Trypanosomes cannot synthesize sialic acids. Infectious stages of the life cycle of the human pathogen Trypanosoma cruzi express a cell-surface glycolipid-anchored trans-sialidase, which can transfer sialic acid between glyco-conjugates. Sialic acid is transferred from host cell-surface and serum sialylglycoproteins to trypanosome cell-surface glycoconjugates. The transfer reaction is specific for donors with terminal alpha-2,3-linked sialic acid, and terminal beta-1,4-linked galactose is the preferred acceptor. In the absence of an acceptor, the enzyme acts as a hydrolase, but cleavage is less efficient than transfer. Trans-sialidase activity is attributable to a few members of a large family of T. cruzi surface glycoproteins, many of which are simultaneously expressed. The functions of the trans-sialidase surface glycoprotein family are unknown but may be important for adhesion, invasion, virulence, or pathogenicity. A trans-sialidase is also expressed in the procyclic forms of Trypanosoma brucei.

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.

Cationic porphyrins form stable complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA, and complexed it with porphyrin. The expression of PKC-alpha protein and mRNA in T24 bladder carcinoma cells was reduced by approximately 80 +/- 10% at a concentration of oligomer of 3 microM, and 9 microM porphyrin. The expression of PKC-beta1, -delta and -straightepsilon isoforms was unaffected by this treatment, but elimination of PKC-zeta protein and mRNA were observed. However, treatment with the porphyrin complex of Isis 3522, an oligomer which is directed at the 5' coding region of the PKC-alpha mRNA, was equally effective as Isis 3521 with respect to PKC-alpha, but did not affect PKC-zeta protein or mRNA levels. Since Isis 3521 has an 11-base region of complementarity with the PKC-zeta mRNA, wheras Isis 3522 has only a 4-base region, the effect of Isis 3521 on PKC-zeta protein and mRNA expression may be due to irrelevant cleavage. Depending upon the desired application, this new strategy may offer several advantages over other methods of antisense oligodeoxynucleotide delivery including efficiency, stability, solubility, relatively low toxicity and serum compatibility. Porphyrins may thus be a potentially useful delivery vehicle for antisense therapeutics and/or target validation.

No abstract

No abstract