Effect of retinol dehydrogenase gene transfer in a novel rat model of Stargardt disease.

Dysfunction of the ATPase-binding Cassette Transporter protein (ABCA4) can lead to early onset macular degeneration, in particular to Stargardt disease. To enable translational research into this form of blindness, we evaluated the effect of Cas9-induced disruptions of the ABCA4 gene to potentially generate new transgenic rat models of the disease. We show that deletion of the short exon preceding the second nucleotide-binding domain is sufficient to drastically knock down protein levels and results in accumulation of retinoid dimers similar to that associated with Stargardt disease. Overexpression of the retinol dehydrogenase enzymes RDH8 and RDH12 can to a limited extent offset the increase in the bisretinoid levels in the Abca4Ex42-/ - KO rats possibly by restricting the time window in which retinal can dimerize before being reduced to retinol. However, in vivo imaging shows that overexpression of RDH8 can induce retinal degeneration. This may be due to the depletion in the outer segment of the cofactor NADPH, needed for RDH function. The translational potential of RDH therapy as well as other Stargardt disease therapies can be tested using the Abca4 knockdown rat model.

Evaluation of a Serum-free Medium for the Production of rAAV-2 using HeLa Derived Producer Cells.

During the last decade, recombinant AAVs have become of increasing interest for gene therapy. Clinical trials have been conducted following promising in vivo evaluations, thus leading laboratories to adapt their production systems for larger and higher quality demands. Classical transfection protocols seem difficult and cumbersome to adapt to a bioreactor scale. The use of stable producer cells appears as an attractive alternative, as this system requires only a single infection step to induce rAAV production. Furthermore, the switch to a serum-free medium is an interesting strategy to increase the biosafety level to satisfy clinical grade requirements for gene therapy products. Here, we have combined both approaches and evaluated different rAAV producer clones in a serum-free medium. We first evaluated the cell growth in a serum-free medium and then did a partial optimisation of the medium composition to obtain vector yields as close as possible to the yields obtained in a classical serum containing medium. Different helper viruses, multiplicity of infection, times of infection and harvest have been compared in small scale cultures in order to determine the optimal settings which were then transferred and evaluated in suspension cultures in spinner flasks. The yields obtained in this system were similar to or at most 2 times lower than those obtained in a serum-containing medium. The scale-up of such a production system as well as the use of high cell density perfusion culture systems will probably lead to considerably higher yields than those obtained in a classical process.