D. Balya

Genetic Reactivation of Cone Photoreceptors Restores Visual Responses in Retinitis Pigmentosa

Let There Be Light Retinitis pigmentosa, a disease that can result from a wide variety of genetic defects, causes degeneration of photoreceptor cells in the retina and leads to blindness. In the course of the disease, it is generally the rod photoreceptor cells that degenerate first. Cone photoreceptor cells may persist, but in a damaged and nonfunctional state. Busskamp et al. (p. 413, published online 24 June; see the cover; see the Perspective by Cepko) have now applied a gene therapy approach to mouse models of retinitis pigmentosa. Inducing expression of a bacterial light-activated ion pump, halorho dopsin, in the damaged cone cells improved visual responses in the diseased mouse retinas. Thus, it may be possible to rescue cone photoreceptors therapeutically, even after they have already been damaged. A bacterial ion pump rescues visual function in damaged cone-photoreceptor cells in mouse models of retinitis pigmentosa. Retinitis pigmentosa refers to a diverse group of hereditary diseases that lead to incurable blindness, affecting two million people worldwide. As a common pathology, rod photoreceptors die early, whereas light-insensitive, morphologically altered cone photoreceptors persist longer. It is unknown if these cones are accessible for therapeutic intervention. Here, we show that expression of archaebacterial halorhodopsin in light-insensitive cones can substitute for the native phototransduction cascade and restore light sensitivity in mouse models of retinitis pigmentosa. Resensitized photoreceptors activate all retinal cone pathways, drive sophisticated retinal circuit functions (including directional selectivity), activate cortical circuits, and mediate visually guided behaviors. Using human ex vivo retinas, we show that halorhodopsin can reactivate light-insensitive human photoreceptors. Finally, we identified blind patients with persisting, light-insensitive cones for potential halorhodopsin-based therapy.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.

High throughput design of Bacterial Artificial Chromosomes (BACs) for cell type specific gene expression in transgenic mice

Recent advances in homologue recombination based bacterial artificial chromosome (BAC) engineering have greatly simplified knock-in, knock-out and random integration based transgenic mice technologies. Despite the rapid advances in ldquowetrdquo BAC engineering, efficient bioinformatics tools for choosing the appropriate BACs and designing the targeting construct with gene specific homology arms has not been described. Here we introduce a novel bioinformatics tool called eBAC (electronic BAC) that designs, in a high throughput way, all the necessary constructs for BAC engineering. The input to eBAC is a list of genes. eBAC automatically locates and analyzes the BACs containing the genes of interest, finds the appropriate homology arms and designs all necessary primers for BAC engineering as well as tests the BAC for critical restriction sites. eBAC shortens the time for designing BAC recombination from many hours to seconds.