Intracellular Oligo Targeting A Theoretical Dissertation T. T. CHUANG Department of Physiopathologv of Receptors Istituto di Ricerche Fannacologiche Mario Negn’ Consorzio Mario Negri Sud 66030 S. Maria Imbaro (Chieti), Italy Antisense technology is potentially a powerful therapeutic method. A major obstacle to its application is the high ratio of antisense to sense sequences required to achieve significant inhibition of gene expression, possibly a reflection of the low efficiency of oligos in reaching their targets. This may be due to two factors: (1) intracellular oligos have a fixed life time due to degradation by nucleases; therefore, oligos may be degraded before having the “luck” of colliding with their target mRNA. On the other hand, (2) it has been demonstrated that intracytoplasmically injected oligos rapidly accumulate in the nucleus (within 60 seconds) to specific, discrete intranuclear sites, where they are bound to unidentified nuclear proteins.’ This apparently specific route of nuclear uptake may represent that of cytoplasmically synthesized deoxyribonucleotides into the nucleus for DNA synthesis.* If so, the intranuclear sites of oligo accumulation may be part of the DNA synthesis machinery and would likely act as quenching sites of oligos. The present paper discusses the feasibility of intracellular oligo targeting as a way to reduce the ratio of antisense to sense sequences needed to achieve significant inhibition. Small nuclear ribonucleoprotein particles (snRNPs), which are involved in splicing of pre-mRNA, are localized to discrete and previously unidentified nucleoplasmic sites where accumulate also labeled pre-mRNA synthesized in sku3 or injected intranuclearly,” probably forming spliceosomes. Thus, targeting of oligos to these snRNP-concentrated sites is sensible because oligos and target mRNAs would become confined to small, defined areas, hence providing maximum possibility for antisense-sense hybridization. Spliceosome targeting would likely involve two major steps, namely, nuclear uptake of oligo and subnuclear localization of the oligo to spliceosomes. The former is likely to be taken care of by natural oligo uptake into the nucleus.’ The latter has to await the elucidation of a subnuclear localization signal for snRNPs; the answer may lie within intron sequences, because they have been shown to be crucial in the colocalization of intranuclearly injected RNA with s ~ R N P s This . ~ signal, once elucidated, can be included in the oligo sequence to enable targeting to snRNPs. Additionally, accessible sequences on U1, U2, and US snRNAs, which are involved in binding to pre-mRNA during splicing, near the sequences complementary to the intron splice site can act as docking receptors for oligos, thereby bringing the antisense and sense sequences face to face. Therefore, a spliceosome-targeted oligo can be envisaged to contain, in addition to the antisense sequence, the snRNA-localization signal and the docking sequence for snRNA. The spliceosomes would act as an “oligoport” where outgoing mRNA and incoming oligo interact, thus economizing on strayed oligos. Obviously, whether spliceosome targeting would interfere with normal snRNP splicing activity is a key factor. 326

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In summary, with increasing knowledge of the highly specific intracellular traffic of various molecules, it may become possible to target antisense oligos to sites of mRNA accumulation. This strategy should reduce dramatically the quantity of oligos needed for antisense inhibition. REFERENCES 1. LEONETI,J.-P., N. MECHTI,D. DEGOLS, C. GAGNOR & B. LEBLEU. 1991. Proc. Natl. Acad. Sci. USA 88: 2702-2706. 2. REICHARD, P. 1988. Ann. Rev. Biochem. 57: 349-374. D. 1990. Proc. Natl. Acad. Sci. USA 87: 147-151. 3. SPECTOR, 4. WANG,J., L.-G. CAO, Y.-L. WANG& T. PEDERSON. 1991. Proc. Natl. Acad. Sci. USA 88: 7391-7395.