Maxine Bauzon

Multigene Expression from a Replicating Adenovirus Using Native Viral Promoters

We have developed a novel therapeutic gene delivery system for oncolytic adenoviruses that takes advantage of the endogenous gene expression machinery (promoters, splicing, polyadenylation signals) of the E3 transcription unit for gene delivery. In this work, we use two sites in the E3 region (6.7 K/gp19K and ADP sites) to demonstrate that (1) multiple therapeutic genes (MCP-3, TNFalpha) can be expressed from a single replicating Ad, (2) timing of expression of these therapeutic genes mimics that of the E3 region genes they replaced, (3) expression of the remaining genes in the complex E3 transcription unit is maintained, and (4) the multigene-expressing virus retains replication competence and ability to induce classical adenovirus cytopathic effects that parallel those of the parental adenovirus (ONYX-320). This system conserves the DNA packaging capacity of the size-constrained viral genome for therapeutic genes and can potentially be used to link therapeutic transgene expression to tumor-restricted viral replication. Potential clinical implications are discussed.

Preclinical Safety Studies of Enadenotucirev, a Chimeric Group B Human-Specific Oncolytic Adenovirus

Enadenotucirev is an oncolytic group B adenovirus identified by a process of bio-selection for the ability to selectively propagate in and rapidly kill carcinoma cells. It is resistant to inactivation by human blood components, potentially enabling intravenous dosing in patients with metastatic cancer. However, there are no known permissive animal models described for group B adenoviruses that could facilitate a conventional approach to preclinical safety studies. In this manuscript, we describe our tailored preclinical strategy designed to evaluate the key biological properties of enadenotucirev. As enadenotucirev does not replicate in animal cells, a panel of primary human cells was used to evaluate enadenotucirev replication selectivity in vitro, demonstrating that virus genome levels were >100-fold lower in normal cells relative to tumor cells. Acute intravenous tolerability in mice was used to assess virus particle-mediated toxicology and effects on innate immunity. These studies showed that particle toxicity could be ameliorated by dose fractionation, using an initial dose of virus to condition the host such that cytokine responses to subsequent doses were significantly attenuated. This, in turn, supported the initiation of a phase I intravenous clinical trial with a starting dose of 1 × 1010 virus particles given on days 1, 3, and 5.

959. Pharmacokinetic Studies of Human IFN-|[szlig]| in Mice Following Gene-Based Delivery

Interferon-|[szlig]| (IFN-|[szlig]|) is an immunoregulatory cytokine that has been approved as a protein therapeutic for multiple sclerosis (MS). Pharmacokinetic studies have shown that IFN-|[szlig]| has an extremely short half-life in the circulation. Within a few hours following bolus delivery of the recombinant protein, hIFN-|[szlig]| levels in serum are undetectable. Gene-based delivery of IFN-|[szlig]| would offer advantages over bolus delivery of protein in providing sustained long-term expression of the protein resulting in therapeutic efficacy while minimizing side effects.