John G. Flannery - US grants

DESCRIPTION (provided by applicant): Gene therapy has been increasingly successful in treating several single-gene defects that cause blindness. In particular, multiple successful clinical trials for Leber's congenital amaurosis type 2 (LCA2) have utilized a 25 year-old viral delivery vehicle, based on adeno-associated virus (AAV) serotype 2, to deliver a functional copy of the rpe65 gene to the retinal pigment epithelium (RPE). These trials have taken landmark strides in enhancing visual function in over 30 patients, success that has established the proof of concept that if a causative gene can be identified in a group of patients, a functional replacement gene can be packaged and safely delivered with AAV. However, as the majority of mutations underlying retinal degenerative diseases have now been identified, it has become clear that almost all encode photoreceptor-specific transcripts, establishing photoreceptors as the primary target for retinal gene therapy. Furthermore, many of these mutations are autosomal dominant, such that gene replacement strategies are not suitable. To build upon the LCA2 trial successes, at least two major hurdles that impede broader application of retinal gene therapy must thus be overcome. First, vectors based on natural AAV variants require a subretinal of the vector to mediate gene delivery to photoreceptors or RPE, with accompanying retinal detachment with the creation of a bleb between the photoreceptors and underlying RPE. This procedure damages the retina, may exacerbate the retinal degeneration, and can induce reactive gliosis. In addition, subretinal injection limits the therapeutic effect to the area of th bleb, beyond which the AAV does not spread. Gene delivery from the vitreous would be considerably less traumatic and would offer the potential for pan-retinal transduction, both of which would represent significant advances. Since no natural AAV serotypes can transduce the photoreceptors from the vitreous in either murine or non-human primate (NHP) models, we developed and implemented a directed evolution approach that, as we have recently published, has yielded a novel AAV capable of photoreceptor transduction from the vitreous in the murine and to an extent in the NHP retina. We now propose to build upon this success and engineer AAV variants for optimal therapeutic gene delivery to the NHP retina. A second problem with photoreceptor gene therapy is that many retinal degenerations are autosomal dominant. While RNAi can yield a partial knockdown of pathological alleles, a full ablation of such genes would be desirable. There have been recent advances in the development of site-specific DNA nucleases that can knock out target genes, and we will build upon these advances to knock out dominant alleles that underlie retinal degeneration. We thus propose a unique blend of molecular virology, protein engineering, and a translationally important animal model to engineer enhanced genetic delivery systems and cargo for treating human retinal disease.

Over 100,000 Americans of all ages suffer from inherited retinal diseases (IRD), which cause a progressive loss of vision. In most IRDs, disease begins in the rods, causing vision loss from the periphery to the center, leaving patients unable to navigate their surroundings. Electronic retinal prosthesis restore useful vision in patients affected by IRDs, and optogenetics is an alternative therapeutic. A major limitation of microbial opsins for restoration of retinal light sensitivity is the high light intensity required for activating channelrhodopsins. A solution to this caveat is the use of opsins with higher light sensitivity but sufficiently fast kinetics for useful motion vision. We propose a novel approach to restore vision to patients using a virus to express a light sensitive protein in specific, second-order retina neurons to make them light sensitive. Our approach uses a common neuronal receptor, modified to add a light receptive function to the remaining light-insensitive retinal neurons that survive after photoreceptor degeneration. The receptor uses either retinal, which is available in the eye, or a synthetic chemical photoswitch delivered by intravitreal injection. In this way, the cells in which the receptor is located respond to light with a change in neural firing. This compensates for their loss of input from photoreceptors, restoring light responsiveness to the retina and sending information to the brain to restore vision. In most cases, this approach is independent of the mutation that caused the photoreceptor degeneration. Exceptions to this approach may be diseases that cause RPE cell death, such as choroideremia. To date, versions of this approach, developed by Co-PIs Isacoff and Flannery, and others in the field, have employed receptors that are rather insensitive to light or very slow in response and so could not support normal vision. We now propose a new strategy that uses the natural amplification properties of GPCR signaling to increase sensitivity (by 1000 times) and speed. GPCR signaling cascades are intrinsic to rods and cones, as well as bipolar, ganglion cells and other cells in the retina. We also pursue a new discovery, emerging from our preliminary experiments, which enables a combinatorial approach that uses more than one optical sensor molecule at a time in order to recreate the natural diversity of natural signaling in the retina that had earlier been missing. Finally, we employ sophisticated behavioral analysis to test not only the restoration of the ability to tell light from dark or flashing from steady light, but to determine if the animal is able to see images. Success of this program would represent a major step in the creation of a retinal prosthetic based on gene therapy.