PROJECT SUMMARY Genome editors have extraordinary potential to become curative, genomic medicines, but require special scrutiny because unintended ‘off-target’ mutations may be permanent and affect long-term patient safety. Of broad concern is that off-target mutations could activate proto-oncogenes, similar to DNA-integrating viral vectors for gene therapy which have caused leukemia in several patients. For increasing numbers of promising therapeutic genome editing strategies, there remains a critical need for optimized and qualified cellular assays to: 1) define the genome-wide activity of targets to inform lead target identification and 2) to functionally characterize the genome-wide activity of editors during the course of investigational new drug (IND) enabling chemistry, manufacturing, and control (CMC) activities. To our knowledge, the most commonly used method to define cellular genome-wide activity of editors for IND applications is GUIDE-seq, a sensitive and unbiased method we previously developed to define the cellular genome-wide activity of genome editing nucleases. As part of our Somatic Cell Genome Editing (SCGE) phase 1 project, we developed GUIDE-seq-2, a new version of GUIDE-seq with improved scalability and accuracy. The objective of this proposal is to optimize, refine, and qualify GUIDE-seq-2 as a fit-for-purpose assay for evaluation of therapeutic genome editors or associated genome edited cellular drug products in regulatory submissions. We propose the following specific aims: 1) Optimize and qualify GUIDE-seq-2 to assess cellular genome-wide activity for IND applications and 2) Apply GUIDE-seq-2 in novel, therapeutically relevant contexts. Together, the expected outcomes of our proposed studies will be an optimized and qualified GUIDE-seq-2 assay that can be directly approlied to establish critical quality attributes of human genome editing products in IND submissions. We envision that the optimized GUIDE- seq-2 protocol we developed and roadmap for assay qualification will become rapidly widely adopted and have a broad positive impact on development and translation of safe and effective genomic medicines.

PROJECT NARRATIVE Genome editing technologies have potential to become safe new genomic medicines to address fundamental genetic causes of human disease. Although fit-for-purpose cellular methods like GUIDE-seq to define the safety and genome-wide off-target activity of human genome editing products are essential during both pre-clinical and IND enabling phases of development, fully optimized and qualified protocols for use in regulatory submissions are not generally available. Here we propose to optimize and qualify GUIDE-seq-2, an improved, highly scalable, and accurate version of GUIDE-seq we recently developed, for use in future therapeutic genome editing IND submissions.