Juan Gallardo Pinera

Program: Molecular Cell Biology

Current advisor: Fumihiko Urano, MD, PhD

Undergraduate university: University of South Florida, 2022

Enrollment year: 2022

Research summary
My research uses in vivo prime editing to fix the Wolfram Syndrome variant in mouse eyes and patient-derived iPSCs differentiated into RGC organoids and neurons

Research Description: In Vivo Prime Editing for Wolfram Syndrome Variant Correction
Project Overview
My research project is focused on the application of in vivo prime editing to address genetic mutations associated with Wolfram Syndrome, a rare, inherited disorder. The study primarily involves two models: mouse eyes and patient-derived induced pluripotent stem cells (iPSCs) differentiated into retinal ganglion cell (RGC) organoids and neurons.
Methodology
1. Prime Editing Technology:
• Prime Editor Selection: Utilizing advanced prime editing systems, likely PE4 or PE5, to enhance editing efficiency and minimize indels.
• epegRNA Design: Designing engineered pegRNAs (epegRNAs) tailored for the specific mutation in Wolfram Syndrome, ensuring stability and efficient editing.
• Delivery Mechanism: Exploring different delivery methods for prime editing components, such as electroporation or viral vectors, suitable for in vivo application in mice and iPSCs.
2. Mouse Model:
• Targeting Mouse Eyes: Implementing prime editing in vivo in mouse models, specifically targeting the eyes to correct the Wolfram Syndrome variant.
• Assessment of Efficacy and Safety: Evaluating the editing efficiency and checking for potential off-target effects or other safety concerns.
3. iPSCs Differentiation and Editing:
• Generation of RGC Organoids and Neurons: Differentiating patient-derived iPSCs into RGC organoids and neurons, replicating the cellular context of Wolfram Syndrome.
• Application of Prime Editing: Applying prime editing techniques to these differentiated cells to correct the Wolfram Syndrome variant.
4. Comparative Analysis:
• Efficiency and Precision: Comparing the efficacy and precision of prime editing in both the in vivo mouse model and the iPSC-derived cells.
• Functional Assessment: Assessing the functional recovery or improvement in the edited cells and tissues.
Objectives and Expected Outcomes
• Primary Objective: To demonstrate that in vivo prime editing can be a viable approach to correct genetic mutations associated with Wolfram Syndrome in both animal models and human-derived cells.
• Anticipated Results: Expectation of high editing efficiency with minimal off-target effects, leading to functional improvement in the disease phenotype.
• Long-term Goal: Paving the way for the development of gene therapy-based treatments for Wolfram Syndrome and similar genetic disorders.
Significance
• Advancement in Gene Therapy: This research could significantly advance the field of gene therapy, particularly for rare genetic diseases like Wolfram Syndrome.
• Methodological Innovation: Establishing a novel application of prime editing in a clinically relevant context, potentially expanding its use in other genetic conditions.
• Contribution to Biomedical Science: Providing insights into the therapeutic potential and limitations of prime editing in living organisms and differentiated human cells.
This research forms a critical step in exploring the translational potential of prime editing, offering hope for gene therapy in treating hereditary diseases.

Graduate publications