2010 Progress Report – Executive Summary
A major challenge for biomedicine is to develop new ways of determining how proteins operate in complexes in cells. This requires molecularly focused methods for dynamic interrogation and manipulation. An attractive approach is to use light as both input and output to probe molecular machines in cells. While there has been significant progress in optical detection of protein function, little advance has been made in control. The NDC for the Optical Control of Biological Function is developing methods for rapidly turning select proteins in cells on and off with light. The strategies are broadly applicable across protein classes.
Biological and disease targets: Three central signaling systems. We are focusing on ion channels, receptors and enzymes. These represent some of the largest protein families, together comprising about 7% of the human genome. The proteins are implicated as causes and drug targets in major human diseases. By enabling rapid, reversible and local switching these tools should make it possible to overcome two major problems in standard drug delivery: a) side effects due to systemic delivery, or systemic leak of locally delivered drugs, and b) long delays between delivery and action, and difficulty to terminate action.
Toolkits of light-gated signaling proteins and of light-gated peptides that selectively modify protein function. We are using structure-based design to chemically synthesize two classes of nanoscale photoswitches: 1) ligand photoswitches that attach to native and engineered target proteins, and remote control their function (with both activating and blocking ligands); and 2) light-gated peptides, which bind to target proteins (at interaction domains or substrates binding sites) in a light-dependent manner, interfering with function only in the bound state.
Addressing specific medical problems. A critical challenge is to transfer our methods from basic research in cell biology to therapeutics. We will focus on animal models of human disease, including using light-gated channels to restore vision to retinae that have lost photoreceptor cells, treatment of retinal disease due to angiogenesis, and prevention of cardiac damage due to ischemia. In Yrs 1-3 we made progress in several areas: a) the fundamental properties of new target proteins, b) new classes of photoswiches, c) design of optical instrumentation for delivering light in vivo and in vitro and d) setting up disease models that can be treated with light-gated proteins, with the greatest progress made toward the restoration of vision in retinae that have lost their photoreceptor cells in models of blinding diseases.
Original goals. Our goal has been to address the major challenge for nanomedicine of developing new methods for remote controlling proteins in cells, with the ultimate goal of applying this to the treatment of human disease through the non-invasive manipulation or restoration of cell and circuit function in vivo. We began with a 3-prong plan:
Aim 1) To engineer three signaling systems to be light sensitive. One of the first successes of this method has been a new computational approach that can design for a particular protein target different attachment sites for a single photoswitch that have opposite effects, thus making it possible to use a single photoswitch with one attachment chemistry to program opponency into cell types that normally behave in an opposite manner. We have also made advances on new photoswitches that can use deeply penetrating 2-photon illumination, new orthogonal methods of protein attachment, tuning the properties of the switches in color and speed, and applying them to new protein targets, including the important NMDA receptor, which is at the heart of learning, memory and circuit development.
Aim 2) To develop the means to deliver genes encoding light-gated signaling proteins, photoswitches and optical stimulation to cells of interest. In model genetic organisms this involves the production of transgenics both via the use of cell-specific promoters and a variant of enhancer trapping in which appropriate targeting is determined functionally in a new approach that we call ”intersectional optogenetics.” In models for humans where gene delivery is only possible via viral transfection we evolve the capsid proteins of the AAV virus and select for targeting to specific cells and search for cell-specific promoters. We also develop a new variant method of photoswitch attachment in which a modified gene does not need to be introduced, and instead the native proteins of the organism are modified (our 1-component PAL system). This has the advantage of not requiring the introduction of a gene, but loses cell-specific targeting. We also develop a new approach that partly compensates for the loss of cell-targeting specificity by using “optical lithography” in which photoswitch attachment is regionally defined by attachment under a pattern of light. Finally, we develop the means for conditioning the stimulating light and for delivering it to the tissue of interest in vivo.
Aim 3) To apply light control of protein function to models of human disease. This includes basic medical research of the biochemical signaling circuits that go awry in disease, including a new effort in cystic fibrosis, to the study of normal and pathological neurodevelopment and to the elucidation of the neural circuit basis of visual processing and behavior. This aim also features animal models of human disease, first and foremost using light-gated channels to restore vision to retinae that have lost photoreceptor cells, an area in which significant progress as already been made. Secondly, the approach will be applied to the treatment of retinal disease due to angiogenesis, to the prevention of cardiac damage due to ischemia.
First disease target- blinding diseases that result from loss of photoreceptor cells. We have begun to pursue a first disease model: blinding diseases that result from loss of photoreceptor cells. Such diseases often spare other cells in the retina for many years. Especially resilient are the output neurons of the retina, the RGCs. Our approach is to endow the surviving cells with an ability to sense light, using our engineered light- gated ion channels. We have successfully expressed light-gated channels selectively in a subset of ON RGCs using viral delivery and cell-specific promoters. We are now in pursuit of other promoter-virus combinations that would target OFF RGCs and other subtypes of ON retinal ganglion cells. We are confident that these will be identified within the next 2 years. While that effort is being pursued, we are already testing the ability of our initial ON RGC expression system to support vision in animals that have lost their photoreceptor cells. Along with new PIs Dan and Baier we will advance to electrophysiological assays in the retina, rodent visual cortex and fish optic tectum, as well as by behavioral analyses in both rodents and fish.
Advancing into higher mammals. Preliminary experiments with our first clinical collaborator, van Gelder, have shown that our photoswitches can be used to drive activity in the RGCs of the isolated primate (macaque) retina. New PI Merigan has preliminary results showing that our promoter-virus system drives expression in macaque RGCs in vivo. Merigan and new PI Siegel will carry out advanced psychophysical and electrophysiological experiments on higher visual processing in macaque. Together with van Gelder this new prong of effort should make it possible to analyze the vision that is mediated in primate by engineered RGCs that rendered directly sensitive to light by our photoswitched channels.
Pathway to Medicine. Blinding diseases such as retinitis pigmentosa (RP) and other blinding diseases result from progressive degeneration of rods and cones. Downstream retinal neurons that receive and process signals from the photoreceptors are preserved for years after the onset of blindness in these diseases , giving hope that visual sensitivity might be restored by allowing the artificial input of information to these surviving cells. Previous retinal prosthetic devices used electrode arrays driven by a CCD video camera to control the firing of surviving retinal interneurons. While electrical stimulation of the retina produced light perception in patients receiving these devices, spatial and temporal resolution was poor, probably because of the relatively large size and imprecise positioning of electrodes in the arrays with relation to individual RGCs. The materials used in building these devices also present serious challenges of long-term biocompatibility. We propose using light-activated ion channels as an alternative therapeutic approach to avoid these problems.
By inserting minimally invasive micro-optical probes directly into deep tissues, cellular structures and dynamics never before visualized in vivo have recently become accessible to imaging with micron-scale resolution. This has begun to allow longitudinal studies in which cells in individual animals are imaged repeatedly over time, reducing the number of animals sacrificed for population statistics and allowing the progression of disease, biological processes, and experimental treatments to be monitored chronically in vivo. However, imaging is a powerful but purely passive technological approach in that it only collects information about the biological state. To alter biological states in active manner, imaging should ideally be combined with analogous capabilities for optical manipulation over protracted time periods, e.g. over the time course of disease progression. In the future, combined optical imaging and control will be performed in an intelligent manner, with the imaging data informing the spatiotemporal patterns of optical stimulation used for cellular control.
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