Gene Therapy to Prevent Blindness
Rajendra Kumar-Singh, PhD, joined the Department of Ophthalmology at Tufts University School of Medicine in spring 2006. He is also a member of the programs in Genetics, Neuroscience, and Cell, Molecular and Developmental Biology at the Sackler School of Graduate Biomedical Sciences. Kumar-Singh’s research group is interested in finding therapies for two major families of diseases that cause retinal degeneration and blindness: age-related macular degeneration (AMD) and retinitis pigmentosa (RP).
After earning a PhD in genetics from the University of Dublin, Kumar-Singh worked with Jeffrey Chamberlain (University of Michigan, Ann Arbor) on adenovirus vectors that can be used to carry genes into cells. His interest in ophthalmology then took him to the Jules Stein Eye Institute (University of California, Los Angeles) where he worked with Debora Farber on animal models of retinal degeneration. Kumar-Singh most recently held positions at the University of Utah in the Department of Ophthalmology and Visual Sciences and the Department of Human Genetics.
Age-Related Macular Degeneration
AMD is associated with the accumulation of cellular debris between the retinal pigment epithelium and Bruch’s membrane. One in three individuals over the age of 65 suffers from AMD and its associated drusen deposits. In 10% of patients with AMD, the disease progresses from a relatively benign “dry” stage to a “wet” stage that is associated with new blood vessel growth from the choroid into the subretinal space. These vessels leak blood and fluid, leading to the death of adjacent photoreceptors and eventual blindness.
Currently there is no therapy to prevent AMD. Progression of AMD can be slowed by antioxidant diet therapy and by therapies in which antibodies to AMD-related proteins (such as vascular endothelial growth factor [VEGF]) are injected directly into the eye about once every six weeks. Current antibody therapies have many drawbacks, including severe side effects such as retinal detachment and an increase in intraocular pressure. Kumar-Singh’s research group is using gene therapy to develop longer-lasting treatments with fewer side effects. In one recent experiment in an in vivo model of AMD, the group used RNA interference (RNAi) techniques to reduce expression of VEGF, resulting in a reduction in retinal angiogenesis. RNAi is a naturally occurring process that allows cells to regulate gene and protein expression by silencing specific genes. RNAi strategies take advantage of this mechanism to selectively alter gene expression within a cell. (For more information on RNAi, please see the links at the end of this article.)
While continuing gene therapy research into ways to reduce the damaging symptoms of wet AMD, Kumar-Singh is also working on ways to prevent the disease. AMD is strongly associated with a specific form of factor H, a protein that is part of the infection-fighting complement system in the blood. “Genetic and biochemical evidence indicates that the complement system is somehow initiating the downstream events that we’re observing,” says Kumar-Singh. “We think a malfunction of the complement system is causing damage to the retinal pigment epithelium and that damage is inadvertently increasing VEGF levels, which are then causing the neovascularization. Angiogenesis drugs treat the downstream effects. We’re interested in how we can address the upstream effects, so instead of treating the symptoms, we may be able to prevent the disease from progressing from the dry to the wet form.”
Kumar-Singh’s research group has begun experiments to develop RNAi techniques for reducing complement activation in the eye. “The first stage is to develop the shRNA [short-hairpin RNA] constructs that we need and the viruses that will carry these constructs into the cells,” he comments. “This has never been done before, so there are many unknowns. But the hypothesis is based on current knowledge. If it works, it will be very exciting.”
RP is a group of hereditary diseases that can cause progressive loss of vision and affects approximately 1 in 3,000 individuals. As with AMD, progression can be slowed with antioxidant diet therapy. However, there are no therapies to prevent or successfully treat these diseases. A major form of RP is caused by any one of hundreds of possible mutations in rhodopsin, the light-absorbing compound present in human rod photoreceptor neurons. Because so many mutations can lead to this form of RP, correcting the disease with the aid of mutation-specific therapies would be very time consuming and costly. Kumar-Singh’s research group hopes to develop a mutation-independent therapy by using a specialized adenovirus vector and RNAi techniques to silence all rhodopsin genes involved—mutant as well as normal—and at the same time add a functional rhodopsin gene that is not subject to silencing. The group has had positive results both in tissue culture and an in vivo model of RP. “This is very exciting because we will be able to treat over one hundred different mutations with the same therapy instead of having to design mutation-specific therapies for each patient,” says Kumar-Singh. While a considerable amount of work needs to be done, the results so far are promising.
Gene Therapy Vectors
One of the biggest obstacles in gene therapy is the need for safe and effective vectors for carrying genes into cells. The development and application of such vectors is another aspect of Kumar-Singh’s research. Although his group has significant expertise in the adenovirus system and is working on several adenovirus vectors for targeting neuronal cells in vivo, Kumar-Singh believes the future of gene therapy lies in nonviral vectors such as zinc finger proteins, which have a zinc atom in the center and finger-like protrusions that grab DNA. “Recombinant zinc fingers targeting very specific sequences in the human genome can be designed to search for and correct specific mutations,” he explains. “We’re designing enzymes that will correct the genetic defect in a cell. This is the holy grail of gene therapy—no transgenes or viruses, just go in there with a pharmacologically safe vector and deliver a reagent that will fix the genetic defect and it’s corrected for the life of the patient. The technology is in very early stages of development, but we think it’s very exciting.”
For more information on the Kumar-Singh group’s research, please go to http://www.tufts.edu/~rkumar02/ and http://www.neec.com/pages/Research_Faculty_KumarSingh.html.
For a review of RNA interference, please click here.
For a video on RNAi, please see http://www.pbs.org/wgbh/nova/sciencenow/3210/02.htm.