OPTOGENETICS INNOVATIONS

A rapidly evolving field of research known as optogenetics offers exciting potential for the restoration of vision in patients with incurable degenerative retinal diseases such as retinitis pigmentosa and advanced macular degeneration, according to Sonja Kleinlogel PhD, head of Optogenetics Group, Institute of Physiology, University of Bern, Switzerland.

“This is an exciting area of research that will hopefully in a few years enable patients with photoreceptor degeneration to get back almost complete vision. The target diseases for this novel treatment are mainly retinitis pigmentosa and age-related macular degeneration, particularly the advanced forms where the photoreceptors have almost completely degenerated,” she told the 13th EURETINA Congress in Hamburg. While research into stem cells and retinal prostheses hold some promise in the treatment of degenerative retinal diseases, optogenetics may offer some advantages over both these approaches, said Dr Kleinlogel.

“The problem with the stem cell approach is that when the photoreceptors have already degenerated, the implanted cells usually do not survive. Similarly, while the retinal prosthesis is undoubtedly a great step forward, there are limitations to maximal achievable resolution so vision will never be restored to normal high-resolution healthy vision allowing the patient, for example, to drive a car or read a book.”

Defining optogenetics as a combination of optics and genetics, Dr Kleinlogel explained that the inception of the discipline began in earnest with the discovery of a rhodopsin-like lightsensitive protein in green algae called channelrhodopsin-2. This visual pigment allows the green algae to perceive light, important to sustain photosynthesis.

Channelrhodopsin-2 reacts to blue light by making the cell membrane permeable to positively charged ions and the resulting influx of ions triggers a nerve impulse that activates the cell. “For neuroscientists, this prompted the idea of isolating this gene and implanting it by targeted genetics into neurons of choice. Researchers then succeeded in isolating halorhodopsin, a chloride pump activated by yellow light, which acts as the perfect counterpart to channelrhodopsin-2 since it lowers the membrane potential effectively switching off the neurons,” said Dr Kleinlogel. Since channelrhodopsin-2 and halorhodopsin react to light of different wavelengths, together they comprise a useful tool for switching cells on and off at will, said Dr Kleinlogel.

The therapeutic concept of Dr Kleinlogel’s optogenetics approach is to target the neurons in the middle layer of the retina – the bipolar cells – which remain intact even when the photoreceptors in the outer layers of the retina have degenerated. Custommade membrane proteins equipped with a rhodopsin-based light antenna are introduced into the bipolar cells using adeno-associated viral (AAV) shuttle vectors as delivery systems. This renders the naturally light-insensitive bipolar cells light-sensitive and capable of detecting visual information, which is subsequently relayed to the brain without receiving input from photoreceptors. Working with channelrhodopsin-2, however, it soon became obvious to Dr Kleinlogel and her team that they would need a next-generation optical genetic tool if the approach were to have any chance of being successful in the clinic.

“There are some limitations to tools such as channelrhodopsin that prevent their use in the clinic. The first is that they require extremely high light intensities to be activated, laser light that is 100 times brighter than sunlight. So when you treat a patient and you have to illuminate the retina with this highintensity blue light you very quickly induce phototoxicity that will kill all of the retinal cells,” she said. The second issue related to the fact that such proteins are isolated from algae and other microbes and will most probably be immunogenic for the human retina. Thirdly, Channelrhodopsin-2 activation of bipolar cells is artificial and does not comply with normal processing of visual information in these cells. The strategy to overcome these deficiencies was to biotechnologically engineer a custom-made protein equipped with a retinal light antenna.

“We came up with a chimeric protein that consists of parts from two retinal proteins. It is based on the bipolar cell specific metabotropic glutamate receptor and contains the light-antenna of melanopsin, thus it will be nonimmunogenic and non-toxic to bipolar cells and the retina. We further profit from the bipolar cell intrinsic and bipolarto- ganglion cell amplification of the visual signal, which makes this novel chimeric protein much more light sensitive compared to channelrhodopsin-2.So now we can actually activate the neurons in normal daylight conditions,” she said.

Studies in transgenic mice have confirmed the “proof of principle” that the therapy can potentially restore vision, said Dr Kleinlogel, with future efforts being geared towards optimising the gene delivery system and setting up phase I clinical trials in humans.

“Naturally we now want to transfer the technology from the mouse tissue into human tissue to see if it works as well, and of course perform toxicology studies. The mice treated with gene therapy that we sacrificed eight months after treatment did not show any obvious immunological or toxic effects within the retina. So it seems quite promising and we are hopeful that positive outcomes can be obtained beyond animal models,” she said.

* At an award ceremony held during the EURETINA Congress, Dr Kleinlogel’s research was awarded first place in the EURETINA Science & Medicine Innovation Awards. Dr Kleinlogel received a cheque for €20,000 on behalf of her research team for its pioneering work in optogenetics research.

Sonja Kleinlogel: kleinlogel@pyl.unibe.ch