Antisense oligonucleotide-mediated inhibition of mutant p53 expression

41 0s

Biochemical Society Transactions (1 996) 24

Antiscnr digonucleotidc-mediated inbibition of mutant p53 expression. CAROLYN J. RUDDELL, JOHN A. GREEN’ AND DAVIDM. TIDD.

Figure I. Activity of chimeric methylphosphonodieer/phosphodi~eranriscnse oligodeoxynucleotides at d o n s 248 and 273 of p53 mRNA in KYOl cells.

Departments of Biochemistry and Medicine’, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK.

The ability to discriminate between point-mutated add normal RNA species may be essential to the development of an antisense oligonucle&id+basej treatment for cancer. This ‘ultimate specificity’ aims to ensure that the expression of point-mutated forms of oncogenes or tumour suppressor genes is inhibited whilst expression of the normal cellular counterparts of these genes is retained. We have r-tly demonstrated single base discrimination for ribonuclease H-dependent antisense effects on p53 mRNA following the introduction of chimeric antisense effectors into living human acute lymphoblastic leukaemia MOLT4 cells by streptolysin 0 cell permeabilization [I]. In this study similar techniques wete used to introduce antisense oligonucleotides of varying structural composltion, directed at mutation sites in d o n s 248 and 273 of p53 mRNA, into intact human chronic myelogenous leukaemia KYOl cells and the activity and specificity of antisense effects were assessed by northern blotting. Chimeric methylphosphonodiester/phosphodiester ‘383’ (3 methylphosphonates at each end of 8 central phosphodiesters) 15-mer antisense oligodeoxynucleotides, directed at the d o n 273 ‘Harlow’ mutation site exhibited good activity and single base mismatch discrimination, reducing p53 mRNA to 40% of the no oligodeoxynucleotide control level with the fully complementary wild-type sequence oligomer, while no reduction in p53 mRNA level was seen with the mutant sequence oligodeoxynucleotide (Fig. 1, lanes I and 2. However, antisense effectors of identical ‘383’ structure centred on a G to A mutation in d o n 248 both caused efficient reduction of p53 mRNA, to approximately 20% of the no oligodeoxynucleotide control level, thus not exhibiting the single base mismatch specificity that the equivalent oligomers had shown at the d o n 273 mutation site (Fig. 1, lanes 5 and 6). The different activities of these oligodeoxynucleotides, which were of similar base composition and identical structure, can be accounted for by differences in mRNA folding at the two target sites. Computer-generated secondary structure models suggested that the two sites were located within areas of secondary structure which were highly dissimilar, d o n 273 being found in a stable ‘hairpin’ of mRNA self-complementarity, wMe the region around codon 248 had low RNA-RNA hybrid stability, and included a number of ‘open loops’. Therefore, the reduction in p53 mRNA seen at the two sites may be related to the ease with which the antisense oligodeoxynucleotides could disrupt the RNA secondary structure and form the RNA-DNA heteroduplexes which are the substates for ribonuclease H. Similar observations have been made previously in our laboratory with respect to bcr-abl mRNA [2]. Increasing the degree of methylphosphonate substitution to give a ‘464’ structure significantly reduced the activity of the fully complementary sequence oligodeoxynucleotide at the d o n 273 mutation site (Fig. 1, lane 3), presumably as a result of the helix-destabilizing effect of the increased number of methylphosphonate linkages [2,3]. However, the reduced RNAoligodeoxynucleotide hybrid stability afforded by the ‘464’ structure was sufficient to increase the specificity of the oligomer directed at the d o n 248 mutation site without compromising the activity of the oligodeoxynucleotide with the fully complementary mutant sequence (Fig. 1, lanes 7 and 8). In order to determine whether the oligodeoxynucleotideduected ribonuclease H activity could be improved at the d o n 273 mutation site, a series of 15-mer oligonucleotides were synthesized with successive Z’-O-methylribonucleotide substitution of the terminal deoxynucleotides, i.e. ‘383’, ‘464’,‘545’ and ‘636’ structures. Oligonucleotides composed entirely of Z-O-methyl sugar-modified ribonucleotides are incapable of directing the activity of ribonuclease H [5], however, their capacity to hybridize strongly with RNA may be exploited to produce chimeric antisense effectors with 2’-(3methylribonucleotide terminals, which have enhanced hybridization characteristics in comparison with unmodified phosphodiester oligodeoxynucleotides [6,7]. Contrary to expectation these compounds did not exhibit greater activity in comparison with the methylphosphonodiesta/phosphodiester chimerics at the d o n 273 mutation site (Figs. 1 and 2). The ‘383’ fully complementary oligonucleotide was the mast active, reducing p53 mRNA to 400h of the no oligomer control level, followed by the ‘464’ and ‘545’, suggesting that the ability of an oligonucleotide to direct the activity of RNase H is not solely dependent on its ability to hybridize with its target RNA, as activity deaeased with increasing hybrid stability along the series of Z’-O-rnethyl oligonucleotides. The same could not be said for the methylphosphonodiester/phosphodieSer chimaics as the activity was lower for the more weakly hybridizing ‘464’ than for the ‘383’ sbuctures. The series of mutant sequence 2’-Omethyi antisense oligonucleotides, which carry a single base mismatch at d o n 273 with respea to the p53 mRNA found in these cells, caused significant non-targeled reduction

7

I, Figure 2. Activity of 2’-O-methyl-modified antisense oligonucleotide structures directed at the d o n 273 ‘Harlow’ site in p53 mRNA in KYOl cells.

Densitometric quantitation of intact pS3 mRNA levels as determined by northern blotting. Total cellular RNA w extracted 4 hours after reversible penneabilization in the presence of 20pU antisense effectors. Values are representative of 4 replicates per$onned in 2 independent experiments. Note: at codon 273. KYOl cells are mutant (Mvl) at codon 248 and wild-type of p53 mRNA, exhibiting less favourable single base mismatch discrimination than the methylphosphonodiester/phosphodiesterstructures at this site. It is not yet entirely clear why the 2’-O-methyl sugar modified oligonucleotides are not as active as was anticipated, however, a n u m b of explanations have been suggested including the fact that the 2’-O-methyl oligonucleotides with all phosphodiester bonds may be less stable to nucleuse attack than the rnethylph~phonodiesterlphosphodiestR chimeric olige deoxynucleotides [7,8]. The lower activity than expected and the apparent decreasing activity of the 2’-Omethyl oligos with increasing number of substitutions seen here is consistent with the hypothesis than an oligo which hybridizes to its target RNA with high affinity may fail to dissociate 6om the degraded RNA fragments after RNase H cleavage, preventing the oligo 6om hctioning in a catalytic manner [3]. Other general considerations not discussed here include the possibility that the size of central ‘deoxy gap’ and nucleotide sequence therein may be an important factor in the efficiency and specificity of RNase H-mediated antisense.’ffe In vivo studies of this type form an important part of the development of an antisense-based cancer treatment by increasing our understanding of how mRNA secondary structure may affect the design of bligonucleotides required in order to achieve maximum activity and specificity for the chosen target in each case. We should like to thank Mrs. J. Wood for laboratory assistance. This work was supported by the Notth West Cancer Research Fund.

I. Giles, RV., Ruddell, C.J., Spiller, D.G., Green, J.A. and Tidd, D.M. (1995) Nuel. AcidF Res., 23,954-%I. 2. Giles, RV., Spiller, D.G., Green, J.A., Clark, RE. and Tidd, D.M. (1995) Blood, 86, 744-754. 3. Giles, RV. and Tidd, D.M. (1992) Anti-Cancer Dnrg Des., 7,3748. 4. Inoue, H., Hayase, Y., Iwai, S. and Ohtsuka, E. (1987) FEES Letters, 215, 327-330. 5. lnoueetal.,(1987)Nucl. AcidcRes., 15,6131-6148 6. Monia, B.P., Lesnik, E.A., Gonzales C., Lima, W.F., MGee, D., Guinosso, C.J., Kawasaki, A.M., Cook, P.D. and Freier, S.M.(1993) J. Biol. Chem., 268, 14514-14522. 7. Tidd, D.M. and Warmius, H.M. (1989) Br. J. Cancer, 60,343-350. 8. Quanin, RS., Brakel, C.L. and Webnur, J.G. (1989) Nuel. AcidF Res., 17, 7253-n62.