Discovery and validation of human genomic safe harbor sites for gene and cell therapies

• Published in: Cell reports methods Vol. 2; no. 1; p. 100154
• Main Authors: Aznauryan, Erik, Yermanos, Alexander, Kinzina, Elvira, Devaux, Anna, Kapetanovic, Edo, Milanova, Denitsa, Church, George M., Reddy, Sai T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.01.2022; Elsevier
• Subjects: criteria; durability; expression; genomic safe harbor; knock-in; safety; transcriptomics; expression; genomic safe harbor; criteria; safety; knock-in; durability; transcriptomics
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2021.100154

Existing approaches to therapeutic gene transfer are marred by the transient nature of gene expression following non-integrative gene delivery and by safety concerns due to the random mechanism of viral-mediated genomic insertions. The disadvantages of these methods encourage future research in identifying human genomic sites that allow for durable and safe expression of genes of interest. We conducted a bioinformatic search followed by the experimental characterization of human genomic sites, identifying two that demonstrated the stable expression of integrated reporter and therapeutic genes without malignant changes to the cellular transcriptome. The cell-type agnostic criteria used in our bioinformatic search suggest widescale applicability of identified sites for engineering of a diverse range of tissues for clinical and research purposes, including modified T cells for cancer therapy and engineered skin to ameliorate inherited diseases and aging. In addition, the stable and robust levels of gene expression from identified sites allow for the industry-scale biomanufacturing of proteins in human cells. [Display omitted] •Provides an integrated pipeline to identify human genomic safe harbor sites•Discovers two sites for long-term expression of genes of interest, Rogi1 and Rogi2•Verifies safety of gene expression from these sites by transcriptome profiling•Suggests use cases for identified sites and proposes further validation experiments The ability to express genes of interest in a reliable and safe manner in clinically relevant cells and tissues is critical for successful gene and cell therapies. Several tools allowing for targeted genomic insertions of DNA sequences have recently been developed; however, genomic regions suitable for safe, long-term hosting, and expression of these sequences have not been identified. We describe a pipeline to computationally predict and experimentally validate human genomic safe harbor sites and characterize two sites, Rogi1 and Rogi2, suitable for integrative gene delivery. Aznauryan et al. establish a pipeline for computational prediction and experimental validation of human genomic safe harbor sites. Two genomic sites, Rogi1 and Rogi2, are characterized as capable of safe and durable expression of genes of interest following targeted insertions in a variety of cellular contexts.