Genomic Safe Harbours and Human Gene Therapy: A Review of Candidate Sites, Evaluation Criteria, and Functional Applications.

Genetic engineering, particularly insertion therapies like CRISPR-Cas9, depends on the stability and predictability of transgene integration, modification, or deletion into human DNA. Unintended outcomes can occur due to random or inadequate targeting, ranging from increased oncogene expression to insertional mutagenesis. Therefore, Genomic Safe Harbours are defined as sites capable of supporting transgene integration, modification, or deletion (i) without disrupting essential cellular functions, and (ii) allowing predictable, stable expression across different contexts. In spite of thorough research across chromosomes, no universally applicable, clinically validated Genomic Safe Harbour site has yet been established. While some widely used sites, such as AAVS1, hRosa26, and CCR5, are often considered Genomic Safe Harbour sites, they do not meet the previously mentioned criteria and show variation in expression and integration outcomes. Furthermore, current identification criteria lack consistency across studies, revealing a fundamental gap: the absence of a standardized framework for identifying and comparing such sites. This review synthesizes current research on Genomic Safe Harbour sites, including their genomic landscape and distribution, covering the number of known sites and their conservation, evaluation criteria such as distinguishing between putative and proven sites, functional applications, prediction tools, and future directions for the standardized and quantitative identification and prioritization of these sites.