WO1991018913A1

WO1991018913A1 - Ribozyme mediated reversal of transformation by cleavage of the hras oncogene rna

Info

Publication number: WO1991018913A1
Application number: PCT/US1990/003218
Authority: WO
Inventors: Kevin J. Scanlon
Original assignee: City Of Hope
Priority date: 1990-06-07
Filing date: 1990-06-07
Publication date: 1991-12-12
Also published as: WO1991018624A1; AU8879991A

Abstract

This invention relates to a ribozyme effective to cleave oncogene RNA. More particularly the invention relates to a ribozyme effective to cleave Hras RNA at a GUC site on codon 12 where an activating point mutation may appear, to a plasmid into which said ribozyme has been cloned, to mammalian cells into which said plasmid has been transfected and which express said ribozyme and to cancer therapy which entails the administration of such ribozymes per se and of such plasmids to induce in vivo expression of such ribozymes. These ribozyme plasmids can reverse the transformation process back to the normal cell phenotype.

Description

RIBOZYME MEDIATED REVERSAL OF TRANSFORMATION BY CLEAVAGE OF THE HRAS ONCOGENE RNA

Field of the Invention

This invention relates to a ribozyme effective to cleave oncogene RNA. More particularly the invention relates to a ribozyme effective to cleave Hras RNA at a GUC site on codon 12 where an activating point mutation may appear, to a plasmid into which said ribozyme has been cloned, to mammalian cells into which said plasmid has been transfected and which express said ribozyme and to cancer therapy which entails the administration of such ribozymes per se are of such plasmids to induce jLn vivo expression of such ribozymes. These ribozyme plasmids can reverse the transformation process back to the normal cell phenotype.

Background of the Invention Ribozyme cleavage of various RNA transcripts is known. See, e.g., published PCT applications no. 89/05852 and no. 88/04300 and pending U.S. Application 9/401,613 filed August 31, 1989.

It is known that a point mutation on codon 12 activates the Hras oncogene to yield a gene product which malignantly transforms cells and may cause a spectrum of neoplasms including human bladder carcinoma: to date no known cancer therapy protocol implicates this phenomenon.

Summary of the Invention Normal Hras gene RNA does not include a ribozyme cleavage site on codon 12. The point mutation which yields a malignancy inducing Hras transcript does include such a cleavage site. This invention provides a ribozyme effective to cleave the mutated gene but not the normal gene. The ribozyme of the invention is effective to cleave the mutated gene both in vitro and in vivo. It accordingly provides unique therapy for bladder carcinoma and other malignancies which may be induced by cells transformed by the mutated Hras gene product. The ribozyme of the invention may be administered by a known delivery system such as a liposome or by other means known to the art. It may also be administered in the form of a vector (i.e., RNA tumor virus) into which it has been cloned and which will express the ribozyme.

The invention also includes a ribozyme which inhibits C-fos expression in response to cis-platin or other stimuli.

Detailed Description of the Invention The DNA sequence flanking codon 12 of the human Hras gene is illustrated by I:

The mutation which activates the Hras gene is shown in brackets above position 2 of codon 12.

The Hras RNA included in mutated gene transcript according includes a GUC site appropriate for ribozyme cleavage as shown in II.

II 5' GUG GUG GGC GCC GUC GGU GUG GGC AAG 3' +8 9 10 11 12 13 14 15 16 A ribozyme effective to catalytically cleave the mutated Hras DNA at the GUC cleavage site in functional association therewith is shown in III. Ill Hras RNA

5'- G GGC GCC GUC GGU GUG GGC - 3 C CCG CGG CA CCA CAC CCG 5' 3' A C^~~ ^G _A A U Ribozyme G A^G

C-G A-U G-C G-C

G A GU

The ribozyme per se may be synthesized in known manner by use of a commercially available synthesizer produced, for example, by Applied Biosystems, Inc. or Milligen.

In the preferred practice of the invention a double stranded DNA molecule having one strand, which upon transformation yields the desired ribozyme, is synthesized. This molecule is then cloned into a plasmid capable of synthesizing the ribozyme _in vivo when transfected into a mammalian cell. Appropriate promoters and terminal sequences may preside and follow the •^•ribozyme" component of the DNA insert to be cloned into the plasmid. Preferably appropriate nucleotide sequences having sufficient overlapping base pairs are amplified by the poly erase chain reaction to provide the insert to be cloned. The 3' and 5' terminus include restriction sites to insure the correct positioning of the insert in the plasmid. A T7 RNA polymerase promoter may be positioned at the 5' terminus of the "ribozyme" to accommodate in vitro cleavage. One appropriate construct is shown by IV.

Sequence-1 T₇ RNA polymerase promoter

5' GGT CGA CTA ATA CGA CTC ACT ATA GGC CCA CAC CCT GAT

Sal I 3' C GTG GGA CTA

In vitro

GA - 3'

CTC AGG CAC TCC TGC TTT GCC GCG GGT TCG AAC - 5'

Seq. 2 Hind III

Hras Ribozyme for I_n Vivo Studies

Primer #1

5'-GGC CCA CAC CCT GAT GA-3'

3^,-CGTG GGA CTA CT CAG GCA CTC CTG CTT TGC CGC GG-5'

Primer #2

PCR ASSAY

Sal I (Coding strand) Hind III

5-GGC CCA CAC CCT GAT GAG TCC GTG AGG ACG AAA GGG CGC CC-3⁷ 3-CCG GGT GTG GGA GTA CTC AGG CAC TCC TGC TTT GCC GCG GG-5' (Template strand)

RNA Polymerase Assay

Ribozyme 3'-CCCGCGG CAA AGC AGG AGU GCC UGA GUA GUC CCA CAC CCG-5'

'-GGGCGCC GUC GGU GUG GGC-3 '

Hras RNA

The T7 RNA polymerase promoter sequence may be omitted if in vitro cleavage of Hras RNA is not contemplated.

The sal I and Hind III sequences are included to insure that the double stranded product which results from PCR amplification of IV appears in the proper orientation when cloned into a plasmid to produce a vector. The selection of an appropriate plasmid is within the skill of the art. A preferred plasmid is pH-0-Apr.l which yields the corresponding vector pE-β Apr.l-Hras-R in which R is the double stranded PCR product, one strand of which is the Hras ribozyme. Other plasmids useful in the invention include pH α pKoneo, the pSV2 cat plasmid and pMAMneo.

The DNA fragments are cloned by sticky end ligation into a selected site of the plasmid.

Cells transfected, e.g., by lipofection or electroporation, with the vector pH-jθ-Apr.l Hras R express the Hras ribozyme shown in III.

The Hras ribozymes or the plasmid vectors which express such ribozymes of the invention may be administered by injection of appropriate delivery systems such as lipomes. Hence, one aspect of the invention includes liposomes in which the Hras ribozymes are encapsulated or are included in the lyposomal bilayers. In vivo ribozymes expressed by cells transfected with a plasmid, as described, express the ribozyme shown in III which cleaves the RNA expressed by malignant Hras genes.

Claims

1. A ribozyme which cleaves Hras RNA at a GUC cleavage site.

2. A ribozyme having the sequence of the ribozyme shown in III.

3. A plasmid having as a cloned insert, a double stranded DNA fragment one strand of said fragment having the sequence of the ribozyme shown in III.

4. A cell including a plasmid, as defined by Claim 3, said cell expressing a ribozyme as shown in III.

5. The plasmid pH-_/9-Apr.1-Hras R.

6. A method of converting a malignant cancer cell to a nonmalignant cell which comprises incorporating a plasmid, as defined by Claim 5, into said cell.

7. A method for treating human bladder carcinoma which comprises administering to a patient in need of such treatment an effective amount of a plasmid, as defined by Claim 5.