PROJECT SUMMARY Genome editors, technologies to modify the genomes of living cells, have extraordinary potential to become safe and effective genomic medicines, direct treatments for the underlying cause of genetic diseases such as sickle cell disease and many others. However, as gene therapy products with novel mechanisms of action, there remains a need for optimized and qualified biochemical IND-enabling assays to assess their safety. We and others have developed sensitive and unbiased research methods for defining the genome-wide activity of editors such as CHANGE-seq and GUIDE-seq. However, they require further optimization and characterization as fit- for-purpose assays to fulfill rigorous regulatory requirements for investigational new drug (IND) submissions. Surprisingly, to our knowledge there are no published methods for high-throughput sequencing characterization of gRNA identity and purity. Thus, there remain urgent unmet needs for publicly available, optimized, and qualified IND-enabling assays to characterize critical genome editing reagents and their associated on- and off- target genome-wide activities. We, therefore, propose the following specific aims: 1) Optimize and qualify CHANGE-seq as IND-enabling biochemical genome-wide activity assay, and 2) Optimize and qualify gRNA sequencing as IND-enabling assay to assess genome editing component identity and purity, and 3) Collaborate to test CHANGE-seq and gRNA sequencing in therapeutic contexts. We anticipate that fulfilling the need for well-characterized assays to identify impurities in critical reagents or characterize key quality attributes of genome editing drug products will have positive impact to accelerate the translation of novel, safe, and effective therapeutic genome editing therapeutic strategies to first-in-human clinical trials.

PROJECT NARRATIVE Genome editing technologies like nucleases, base editors, and prime editors may become safe and effective genomic medicines to treat monogenic human diseases either ex vivo or in vivo. Although fit-for-purpose biochemical methods like CHANGE-seq and gRNA sequencing have advantages as highly-sensitive IND- enabling assays for nucleases and base editors, to our knowledge, fully optimized and qualified protocols for use in regulatory submissions are not generally available. Here we propose to optimize and qualify CHANGE- seq adapted for nucleases and base editors, and associated gRNA sequencing, for use in future therapeutic genome editing INDs.