Cancer-selective antiproliferative activity is a general property of some G-rich oligodeoxynucleotides.

Oligodeoxynucleotide libraries containing randomly incorporated bases are used to generate DNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX). We predicted that combinatorial libraries with alternative base compositions might have innate properties different from the standard library containing equimolar A + C + G + T bases. In particular, we hypothesized that G-rich libraries would contain a higher proportion of quadruplex-forming sequences, which may impart desirable qualities, such as increased nuclease resistance and enhanced cellular uptake. Here, we report on 11 synthetic oligodeoxynucleotide libraries of various base combinations and lengths, with regard to their circular dichroism, stability in serum-containing medium, cellular uptake, protein binding and antiproliferative activity. Unexpectedly, we found that some G-rich libraries (composed of G + T or G + C nucleotides) strongly inhibited cancer cell growth while sparing non-malignant cells. These libraries had spectral features consistent with G-quadruplex formation, were significantly more stable in serum than inactive libraries and showed enhanced cellular uptake. Active libraries generally had strong protein binding, while the pattern of protein binding suggested that G/T and G/C libraries have distinct mechanisms of action. In conclusion, cancer-selective antiproliferative activity may be a general feature of certain G-rich oligodeoxynucleotides and is associated with quadruplex formation, nuclease resistance, efficient cellular uptake and protein binding.

G-rich oligonucleotides for cancer treatment.

Oligonucleotides with guanosine-rich (G-rich) sequences often have unusual physical and biological properties, including resistance to nucleases, enhanced cellular uptake, and high affinity for particular proteins. Furthermore, we have found that certain G-rich oligonucleotides (GROs) have antiproliferative activity against a range of cancer cells, while having minimal toxic effects on normal cells. We have investigated the mechanism of this activity and studied the relationship between oligonucleotide structural features and biological activity. Our results indicate that the antiproliferative effects of GROs depend on two properties: the ability to form quadruplex structures stabilized by G-quartets and binding affinity for nucleolin protein. Thus, it appears that the antiproliferative GROs are acting as nucleolin aptamers. Because nucleolin is expressed at high levels on the surface of cancer cells, where it mediates the endocytosis of various ligands, it seems likely that nucleolin-dependent uptake of GROs plays a role in their activity. One of the GROs that we have developed, a 26-nucleotide phosphodiester oligodeoxynucleotide now named AS1411 (formerly AGRO100 or GRO26B-OH), is currently being tested as an anticancer agent in Phase II clinical trials.

Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies.

Dog and rat animal models have been developed for repeated intravascular administrations to the liver. However, mice have generally been considered too small to use for these models. This study describes the development of mouse models that permit the establishment of liver metastases that can be subsequently treated by repeated injections into the portal venous system. A mini-laparotomy is done to mobilize the spleen and transpose it to a s.c. pocket with its vascular pedicle intact. A suspension of single tumor cells is then inoculated into the portal vein to establish diffuse liver metastases. These tumors may be treated by simple percutaneous injections directly into the s.c. whole spleen reservoir. The ease of injection into the s.c. spleen permits repeated injections into the portal venous system. The usefulness of this model was shown in experiments revealing that multiple portal venous administrations of a replication-conditional, oncolytic herpes simplex virus mutant are more effective than a single portal venous administration. In a modification of this model, the spleen is first split into two, leaving intact the vascular pedicle for each half of the spleen. Tumor cells are inoculated into one hemi-spleen, which is then resected 10 minutes later. The other hemi-spleen is transposed to the s.c. position, thereby permitting subsequent repetitive portal venous injections via percutaneous injections into the s.c. hemi-spleen. These mouse models are useful for a wide range of studies.