized strand and the corresponding 23rd base on the complementary strand hybridized to the crRNA.

Fourth, in Cpf1 complexes the seed region is located within the first 5 nucleotides of the guide sequence. The Cpf1 crRNA seed regions are very sensitive to mutations, and even single base substitutions in this region can dramatically reduce cleavage activity (see Zetsche B. et al. 2015, Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System) Cell, 163, 759-771). Of particular note is the fact that, unlike the CRISPR target Cas9, the cleavage sites and seed region of the Cpf1 systems do not overlap. Additional Guide to Creating Oligos

- 51,046,334 nucleotides targeting the crRNA Cpfl, available at (Zetsche B. et al. 2015. Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Cell, 163, 759-771).

Those skilled in the art will understand that Cpf1 as disclosed herein can be any variant obtained or isolated from any source, many of which are known in the art. For example, in some embodiments, the Cpf1 peptide of the present invention may include FnCPF1 (eg, SEQ ID NO: 2) shown in FIG. 2, AsCpf1 (eg, FIG. 16), LbCpf1 (eg, FIG. 17), or any other of the many known Cpf1 proteins from various other microbial species and synthetic variants thereof.

In some embodiments, the composition comprises a Cpf1 polypeptide. In some embodiments, the Cpf1 polypeptide is enzymatically active, eg, the Cpf1 polypeptide, when bound to a guide RNA, cleaves a target nucleic acid. In some embodiments, the Cpf1 polypeptide exhibits reduced enzymatic activity compared to a wild-type Cpf1 polypeptide (eg, compared to a Cpf1 polypeptide comprising the amino acid sequence depicted in FIGS. 2, 16, or 17) and retains DNA binding activity.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the amino acid sequence shown in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with a contiguous region containing from 100 to 200 amino acids (aa), from 200 to 400 aa, from 400 to 600 aa, from 600 to 800 aa, from 800 to 1000 aa, from 1000 to 1100 aa, 1100 to 1200 aa or 1200 to 1300 aa, the amino acid sequence shown in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the RuvCI domain of the Cpf1 polypeptide with the amino acid sequence depicted in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the RuvCII domain of the Cpf1 polypeptide with the amino acid sequence depicted in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the RuvCIII domain of the Cpf1 polypeptide with the amino acid sequence depicted in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide exhibits reduced enzymatic activity compared to a wild-type Cpf1 polypeptide (eg, compared to a Cpf1 polypeptide containing the amino acid sequence depicted in Fig. 2, 16, or 17) and retains DNA binding activity. In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the amino acid sequence shown in FIG. 2, 16 or 17; and includes an amino acid substitution (for example, a D^A substitution) at the amino acid residue corresponding to amino acid 917 amino acid after

- 52 046334 of the sequence shown in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the amino acid sequence shown in FIG. 2, 16 or 17; and includes an amino acid substitution (eg, an E^A substitution) at an amino acid residue corresponding to amino acid 1006 of the amino acid sequence depicted in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide comprises an amino acid sequence comprising at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90% or 100% amino acid sequence identity with the amino acid sequence shown in FIG. 2, 16 or 17; and includes an amino acid substitution (eg, a D^A substitution) at the amino acid residue corresponding to amino acid 1255 of the amino acid sequence depicted in FIG. 2, 16 or 17.

In some embodiments, the Cpf1 polypeptide is a fusion polypeptide, for example, a Cpf1 fusion polypeptide comprising a) a Cpf1 polypeptide; and b) a heterologous merger 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 within a Cpf1 polypeptide.

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

Bound guide RNA and donor nucleic acid.

In one aspect, the invention relates to a complex comprising a CRISPR system comprising an RNA-guided endonuclease (eg, a Cas9 or Cpf1 polypeptide), a guide RNA, and a donor polynucleotide, wherein the guide RNA and the donor polynucleotide are linked. As shown herein, the guide RNA and the donor polynucleotide can be linked either covalently or non-covalently. In one embodiment, the guide RNA and the donor polynucleotide are chemically ligated. In another embodiment, the guide RNA and the donor polynucleotide are enzymatically ligated. In one embodiment, the guide RNA and the donor polynucleotide hybridize to each other. In another embodiment, both the guide RNA and the donor polynucleotide are hybridized through the bridging sequence. Any number of such hybridization schemes is possible.

Deaminase.

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

In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). Adenosine deaminases (eg, engineered adenosine deaminases, evolved adenosine deaminases) presented herein can be derived from any organism, such as a bacterium. 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.

In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%,

- 53 046334 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% or at least 99.5% identical to natural deaminase.

In some embodiments, the adenosine deaminase is derived from a bacterium, such as E. coli, S. aureus, S. typhi, S. putrefaciens, H. influenzae, or C. crescentus. In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, truncated ecTadA may lack one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N- terminal amino acid residues 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 C- terminal amino acid residues relative to full-length ecTadA. In some embodiments, the ecTadA deaminase does not contain an N-terminal methionine. In some embodiments, the deaminase is APOVEC1 or a variant thereof.

The deaminase can be used in conjugation with any other CRISPR element disclosed herein (i.e., as a composition), or the deaminase can be fused to any other CRISPR element (e.g., Cas9 or Cpf1) disclosed herein (i.e. i.e. in the form of a complex). In some embodiments, the deaminase is fused to Cas9, Cpfl, or variants thereof.

Other components.

The composition may further contain any other components commonly used in nucleic acid or protein delivery formulations. For example, the composition may further contain lipids, lipoproteins (eg, cholesterol and derivatives), phospholipids, polymers or other components of liposomal or micellar delivery vehicles. The composition may also contain a solvent or carrier suitable for administration to cells or hosts, such as a mammal or human.

In some embodiments, the composition contains a second polymer that includes polyethylene oxide (PEG). For example, the composition may contain PEG-pAsp (DET), PEG-pAsp, PEG-pAsp derivatives (DET), PEG-pAsp derivatives, or a combination thereof. Without being bound by any particular theory, it is believed that these PEGylated polymers can control the size of the nanoparticles and their interaction with target serum proteins and cells. The polyethylene oxide polymer can be combined with other components in any way and in any order.

In some embodiments, the composition further contains one or more surfactants. The surfactant may be a nonionic surfactant and/or a zwitterionic surfactant. A list of typical surfactants includes, but is not limited to: polyoxyethylene sorbitan ester surfactants (commonly referred to as Tweens), especially polysorbate 20 and polysorbate 80; ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO) copolymers sold under the trade name DOWFAX™, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (hydroxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100 or tert-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-6301NP-40); phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether and sorbitan esters (commonly known as Spans), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. In some embodiments, the surfactant is an anticoagulant (eg, heparin or the like). In some embodiments, the composition further includes one or more pharmaceutically acceptable carriers and/or excipients.

In some cases, a component (eg, a nucleic acid component (eg, a guide nucleic acid, etc.); a protein component (eg, a Cas9 or Cpf1 polypeptide, a variant of a Cas9 or Cpf1 polypeptide); etc.) includes a tag. The terms label, detectable label or fragment label as used herein refer to any fragment that allows the detection of a signal, and can vary widely depending on the specific nature of the analysis. Tag moieties of interest include both directly detectable tags (direct tags) (eg, fluorescent tags) and indirectly detectable tags (indirect tags) (eg, a member of a binding pair). The fluorescent label may be any fluorescent label (e.g., fluorescent dye (e.g., fluorescein, Texas red, rhodamine,

- 54 046334

ALEXAFLUOR®, etc.), fluorescent protein (e.g. green fluorescent protein (GFP), enhanced GFP (EGFP)), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), Cherry , Tomato, Tangerine and any of their fluorescent derivatives), etc.). Suitable groups of detectable (directly or indirectly) labels for use in the methods include any group that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical or other means. For example, suitable indirect labels include biotin (a member of a binding pair), which can bind to streptavidin (which itself can be directly or indirectly labeled). Labels may also include: a radioactive label (direct label) (eg, ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P); enzyme (indirect label) (eg, peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, etc.); fluorescent protein (direct label) (eg, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivatives thereof); metal mark (straight mark); colorimetric mark; member of a binding pair, etc. By a binding pair partner or a member of a binding pair is meant one of the first and second moieties, wherein the first and second moieties have a specific binding affinity for each other. Suitable binding pairs include, but are not limited to: antigen/antibodies (e.g., digoxigenin/anti-digoxigenin, dinitrophenyl TNO)/anti-OHN, dansyl-Hanti-dansyl, fluorescein/anti-fluorescein, Lucifer yellow/anti-Lucifer yellow, and rhodamine antirhodamine), biotin/avidin (or biotin/streptavidin) and calmodulin binding protein (CBP)/calmodulin. Any member of a binding pair may be suitable for use as an indirectly determined label.

Any given component or combination of components may be unlabeled or labeled with a detectable label. In some embodiments, when two or more components are marked, they may be marked with tag fragments that can be distinguished from each other.

Encapsulation and nanoparticles.

In some embodiments of the composition, a polymer is combined with a nucleic acid and/or polypeptide and partially or completely encapsulates the nucleic acid and/or polypeptide. In some formulations, the composition may contain nanoparticles containing a polymer and a nucleic acid and/or polypeptide.

In some embodiments, the composition may contain a core nanoparticle in addition to a polymer of the invention and a nucleic acid or polypeptide. Any suitable nanoparticles can be used, including metal nanoparticles (eg gold) or polymer nanoparticles.

The polymer disclosed herein and the nucleic acid (eg, guide RNA, donor polynucleotide, or both) or polypeptide can be conjugated to the surface of the nanoparticles directly or indirectly. For example, a polymer disclosed herein and a nucleic acid (eg, guide RNA, donor polynucleotide, or both) or polypeptide can be conjugated to the surface of a nanoparticle directly or indirectly through an intermediate linker.

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

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 disclosed herein (e.g., Cas9 polypeptide and guide RNA, donor polynucleotide and Cpf1 polypeptide) to the nanoparticle conjugate -nucleic acid. For example, the linker nucleic acid may have a sequence that hybridizes to a guide RNA or donor polynucleotide.

The nucleic acid conjugated to a nanoparticle (eg, a colloidal metal (eg, gold) nanoparticle; a biocompatible polymer-containing nanoparticle) may be of any suitable length. When the nucleic acid is a guide RNA or a donor polynucleotide, the length is usually suitable for such molecules, for example, as described herein and known in the art. If the nucleic acid is a linker nucleic acid, it may have any length suitable for the linker, for example, from 10 to 1000 nucleotides (nt), for example, from about 1 to about 25 nt, from about 25 to about 50 nt, from about 50 to about 100 nt, from about 100 to about 250 nt, from about 250 to about 500 nt, or from about 500 to about 1000 nt. In some cases

- 55 046334 a nucleic acid conjugated to a nanoparticle (eg, a colloidal metal (eg, gold) nanoparticle; a nanoparticle containing a biocompatible polymer) may have a length of more than 1000 nt.

When a nucleic acid bound (e.g., covalently linked; non-covalently linked) to a nanoparticle contains a nucleotide sequence that hybridizes to at least a portion of the guide RNA or donor polynucleotide present in the complex of the invention, it has a region with sequence identical to the complement region of the guide An RNA or donor polynucleotide sequence sufficient to facilitate hybridization. In some embodiments, the nucleic acid associated with the nanoparticle in the complex of the present invention is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99, or 100% nucleotide sequence identity with a complement of 10 to 50 nucleotides in length (e.g., 10 to 15 nt, 15 to 20 nt, 20 to 25 nt, 25 up to 30 nt, 30 to 40 nt, or 40 to 50 nt) guide RNA or donor polynucleotide present in the complex.

In some embodiments, the nucleic acid bound (eg, covalently linked; non-covalently linked) to the nanoparticle is a donor polynucleotide or has the same or substantially the same nucleotide sequence as the donor polynucleotide. In some embodiments, the nucleic acid bound (eg, covalently linked; non-covalently linked) to the nanoparticle contains a nucleotide sequence that is complementary to the donor DNA template.

Mode of application.

The polymers provided herein can be used for any purpose, but are contemplated to be particularly useful for combining and, in some embodiments, encapsulating biological molecules (eg, nucleic acids and polypeptides) for various purposes. In one aspect, a method is provided for encapsulating a biological molecule, such as a polypeptide or nucleic acid, by combining a polymer of the invention as provided herein with a biological molecule, whereby the polymer partially or completely encapsulates the biomolecule. A biomolecule partially or completely encapsulated by a polymer is sometimes called a nanoparticle.

Also provided herein is a method for delivering a nucleic acid and/or polypeptide into a cell, where the cell may be in vitro or in vivo. The method includes introducing a composition containing a polymer and a nucleic acid and/or polypeptide of the present invention into a cell or subject containing the cell. The method can be used with any type of cell or subject, but is particularly useful for mammalian cells (eg, human cells). In some embodiments, the polymer includes a targeting agent such that the nucleic acid and/or polypeptide is delivered predominantly or exclusively to the target cells or tissues (e.g., cells or tissues of the peripheral nervous system, central nervous system, eye of the subject, liver, muscle, lung, bone (eg hematopoietic cells) or tumor cells or tissues).

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 includes homology directed repair (HDR). In some embodiments, use of the complex of the present invention for performing HDR provides an HDR efficiency of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10% , at least 15%, at least 20%, at least 25% or more than 25%. In some embodiments, the method of modifying a target nucleic acid involves non-homologous end joining (NHEJ). In some embodiments, use of the complex of the present invention to perform HDR provides an NHEJ efficiency of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10% , at least 15%, at least 20%, at least 25 or more than 25%.

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

Example 1.

The present example provides guidance for the synthesis of the polymer of the invention. The synthesis involves the Michael addition reaction of acrylate. The example procedure is as follows.

- 56 046334

Scheme 1

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 suspension was stirred until all polymers were completely dissolved. Acrylate 1 (1.15 mg, 5.5 µmol) was added to the reaction mixture and the reaction mixture was stirred for 40 hours at room temperature. The crude product was purified by precipitation with acetonitrile and washed three times with acetonitrile to obtain 4 mg of polymer 5, where (a+b) is 55 and (c+d) is 25.

^1H NMR (400 MHz, D2O): δ 4.8-4.6 (bs, 4H), 4.2 (t, 2H), 3.4-2.4 (m, 40H).

As shown in Scheme 1, the Michael addition reaction of acrylate 1 results in the formation of an amine-based bond (see, for example, polymer 5). Thus, the original amino functional group in pAsp (DET) remains intact.

A similar procedure can be used to prepare additional polymers of formula 1 (eg, polymers 1-4 and 6-12). Nucleophilic substitution can be used to prepare polymers 28 and 29.

Additionally, this procedure can be applied to another starting polymer, such as the polymer prepared in Example 7, to produce other polymers of formula 1 (e.g., polymers 13-24 can be prepared by this method from polymer 30 or 32 demethylated at the terminal nitrogen to produce primary or secondary amine).

Example 2.

The present example provides guidance for the synthesis of the polymer of the invention. The synthesis involves a Michael addition reaction of acrylate. The example procedure is as follows.

Scheme 2

- 57 046334

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 stirred at room temperature for 10 min until the polymer was completely dissolved. The solution was diluted with 300 μl of DCM. Acrylate 2 (1.05 mg, 5.5 μmol) in 30 μl DCM was added to the reaction mixture and the reaction mixture was stirred at room temperature for 48 h. The crude product was purified by precipitation in large excess of diethyl ether to obtain 4.3 mg of polymer 8 , where (a+b) is equal to 55 and (c+d) is equal to 25.

¹ H NMR (400 MHz, D2O): δ 4.8-4.6 (br.s, 4H), 4.03 (t, 2H), 3.8-2.5 (m, 27H), 1, 5 (t, 2H), 1.25 (s, 12H), 0.7 (t, 3H).

As shown in Scheme 2, the Michael addition reaction of acrylate 2 results in the formation of an amine-based bond (see, for example, polymer 8). Thus, the original amine functional group in pAsp (DET) remains intact.

Example 3.

This example demonstrates the ability of the polymers disclosed herein to deliver Cas9 ribonucleoprotein (Cas9 RNP) into a cell. The level of Cas9 RNP delivery was assessed in green fluorescent protein (GFP)-inducible HEK293T (GFP-HEK) cells.

pmol of Cas9 RNP was mixed with (i) polymer 5, (ii) polymer 8, and (iii) pAsp (DET) used as a control. GFP-HEK cells were treated with the resulting mixture under serum-free conditions. Polymers were added at doses of 0.375, 0.75, 1.25, 2.5, 5 and 10 μg to select the optimal dose that provides the highest efficacy and minimal toxicity. The results are presented in Fig. 3.

In fig. Figure 3 shows the level of Cas9 RNP delivery measured as the percentage of GFP(-) in GFP-HEK cells treated with the three mixtures. All three polymers (i.e., polymer 5, polymer 8, and pAsp (DET)) showed similar levels of gene editing at polymer doses ranging from 0.375 to 2.5 μg. However, polymer 8 and pAsp (DET) demonstrated high levels of cell toxicity (less than 50% cell viability) at polymer doses of 5 and 10 μg. Moreover, polymer 5 provided optimal delivery at a polymer dose of 5 μg, as evidenced by the GFP% level, without significant toxicity.

Example 4.

This example demonstrates the ability of the polymers disclosed herein to deliver Cre recombinase, which can alter loxP sequences, into cells.

Using primary ai9 mouse myoblasts that contain a stop sequence between loxP (gray arrow in Fig. 4), the level of delivered Cre recombinase can be measured by RFP expression. The Ai9 reporter allele contains a STOP cassette flanked by the loxP sequence, which prevents RFP transcription. Cre recombinase removes the STOP cassette flanked by the loxP sequence, thereby allowing transcription of RFP.

Cre recombinase (2 μg) was mixed with (i) polymer 5 and (ii) pAsp (DET) used as a control to prepare nanoparticles. Polymers were added at doses of 1.25, 2.5, 5 10 and 20 μg to determine the optimal dose that provides the highest efficacy and minimal toxicity. Ai9 myoblasts were treated with the resulting nanoparticles, and RFP+ was quantified by flow cytometry 4 days after treatment. The results are presented in Fig. 5.

In fig. Figure 5 shows the level of delivery of Cre recombinase to primary myoblasts as measured by the level of RFP+ expression. As shown in FIG. 5, polymer 5 provided higher levels of RFP+ expression than the control (pAsp (DET)) at polymer doses of 5, 10, and 20 μg, indicating that at higher doses, polymer 5 provides more efficient delivery of Cre recombinase compared to pAsp(DET).

Example 5.

This example demonstrates the ability of the polymers disclosed herein to deliver Cre recombinase in vivo to mice.

Ai9 mice were administered (i) Cre recombinase alone and (ii) PEGylated polymer nanoparticles (i.e., a mixture of Polymer 5 and PEG polymer) encapsulating Cre recombinase. Cre recombinase (35 μg) was delivered with a mixture of polymer 5 and PEG-PAsp (DET) (100 μg). Blending polymer 5 with PEG-PAsp (DET) allows for control of the size of polymer nanoparticles. Two weeks after injection, gastrocnemius muscles of ai9 mice were collected. The resulting muscle cross sections were imaged to detect red fluorescent protein, which was generated by DNA recombination. The results are presented in Fig. 6.

In fig. Figure 6 shows that polymer nanoparticles improve the delivery of Cre recombinase into mouse muscle. Injection of Cre recombinase resulted in RFP expression only in limited areas of muscle, as evidenced by fewer areas of red fluorescence protein visualized. At the same time, injection of Cre recombinase encapsulated in a polymer nanoparticle resulted in RFP expression in most areas of the gastrocnemius muscle.

The challenge of protein delivery is how widely the protein can be delivered and affect more tissue sites. Nanoparticles obtained from a mixture of polymer 5 and pegpolymer,

- 58 046334 can effectively deliver Cre recombinase and promote its distribution in the body of ai9 mice.

Example 6.

This example demonstrates the ability of the polymers disclosed herein to deliver Cas9 to neuronal cells expressing GFP.

To determine whether polymer 5 could deliver Cas9 RNP to neuronal cells, GFP-expressing neuronal cells that were differentiated from neuronal progenitor cells were used. Neuronal cells expressing GFP were treated with OGRG and Cas9 protein using the polymers disclosed herein as delivery vehicles. Seven days after treatment, genomic DNA was extracted from the cells and PCR amplification of the GFP gene was performed. To measure the frequency of indel mutations resulting from Cas9 gene editing, TIDE analysis was performed (TIDE software from Desktop Genetics, Netherlands Cancer Institute). The results are presented in Fig. 7.

As shown in FIG. 7, polymer 5 was able to deliver Cas9 RNP and induce 11% indel mutations in neuronal cells.

Example 7.

This example provides guidance for the synthesis of the polymer disclosed herein. The synthesis involves ring opening polymerization followed by modification to obtain the polymer of formula 4. The exemplary procedure is as follows.

Scheme 3

Synthesis of polymer 33: (a) tosyl chloride, TEA, DCM; (b) N, ^Ntrimethylethylenediamine, K2CO ₃ , acetonitrile, reflux; (c) LiAlH4, THF; (d) compound 7, K2CO3, acetonitrile, reflux; (e) LiAlH4, THF; (f) butylamine, DCM-DMF (9:1), 48 h; (g) compound 9, NMP, 6 hours.

As shown in Scheme 3, ring opening polymerization of compound 10 results in compound 11 with n=63, which can be further modified to form polymer 33 after treatment with compound 9.

- 59 046334

A similar procedure can be used to prepare other polymers of Formula 4 (eg, polymers 30-32). Additionally, the terminal tertiary amine on the polymer side chains can be demethylated by conventional methods to yield polymers 39-42.

Example 8.

This example demonstrates the ability of the polymers disclosed herein to deliver Cre recombinase into the cell, which can alter loxP sequences.

Using cells containing the Traffic Light Reporter ((TLR)-HEK 293T), which were created by viral transduction of Traffic Light Reporter into HEK 293T (see Fig. 8), the level of expression of the delivered Cre recombinase can be measured using red fluorescent protein (RFP). The Traffic Light Reporter in HEK 293T contains a STOP cassette and two loxP sequences flanking the GFP sequence, thereby preventing RFP transcription and in turn expressing GFP in the absence of Cre. Cre recombinase removes the loxP sequences and allows RFP transcription.

Cre recombinase (1 μg) was mixed with polymer 33 obtained in Example 7 or pAsp (DET) used as a reference sample to obtain nanoparticles. Polymers were added in doses of 1.25 μg or 2.5 μg to select the optimal dose that provides the highest efficacy and minimal toxicity. TLR HEK293T cells were treated with the resulting nanoparticles, and RFP+ quantification was performed by flow cytometry 3 days after treatment. The results are presented in Fig. 9.

In fig. 9 shows the level of Cre recombinase delivery into (TLR)-HEK 293T cells as measured by the level of RFP+ expression. As shown in FIG. 9, polymer 33 provides improved delivery compared to Cre-only samples and reference pAsp (DET) at 1.25 and 2.5 μg polymer doses.

Example 9.

This example demonstrates the ability of the polymers disclosed herein to deliver Cas9 ribonucleoprotein (Cas9 RNP) into cells. The level of Cas9 RNP delivery was assessed in green fluorescent protein (GFP)-inducible HEK293T (GFP-HEK) cells.

pmol of Cas9 RNP (sgRNA+Cas9 protein) was mixed with polymer 33 obtained in Example 7 or pAsp (DET) used as a reference sample to obtain nanoparticles. Polymers were added at a dose of 2.5 μg. GFP-HEK cells were treated with the resulting mixture under serum-free conditions. The results are presented in Fig. 10.

In fig. 10 shows the level of Cas9 RNP delivery measured by GFP% in GFP-HEK cells treated with the two mixtures. Both polymers (i.e., polymer 33 and pAsp (DET)) showed the ability to deliver Cas9 RNP at a polymer dose of 2.5 μg compared to the control. However, at a dose of 2.5 μg, polymer 33 was more effective than pAsp (DET).

Example 10.

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

eGFP mRNA (200 ng) was mixed with polymer 33 (600 ng) prepared in Example 7 and incubated for 5 min. The resulting polymer nanoparticles were introduced into HEK 293T cells in OptiMEM medium. After 24 h, cells were separated from the plate and analyzed by flow cytometry. Lipofectamine was used as a positive control for eGFP mRNA delivery. The results are presented in Fig. eleven.

In fig. Figure 11 shows that polymer 33 provides improved delivery of eGFP mRNA to HEK 293T cells compared to the control.

Example 11.

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

eGFP mRNA (200 ng) was mixed with polymer 33 (1.2 μg) prepared as described in Example 7 and incubated for 2, 5, 10 and 30 min. The resulting polymer nanoparticles were introduced into HEK 293T cells in OptiMEM medium. After 24 h, cells were separated from the plate and analyzed by flow cytometry. Lipofectamine was used as a positive control for eGFP mRNA delivery. The results are presented in Fig. 12.

In fig. Figure 12 shows that nanoparticles formed within 2 min of incubation and polymer 33 provided effective delivery of eGFP mRNA at all incubation time intervals.

Example 12.

This example demonstrates the ability of the polymers disclosed herein to deliver mRNA. The level of red fluorescent protein (RFP) mRNA delivery into HEK 293T cells was assessed by the level of RFP+ expression.

RFP mRNA (200 ng) was mixed with polymer 33, prepared as described in example 7, or pAsp

- 60 046334 (DET), used as a reference sample to obtain nanoparticles. Polymer 33 and pAsp (DET) were added at doses of (i) 600 ng or (ii) 480 ng in combination with 120 ng of 1.5 kDa PEG-PAsp (DET) polymer. The 1.5 kDa PEG-PAsp (DET) polymer allows for controlled nanoparticle size. TLR HEK293T cells were treated with the resulting nanoparticles, and RFP+ was quantified by flow cytometry 24 hours after treatment. The results are presented in Fig. 13.

In fig. 13 shows that compared to the reference polymer (Pasp (DET)), polymer 33 improved delivery of mRNA to HEK 293T cells in the absence of the 1.5 kDa PEG-PAsp (DET) polymer. In addition, in FIG. 13 shows that 600 ng of polymer 33 provides more efficient delivery of mRNA to HEK 293T cells than 480 ng of polymer 33 in combination with 120 ng of 1.5 kDa PEG-PAsp (DET) polymer.

Example 13.

This example demonstrates the ability of the polymers disclosed herein to deliver mRNA. The level of red fluorescent protein (RFP) mRNA delivery into HEK 293T cells was assessed by the level of RFP+ expression.

RFP mRNA (200 ng) was mixed with polymer 33 obtained in Example 7 or pAsp (DET) used as a reference sample to prepare nanoparticles. Polymer 33 and pAsp (DET) were added at doses of (i) 1 μg or (ii) 800 ng in combination with 200 ng of 1.5 kDa PEG-PAsp (DET) polymer. The 1.5 kDa PEG-PAsp (DET) polymer allows for controlled nanoparticle size. TLR HEK293T cells were treated with the resulting nanoparticles and RFP+ quantification was performed by flow cytometry 24 hours after treatment. The results are presented in Fig. 14.

In fig. 14 shows that compared to the reference polymer (PAsp (DET)), polymer 33 provides comparable delivery of mRNA to HEK 293T cells in both the presence and absence of PEG-PAsp (DET) 1.5 kDa polymer at doses of (i) 1 µg or (ii) 800 ng in combination with 200 ng of 1.5 kDa PEG-PAsp (DET) polymer.

Example 14.

This example demonstrates the ability of the polymers disclosed herein to deliver Cas9 ribonucleoprotein (Cas9 RNP) into cells using various buffers. The level of Cas9 RNP delivery was assessed in green fluorescent protein (GFP)-inducible HEK293T (GFP-HEK) cells.

pmol of Cas9 RNP (sgRNA+Cas9 protein) was mixed with 4 μg of polymer 33, prepared as described in example 7, or with 5 μg of pAsp (DET), used as a reference, to obtain nanoparticles. GFP-HEK cells (200,000 cells) were treated in media with three different buffer systems, namely (i) (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES; 20 mM), (ii) Opti-MEMTM ( commercially available from Thermo Fisher Scientific; Waltham, MA), or Dulbecco's modified Eagle's medium (DMEM; commercially available from Thermo Fisher Scientific; Waltham, MA). The results are presented in Fig. 15.

In fig. 15 shows the level of Cas9 RNP delivery as measured by the percentage of GFP(-) in GFP-HEK cells treated with two mixtures (ie, 4 μg of polymer 33 or 5 μg of pAsp (DET)) in three different buffer systems. Both polymers (ie polymer 33 and pAsp (DET)) had the same trend in the three different buffer systems: HEPES medium was more preferable than Opti-MEM™ and Opti-MEM™ was more preferable than DMEM. In addition, polymer 33 was more effective than pAsp (DET) in all three buffer systems.

Example 15.

This example demonstrates the ability of the polymers disclosed herein to stably encapsulate nucleic acid.

Polymers 2, 5 and 33 of the present invention (1 μg/μl, 10 mM HEPES) were pipetted with oligonucleotides (1 μg/μl, 10 mM HEPES) in a mass ratio of 5:1, and then left for 10 minutes at room temperature. temperature for the formation of nanoparticles. The resulting nanoparticles were stored at 4°C. Nanoparticles from the starting material were diluted ∼1:20 using 10 mM HEPES, and dynamic light scattering (DLS) was determined at 0, 3, 5, and 7 days (n=1) after preparation. The average particle size determined from the scattered light intensity is shown in FIG. 18.

The observed nanoparticle size ranged from 150 to 350 nm. Over a 7-day observation period, the polymers showed minimal size change, indicating the stability of the nanoparticles. The change in size of pAsp[DET] nanoparticles used as a control (data not shown) at day 7 was slightly greater than that of the other nanoparticles tested.

Example 16.

This example shows the preparation of a polymer according to the invention.

100 mg (0.0074 mmol) poly(β-benzyl-1-aspartate) (PBLA) was dissolved in 3 ml NMP. 1.5 g of 1,4,7,10-tetramethyltriethylenetetraamine was added to this reaction mixture, and the reaction mixture was stirred for 16 hours at room temperature. The crude reaction mixture was precipitated with diethyl

- 61 046334 ether and the crude product was collected by centrifugation (5000 g for 15 min). The crude product was dissolved in 3 ml of 1 M HCl and dialyzed against water to achieve pH 6. The resulting polymer solution was lyophilized to obtain polymer 41 as a white powder.

Example 17.

This example demonstrates the use of the polymers disclosed herein to deliver mRNA into a cell.

mRNA encoding green or red fluorescent protein was mixed with the test polymer and combined with one of several different cell types, as indicated in the table. 2. Transfection was measured as a function of fluorescence. The results are presented in table. 2, which show that almost all polymers provide some level of transfection in at least one cell type.

table 2

Polymer Hepatocyte primary mouse myoblast nervous stem cell HEK2 93 Hematopoietic kaya stem person cell nin -7 NER G2 Without cheese serum ka Without cheese serum ka Without syvo R· serum 1 0 0 0 0 0 1 0 0.0 2 0 0 0 0 0 1 0 4.1 3 0 0 0 1 12 1 0 0.3 4 0 0 0 0 0 1 1 0.0 5 0 0 3 1 19 3 12 0.2 6 0 0 0 0 0 0 0 0.0 7 9 0 0 1 7 3 6 1.9 8 7 0 N/A N/A N/A N/A N/A N/A 9 0 0 25 0 4 2 3 0.9 10 0 0 8 0 25 3 19 0.7 AND 0 0 23 0.5 18 4 25 0.2 12 N/A N/A N/A N/A N/A N/A 9 N/A 33 0 0 26 2 41 4 27 N/A Lipofect amine 84 43 39 52 49 N/A 26 0.0 Mosk 0 0 0 0 N/A N/A N/A N/A pAspfDET ] 0 0 N/A N/A 39 3 28 0.0

N/A = Not tested.

Mock = polymer without mRNA.

Murine primary myoblasts were tested using mRNA encoding green fluorescent protein. All other cell types were tested using mRNA encoding red fluorescent protein.

The present description presents preferred embodiments of the invention, including the best method known to the inventors for carrying out the invention. Upon examination of the foregoing description, modifications to these preferred embodiments may become apparent to those skilled in the art. The inventors expect that those skilled in the art will use such variations as appropriate, and that the invention will be practiced in a manner other than that disclosed herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter set forth in the appended claims to the extent permitted by applicable law. Moreover, any combination of the above-described elements in all their possible variations is covered by the invention, unless otherwise indicated in the present description or clearly contrary to the context.

If a range of values is provided, it is understood that every intervening value, down to the decimal unit of the lower limit, unless the context clearly requires otherwise, is between the upper and lower limits of that range and any other specified or intervening

-

Claims (27)

a value in the specified range is within the scope of the invention. The upper and lower limits of these narrower ranges may be independently included within the narrower ranges and are also within the scope of the invention, subject to any specifically excluded limit within said range. If the specified range includes one or both limits, ranges excluding one or both of these included limits are also included within the scope of the invention. Unless otherwise specified, all technical and scientific terms used in this description have the same meaning as they are commonly understood by one skilled in the art to which the present invention relates. Although any methods and materials similar or equivalent to those disclosed herein may also be used in the practice or testing of the present invention, the preferred methods and materials are presented herein. All publications referred to herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which such publications are referenced. It should be noted that as used in this specification and in the accompanying claims, the singular forms of the singular number include references to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to a complex includes a plurality of such complexes, and a reference to a Cas9 polypeptide includes a reference to one or more Cas9 polypeptides and equivalents thereof known to those skilled in the art, etc. In addition, it should be noted that the claims may be drafted so as to exclude any optional element. Thus, this statement is intended as an a priori basis for the use of exclusionary terminology such as exclusively, only, etc. in connection with the enumeration of the elements of a claim or for the use of a negative limitation. It is understood that certain features of the invention, which for clarity are described in the context of individual embodiments, may also be presented in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be presented separately or in any suitable subcombination. All combinations of embodiments related to the invention are specifically embraced by the present invention and are disclosed herein as if each combination were separately and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each such subcombination were separately and explicitly disclosed herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the present invention cannot be dated earlier than the date of publication by reason of an earlier invention. In addition, published dates may differ from actual publication dates, which may require independent confirmation. CLAIM 1. Polymer containing the structure of formula 4: oh oh where each of m ¹ and n ¹ is an integer from 0 to 1000, provided that the sum of m ¹ +n ¹ is greater than 2; the symbol / indicates that the links separated in this way are connected randomly or in any order; each of A ¹ and A ² independently represents a group of the formula: -(CH ₂ ) _pl -[NR ² -(CH2)ql-]rlNR ² 2; -(CH ₂ ) _p2 -N[-(CH ₂ ) _q2 -NR ² ₂ ] ₂ ; -(CH ₂ ) _p3 -N{[-(CH ₂ ) _q3 -NR ² 2][-(CH2)q4-NR ² -]r2R ² }; or -(CH ₂ ) _p4 -N{-(CH ₂ ) _q5 -N[-(CH ₂ ) _q6 -NR ² ₂ ] ₂ } ₂ , where each of p1-p4, q1-q6 and r1 and r2 independently represents integer from 1 to 5; each R ² independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group, with the proviso that A ¹ and A ² each contain at least one tertiary amine; each of R ^3a and R ^3b independently represents a methylene or ethylene group; each R ¹³ independently represents hydrogen or a C ₁ -C ₁₂ alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group. - 63 046334 2. A polymer according to claim 1, wherein groups A ¹ and A ² each include at least two tertiary amines. 3. The polymer according to claim 1, wherein in groups A ¹ and A ² each nitrogen atom containing R ² substituents is a tertiary amine, except that the terminal amine may be a primary, secondary or tertiary amine. 4. A polymer according to claim 1, wherein in groups A ¹ and A ² each of R ² is ethyl or methyl, except that the terminal amine of A ¹ and A ² may be a primary, secondary or tertiary amine. 5. A polymer according to any one of claims 1 to 4, wherein in groups A ¹ and A ² the terminal amine is a secondary or tertiary amine. 6. The polymer according to claim 1, wherein each of A ¹ and A ² represents a group of the formula -(CH ₂ )-NR ² -(CH2)-NR ² 2, wherein R ² in each case independently represents a C ₁ -C ₁₂ -alkyl group, alkenyl group, cycloalkyl group or cycloalkenyl group, except that the terminal the amine is a primary, secondary or tertiary amine. 7. The polymer of claim 6, wherein R ² of groups A ¹ and A ² is in each case ethyl or methyl, except that the terminal amine of groups A ¹ and A ² may be a primary, secondary or tertiary amine. 8. Polymer according to any one of claims 1 to 7, where R ¹³ represents hydrogen. 9. Polymer according to any one of claims 1 to 7, where R ¹³ represents methyl. 10. Polymer according to any one of claims 1-9, having the structure of formula 4A: where c is an integer from 0 to 50; Y is optionally present and is a cleavable linker; R ⁶ represents hydrogen, an amino group, an aryl group, a heterocyclic group, a C ₁ -C ₁₂ alkyl group, an alkenyl group, a cycloalkyl group or a cycloalkenyl group, a linear or branched C ₁ -C ₁₂ alkyl group, optionally substituted by one or more amines ; or a tissue-specific or cell-specific targeting moiety. 11. A polymer according to any one of claims 1 to 10, which is a cationic polymer. 12. Polymer according to claim 1, having the formula: - 64 046334 - 65 046334 Polymer 42 where (a+b) ranges from 5 to 160. 13. Polymer according to claim 1, having the formula: - 66 046334 - 67 046334 - 68 046334 or - 69 046334 - 70 046334 or or where (a+b) is from 5 to 75 and (c+d) is from 5 to 80. 14. A composition for delivering a nucleic acid and/or polypeptide into a cell, containing a polymer according to any one of claims 1 to 13 and a nucleic acid and/or polypeptide. 15. The composition according to claim 14, wherein the cell is located in the body of the subject. 16. The composition according to claim 14 or 15, where the polymer partially or completely encapsulates the nucleic acid and/or polypeptide. 17. The composition according to any one of claims 14-16, containing a guide nucleic acid and/or a donor nucleic acid. 18. The composition according to any one of claims 14-17, containing an endonuclease. 19. The composition according to claim 18, containing an RNA-guided endonuclease or a nucleic acid encoding it. 20. The composition of claim 19, wherein the RNA-guided endonuclease is Cas9, Cpf1, or a combination thereof. 21. Composition according to any one of claims 14-20, containing DNA recombinase. 22. The composition according to claim 21, wherein the DNA recombinase is a Cre recombinase. 23. The composition according to any one of claims 14-22, containing zinc finger nuclease. 24. The composition according to any one of claims 14 to 23, containing an effector nuclease like a transcription activator. 25. The composition according to any one of claims 14 to 24, containing a second polymer which contains polyethylene oxide. 26. A method for delivering a nucleic acid and/or a polypeptide into a cell, including introducing into the cell a composition according to any one of claims 14-25. 27. The method according to claim 26, where the cell is in the body of the subject and the subject is administered the composition according to -