Ultra-sensitive, unbiased, high-throughput, biochemical CHANGE-seq genome-wide activity and gRNA sequencing assays for therapeutic genome editing INDs

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.