Project summary We propose to develop a method of non-surgical, spatially precise, ultrasound-enhanced delivery of macromolecules to the retina. The need for such delivery methods stems from the prevalence of recent success in the treatment of retinal disorders by gene therapy. Inherited retinal diseases affect several million people worldwide and often result in vision loss and blindness. Most retinal disorders are incurable, but recent advances in gene therapy have restored vision and hope to many. In animal research, gene delivery to the eye has also helped uncover mechanisms of retinal disease. However, in both the clinic and animal research, delivery of genes to the eye is challenging. Currently, the delivery of genetic material requires surgery performed by a vitreo-retinal surgeon to access the subretinal space for the injection of genetic material. This process is technically difficult and can result in serious complications. Here, we will develop a new method of macromolecule and gene delivery to the retina that does not require eye surgery or intraocular injection of genetic material. In this method called, Enhanced Transretinal Ultrasound Delivery (ETUDE), we combine our experience in focused-ultrasound gene delivery and retinal disorders. In ETUDE, focused ultrasound (FUS) is targeted with high precision to a small region of retina. Then, a clinically-approved microbubble contrast agent is injected through a peripheral vein. In the brain, such a contrast agent responds to ultrasound and exerts mild pressure on interior lumen of capillaries. This pressure then opens the tight junctions in blood-brain barrier (BBB) and allows for free passage of molecules up to ~20 nanometers in diameter. This BBB opening lasts for several hours and has was previously used for delivery of small molecules, proteins, and viral vectors. The retina contains a similar vasculature referred to as a blood-retinal barrier (BRB). We hypothesize that BRB and BBB react similarly to the FUS in presence of an ultrasound contrast agent and will similarly enable site-specific delivery of molecules to the eye. To enable high-precision, high-safety gene delivery to the eye we propose to use an innovative method of spatial targeting of ultrasound. We will use high-frequency ultrasound and record the ultrasound echo of the microbubble contrast agent in the retina. We expect to target retinal ~300-micron sized regions spanning the retinal thickness in mice. Throughout this project, we will develop safe protocols for ETUDE, and quantitatively characterize its efficiency, spatial precision, and any potential tissue damage in gene delivery of intravenously applied viral vectors, and enable cell-type specific gene delivery to retinal ganglion cells (RGC). If successful, we will have enabled a safe, non-surgical, site-specific, gene and macromolecule delivery to specific retinal cell-types.