# US 11,304,970 B2 · EGFR gene expression-suppressing siRNA, precursor of same, and applications thereof

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- Number: US 11,304,970 B2
- Document ID: US11304970B2
- Status: Granted
- Kind: B2
- Application number: 16/099,006
- Filed: 2017-05-05
- Published: 2019-08-22
- Granted: 2022-04-19
- Earliest claimed: 2016-05-05
- Assignees: Jiangsu Micromedmark Biotech Co., Ltd.
- Applicants: Jiangsu Micromedmark Biotech Co., Ltd.
- Inventors: Chenyu Zhang; Xi Chen; Hongwei Liang; Uzair Ur-Rehman
- Web page: https://patentgrep.com/patent/US11304970B2

## CPC

- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P 35/02: specific for leukemia
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 2310/122: Hairpin
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/30: Special therapeutic applications
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are an EGFR gene expression-suppressing siRNA, a precursor sequence of same, and uses thereof.

## Claims

### Claim 1

A precursor sequence, wherein the sequence has a structure from the 5′ terminus to the 3′ terminus as shown in formula I:
wherein
B1 is a first ribonucleic acid sequence comprising an EGFR siRNA sense strand sequence;
B2 is a sequence substantially or completely complementary to B1, and B2 is not complementary to C, wherein substantially complementary means there are 2-8 non-complementary bases between B2 and B1;
C is a stem-loop structure sequence; and
A1 is UGCUG and/or A2 is CAGG or CAGGA,
wherein, the nucleotide sequence of the EGFR siRNA sense strand is selected from the group consisting of: SEQ ID NO: 197, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 47, SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 68 and SEQ ID NO: 72;
and the precursor sequence as shown can be processed in a host to form the EGFR siRNA.

### Claim 2 (depends on 1)

The precursor sequence of claim 1, wherein substantially complementary means that there are 3-5 non-complementary bases between the B2 and B1.

### Claim 3 (depends on 1)

A polynucleotide wherein the polynucleotide can be transcribed by a host to form the precursor sequence of claim 1.

### Claim 4 (depends on 1)

An expression vector containing the precursor sequence of claim 1 or a polynucleotide which can be transcribed by a host to form said precursor sequence.

### Claim 5 (depends on 1)

A pharmaceutical preparation comprising:
(a) an expression vector for expression of an siRNA that inhibits EGFR gene expression; and
(b) a pharmaceutically acceptable carrier;
wherein the expression vector expresses the precursor sequence of claim 1.

### Claim 6 (depends on 5)

A method for administering a medicament to a mammal in need thereof, comprising:
administering the pharmaceutical preparation of claim 5 at a first site of the mammal, whereby the expression vector is processed to form a microvesicle in the mammal which is transported to a second site on the mammal where the siRNA is expressed.

### Claim 7 (depends on 1)

A pharmaceutical composition, comprising
the precursor sequence of claim 1 or
an expression vector comprising said precursor sequence or a polynucleotide which can be transcribed by a host to form said precursor sequence, and
a pharmaceutically acceptable carrier.

### Claim 8 (depends on 7)

The pharmaceutical composition of claim 7, comprising an expression vector wherein the expression vector contains said precursor sequence or a polynucleotide which can be transcribed by a host to form said precursor sequence; and optionally, wherein
the dosage form of the pharmaceutical composition comprises a tablet, a capsule, a powder, a pill, a granule, a syrup, a solution, a suspension liquid, an emulsion, a suspension, an injection solution, or an injectable powder.

### Claim 9 (depends on 7)

The pharmaceutical composition of claim 7, adapted for oral, respiratory tract, injection, transdermal, mucosal, or cavity administration.

### Claim 10

An siRNA for inhibiting expression of an EGFR gene, wherein the nucleotide sequence of the sense strand of the siRNA is SEQ ID NO: 197.

### Claim 11 (depends on 1)

A method for inhibiting EGFR or for treating a malignant tumour highly expressing EGFR, wherein the malignant tumour is selected from the group consisting of liver cancer, lung cancer, stomach cancer, oesophageal cancer, ovarian cancer, colorectal cancer, cervical cancer, pancreatic cancer, prostatic cancer, leukaemia, breast cancer, kidney cancer, bladder cancer, oral epithelial cancer, head and neck cancer, brain tumour and glioblastoma, comprising administering to a subject in need thereof,
an effective amount of the precursor sequence of claim 1, or
an expression vector comprising said precursor sequence or a polynucleotide which can be transcribed by a host to form said precursor sequence.

### Claim 12 (depends on 8)

The pharmaceutical composition of claim 8, wherein the dosage form is an injection.

### Claim 13 (depends on 12)

The pharmaceutical composition of claim 12, wherein the injection is an intravenous injection or an intraperitoneal injection.

### Claim 14 (depends on 9)

The pharmaceutical composition of claim 9, adapted for administration by direct injection of a plasmid.

### Claim 15 (depends on 1)

The precursor sequence of claim 1, wherein substantially complementary means that there are 1-2 bases deleted in B2 as compared with B1.

### Claim 16 (depends on 10)

A method for inhibiting EGFR or for treating a malignant tumour highly expressing EGFR, wherein the malignant tumour is selected from the group consisting of liver cancer, lung cancer, stomach cancer, oesophageal cancer, ovarian cancer, colorectal cancer, cervical cancer, pancreatic cancer, prostatic cancer, leukaemia, breast cancer, kidney cancer, bladder cancer, oral epithelial cancer, head and neck cancer, brain tumour and glioblastoma, comprising administering to a subject in need thereof an effective amount of an siRNA of claim 10.