Stephen Tsang, M.D., Ph.D.'s research efforts are to find new treatments for photoreceptor degeneration in retinitis pigmentosa (RP), age-related macular degeneration (AMD) and related retinal dystrophies, the most common forms of degenerative disease in the central nervous system and have profound impact on quality of life. Over 9 million Americans are affected with photoreceptor degenerations, far exceeding the number with Alzheimer disease. Inherited forms of photoreceptor degeneration affect about one in 2000 people. Presently there is no cure.
RP is the most common cause of inherited blindness, named for the increased pigmentation that appears in the areas of retinal cell death during late manifestation of the disease. Initial symptoms include night blindness, due to the death of rod photoreceptor cells - the light-sensing neurons at the peripheral retina - resulting in "tunnel vision." In later stages, RP destroys cone photoreceptor cells in the macula, responsible for fine central vision. One in ten Americans is a carrier for a defect in one of the 180 genes associated with RP. Our research has illuminated the mechanisms by which the phosphodiesterase (PDE6) signaling network regulates rod and cone survival. Defects in the PDE6 gene account for approximately 75,000 yearly cases of RP worldwide.
In related research initiatives, we study and manipulate photoreceptor degeneration gene expression in mice, which closely parallels similar conditions in humans. Their goal is to control the expression of this faulty PDE6 gene by using inducible gene targeting that allows the activity of a gene in a specific tissue to be disrupted at any time during the life of a mouse. By following the effects of the genetic abnormality after the photoreceptors have fully developed, they hope to gain an understanding of the early events controlling photoreceptor signaling and degeneration in mice, which could lead to new drug targets for the prevention or delay of human retinal degenerations.
Gaining temporal and spatial control of gene expression is essential for the elucidation of gene function in the whole organism. The reagents that we develop can be built into gene therapy vector to provide temporal and spatial control of gene expression of any therapeutic gene. An inducible gene targeting system can be used to address several previously unapproachable problems in sensory biology as well as gene therapy.
Furthermore, we believe that cell transplantation in the human retina has the potential to restore lost vision and provide treatment for advanced stages of retinal degeneration featuring significant photoreceptor neuronal loss, noting that a major obstacle for this approach is the ability to produce sufficient patient specific photoreceptor cells for transplantation. Adult retinal stem cells, which reside in the ciliary body of the adult human eye, are one potential source of photoreceptors. Fish regenerate retinal neurons from a population of stem cells that are intrinsic in the ciliary body, which surrounds the lens of the eye and maintains proper pressure in the eyeball; these cells reside within the differentiated retina throughout the lifetime of the animal. The progeny of fish stem cells can divide and migratory progenitors are the antecedents of photoreceptor precursors. It is these intrinsic adult retinal stem cells that allow the fish to regenerate photoreceptor neurons spontaneously when existing neurons are killed. Stem cell transplantation therefore has the potential to restore lost vision and provide treatment for advanced stages of retinal degeneration even in cases of significant photoreceptor loss in humans.
Our research paves the way toward
"retinoplasty," reconstruction of interfaces between photoreceptors and their environment after the onset of retinal degeneration. Our approach involves the culture of human retinal stem cells from the ciliary body in eye-bank globes, and using those cultured cells to determine the combination of transcription factors involved in regulating their proliferation and differentiation into light-sensing photoreceptor neurons. These experiments will identify the effectors regulating human retinal stem cell differentiation and proliferation, as well as testing the ability of in vitro generated stem cells to repopulate the diseased retina. Future applications may include patient-specific stem cells obtained from fine-needle aspiration of their ciliary bodies in the operating room. Based on our findings, we foresee the ability to manipulate the patients' own stem cells to cure their specific disease. This approach will solve the problem of limited supply of allograft rejection by using a patient's own cells.
We established a stem cell line engineered to express green fluorescent protein (GFP) under control of the rod photoreceptor-specific Pde6g promoter through an internal ribosome entry site (IRES), Pde6g-IRES-GFP. The Pde6g-IRES-GFP cassette was introduced into mouse stem cells. The GFP marker is transcribed as a bicistronic message in conjunction with Pde6g. The GFP marker will only be expressed when these stem cells differentiate into rod photoreceptors (Fig. 2). Control retina is shown in the left (Fig. 1); whereas GFP marked photoreceptors are derived from stem cells found in Fig. 2. The Pde6g-IRES-GFP retinas show specific GFP marker expression in the outer nuclear layer marked by white arrows (Fig. 2).
