Preserving the Precious Gift of Sight

Graeme Mardon, PhD

MILLER RESEARCH PROJECT

Department of Pathology

Baylor College of Medicine

Houston, TX

 

Dr. Mardon’s Research Project

Genetic and Molecular Analysis of Retinal Development

Current Research Interests

The long-term goal of this project is to improve both the diagnoses and treatments of Leber congenital amaurosis (LCA), which accounts for more than 5% of all retinal diseases. LCA is characterized by severe blindness at birth or within the first year of life. The clinical phenotypes of LCA classically follow autosomal recessive modes of inheritance. The molecular basis for LCA is heterogeneous, with mutations in 19 different genes associated with LCA. Since the LCAs are early-onset, recessive diseases, it is perhaps not surprising that nearly all mouse knockouts of homologs of these genes also result in severe retinal phenotypes. Dr. Mardon’s laboratory recently identified a new gene associated with LCA (Kcjn13), which encodes inwardly rectifying potassium channel but for which no animal models have been established. They have created a new mouse model for LCA by knocking out the mouse Kcnj13 gene using CRISPR technology. These mouse models will serve as an important basis for understanding the mechanism of disease in human and developing gene therapy approaches.

Progress in 2016

Dr. Mardon generated a conditional allele of Kcnj13 and demonstrated that loss of Kcnj13 in the RPE causes strong loss of photoreceptors by 3-5 months of age. These data show that his conditional allele is functioning efficiently and that he is now poised for a full developmental study of Kcnj13 function.

Plans for 2017

Specific Aim 1) Study mouse models for KCNJ13 to understand LCA disease pathology. Dr. Mardon has created new mouse models for LCA by generating Kcnj13 null alleles by gene targeting and CRISPR technology. He has now also built a conditional Kcnj13 allele that allows him to bypass early lethality, and he observes strong loss of photoreceptors using this conditional allele. During the next year, Dr. Mardon proposes to use this model extensively to understand LCA pathogenesis. In particular, he observes defects in melanosome aggregation and hypothesizes that phagocytosis or photoreceptor outer segments (OS) by the retinal pigment epithelium (RPE) may be impaired. Dr. Mardon has shown that strong loss of photoreceptors is observed by 3-5 months of age, and will now examine phagocytosis in at least six animals at 1, 2, 3, and 4 months of age by using immunohistochemistry to determine if and when defects in phagocytosis can be observed. These studies will provide crucial information regarding the best time course for treat by gene therapy.

Progress in 2015

To create a new mouse model for LCA, Dr. Mardon knocked out the mouse Kcnj13 gene by gene targeting, and is analyzing the phenotype of Kcnj13 mutants by histology, immunohistochemistry, electrophysiology, and transmission electron microscopy. In 2015, Dr. Mardon found that conditional loss of Kcnj13 function in his mouse model causes strong loss of photoreceptors. He is now poised for detailed studies in the next award year.

Progress in 2014

Dr. Mardon made a significant breakthrough in his research concerning the Kcnj13 retinal disease gene. Specifically, his laboratory found that loss of Kcnj13 function in their mouse models causes strong loss of photoreceptors and is now poised for full-scale analysis in the next award year. Dr. Mardon generated and characterized null and conditional mutations in this critical human disease gene in mice and has shown that homozygous mutant mice recapitulate the human disease phenotype.

Progress in 2013

Dr. Mardon’s laboratory has recently identified a new gene associated with LCA (named Kcjn13), which encodes an inwardly rectifying potassium channel but for which no animal models have been established. His preliminary evidence suggests that his mouse mutation may be homozygous lethal. Therefore, in addition to characterizing this allele in more detail, he will also generate conditional alleles of Kcnj13. A detailed understanding of Kcnj13 function could have broad implications for our ability to diagnose, prevent, and treat retinal diseases.

Progress in 2012

To create a new mouse model for LCA, Dr. Mardon has knocked out the mouse SPATA7 gene by gene targeting. SPATA7 mutants are homozygous viable but have severe defects in retinal development, closely mimicking the human disease. Dr. Mardon has analyzed the phenotype of SPATA7 mutants by histology, immunohistochemistry, electrophysiology, and transmission electron microscopy. A Manuscript is being prepared reporting this work. Dr. Mardon is now making rapid progress using this model to develop gene therapy approaches with the ultimate goal of treating human patients with mutations in SPATA7.

Dr. Mardon’s laboratory recently identified the causative gene associated with LCA3, named SPATA7, which encodes a highly conserved but novel protein of unknown function and for which no animal models have been established. Significantly, SPATA7 mutations are associated with both LCA and early-onset retinitis pigmentosa (RP), suggesting that a detailed understanding of SPATA7 function could have broad implications for our ability to diagnose, prevent, and treat human retinal diseases.

One of the most promising methods for treating human retinal disease is the use of gene therapy. Dr. Mardon uses the mouse model of LCA that his laboratory developed in 2011 as a system for testing the efficacy of gene therapy.

Specifically, they test how long after disease symptoms appear can mice be treated by gene therapy and still show significant improvement in visual function. They use the adeno-associated virus (AAV) system to restore Spata7 gene function specifically in the eyes of Spata7 mutant mice. They test for response to light using electroretinograms.  Dr. Mardon also tests for restoration of photoreceptor cells using histology and light microscopy. Data from these experiments is expected to have important implications for human gene therapy approaches in the future.

By one year of age, Spata7 mutant mice present with a severe loss of photoreceptors, as seen by the large reduction in the outer nuclear layer (ONL) of the retina.  These mice fully recapitulate the human LCA disease phenotype and are now being used for gene therapy studies.

Progress in 2011

Dr. Mardon’s laboratory uses a mouse model they created for LCA3 to decipher the mechanism of disease in humans and to validate this model for gene therapy studies. They successfully completed these goals and have shown that the severe reduction in the number of photoreceptor cells and little or no response to light in the absence of Spata7 function is due to the mislocalization of the visual pigment Rhodopsin.

Normally, virtually all Rhodopsin protein is found in the outer segment of photoreceptors. In Spata7 mutants, Rhodopsin is found throughout the cells, which then later die.  If they remove both Spata7 and Rhodopsin, cell death is blocked, demonstrating that mislocalization of Rhodopsin is the key event leading to photoreceptor loss.  This model is now validated for the next major goal: to develop gene therapy approaches.

The long-term goal of Dr. Mardon’s research is to improve our ability to prevent, diagnose, and treat human retinal disease.  His laboratory employs a three-pronged approach.  First, they are actively mapping and identifying new human retinal disease genes using cutting-edge genomic technologies.  Specifically, they are mapping new genes that cause Leber Congenital Amaurosis (LCA), the most common form of congenital blindness in humans.

Second, Dr. Mardon’s laboratory uses the mouse as a model system to study the function of conserved genes required for normal retinal development, including genes identified in their screen of LCA patients.  Finally, they use their mouse models to test new treatments to cure blindness, including gene therapy.  This combination of approaches comprises an efficient and comprehensive plan to advance our understanding of the molecular and genetic mechanisms of human retinal disease.

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The mission of the Retina Research Foundation is to reduce retinal blindness worldwide by funding programs in research and education. As a public charity, RRF raises funds from the private sector and the investment of its endowment funds.

RRF’s 48th Anniversary

October 1, 2017