1. Field of Invention
The present invention relates to methods of intracellular delivery of oligonucleotides. Yet further, the present invention relates to the use of the delivery system to deliver G-quartet oligonucleotides as a cancer therapy or an anti-viral therapy.
2. Related Art
The G-rich oligonucleotides have been identified, cloned and characterized in the telomeric sequences of many organisms, such as fungi, ciliates, vertebrates and insects (Henderson, 1995). The main structural motif of telomeric DNA is the G-quartet structure, which was first proposed by Cellert et al., 1962. The G-quartet consists of four guanine bases in a sequence array arranged into a cyclic Hoogsteen H-bonding structure and each G-base makes two H-bonds with its neighbor G-base (N1 to O6 and N2 to N7). G-quartets stack on top of each other to form tetrad-helical structures. The uniquely structural feature of G-quartet is a pocket in the center lined by electronegative carbonyl oxygenes to be the site of interaction with a cation. G-quartet structures exhibit some specific behaviors as nucleic acids (Williamson, 1994; Rhodes, et al., 1995; Gilber et al., 1999). They are very polymorphic. A family of related G-quartet structures, such as a single-stranded monomer, hairpin dimmers and parallel stranded tetramers, can be formed based upon sequence, concentration and base composition of the nucleic acids. Also, they can readily discriminate between different monovalent cations. G-tetrad forming oligonucleotides have specific affinity for monovalent cations and G-quartet formation strongly depends on the presence of cations. The order of preference that has been proposed is K+>Rb+>Na+>Li+ or Cs+ (Sen et al., 1990; Jing et al., 1997). The selectivity of G-quartet structures for cations is due to the ionic radius, and potassium appears to have the optimal size to interact within a G-octamer. The folding and unfolding transitions for G-quartet structures are extremely slow, so that the G-quartet structures are both thermodynamically and kinetically stable. The stability and slow kinetic transition of G-quartet structures have some important consequences for their biological rules.
Several groups have demonstrated that G-rich oligonucleotides forming G-quartet structures can be potential therapeutic drugs, such as potent HIV inhibitors (Jing et al., 2000; Jing et al., 1998; Jing et al., 2000b) and inhibitor of human nuclear top 1. The previous studies demonstrated that the G-rich oligonucleotides form a stable intramolecular G-quartet structure to inhibit HIV-1 integrase by binding it into the active site of the target protein (Katchalski-Katzri et al., 1992; Vakser et al., 1996).
The delivery of G-quartet oligonucleotides to the designed targets is a critical issue for oligonucleotides as pharmaceuticals. To utilize this structure as a pharmaceutical, it is imperative that the G-quartet oligonucleotides be delivered into the nuclei of target cells. The present invention is the first to deliver these G-quartet oligonucleotides into the nuclei of the target cells.
USPTO Backgrounds
USPTO Backgrounds