Research Description

Ribozyme Gene Therapy for Retinitis Pigmentosa

Autosomal Dominant Retinitis Pigmentosa, or ADRP, is an eye disease that affects an estimated 100,000 Americans and 1 in every 3000 people worldwide. The effects of Retinitis Pigmentosa begin with nyctalopia, or night blindness, followed by tunnel vision and ultimately complete loss of vision. Effects vary in individuals some do not become legally blind until their 50s, others become completely blind in their childhood. In rare cases, some never completely go blind. The disease is caused by genetic mutations in cells of the retina. The affected genes are translated by the cell into proteins which become toxic to the photoreceptor cells that sense light in the retina: the rod and cone cells. In ADRP’s treatment, replacing the mutant gene with a functional gene is not adequate expression of the mutant gene must be shut down so the poisonous protein is no longer produced. The approach is two fold: 1) Mute the translation of mutant and toxic proteins and, 2) Introduce p! roduction of normal and healthy proteins.

My work under Dr. Lewin will focus on testing the first half of the experiment, muting expression of the mutant gene, in mouse models of ADRP. The gene therapy is delivered by Adeno-associated virus (AAV), which is non pathogenic. The focal point of this investigation is correcting a mutation in the gene that codes for rhodopsin, the main visual pigment of rod photoreceptors. My proposed research will be focused on investigating methods of silencing expression of the mutant rhodopsin gene.

To knock down the expression of the mutant gene for rhodopsin, I will design hairpin and hammerhead ribozymes (small RNA’s that act as enzymes) that splice the mRNA carrying the mutant rhodopsin gene. These ribozymes will therefore prevent the translation and synthesis of both normal and mutated rhodopsin. The ribozymes will be packaged into AAV and injected into mice retinas. I will observe the progress of these injections using electroretinography (ERG). Ultimately, mice will be humanely sacrificed and their retinas will be studied under microscope to determine if the therapy was effective.

Outside of my work, the project will also construct versions of the rhodopsin gene that are resistant to the small RNA inhibitors. The ultimate goal is to combine the mRNA splicing ribozymes that I cloned with a supplementary wild type rhodopsin gene in a single virus vector and deliver this combination to mice carrying mutated forms of rhodopsin leading to ADRP. Thus, the mutant gene would be silenced and the wild type gene would be expressed.