# US 8,569,065 B2 · Compositions and methods for the delivery of biologically active RNAs

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 8,569,065 B2
- Document ID: US8569065B2
- Status: Granted
- Kind: B2
- Application number: 12/724,408
- Filed: 2010-03-15
- Published: 2010-09-16
- Granted: 2013-10-29
- Earliest claimed: 2009-03-13
- Assignees: Egen, Inc.
- Applicants: Kevin Polach; Jason Fewell; Khursheed Anwer
- Inventors: Kevin Polach; Jason Fewell; Khursheed Anwer
- Web page: https://patentgrep.com/patent/US8569065B2

## CPC

- 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 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/85: for animal cells
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides novel compounds, compositions, and methods for the delivery of biologically active RNA molecules to cells. Specifically, the invention provides novel nucleic acid molecules, polypeptides, and RNA-protein complexes useful for the delivery of biologically active RNAs to cells and polynucleotides encoding the same. The invention also provides vectors for expressing said polynucleotides. In addition, the invention provides cells and compositions comprising the novel compounds and vectors, which can be used as transfection reagents. The invention further provides methods for producing said compounds, vectors, cells, and compositions. Additionally, vectors and methods for delivering biologically active RNA molecules to cells and/or tissues are provided. The novel compounds, vectors, cells, and compositions are useful, for example, in delivering biologically active RNA molecules to cells to modulate target gene expression in the diagnosis, prevention, amelioration, and/or treatment of diseases, disorders, or conditions in a subject or organism.

## Claims

### Claim 1

An expression vector comprising a first polynucleotide and a second polynucleotide, wherein
the first polynucleotide encodes a biologically active RNA sequence and a recognition RNA sequence; and
the second polynucleotide encodes a polypeptide comprising (i) an RNA binding domain sequence that binds the recognition RNA sequence and (ii) a non-classical secretory domain sequence that facilitates secretion of a RNA-polypeptide complex from a cell via an ER-Golgi independent pathway, the RNA-polypeptide complex comprising the biologically active RNA sequence, the recognition RNA sequence, and the polypeptide.

### Claim 2 (depends on 1)

A cell comprising the expression vector of claim 1.

### Claim 3 (depends on 1)

A method for secreting a biologically active RNA molecule from a cell comprising transfecting the cell with the expression vector of claim 1, transcribing the first polynucleotide, expressing the polypeptide, forming the complex, and secreting the complex from the cell, thereby secreting the biologically active RNA molecule from the cell.

### Claim 4 (depends on 1)

The expression vector of claim 1, wherein the first polynucleotide further encodes a terminal minihelix sequence.

### Claim 5 (depends on 1)

The expression vector of claim 1, wherein the second polynucleotide further encodes a cell penetrating peptide.

### Claim 6 (depends on 1)

The expression vector of claim 1, wherein the biologically active RNA sequence of the first polynucleotide is selected from the group consisting of an RNA ribozyme, an antisense RNA, an RNA aptamer, a small interfering RNA (siRNA), a double stranded RNA (dsRNA), a micro RNA (miRNA), and a small hairpin RNA (shRNA).

### Claim 7 (depends on 1)

The expression vector of claim 1, wherein the non-classical secretory domain sequence is selected from the group consisting of Galectin-1, Galectin-3, interleukin 1α(IL-1α), interleukin 1β(IL-1β), hydrophilic acylated surface protein B (HASPB), high mobility group box protein 1 (HMGB1), fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), interleukin 2 (IL-2), Thioredoxin, and Rhodanese.

### Claim 8 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is a U1 loop sequence and the RNA binding domain sequence is a U1A domain.

### Claim 9 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is a nucleon recognition element (NRE) stem loop sequence and the RNA binding domain sequence is a Nucleolin RNA binding domain.

### Claim 10 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is a S1A stem loop sequence and the RNA binding domain sequence is a hRBMY domain.

### Claim 11 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is an AU rich element (ARE) sequence and the RNA binding domain sequence is a Tristetrapolin TTP73 domain.

### Claim 12 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is a Box B sequence and the RNA binding domain sequence is a Bacteriophage Protein N domain.

### Claim 13 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is a Rev sequence and the RNA binding domain sequence is a Rev protein domain.

### Claim 14 (depends on 1)

The expression vector of claim 1, wherein the recognition RNA sequence of the first polynucleotide is an alpha mosaic virus (AMV) sequence and the RNA binding domain sequence is a AMV coat domain.

### Claim 15 (depends on 2)

The cell of claim 2, wherein the biologically active RNA sequence of the first polynucleotide is selected from the group consisting of an RNA ribozyme, an antisense RNA, an RNA aptamer, a siRNA, a dsRNA, a miRNA, and an shRNA.

### Claim 16 (depends on 15)

The cell of claim 15, wherein the first polynucleotide further encodes a terminal minihelix sequence.

### Claim 17 (depends on 5)

The expression vector of claim 5, wherein the biologically active RNA sequence of the first polynucleotide is selected from the group consisting of an RNA ribozyme, an antisense RNA, an RNA aptamer, a siRNA, a dsRNA, a miRNA, and an shRNA.

### Claim 18 (depends on 5)

The expression vector of claim 5, wherein the cell penetrating peptide sequence is selected from the group consisting of penetratin, transportin, model amphiphatic peptide (MAP), trans-activator of transcription (TAT), homeodomain of antennepedia (AntP), feline herpes virus (FHV) coat protein, and transportin 10 (TP10).

### Claim 19

An expression vector comprising a first polynucleotide and a second polynucleotide, wherein
the first polynucleotide encodes a biologically active RNA sequence and a recognition sequence, operably linked to a first promoter sequence and a first polyA addition sequence; and
the second polynucleotide encodes a polypeptide comprising (i) an RNA binding domain sequence that binds the recognition RNA sequence, and (ii) a non-classical secretory domain sequence that facilitates secretion of a RNA-protein complex from a cell via an ER-Golgi independent pathway, operably linked to a second promoter sequence and a second polyA addition sequence, wherein the RNA-polypeptide complex comprises the biologically active RNA sequence, the recognition RNA sequence, and the polypeptide.

### Claim 20 (depends on 19)

The expression vector of claim 19, wherein the first polynucleotide further encodes a terminal minihelix sequence.

### Claim 21 (depends on 19)

The expression vector of claim 19, wherein the second polynucleotide further encodes a cell penetrating peptide.

### Claim 22 (depends on 19)

The expression vector of claim 19, wherein the biologically active RNA sequence of the first polynucleotide is selected from the group consisting of an RNA ribozyme, an antisense RNA, an RNA aptamer, a siRNA, a dsRNA, a miRNA, and an shRNA.

### Claim 23 (depends on 19)

A cell comprising the expression vector of claim 19.

### Claim 24 (depends on 19)

A method for secreting a biologically active RNA molecule from a cell transfecting the cell with the expression vector of claim 19, transcribing the first polynucleotide, expressing the polypeptide, forming the complex, and secreting the complex from the cell, thereby secreting the biologically active RNA molecule from the cell.

### Claim 25 (depends on 23)

The cell of claim 23, wherein the biologically active RNA sequence of the first polynucleotide is selected from the group consisting of an RNA ribozyme, an antisense RNA, an RNA aptamer, a siRNA, a dsRNA, a miRNA, and an shRNA.

### Claim 26 (depends on 25)

The cell of claim 25, wherein the first polynucleotide further encodes a terminal minihelix sequence.

### Claim 27

A pharmaceutical composition comprising:
a first expression vector comprising a first polynucleotide encoding a biologically active RNA sequence and a recognition RNA sequence;
a second expression vector comprising a second polynucleotide encoding a polypeptide comprising (i) an RNA binding domain sequence that binds the recognition RNA sequence and (ii) a non-classical secretory domain sequence that facilitates secretion of a RNA-polypeptide complex from a cell via an ER-Golgi independent pathway, the RNA-polypeptide complex comprising the biologically active RNA sequence, the recognition RNA sequence, and the polypeptide, and
a pharmaceutically acceptable carrier.

### Claim 28 (depends on 27)

The pharmaceutical composition of claim 27, wherein the first polynucleotide further encodes a terminal minihelix sequence.

### Claim 29 (depends on 27)

The pharmaceutical composition of claim 27, wherein the second polynucleotide further encodes a cell penetrating peptide.

### Claim 30 (depends on 27)

The pharmaceutical composition of claim 27, wherein the biologically active RNA sequence of the first polynucleotide is selected from the group consisting of an RNA ribozyme, an antisense RNA, an RNA aptamer, a siRNA, a dsRNA, a miRNA, and an shRNA.