Protamine-Fragment Peptides Fused to an SV40 Nuclear Localization Signal Deliver Oligonucleotides That Produce Antisense Effects in Prostate and Bladder Carcinoma Cells

Abstract

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.

Peptide antisense nanoparticles

“…Therefore, in this example, the addition of peptides to antisense nanoparticles increases efficacy by Ϸ50%. This antisense activity is comparable to using molecular peptide-oligonucleotide conjugates at more than 5 times the concentration of the agent described here (49,50). All control nanoparticles with noncomplementary oligonucleotide sequences, including those containing Tat and NLS peptides, had no visible effect on GAPDH levels.…”

“…Fluorescence imaging experiments were conducted to examine the intracellular localization pattern of the particles because translocation into the nucleus has been shown to increase efficacy of antisense agents (39,50). For these experiments, nanoparticles were functionalized with fluorescently labeled oligonucleotides.…”

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

“…Cloroquine is a weak base that accumulates in acidic organelles such as late endosomes and lysosomes, raising the luminal pH of the organelles and avoiding enzymatic degradation of non-viral vectors [45]. This drug is frequently used to investigate the effect of the endosomal/lysosomal entrapment as a barrier to gene delivery, since its action causes a rise in intraendosomal osmolarity and its eventual lysis [46,47]. In our case, pre-treatment of HeLa cells with chloroquine (100 μM) enhanced the LD4 mediated transfection efficiency by 383%.…”

Low molecular weight protamine as an efficient and nontoxic gene carrier: in vitro study

“…Peptide carriers are one of the most promising tools for delivering biologically active molecules into cells 11, 12, 27–29. In the present study, LMWP fractions were evaluated as possible carriers for gene delivery purposes.…”