Because of the hereditary nature of LCA, current emphasis is on understanding the genetics of the LCA family of diseases and identifying the various genes whose mutations lead to LCA pathology. Along with this, studying the clinical manifestations of the disease process as expressed in the retina is also very important.
Since there are now several possibilities for treatment of LCA on the horizon, the FRR is actively supporting some of these efforts directly and indirectly. It is hoped, for example, that our efforts in funding leading researchers in the genetics field will find all the LCA genes and lead to gene replacement therapy for LCA patients.
Another important area is that of sight restoration through retinal implantation of natural photoreceptor cells, stem cells or an artificial retinal prosthetic device. In retinal cell transplantation, freshly implanted retinal tissue or stem cells might take the place of degenerated photoreceptors. Here, the FRR funds the leading transplant laboratory in the world. Similarly, the FRR supports researchers involved in developing the retinal prosthetic device – the “retinal chip”. This device will mimic the function of the photoreceptors in a patient’s eye and has the potential of restoring a high degree of functional vision.
Finally, the FRR leads international efforts to unite clinicians in their efforts to move to clinical trials for LCA. Disease classification, patient registries and aid in establishing sites for clinical trials are all part of the FRR game plan for conquering LCA.
Grants Funded by the FRR: Progress in Curing LCA
Over the last few years, the FRR has taken the lead in funding:
a) research that identifies gene mutations that cause LCA
b) research on understanding the factors that cause photoreceptor cell death and c) work such as genotyping that is needed in planning LCA clinical trials
More recently, we have taken advantage of scientific advances in several fields to also move to preclinical work that could directly lead to clinical trials. Following are 5 grants funded by the FRR that move us to treatments for LCA. The first two grants are mainly involved in assessing patient LCA pools in preparation for clinical trials and the last three grants outline strategies for preclinical testing that could lead to effective therapies.
Genetic Deciphering of LCA: Identification of Different Physiopathologic Pathways and Genotype-Phenotype Correlations
Dr. Josseline Kaplan, Hopital des Enfants-Malades, Paris, France
Before a specific therapy can be considered for a form of LCA, much has to be known about the disease process as to genetics and pathophysiology. For almost 20 years now, Dr. Josseline Kaplan and her group in Paris, France have been in the forefront in supplying this knowledge. For example, she has used sophisticated genetic mapping and screening techniques to identify several genes whose mutations are now known to cause forms of LCA. This work continues in the discovery of new gene mutation. Although about 60-70% of the disease-causing mutations are now though to be known, it is critical to identify these mutations for all LCA patients and Dr. Kaplan’s group is one that is leading the way in this research area. A parallel effort funded by the FRR is in identifying the function of the protein products of the new LCA genes. Each LCA gene acts as a blueprint for an important protein and, if that protein is defective or missing, the photoreceptor cell will dysfunction and die. Understanding the function of the specific protein could lead to the development of inhibitor or activator agents that would restore function of the protein or the biochemical pathway which it controls.
Finally, the FRR continues to support Dr. Kaplan’s outstanding efforts in establishing genotype-phenotype correlations in LCA patients. Genotyping, as you know, is uncovering the gene mutation that underlies a form of LCA. Phenotyping is documenting the characteristics of the disease process itself in a particular patient. For example, age of onset, clinical manifestations, etc. It can be very useful to know which set of clinical characteristics (phenotype) are associated with which gene mutation (genotype). This can greatly help to speed up future genotyping (i.e., strongly implicating a particular gene mutation in a patient with a specific phenotype) and also in predicting the course of the disease in patients with known gene mutations.
LCA Gene Identification and Patient Genotyping
Dr. Ed Stone, Dept. of Ophthalmology, University of Iowa, Iowa City, IA USA
The genome screening of candidate genes for genetic diseases such as LCA and the ultimate identification and characterization of these genes has been a main focus of Dr. Stone’s research effort for many years now. This is a herculean effort since a typical candidate gene experiment can easily involve tens of thousands of individual assays. To aid him in this “gene search” effort, Dr. Stone has employed the emerging field of “bioinformatics” which allows him to screen the most promising segments of a great many genes for disease-causing variations instead of the more conventional method of screening only a small number of genes in their entirety. This novel method of screening is called “Prioritization of Annotated Regions (PAR) and has allowed Dr. Stone and his coworkers to much more efficiently screen genes that might harbor LCA disease-causing mutations.
In addition to discovering new genes whose mutations lead to LCA, an important ongoing goal of Dr. Stone’s clinical effort is to identify as many individual patients with variations in each known LCA gene as possible. The identification of patient groupings with mutations in specific genes is especially important as we are reaching the era of effective human gene therapy. Gene therapy is certainly not possible if the mutated gene is not known. One part of this effort would be to conduct a comprehensive survey of populations such as in the state of Iowa where there is a relatively large population of patients with LCA. Benefits would be to gain a better understanding of the “natural history” of the different molecular forms of LCA and to establish a well characterized pool of patients that could be used in future clinical trials.
The final area of concentration of FRR funding of Dr. Stone’s work is the continuation of his genotyping efforts. This has been an extremely successful project to date and has made the Carver Laboratory the premier genotyping lab in the world. Dr. Stone has worked tirelessly in seeing that every LCA patient has an opportunity to be genotyped. This work must continue to make sure that treatments are ultimately afforded to every LCA patient.
Production of Photoreceptor Cells by Mueller Cells
Dr. Connie Cepko, Dept. Of Genetics, Harvard University, Boston, MA USA
Stem cell therapy offers the potential of restoring photoreceptor and other cells in the retina in a degenerative disease such as LCA. One problem with the use of stem cells has been the source of cells used as the replacement “progenitor” cells. One possibility has been the use of stem cells harvested from nearby tissues such the ciliary body, iris or even from more peripheral tissues. However, use of such cells could lead to severe immunological complications. A more attractive choice would be to use cells already residing in the retina as endogenous progenitor/stem cells. A cell that fits this description is the Muller glial cell. Muller cells are a specialized type of cell in the retina that are called “glia”. Glian cells are not neurons like photoreceptor cells but have many functions that support neurons (e.g., nutrition, etc.). Dr. Cepko and her team at Harvard have found that Muller cells have many characteristics of retinal progenitor cells like the ability to multiply under proper conditions and attaining some properties of photoreceptor cells.
A first step in this process would be to induce endogenous Muller cells to produce photoreceptor cells using a specialized form of viral gene transfer. This type of gene therapy has already been shown to direct formation of rod photoreceptor cells in test mice. The second step would be to repair the new photoreceptor cells produced by the Muller glia. Even these cells would contain the mutant gene and would need to receive a normal copy of the defective gene.
This research is a novel approach to restoring photoreceptors in many cases such as in rapidly advancing disease or in last, end-stage disease. It has the potential to repair as well as replace dying or dead photoreceptors in one manipulation.
RPGRIP Gene Therapy
Dr. Tiansen Li and Dr. Eliot Berson, Dept. of Ophthalmology,
Harvard Medical School, Boston, MA USA
Gene Therapy has the potential of long-term restoration of vision in situations where retinal photoreceptor cells yet remain and the gene mutation is known such that the correct replacement can be made. This was spectacularly shown through sight restoration in the dog model of LCA caused by mutation of the RPE65 gene. RPE65 gene mutations lead to only a small percentage of LCA cases though so gene therapy of other forms of LCA must be planned. One form of LCA that has now been well studied is that caused by mutation in the RPGRIP gene. This gene produces a protein that is essential for normal functioning of both rod and cone photoreceptor cells. Work in this area has been led by Dr. Tiansen Li and his colleague Dr. Eliot Berson at the Massachusetts Eye & Ear Infirmary.
A critical step in moving to any clinical trial is the development of a good animal model of the disease that has the same gene mutation as that in the human. This was established in a mouse ”knockout” model in which the normal RPGRIP gene in the animals was deleted using sophisticated molecular biological techniques. With a specialized vector system, a copy of the normal RPGRIP gene was then delivered to the eye of the mutant mouse. The results were excellent in that Dr. Li and his associates found that the new gene was functional and produced the normal GRIP protein. Biologically, photoreceptor preservation was a result with well developed photoreceptors in the treated eye.
With this positive initial finding, Dr. Li and coworkers have continued to study the effects of gene replacement therapy in the RPGRIP-deficient mouse eye. An important question is whether such gene therapy rescues both rods and cone photoreceptor neurons. Although rods dominate in a mouse retina, cones are the most important photoreceptor type in the human serving central, sharp and color vision. Even though it is clear that the mouse form of the RPGRIP gene is functional after replacement therapy, functionality of the human form of the gene must also be tested in the model. For this, the human gene will be combined with other proper vector elements in a “construct” that hopefully will be effective when delivered to the mutant mouse retina. This is critical in establishing a prototype RPGRIP gene construct that assures safety and efficacy and can be used in future human clinical trials.
LCA5 Gene Therapy
The LCA5 Consortium of Investigators:
Drs. F. Cremers, A. den Hollander, R. Roepman, The Netherlands;
Dr. R. Koenekoop, Canada; Dr. P. Nishina, USA; Dr. J. Bennett, USA.
A stellar grouping of investigators has been assembled by the FRR to plan for possible gene therapy in LCA5. LCA5 is caused by a mutation in the gene for a protein called “Lebercillin”, the name derived from a combination of “Leber”, the Ophthalmologist who first described LCA and “cilium” a critical structure of the photoreceptor cell in which the protein functions. The Dutch and Canadian investigators as well as others first identified the LCA mutation in the lebercillin gene in 2007. Since then, a large amount of basic work has been done in isolating the gene (“cloning”), studying its characteristics and preparing for gene replacement therapy of LCA5 patients.
To get to a human LCA clinical trial, much work need to be done as described above. The first order of business would be to “construct” an animal model of LCA5 with a mutation in the lebercillin gene. This is currently being done by one of the world’s experts in this area, Dr. P. Nishina. As the construction of the animal model is in progress, parallel efforts are underway in identification of patients with LCA5 who might be eligible for participation in the clinical trial or be in line for therapy if the trial is indeed successful. This involves genotyping and phenotyping, i.e., a thorough eye examination. Another parallel effort is in trying to understand the basic biological function and interaction of the lebercillin protein in the photoreceptor cell. The protein is localized in a tubular structure within the photoreceptor cell called the “cilium” that plays a major and critical role in bulk protein transport within the cell. Its exact function though is not well understood so further investigations are warranted and could point the way to other therapies in the future.
Finally, preparations for gene therapy experiments in the animal model must be made. To do this, again one of the world’s top experts, Dr. Jean Bennett has agreed to do this work. In fact, the work has already begun in preparing the “vector” system that will act as a vehicle/truck to transport the new, replacement lebercillin gene into the photoreceptor. This molecular work is specialized and intense but is an area with which Dr. Bennett is very familiar since she is currently leading one of the teams doing the clinical trial on the RPE65 gene. When Dr. Nishinais successful in producing the LCA5 animal model, animal s will be shipped to Dr. Bennett for experiments on gene replacement. In this way, the path will be cleared for conducting human clinical trial for LCA5 therapy.
