Gene-directed enzyme prodrug therapy for localized chemotherapeutics in allograft and xenograft tumor models

Abstract

Most chemotherapy regimens rely on systemic administration of drugs leading to a wide array of toxicities. Using viral-vector-mediated gene modification of muscle tissues, we have developed a method for gene-directed enzyme prodrug therapy that allows for localized drug administration. An inactive prodrug of geldanamycin was activated locally for inhibition of tumor growth without systemic toxicities. A recombinant adeno-associated virus (rAAV) was used to deliver β-galactosidase (LacZ) to the treatment group and green fluorescent protein to the control group. After 1 week, both groups received adenocarcinoma cells in the same location as the previous rAAV injection. The geldanamycin prodrug was administered 1 h later via intraperitoneal injection. Tumor growth was significantly suppressed in animals whose muscles were gene modified to express β-galactosidase compared with the control. Serum assay to access hepatotoxicity resulted in no significant differences between the animals treated with the inactive or activated form of geldanamycin, indicating minimal damage to non-target organs. Using gene-directed enzyme prodrug therapy, in combination with novel recombinant AAV vectors, we have developed a method for localized activation of chemotherapeutic agents that limits the toxicities seen with traditional systemic administration of these potent drugs.

A Therapeutic Role for Survivin in Mitigating the Harmful Effects of Ionizing Radiation

“…Adenoassociated viruses (AAV), which have consistently been the choice for gene therapy regimens due to their inability to transmit human disease, were modified to create unique recombinant AAV vectors (rAAV) which have proven to exhibit enhanced tissue tropism [ 28 , 29 ]. The novel rAAV, rAAVrec2, which is specific to our laboratory has been employed in our previously published experiments which demonstrated its advantages over traditional viral serotypes [ 24 , 25 ]. Using rAAVrec2, we were able to successfully localize the expression of survivin to the targeted radiation area, while preventing any systemic effects.…”

“…A recombinant adenoassociated virus (rAAV) was used for local delivery of the chosen protein to the left hind leg of each animal. This rAAV vector, known as serotype rAAVrec2, was derived in our laboratory using a PCR shuffling technique from human and novel nonhuman primate viral isolates and has been successfully employed in other gene therapy protocols, including multiple studies previously published by our laboratory [ 24 , 25 ]. Specifically, a rAAV vector containing the gene for either murine-sourced survivin or yellow fluorescent protein (YFP) was constructed.…”

“…Gene-modification was accomplished using direct injection of the viral vector using a 50 μ L Hamilton syringe with a 30-gauge needle. Previous experiments conducted in our laboratory have identified this as an effective method for localizing the viral vector gene products while limiting the operative time required for transduction [ 24 , 25 ]. Viral vectors were titered using real-time PCR.…”

ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Introduction)

“…Gene therapies constitute a separate entity because genes can be manipulated in different ways [12]: replacing the defective gene with a normal gene (this approach mainly works against nonmalign disorders with a single gene aberration [13]), simulating the immune response against cancer cells [14], sensitizing cancer tissues to conventional chemotherapy and radiotherapy [15], delivering genes that change drugs from an inactive prodrug to the active form to cancer cells [16], blocking processes that protect cancer cells such as antiapoptotic mechanisms [17] and using altered viruses (oncolytic virus therapy) to kill cancer cells directly [18], or by means of DNA or RNA oligonucleotide therapies [19]. Targeted therapies are the most common biological approach not only to malignancies but also to inflammatory disorders.…”

Targeting assay of a fusion protein applied in enzyme prodrug therapy

“…The enzymes are directed to the tumor sites by targeted molecules, including antibodies (5), mesenchymal stem cells (6), polymers (7) and genes (8,9). Nontoxic prodrugs, which are substrates of the enzymes, are hydrolyzed by these enzymes in order to release the cytotoxic agents (3,10).…”