We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5-g*tggtgggtgggtggg*t-3, where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 M, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with t 1 ⁄2 of 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (t 1 ⁄2 from ϳ3 to 7 min), low cellular uptake, and no sequencespecific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.Guanine-rich nucleic acid strands, under physiological salt and pH conditions, can adopt a higher order, thermodynamically stable conformation containing square-planar arrangement of four guanines that are hydrogen-bonded in the Hoogsteen manner and stabilized by a monovalent cation (1-4). Depending upon the base composition, sequence, and concentration of the nucleic acids, guanine quartet-containing structures (or G-quartets) 1 can be generated from DNA or RNA, either by the intramolecular folding of a single G-rich strand, or by the association of multiple strands (1-7). Believed to be ubiquitous in nature, G-quartets are proposed to participate in diverse biological processes including the modulation of telomere activity, dimerization of HIV RNA, and site-specific genetic recombination in immunoglobulin switch regions (5-9). In addition, using a combination of rational drug design and combinatorial screening methods, several biologically active oligonucleotides have been described, each of unique specificity and the potential to form G-quartet motifs (10 -15). In particular, we have identified a family of deoxyguanosine-and thymidine-rich (deoxyribo)oligonucleotides that are potent inhibitors of HIV-1 expression in standardized cell culture-based assays (16,17). One such inhibitor is T30175, a 17-mer oligonucleotide synthesized with a natural phosphodiester backbone (Table I). A more potent version, T30177, has the same sequence, but contains a single phosphorothioate internucleosid...

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A plasmid expression system encoding murine IFN-alpha4 and complexed with a protective interactive noncondensing polymeric (PINC) delivery system was used for in vivo immunotherapy treatment of an immunogenic murine renal cell carcinoma, Renca, and a nonimmunogenic mammary adenocarcinoma, TS/A. Mice bearing established tumors were treated with IFN-alpha/polyvinylpyrrolidone (PVP) expression complexes via direct intratumoral injection. Up to 100% inhibition of tumor growth was observed in the treated mice. By using an optimal dose of 96 and 48 microg of formulated IFN-alpha plasmid for the treatment of Renca and TS/A, respectively, 30% (Renca) and 10% (TS/A) of the treated animals remained tumor free. Inhibition of tumor growth was dependent on activation of the immune system. The antitumor activity elicited by IFN-alpha gene therapy was abrogated when mice were selectively depleted of CD8+ T cells. By contrast, depletion of CD4+ T cells resulted in enhanced tumor rejection following IFN-alpha/PVP treatments. Finally, mice that remained tumor free following IFN-alpha gene therapy displayed immune resistance to a subsequent tumor challenge. These data provide evidence that IFN-alpha gene therapy can be used to induce an efficient antitumor response in vivo.

The present study demonstrates that mIL-2 or mIL-12 augments tumor inhibition from mIFN-alpha and increases activation of NK and CD8+ T cells. These data support further investigation of polymer and lipid mediated delivery of cytokine genes for head and neck cancer.

The nonviral gene delivery system was well tolerated, and combined mIL-2 and mIL-12 gene transfer generated potent antitumor immune responses against HNSCC in our murine model. Combined nonviral IL-2 and IL-12 gene therapy may have great potential as a primary or adjuvant treatment for HNSCC in humans.