Understanding and treating CRX-linked retinopathies

PROJECT SUMMARY
Precisely regulated gene expression is essential for photoreceptor development and maintenance. This process
is governed by a genetic program centered on the cone-rod homeobox transcription factor CRX. Mutations in
the human CRX gene have been associated with dominant retinopathies with a wide-range of phenotypes and
ages of onset. A poor understanding of the mechanism of each individual mutation has made it difficult to develop
treatment strategies. To address these challenges, our lab has defined four classes of disease-causing CRX
mutations and made mouse models carrying a representative mutation(s) of each class. Up to now, we and
others have characterized and reported findings on mouse models for three such classes, proving concordance
between the mouse and human conditions due to each mutation. These studies have already provided a deep
knowledge of disease pathogenesis. However, the pathogenic mechanism of mutations in the remaining class
(Class II) remains to be determined. Class II mutations are linked to the early-onset dominant retinopathies Leber
congenital amaurosis (adLCA) and cone rod dystrophy (adCoRD). We have generated mouse lines carrying two
individual Class II mutations, Crx-K88N and Crx-E80A, and find that each develops a dominant LCA or CoRD-
like phenotype associated with misregulation of photoreceptor gene expression. Because these mutations are
located in the CRX homeodomain responsible for DNA binding, we hypothesize that the disease proteins
misregulate gene expression by altering CRX’s DNA binding specificity, leading to CRX malfunction at target
sites. In Aim 1 of this proposal, we will test our hypothesis in both cell culture and mouse models using cell
biology, molecular and functional genomics approaches. Using unbiased high-throughput DNA binding and
regulatory function assays, we will determine how these mutations alter CRX’s regulatory activity, leading to
misregulation of gene expression and functional deficits in photoreceptors. In Aim 2, we will address the lack of
treatment strategies for CRX diseases. We hypothesize that exogenous introduction of the proper amount of
normal CRX during a therapeutic window can improve the photoreceptor phenotype in diseased retinae. We
have designed a tunable gene augmentation approach that incorporates a tetracycline (doxycycline) switch to
turn-on or turn-off therapeutic CRX produced by a transgene integrated within the genome or carried by an adeno
associated virus (AAV). We will evaluate phenotypic improvement using established multidisciplinary
approaches and expect to see varying degrees of phenotype rescue in different mouse models by CRX
augmentation. The outcome of this research will advance our understanding of CRX disease and photoreceptor
development, and inform future efforts to treat patients with CRX disease.

Grant Number: 5R01EY032136-05
NIH Institute/Center: NIH
Principal Investigator: SHIMING CHEN