# US 8,541,384 B2 · Compositions and methods for siRNA inhibition of angiogenesis

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- Number: US 8,541,384 B2
- Document ID: US8541384B2
- Status: Granted
- Kind: B2
- Application number: 11/423,025
- Filed: 2006-06-08
- Published: 2007-02-15
- Granted: 2013-09-24
- Earliest claimed: 2002-07-24
- Assignees: The Trustees of the University of Pennsylvania
- Applicants: Michael J. Tolentino; Samuel Jotham Reich
- Inventors: Michael J. Tolentino; Samuel Jotham Reich
- Web page: https://patentgrep.com/patent/US8541384B2

## CPC

- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 15/86: Viral vectors
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N 2310/111: spanning the whole gene, or a large part of it
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/53: partially self-complementary or closed
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

RNA interference using small interfering RNAs which are specific for the vascular endothelial growth factor (VEGF) gene and the VEGF receptor genes Flt-1 and Flk-1/KDR inhibit expression of these genes. Diseases which involve antiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

## Claims

### Claim 1

A pharmaceutical composition comprising an effective amount of a double-stranded short interfering ribonucleic acid (siRNA) and a pharmaceutically acceptable carrier, wherein the short interfering ribonucleic acid (siRNA) comprises a sense RNA strand and an antisense RNA strand, wherein the sense and the antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence identical to a target sequence in human vascular endothelial growth factor (VEGF) mRNA, wherein the nucleotide sequence identical to a target sequence consists of a sequence selected from the group consisting of: SEQ ID NO. 13, 19, 20, 47, 48, 50 , 51, 52, 56, 16, 23, 28, 29, 34, 57, 66, 71, 75, 84, and 88.

### Claim 2 (depends on 1)

The pharmaceutical composition of claim 1, further comprising lipofectin, lipofectamine, cellfectin, polycations, or liposomes.

### Claim 3 (depends on 1)

The pharmaceutical composition of claim 1, wherein the sense and antisense RNA strands forming the RNA duplex are covalently linked by a single-stranded hairpin.

### Claim 4 (depends on 1)

The pharmaceutical composition of claim 1, wherein the short interfering ribonucleic acid (siRNA) further comprises non-nucleotide material.

### Claim 5 (depends on 1)

The pharmaceutical composition of claim 1, wherein the sense and antisense RNA strands are stabilized against nuclease degradation.

### Claim 6 (depends on 1)

The pharmaceutical composition of claim 1, further comprising a 3′ overhang.

### Claim 7 (depends on 6)

The pharmaceutical composition of claim 6, wherein the 3′ overhang comprises from 1 to about 6 nucleotides.

### Claim 8 (depends on 6)

The pharmaceutical composition of claim 6, wherein the 3′ overhang comprises about 2 nucleotides.

### Claim 9 (depends on 1)

The pharmaceutical composition of claim 1, wherein the sense RNA strand comprises a first 3′ overhang, and the antisense RNA strand comprises a second 3′ overhang.

### Claim 10 (depends on 9)

The pharmaceutical composition of claim 9, wherein the first and second 3′ overhangs separately comprise from 1 to about 6 nucleotides.

### Claim 11 (depends on 9)

The pharmaceutical composition of claim 9, wherein the first 3′ overhang comprises a dinucleotide and the second 3′ overhang comprises a dinucleotide.

### Claim 12 (depends on 11)

The pharmaceutical composition of claim 11, where the dinucleotide comprising the first and second 3′ overhang is dithymidylic acid (TT) or diuridylic acid (uu).

### Claim 13 (depends on 6)

The pharmaceutical composition of claim 6, wherein the 3′ overhang is stabilized against nuclease degradation.

### Claim 14 (depends on 6)

The pharmaceutical composition of claim 6, wherein the sense and antisense RNA strands forming the RNA duplex are covalently linked by a single stranded hairpin.

### Claim 15 (depends on 1)

The pharmaceutical composition of claim 1, wherein said short interfering ribonucleic acid (siRNA) is naked siRNA.

### Claim 16 (depends on 15)

The pharmaceutical composition of claim 15, wherein said pharmaceutically acceptable carrier consists essentially of saline.

### Claim 17 (depends on 1)

The pharmaceutical composition of claim 1, wherein said pharmaceutically acceptable carrier consists essentially of saline.

### Claim 18

A pharmaceutical composition comprising an effective amount of a double-stranded short interfering ribonucleic acid (siRNA) and a pharmaceutically acceptable carrier, wherein the short interfering ribonucleic acid (siRNA) comprises a sense RNA strand and an antisense RNA strand, wherein the sense and the antisense RNA strands form an RNA duplex, and wherein the sense RNA strand comprises a nucleotide sequence identical to a target sequence of about 19 to about 25 contiguous nucleotides in human vascular endothelial growth factor (VEGF) mRNA, wherein said effective amount is an amount effective to inhibit the expression of overexpressed VEGF, wherein the nucleotide sequence identical to a target sequence consists of a sequence selected from the group consisting of: SEQ ID NO. 13, 19, 20, 47, 48, 50, 51, 52, 56, 16, 23, 28, 29, 34, 57, 66, 71, 75, 84, and 88.

### Claim 19 (depends on 1)

The pharmaceutical composition of claim 1, wherein said effective amount is effective to inhibit angiogenesis.