# US 2020/0399660 A1 · Modified polyamine polymers for delivery of biomolecules into cells

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- Number: US 2020/0399660 A1
- Document ID: US20200399660A1
- Status: Published application
- Kind: A1
- Application number: 16/909,475
- Filed: 2020-06-23
- Published: 2020-12-24
- Earliest claimed: 2019-06-24
- Applicants: Promega Corporation
- Inventors: Wenhui Zhou; Trish Hoang; Christopher Todd Eggers; Hui Wang; Thomas Machleidt; Brock Binkowski; Poncho Meisenheimer; Frank Fan; Keith Wood
- Web page: https://patentgrep.com/patent/US20200399660A1

## CPC

- C12N 15/907: {in mammalian cells}
- C08G 73/0206: {Polyalkylene(poly)amines}
- C08G 73/028: {Polyamidoamines}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS

## Abstract

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

## Claims

### Claim 1

A compound or a salt thereof, the compound comprising:
a polyethyleneimine polymer; and
a plurality of substituents bound to amino groups of the polyethyleneimine polymer, wherein each substituent independently has a formula (I):
—X—(CH2)n—Z (I),
wherein:
X is a bond or —C(O)—O—;
n is 0, 1, 2, 3, 4, or 5; and
Z is selected from a haloalkyl group, an aryl group, a substituted aryl group, a heteroaryl group, and a substituted heteroaryl group.

### Claim 2 (depends on 1)

The compound of claim 1, or a salt thereof, wherein the polyethyleneimine polymer has a weight average molecular weight of about 500 Da to about 250000 Da.

### Claim at position 3

-4. (canceled)

### Claim 5 (depends on 1)

The compound of claim 1, or a salt thereof, wherein the polyethyleneimine polymer is a branched polyethyleneimine polymer.

### Claim 6 (depends on 1)

The compound of claim 1, or a salt thereof, wherein the polyethyleneimine polymer is a linear polyethyleneimine polymer.

### Claim 7 (depends on 1)

The compound of claim 1, or a salt thereof, wherein Z is a haloalkyl group having the following formula:
—(CF2)m—CF3, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

### Claim 8 (depends on 1)

The compound of claim 1, or a salt thereof, wherein Z is a pentafluorophenyl group or an unsubstituted pyridyl group.

### Claim 9

(canceled)

### Claim 10 (depends on 1)

The compound of claim 1, or a salt thereof, wherein:
X is —C(O)O—; and
n is 1 or 2.

### Claim 11

(canceled)

### Claim 12 (depends on 1)

The compound of claim 1, or a salt thereof, wherein
X is a bond; and
n is 1 or 2.

### Claim 13 (depends on 1)

The compound of claim 1, or a salt thereof, wherein about 0.1 mol % to about 60 mol % of the amino groups of the polyethyleneimine polymer are bound to a substituent of formula (I).

### Claim 14

(canceled)

### Claim 15 (depends on 13)

The compound of claim 13, or a salt thereof, wherein about 8 mol % to about 40 mol % of the amino groups of the polyethyleneimine polymer are bound to a substituent of formula (I).

### Claim 16

A compound or a salt thereof, the compound comprising:
a poly(amidoamine) dendrimer; and
a plurality of substituents bound to amino groups of the poly(amidoamine) dendrimer, wherein each substituent independently has a formula (I):
—X—(CH2)n—Z (I),
wherein:
X is a bond or —C(O)—O—;
n is 0, 1 or 2; and
Z is selected from a haloalkyl group, an aryl group, a substituted aryl, a heteroaryl group, and a substituted heteroaryl group.

### Claim at position 16

17-27. (canceled)

### Claim 28 (depends on 1)

A method of delivering a biomolecule to a cell, comprising:
contacting the cell with an effective amount of a compound of claim 1, or a salt thereof; and
contacting the cell with the biomolecule.

### Claim 29

(canceled)

### Claim 30 (depends on 28)

The method of claim 28, wherein 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 combinations or derivatives thereof.

### Claim at position 20

-32. (canceled)

### Claim 33 (depends on 28)

The method of claim 28, wherein the biomolecule comprises a polypeptide sequence of SEQ ID NO: 4.

### Claim 34 (depends on 28)

The method of claim 28, wherein the biomolecule is a ribonucleoprotein complex comprising a Cas9 protein.

### Claim at position 23

-36. (canceled)

### Claim 37 (depends on 1)

A kit comprising a compound of claim 1, or a salt thereof.

### Claim 38

(canceled)

### Claim 39 (depends on 37)

The kit of claim 37, further comprising at least one of a DNA molecule, an RNA molecule, a peptide, a polypeptide, a protein, or any combinations or derivatives thereof.

### Claim at position 27

-41. (canceled)

### Claim 42 (depends on 37)

The kit of claim 37, further comprising instructions for using the compound or the salt thereof for transfection of a biomolecule.

### Claim 43 (depends on 1)

A method for altering a sequence of 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; and
delivering the RNP complex into a cell using a compound of claim 1.

### Claim 44 (depends on 1)

A method for tagging 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 or polypeptide tag sequence; and
delivering the RNP complex into a cell using a compound of claim 1.

### Claim at position 31

-46. (canceled)