Antisense Oligonucleotides as Antiviral Agents” JUDY COULSON AND ALAN D. B. MALCOLM Department of Biochemistry Charing Cross and WestminsterMedical School London, W68RF, UK Antisense oligodeoxynucleotides (ODNs) have shown tremendous potential as antiviral agents. Many problems, however, remain to be solved. Of these, delivery to the affected tissue is one of the most important. With epitheliotropic viruses such as the human papilloma viruses (HPV), problems of delivery are greatly reduced. We report here our studies in the development of antisense oligodeoxynucleotides as anti-HPV agents. HPV only divide in differentiating epithelial tissue. The DNA of the genome is a supercoiled circular molecule of just under 8 kb in length. Many different strains of HPV have been identified. HPV 1 appears to be principally responsible for ordinary skin warts, HPV 6 for genital warts, and HPV 16 and 18 for genital lesions (warts) that show a high frequency for conversion to the malignant state.’ The HPV genome contains nine ORFs. Seven of these are expressed during the early part of the life cycle of the virus and two during the late part. Two of the early genes in particular have been shown to play a major role in virus-induced tumorigenesis. E6 affects transformation by binding to and causing degradation of the tumor suppressor gene ~ 5 3whereas ,~ E7 binds to the retinoblastoma gene product ~ 1 0 5 . ~ These genes are frequently found to be integrated into the cellular genome in biopsies from cervical carcinomas, and E6 and E7 gene products are detectable. In HPV16 E7/EJ rus transformed cell lines, the continued expression of E7 is required to maintain the transformed state.4 We have therefore chosen the E6 and E7 sequences as targets for our antisense work. We have cloned the E6 and E7 genes of HPV16 into the transcription vector Bluescript and used these as templates for in vitro transcription. This was carried out using T3 RNA polymerase to produce sense RNA and T7 RNA polymerase for antisense RNA synthesis. A 5’ 7meGpppS’G cap analog was included in the reaction mix and is used to initiate transcription, yielding RNA that is efficiently translated in a rabbit reticulocyte lysate. Unmodified antisense oligonucleotides HPVAS1, HPVAS2, and HPVAS3 (FIG. 1) have been synthesized and tested for their effectiveness in causing translational arrest of E6 and E7 RNAs in vitro. The first two are 16 and 32 mers, respectively, directed against the initiation codon region of E7; the third is a 16 mer directed against the initiation codon region of E6. Micrococcal nuclease-treated rabbit reticulocyte lysate was used to translate the E6 and E7 RNAs. Synthesized proteins were labeled by the incorporation of (35S)-methionine. In translational reactions containing capped sense E6 RNA in the absence of antisense, a discrete band of labeled protein is seen on an SDS polyacrylamide gel. “This work was supported by Stiefel International. J. C. holds a studentship from SERC. 339

ANNALS NEW YORK ACADEMY OF SCIENCES

Taraet Reaion of HPV16 E6 Gene HPV Genome

E6 NONSENSE ATGCATCAGTACTGAT

E6 SENSE GC AGA CAT TTT ATG CA

GGC GTA ACC GAA TTC GGT TG

GGC GTA ACC GAA ATC GGT TGA ACC - - 1Sbp--AAA AGC AGA CAT TTT ATG CAC CAA ... CG TCT GTA AAA TAC GT HPV AS3

.............450bo.

-AGA GAA ACC CAG CTG TAA TCA CT CTT TGG GTC GAC ATT AGT

W2)

FIGURE lk The E6 coding region showing the target site of the antisense ODN HPV AS3, along with sequences for the sense and nonsense control ODNs and the PCR primers: forward sense primer E6(1) and reverse antisense primer E6(2) used in the construction of pBS-E6.

This runs at a size slightly larger than that of globin (16 kD). The E6 band migrates at between 17 and 21 kD, the actual position being concentration dependent. An additional band is seen in the E6 lane which is likely to be a dimer of E6, induced by high concentrations and not denatured in this system; it cannot be due to the translation of longer transcripts, as these are not seen on a glyoxal RNA gel. In the presence of 25 FM HPVAS3 the translation of the E6 RNA is inhibited. The higher molecular weight band also disappears in the presence of the antisense oligo, supporting the hypothesis that it is a dimer of the E6. In the presence of 25 pM sense oligonucleotide, the E6 bands remain unaffected, showing that the effect of the antisense oligo is specific. The antisense RNA, transcribed from the construct using T7 RNA polymerase, also results in the disappearance of the HPV specific bands, lending further support to the sequence-specific translational arrest induced by HPVAS3. Similar results have been obtained using HPVASl and HPVAS2 to modulate the in virro translation of the E7 gene in a cell-free system.

COULSON & MALCOLM: ANTISENSE OLIGONUCLEOTIDES

34 1

Taraet Reaion of HPV 16 E7 Gene

HPV 16 GENOME

TAC GTA CGT GAT CTA

G

E7 NONSENSE

E 7 SEN ( 3 2 ) G AGA AAC CCA GCT GTA ATC ATG CAT GGA GAT A GCT GTA ATC ATG CAT G

AAGAACACGTAGAGAAACCCAGCTGTAATCATGCATGGAGATACACCTACATTGCATGAA CGA CAT TAG TAC GTA C

HPV AS1

C TCT TTG GGT CGA CAT TAG TAC GTA CCT CTA T

HPV AS2

FIGURE 1B. The E7 coding region showing the target site of the antisense ODNs HPVASl and HPVAS2 along with sequences for sense and nonsense control ODNs.

ACKNOWLEDGMENT

We thank Professor Mike Clemens for providing the rabbit reticulocyte lysate. REFERENCES

1. ARENDS,M. J., A. H. WYLLIE & C. C. BIRD.1990. Human Pathol. 21: 686-698. M., B. A. WERNESS, J. M. HUIBREGTSE, A. J. LEVINE & P. M. HOWLEY. 1990. 2. SCHEFFNER, Cell 63: 1129-1136. K. MUNGER & E. HARLOW. 1989. Science 243: 934-937. 3. DYSON,N., P. M. HOWLEY, 4. CROOK,T., J. P. MORGERNSTERN, L. CRAWFORD & L. BANKS. 1989. EMBO J. 8: 513-519.