Modified polyamine polymers for delivery of biomolecules into cells

Abstract

Provided herein are compounds, compositions and methods for delivering biomolecules into cells. Specifically, the present disclosure provides modified polyamine polymers, including polyamine polymers having fluorinated substituents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 865,638, filed on June 24, 2019, the entire contents of which are fully incorporated herein by reference.

[0003] Materials Submitted Electronically Are Incorporated by Reference

[0004] The computer-readable nucleotide / amino acid sequence listing filed concurrently with this document is incorporated herein by reference in its entirety and is identified as follows: a 10,000-byte ASCII (text) file named "36754-601_ST25", created on June 23, 2020. Technical Field

[0005] Provided herein are compounds, compositions and methods for delivering biomolecules into cells. Specifically, the present disclosure provides modified polyamine polymers, including polyamine polymers having fluorinated substituents. Background Art

[0006] Cationic polymers, such as polyethyleneimine (PEI) and poly(amidoamine) (PAMAM) dendrimers, are widely used as vectors to introduce foreign genes into cells. These materials are easy to manufacture and have excellent safety compared to viral gene delivery. However, their commercial and clinical applications are limited by relatively low transfection efficiency and poor cell viability. Summary of the invention

[0007] Provided herein are compounds, compositions and methods for delivering biomolecules into cells. Specifically, the present disclosure provides modified polyamine polymers, including polyamine polymers having fluorinated substituents.

[0008] Embodiments of the present disclosure include a compound or a salt thereof, the compound comprising:

[0009] Polyethyleneimine polymers; and

[0010] A plurality of substituents bonded to the amino groups of the polyethyleneimine polymer, wherein each substituent independently has formula (I):

[0011] -X-(CH2) n -Z

[0012] (I)

[0013] in:

[0014] X is a bond or -C(O)-O-;

[0015] n is 0, 1, 2, 3, 4 or 5; and

[0016] Z is selected from the group consisting of haloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0017] In some embodiments, the polyethyleneimine polymer has a weight average molecular weight of about 500Da to about 250000Da. In some embodiments, the polyethyleneimine polymer has a weight average molecular weight of about 500Da to about 2000Da. In some embodiments, the polyethyleneimine polymer has a weight average molecular weight of about 5000Da to about 25000Da.

[0018] In some embodiments, the polyethyleneimine polymer is a branched polyethyleneimine polymer. In some embodiments, the polyethyleneimine polymer is a linear polyethyleneimine polymer.

[0019] In some embodiments, Z is a haloalkyl group having the formula: -(CF2) m -CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, Z is pentafluorophenyl. In some embodiments, Z is unsubstituted pyridinyl.

[0020] In some embodiments, X is -C(O)O-, and n is 1 or 2.

[0021] In some embodiments, Z is a haloalkyl group having the formula: -(CF2) m -CF3, wherein m is 1, 2, 3, 4 or 5.

[0022] In some embodiments, X is a bond, and n is 1 or 2.

[0023] In some embodiments, about 0.1mol% to about 60mol% of the amino groups of the polyethyleneimine polymer are combined with the substituents of formula (I). In some embodiments, about 5mol% to about 50mol% of the amino groups of the polyethyleneimine polymer are combined with the substituents of formula (I). In some embodiments, about 8mol% to about 40mol% of the amino groups of the polyethyleneimine polymer are combined with the substituents of formula (I).

[0024] Embodiments of the present disclosure also include a compound or a salt thereof, the compound comprising:

[0025] Poly(amidoamine) dendrimers; and

[0026] A plurality of substituents bonded to the amino groups of the poly(amidoamine) dendrimer, wherein each substituent independently has formula (I):

[0027] -X-(CH2) n -Z

[0028] (I)

[0029] in:

[0030] X is a bond or -C(O)-O-;

[0031] n is 0, 1 or 2; and

[0032] Z is selected from the group consisting of haloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0033] In some embodiments, the poly(amidoamine) dendrimer is a 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th generation poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a 1st, 2nd, 3rd, or 4th generation poly(amidoamine) dendrimer.

[0034] In some embodiments, Z is a haloalkyl group having the formula: -(CF2) m -CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, Z is pentafluorophenyl. In some embodiments, Z is unsubstituted pyridinyl.

[0035] In some embodiments, X is -C(O)O-. In some embodiments, Z is a haloalkyl group having the formula: -(CF2) m -CF3, wherein m is 1, 2, 3, 4 or 5.

[0036] In some embodiments, X is a bond, and n is 1.

[0037] In some embodiments, from about 0.1 mol% to about 80 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bound to substituents of formula (I). In some embodiments, from about 10 mol% to about 70 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bound to substituents of formula (I). In some embodiments, from about 20 mol% to about 70 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bound to substituents of formula (I).

[0038] Embodiments of the present disclosure also include a method of delivering a biomolecule into a cell, the method comprising: contacting the cell with an effective amount of a compound described herein (i.e., a compound comprising a polyethyleneimine polymer and a plurality of substituents of formula (I), or a compound comprising a poly(amidoamine) dendrimer and a plurality of substituents of formula (I)) or a salt thereof; and contacting the cell with the biomolecule. In some embodiments, the method comprises contacting the cell with an effective amount of two or more different compounds or salts thereof.

[0039] In some embodiments, the biomolecule is at least one of a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, a peptide, a polypeptide, a protein, or any combination or derivative thereof. In some embodiments, the biomolecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. In some embodiments, the biomolecule is a peptide or polypeptide capable of having luminescent activity. In some embodiments, the biomolecule comprises a polypeptide sequence (LgBiT) of SEQ ID NO:4. In some embodiments, the biomolecule is a ribonucleoprotein complex comprising a Cas9 protein. In some embodiments, the ribonucleoprotein complex further comprises a guide RNA (gRNA) and a donor DNA template, wherein the donor DNA template comprises a sequence encoding a polypeptide from SEQ ID NO:3.

[0040] In some embodiments, the method comprises mixing the compound and the biomolecule to form a mixture and then contacting the cell with the mixture.

[0041] Embodiments of the present disclosure also include a kit comprising a compound or a salt thereof, wherein the compound or the salt thereof is a compound described herein (i.e., a compound comprising a polyethyleneimine polymer and a plurality of substituents of formula (I), or a compound comprising a poly(amidoamine) dendrimer and a plurality of substituents of formula (I)).

[0042] In some embodiments, the kit comprises a compound or a salt thereof in a container.

[0043] In some embodiments, the kit further comprises at least one of a DNA molecule, an RNA molecule, a peptide, a polypeptide, a protein, or any combination or derivative thereof. In some embodiments, the biomolecule is a peptide or polypeptide capable of having luminescent activity. In some embodiments, the biomolecule comprises a polypeptide sequence (LgBiT) of SEQ ID NO:4. In some embodiments, the peptide comprises a Cas9 protein. In some embodiments, the DNA molecule is a donor DNA template comprising a sequence encoding a polypeptide from SEQ ID NO:3.

[0044] In some embodiments, the kit further comprises instructions for transfecting a biomolecule using the compound or salt thereof.

[0045] Embodiments of the present disclosure also include a method of changing the sequence of an endogenous protein in a cell, the method comprising:

[0046] Assembling a ribonucleoprotein (RNP) complex comprising a Cas9 protein, a donor DNA template, and a guide RNA; and

[0047] The RNP complex is delivered into cells using the compounds described herein.

[0048] Embodiments of the present disclosure also include a method for labeling an endogenous protein in a cell, the method comprising:

[0049] Assembling a ribonucleoprotein (RNP) complex comprising a Cas9 protein, a donor DNA template, and a guide RNA, wherein the donor DNA template comprises a sequence encoding a peptide or polypeptide tag sequence; and

[0050] The RNP complex is delivered into cells using the compounds described herein.

[0051] In some embodiments, the donor DNA template comprises a sequence encoding a peptide tag selected from SEQ ID NO: 3 and SEQ ID NO: 5. In some embodiments, the donor DNA template further comprises homology arms flanking the sequence encoding the peptide or polypeptide tag sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1A-1B Depicted are the results of transfection of HEK293 cells with a constant molar ratio of modified PEI polymer as described in Example 2. FIG1A shows the results of transfection of HEK293 cells with a constant molar ratio of modified PEI polymer as described in Example 2. The expression measured The transfection efficiency of the vector is shown in Figure 1B. Measurement of cell viability using luminescent cell viability assay.

[0053] Figure 2A-2B Depicted are the results of transfection of HEK293 cells with modified PEI compounds compared to unmodified polymer and FuGENE HD as a control as described in Example 2. FIG2A shows the ratio of luminescent signal from NanoLuc relative to the control, and FIG2B shows the ratio of luminescent signal from NanoLuc relative to the control using a transfection agent compared to a sample without a transfection agent. Measurement of cell viability using luminescent cell viability assay.

[0054] Figures 3A-3CDepicted are the results of titrating modified PEI polymers and incubating with a constant amount of DNA prior to transfection of HEK cells as described in Example 2. FIG3A shows data for transfections performed in the presence of serum, and FIG3B shows data for transfections performed in the absence of serum. FIG3C shows data for transfections performed in the presence of serum followed by incubation with a constant amount of DNA. Measurement of cell viability using luminescent cell viability assay.

[0055] Figures 4A-4I Depicted is the use of Reagents, luminescence results of titration of modified PEI and PAMAM polymers prior to transfection of nine different cell types in the presence of serum and incubation with a constant amount of DNA.

[0056] Figures 5A-5H The results of the present invention demonstrate the use of modified PEI and PAMAM polymers in nine different cell types in the presence of serum after transfection as described in Example 2. Measurement of cell viability using luminescent cell viability assay.

[0057] Figures 6A-6E Depicted are the results of titrating modified PEI and PAMAM polymers prior to transfection of five different cell types in the presence of serum as described in Example 2 and incubation with a constant amount of DNA.

[0058] Figures 7A-7D The results of the present invention demonstrate the use of modified PEI and PAMAM polymers in four different cell types after transfection in the presence of serum as described in Example 2. Measurement of cell viability using luminescent cell viability assay.

[0059] Figures 8A-8E Depicted are the results of titrating modified PEI and PAMAM polymers prior to transfection of five different cell types in the presence of serum as described in Example 2 and incubation with a constant amount of DNA.

[0060] Figures 9A-9D The results of the present invention demonstrate the use of modified PEI and PAMAM polymers in five different cell types after transfection in the presence of serum as described in Example 2. Measurement of cell viability using luminescent cell viability assay.

[0061] Figures 10A-10BThe results of delivering LgBiT to clones stably expressing HiBiT fusions are depicted. Figure 10A depicts the results of delivering LgBiT to PKCα-HiBiT or HDAC6-HiBiT clones of HeLa cells or CDK6-HiBiT clones of HEK293 cells using modified PEI compounds, compared with direct transduction of unmodified polymers and BacMam CMV-LgBiT to the corresponding clones above. For each PEI compound tested, the percentage of luminescent signal from LgBiT-delivered PKCα-HiBiT clones, HDAC6-HiBiT or CDK6-HiBiT clones relative to the luminescent signal from the corresponding BacMam CMV-LgBiT-transduced HiBiT clones is shown. Figure 10B depicts the results of delivering LgBiT to HDAC2-HiBiT clones of HeLa cells or CDK12-HiBiT clones of HEK293 cells using modified PEI compounds, compared with direct transduction of unmodified polymers and BacMamCMV-LgBiT to the corresponding clones above. The percentage of the luminescent signal from the HDAC2-HiBiT clone of HeLa cells or the CDK12-HiBiT clone of HEK293 cells relative to the luminescent signal from the corresponding BacMam CMV-LgBiT-transduced HiBiT clone is shown for each PEI compound tested.

[0062] Figures 11A-11B Depicted are measurements of cell viability of HiBiT clones derived from HeLa and HEK293 cells after delivery of LgBiT. FIG. 11A includes PKCα-HiBiT, HDAC6-HiBiT, and CDK6-HiBiT, and FIG. 11B includes HDAC2-HiBiT and CDK12-HiBiT. LgBiT was delivered to these cells via modified PEI compounds or unmodified polymers. Negative controls were those in which PEI was not added to the cells. Luminescence signal (RLU) represents the amount of light emitted by the cells as described using Cell viability measured by luminescent cell viability assay.

[0063] Figures 12A-12C Depicted are the bioluminescent imaging results of delivering LgBiT to clones stably expressing HDAC2-HiBiT in HeLa cells or stably expressing HDAC6-HiBiT in HeLa cells using modified PEI compounds 7666-3 ( FIG. 12A ), 7668-1 ( FIG. 12B ), 7669-2 ( FIG. 12C ).

[0064] Figures 13A-13BScreening results of modified PEI compounds and unmodified PEI to examine the delivery of LgBiT to different HiBiT edited cells are depicted. Figure 13A depicts the percentage of LgBiT delivered by PEI compared to BacMam-LgBiT transduction. Figure 13B represents the viability of cells treated with PEI-LgBiT complexes for 24 hours.

[0065] 14A-C depict the results of bioluminescent imaging of different cell lines after delivery of LgBiT to HiBiT marker proteins located on the cell surface, cytoplasm, and nucleus.

[0066] Figures 15A-15C Depicted are the results of fluorescence imaging of HeLa cells after delivery of Halo Tag-LgBiT using modified PEI compared to unmodified PEI.

[0067] Figures 16A-16B The results are shown in Figure 2. TM Results of RNP delivery of VS-HiBiT tag to the C-terminus of GAPDH in HEK293 cells using modified PEI compound 7667-4 compared with , CRISPRMax and nucleofection. The luminescence signal (RLU) normalized to the number of cells represents the efficiency of RNP delivery. The number of cells was determined by The results were determined by a luminescent cell viability assay (Figure A). Increasing concentrations of PEI showed improved RNP delivery but resulted in more cytotoxicity (Figure 16B).

[0068] Figures 17A-17E Describe that constant amount of ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor template are delivered to HEK293 cells (HEK293 / Fluc cells) stably expressing firefly luciferase, and the donor template is designed to insert HiBiT into the N-terminus of Fluc via CRISPR / Cas9. RNP / ssODN mixture is delivered to cells by modified PEI and PAMAM compounds of the final concentration listed by titration. Two days later, the viability (CTF; Figure 17A) of cells is measured using CellTiter-Fluor, Fluc expression (Figure 17B) is measured using ONE-Glo EX, and HiBiT signal (Figure 17C) is measured using HiBiT NoDLR assay. HiBiT signal is normalized to cell number (Figure 17D) using HiBiT / CTF ratio and normalized to Fluc expression (Figure 17E) using HiBiT / Fluc ratio.

[0069] Figures 18A-18BHEK293 / Fluc cell pools are depicted, wherein RNP / ssODN mixtures for knocking in HiBiT through CRISPR have been delivered using nuclear transfection or modified PEI or PAMAM polymers. Cell pools were expanded for many days before measurement to eliminate the complicating factors of cell death during treatment. After knocking in HiBiT at the N-terminal of Fluc, viability, Fluc expression and HiBiT signal (Figure 18 A) were measured using CellTiter-Fluor+HiBiT NanoDLR. The Fluc / CTF ratio of nuclear transfection cells significantly decreased and may indicate that InDel mutations cause expression loss. As indicated by the HiBiT / CTF ratio, the RNP / ssODN delivery mediated by modified polymers causes higher normalized Fluc expression, but also causes higher normalized HiBiT signal. Similarly, after delivery of an RNP / ssODN mixture designed to knock-in HiBiT at the C-terminus of GAPDH, the modified PEI polymer showed a higher normalized HiBiT signal compared to nucleofection ( FIG. 18B ).

[0070] Figures 19A-19B Protein degradation assay results are presented to demonstrate that the modified PEI polymers described herein deliver functional LgBiT into cells as described in Example 6.

[0071] Figures 20A-20D Results of an assay to distinguish intracellular from extracellular luminescence systems in four different cell lines (HEK293, A549, H1299 and Mia-PaCa2) as described in Example 7 are presented. DETAILED DESCRIPTION

[0072] Provided herein are modified polyethyleneimine polymers and modified poly(amidoamine) dendrimers for delivering biomolecules into cells.

[0073] In some embodiments, the modified polymers and dendrimers include fluorinated substituents. Fluorinated compounds are both hydrophobic and oleophobic, and have a high tendency to phase separate in polar and non-polar environments, so fluorination can increase the affinity of the polymer for the cell membrane and help the molecule to cross the lipid bilayer of the cell membrane, as well as the endosomal / lysosomal membrane, and promote the intracellular detachment of the polymer. In addition, fluorinated polymers have low surface energy and tend to associate with each other at low concentrations, thereby allowing the formation of polymer complexes with nucleic acids or proteins at low concentrations.

[0074] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0075] 1. Definition

[0076] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. For example, any nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used nomenclature and techniques in the art. The meaning and scope of the terms should be clear; however, if there is any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0077] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry and specific functional moieties and reactivity are described in Sorrell, Organic Chemistry, 2nd edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th edition, John Wiley & Sons, New York, 2013; Larock, Comprehensive Organic Transformations, 3rd edition, John Wiley & Sons, New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 3rd edition, Cambridge University Press, Cambridge, 1987; The entire contents of each are incorporated herein by reference.

[0078] As used herein, the term "alkyl" refers to a group containing 1 to 30 carbon atoms, for example 1 to 16 carbon atoms (C1-C 16 alkyl), 1 to 14 carbon atoms (C1-C 14 alkyl), 1 to 12 carbon atoms (C1-C 12 alkyl), 1 to 10 carbon atoms (C1-C 10The alkyl group may be a straight or branched saturated hydrocarbon chain of 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl) or 1 to 4 carbon atoms (C1-C4 alkyl). Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylphenyl, 2,3-dimethylphenyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl.

[0079] As used herein, the term "aryl" refers to phenyl, or a bicyclic or tricyclic aromatic fused ring system. An example of a bicyclic fused ring system is a phenyl attached to a parent molecular moiety and fused to a phenyl. An example of a tricyclic fused ring system is a phenyl attached to a parent molecular moiety and fused to two other phenyls. Representative examples of bicyclic aryls include, but are not limited to, naphthyl. Representative examples of tricyclic aryls include, but are not limited to, anthracenyl.

[0080] As used herein, the term "cycloalkyl" refers to a saturated carbocyclic ring system containing three to ten carbon atoms and zero heteroatoms. The cycloalkyl group can be monocyclic, bicyclic, bridged, fused or spirocyclic. Representative examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl and bicyclo[5.2.0]nonyl.

[0081] As used herein, the term "halogen" or "halo" means F, Cl, Br or I.

[0082] As used herein, the term "haloalkyl" means an alkyl as defined herein, wherein one or more hydrogen atoms are replaced by halogen. For example, one, two, three, four, five, six, seven or eight hydrogen atoms may be replaced by halogen. Representative examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, 2-fluoroethyl, 2,2-difluoroethyl and 2,2,2-trifluoroethyl.

[0083] As used herein, the term "heteroaryl" refers to an aromatic monocyclic or aromatic bicyclic or aromatic tricyclic system. An aromatic monocyclic ring is a five-membered or six-membered ring containing at least one heteroatom independently selected from the group consisting of N, O and S (e.g., 1, 2, 3 or 4 heteroatoms independently selected from O, S and N). A five-membered aromatic monocyclic ring has two double bonds, and a six-membered six-membered aromatic monocyclic ring has three double bonds. An example of a bicyclic heteroaryl is a monocyclic heteroaromatic ring fused to a monocyclic aryl as defined herein or a monocyclic heteroaromatic as defined herein. An example of a tricyclic heteroaryl is a monocyclic heteroaromatic ring fused to two rings independently selected from a monocyclic aryl as defined herein or a monocyclic heteroaromatic as defined herein. Representative examples of monocyclic heteroaryl groups include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. The representative example of bicyclic heteroaryl includes but is not limited to benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolyl, quinoxalinyl, thiazolopyridyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl and thienothienyl. The representative example of tricyclic heteroaryl includes but is not limited to dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic and tricyclic heteroaryl are connected to the parent molecular part by any carbon atom or any nitrogen atom contained in the ring.

[0084] As used herein, the term "heterocycle" or "heterocyclic" means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. A monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A three- or four-membered ring contains 0 or 1 double bond and 1 heteroatom selected from the group consisting of O, N, and S. A five-membered ring contains 0 or 1 double bond and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. A six-membered ring contains 0, 1, or 2 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. A seven- and eight-membered ring contains 0, 1, 2, or 3 double bonds and 1, 2, or 3 heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, dioxepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithiolanyl, 1,3-dithiacyclyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxadiazolyl, oxazolidin ... Alkyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiophenyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinyl, 1,3-thiazinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl (thiomorpholine sulfone), thiopyranyl and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle or spiroheterocyclyl, or a bridged monocyclic heterocycle system in which two non-adjacent atoms of the ring are connected by an alkylene bridge of 1, 2, 3 or 4 carbon atoms or an alkenylene bridge of two, three or four carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothiophenyl, 2,3-dihydroisoquinolinyl, 2-azaspiro[3.3]heptan-2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-1H-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Examples of tricyclic heterocycles are bicyclic heterocycles fused to a phenyl group, or to a monocyclic cycloalkyl group, or to a monocyclic cycloalkenyl group, or to a monocyclic heterocycle, or to a bicyclic heterocycle wherein two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3 or 4 carbon atoms or an alkenylene bridge of two, three or four carbon atoms.Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H-1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.1. 3,7 ]decane) and oxa-adamantane (2-oxatricyclo[3.3.1.1 3,7 The monocyclic, bicyclic, and tricyclic heterocycles are attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings.

[0085] As used herein, the term "perfluoroalkyl" refers to an alkyl group in which each hydrogen is replaced by fluorine. Representative examples of perfluoroalkyl groups include, but are not limited to, trifluoromethyl, perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluoropentyl, and perfluorohexyl.

[0086] As used herein, the term "poly(amidoamine)" (or "PAMAM") refers to an art-recognized class of dendritic polymers having a diamine core and repeating branching subunits having amide and amine functional groups formed by reacting a diamine with methyl acrylate and then with another diamine. These dendritic polymers are defined by the number of "layers" of amidoamine groups extending from the core, with each "layer" being referred to as a "generation." For example, ethylenediamine can be reacted with methyl acrylate to form methyl 3-[2-[bis(3-methoxy-3-oxopropyl)amino]ethyl-(3-methoxy-3-oxopropyl)amino]propanoate, which can then be reacted with more ethylenediamine to form a "generation 0" PAMAM, N-(2-aminoethyl)-3-[[3-(2-aminoethylamino)-3-oxopropyl]-[2-[bis[3-(2-aminoethylamino)-3-oxopropyl]amino]ethyl]amino]propionamide. The same reaction sequence is repeated to similarly functionalize each primary amino group of the 0th generation PAMAM, which can form the 1st generation PAMAM, and so on. The chemical structure of the 1st generation PAMAM is shown below, with the "core" 0th generation PAMAM structure highlighted:

[0087]

[0088] As used herein, the term "polyethyleneimine" (or "PEI") refers to a polymer based on repeating iminoethylene groups. "Linear polyethyleneimine" is a polymer having the formula -(CH2-CH2-NH) n- is a linear polymer, and therefore includes only secondary amino groups in the polymer chain. "Branched polyethyleneimine" is a branched polymer synthesized by aziridine ring-opening polymerization, and includes primary, secondary and tertiary amino groups. Branched polyethyleneimine is generally depicted as shown below, but those skilled in the art will understand that branched polyethyleneimine is not a polymer with the exact type of repeating units shown in brackets; rather, this merely illustrates the different ways in which individual -CH2-CH2-NH- groups can be linked together, and additional connection and branching points are possible:

[0089]

[0090] In some cases, the number of carbon atoms in a group (e.g., alkyl, haloalkyl, or cycloalkyl) is indicated by the prefix "C x -C y ” means, where x is the minimum number of carbon atoms in the group and y is the maximum number. Thus, for example, “C1-C3 alkyl” refers to an alkyl group containing 1 to 3 carbon atoms, while “C1-C6 haloalkyl” refers to a haloalkyl group containing 1 to 6 carbon atoms.

[0091] The term "substituent" refers to a group that replaces an atom in the indicated group.

[0092] When a group or moiety can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using "substituted" can be replaced by a list of listed indicated groups or by suitable groups known to those skilled in the art (e.g., one or more of the groups listed below). Substituents include, but are not limited to, halogen, =O, =S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroaralkyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, -COOH, ketone, amide, carbamate, and acyl.

[0093] As used herein, the terms "Oplophorus luciferase" and "Oplophorus-derived luciferase" are used interchangeably and refer to a luciferase secreted from the deep-sea shrimp Oplophorus gracilirostris (e.g., SEQ ID NO: 1), including wild-type, variants, and mutants thereof. For example, suitable Oplophorus luciferase variants are described in U.S. Pat. Nos. 8,557,970 and 8,669,103, each of which is incorporated herein by reference in its entirety. Exemplary Oplophorus-derived luciferases include, for example, the luciferase of SEQ ID NO: 2 (also referred to interchangeably herein as "NanoLuc," "Nluc," "Nluc luciferase," and "Nluc enzyme").

[0094] For the compounds described herein, radicals and substituents may be chosen based on the allowed valences of atoms and substituents such that selection and substitution results in stable compounds, e.g., which do not spontaneously transform, e.g., by rearrangement, cyclization, elimination, and the like.

[0095] As used herein, the terms "comprise", "including", "having", "has", "may", "include" and variations thereof are intended to be open-ended transitional phrases, terms or words that do not exclude the possibility of additional actions or structures. Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Many embodiments of this document are described using open "comprising" language. Such embodiments encompass multiple closed "consisting of" and / or "consisting essentially of" embodiments that can alternatively be claimed or described using such language. The present disclosure also contemplates other embodiments that "comprise" the embodiments or elements presented herein, "consisting of", and "consisting essentially of", whether or not explicitly set forth.

[0096] For the description of numerical ranges herein, each number therebetween with the same degree of precision is expressly contemplated. For example, for the range of 6-9, in addition to 6 and 9, the numbers 7 and 8 are also contemplated, and for the range of 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are expressly contemplated.

[0097] 2. Polymer

[0098] The present disclosure includes modified polyamine polymers suitable for delivering biomolecules, such as genes, proteins, and ribonucleoproteins and related complexes, into cells.

[0099] In some embodiments, the present disclosure provides a compound comprising:

[0100] Polyethyleneimine polymers; and

[0101] A plurality of substituents bonded to the amino groups of the polyethyleneimine polymer, wherein each substituent independently has formula (I):

[0102] -X-(CH2) n -Z

[0103] (I)

[0104] in:

[0105] X is a bond or -C(O)-O-;

[0106] n is 0, 1, 2, 3, 4 or 5; and

[0107] Z is selected from the group consisting of haloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0108] In some embodiments, the polyethyleneimine polymer has a weight average molecular weight of about 500 Da to about 250,000 Da, or about 500 Da to about 30,000 Da, or about 500 Da to about 2000 Da, or about 5000 Da to about 25,000 Da. For example, the polyethyleneimine polymer can have a weight average molecular weight of about 500 Da, about 600 Da, about 700 Da, about 800 Da, about 900 Da, about 1000 Da, about 1200 Da, about 1400 Da, about 1600 Da, about 1200 Da, about 1400 Da, about 1600 Da, about 1800 Da, about 2000 Da, about 3000 Da, about 4000 Da, about 5000 Da, about 6000 Da, about 7000 Da, about 8000 Da, about 9000 Da, about 1 ...8000 Da, about 8000 Da, about 9000 Da, about 1000 Da, about 1200 Da, about 1400 Da, about 1600 Da, about 180 In some embodiments, the polyethyleneimine polymer has a weight average molecular weight of about 600Da, about 800Da, about 1200Da, about 1800Da, about 5000Da, about 10000Da, about 20000Da, about 25000Da, about 30000Da, about 35000Da, about 40000Da, about 45000Da, about 50000Da, about 100000Da, about 150000Da, about 200000Da or about 250000Da. In certain embodiments, the polyethyleneimine polymer has a weight average molecular weight of about 600Da, about 800Da, about 1200Da, about 1800Da, about 5000Da, about 10000Da or about 25000Da.

[0109] The polyethyleneimine polymer can be linear or branched. In some embodiments, the polyethyleneimine polymer is a linear polyethyleneimine polymer. In some embodiments, the polyethyleneimine polymer is a branched polyethyleneimine polymer.

[0110] In some embodiments, the polyethyleneimine polymer can be crosslinked. For example, the polyethyleneimine polymer can be crosslinked with urea (i.e., so that two amino groups on two different polymers are linked together through a C=O group) before or after functionalization so that the polymer includes multiple substituents of formula (I).

[0111] In various substituents of Formula (I), X is a bond or -C(O)-O-. In some embodiments, X is a bond. In some embodiments, X is -C(O)-O-.

[0112] In various substituents of Formula (I), n is 0, 1, 2, 3, 4, or 5. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 1 or 2.

[0113] In some embodiments, X is -C(O)-O- and n is 1 or 2. In some embodiments, X is a bond and n is 1 or 2.

[0114] In the plurality of substituents of formula (I), Z is selected from haloalkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl. In some embodiments, Z is selected from haloalkyl, substituted aryl and unsubstituted heteroaryl.

[0115] In some embodiments, Z is a haloalkyl group. In some embodiments, the haloalkyl group is a perfluoroalkyl group. In some embodiments, Z is a group having the formula -(CF2) m -CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 3, 4, 5, 6, or 7.

[0116] In some embodiments, Z is aryl or substituted aryl. In some embodiments, Z is substituted aryl. In some embodiments, Z is substituted phenyl. In some embodiments, Z is phenyl substituted with 1, 2, 3, 4 or 5 substituents selected from halo. In some embodiments, Z is pentafluorophenyl.

[0117] In some embodiments, Z is heteroaryl or substituted heteroaryl. In some embodiments, Z is unsubstituted heteroaryl. In some embodiments, Z is unsubstituted monocyclic heteroaryl. In some embodiments, heteroaryl is a monocyclic heteroaryl having 1, 2 or 3 heteroatoms independently selected from N, O and S. In some embodiments, Z is unsubstituted pyridyl.

[0118] In some embodiments, X is -C(O)O-, and n is 1 or 2. In some embodiments, X is a bond, and n is 1 or 2.

[0119] In some embodiments, about 0.1 mol% to about 60 mol% of the amino groups of the polyethyleneimine polymer are combined with the substituents of formula (I). For example, about 10 mol% to about 60 mol%, about 5 mol% to about 50 mol%, or about 8 mol% to about 40 mol% of the amino groups of the polyethyleneimine polymer are combined with the substituents of formula (I). In some embodiments, about 0.1 mol%, about 0.5 mol%, about 1 mol%, about 2 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, or about 80 mol% of the amino groups of the polyethyleneimine polymer are combined with the substituents of formula (I). For example, as discussed further below, the degree of modification can be adjusted by using different amounts of the reagent. 1 The degree of modification was determined by HNMR spectroscopy.

[0120] In some embodiments, the present disclosure provides a compound comprising:

[0121] Poly(amidoamine) dendrimers; and

[0122] A plurality of substituents bonded to the amino groups of the poly(amidoamine) dendrimer, wherein each substituent independently has formula (I):

[0123] -X-(CH2) n -Z

[0124] (I)

[0125] in:

[0126] X is a bond or -C(O)-O-;

[0127] n is 0, 1 or 2; and

[0128] Z is selected from the group consisting of haloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0129] In some embodiments, the poly(amidoamine) dendrimer is a 0th, 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th generation poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th generation poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a 1st, 2nd, 3rd, or 4th generation poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a 1st generation poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a 2nd generation poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a generation 3 poly(amidoamine) dendrimer. In some embodiments, the poly(amidoamine) dendrimer is a generation 4 poly(amidoamine) dendrimer.

[0130] In various substituents of Formula (I), X is a bond or -C(O)-O-. In some embodiments, X is a bond. In some embodiments, X is -C(O)-O-.

[0131] In various substituents of Formula (I), n is 0, 1, 2, 3, 4, or 5. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 0, 1, or 2. In some embodiments, n is 1 or 2.

[0132] In some embodiments, X is a bond and n is 1.

[0133] In the plurality of substituents of formula (I), Z is selected from haloalkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl. In some embodiments, Z is selected from haloalkyl, substituted aryl and unsubstituted heteroaryl.

[0134] In some embodiments, Z is a haloalkyl group. In some embodiments, the haloalkyl group is a perfluoroalkyl group. In some embodiments, Z is a group having the formula -(CF2) m-CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 3, 4, 5, 6, or 7.

[0135] In some embodiments, Z is aryl or substituted aryl. In some embodiments, Z is substituted aryl. In some embodiments, Z is substituted phenyl. In some embodiments, Z is phenyl substituted with 1, 2, 3, 4 or 5 substituents selected from halo. In some embodiments, Z is pentafluorophenyl.

[0136] In some embodiments, Z is heteroaryl or substituted heteroaryl. In some embodiments, Z is unsubstituted heteroaryl. In some embodiments, Z is unsubstituted monocyclic heteroaryl. In some embodiments, heteroaryl is a monocyclic heteroaryl having 1, 2 or 3 heteroatoms independently selected from N, O and S. In some embodiments, Z is unsubstituted pyridyl.

[0137] In some embodiments, X is -C(O)O-, and n is 1 or 2. In some embodiments, X is a bond, and n is 1 or 2.

[0138] In some embodiments, about 0.1 mol% to about 80 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bound to substituents of formula (I). For example, about 10 mol% to about 70 mol% or about 20 mol% to about 70 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bound to substituents of formula (I). In some embodiments, about 0.1 mol%, about 0.5 mol%, about 1 mol%, about 2 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, about 70 mol%, about 75 mol%, or about 80 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bound to substituents of formula (I). For example, as discussed further below, the degree of modification can be adjusted by using different amounts of the reagents. For example, the amount of the primary amino groups of the poly(amidoamine) dendrimer can be adjusted by using different amounts of the reagents. For example, the amount of the primary amino groups of the poly(amidoamine) dendrimer can be adjusted by using different amounts of the reagents. 1 H NMR spectroscopy was used to determine the degree of modification.

[0139] The polymer includes a functional group that can be a cationic group (e.g., -NH2 can be -NH3 + ), so in some embodiments, salts can be formed with suitable anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. In some embodiments, the compound is a hydrochloride.

[0140] Compounds can be prepared by a variety of methods. For example, carbamate-modified linear or branched PEI polymers or PAMAM dendrimers can be prepared by reacting with fluorinated alkyl p-nitrophenyl carbonate compounds, as shown in Schemes 1A, 1B, and 1C. PEI polymers and PAMAM dendrimers can also be modified by reacting with fluorinated aldehydes to form imine adducts, followed by reduction of the imine with NaBH4 to obtain the desired alkylated polymers, as shown in Scheme 2. The amine modification percentage can be adjusted by adding different amounts of reagents. In addition, the primary amines of branched PEI can be protected to a certain extent by BOC, followed by alkylation and deprotection with fluorinated alkyl halides to obtain free primary amine-alkylated PEI compounds, as shown in Scheme 3.

[0141] In the following schemes, the following abbreviations are used: Boc is tert-butyloxycarbonyl; DCM is dichloromethane; THF is tetrahydrofuran; TFA is trifluoroacetic acid; and TIPS is triisopropylsilane.

[0142] Scheme 1. Synthesis of modified polymers via carbamate modification

[0143] A. Synthesis of Carbamate-Modified Branched PEI Compounds

[0144]

[0145] B. Synthesis of Carbamate-Modified Linear PEI Compounds

[0146]

[0147] C. Synthesis of the first generation of carbamate-modified PAMAM

[0148]

[0149] Scheme 2. Synthesis of modified PEI polymers via imine reduction

[0150]

[0151] Scheme 3. Alkylated PEI with free primary amine via BOC protection, alkylation and deprotection

[0152]

[0153] The compounds and intermediates herein can be separated and purified by methods well known to those skilled in the art of organic synthesis. Examples of conventional methods for separating and purifying compounds may include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilyl groups; by recrystallization at elevated or low temperatures and optionally pre-treated with activated carbon; thin layer chromatography; distillation at various pressures; vacuum sublimation; and grinding, as described, for example, in "Vogel's Textbook of Practical Organic Chemistry," 5th edition (1989), Furniss, Hannaford, Smith, and Tatchell, publishers Longman Scientific & Technical, Essex CM202JE, England.

[0154] The reaction conditions and reaction times of each individual step can vary according to the specific reactant used and the substituent present in the reactant used. Specific procedures are provided in the Examples section. The reaction can be carried out in a conventional manner, for example, by removing the solvent from the residue for post-treatment, and further purified according to methods well known in the art, such as but not limited to crystallization, distillation, extraction, grinding and chromatography. Unless otherwise described, starting materials and reagents are commercially available or can be prepared from commercially available materials by those skilled in the art using the methods described in the chemical literature. If the starting material is not commercially available, it can be prepared by being selected from the following program: standard organic chemistry techniques, techniques similar to the synthesis of compounds with known structures, or techniques similar to the procedures described in the above schemes or the Synthesis Examples section.

[0155] Routine experiments, including appropriate manipulation of reaction conditions, reagents, and synthetic route sequences, protection of any chemical functional groups incompatible with the reaction conditions, and removal of protection at appropriate points in the method reaction sequence are all included within the scope of the present invention. Suitable protecting groups and methods for protecting and removing different substituents using such suitable protecting groups are well known to those skilled in the art; examples thereof can be found in PGM Wuts's paper entitled "Greene's Protective Groups in Organic Synthesis" (5th edition), John Wiley & Sons (2014), which is incorporated herein by reference in its entirety. The synthesis of the compounds of the present invention can be accomplished by methods similar to those described in the synthetic schemes and specific embodiments described above.

[0156] The synthetic schemes and specific examples described are illustrative and should not be construed as limiting the scope of the invention, as the scope of the invention is defined in the claims. All alternatives, modifications and equivalents of the synthetic methods and specific examples are included within the scope of the claims.

[0157] Exemplary polymers that have been prepared include those listed in Table 1. Further details on the synthesis of these polymers, including specific examples with actual and / or theoretical percentage modifications, can be found in the Examples.

[0158] Table 1. Exemplary polymers

[0159]

[0160]

[0161] 3. Methods

[0162] Embodiments of the present disclosure include various compositions and methods for delivering biomolecules of interest into cells. According to these embodiments, the compositions and methods include using modified polyamine polymers, such as PEI and PAMAM dendrimers, to deliver deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, peptides, polypeptides, proteins, ribonucleoproteins, or any combination or derivative thereof into cells, and in some cases, electroporation is not required.

[0163] Compositions comprising PEI or PAMAM dendrimers or any combination thereof as disclosed herein can be used to deliver nucleic acid molecules into cells (e.g., gene transfection). In some embodiments, nucleic acids are polynucleotides comprising DNA, RNA, or a combination and derivative thereof. The terms "nucleic acid molecule," "nucleic acid sequence," and "polynucleotide" are synonyms, intended to encompass polymers of DNA or RNA, which can be single-stranded or double-stranded, and can contain non-natural or altered nucleotides. As equivalents, the terms include analogs and modified polynucleotides of RNA or DNA made from nucleotide analogs, such as, but not limited to, methylated and / or capped polynucleotides. Nucleic acids are typically connected via phosphate bonds to form nucleic acid sequences or polynucleotides, but many other connections (e.g., phosphorothioate, boric acid phosphate, etc.) are known in the art.

[0164] One or more nucleic acid molecules can be DNA, RNA or a combination thereof (e.g., DNA / RNA hybrids). In some embodiments, the nucleic acid molecule is a plasmid. As used herein, the term "plasmid" refers to a small DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently (i.e., as an "episome"). Plasmids are naturally present in bacteria, archaea, and other eukaryotic organisms, usually in the form of small circular double-stranded DNA molecules. Synthetic plasmids are widely used in the art as vectors in molecular cloning to drive the replication of recombinant DNA sequences in host organisms. Plasmid DNA can be produced using conventional molecular biology techniques, such as described in, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press (June 15, 2012), or can be obtained from commercial sources.

[0165] Plasmid can be any suitable recombinant DNA or RNA plasmid, which contains a heterologous nucleic acid sequence to be delivered to a target cell in vitro or in vivo. For purposes such as disease treatment or prevention, the heterologous nucleic acid sequence can encode a gene product (e.g., protein) of interest, and can optionally be in the form of an expression cassette. The term "recombinant" refers to a polynucleotide of semisynthetic or synthetic origin that does not exist in nature or is connected to another polynucleotide in an arrangement not found in nature. As used herein, the term "heterologous" refers to a nucleic acid sequence obtained or derived from an entity that is genetically different from the rest of the entity to which it is compared.

[0166] In some embodiments, PEI or PAMAM dendrimers as disclosed herein can be used to deliver proteins, peptides or antibodies into cells. For example, PEI or PAMAM dendrimers can be used to deliver proteins or peptides (or polynucleotides thereof) that are capable of forming a bioluminescent complex (e.g., complementation), such as but not limited to NanoLuc (SEQ ID NO: 2), HiBiT (SEQ ID NO: 3), LgBiT (SEQ ID NO: 4), SmBiT (SEQ ID NO: 5), DarkBiT (SEQ ID NO: 6), DarkBiT (SEQ ID NO: 7; SEQ ID NO: 8), LgTrip 3092 (SEQ ID NO: 9), LgTrip 3546 (SEQ ID NO: 10), LgTrip 2098 (SEQ ID NO: 11), and SmTrip9 (SEQ ID NO: 12). In some embodiments, complementary bioluminescent proteins or peptides can be used to label one or more proteins of interest or peptides of interest for subsequent evaluation or monitoring based on the detection or non-detection of bioluminescence (e.g., the formation of a bioluminescent complex).

[0167] According to these embodiments, proteins or peptides delivered using modified PEI or PAMAM dendrimers can be used to study protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking, and biological functions of proteins or peptides. For example, peptides or proteins delivered using modified PEI or PAMAM dendrimers can form complementary proteins with target proteins labeled with other fragments for protein quantification in living cells. In one embodiment, HiBiT-tagged protein targets (e.g., proteins of interest containing HiBiT tags) can be quantified by directly delivering LgBiT in cells without the need for a separate gene transfection step (e.g., BacMam transfection, nuclear transfection, etc.).

[0168] In some embodiments, the nucleic acid molecule is a DNA plasmid comprising one or more nucleic acid sequences that mediate target gene editing or modification. For example, the DNA plasmid may comprise components of the CRISPR / Cas9 gene editing system, including but not limited to the Cas9 gene or protein. The CRISPR / Cas gene editing system has been developed to achieve targeted modification of a specific gene of interest in a cell. The CRISPR / Cas gene editing system is based on the RNA-guided Cas9 nuclease from the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system (see, e.g., Jinek et al., Science, 337:816 (2012); Gasiunas et al., Proc. Natl. Acad. Sci. USA, 109, E2579 (2012); Garneau et al., Nature, 468:67 (2010); Deveau et al., Annu. Rev. Microbiol., 64:475 (2010); Horvath and Barrangou, Science, 327:167 (2010); Ma ... Karova et al., Nat. Rev. Microbiol., 9, 467 (2011); Bhaya et al., Annu. Rev. Genet., 45, 273 (2011); Cong et al., Science, 339:819-823 (2013); and U.S. Patents 8,697,359, 8,795,965 and 9,322,037; all of which are incorporated herein by reference in their entirety).

[0169] In some embodiments, the nucleic acid is a guide RNA (gRNA) compatible with the CRISPR / Cas gene editing system, for example, a gRNA comprising a CRISPR targeting RNA (crRNA) and a trans-activating crRNA (tracrRNA). In some embodiments, the crRNA and tracrRNA components of the CRISPR / Cas system are fused into one RNA molecule to target a specific sequence of genomic DNA (gRNA is not found in nature). The crRNA sequence is typically synthesized to target the desired genomic DNA, while the tracrRNA sequence is derived from the bacterial sequence required for complexing with the Cas protein. Guide RNA may also be referred to as single guide RNA (sgRNA).

[0170] In some embodiments, gRNA and Cas9 protein can include ribonucleoprotein (RNP) complex. RNP generally includes purified Cas9 protein compounded with gRNA. Such RNP can be assembled in vitro and can be delivered directly to cells using modified polyamine polymers disclosed herein, such as PEI and PAMAM dendrimers, and in some cases, without electroporation. Compared with plasmid-based Cas9 / gRNA expression, Cas9 RNP can cleave genomic targets with similar efficiency, and can be used for most current genome engineering applications of CRISPR, including but not limited to single gene or multi-gene knockout in various cell types, gene editing using homology-directed repair (HDR), and generation of a large number of genomic deletions.

[0171] In some embodiments, as further disclosed herein, modified polyamine polymers, such as PEI and PAMAM dendrimers, are used to deliver Cas9 RNPs to cells to facilitate the insertion of luminescent peptides or polypeptide tags into the protein of interest in the cell. In some embodiments, such RNPs are delivered to cells to cause the expression of the protein of interest and the luminescent tag (e.g., as measured by the luminescence of the tag) and / or may cause the generation of clones showing stable expression of the protein of interest and the luminescent tag. For example, Cas9 RNPs may include Cas9 protein, gRNA, and donor DNA templates. The donor DNA template may include polynucleotides encoding a peptide or polypeptide that can have luminescent activity and a genomic sequence that facilitates the insertion of the peptide or polypeptide into the vicinity of the endogenous protein of interest (e.g., homology arm). RNPs comprising Cas9 protein, gRNA, and donor DNA templates may be incorporated into a composition comprising a modified polyamine polymer of the present disclosure to facilitate delivery into cells, thereby instantaneously and stably quantifying protein expression (e.g., luminescence).

[0172] In some embodiments, the donor DNA template includes a sequence encoding HiBiT (SEQ ID NO: 3), which is an 11 amino acid polypeptide that can produce bright quantitative luminescence through high affinity complementation with the 18kDa subunit (LgBiT) derived from NanoLuc. According to these embodiments, RNPs having donor DNA comprising a sequence encoding HiBiT can be delivered to cells using the modified polyamine polymers of the present disclosure. In some embodiments, in order to produce luminescence, a luminescent substrate and LgBiT (SEQ ID NO: 4) are added to a cell lysate or delivered directly to a cell using the modified polyamine polymers of the present disclosure. In the presence of the substrate, complementation of HiBiT and LgBiT will produce a luminescent signal proportional to the expression of the protein of interest. In some cases, the delivery of RNPs and / or luminescent peptides or polypeptides to cells using the modified polyamine polymers of the present disclosure does not require the use of other delivery methods, such as electroporation, and does not cause significant cytotoxicity.

[0173] In some embodiments, the donor DNA template includes a sequence encoding an 11 amino acid polypeptide (e.g., SmBiT (SEQ ID NO: 5)), which is capable of producing bright quantitative luminescence through high affinity complementation with an 18 kDa subunit (e.g., LgBiT) derived from NanoLuc. According to these embodiments, RNPs having donor DNA comprising a sequence encoding SmBiT can be delivered to cells using the modified polyamine polymers of the present disclosure. In some embodiments, to produce luminescence, a luminescent substrate and LgBiT (SEQ ID NO: 4) are added to a cell lysate or delivered directly to a cell using the modified polyamine polymers of the present disclosure. In the presence of the substrate, complementation of SmBiT and LgBiT will produce a luminescent signal proportional to the expression of the protein of interest. In some cases, the delivery of RNPs and / or luminescent peptides or polypeptides to cells using the modified polyamine polymers of the present disclosure does not require the use of other delivery methods, such as electroporation, and does not cause significant cytotoxicity.

[0174] In some embodiments, the donor DNA template comprises a sequence encoding any peptide or polypeptide disclosed in U.S. Patent No. 9,797,890, which is incorporated herein by reference in its entirety and for all purposes. For example, the donor template may include a polynucleotide encoding a peptide or polypeptide comprising a single amino acid difference from MGVTGWRLCERILA (SEQ ID NO: 8). In some embodiments, the donor template may include a polynucleotide encoding a peptide or polypeptide comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from MGVTGWRLCERILA (SEQ ID NO: 2), or any other peptide or polypeptide disclosed in U.S. Patent No. 9,797,890.

[0175] In some embodiments, the donor DNA template comprises a sequence encoding any peptide or polypeptide disclosed in U.S. Provisional Patent Serial No. 62 / 684,014, which is incorporated herein by reference in its entirety and for all purposes. For example, the donor template may include a polynucleotide encoding a peptide or polypeptide comprising a single amino acid difference from LgTrip3092 (SEQ ID NO: 9), LgTrip 3546 (SEQ ID NO: 10), LgTrip 2098 (SEQ ID NO: 11), or SmTrip9 (SEQ ID NO: 12). In some embodiments, the donor template may include a polynucleotide encoding a peptide or polypeptide comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from SEQ ID Nos: 9-12, or any other peptide or polypeptide disclosed in U.S. Provisional Patent Serial No. 62 / 684,014.

[0176] Embodiments of the present disclosure also include identifying the best modified PEI or PAMAM concentration or a combination thereof at the optimal concentration of the biomolecule of interest, which can provide acceptable results in the context of, for example, transfection efficiency and toxicity levels. These ratios or concentrations can be determined empirically and depend on a variety of factors, including but not limited to the amine modification percentage in PEI and PAMAM, the biomolecule of interest (e.g., polynucleotides, polypeptides), cell types, cell density, the nature of the assay, etc. In some embodiments, the optimal concentration is in the range of nM to μM. In some embodiments, the optimal concentration is in the range of 1 μM to 20 μM.

[0177] Cells that can be used together with the compositions and methods of the present disclosure include any suitable prokaryotic or eukaryotic cells. Suitable cells can include those that grow easily and reliably, have a fairly fast growth rate, have a well-characterized expression system, and can be easily and effectively transformed or transfected. Examples of suitable prokaryotic cells include, but are not limited to, cells from Bacillus (Bacillus) (e.g., Bacillus subtilis and Bacillus brevis), Escherichia (Escherichia) (e.g., Escherichia coli), Pseudomonas (Pseudomonas), Streptomyces (Streptomyces), Salmonella (Salmonella) and Erwinia (Erwinia). Particularly useful prokaryotic cells include various Escherichia coli strains (e.g., K12, HB101 (ATCC No.33694), DH5a, DH10, MC1061 (ATCC No.53338) and CC102). Suitable eukaryotic cells are known in the art and include, for example, yeast cells, insect cells and mammalian cells, including primary cells and transformed cells. In some embodiments, the cell is a mammalian cell. Many suitable mammalian host cells are known in the art, and many can be obtained from the American Type Culture Collection (ATCC, Manassas, VA). The example of suitable mammalian cells includes, but is not limited to, Chinese hamster ovary cells (CHO) (ATCC No. CCL61), CHO DHFR- cells (Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573) and 3T3 cells (ATCC No. CCL92). Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and CV-1 cell line (ATCC No. CCL70).

[0178] Embodiments of the present disclosure also include kits comprising various components described herein. For example, the kit may include any modified polyamine polymer compound or its salt described herein, as well as a container and / or instructions. The kit may also include at least one of a DNA molecule, an RNA molecule, a peptide, a polypeptide, a protein, or any combination or derivative thereof. In some embodiments, the kit includes a peptide or polypeptide of a Cas9 protein. In some embodiments, the kit includes a donor DNA template, and the donor DNA template includes a sequence encoding a luminescent peptide or polypeptide (e.g., HiBiT, LgBiT). In some embodiments, the kit includes gRNA. In some embodiments, the kit includes an RNP complex, and the RNP complex includes, for example, a peptide or polypeptide of a Cas9 protein, a donor DNA template and a gRNA comprising a sequence encoding a luminescent peptide or polypeptide (e.g., HiBiT, LgBiT).

[0179] 4. Examples

[0180] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and understandable, and may be performed using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the present disclosure will be more clearly understood by reference to the following examples, which are merely intended to illustrate some aspects and embodiments of the present disclosure and should not be construed as limiting the scope of the present disclosure. The disclosures of all journal references, U.S. patents, and publications cited herein are incorporated herein by reference in their entirety.

[0181] The present disclosure has many aspects, as illustrated by the following non-limiting examples:

[0182] In the Examples, the following abbreviations are used: Boc is tert-butyloxycarbonyl; DMEM is Dulbecco's Modified Eagle Medium; DMF is N,N-dimethylformamide; EtOH is ethanol; FBS is fetal bovine serum; MeOH is methanol; THF is tetrahydrofuran;

[0183] Example 1

[0184] Compound synthesis

[0185] I. Urethane modification

[0186] Synthesis of fluorinated alkyl p-nitrophenyl carbonate.

[0187]

[0188] To a solution of fluorinated alcohol (1 eq.) and p-nitrophenyl chloroformate (1.5 eq.) in anhydrous THF was added anhydrous pyridine (3 eq.) at 0°C. The mixture was stirred at room temperature overnight. After filtering to remove the white solid, the solvent in the filtrate was evaporated and the residue was purified by passing through a silica gel column using heptane / ethyl acetate as eluent to give the desired compound in 80-90% yield.

[0189] (3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl) 4-nitrophenyl carbonate (WZ-0845): 1 H NMR (CD2Cl2, δppm): 8.31 (d, 2H), 7.46 (d, 2H), 4.62 (t, 2H, CH2O), 2.69 (m, 2H, CH2). MS(m / e)[M+H](C 15 H8F 13 NO5): Calculated value 529.21, observed value 529.4.

[0190] 4-Nitrophenyl (3,3,4,4,5,5,6,6,6-nonafluorohexyl) carbonate (WZ-0921). 1 HNMR (CD2Cl2, δppm): 8.33 (d, 2H), 7.47 (d, 2H), 4.64 (t, 2H, CH2O), 2.70 (m, 2H, CH2). MS(m / e)[M+H](C 13 H8F9NO5): Calculated 429.19, observed 430.2.

[0191] 4-Nitrophenyl (2,2,3,3,4,4,5,5,6,6,6-Undecafluorohexyl)carbonate (WZ-0851). 1 HNMR (CD2Cl2, δppm): 8.34 (d, 2H), 7.47 (d, 2H), 4.84 (t, 2H, CH2). MS(m / e)[M+H](C 13 H6F 11 NO5): Calculated value 465.18, observed value 465.2.

[0192] 4-Nitrophenyl (2,2,3,3,4,4,5,5,5-nonafluoropentyl) carbonate (WZ-0886). 1 HNMR (CD2Cl2, δppm): 8.34 (d, 2H), 7.47 (d, 2H), 4.85 (t, 2H, CH2). MS(m / e)[M+H](C 12 H6F9NO5): Calculated 415.17, observed 415.3.

[0193] 4-Nitrophenyl (3,3,4,4,5,5,5-heptafluoropentyl) carbonate (WZ-0887). 1 H NMR (CD2Cl2, δppm): 8.32 (d, 2H), 7.45 (d, 2H), 4.64 (t, 2H, CH2O), 2.68 (m, 2H, CH2). MS(m / e)[M+H](C 12 H8F7NO5): Calculated 379.19, observed 379.2.

[0194] Synthesis of carbamate-modified PEI 7677. As shown in Scheme 4, 0.1 g / ml THF solution of 4-nitrophenyl (3,3,4,4,5,5,6,6,6-nonafluorohexyl)carbonate (WZ-0850) was added to 4 vials containing PEI (MW 800) and 150 mg (3.49 mmol monomer) in 5 ml THF / 1 ml MeOH in an amount equal to 16% (0.24 g, 0.56 mmol), 27% (0.40 g, 0.94 mmol), 42% (0.63 g, 1.46 mmol) and 53% (0.79 g, 1.84 mmol) of monomer mole percentage, respectively. The resulting solution was stirred for two days. The four separate solutions were then transferred to four Float-A-Lyzer G2 dialysis devices (0.5-1.0 kD, 10 ml) and dialyzed sequentially with methanol, 0.02 M HCl in methanol, and 0.02 M HCl in H2O for three days. The mixture was further transferred to four test tubes filled with tert-butyl alcohol (10 ml). The resulting four mixtures were filtered into four vials, and the filtrate was freeze-dried for two days to obtain white powders 7667-a-1, 7667-a-2, 7667-a-3, and 7667-a-4 with four different theoretical modification percentages. By 1 The actual modification percentages obtained by H NMR were 10.2%, 16.4%, 22.0% and 30.0%, respectively.

[0195] Scheme 4. Synthesis of carbamate-modified #7667.

[0196]

[0197]

[0198] Other urethane modified branched PEI, linear PEI or PAMAM were prepared by using similar methods described for 7667. The theoretical and actual modification percentages of the PEI monomer or PAMAM primary amine are listed in Table 2.

[0199] Table 2. Theoretical and actual modification percentages of PEI monomers of different sizes or PAMAM free amines of different generations modified by different fluorinated carbamates.

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] II. Imine reduction modification

[0206] Synthesis of PEI 7636 modified by imine reduction. As shown in Scheme 5, 4 vials containing 100 mg (2.33 mmol monomer) of PEI (MW 800) in 2 ml MeOH were added with 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanal (0.5 g / ml) in THF in amounts equal to 16% (0.135 g, 0.37 mmol), 21% (0.181 g, 0.50 mmol), 27% (0.226 g, 0.62 mmol) and 38% (0.315 g, 0.87 mmol) of monomer molar percentage, respectively. The resulting solution was stirred overnight. 43 mg, 57 mg, 71 mg, 85 mg and 99 mg of NaBH4 were slowly added to the above 4 corresponding solutions at 0°C, respectively. The mixture was stirred at room temperature for 2 hours until the bubbles disappeared. Then four independent solutions are transferred in four Float-A-Lyzer G2 dialysis devices (0.5-1.0 kD, 10 ml), and successively with methanol, 0.02 M HCl methanol solution and 0.02 M HCl H O solution dialyzed for three days. The mixture is further transferred in 4 different test tubes equipped with tert-butyl alcohol (10 ml). The resulting 4 kinds of mixtures are filtered in 4 vials, and the filtrate is freeze-dried for two days to obtain light yellow powder 7636-b-1, 7636-b-2, 7636-b-3 and 7636-b-4 with 4 different theoretical modification percentages.

[0207] Scheme 5. Synthesis of PEI7636-b modified by imine reduction.

[0208]

[0209]

[0210] Other imine reduction modified branched PEIs were prepared by using a similar method as described for 7636. The theoretical modification percentages of the PEI monomers are listed in Table 3.

[0211] Table 3. Theoretical modification percentages of PEI monomers of different sizes after being modified by different fluorinated aldehydes via imine reduction.

[0212]

[0213]

[0214] Pentafluorobenzyl-modified PEI 7669 and 7677 and picolyl-modified PEI 7676 can be prepared by similar methods via imine reduction. Table 4 lists the theoretical and actual modification percentages of PEI monomers.

[0215]

[0216] Table 4. Aromatic ring modified PEI via imine reduction

[0217]

[0218]

[0219] III. Alkylated PEI via BOC protection, alkylation and deprotection.

[0220] BOC protected PEI. 2.5 g (58.1 mmol monomer) of PEI 1200 or PEI 25000 and NaHCO3 were dissolved in 20 ml of THF / 40 water. THF was added to a 20 ml solution of BOC-anhydride (5.07 g, 23.3 mmol) at 0°C. The mixture was stirred at room temperature overnight. The mixture was then heated to 65°C overnight. TLC confirmed the complete disappearance of BOC-anhydride by iodine staining. After removing THF, the solution was frozen and dried by lyophilization. BOC protected PEI was purified by HPLC. 1 HNMR confirmed.

[0221] Alkylation of BOC-protected PEI. BOC-PEI (0.20 g, 4.65 mmol) was suspended in EtOH or DMF. 1,1,1,2,2,3,3,4,4-nonafluoro-6-iodohexane was added to the mixture in an amount equal to 30%, 40% and 50% of the PEI monomer, and the mixture was heated to 95°C for 48 hours. After removal of EtOH, the residue of each reaction was triturated with ether, or the ether was poured into each individual reaction DMF solution and the solvent was removed by centrifugation. The light yellow solid from each reaction was then washed with ether and removed by centrifugation twice more and dried in vacuo. FNMR confirmed the alkylated BOC-PEI.

[0222] Deprotection of alkylated BOC-PEI. 12 ml of TFA / DCM (1:1) was added to each of the above white solids in the presence of TIPS (0.1 ml). The mixture was stirred at room temperature for 3 hours. After removal of the solvent, the residual solid was ground with ether, centrifuged 3 times, and dried in vacuo. The solid was dissolved in water and dialyzed three times in H2O for three days. The solution was lyophilized to obtain a light yellow powder. The percentage of modification was calculated by HNMR and FNMR using CF3CH2OH as an internal control (Table 5).

[0223] Table 5. Alkylation percentage of PEI monomers of different sizes via BOC protection and deprotection

[0224]

[0225]

[0226] Example 2

[0227] Gene delivery via modified PEI polymers

[0228] In transfection, 90 ng / well of vector DNA (Promega catalog number E4881) was diluted with 10 ng / well One day before the expression construct (Promega catalog # N1501), HEK293 cells were cultured in 100 μl DMEM / FBS at 1.5×10 5 Cells were transfected with various concentrations of various modified PEI polymers and compared with a standard 3:1 ratio of FuGENE HD positive control (Promega catalog number E2311) and a negative control of buffer alone. luciferase assay reagent (Promega catalog # N1110), these conditions provide a wide dynamic range for measuring transfection efficiency. In some cases, transfections can also be performed in serum-deficient medium, with serum added several hours after transfection. Reagent (Promega catalog # G7570) was added to duplicate wells to measure toxicity.

[0229] A 9:1 ratio of PEI monomers (N atoms) to DNA phosphorus atoms was initially chosen as the default 1x concentration rather than using a weight-to-weight ratio. Both transfection efficiency and toxicity are affected by this ratio. The different concentrations of PEI used relative to the standard 1x molar concentration are reported.

[0230] Initial experiments using this fixed ratio of monomer to DNA showed that the modified PEI polymer was as effective as FuGENEHD in transfecting HEK293 cells. Transfection efficiencies as measured by expression can be as high as with FuGENE HD, with no DNA uptake in the absence of the reagent, as shown in Figure 1A. The degree of modification of the base polymer has a dramatic effect on both transfection efficiency and toxicity, highlighting the importance of fine-tuning the chemical synthesis for this application. At least at this constant ratio of PEI monomers, compounds based on the 800 Da polymer were more effective than those based on the 25,000 Da polymer, with 7636-2 and 7633-5 performing particularly well. PEI compounds based on the 25,000 Da polymer were generally more sensitive to the presence of serum during transfection, indicating relatively low transfection, while the 800 Da polymer showed less of this effect.

[0231] Cell viability data are shown in Figure 1 B. Most compounds were well tolerated.

[0232] Further experiments showed that additional modifying groups could be used to increase transfection efficiency or reduce toxicity, particularly compared to MW 800Da polymer starting material, which showed negligible transfection and high toxicity. As shown in Figure 2, the unmodified 25,000Da polymer already showed relatively high transfection efficiency and low toxicity. The 7669 series of modified 25,000Da polymers were likewise very efficient at all modification levels. On the other hand, the efficiency of the 7666 series decreased with increasing modification levels. In the case of 7666-1, increasing the polymer to DNA ratio decreased transfection efficiency. However, for 7667-2, which is based on an 800Da polymer, efficient transfection was only seen at concentrations at least twice that of the standard 1x conditions. 7636-2, 7676-3, and 7677-2 all gave good results at 1x concentrations.

[0233] To better determine the optimal concentration of each compound, titrations of various modified PEIs were added to a constant amount of DNA and used to transfect HEK293 cells. The data are shown in Figure 3, where Figure 3A shows the data in the presence of serum and Figure 3B shows the data in the absence of serum. Figure 3C shows the data in the presence of serum using Cell viability measured by luminescent cell viability assay (Promega catalog number G7570). In the legend, MW800 and MW25000 refer to unmodified PEI polymers with specified molecular weights. In this figure, the relative PEI concentration of 1 indicates that the default ratio of PEI monomer to DNA phosphorus is 9:1. When comparing transfection efficiency, the experiment determined the optimal molar ratio of PEI monomer to DNA for each compound. At twice the standard ratio, 7636-2 and 7667-4 both showed higher transfection efficiency than FuGENE HD without significant toxicity. In the absence of serum, 7676-1 was also able to compete with FuGENE HD.

[0234] A panel of polymers, including PEI polymers or PAMAM polymers of different molecular weights, was used to transfect nine cell lines at different polymer concentrations. 10 ng / well of vector DNA (Promega catalog number E4881) diluted with 90 ng / well was used for transfection. One day before expression of the construct (Promega catalog number N1501), HEK293, HeLa, MDA-MB-231, HepG2, A375, HCT116, U2-OS, Jurkat, and A549 cells were cultured in 100 μl DMEM / FBS (HEK293, HeLa, MDA-MB-231, HepG2, A375, HCT116, U2-OS), RPMI / FBS (Jurkat), or F12 / FBS (A549) at 1.0 × 10 5 Cells were transfected with various modified PEI or PAMAM polymers at different concentrations and incubated with FuGENEHD and ViaFect at a 3:1 ratio. TM Positive controls (Promega catalog numbers E2311 and E4981) and a negative control of buffer alone were compared. Luciferase Assay Reagent (Promega Catalog # N1110). These conditions provide a wide dynamic range for measuring transfection efficiency ( Figures 4A-4I ). By converting the restored Reagent (Promega catalog # G7570) was added to duplicate wells to measure toxicity ( Figures 5A-5H At the optimal concentration, the third generation PAMAM polymer 7767-4 showed good More than ViaFect in expression TM The performance of the FuGENE HD control is comparable or nearly comparable.

[0235] To further investigate the ability of PAMAM polymers to transfect a variety of cell types, titrations of PAMAM polymers of varying sizes and modification levels were used to transfect five different cell lines (Figures 6-9). One day before the expression construct (Promega catalog number N1501), HEK293, HeLa, MDA-MB-231, HepG2, and Jurkat cells were cultured in 100 μl DMEM / FBS at 1.5×10 5 Cells were plated at 10 cells / mL (all cell types except Jurkat plated in RPMI / FBS). Cells were transfected with titrations of various PAMAM polymers: 7766 (-1 to -4), 7767 (-1 to -4) and 7768-1b (FIGS. 6 and 7) and 7768 (-2 to -4), 7825 (-1 to -4) and 7826 (-1 to -4) (FIGS. 8 and 9). FuGENE HD and ViaFect were used in a 3:1 ratio. TM (Promega catalog numbers E2311 and E4981) were used as positive controls, while buffer alone was used as a negative control. Transfection efficiency was measured by luciferase assay (Promega catalog number N1110). Figures 6A-6E and 8A-8E ). By adding the restored Reagent (Promega Cat. No. G7570) was added to duplicate wells of all cells except HeLa cells to measure toxicity ( Figures 7A-7D and 9A-9D ). The degree of modification of the polymer has an impact on the optimal concentration and transfection efficiency. While 7767-4 again showed effective transfection of all cell types over a wide range of concentrations, the less modified versions 7767-2 and 7767-3 required higher concentrations, but those higher concentrations could deliver similar or even greater amounts of DNA than 7767-4.

[0236] Example 3

[0237] LgBiT delivery

[0238] This example involves CRISPR-mediated tagging, as outlined in Schwinn et al., "CRISPR-Me diated Tagging of Endogenous Proteins with a Luminescent Peptide," ACS Chem. Biol. 2018, 13, 467-474. HiBiT-edited cells were generated by CRISPR, and clones were isolated by single-cell sorting. The clones were plated in 96-well plates at 10,000 cells per well. After 24 hours, the cells were transfected with complexes of different modified PEI (1 μg / ml) and LgBiT protein (170 nM). The complex was prepared by incubating modified PEI (100 μg / ml) with LgBiT (17 μM) in Opti-MEM I reduced serum medium for 30 minutes at room temperature. Cells treated with 10% BacMam-LgBiT to transduce CMV / LgBiT expression constructs were used as benchmark controls. After 24 hours, cells were treated with DarkBiT (SEQ ID NO: 6 or SEQ ID NO: 7) to quench the luminescent activity of extracellular LgBiT. DarkBiT is a peptide with high affinity for LgBiT (the LgBiT / DarkBiT complex produces little or no luminescence) (1 μM). Live Cell Assay System (Promega Catalog No. N2011) and The cells were assayed using the 2.0 assay (Promega catalog number G7570). Luminescence was measured on a Discover. Data were analyzed using Prism 5.0 software (GraphPad). The results are shown in Figures 10-11.

[0239] The brightness of the luminescent signal is proportional to the amount of intracellular LgBiT delivered, which may be related to the efficiency of protein uptake. In general, large-size PEI (MW = 25,000), whether modified or not, performs better than its corresponding small-size PEI (MW = 800) in LgBiT delivery. Modified large-size PEI is better than unmodified PEI. Among the top hits, 7666-3, 7668-1, 7669-2, 7675-1, and 7709-3 were the best in promoting LgBiT uptake in 5 different HiBiT clones (Figures 10 and 11). Among the 6 best candidates, 7669-2 and 7675-1 caused marginal cytotoxicity. No toxicity was observed for 7666-3, 7668-1, and 7709-3 (Figures 11A and 11B).

[0240] HDAC2-HiBiT and HDAC6-HiBiT clones of HeLa cells were seeded in 8-well chamber slides at 25,000 cells per well. After 24 hours, cells were transfected with complexes of different PEI (1 μg / ml) and LgBiT protein (170 nM). Complexes were prepared by incubating 17 μM LgBiT with 100 μg / ml 7666-4, 7668-1, or 7669-2 in Opti-MEM I reduced serum medium for 30 minutes at room temperature. After 24 hours, cells were switched to CO2-independent medium and added Living cell substrates. Cells were imaged using a bioluminescence imager ( Figures 12A-12C ).

[0241] HiBiT-edited HeLa or HEK293 cells were plated at 10,000 cells / well in the wells of a 96-well plate. After 24 hours, cells were transfected with complexes of different PEIs (modified and unmodified; 5ug / ml) and LgBiT protein (200nM). Complexes were prepared by incubating PEI with LgBiT in Opti-MEM reduced serum medium for 30 minutes at room temperature. Cells transduced with CMV-LgBiT expression constructs treated with 10% BacMam-LgBiT were used as controls. After 24 hours, the cell culture medium was changed and the cells were transfected with Live Cell Assay System (Promega Catalog No. N2011) and The cells were assayed using the 2.0 assay (Promega catalog number G7570). Luminescence was measured on a 100 μM RT-PCR platform. Figure 13A depicts the percentage of LgBiT protein delivered compared to LgBiT protein delivered via BacMam transduction. The brightness of the luminescent signal is proportional to the amount of intracellular LgBiT delivered into the cells, which may be related to protein uptake efficiency. Figure 13B depicts the percentage of viable cells relative to untreated cells.

[0242] HiBiT-edited HeLa or HEK293 cells were plated at 25,000 cells / well in 400uL in 8-chamber slides. After 24 hours, cells were transfected with PBI-7666-3·LgBiT complex. Complexes were prepared by incubating 2uM LgBiT with 100ug / mL PBI-7666-3 in Opti-MEM I Reduced Serum Medium for 30 minutes at room temperature. 40 microliters of complex was added to each well. After 24 hours, cells were switched to CO2-independent medium and added Live cell substrate (Promega catalog number N2011). Cells were imaged with a bioluminescent imager. Cells treated with 10% BacMam-LgBiT to transduce the CMV-LgBiT expression construct were used as controls. FIG. 14A shows images of CASP3-HiBiT and EGFR-HiBiT clones of HeLa cells; FIG. 14B shows images of GSK3b-HiBiT and HDAC6-HiBit clones of HeLa cells; and FIG. 14C shows images of HDAC2-HiBiT clones of HeLa cells and CDK11-HiBiT clones of HEK293 cells.

[0243] Example 4

[0244] HaloTag-LgBiT delivery

[0245] HeLa cells were plated at 25,000 cells per well in 8-well chamber slides. After 24 hours, cells were transfected with different complexes of PEI (1 μg / ml) and HaloTag-LgBiT protein (200 nM). Complexes were prepared by incubating PEI (100 μg / ml) with HaloTag-LgBiT (20 μM) in Opti-MEM I reduced serum medium for 30 minutes at room temperature. After 24 hours, cells were switched to Opti-MEM I reduced serum medium and then incubated with Oregon The cells were incubated with ligand (1 μM) for 30 minutes. The cells were washed 3 times with Opti-MEM I reduced serum medium. The last wash was completed by incubating the cells in the medium at 37°C under 5% (v / v) CO2(g) for 30 minutes. During the last 5 minutes, the cells were probed with nuclear probe at 37°C. Live The cells were counterstained with 5% paraformaldehyde and 1% paraformaldehyde reagent. The cells were imaged using a C2 laser scanning confocal microscope (Nikon).

[0246] The green fluorescence intensity is proportional to the amount of intracellular HaloTag-LgBiT delivered, which may be related to the protein uptake efficiency. The modified large-size PEI (MW = 25000) is superior to the unmodified PEI in delivering HaloTag-LgBiT. Among the top hits, 7636-2, 7637-3, 7666-3, 7668-1 and 7676-5 have the best effect ( Figures 15A-15C Among them, three were also the best candidates for LgBiT delivery (7666-3 and 7668-1), suggesting that the size of the cargo affects the uptake efficiency.

[0247] Punctate staining was observed in all treatments, indicating that most of the HaloTag-LgBiT protein was localized in endosomes. The brightness in endosomes poses a technical challenge for quantifying the fraction of protein released into the cytosol.

[0248] Example 5

[0249] RNP delivery

[0250] HiBiT knocked into the C-terminus of GAPDH in HEK293 cells. RNP complex assembly and preparation for electroporation of complexes into cells were performed as previously described (Schwinn et al. "CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide," ACS Chem. Biol. 2018, 13, 467-474). Briefly, in a 5 μl reaction, a duplex of tracer RNA and guide RNA was prepared (to 24 μM), which was then combined with 5 μl Cas9 (20 μM) for further incubation at room temperature for 10 minutes. For HEK293, cells (2×10 5 ) were resuspended in 20 μl of 4D Nucleofector solution SF. RNP complexes (10 μl) and donor DNA (1 μl of 100 μM single-stranded oligodeoxynucleotide, ssODN) were then added to the cells. The donor DNA was designed to add the VS-HiBiT sequence to the C-terminus of the GAPDH gene. The cells were electroporated using the 4D Nucleofector system. The cells were then incubated at room temperature for 5 minutes and then transferred to a 6-well plate for culture.

[0251] A similar procedure was used for the initial RNP delivery experiments using modified PEI. Briefly, the RNP mixture (10 μl of 12 μM double-stranded guide RNA and 10 μM Cas9) was prepared as described above. After adding donor DNA (1 μl, 100 μM) to the RNP mixture, the mixture was made up to 98 μl using Opti-MEM I reduced serum medium. Modified PEI 7667-4 (2 μl, 10 mg / ml) was then added to the RNP reaction and incubated at room temperature for 30 minutes. The complex (100 μl) was then added to the cells (2×10 5 The cells were cultured in serum-free medium for 1 hour. Afterwards, the cells were supplemented with FBS (10%) for growth and measurement. After 24-48 hours, the cells were cultured using HiBiT Lysis Reagent and 2.0 Determination of edited cells. Use The percentage of live cells was calculated relative to untreated cells using a luminescent cell viability assay (Promega Cat. No. G7570) ( FIG. 16B ).

[0252] The HiBiT luminescence signal normalized to the number of viable cells should be proportional to the HiBiT insertion efficiency at the GAPDH locus, which may be correlated with the RNP delivery efficiency. Nucleofection is expected to provide the most efficient RNP / ssODN delivery. As shown in Figure 16A, under initial conditions, nucleofection showed a significantly higher efficiency than FuGENE HD, ViaFect TM , CRISPRMax, or 7667-4. However, even without optimizing the modified PEI compound or delivery conditions, the RNP delivery efficiency of 7667-4 was higher than that of FuGENE HD and ViaFect TM Much better, and only slightly lower than CRISPRmax.

[0253] Model system for measuring HiBiT knock-in, target expression, and viability in the same sample. To screen many compounds and delivery conditions, a plate-based system that provides more information is needed. HiBiT is knocked into the N-terminus of the firefly luciferase (Fluc) gene in the HEK293 / CMV-Fluc stable cell line. The gRNA is designed to generate a double-strand break within the start methionine codon so that non-homologous end joining (NHEJ) repair will tend to generate insertion / deletion (InDel) mutations that knock out expression, while homology-directed repair (HDR) will generate HiBiT signal and retain Fluc signal (Figure 1A). For measuring cell viability, CellTiter-Fluor (Promega catalog number G6080) is multiplexed so that both Fluc and HiBiT signals can be normalized to the fluorescence of live cells. The normalized ratio of Fluc signal and HiBiT signal can be used to estimate the extent of Fluc knock-out caused by InDel mutations and HiBiT knock-in caused by HDR.

[0254] When optimizing the conditions for delivering RNP / ssODN to cells using modified PEI and PAMAM polymers, various concentrations of RNP, ssODN and polymers are used. Typically, 1.25 μM Cas9 is incubated at room temperature for 10 minutes with 1.5 μM gRNA to produce RNP. Donor ssODN and modified PEI or PAMAM polymers are then added to produce 11x solutions of 250nM RNP, 1 μM ssODN and 22-110 μg / ml polymers. After incubation at room temperature for 30 minutes, the complex is added to the cells and they are diluted 11 times (e.g., 10 μl+100 μl culture medium in 96-well plates) in culture medium. Two days later, HEK293 / Fluc cells are analyzed by replacing cell culture medium with OptiMEM-I containing CellTiter-Fluor reagent (Promega catalog number G6080) to measure the number of viable cells in the well (Figure 17A). Fluc expression was measured using ONE-Glo EX (FIG. 17B), and HiBiT signal was measured using HiBiT NanoDLR assay (FIG. 17C). HiBiT signal was normalized to cell number using HiBiT / CTF ratio (FIG. 17D). HiBi Reporter system (HiBiTNanoDLR TM ) Quantification of Fluc and HiBiT signals in the wells. ( FIG17E ).

[0255] Figures 18A-18B HEK293 / Fluc cell pools are depicted, wherein RNP / ssODN mixtures for knocking in HiBiT through CRISPR have been delivered using nuclear transfection or modified PEI or PAMAM polymers. Cell pools were expanded for many days before measurement to eliminate the complicating factors of cell death during treatment. After knocking in HiBiT at the N-terminal of Fluc, viability, Fluc expression and HiBiT signal (Figure 18 A) were measured using CellTiter-Fluor and HiBiT NanoDLR. The Fluc / CTF ratio of nuclear transfection cells significantly decreased and may indicate that InDel mutations cause expression loss. As indicated by the HiBiT / CTF ratio, the RNP / ssODN delivery mediated by modified polymers causes higher normalized Fluc expression, but also causes higher normalized HiBiT signal. Similarly, after delivery of an RNP / ssODN mixture designed to knock-in HiBiT at the C-terminus of GAPDH, the modified PEI polymer showed a higher normalized HiBiT signal compared to nucleofection ( FIG. 18B ).

[0256] For other cell types, use The HiBiT lysis assay measures the extent of HiBiT insertion and is expressed using CellTiter-Fluor in the same well or in duplicate wells. Viability was measured using the ELISA kit (Promega Catalog No. G7570).

[0257] Scheme 1A. High-throughput model system for measuring HiBiT knock-in and Fluc knock-out.

[0258]

[0259] Figure 1B. gRNA and ssODN design for HiBiT knock-in in HEK293 / CMV-Fluc stable cell line.

[0260]

[0261]

[0262] Table 6. Percentage of clones with significant Fluc and HiBiT signals after single cell sorting of HEK293 / Fluc cell pools with HiBiT knocked in at the Fluc or GAPDH locus. When gRNA targets Fluc instead of GAPDH, InDel mutations generated by NHEJ-mediated repair are expected to disrupt Fluc expression.

[0263] % High Fluc / CTF % High HiBiT / CTF #clone Using nuclear transfection of Fluc 12.93 6.61 234 Using 7740-2's Fluc 16.28 14.73 129 Using 7740-1's Fluc 17.44 18.02 172 Using 7666-5's Fluc 69.39 7.48 147 GAPDH 98.88 2.79 179 Using GAPDH from 7741-2 99.51 6.34 205 Using GAPDH from 7759-3 98.96 7.81 192

[0264] Example 6

[0265] PROTAC degradation of HiBiT-BRD4

[0266] A protein degradation assay was used to demonstrate that PEI delivered functional LgBiT into cells. HiBiT-BRD4 HEK293 cells were plated into wells of a 96-well plate at 10,000 cells / well. After 24 hours, the cells were transfected with a complex of PBI-7666-3 (10ug / ml) and LgBiT protein (200nM). The complex was prepared by incubating PEI with LgBiT in Opti-MEM reduced serum medium for 30 minutes at room temperature. Cells treated with 10% BacMam-LgBiT to transduce the CMV-LgBiT expression construct were used as a control. After 21 hours, the cell culture medium was changed to a medium containing Endurazine substrate (Promega catalog number N2571) fresh medium and incubate for 3 hours. Next, the cells were treated with titration of the bromodomain BET inhibitor MZ1 to target BRD4 for degradation. The Clariostar instrument was used for 24 hours of kinetic measurements. Figure 19A depicts the percentage of degraded BRD4, which is drawn to show the response similarity between 2 delivery methods: BacMam and PEI. Figure 19B depicts the percentage of degraded BRD4 after MZ1 treatment for 24 hours, and it responds in a dose-dependent manner.

[0267] Example 7

[0268] Removal of extracellular LgBiT

[0269] HiBiT-KRAS clones from 4 different cell backgrounds (HEK293, A549, H1299 or MiaPaCa-2) were plated into wells of a 96-well plate at 10,000 cells / well. After 24 hours, the cells were transfected with a complex of PBI-7666-3 (5ug / ml) and LgBiT protein (200nM). The complex was prepared by incubating PEI with LgBiT in Opti-MEM reduced serum medium for 30 minutes at room temperature. Cells treated with 10% BacMam-LgBiT to transduce CMV-LgBiT expression constructs were used as controls. Cells were treated with digitonin (50ug / mL) and LgBiT protein (200nM). Four conditions were examined to distinguish intracellular from extracellular luminescent signals. "No replacement conditions" refer to when cells are treated with LgBiT·7666-3 for 4 hours or 24 hours, and luminescence is measured at a given time without replacing the medium. "Replacement conditions" refer to when cells after LgBiT delivery are replaced with fresh medium in the absence or presence of DarkBiT (SEQ ID NO:6) (1uM). DarkBiT peptide is a cell-impermeable peptide that is required to inactivate extracellular LgBiT. "Washing conditions" refer to when cells after LgBiT delivery are washed once with fresh medium, replaced with fresh medium, and then luminescence is measured. Washing conditions reflect intracellular measurements of NanoBiT. Replacement conditions remove some but not all extracellular LgBiT; therefore, the luminescence signal is lower than no replacement conditions, but higher than wash conditions. At a given time, using Live Cell Assay System (Promega Catalog No. N2011) and The cells were analyzed using the 2.0 assay (Promega catalog number G7570). The luminescence was measured on the Discover. The results are shown in Figures 20A-20D middle.

[0270] The PEI-based system delivers functional LgBiT into cells. The intracellular signals obtained from the PEI method were similar between the incubation times of 4 hours and 24 hours, indicating that this method requires at least 4 hours of incubation time to reach its saturation signal. In contrast, BacMam transduction delivers nucleic acids. For each cell line tested, after a 4-hour incubation time, the signal derived from BacMam was much lower than that of the PEI method. After 24 hours of incubation, in the case of HEK293 cells, the signals between the PEI method and BacMam transduction were comparable. In the other three cases, BacMam transduction was superior to the modified PEI compound in delivering LgBiT.

[0271] It should be understood that the foregoing detailed description and the accompanying examples are illustrative only and should not be taken as limiting the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0272] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present disclosure, including but not limited to those changes and modifications associated with the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the present disclosure.

[0273] The sequences provided below are referenced herein and are provided as part of the accompanying sequence listing and sequence listing statement:

[0274] WTOgLuc (SEQ ID NO: 1)

[0275] MFTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVTINGVTGWRLCENILA

[0276] NanoLuc (SEQ ID NO: 2)

[0277] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0278] HiBiT(SEQ ID NO:3)

[0279] VSGWRLFKKIS

[0280] LgBiT(SEQ ID NO:4)

[0281] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN

[0282] SmBiT(SEQ ID NO:5)

[0283] VTGYRLFEEIL

[0284] DarkBiT(SEQ ID NO:6)

[0285] VSGWALFKKIS

[0286] DarkBiT(SEQ ID NO:7)

[0287] MVSGWALFKKIS

[0288] DarkBiT(SEQ ID NO:8)

[0289] MGVTGWRLCERILA

[0290] LgTrip 3092(SEQ ID NO:9)

[0291] VFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPD

[0292] LgTrip 3546(SEQ ID NO:10)

[0293] VFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD

[0294] LgTrip 2098(SEQ ID NO:11)

[0295] VFTLDDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIMRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNKLNYFGRPYEGIAVFDGKKITTTGTLWNGNKIIDERLITPD

[0296] SmTrip9(SEQ ID NO:12)

[0297] GSMLFRVTINS

Claims

1. A compound or a salt thereof, the compound comprising: Polyethyleneimine polymers; and A plurality of substituents bonded to the amino groups of the polyethyleneimine polymer, wherein each substituent independently has formula (I): -X-(CH2) n -Z (I)， in: X is -C(O)-O-; n is 1, 2, 3, 4 or 5; and Z is a haloalkyl group having the formula: -(CF2) m -CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. 2 . The compound or salt thereof according to claim 1 , wherein the polyethyleneimine polymer has a weight average molecular weight of 500 Da to 250,000 Da. 3 . The compound or salt thereof according to claim 2 , wherein the polyethyleneimine polymer has a weight average molecular weight of 500 Da to 2000 Da.

4. The compound or salt thereof according to claim 2, wherein the polyethyleneimine polymer has a weight average molecular weight of 5000 Da to 250000 Da.

5. The compound or salt thereof according to any one of claims 1 to 4, wherein the polyethyleneimine polymer is a branched polyethyleneimine polymer.

6. The compound or salt thereof according to any one of claims 1 to 4, wherein the polyethyleneimine polymer is a linear polyethyleneimine polymer.

7. The compound or salt thereof according to any one of claims 1 to 4, wherein: n is 1 or 2.

8. The compound or salt thereof according to claim 7, wherein m is 1, 2, 3, 4 or 5.

9. The compound or salt thereof according to any one of claims 1 to 4 and 8, wherein 0.1 mol% to 60 mol% of the amino groups of the polyethyleneimine polymer are bonded to a substituent of formula (I).

10. The compound or a salt thereof according to claim 9, wherein 5 mol% to 50 mol% of the amino groups of the polyethyleneimine polymer are bonded to the substituent of formula (I).

11. The compound or a salt thereof according to claim 10, wherein 8 mol% to 40 mol% of the amino groups of the polyethyleneimine polymer are bonded to the substituent of formula (I).

12. A compound or a salt thereof, the compound comprising: 1st, 2nd, 3rd or 4th generation poly(amidoamine) dendrimers; and A plurality of substituents bonded to the amino groups of the poly(amidoamine) dendrimer, wherein each substituent independently has formula (I): -X-(CH2) n -Z (I)， in: X is -C(O)-O-; n is 1 or 2; and Z is a haloalkyl group having the formula: -(CF2) m -CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

13. The compound or salt thereof according to claim 12, wherein the poly(amidoamine) dendrimer is a third generation poly(amidoamine) dendrimer.

14. The compound or salt thereof according to any one of claims 12 to 13, wherein m is 1, 2, 3, 4 or 5.

15. The compound or salt thereof according to any one of claims 12 to 14, wherein 0.1 mol% to 80 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bonded to substituents of formula (I).

16. The compound of claim 15, wherein 10 mol% to 70 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bonded to substituents of formula (I).

17. The compound of claim 16, wherein 20 mol% to 70 mol% of the primary amino groups of the poly(amidoamine) dendrimer are bonded to substituents of formula (I).

18. A method for delivering a biomolecule into a cell, the method comprising: Contacting the cell with an effective amount of a compound or a salt thereof according to any one of claims 1 to 17; as well as The cell is contacted with the biomolecule.

19. The method of claim 18, wherein the method comprises contacting the cell with an effective amount of two or more different compounds of any one of claims 1 to 18, or salts thereof.

20. The method of claim 18 or 19, wherein the biomolecule is at least one of a deoxyribonucleic acid (DNA) molecule, a ribonucleic acid (RNA) molecule, a peptide, a protein, or any combination thereof.

21. The method of claim 18 or 19, wherein the biomolecule is a polypeptide.

22. The method of claim 18 or 19, wherein the biomolecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule.

23. The method of claim 18 or 19, wherein the biomolecule is a peptide capable of luminescent activity.

24. The method of claim 18 or 19, wherein the biomolecule is a polypeptide capable of luminescent activity.

25. The method of claim 18 or claim 19, wherein the biomolecule comprises the polypeptide sequence of SEQ ID NO:

4.

26. The method of claim 18 or 19, wherein the biomolecule is a ribonucleoprotein complex comprising a Cas9 protein.

27. The method of claim 26, wherein the ribonucleoprotein complex further comprises a guide RNA (gRNA) and a donor DNA template, wherein the donor DNA template comprises a sequence encoding a polypeptide from SEQ ID NO:

3.

28. The method of any one of claims 18-19 and 27, comprising mixing the compound and the biomolecule to form a mixture, and subsequently contacting the cell with the mixture.

29. A kit comprising the compound or salt thereof according to any one of claims 1 to 17.

30. The kit of claim 29, wherein said kit comprises said compound or said salt thereof in a container.

31. The kit of claim 29 or 30, further comprising at least one of a DNA molecule, an RNA molecule, a peptide, a protein, or any combination thereof.

32. The kit of claim 29 or 30, further comprising a polypeptide.

33. The kit of claim 31, wherein the peptide comprises a Cas9 protein.

34. The kit of claim 31, wherein the DNA molecule is a donor DNA template comprising a sequence encoding a polypeptide from SEQ ID NO:

3.

35. The kit of any one of claims 29-30 and 33-34, further comprising instructions for transfecting a biomolecule using the compound or the salt thereof.

36. A method for changing the sequence of an endogenous protein in a cell, the method comprising: Assembling a ribonucleoprotein (RNP) complex containing the Cas9 protein, donor DNA template, and guide RNA; as well as The RNP complex is delivered into a cell using the compound of any one of claims 1-17.

37. A method for labeling an endogenous protein in a cell, the method comprising: Assembling a ribonucleoprotein (RNP) complex comprising a Cas9 protein, a donor DNA template, and a guide RNA, wherein the donor DNA template comprises a sequence encoding a peptide tag sequence; as well as The RNP complex is delivered into a cell using the compound of any one of claims 1-17.

38. The method of claim 37, wherein the donor DNA template comprises a sequence encoding a peptide tag selected from SEQ ID NO: 3 and SEQ ID NO:

5.

39. The method of claim 37, wherein the donor DNA template further comprises homology arms flanking the sequence encoding the peptide tag sequence.

40. A method for labeling an endogenous protein in a cell, the method comprising: Assembling a ribonucleoprotein (RNP) complex comprising a Cas9 protein, a donor DNA template, and a guide RNA, wherein the donor DNA template comprises a sequence encoding a polypeptide tag sequence; and The RNP complex is delivered into a cell using the compound of any one of claims 1-17.

41. The method of claim 40, wherein the donor DNA template further comprises homology arms flanking the sequence encoding a polypeptide tag sequence.

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