Gene Transfer Studies for Cystinosis
GENE TRANSFER STUDIES FOR CYSTINOSIS
I) Validating in vitro gene transfer studies by in vivo gene transfer
We finished our preliminary in vivo gene transfer studies where we targeted the liver using a human adenovirus vector expressing CTNS. We significantly reduced cystine levels (by 1.7-fold compared to control levels, p < 0.05) in 2 and 3 month-old mice whereas we did not reduce cystine levels in 6 and 9 month-old mice. We determined that the efficiency of transduction was equivalent for all ages (in the range of 40-50%). Thus, this study raised two possibilities: i) either longer transduction times (> 1 week) are required to reduce the higher cystine levels in older mice or ii) beyond a certain age we cannot use gene transfer to reduce cystine levels. To differentiate between these two possibilities, we need to extend the post-transduction period (from 1 to 4 weeks) by immunosuppression of the mice. We have performed matings to have two groups of mice (each containing > 12 mice), one group to be aged 2 months and the other to be aged 6 months at the time of the experiments. We have just received the cyclosporin A that we require from the Hospital “Arnaud de Villeneuve” in . Thus, this experiment is now programmed for the end of August when the mice reach the required age.
II) Ocular gene transfer studies
Our article detailing the ocular anomalies in the cystinosis animal model was accepted in March 2007 for publication in the journal “Pediatric Research” and will be published in August.
Prior to beginning eye gene transfer studies, we wanted to generate more clinically relevant CTNS-expressing vectors, which can readily transduce the cornea: a helper-dependent canine adenovirus (HD CAV) and an adeno-associated virus (AAV8) vector. Concerning HD CAV, we are still in the optimisation steps of vector production. In contrast, the production of the AAV8 vector has been achieved by the Vector Production Platform at the Centre of Biotechnology and Animal Gene Therapy (, Spain). Claire Hippert, the recipient of the CRF Ph.D. fellowship, spent a week in Barcelona learning AAV production steps. We received our CTNS-expressing AAV8 vector last week and are currently testing its activity in vitro before beginning in vivo studies.
III) Characterisation of the CNS anomalies in cystinosis mice
To complement our behavioural studies, we assayed the cystine levels in the hippocampus, residual forebrain, cerebellum and brainstem of Ctns-/- mice and found that the hippocampus contained the highest cystine levels consistent with the memory and learning defects that we detected. Moreover, we performed a histological study to detect the presence of cystine crystals and showed that the cystine levels in this structure were not high enough to form crystals. In contrast, we detected abundant crystals within or adjacent to the choroid plexus and in the brain parenchyma mainly clustered around capillaries. This study was submitted to the journal “Neurobiology of Aging” and was accepted for publication pending minor revision at the beginning of June 2007. We are currently performing additional experiments and revising the manuscript according to the reviewer’s comments.
