Gerald W Both Fiona Cameron Anne Collins Linda J Lockett and Jan Shaw


Gene-directed enzyme prodrug therapy (GDEPT) is an emerging approach for the treatment of cancers. A variety of viral vectors have been used to deliver genes that encode the relevant enzymes, and some have been tested in clinical trials. To ensure the potency and efficacy of such vectors and to obtain regulatory approval to administer them to humans, it is necessary to develop a suite of assays that provide quality assurance. New GDEPT vectors based on ovine atadenovirus and Escherichia coli purine nucleoside phosphorylase (PNP) have been developed for first time use in humans in a phase I trial for the treatment of prostate cancer. Here we describe methods for their production together with several quality-control assays. In particular, a functional cell killing assay was devised to measure the potency of PNP-GDEPT vectors, the principles of which could easily be adapted to other systems.

Key Words: Ovine atadenovirus; vectors; PNP-GDEPT; prostate cancer. 1. Introduction

Human adenovirus vectors have been widely used in the laboratory and clinic, and procedures for their construction and manufacture are well known (1). However, attention is now also being directed to nonhuman adenovirus vectors partly because they are not neutralized by immunity that exists in humans as a result of natural infection, which may provide an advantage for gene delivery in vivo. The nonhuman vector derived from an ovine adenovirus isolate OAdV287 (OAdV) has been developed as a gene delivery vector (2). OAdV is the prototype of a new genus known as the atadenoviruses (3). These viruses have very different biological properties compared with mastadeno-viruses, the best studied group. Some clues as to the function of certain genes

From: Methods in Molecular Medicine, Vol. 130: Adenovirus Methods and Protocols, Second Edition, vol. 1: Adenoviruses Ad Vectors, Quantitation, and Animal Models Edited by: W. S. M. Wold and A. E. Tollefson © Humana Press Inc., Totowa, NJ

have been obtained, but there are numerous genes with no known homologs whose functions are still being elucidated (4).

Vectors OAdV220 and OAdV623 express the Escherichia coli PNP gene from different promoters (see Subheading 2.1., item 3). These were constructed and tested in preclinical GDEPT studies for prostate cancer (5-8). Procedures for the construction of plasmids and rescue of infectious OAdV vectors from transfected DNA have been described (9,10). Because OAdV replication is abortive in nonovine and human cell lines, these vectors were propagated in fetal ovine skin (HVO156) or lung (CSL503) cells (11), which are established, adherent cell lines that have not been immortalized. In this work we have used CSL503 cells because master and working cell banks have been established and tested for adventitious agents.

OAdV623 and OAdV220 lysed the production cells very efficiently, necessitating changes to earlier methods of harvest. Here we describe the modified manufacturing procedure together with quality-control assays that were used routinely. Additional assays were used by a contract manufacturer to characterize vector suitable for a phase I clinical trial.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment