Sensitive, unbiased, high-throughput, cellular GUIDE-seq-2 genome-wide activity assay for therapeutic genome editing INDs - 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.
