MODULATION OF CELL GROWTH BY ANTISENSE AND ANTIGENE REAGENTS

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"The focus of this project is three-fold: (1) to characterize uptake and intra- cellular processing of unmodified and modified oligonucleotides; (2) to utilize antisense and antigene technology in several in vitro model systems to identify critical elements in cell proliferation/viral replication; and (3) to study the efficacy of antisense and antigene reagents as in vivo modulators of gene express- ion. (1) We have characterized the uptake of modified oligos as an energy-depend- ent, endocytic process, mediated by at least one cell surface-binding protein. We have devised a novel technique to study olig uptake, intracellular localization, and association with protein and nucleic acids. This non-invasive technique will permit subcellular localization over time of an internalized oligo. (2) We have confirmed that c-myc inhibition is cytostatic for normal and malignant lymphoid cells and some Burkitt lymphoma cells can be specifically growth-arrested in vitro with a novel c-myc antisense. We have confirmed that N-myc inhibition leads to re- duction in growth secondary to alteration in differentiative status of neuroecto- derm-derived cell lines. (3) We have demonstrated that c-myc antisense is particularly effective against several solid tumors, including human and rat glio- blastoma. In solid tumors, the c-myc antisense oligonucleotide has, in addition to its antisense effects, a sequence-specific, non-antisense mediated, effect on cellular attachment to extracellular matrix. (4) We have identified sequences capable of specifically inhibiting bcr-abl tyrosine kinase and other tyrosine kinases via direct interaction with the proteins. (5) We have been able to demonstrate significant prolongation of animal survival following injection of tumor cells treated with antisense to c-myc. At the same time such antisense treatment has no effect on normal bone marrow cells. These last findings have led to initiation of the process required to undertake a Phase I clinical trial using antisense (c-myc-targeted) and anti-protein (bcr-abl-targeted) oligonucleotides as bone marrow purging agents. Most recently, we have been investigating the efficacy of in vivo delivery of synthetic ribozymes targeting VEGF and FGF for treatment of glioblastoma. We are using a rat CNS model in which ribozymes are delivered intracranially via osmotic pump. Preliminary data suggest that a combination of anti-VEGF and anti-FGF ribozymes significantly prolongs animal survival if administration is begun at the same time as tumor cell inoculation (using a human glioblastoma, U87). Ongoing studies will determine the distribution of ribozymes in brain tissue and tumor, and in vitro effectiveness of these ribozymes toward VEGF and FGF mRNA in the tumor cells. In additional animal studies, we have demonstrated significant anti-tumor activity of a unique CpG oligonucleotide. A single inoculation of mice with this oligonucleotide either pre- or post-tumor inoculation is able to prevent tumor growth, or cause tumor stasis/regression, in greater than 70 percent of animals. The mechanism of action appears to involve the host immune system, but NK cells do not appear to be required."