# US 7,655,787 B2 · pRNA chimera

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- Number: US 7,655,787 B2
- Document ID: US7655787B2
- Status: Granted
- Kind: B2
- Application number: 10/373,612
- Filed: 2003-02-24
- Published: 2004-07-01
- Granted: 2010-02-02
- Earliest claimed: 2000-08-23
- Assignees: Purdue Research Foundation
- Inventors: Peixuan Guo; Stephen M. Hoeprich; Dan Shu
- Web page: https://patentgrep.com/patent/US7655787B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- 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 15/1131: {against viruses}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12Y 113/11031: Arachidonate 12-lipoxygenase (1.13.11.31), i.e. lipoxygenase-type-12
- 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 2310/11: Antisense
- C12N 2310/111: spanning the whole gene, or a large part of it
- C12N 2310/121: Hammerhead
- C12N 2310/16: Aptamers
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2310/53: partially self-complementary or closed
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2799/022: where the vector is derived from an adenovirus
- 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
- C12Y: ENZYMES
- 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)

## Abstract

A circularly permuted chimeric pRNA molecule carrying a stabilized biologically active RNA, such as a ribozyme.

## Claims

### Claim 1

A pRNA chimera comprising:
(a) a pRNA region; and
(b) a spacer region comprising a biologically active RNA, the spacer region covalently linked at its 5′ and 3′ ends to the pRNA region, wherein the biologically active RNA specifically binds a pre-identified substrate;
wherein the pRNA region comprises:
(i) in the 5′ to 3′ direction beginning at the covalent linkage of the pRNA with the 3′ end of the spacer region
a first loop;
a second loop; and
a lower stem-loop structure comprising a bulge, a first stem section and a third loop;
(ii) a second stem section interposed between the spacer region and the stem-loop structure;
(iii) a third stem section interposed between the stem-loop structure and the first loop;
(iv) a fourth stem section interposed between the first loop and the second loop; and further comprises
(v) an opening defining 5′ and 3′ ends of the pRNA chimera.

### Claim 2 (depends on 1)

The pRNA chimera of claim 1 wherein the biologically active RNA is selected from the group consisting of a ribozyme, a siRNA, an RNA aptamer, an antisense RNA and a peptide nucleic acid (PNA).

### Claim 3 (depends on 1)

The pRNA chimera of claim 1 wherein
the pRNA region comprises a circularly permuted pRNA of a bacteriophage selected from the group consisting of bacteriophage φ29, SF5′, B103, PZA, M2, NF and GA1.

### Claim 4 (depends on 3)

The pRNA chimera of claim 3 wherein the biologically active RNA is selected from the group consisting of a ribozyme, a siRNA, an RNA aptamer, an antisense RNA and a peptide nucleic acid (PNA).

### Claim 5 (depends on 1)

A DNA molecule comprising a nucleotide sequence that encodes the pRNA chimera of claim 1.

### Claim 6 (depends on 1)

A method for making a pRNA chimera comprising:
providing a DNA encoding the pRNA chimera of claim 1; and
transcribing the DNA in vitro to yield the pRNA chimera.

### Claim 7 (depends on 6)

The method of claim 6 wherein the step of providing comprises generating the DNA by a polymerase chain reaction.

### Claim 8 (depends on 6)

The method of claim 6 wherein the step of providing comprises (i) cloning the DNA into a plasmid; and (ii) replicating the plasmid.

### Claim 9 (depends on 1)

A method for delivering a biologically active RNA to a cell comprising:
introducing into the cell a DNA molecule comprising a nucleotide sequence that operably encodes the pRNA chimera of claim 1; and
causing transcription of the DNA to yield the biologically active RNA.

### Claim 10 (depends on 9)

The method of claim 9 wherein the biologically active RNA is selected from the group consisting of a ribozyme, a siRNA, an RNA aptamer, an antisense RNA and a peptide nucleic acid (PNA).

### Claim 11 (depends on 9)

The method of claim 9 wherein the cell is present in a cell culture, a tissue, an organ or an organism.

### Claim 12 (depends on 9)

The method of claim 9 wherein the cell is a mammalian cell.

### Claim 13 (depends on 12)

The method of claim 12 wherein the cell is a human cell.

### Claim 14

A pRNA chimera comprising:
(a) a pRNA region; and
(b) a spacer region comprising a biologically active RNA that specifically binds a pre-identified ligand or receptor, the spacer region covalently linked at its 5′ and 3′ ends to the pRNA region; wherein the pRNA region comprises:
(i) in the 5′ and 3′ direction beginning at the covalent linkage of the pRNA with the 3′ end of the spacer region
a first loop;
a second loop; and
a lower stem-loop structure comprising a bulge, a first stem section and a third loop;
(ii) a second stem section interposed between the spacer region and the stem-loop structure;
(iii) a third stem section interposed between the stem-loop structure and the first loop;
(iv) a fourth stem section interposed between the first loop and the second loop; and further comprises
(v) an opening defining 5′ and 3′ ends of the pRNA chimera.

### Claim 15

A pRNA chimera comprising:
(a) a pRNA region; and
(b) a spacer region comprising a biologically active RNA that comprises at least one of a specific binding activity and an enzymatic activity, the spacer region covalently linked at its 5′ and 3′ ends to the pRNA region;
wherein the pRNA region comprises:
(i) in the 5′ and 3′ direction beginning at the covalent linkage of the pRNA with the 3′ end of the spacer region
a first loop;
a second loop; and
a lower stem-loop structure comprising a bulge, a first stem section and a third loop;
(ii) a second stem section interposed between the spacer region and the stem-loop structure;
(iii) a third stem section interposed between the stem-loop structure and the first loop;
(iv) a fourth stem section interposed between the first loop and the second loop; and further comprises
(v) an opening defining 5′ and 3′ ends of the pRNA chimera.

### Claim 16 (depends on 1)

The pRNA chimera of claim 1 wherein (i) the biologically active RNA comprises an aptamer and (ii) the pre-identified substrate comprises a cell surface receptor.

### Claim 17

A pRNA chimera comprising:
(a) a pRNA region; and
(b) a spacer region comprising a biologically active RNA that does not comprise the nucleotide sequence UAAUACGACUCACUAUA as set forth in SEQ ID NO:8, the spacer region covalently linked at its 5′ and 3′ ends to the pRNA region, wherein the biologically active RNA specifically binds a pre-identified substrate;
wherein the pRNA region comprises:
(i) in the 5′ to 3′ direction beginning at the covalent linkage of the pRNA with the 3′ end of the spacer region
a first loop;
a second loop; and
a lower stem-loop structure comprising a bulge, a first stem section and a third loop;
(ii) a second stem section interposed between the spacer region and the stem-loop structure;
(iii) a third stem section interposed between the stem-loop structure and the first loop;
(iv) a fourth stem section interposed between the first loop and the second loop; and further comprises
(v) an opening defining 5′ and 3′ ends of the pRNA chimera.

### Claim 18

A pRNA chimera comprising:
(a) a pRNA region; and
(b) a spacer region comprising a biologically active RNA, the spacer region covalently linked at its 5′ and 3′ ends to the pRNA region, wherein the biologically active RNA binds a pre-identified substrate and comprises a polyribonucleotide selected from the group consisting of an RNA of at least 25-100, 46, 47, 63, 66, 94, 120, 168 and 188 nucleotides,
wherein the pRNA region comprises:
(i) in the 5′ to 3′ direction beginning at the covalent linkage of the pRNA with the 3′ end of the spacer region
a first loop;
a second loop; and
a lower stem-loop structure comprising a bulge, a first stem section and a third loop;
(ii) a second stem section interposed between the spacer region and the stem-loop structure;
(iii) a third stem section interposed between the stem-loop structure and the first loop;
(iv) a fourth stem section interposed between the first loop and the second loop; and further comprises
(v) an opening defining 5′ and 3′ ends of the pRNA chimera.