Reference standard for gene therapy closer

Abstract

On both sides of the Atlantic, new initiatives to establish gene therapy benchmarks could stimulate research into rare conditions and allow researchers to compare their results with unprecedented accuracy.Certainly, there is a need to standardize gene therapy vectors. Terence Flotte (University of Florida, Gainesville, FL, USA) notes that there was a consistent five-to-tenfold difference in the potency of adeno-associated virus (AAV), one of the most common gene therapy platforms, between his lab and just one of his collaborators. ‘The range is probably much more than this,’ he admits. ‘There is no comprehensive data. However, assays taken on the spot suggest a 100-fold difference in potency. And that doesntake into account differences in the biological potency – the virus's ability to infect cells.’ Clearly, this makes comparing studies difficult.To accurately compare studies, Dominic Wells (Imperial College School of Medicine, London, UK) proposes that researchers need to know three key measurements: (1) the number of virus particles, obtainable from DNA based measurements; (2) the number of infectious units, based on cell culture assays; and (3) the number of in vivo infectious units. ‘Cell culture and in vivo-based assays are difficult to standardize due to individual differences in handling cells and in vivo experiments,’ he notes. ‘In addition, gene therapy reagents show different efficiencies of infection of different cells and tissues. These differences can be compensated for by parallel testing of a known reference standard with a known outcome.’This principle underlines attempts on both sides of the Atlantic to standardize gene delivery. For instance, researchers at the University of Florida, supported by a $75 000 grant from the National Institutes of Health, are culturing a batch of high-concentration, high-purity AAV that will be stored at a national repository after ‘extensive scrutiny’ by universities and biotechnology companies. The benchmark will allow scientists to compare the strength of their investigational therapies.Flotte predicts that the new reference standard could have its greatest impact in the management of rare diseases using gene therapy. Taking a vector through phase I, II and III trials is impractical in such cases – there is neither the patient population nor an adequate return on investment. ‘The new benchmarked vector offers a platform for the management of rare disease that has an established, characterized toxicological profile,’ he says.‘This is an important step forward in safety testing and will allow a more direct comparison of the results from different groups,’ Wells comments. But he adds: ‘Clearly, reference standards will be needed for each gene delivery system. The University of Florida work just deals with AAV. As each vector system is developed, there will be a need to produce new reference standards. However, AAV is the agent with the most immediate need for a standard as quantitation of this vector has been the most variable.’The European initiative aims to standardize a wider range of vectors. Over the last few years, Genethon (Evry, France) provided standardized gene therapy reagents, such as cell lines, plasmids and tailored viral vector preparations both within France, via the Gene Vector Production Network (GVPN1Gene Vector Production Network: http://www.genethon.fr/gvpn11), and across Europe (Gene vector Database and Repository: EGDR2European Gene vector Database and Repository: http://www.egdr.org22). But, while there is considerable demand, standardization remains a problem.‘The quantity of gene therapy products delivered is doubling each year,’ says Mauro Mezzina, Research Director at Genethon. ‘We are still far from standard methods to produce reagents and standard samples to be used as internal positive controls. Even though the three GVPN laboratories in France produce vectors and other related material using similar procedures, these are still non-homogeneous, and no reference material has been yet produced or validated. Therefore, gene therapy medicine is still very far from establishing ‘‘which dose of vector and how often’’ to obtain a therapeutic benefit.’The next stage, which is just underway, will make standardized preparations and calibrated vectors available to European researchers. EGDR brings together 15 European laboratories, spanning academia and biotechnology, to establish reference standard samples and procedures for gene therapy, covering, for example, all viral and non-viral vectors, cell lines, other reagents, production and purification procedures. Olivier Danos, Scientific Director of Genethon and President of the European Society of Gene Therapy, adds that EGDR also acts as a repository for knowledge in the literature as well as unpublished practical details (see also Nevin and Spink, this issue).Eventually, there is likely to be a single repository for the entire world. Danos notes there is already collaboration across the Atlantic and he predicts that EGDR will probably use the American vector as the standard. Eventually, he expects systems to merge. ‘We would like to have a common world reference,’ he says. ‘The European initiative is the first step towards this.’