Re-engineering adenovirus regulatory pathways to enhance oncolytic specificity and efficacy.

Replicating adenoviruses may prove to be effective anticancer agents if they can be engineered to selectively destroy tumor cells. We have constructed a virus (01/PEME) containing a novel regulatory circuit in which p53-dependent expression of an antagonist of the E2F transcription factor inhibits viral replication in normal cells. In tumor cells, however, the combination of p53 pathway defects and deregulated E2F allows replication of 01/PEME at near wild-type levels. The re-engineered virus also showed significantly enhanced efficacy compared with extensively studied E1b-deleted viruses such as dl1520 in human xenograft tumor models.

Specific depletion of human anti-adenovirus antibodies facilitates transduction in an in vivo model for systemic gene therapy.

Recombinant adenoviral (rAd) vectors are capable of mediating high-efficiency gene transfer in vivo. Under conditions requiring systemic administration, however, the use of rAd vectors can be problematic due to the presence of circulating anti-adenovirus antibodies developed either through natural infection or during the course of treatment. We developed a passive immunization model in SCID/Beige mice to assess the effect of human and mouse anti-adenovirus antibodies on systemic administration of a rAd vector expressing beta-galactosidase (rAd-betagal). In this model, the in vitro neutralizing activity of human or mouse antibodies used for passive immunization correlated well with inhibition of transduction of the liver following i.v. administration of rAd-betagal. Depletion of antibodies to individual adenovirus structural proteins (hexon, penton, fiber) by affinity chromatography demonstrated that antibodies to each of the three virion components contributed to neutralization of infectivity in vitro and to inhibition of transduction in vivo. Depletion of antibodies against all three structural proteins from human or mouse immune serum prior to passive immunization restored in vivo transduction activity to levels comparable to those obtained with nonimmune serum. Our data suggest that depletion of both murine and human anti-adenoviral antibodies can restore transduction in vivo during systemic rAd gene therapy in hosts previously exposed to adenovirus.

Calpain inhibitor 1 activates p53-dependent apoptosis in tumor cell lines.

Reports suggest a role of calpains in degradation of wild-type p53, which may regulate p53 induction of apoptosis. A calpain inhibitor, n-acetyl-leu-leu-norleucinal (calpain inhibitor 1), was assessed for ability to enhance p53-dependent apoptosis in human tumor cell lines with endogenous wild-type p53 and in altered p53 cell lines with the replacement of wild-type p53 by a recombinant adenovirus (rAd-p53). Calpain inhibitor 1 treatment resulted in increased levels of activated p53, increased p21 protein, and activation of caspases. Cell lines with wild-type, but not mutated or null, p53 status arrested in G0/G1 and were sensitive to calpain inhibitor-induced apoptosis. Regardless of endogenous p53 status, calpain inhibitor treatment combined with rAd-p53, but not empty vector virus, enhanced apoptosis in tumor cell lines. These results demonstrate p53-dependent apoptosis induced by a calpain inhibitor and further suggest a role for calpains in the regulation of p53 activity and induction of apoptotic pathways.

Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer.

Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCB-infected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with this recombinant adenoviral vector has a potential use in cancer gene therapy.

Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles.

The expanding use of adenoviral vectors for gene therapy has brought about the need for new analytical tools. We have developed an anion-exchange high-performance liquid chromatography method to analyze recombinant adenovirus serotype 5 samples. Before this assay, available analytical methods consisted of either long-term biological assays or required highly purified test articles. These methods were inadequate for optimizing adenovirus production and purification. This assay can quantitate viral particles in either crude lysates or highly pure samples. It can be used to assess particles in both dilute and concentrated samples over a wide dynamic range. Moreover, the population of viral particles eluted in the peak contains most of the infectious virions. This assay is a sensitive technique that overcomes the limitations of previous methods. It provides an essential tool to accomplish process optimization.

Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography.

We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications.