Validated Immunoassays to Accelerate Therapeutic Genome Editing INDs

PROJECT SUMMARY / ABSTRACT This application is submitted in response to RFA-RM-24-007 with the overriding objective of providing validated assays to understand the immunology of gene editors in pre-clinical studies supporting investigational new drugs (INDs) and eventually in clinical trials. Immunomonitoring is critically important to ensure safety as gene editing therapies transition to the clinical setting for a wide range of applications and diseases. The goals of these studies will be accomplished through the following Specific Aims: (1) Validate a cytokine flow cytometry assay measuring T-cell immunity to commonly used gene editors, vectors, and related peptides from the human peptidome; and (2) Provide a validated multiplex assay for binding antibodies against the domains of relevant gene-editing proteins. We will validate these assays by demonstrating accuracy, precision, specificity, linearity, and range with Quality Assurance monitoring. The assays proposed are based on cytokine flow cytometry and the adaptation of a multiplex microbead immunoassay to accommodate multiple antigens of interest for genome editors being tested for future human use. We will also track the implications of T-cell responses to editors or self-proteins in vivo using total-body positron emission tomography (PET) to assist in fully characterizing the assays proposed. These studies will accelerate the progress of safe and effective somatic cell genome editing therapies to the clinic by providing rigorously validated assays for multiple editing strategies. The assays are designed to address many Cas9-only editors, prime editors, and base editors— while in addition providing a rigorous framework that can be adapted to future gene editors. FDA approved PET imaging methods will also provide a powerful tool for accurately understanding in vivo events and best practices for IND-enabling studies and clinical trials.