# US 5,707,835 A · Ribozymes

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- Number: US 5,707,835 A
- Document ID: US5707835A
- Status: Granted
- Kind: A
- Application number: 08/446,884
- Filed: 1995-05-17
- Granted: 1998-01-13
- Earliest claimed: 1987-12-15
- Assignees: Gene Shears Pty. Limited
- Inventors: James Phillip Haseloff; Wayne Lyle Gerlach; Philip Anthony Jennings; Fiona Helen Cameron
- Web page: https://patentgrep.com/patent/US5707835A

## 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)}
- A01N 57/16: containing heterocyclic radicals
- A01N 63/50: Isolated enzymes; Isolated proteins (peptides A01N37/46)
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- 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 2310/12: catalytic nucleic acids, e.g. ribozymes
- C12N 2310/121: Hammerhead
- C12N 2310/124: based on group I or II introns
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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

Compounds having highly specific endoribonuclease activity are described. The compounds of this invention, also known as ribozymes, comprise ribonucleotides having two hybridizing regions with predetermined sequences capable of hybridizing with a plant, animal or viral target RNA, a region of defined sequence and a base paired stem region.

## Claims

### Claim 1

A compound having the formula:
3'--›--(Y).sub.r --Q--(Y).sub.s --!.sub.z --5'
wherein each Q represents the structure ##STR10## wherein each X represents a ribonucleotide which may be the same or different;
wherein each of (X).sub.n and (X).sub.n' represents an oligoribonucleotide having a predetermined sequence which hybridizes with an RNA target sequence to be cleaved, and (b) does not naturally occur covalently bound to the sequences A--A--A--G--C-- and X--C--U--G--A--, respectively, such RNA target sequence not being present within the compound;
wherein each of n and n' represents an integer which defines the number of ribonucleotides in the oligoribonucleotide with the proviso that the sum of n+n' is greater than 6 and sufficient to allow the compound to stably interact with the RNA target sequence through base pairing;
wherein each * represents base pairing between the nucleotides located on either side thereof;
wherein each solid line represents a chemical linkage providing covalent bonds between the ribonucleotides located on either side thereof;
wherein a represents an integer which defines a number of ribonucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (X).sub.a is bonded to the G located 3' of (X).sub.a ;
wherein each of m and m' represents an integer which is greater than or equal to 1;
wherein each of the dashed lines independently represents either a chemical linkage providing covalent bonds between the ribonucleotides located on either side thereof or the absence of any such chemical linkage;
wherein (X).sub.b represents an oligoribonucleotide which may be present or absent with the proviso that b represents an integer which is greater than or equal to 4 if (X).sub.b is present;
wherein one or more of the ribonucleotides may be a ribonucleotide derivative;
wherein each Q may independently be the same or different;
wherein each Y represents a ribonucleotide which may be the same or different;
wherein each of r and s represents an integer greater than or equal to 0; and
wherein z represents an integer greater than or equal to 1.

### Claim 2 (depends on 1)

A transfer vector comprised of RNA or DNA or a combination thereof containing a nucleotide sequence which on transcription gives rise to a compound of claim 1.

### Claim 3 (depends on 2)

A transfer vector according to claim 2 which is a bacterial plasmid, viral RNA, viral DNA or phage DNA.

### Claim 4 (depends on 2)

A method of using the transfer vector of claim 2 to produce a compound which comprises transcribing a nucleotide sequence which corresponds to the transfer vector with RNA polymerase under suitable conditions so as to produce and recover said compound.

### Claim 5 (depends on 2)

A method of using the transfer vector of claim 2 which comprises introducing the vector into a suitable eukaryotic host cell and culturing the host cell so as to effect transcription of the nucleotide sequence contained within the transfer vector and thereby produce the compound encoded by such nucleotide sequence, which compound which hybridizes with and specifically cleaving an RNA target sequences within the eukaryotic host cell.

### Claim 6 (depends on 2)

A method of using the transfer vector of claim 2 to produce a compound in a host cell which comprises introducing the transfer vector into a suitable host cell and culturing the host cell under conditions to express the transfer vector so as to thereby produce said compound in the host cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein the host cell is a prokaryote.

### Claim 8 (depends on 6)

The method of claim 6, wherein the host cell is a plant cell.

### Claim 9 (depends on 6)

The method of claim 6, wherein the host cell is an animal cell.

### Claim 10 (depends on 1)

A prokaryotic or cultured eukaryotic cell containing a nucleotide sequence which on transcription gives rise to a compound of claim 1.

### Claim 11

A compound having the formula:
3'--›--(Y).sub.r --Q--(Y).sub.s --!.sub.z --5'
wherein each Q represents the structure ##STR11## wherein each X represents a ribonucleotide which may be the same or different;
wherein each of (X).sub.n and (X).sub.n' represents an oligoribonucleotide having a predetermined sequence which hybridizes with an RNA target sequence to be cleaved, and (b) does not naturally occur covalently bound to the sequences A--A--A--G--C-- and X--C--U--G--A--, respectively, such RNA target sequence not being present within the compound;
wherein each of n and n' represents an integer which defines the number of ribonucleotides in the oligoribonucleotide with the proviso that the sum of n+n' is greater than 6 and sufficient to allow the compound to stably interact with the RNA target sequence through base pairing;
wherein each * represents base pairing between the nucleotides located on either side thereof;
wherein each solid line represents a chemical linkage providing covalent bonds between the ribonucleotides located on either side thereof;
wherein a represents an integer which defines a number of ribonucleotides with the proviso that a may be 0 or 1 and if 0, the A located 5' of (X).sub.a is bonded to the G located 3' of (X).sub.a ;
wherein each of m and m' represents an integer which is greater than or equal to 3;
wherein each of the dashed lines independently represents either a chemical linkage providing covalent bonds between the ribonucleotides located on either side thereof or the absence of any such chemical linkage;
wherein (X).sub.b represents an oligoribonucleotide which may be present or absent with the proviso that b represents an integer which is greater than or equal to 4 if (X).sub.b is present;
wherein one or more of the ribonucleotides may be a ribonucleotide derivative;
wherein each Q may independently be the same or different;
wherein each Y represents a ribonucleotide which may be the same or different;
wherein each of r and s represents an integer greater than or equal to 0; and
wherein z represents an integer greater than or equal to 1.