# US 8,785,618 B2 · Method of delivering RNA interference and uses thereof

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- Number: US 8,785,618 B2
- Document ID: US8785618B2
- Status: Granted
- Kind: B2
- Application number: 13/948,435
- Filed: 2013-07-23
- Published: 2013-11-14
- Granted: 2014-07-22
- Earliest claimed: 2004-08-16
- Assignees: Children's Medical Center Corporation
- Applicants: Children's Medical Center Corporation
- Inventors: Judy Lieberman; Erwei Song
- Web page: https://patentgrep.com/patent/US8785618B2

## CPC

- C12N 15/1138: {against receptors or cell surface proteins}
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- C07K 14/46: from vertebrates
- C07K 16/1145: {Env proteins, e.g. gp41, gp110/120, gp160, V3, principal neutralising domain [PND] or CD4-binding site}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- 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 15/1132: {against retroviridae, e.g. HIV}
- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1136: {against growth factors, growth regulators, cytokines, lymphokines or hormones}
- C07K 2317/77: Internalization into the cell
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C07K 2319/85: containing an RNA binding domain
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/3513: Protein; Peptide
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention provides a method of RNA interference, which comprises contacting the cell with a fusion protein-double stranded RNA complex, the complex comprising the double stranded RNA segment containing a double stranded RNA of interest and a fusion protein, the fusion protein comprising (1) a targeting moiety, which will specifically binds to a site on a target cell, and (2) a binding moiety, which will bind to the double stranded RNA, wherein the double stranded RNA segment initiates RNA interference in the cell.

## Claims

### Claim 1

A composition that selectively binds to an antigen on the surface of a target cell and selectively initiates RNA interference in the cell comprising:
a) an siRNA molecule comprising a double stranded RNA segment of about 15-50 base pairs in length, wherein one of one strands is complementary and the other strand identical to a RNA interference target RNA; and
b) a fusion protein, comprising (1) an antibody that binds specifically to an antigen on the surface of a target cell, and (2) a protamine protein, or fragment thereof, capable of binding to double stranded RNA without chemical conjugation.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the antibody is an antibody to a viral envelope protein, a cellular receptor, or an extracellular domain of an activated receptor.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the antibody is a single chain antibody, a Fab portion of an antibody or a (Fab')2 segment.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the antibody recognizes Gp120 or gp160.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the-protamine protein, or portion thereof, is fused to the carboxyl portion of the antibody.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the siRNA targets mRNA encoding c-myc, VEGF, CD4, CCR5, gag, MDM2, Apex, Ku70, or ErbB2.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the cell is selected from the group consisting of hepatocytes, myocytes, neural cells, lipocytes, lymphocytes, macrophages, cardiac cells, endothelial cells, and epithelial cells.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the target cell is a malignant cell.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the malignant cell is selected from the group consisting of a lung cancer cell, a retinal cancer cell, a breast cancer cell, a ovarian cancer cell, a prostate cancer cell, a head and neck cancer cell, a lymphoma cell, a melanoma cell, a glioma cell, a bladder cancer cell, a genital-urinary cancer cell, a stomach cancer cell, a pancreatic cancer cell, a liver cancer cell, a kidney cancer cell, and a gastrointestinal cancer cell.

### Claim 10 (depends on 1)

The composition of claim 1, comprising further comprising a pharmaceutical carrier.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the pharmaceutical carrier is formulated for oral, rectal, vaginal, parenteral, topical, or intravenous administration.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the protamine fragment thereof is encoded by a nucleic acid having a sequence selected from the group consisting of:
SEQ ID NO:1, SEQ ID NO:2; SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, and nucleic acids 10-76 of SEQ ID NO:1.

### Claim 13 (depends on 1)

A kit comprising the composition of claim 1.

### Claim 14

A kit comprising:
a) an siRNA molecule comprising double stranded RNA segments of about 15-50 base pairs in length; and
b) a fusion protein, comprising (1) an antibody that binds specifically to an antigen on the surface of a target cell, and (2) a protamine protein, or fragment thereof, capable of binding to double stranded RNA without chemical conjugation.

### Claim 15 (depends on 14)

The kit of claim 14, further comprising a plurality of different siRNA molecules.