A detailed understanding of the genetic basis of inherited and acquired hematologic diseases has emerged during recent decades, encouraging efforts to develop genetically targeted reagents as both experimental tools and novel therapeutics. Peptide nucleic acid (PNA), a DNA mimic in which the phosphate deoxyribose backbone of DNA has been replaced by a pseudopeptide polymer, first described in 1991, has attracted particular interest as a gene-targeting reagent, since it is highly stable and binds to complementary RNA and DNA with high affinity and specificity. However, because PNA oligomers resist cellular uptake, their development as tools for modifying gene expression in whole animal studies or as potential therapeutic agents has been limited. To explore the possibility that the transmembrane "transport" domains of microbial toxin proteins might serve as vehicles for cellular delivery of PNA, we studied the ability of recombinant Anthrax "protective antigen" (PA), the non-toxic component of Anthrax toxin that mediates cell binding and delivery, to transport antisense PNA oligomers effectively into cells. For these studies, we first generated CHO-K1 cell lines (CHO-Luc654) that had been engineered by stable transfection to express a modified luciferase gene (Luc-βIVS2-654) interrupted by a mutant β-globin intron-2 with an aberrant splice site that could be blocked by antisense PNA, thereby inducing luciferase expression as an indicator of antisense activity (ref.
Sazani & Kole, J Clin Invest 112:481, 2003
). We then synthesized PNA oligomers with poly-lysine tails and 18-mer nucleobase sequences antisense to a region of Luc-βIVS2-654 pre-mRNA flanking the aberrant IVS2-654 splice site. As anticipated from prior reports (Nucleic Acids Res 29:3965, 2001
), antisense PNA-(Lys)8 oligomers demonstrated detectable sequence-specific activity in inducing luciferase expression in CHO-Luc654 cells when incubated with the cells for 48–72 hrs at micromolar concentrations. However, this activity was greatly and significantly amplified when CHO-Luc654 cells were incubated with antisense PNA-(Lys)8 together with Anthrax PA (0.3–1.0 μg/mL), which had no effects on the cells by itself, such that antisense activity could be detected at PNA concentrations as low as 30 nM. Antisense PNA-(Lys)8 (300 nM), with but not without Anthrax PA (0.3 μg/mL), was also found by rtPCR to induce correctly spliced β-globin transcripts in cultured erythroid progenitor cells obtained from a patient with β-thalassemia [genotype, IVS2-654(βo/βE), kindly provided to us by Dr. Edmond Ma, Queen Mary Hospital, Univ. of Hong Kong]. These studies provide proof-of-principle evidence that the transmembrane transport function of microbial toxins (e.g. Anthrax PA) can be harnessed to deliver antisense PNA oligomers effectively into cells. Since microbial toxin proteins can be molecularly engineered to achieve cell selective binding (as exemplified by the IL2-diphtheria fusion toxin, ONTAK®), our findings support the possibility that novel therapeutic agents, based on PNA oligomer constructs and modified microbial toxin proteins, can be developed that combine both genetic targeting and cell selectivity.
