Cellular pharmacology of oligonucleotide analogs
Oligonucleotide therapeutics show great potential for gene-specific therapy of a wide variety of diseases. Oligonucleotides have been modified in many ways to improve nuclease stability and cell membrane permeability. Study of the cellular pharmacology of oligonucleotide analogs is vital not only for understanding of their biological function but also for designing of more potent analogs. This work investigated the cellular pharmacology of several modified oligonucleotide analogs and also demonstrated novel strategies to improve cell specific targeting and cellular uptake using biologically stable analogs. Two anti c-myc and anti c-myb sequences used for antisense experiments contain contiguous guanosines capable of forming tetraplex structures which might lead to non-specific effects. Native gel electrophoresis revealed, that, although these sequences can form tetraplex structures at room temperatures and below, they do not do so under physiological conditions. Thus, these particular sequences should be available for cellular uptake as single strands. Cellular uptake of five different modified oligonucleotide analogs, in a variety of sequences was studied in several transformed and untransformed cell lines. The observations provide a direct quantitative comparison of cellular uptake as a function of oligonucleotide modification, and imply that transformation enhances cellular uptake. The cellular uptake of oligonucleotide analogs is inefficient and typically independent of cell type. Cell specific targeting and better cellular uptake may lead to better efficacy due to higher accumulation of the molecules inside the targeted cells. For this purpose a novel strategy was devised in which a peptide moiety designed to bind to a cell surface receptor, which is overexpressed in several oncogenic cell lines, was conjugated to different modified analogs to achieve cell specific targeting and better cellular uptake. A novel automated solid phase synthesis method was developed for synthesizing DNA-peptide conjugates. This synthesis strategy can easily be extended to other nucleic acid analog-peptide conjugates. A semi-automated strategy is described where two different chemistries, Fmoc and Boc, were used to create a PNA-peptide conjugate. Based on the cellular uptake, biophysical, and biological stability data, the peptide nucleic acid-peptide conjugates seem to be the most suitable analog for therapeutic purposes.
Basu, Soumitra, "Cellular pharmacology of oligonucleotide analogs" (1996). ProQuest ETD Collection - Thomas Jefferson University. AAI9727328.