Title: Prototype foamy virus: vector manipulation and integration.
Funder: Wellcome Trust

Abstract: Foamy viruses, the second subfamily of the Retroviridae, have been studied in our laboratory for over 2 decades. In that time, we have contributed significantly to understanding the biology of the prototype foamy virus (PFV), previously called the human foamy virus (HFV). Indeed, we demonstrated that it was almost identical in sequence to the chimpanzee isolate (1) and that there was no human reservoir of infection (2). When we realised the potential of PFV as a vector {3,4,5}, we were the first to produce replication-defective foamy virus vectors (6) which functioned efficiently in vitro (7), but performed disappointingly in vivo, mainly because titre was limiting. Much of our work has focussed on elucidating the replication pathway of PFV (8-1 6), with a view to optimising virus release from cells (17) and vector development (18-21). Recently, using a novel 4-plasmid vector production system, Trobridge et al (22,23) have produced higher titre (>107 transducing units/ml) PFV vectors than our own (at best, 1 06 ), which, when they express CD18, are capable of reversing the cellular and disease phenotype of 4/5 dogs with CLAD (a genetic disease resulting in integrin, CD18, deficiency) (24). The dogs remain well almost 5 years post-PFV infusion. Analysis of integration sites showed no evidence of vector integration near genes influencing proliferation, supporting the premise that PFV vectors may be safer than gammaretroviruses. However, insertions were noted close to the MDS-EV1 locus, previously implicated in insertional mutagenesis. It is, therefore, critical that PFV integration is understood before these vectors can proceed to clinical trial. Integration is orchestrated by the viral integrase (IN). Following reverse transcription (conversion of the diploid viral RNA genome into a linear double-stranded DNA form), IN catalyses two essential reactions, 3'processing and strand transfer, acting upon both ends of the linear proviral DNA. The IN carries out these reactions within the pre-integration complex (PIC), a large nucleoprotein assembly. At the heart of the PIC is a tetramer of IN assembled on a pair of viral DNA ends, termed the intasome. Work in our department has lead to detailed structural characterization of the intasome from PFV (25,26). However, beyond this core component, the architecture of the retroviral PIC remains uncharted. Because PFV contains reversetranscribed DNA already in viral particles (27), it may allow easy access to large amounts of PIGs for further characterization. Fortuitously, PFV presents the most elegant system in which to study retroviral integration, an understanding of which is critical for clinical exploitation of PFV vectors. Nathan already has some experience with PFV vectors from his project, so he starts from a strong position and we already have some data on PFV vector usage in metachromatic leukodystrophy (MLD). If he can add to this, an early publication would be encouraging. Once mesenchymal stem cell (MSC) transduction has been established and his vector stock made, the in vivo work will not be labour intensive for him and he can investigate the mechanism of integration.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION