Project Summary/Abstract Retinoblastoma (Rb) is the most common eye cancer in children and a significant contributor to childhood cancer deaths worldwide. Rb tumors arise from biallelic loss of the RB1 gene during retinal development in-utero, and are uniformly lethal if left untreated. While clinical management of the tumor has advanced in recent decades, the non-specific and highly toxic nature of current therapies frequently result in life-long visual compromise and health complications. Similarly, the exorbitant cost and limited access to advanced procedures render the best treatment options inaccessible to most children worldwide, where eye removal remains the only life-saving option for patients. However, the social stigma and psychological effects of eye removal often lead parents to forego treatment, resulting in survival rates below 50% in many regions of the world. The most significant barrier to progress in developing targeted selective therapies for retinoblastoma is the gap in knowledge regarding the molecular drivers of retinoblastoma progression. Currently, no laboratory models exist, animal or otherwise, which faithfully recapitulate the human manifestation of this disease, and the limited amount of information available comes from tumors which necessitate removal, thus only providing a snapshot of the molecular mechanisms present in advanced cases. The ability to study these tumors in earlier stages, including their pre- malignant phase would shed light into the molecular drivers of Rb progression, lead to more targeted therapies which obviate the need for eye removal, ideally preserving vision but, more importantly, save lives. Recently, we have developed a human iPSC-based 3-Dimensional retinal organoid differentiation protocol and tissue culture bioreactor system in which we can control various environmental parameters critical for proper retinal development. Using this platform, we can use gene editing to introduce the most prevalent mutations found in Rb tumors and evaluate their effect on retinal development and tumor formation. We have shown how some of these mutations lead to amplification of a pre-malignant undifferentiated mutant cell clone, and disrupt terminal differentiation of other retinal cell types. We now propose to establish this platform as the first model system in which the various aspects of human Rb tumors can be elucidated, from their initiation through the drivers of malignant transformation. In order to achieve this, we aim to 1) Optimize, validate, and deploy our tissue bioreactor system to recapitulate the microenvironment of the developing retina, 2) use gene editing technology to establish the physiologic pattern of mutation acquisition during retinogenesis which leads to Rb tumor formation, and 3) use our 3D retinal organoid platform to screen pharmacological compounds that can address the molecular determinants of Rb tumor progression.

Project Narrative Retinoblastoma (Rb) is a developmental tumor of the retina and a significant contributor to childhood cancer death worldwide. For many patients, the only life-saving treatment option is removal of the affected eye. A major obstacle to the development of targeted therapies that spare the normal tissues in the eye is the lack of laboratory models in which these tumors can be studied. Emerging 3-Dimensional organoid culture techniques, tissue engineering applications, gene editing, and single-cell resolution genetic analysis have the potential to create platforms in which Rb tumor pathobiology can be fully elucidated. We have developed a 3D Retinal Organoid platform which incorporates all of these new technologies and is capable of recapitulating the biological nuances of human retinoblastoma.