Quantification of high-capacity helper-dependent adenoviral vector genomes in vitro and in vivo, using quantitative TaqMan real-time polymerase chain reaction.

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host's immune system. Another advantage is their large cloning capacity (up to approximately 35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials.

Regulatable gene expression systems for gene therapy applications: progress and future challenges.

Gene therapy aims to revert diseased phenotypes by the use of both viral and nonviral gene delivery systems. Substantial progress has been made in making gene transfer vehicles more efficient, less toxic, and nonimmunogenic and in allowing long-term transgene expression. One of the key issues in successfully implementing gene therapies in the clinical setting is to be able to regulate gene expression very tightly and consistently as and when it is needed. The regulation ought to be achievable using a compound that should be nontoxic, be able to penetrate into the desired target tissue or organ, and have a half-life of a few hours (as opposed to minutes or days) so that when withdrawn or added (depending on the regulatable system used) gene expression can be turned "on" or "off" quickly and effectively. Also, the genetic switches employed should ideally be nonimmunogenic in the host. The ability to switch transgenes on and off would be of paramount importance not only when the therapy is no longer needed, but also in the case of the development of adverse side effects to the therapy. Many regulatable systems are currently under development and some, i.e., the tetracycline-dependent transcriptional switch, have been used successfully for in vivo preclinical applications. Despite this, there are no examples of switches that have been employed in a human clinical trial. In this review, we aim to highlight the main regulatable systems currently under development, the gene transfer systems employed for their expression, and also the preclinical models in which they have been used successfully. We also discuss the substantial challenges that still remain before these regulatable switches can be employed in the clinical setting.