The leading cause of visual loss in Americans over the age of 65 is age related macular degeneration (AMD) which occurs in both a "wet" and a "dry" form. Both forms of the disease are associated with loss of cells called retinal pigmented epithelium (RPE) which can lead to profound loss of central vision. Currently, there is no treatment that will reverse or prevent the loss of these cells and associated blindness. Nutritional supplementation with antioxidants, macular pigments and long chain PUFAs was shown to slightly reduce disease progression but the clinical reality is that 6-8% of people over the age of 75 are legally blind from this disease. Others have observed that RPE cells can be obtained from human embryonic stem cells and that these cells may be transplanted into eyes of animals with diseases that resemble human macular degeneration with RPE dysfunction. Potential problems with this approach to treating humans with this disease include the possibility that the embryonic stem cells from which the RPE are derived may be carried over into the eye and form tumors or elicit an immune response in the recipient eye since the cells are not from the same individual receiving the transplant. Recent advances in stem cell biology now make it possible to induce the formation of pluripotent stem cells (iPSC) from adult, or somatic, tissues of individuals; these cells, in turn, may be stimulated to form RPE cells. Thus, it would be possible to produce RPE cells from the skin or hair of the same individual who would receive the transplant. In order to do this, current technology requires the use of lentiviral vectors that integrate into the genetic material of the recipient cells. In addition, the efficiency with which the iPSC are formed is not very high and to produce these cells, and the derived RPE cells, it is necessary to use "feeder" cell layers and molecules derived from animals. In this research proposal, we will take advantage of novel chemistries and molecular biological techniques to develop iPSC from somatic tissues of patients with AMD and produce RPE cells that could then be used to replace damaged tissue in these patients' eyes. Using small molecules obtained from unique chemical libraries, we will enhance the efficiency of iPSC and RPE production from somatic cells and eliminate the use of animal cell "feeder" layers and supplements. Furthermore, we will use a unique procedure ("an episomal vector") to produce iPSC from somatic cells that does not require the use of potentially dangerous viruses and permanently integrated genetic material. If we are successful, a patient with early signs of AMD could come into their ophthalmologist's office, have a skin biopsy performed that could be used to obtain RPE cells that could then be transplanted into that individual's eye at a later date when their own RPE begin to degenerate, but before they have visual loss.
As the population ages, individuals are prone to develop diseases of aging that significantly impact the quality of life in the "golden years." Foremost among these diseases is age related macular degeneration (AMD), a disease that affects the tissue in the back of the eye (the retina) used for vision. The central portion of this tissue, called the macula, is most affected in AMD and can lead to loss of fine, or "reading", vision. Vision loss occurs from two principal forms of the disease; the "wet" type involving the abnormal growth of new blood vessels and the "dry" type, involving degeneration and scarring of the macula. In both forms, special types of cells under the retina, called retinal pigmented epithelial (RPE) cells degenerate and contribute to the loss of vision. It is estimated that 15-20 million Americans over the age of 65 have AMD and 10-15% of these have vision loss secondary to the disease. Geographic atrophy (the dry type) affects 0.81% of the US population, equivalent to a prevalence of approximately 300,000 in California. Another 6.12% of the population (2.2 million Californians) suffer from early-stage AMD which puts them at high risk for developing geographic atrophy within 5 years. Currently there are some drugs available to help a certain portion of patients with the "wet" form of the disease, but there are no treatments for the "dry" or atrophic, form of the disease. We propose the use of transplants of healthy RPE cells into the eyes of patients with the atrophic form of AMD; these cells would be derived from the patients own skin or hair cells after inducing the production of pluripotent stem cells which could then be stimulated to become RPE cells. If successful, this approach would potentially provide a treatment for the leading cause of vision loss in Californians over the age of 65 with cells derived from their own tissue, avoiding potential complications associated with (1) repeated injections into the eye, (2) using cells from animals or other individuals and (3) deriving cells for transplant that have been exposed to animal cells or molecules and viral genetic material. Preserving vision in the elderly population not only would immensely improve the quality of life for these individuals, but it would also greatly facilitate their ability to function independently and productively.
This application focuses on the development of regenerative therapies to treat age-related macular degeneration (AMD), a leading cause of impaired vision and blindness. The central premise of this work is that stem cell therapies, particularly therapies using autologous cell products, can be used to restore or repair the retinal pigment epithelium (RPE) that degenerates in AMD. The therapeutic plan is to collect autologous somatic cells, reprogram them into pluripotent stem cells, and then terminally differentiate them into a "purified" population of RPE cells to be transplanted into diseased retina. First, the applicants propose to use an episomal vector to generate induced pluripotent stem cells (iPSCs), although they are also developing a small molecule mixture to enhance reprogramming of somatic cells. In the second aim, the applicants will differentiate these iPSCs into RPE cells and characterize them using a variety of morphologic, functional, and immunohistochemical assays. In the third aim, the applicants propose to optimize the RPE differentiation procedures using small molecules to enhance the efficiency of differentiation. Next, they will assess the ability of RPE generated from rodent and human iPSCs to provide structural and functional rescue in rodent models of retinal degeneration. In the fifth aim, the applicants propose to demonstrate that human somatic cells, obtained from individuals of varying ages and including patients with retinal disease, can be induced to form iPSC-derived RPE. In the sixth and final aim, a series of GMP compliant processes will be devised to produce sufficient human RPE for use in preclinical development studies and in clinical trials. The applicants intend to address a number of safety issues with the use of such stem cells, especially the potential for uncontrolled growth and tumor formation. Therefore, effort will be spent on developing means for deriving a homogeneous population of differentiated RPEs without contamination by undifferentiated iPSCs. Further, they will develop methods of delivering the cells into the subretinal space of humans in an efficient manner without injuring the overlying retina. Dose and frequency of dosing will be determined from preclinical efficacy studies.
The reviewers agreed that the proposed research addresses a critical medical need. Macular degeneration is a life-altering disease that negatively affects the quality of life of many older individuals, and will impact many more in the near future due to rapid increase in the numbers of older people. For many patients with AMD, current treatment options have limited effectiveness. While embryonic stem cell therapy has shown promise, the issues with immune rejection from allogeneic donors are substantial. The rationale underlying this effort, which is to engineer a patient’s own tissues to restore RPE function with a lower chance of rejection, is very strong and would likely have high impact if brought to fruition. Furthermore, reviewers highlighted the many advantages of the eye as a testing ground for stem cell therapies including its relative immune privilege, its ready accessibility for monitoring and imaging purposes, and the fact that even removal of a diseased eye, (in case of serious complications) is not a life-threatening event
Reviewers were very enthusiastic about the experimental approaches proposed in this application. They found the research plan to be well-conceived and feasible, and commented that the proposal is straightforward, well written, and based on convincing precedents that were established in rodent models. Several key strengths were identified that contributed to the reviewers’ enthusiasm. Most importantly, the applicants provided a wealth of convincing preliminary data to support the merits of the scientific approach, such as thorough characterization of the episomal reprogramming factor and successful use of small molecules to enhance hESC growth in the absence of feeder cells. Reviewers were supportive of the use of drug discovery approaches for further identifying and optimizing small molecules to enhance derivation of iPSC from both rodent and human somatic cells (in combination with the unique episomal vector). They also appreciated proposed efforts to characterize the RPE cells that were derived from human and rodent iPSCs, and the emphasis on developing truly homogeneous RPE cell populations. A pure RPE transplant product would decrease the risk of introducing undifferentiated iPSCs into the retina, and therefore decrease the potential for teratoma formation. In addition, the reviewers expressed great confidence in the strength and qualifications of the research team. The principal investigator is an established and respected clinician scientist with an excellent track record in the appropriate field. Furthermore, the outstanding research environment, including the specialized expertise of the collaborators, convinced the reviewers that this team would be well poised to succeed.
While these merits were deemed considerable enough to recommend the proposal be funded, the reviewers were concerned about the scale of the proposal and questioned whether it could be realized in its entirety. Several reviewers indicated that due to the highly ambitious end-points, it was unlikely that every aim would be achieved within the limited time frame of this funding program. In particular, the “omics” characterizations were regarded as being too open ended, and applicants were not sufficiently clear in describing how the results of these studies would necessarily advance progress toward human trials. Some reviewers also questioned the long-term fate of the transplanted RPE cells in the damaged retina – they may degenerate due to the diseased environment. The reviewers were also uncertain whether the RPE transplantation would be therapeutic, as rodents do not have a macula and so are limited as a disease model. As a final point, the reviewers wished that more details had been provided about the episomal targeting vector and the manner in which safety of this gene therapy would be addressed. Experiments directly addressing the eye as an environment for teratoma formation (using the proposed cell product) would have strengthened the work, to detect leakiness of the cre-lox strategy.
In summary, reviewers felt that the research proposal was of sound rationale, feasible design, and addressed an important unmet need in the field of regenerative medicine. While the end points might be overly ambitious, the reviewers remained generally enthusiastic based on the formidable strength of the research team as well as the merits of the intermediate goals.