Research Supported by CGF

Tufts University School of Medicine

Aphakic Glaucoma Research

“In last year’s newsletter of The Children’s Glaucoma Foundation (2019), you read about a new research initiative to develop a gene therapy approach for the treatment of infantile aphakic glaucoma. I am excited to report that since the initiation of those studies, we have made tremendous progress. Specifically, we have completed development and testing of a novel adeno-associated virus (AAV) that expresses an inhibitor of a process known as epithelial to mesenchymal transition (EMT) of lens epithelial cells. AAV is currently an FDA-approved gene delivery method for a disorder in children that causes blindness and hence we believe that while the path we are taking is highly novel, it is de risked by prior success in this field.

The process of EMT leads to a blockage of the outflow pathway of fluids in the front of the eye due to ‘clogging’ of tissues known as the trabecular meshwork. This blockage to outflow results in a build-up of pressure in the eye that ultimately results in the death of cells in the back of the eye and consequently, blindness. The FDA requires that novel therapies be tested for safety and efficacy prior to testing in humans. During the previous year, we have modified the local environment of the eyes of small rodents such as to increase the pressure within the eye. This surprisingly led to very high levels of EMT at the trabecular meshwork and a significant increase in pressure within the eye. We essentially generated a non human model of infantile aphakic glaucoma. We also tested this same disease process in the presence of our therapeutic AAV and we found that both the EMT and pressure within the eye was reduced. These results are extremely exciting. Before proceeding, we need to test various doses of the therapy and in enough examples such that they are highly statistically significant. We had anticipated completion of these studies by Summer of 2020 and although we are still on target, the temporary shutdown of our laboratories due to Covid-19 has set us back by about three months. As of writing, I am pleased to report that the studies have been re initiated and we are moving forward. Additional excitement comes from having tested and proven our AAV vector in a larger eye akin to that of a human. We essentially already have proof-of-concept of our hypothesis. During the next 4 to 6 months we expect to complete these studies and start planning the generation of GMP (good manufacturing practice) grade AAV vector needed for obtaining safety data that would be used for applying to the FDA to test a new investigational drug (IND) in humans. These latter studies will collectively take two to three years but our unprecedented success thus far gives us hope that we can complete them sooner rather than later.”

-Rajendra Kumar-Singh, PhD
Professor, Developmental, Molecular and Chemical Biology
Tufts University School of Medicine

University of Georgia

Aniridia Corneal repair research

“Current approaches for functionally restoring the ocular surface for someone who has Aniridia or other problems with their corneas involves grafting cells from a healthy corneal limbus or other tissues. Conceptually, it makes sense to use limbal tissues because the corneal limbus is the part of the eye where the cells that normally make the corneal surface are located, and so this approach was developed first. However, there are problems using limbal cells to fix corneal problems. First, it may not be possible to obtain any limbal cells if the patient has damage to both of their eyes. Second, cells from a donor can be used, but donors are in short supply, success rates are lower, and donor cells are usually only effective in the short to medium term. Third, there are risks to the healthy donor eye associated with removing limbal stem cells and there are risks to the patient from receiving cells from somebody else. These limitations and risks have prompted doctors and researchers to look for alternative sources of cells that could be used to repair the corneal surface. Two alternative sources of cells that are currently being used clinically are cells taken from inside the cheek or from wisdom teeth.

While the use of cells from the inside of the cheek or tooth to repair to corneal surface works well for many people, this approach works less well for individuals who have problems with their corneas due to mutations in genes such as PAX6. The reason for this is because the mutation is in all the cells regardless of where they come from in the individual's body. This makes the problem of repairing the corneal surface in someone with a genetic mutation more difficult.

Induced pluripotent stem cells (iPSCs) are a type of progenitor cell that can be created in the laboratory directly from many different cells in the body. This technology holds great promise in regenerative medicine because iPSCs represent an unlimited source of cells that can be used to replace cells or repair tissues lost to damage or disease. Some examples of iPSC-based therapies being developed include treatment of diseases of the liver, heart, and pancreas (diabetes). A handful of groups around the world, including the Lauderdale lab, are developing this technology to repair the cornea.

With financial support from the Children's Glaucoma Foundation, the Lauderdale lab completed in 2019 a pilot project showing that it was 1) possible to create iPSCs from skin cells taken from a person with aniridia, 2) that the person's PAX6 mutation could be repaired in the iPSC cells, and 3) that these cells could be used clinically to repair the cornea in a rabbit model of limbal stem cell deficiency. While very exciting results were obtained, this pilot study also highlighted the need for a better method of coaxing the iPSCs to a type of cell that could be used clinically to repair the cornea in a person.

This past year, Dr. Heike Kroger and Ms. Sneha Kotagudda Murali Mohan joined the Lauderdale lab to work on a solution to this problem and help move this project across the finish line. Dr. Kroger brings expertise in generating and working with human iPSCs and has an extensive track record in working on human genetic eye disorders. Sneha holds a Master of Science degree for her work on human iPSCs in the context of neural development and joined the Lauderdale lab to work on converting iPSCs into corneal progenitor cells as her doctoral project.

iPSCs do not exist naturally and are instead generated (“induced” or “reprogrammed”) in culture from somatic (adult) cells through ectopic co-expression of defined pluripotency factors. This reprogramming is what gives the cells the potential to become any cell type in the body; essentially the cell's identity is erased and the cell is reset to a generic identity that can be manipulated. Cells are manipulated by adding specific factors to their growth media. The trick is to find the right factors/conditions to coax iPSCs from a generic iPSC state to the desired cell type. Although the recipes to make neurons and glia are well established, the recipes to make corneal cells are few. In large part, this is due to the fact that we don't really understand how corneal progenitor cells are normally made by the body.

This past year, "team cornea" worked on developing new "how to make corneal progenitor cell" recipes with success. Our current recipes appear to be producing corneal progenitor cells from iPSCs at higher numbers than the recipe we used for the pilot experiments and those recipes that have been published. Unlike baked products, the batches of cells being produced in the lab are complex mixtures and require careful assessment before they can be used to treat people. Therefore, the research team is working hard on testing to make sure that the cells that are being made in the lab can be used treat people. While we still have some distance to go, the finish line is now in sight.”

-Dr. James D Lauderdale
Associate Professor
The University of Georgia
Department of Cellular Biology

For more information about the CRISPR technique for genome editing Dr. Lauderdale’s lab is using to restore PAX-6 function, please view the nature video below: