The premise of gene therapy is to introduce a functional copy of a gene in to cells where one or both copies are missing, meaning that this approach is only appropriate for those diseases caused by a straightforward loss of gene function.
The greatest challenge facing the field is how to deliver a gene to the correct cells or organs in a patient, and to get stable, prolonged expression of the gene. Two thirds of clinical trials currently use viral vectors (Edelstein et al., 2007; updated 2013) which deliver genes to cells by integrating into the genome (Vannucci et al., 2013). This approach can be highly problematic; by integrating in to the target genome, the virus can perturb normal expression of the genes surrounding the integration site. Indeed, during an early clinical trial of a gene therapy for SCID-X1, two patients in a cohort of ten developed T-cell leukaemia due to insertion of the retrovirus close to the LMO2 promoter, causing overexpression of this gene (Hacein-Bey-Abina et al., 2003).
Recently, Dr Suet Ping Wong and Dr Richard Harbottle, from Imperial College London, have successfully reintroduced a functional copy of the BHD gene, FLCN, into FLCN-deficient UOK257 cells derived from the kidney tumour of a BHD patient (Wong and Harbottle, 2013). Instead of using a viral vector, they used a Scaffold/Matrix Attachment Region (S/MAR) DNA vector, which does not integrate into the host cell’s genome, thus avoiding complications that arise from using viral vectors. This group had previously shown that these 1 – 2 vector copies per cell are maintained episomally through countless cell divisions, by direct attachment of the vector to the intracellular matrix via its S/MAR element (Argyros et al., 2012).
Characterisation of the UOK257 cells carrying the S/MAR plasmid (UOK257-FS) showed that expression of the FLCN protein was 16-fold higher than the parental cell line, meaning that FLCN expression had been successfully reconstituted in these cells. UOK257-FS cells displayed reduced cell-cell contact, increased TGF-β signalling, reduced cell proliferation rates, and reduced mTOR signalling under serum-starved conditions, compared to the parental UOK257 cell line, indicating that FLCN function had also been reinstated in these cells.
Furthermore, while UOK257 cells formed peritoneal tumours between 0.5 – 1.5 cm in diameter in xenograft experiments, UOK257-FS cells had not formed tumours at 150 days post injection, although cell spheroids between 0.2 – 0.5 cm2 were isolated from one animal. Subsequent RT-PCR analysis of the UOK257-FS spheroids showed that FLCN expression had been retained and that TGF-β signalling also remained elevated over at least 50 cell divisions.
While it is important to note that this study was undertaken in cells, it is a significant first step towards developing a gene therapy for BHD as it shows that S/MAR vectors can be used to stably reinstate wildtype FLCN function in FLCN-deficient cells. Effective methods of delivery to the kidneys, lungs and skin need to be developed and this therapy tested in animal models of BHD before this treatment can be tested in a clinical trial. However, Glybera, the first gene therapy to gain regulatory approval, became available to lipoprotein lipase deficiency patients in November 2012, and there are currently more than 3000 clinical trials testing gene therapies, indicating that gene therapy may prove to be a common and effective approach to treat genetic diseases, including BHD, in the future.
Argyros O, Wong SP, Gowers K, & Harbottle RP (2012). Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modelling. PloS one, 7 (10) PMID: 23110132
Edelstein ML, Abedi MR, & Wixon J (2007). Gene therapy clinical trials worldwide to 2007–an update. The journal of gene medicine, 9 (10), 833-42 PMID: 17721874
Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, Sorensen R, Forster A, Fraser P, Cohen JI, de Saint Basile G, Alexander I, Wintergerst U, Frebourg T, Aurias A, Stoppa-Lyonnet D, Romana S, Radford-Weiss I, Gross F, Valensi F, Delabesse E, Macintyre E, Sigaux F, Soulier J, Leiva LE, Wissler M, Prinz C, Rabbitts TH, Le Deist F, Fischer A, & Cavazzana-Calvo M (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science (New York, N.Y.), 302 (5644), 415-9 PMID: 14564000
Vannucci L, Lai M, Chiuppesi F, Ceccherini-Nelli L, & Pistello M (2013). Viral vectors: a look back and ahead on gene transfer technology. The new microbiologica, 36 (1), 1-22 PMID: 23435812
Wong SP, & Harbottle RP (2013). Genetic modification of dividing cells using episomally maintained S/MAR DNA vectors. Molecular therapy. Nucleic acids, 2 PMID: 23941867