Sidney Kimmel Medical College, Thomas Jefferson University
Dr. Budd Tucker was awarded the 2023 Cogan Award and delivered the final award lecture at this year’s ARVO conference, titled “Treating Inherited Retinal Degenerative Blindness.” Following an introduction by his colleague and friend, Dr. Edwin Stone, Dr. Tucker began by discussing promising treatment options for inherited retinal diseases, including drug therapies, gene therapies, and cell replacement therapies. He explained that the treatment most appropriate for a patient depends on our knowledge of the disease mechanism and the patient’s stage of disease.
To illustrate how his group uses patient-derived induced pluripotent stem cells (iPSCs) to generate tissue for the evaluation of disease pathology, development of gene-based therapeutics, and development of an autologous photoreceptor cell replacement approach, he presented two case studies.
The first was a 37-year-old man with 20/20 vision in both eyes and ample photoreceptors remaining. In such an individual, Dr. Tucker believes the ideal treatment would be gene therapy. The first step would be to determine what gene is causing the person’s retinal dystophy. A homozygous ALU insertion in exon 9 of the MAK (male germ cell-associated kinase) gene was found in this individual. After this mutation was identified and the gene was confirmed to be expressed in the retina, Dr. Tucker’s lab generated iPSCs from the proband and controls.
Employing 2D differentiation, they generated sheets of retinal cells, which were used to investigate the effect of the mutation on the corresponding mRNA transcript and protein. These studies revealed that the proband lacked expression of the exon 9 containing transcript, resulting in an absence of MAK protein in the retina. After a functional assay was developed, a gene therapy-based approach could be pursued for this patient, which involved creating vectors expressing the wildtype gene and testing the vector in vitro and in vivo in animal models to ensure its functionality.
The next step was clinical production. In 2014/2015, Dr. Tucker and his team envisioned building their own GMP facility, which would allow them to complete all the studies required for the initiation of clinical trials rather than outsourcing this work. Using this facility, they have now made multiple batches of a variety of virus vectors. Regarding the MAK vector, they have made three clinical batches of the virus, have completed local and systemic toxicology studies, and will be starting the clinical trial soon.
The second case was a 58-year-old man with massive bone spicule pigmentation, constricted inner retinal vasculature, and very few photoreceptors remaining. This was in fact the same patient as the first case but 20 years later, demonstrating the progressive nature of these conditions if left untreated. Rather than gene therapy at this point, the patient would now benefit more from cell replacement, the goal of which is to engineer a photoreceptor cell layer that will make synaptic connections with bipolar neurons when transplanted into the subretinal space.
Over the years, many cell types ranging from embryonic stem cells to retinal progenitor cells and photoreceptor precursor cells have been used for photoreceptor cell replacement with varying degrees of success. George Daley, one of Dr. Tucker’s mentors, generated one of the first mouse iPSC lines. They used these iPSCs to generate photoreceptor precursor cells, which partially restored electro retinal function when transplanted into the subretinal space of rhodopsin-deficient animals.
Since then, they have shifted to focus on human iPSCs and to using 3D differentiation rather than 2D. Prior to differentiation and transplantation, CRISPR is used to correct the genetic mutations in the iPSCs. To increase cell survival rates following transplantation, Dr. Tucker’s group uses a two-photon lithography system to functionalize polycaprolactone (PCL) and produce a 3D cell delivery scaffold.
To increase iPSC production efficiency, Dr. Tucker has automated many aspects of the process, including implementing a robotic device that can identify iPSC colonies and move them to another dish, which is one of the most challenging tasks to manually perform in this process. Additionally, their system uses an automated incubator and a robotic arm to replace the technician’s arms. The iPSCs generated can differentiate into well-structured retinal organoids, and these grafts have demonstrated excellent biocompatibility in systemic toxicology studies in animal models.
Dr. Tucker’s engaging lecture depicted a promising future for the development of new therapies for inherited retinal degenerations.